Optimized Liver-Specific Expression Systems for FVIII and FIX

Abstract

The present invention relates to nucleic acid expression cassettes and vectors containing liver-specific regulatory elements and codon-optimized factor IX or factor VIII transgenes, methods employing these expression cassettes and vectors and uses thereof. The present invention is particularly useful for applications using liver-directed gene therapy, in particular for the treatment of hemophilia A and B.

Description

FIELD OF THE INVENTION

[0001] The invention relates to nucleic acid expression cassettes and expression vectors for gene therapy with improved liver-specific expression capabilities, particularly for use as a gene therapy means for the treatment of hemophilia, more particularly for restoring coagulation factor IX (FIX) and/or coagulation factor VIII (FVIII) deficiency in liver-directed gene therapy of respectively, hemophilia B and hemophilia A.

BACKGROUND OF THE INVENTION

[0002] Hemophilia B is an X-linked, recessive bleeding disorder caused by deficiency of clotting factor IX (FIX). Hemophilia A is a serious bleeding disorder caused by a deficiency in, or complete absence of, the blood coagulation factor VIII (FVIII). The clinical presentation for hemophilia A and B is characterized by episodes of spontaneous and prolonged bleeding. There are an estimated 1 in 5,000 and 1 in 20,000 individuals suffer from hemophilia A and B, respectively.

[0003] Currently, hemophilia A and B is treated with protein replacement therapy using either plasma-derived or recombinant FVIII or FIX. Although protein replacement markedly improved the life expectancy of patients suffering from hemophilia, they are still at risk for severe bleeding episodes and chronic joint damage, since prophylactic treatment is restricted by the short half-life, the limited availability and the high cost of purified clotting factors, which can approach 100.000$/patient/year. In addition, the use of plasma-derived factors obtained from contaminated blood sources increases the risk of viral transmission. Gene therapy offers the promise of a new method of treating hemophilia B, since the therapeutic window is relatively broad and levels slightly above 1% of normal physiologic levels are therapeutic. If successful, gene therapy could provide constant FVIII or FIX synthesis which may lead to a cure for this disease. The different modalities for gene therapy of hemophilia have been extensively reviewed (Chuah et al., 2012a, 2012b, 2012c; VandenDriessche et al., 2012; High 2001, 2011; Matrai et al., 2010a, 2010b).

[0004] The severity of hemophilia A and hemophilia B has been classified by the subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis into three forms, depending on respectively, the FVIII level and the FIX level: 1) severe form (FVIII or FIX level less than 0.01 international units (IU)/ml, i.e. less than 1% of normal FVIII or FIX level), 2) moderate form (FVIII or FIX level from 0.01 to 0.05 IU/ml, i.e. from 1 to 5% of normal FVIII or FIX level), and 3) mild from (FVIII or FIX level higher than 0.05 to 0.4 IU/ml, i.e. higher than 5 to 40% of normal FVIII or FIX level). Hemophilia A is the most common hereditary coagulation disorder with an incidence approaching approximately 1 in 5000 males.

[0005] Protein substitution therapy (PST) with purified or recombinant FVIII and FIX has significantly improved the patients' quality of life. However, PST is not curative and patients are still at risk of developing potentially life-threatening hemorrhages and crippling joint inflammation. Unfortunately, many patients suffering from hemophilia A (up to 40%) develop neutralizing antibodies to FVIII (i.e. "inhibitors") following PST. Similarly, an estimated 10% of patients suffering from hemophilia B develop "inhibitors" to FIX. These inhibitors complicate the management of bleeding episodes and can render further PST ineffective. These limitations of PST, justify the development of gene therapy as a potential alternative for hemophilia treatment. Furthermore, only a modest increase in FIX or FVIII plasma concentration is needed for therapeutic benefit, with levels of more than 1% of normal levels able to achieve markedly reduced rates of spontaneous bleeding and long-term arthropathy.

[0006] The liver is the main physiological site of FIX and FVIII synthesis and hence, hepatocytes are well suited target cells for hemophilia gene therapy. From this location, FIX or FVIII protein can easily enter into the circulation. Moreover, the hepatic niche may favor the induction of immune tolerance towards the transgene product (Annoni et al., 2007; Follenzi et al., 2004; Brown et al., 2007; Herzog et al., 1999; Matrai et al., 2011; Matsui et al., 2009). Liver-directed gene therapy for hemophilia can be accomplished with different viral vectors including retroviral (Axelrod et al., 1990; Kay et al., 1992; VandenDriessche et al., 1999, Xu et al., 2003, 2005), lentiviral (Ward et al., 2011, Brown et al., 2007, Matrai et al., 2011), adeno-associated viral (AAV) (Herzog et al., 1999) and adenoviral vectors (Brown et al., 2004; Ehrhardt & Kay, 2002). In particular, AAV is a naturally occurring replication defective non-pathogenic virus with a single stranded DNA genome. AAV vectors have a favorable safety profile and are capable of achieving persistent transgene expression. Long-term expression is predominantly mediated by episomally retained AAV genomes. More than 90% of the stably transduced vector genomes are extra-chromosomal, mostly organized as high-molecular-weight concatamers. Therefore, the risk of insertional oncogenesis is minimal, especially in the context of hemophilia gene therapy where no selective expansion of transduced cells is expected to occur. Nevertheless, oncogenic events have been reported following AAV-based gene transfer (Donsante et al., 2007) but it has been difficult to reproduce these findings in other model systems (Li et al., 2011). The major limitation of AAV vectors is the limited packaging capacity of the vector particles (i.e. approximately 5.0 kb, including the AAV inverted terminal repeats), constraining the size of the transgene expression cassette to obtain functional vectors (Jiang et al., 2006). Several immunologically distinct AAV serotypes have been isolated from human and non-human primates (Gao et al., 2002, Gao et al. 2004), although most vectors for hemophilia gene therapy were initially derived from the most prevalent AAV serotype 2. The first clinical success of AAV-based gene therapy for congenital blindness underscores the potential of this gene transfer technology (Bainbridge et al., 2008). AAV-mediated hepatic gene transfer is an attractive alternative for gene therapy of hemophilia for both liver and muscle-directed gene therapy (Herzog et al., 1997, 1999, 2002; Arruda et al., 2010; Fields et al., 2001; Buchlis et al., 2012; Jiang et al., 2006; Kay et al., 2000). Preclinical studies with the AAV vectors in murine and canine models of hemophilia or non-human primates have demonstrated persistent therapeutic expression, leading to partial or complete correction of the bleeding phenotype in the hemophilic models (Snyder et al., 1997, 1999; Wang et al., 1999, 2000; Mount et al., 2002; Nathwani et al., 2002). Particularly, hepatic transduction conveniently induces immune tolerance to FIX that required induction of regulatory T cells (Tregs) (Mingozzi et al., 2003; Dobrzynski et al., 2006). Long-term correction of the hemophilia phenotype without inhibitor development was achieved in inhibitor-prone null mutation hemophilia B dogs treated with liver-directed AAV2-FIX gene therapy (Mount et al, 2002). In order to further reduce the vector dose, more potent FIX expression cassettes have been developed. This could be accomplished by using stronger promoter/enhancer elements, codon-optimized FIX or self-complementary, double-stranded AAV vectors (scAAV) that overcome one of the limiting steps in AAV transduction (i.e. single-stranded to double-stranded AAV conversion) (McCarty, 2001, 2003; Nathwani et al, 2002, 2006, 2011; Wu et al., 2008). Alternative AAV serotypes could be used (e.g. AAV8 or AAV5) that result in increased transduction into hepatocytes, improve intra-nuclear vector import and may reduce the risk of T cell activation (Gao et al., 2002; Vandenberghe et al., 2006) though it is not certain that this would necessarily also translate to human subjects since the epitopes are conserved between distinct AAV serotypes (Mingozzi et al., 2007). Liver-directed gene therapy for hemophilia B with AAV8 or AAV9 is more efficient than when lentiviral vectors are used, at least in mice, and resulted in less inflammation (VandenDriessche et al., 2007, 2002). Furthermore, recent studies indicate that mutations of the surface-exposed tyrosine residues allow the vector particles to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation, which resulted in a 10-fold increase in hepatic expression of FIX in mice (Zhong et al., 2008).

[0007] These liver-directed preclinical studies paved the way toward the use of AAV vectors for clinical gene therapy in patients suffering from severe hemophilia B. Hepatic delivery of AAV-FIX vectors resulted in transient therapeutic FIX levels (maximum 12% of normal levels) in subjects receiving AAV-FIX by hepatic artery catheterization (Kay et al., 2000). However, the transduced hepatocytes were able to present AAV capsid-derived antigens in association with MHC class I to T cells (Manno et al., 2006, Mingozzi et al., 2007). Although antigen presentation was modest, it was sufficient to flag the transduced hepatocytes for T cell-mediated destruction. Recently, gene therapy for hemophilia made an important step forward (Nathwani et al., 2011; Commentary by VandenDriessche & Chuah, 2012). Subjects suffering from severe hemophilia B (<1% FIX) were injected intravenously with self-complementary (sc) AAV8 vectors expressing codon-optimized FIX from a liver-specific promoter. This AAV8 serotype exhibits reduced cross-reactivity with pre-existing anti-AAV2 antibodies. Interestingly, its uptake by dendritic cells may be reduced compared to conventional AAV2 vectors, resulting in reduced T cell activation (Vandenberghe et al., 2006). In mice, AAV8 allows for a substantial increase in hepatic transduction compared to AAV2, though this advantage may be lost in higher species, like dog, rhesus monkeys and man. Subjects received escalating doses of the scAAV8-FIX vector, with two participants per dose. All of the treated subjects expressed FIX above the therapeutic 1% threshold for several months after vector administration, yielding sustained variable expression levels (i.e. 2 to 11% of normal levels). The main difference with the previous liver-directed AAV trial is that for the first time sustained therapeutic FIX levels could be achieved after gene therapy. Despite this progress, T-cell mediated clearance of AAV-transduced hepatocytes remains a concern consistent with elevated liver enzyme levels in some of the patients. Transient immune suppression using a short course of glucocorticoids was used in an attempt to limit this vector-specific immune response.

[0008] One of the significant limitations in the generation of efficient viral gene delivery systems for the treatment of hemophilia A by gene therapy is the large size of the FVIII cDNA. Previous viral vector-based gene therapy studies for hemophilia A typically relied on the use of small but weak promoters, required excessively high vector doses that were not clinically relevant or resulted in severely compromised vector titers. Several other ad hoc strategies were explored, such as the use of split or dual vector design to overcome the packaging constraints of AAV, but these approaches were overall relatively inefficient and raised additional immunogenicity concerns (reviewed in Petrus et al., 2010). It has been found that the FVIII B domain is dispensable for procoagulant activity. Consequently, FVIII constructs in which the B domain is deleted are used for gene transfer purposes since their smaller size is more easily incorporated into vectors. Furthermore, it has been shown that deletion of the B domain leads to a 17-fold increase in mRNA and primary translation product. FVIII wherein the B domain is deleted and replaced by a short 14-amino acid linker is currently produced as a recombinant product and marketed as Refacto.RTM. for clinical use (Wyeth Pharma) (Sandberg et al., 2001). Miao et al. (2004) added back a short B domain sequence to a B domain deleted FVIII, optimally 226 amino acids and retaining 6 sites for N-linked glycosylation, to improve secretion. McIntosh et al. (2013) replaced the 226 amino-acid spacer of Miao et al. with a 17 amino-acid peptide in which six glycosylation triplets from the B-domain were juxtaposed. Yet, production was still not sufficient for therapeutic purposes.

[0009] Non-viral vectors typically rely on a plasmid-based gene delivery system, where only the naked DNA is delivered, potentially in conjunction with physicochemical methods that facilitate transfection. Consequently, the non-viral approach may be less immunogenic and potentially safer than viral vectors, though innate immune response may still occur. The non-viral gene transfer method is simple, but the efficiency is generally low compared to most viral vector-mediated gene transfer approaches. Efficient in vivo gene delivery of non-viral vectors remains a bottleneck. Typically, for hepatic gene delivery, plasmids are administered by hydrodynamic injection. In this case, a hydrodynamic pressure is generated by rapid injection of a large volume of DNA solution into the circulation, in order to deliver the gene of interest in the liver (Miao et al., 2000). Efforts are being made to adapt hydrodynamic injection towards a clinically relevant modality by reducing the volume of injection along with maintaining localized hydrodynamic pressure for gene transfer. Alternative approaches based on targetable nanoparticles are being explored to achieve target specific delivery of FIX into hepatocytes. Expression could be prolonged by removing bacterial backbone sequences which interfere with long term expression (i.e. mini-circle DNA). Finally, to increase the stability of FIX expression after non-viral transfection, transposons could be used that result in stable genomic transgene integration. We and others have shown that transposons could be used to obtain stable clotting factor expression following in vivo gene therapy (Yant et al., 2000; Mates, Chuah et al., 2009, VandenDriessche et al., 2009; Kren et al.,2009; Ohlfest et al., 2004).

[0010] An exemplary state of the art vector for liver-specific expression of FIX is described in WO 2009/130208 and is composed of a single-stranded AAV vector that contains the TTR/Serp regulatory sequences driving a factor cDNA. A FIX first intron was included in the vector, together with a poly-adenylation signal. Using said improved vector yielded about 25-30% stable circulating factor IX.

[0011] In order to translate viral-vector based gene therapy for hemophilia to the clinic, the safety concerns associated with administering large vector doses to the liver and the need for manufacturing large amounts of clinical-grade vector must be addressed. Increasing the potency (efficacy per dose) of gene transfer vectors is crucial towards achieving these goals. It would allow using lower doses to obtain therapeutic benefit, thus reducing potential toxicities and immune activation associated with in vivo administration, and easing manufacturing needs.

[0012] One way to increase potency is to engineer the transgene sequence itself to maximize expression and biological activity per vector copy. We have shown that FIX transgenes optimized for codon usage and carrying an R338L amino acid substitution associated with clotting hyperactivity and thrombophilia (Simioni et al., 2009), increase the efficacy of gene therapy using lentiviral vector up to 15-fold in hemophilia B mice, without detectable adverse effects, substantially reducing the dose requirement for reaching therapeutic efficacy and thus facilitating future scale up and its clinical translation (Cantore et al., 2012, Nair et al., 2014).

[0013] Also codon optimization of human factor VIII cDNAs leads to high-level expression. Significantly greater levels (up to a 44-fold increase and in excess of 200% normal human levels) of active FVIII protein were detected in the plasma of neonatal hemophilia A mice transduced with lentiviral vector expressing FVIII from a codon-optimized cDNA sequence, thereby successfully correcting the disease model (Ward et al., 2011).

[0014] In WO 2014/064277 expression vectors are described which combine the robust Serpin enhancer with codon-optimized transgenes encoding FIX or FVIII, resulting in increased liver-specific expression of FIX and FVIII, respectively.

[0015] It is an object of the present invention to further increase the efficiency and safety of liver-directed gene therapy for hemophilia A and B.

SUMMARY OF THE INVENTION

[0016] It is an object of the present invention to increase the efficiency and safety of liver-directed gene therapy for hemophilia B. The above objective is accomplished by providing a nucleic acid expression cassette and a vector, either a viral vector, in particular an AAV-based vector, or a non-viral vector, comprising specific regulatory elements that enhance liver-directed gene expression, while retaining tissue specificity, in conjunction with the use of a transgene, preferably a codon-optimized transgene, encoding human FIX, preferably human FIX containing a hyper-activating mutation.

[0017] The resulting vector and nucleic acid expression cassette result in unexpectedly high expression levels of FIX in the liver, due to its unique combination of regulatory elements. The combined effect of these elements could not have been predicted. Previously, we reported on a new regulatory element that in combination with other vector elements represented a more than 20-fold increase in FIX levels (cf. WO2014/064277). In the present invention, it is shown that combining 3 copies of the serpin enhancer with the known natural TTR enhancer further increases FIX expression by 6 to 10 fold. This increase in hFIX activity was shown to be synergistic. It is another object of the present invention to increase the efficiency and safety of liver-directed gene therapy for hemophilia A. As shown in the experimental section, this objective is accomplished by providing a vector, either a viral vector, in particular an AAV-based vector, or a non-viral vector, comprising a nucleic acid expression cassette with specific regulatory elements that enhance liver-directed gene expression, while retaining tissue specificity, in conjunction with the use of a codon-optimized human FVIII construct, in particular a codon-optimized B domain deleted FVIII construct driven from a minimal transthyretin promoter. The resulting AAV-based vector and nucleic acid expression cassette resulted in unprecedented, supra-physiologic FVIII expression levels (cf. WO2014/064277). The inventors now demonstrated that the specific combination of three copies of the Serpin enhancer with the natural TTR enhancer, and the codon-optimized B domain deleted FVIII transgene or the codon optimized padua mutant FIX transgene driven from a minimal transthyretin promoter provides for a synergistic effect on FVIII or FIX expression levels compared to expression cassettes containing the natural TTR enhancer and the codon-optimized B domain deleted FVIII transgene or the codon optimized padua mutant FIX transgene driven from a minimal transthyretin promoter.

[0018] The combination of the triple repeat of the Serpin enhancer defined by SEQ ID NO:5 and the transthyretin enhancer defined by SEQ ID NO:12 has been shown to be unexpectedly potent in increasing expression of a transgene operably linked to it. Said regulatory element is defined by SEQ ID NO:13. Said regulatory element can further be combined with the transthyretin minimal promotor as defined by SEQ ID NO.6. This creates a combination of 3x the SerpEnh (3.times. SEQ ID NO.5, e.g. such as in SEQ ID NO.11) with the TTRe and TTRm nucleic acid sequence e.g. as defined by SEQ ID NO.69. For example, such a construct results in a regulatory element as defined by SEQ ID NO:58, which has been tested to increase the expression of both FVIII and FIX transgenes as shown herein.

[0019] The invention therefore provides the following aspects:

[0020] Aspect 1. A nucleic acid expression cassette comprising a triple repeat, preferably a tandem repeat, of a liver-specific nucleic acid regulatory element comprising or consisting of the nucleic acid fragment defined by SEQ ID NO:5 or a sequence having at least 95% identity to said sequence, preferably a liver-specific nucleic acid regulatory element of 150 nucleotides or less, comprising or consisting of the nucleic acid fragment defined by SEQ ID NO:5 or a sequence having at least 95% identity to said sequence, more preferably the regulatory element (3.times. SERP) as defined by SEQ ID NO:11, operably linked to a promoter and a transgene, preferably a codon-optimized transgene, wherein the promoter is a liver-specific promoter.

[0021] Preferably said promoter is derived from the transthyretin (TTR) promoter, more preferably said promoter is the minimal TTR promotor (TTRm) as defined by SEQ ID NO:6.

[0022] In another preferred embodiment, said liver-specific promoter is derived from the AAT promoter, e.g. as defined by SEQ ID NO.64.

[0023] In further embodiments, the liver-specific promotor is selected from the group comprising: the transthyretin (TTR) promoter or TTR-minimal promoter (TTRm), the alpha 1-antitrypsin (AAT) promoter, the albumin promotor (ALB) or minimal albumin promoter (ALBm), the apolipoprotein Al (APOA1) promoter, the complement factor B (CFB) promoter, the ketohexokinase (KHK) promoter, the hemopexin (HPX) promoter, the nicotinamide N-methyltransferase (NNMT) promoter, the (liver) carboxylesterase 1 (CES1) promoter, the protein C (PROC) promoter, the apolipoprotein C3 (APOC3) promoter, the mannan-binding lectin serine protease 2 (MASP2) promoter, the hepcidin antimicrobial peptide (HAMP) promoter, or the serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) promoter.

[0024] Aspect 2. The nucleic acid expression cassette according to aspect 1, further comprising a nucleic acid regulatory element comprising or consisting of the nucleic acid fragment defined by SEQ ID NO:12 (TTRe), or a sequence having at least 95% identity to said sequence, preferably a nucleic acid regulatory element of 150 nucleotides or less comprising or consisting of the nucleic acid fragment defined by SEQ ID NO:12, or a sequence having at least 95% identity to said sequence. In a preferred embodiment, the combination of the TTRe and TTRm nucleic acid modules is defined by SEQ ID NO.69.

[0025] Aspect 3, The nucleic acid expression cassette according to aspect 2, comprising the nucleic acid regulatory element as defined by SEQ ID NO:13, preferably the nucleic acid regulatory element as defined by SEQ ID NO:57, operably linked to a promotor and a transgene, or the nucleic acid regulatory element as defined by SEQ ID NO:58, operably linked to a transgene.

[0026] Aspect 4. The nucleic acid expression cassette according to any one of aspects 1 to 3, wherein said transgene encodes for coagulation factor VIII or coagulation factor IX.

[0027] Aspect 5. The nucleic acid expression cassette according to aspect 4, wherein said coagulation factor VIII has a deletion of the B domain.

[0028] Aspect 6. The nucleic acid expression cassette according to aspect 5, wherein said B domain of said FVIII is replaced by a linker defined by SEQ ID NO:59.

[0029] Aspect 7. The nucleic acid expression cassette according to any one of aspects 4 to 6, wherein said transgene encoding for coagulation factor VIII is defined by SEQ ID NO: 18.

[0030] Aspect 8. The nucleic acid expression cassette according to aspect 4, wherein said coagulation factor IX contains a hyper-activating mutation.

[0031] Aspect 9. The nucleic acid expression cassette according to aspect 8, wherein said hyper-activating mutation in coagulation factor IX corresponds to an R338L amino acid substitution.

[0032] Aspect 10. The nucleic acid expression cassette according to any one of aspects 4, 8 or 9, wherein said transgene encoding for coagulation factor IX is defined by SEQ ID NO:9.

[0033] Aspect 11. The nucleic acid expression cassette according to any one of aspects 1 to 10, wherein said promoter is a liver-specific promoter, preferably a promoter derived from the transthyretin (TTR) promoter, preferably the minimal TTR promotor as defined by SEQ ID NO:6.

[0034] Aspect 12. The nucleic acid expression cassette according to any one of aspects 1 to 11, further comprising a minute virus of mouse (MVM) intron, preferably the MVM intron as defined by SEQ ID NO:8.

[0035] Aspect 13. The nucleic acid expression cassette according to any one of aspects 1 to 12, further comprising a transcriptional termination signal derived from the bovine growth hormone polyadenylation signal (BGHpA), preferably the BGHpA as defined by SEQ ID NO:10, or derived from the Simian virus 40 polyadenylation signal (SV40pA), preferably the SV40pA as defined by SEQ ID NO:19, or a synthetic polyadenylation signal, preferably the synthetic polyadenylation signal as defined by SEQ ID NO: 56.

[0036] Aspect 14. A vector comprising the nucleic acid expression cassette according to any one of aspects 1 to 13.

[0037] Aspect 15. The vector according to aspect 14, wherein said vector is a viral vector.

[0038] Aspect 16. The vector according to aspect 15, wherein said vector is derived from an adeno-associated virus (AAV).

[0039] Aspect 17. The vector according to aspect 16, wherein said vector is a single-stranded AAV vector, preferably a single-stranded AAV serotype 8 vector.

[0040] Aspect 18. The vector according to any one of aspects 14 to 17, defined by SEQ ID NO: 16, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23, preferably SEQ ID NO:22 or SEQ ID NO:23, more preferably SEQ ID NO:22.

[0041] Aspect 19. The vector according to aspect 16, wherein said vector is a self-complementary AAV vector, preferably a self-complementary AAV serotype 9 vector.

[0042] Aspect 20. The vector according to any one of aspects 14 to 16, or 19, defined by SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO:25, preferably SEQ ID NO:4 or SEQ ID NO:25, more preferably SEQ ID NO:4, or the vector according to any one of aspects 14 to 16, or 19, defined by SEQ ID NO: 65, SEQ ID NO:66, or SEQ ID NO:68, preferably SEQ ID NO:65 or 68, more preferably SEQ ID NO:65.Aspect 21. The vector according to aspect 14, wherein said vector is a non-viral vector, such as a transposon-based vector (e.g. a PiggyBac(PB)-based vector or a Sleeping Beauty(SB)-based vector).

[0043] Aspect 22. A method to obtain levels of factor VIII in plasma equal to or higher than the therapeutic threshold concentration of 10 plasma in a subject, comprising the transduction or transfection of the vector according to any one of aspects 14 to 18, or 21 into a subject.

[0044] Aspect 23. The method according to aspect 22, wherein the transduction of the vector according to any one of aspects 14 to 18, or 21 into the subject is done at a dose lower than 2.5.times.10.sup.11 vg/kg.

[0045] Aspect 24. A method to obtain levels of factor IX in plasma equal to or higher than the therapeutic threshold concentration of 10 mU/ml plasma in a subject, comprising the transduction or transfection of the vector according to any one of aspects 14 to 16, or 19 to 21 into a subject.

[0046] Aspect 25. The method according to aspect 24, wherein the transduction of the vector according to any one of aspects 14 to 16, or 19 to 21 into the subject is done at a dose lower than 2.times.10.sup.11 vg/kg.

[0047] Aspect 26. The method according to any one of aspects 22 to 25, wherein said transduction or transfection is by intravenous administration.

[0048] Aspect 27. The method according to any one of aspects 22 to 26, wherein said subject is a mammalian subject, preferably a human subject.

[0049] Aspect 28. A method for treating hemophilia A in a mammalian subject, comprising performing the method according to any one of aspects 22, 23, 26 or 27.

[0050] Aspect 29. The use of the vector according to any one of aspects 14 to 18, or 21 for the manufacture of a medicament to treat hemophilia A.

[0051] Aspect 30. The vector according to any one of aspects 14 to 18, or 21 for use in the treatment of hemophilia A.

[0052] Aspect 31. A method for treating hemophilia B in a mammalian subject, comprising performing the method according to any one of aspects 24 to 27.

[0053] Aspect 32. The use of the vector according to any one of aspects 14 to 16, or 19 to 21 for the manufacture of a medicament to treat hemophilia B.

[0054] Aspect 33. The vector according to any one of aspects 14 to 16, 19 to 21 for use in the treatment of hemophilia B.

[0055] Aspect 34. A pharmaceutical composition comprising a vector according to any one of aspects 14 to 18, or 21 and a pharmaceutically acceptable carrier, optionally further comprising an active ingredient for treating hemophilia A.

[0056] Aspect 35. The pharmaceutical composition according to aspect 34 for use in treating hemophilia A.

[0057] Aspect 36. The pharmaceutical composition for use according to aspect 35, or the vector for use according to aspect 30, wherein said treatment results in levels of factor VIII in plasma of the treated subject that are equal to or higher than the therapeutic threshold concentration of 10 mU/ml plasma in a subject.

[0058] Aspect 37. The pharmaceutical composition for use according to any one of aspects 35 or 35, or the vector for use according to any one of aspects 30 or 36, wherein said treatment comprises the transduction of the vector according to any one of aspects 14 to 18 into the subject at a dose lower than or equal than 2.5.times.10.sup.11 vg/kg.

[0059] Aspect 38. A pharmaceutical composition comprising a vector according to any one of aspects 14 to 16, 19 to 21 and a pharmaceutically acceptable carrier, optionally further comprising an active ingredient for treating hemophilia B.

[0060] Aspect 39. The pharmaceutical composition according to aspect 38, for use in treating hemophilia B.

[0061] Aspect 40. The pharmaceutical composition for use according to aspect 39, or the vector for use according to aspect 33, wherein said treatment results in levels of factor IX in plasma of the treated subject that are equal to or higher than the therapeutic threshold concentration of 10 mU/ml plasma in a subject, preferably equal to or higher than the therapeutic concentration of 50 mU/ml plasma in a subject, more preferably equal to or higher than the therapeutic concentration of 100 mU/nnl plasma in a subject, even more preferably equal to or higher than the therapeutic concentration of 150 mU/ml plasma in a subject and even more preferably equal to or higher than the therapeutic concentration of 200 mU/ml plasma in a subject.

[0062] Aspect 41. The pharmaceutical composition for use according to aspect 39 or 40, or the vector for use according to aspect 33 or 40, wherein said treatment comprises the transduction of the vector according to any one of aspects 14 to 16, 19 or 20 into the subject at a dose lower than or equal than 2.times.10.sup.12 vg/kg, preferably at a dose lower than or equal than 6.times.10.sup.11 vg/kg, more preferably ata dose lower than or equal than 2.times.10.sup.11vg/kg.

BRIEF DESCRIPTION OF THE FIGURES

[0063] The present invention is illustrated by the following figures which are to be considered for illustrative purposes only and in no way limit the invention to the embodiments disclosed therein:

[0064] FIG. 1: Design of the self-complementary (sc), double-stranded adeno-associated viral (AAV) vectors AAVsc-SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA (2510 bp). FIG. 1A: AAVsc-3.times.SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA (2683 bp). FIG. 1B: AAVsc-TTREnh-TTRm-MVM-co-FIX-R338L-BGHpA (2540 bp). FIG. 1C: AAVsc-3.times.SerpEnh-TTREnh-TTRm-MVM-co-FIX-R338L-BGHpA (2760 bp). FIG. 1D: and AAVsc-3.times.SerpEnh-TTREnh-TTRm-MVM-co-FIX-R338L-Synt.pA (2519 bp). FIG. 1E: The minimal transthyretin promoter (TTRm) is driving the expression of the codon-optimized human factor IX (co-FIX-R338L) containing the hyper-activating, thrombophilic FIX mutation (R338L). Either the native TTR enhancer (TTRe), the Serpin enhancer (SERP), a triplet of the Serpin enhancer (3.times.SERP), or a combination of a triplet of the Serpin enhancer (3.times.SERP) and the native TTR enhancer (TTRe) is cloned upstream of TTRm. The minute virus of mouse intron (MVM), the bovine growth hormone polyadenylation site (BGHpA) or a synthetic polyadenylation site (Synt.pA), and the inverted terminal repeats (ITR) are indicated. The sequences flanking/linking the different elements are indicated. The indicated vector sizes includes both ITR's.

[0065] FIG. 2: Design of the single-stranded (ss) adeno-associated viral (AAV) vectors AAVss-TTRm-MVM-co-hFVIII-.DELTA.B-Sv40pA (5160 bp). FIG. 2A: AAVss-SerpEnh-TTRm-MVM-co-hFVIII-.DELTA.B-Sv40pA (5252 bp). FIG. B: AAVss-3.times.SerpEnh-TTRm-MVM-co-hFVIII-.DELTA.B-Sv40pA (5410 bp). FIG. C: AAVss-TTREnh-TTRm-MVM-co-hFVIII-.DELTA.B-Sv40pA (5272 bp). FIG. D: AAVss-3.times.SerpEnh-TTRm-MVM-co-hFVIII-.DELTA.B-Synt.pA (5325 bp). FIG. E: AAVss-3.times.SerpEnh-TTRm-co-hFVIII-AB-Synt .pA (5217 bp). FIG. F: AAVss-3.times.SerpEnh-TTREnh-TTRm-MVM-co-hFVIII-AB-Sv40pA (5493 bp). FIG. G: AAVss-3.times.SerpEnh-TTREnh-TTRm-co-hFVIII-.DELTA.B-Synt.pA (5307 bp). FIG. H: and AAVss-TTREnh-TTRm-co-hFVIII-AB-Synt.pA (5083 bp). FIG. I: The minimal transthyretin promoter (TTRm) is driving the expression of the human codon-optimized B-domain deleted factor VIII (cohFVIIIdeltaB). The native TTR enhancer (TTRe), the Serpin enhancer (SERP), a triplet of the Serpin enhancer (3.times.SERP), or a combination of a triplet of the Serpin enhancer (3.times.SERP) and the native TTR enhancer (TTRe) may be cloned upstream of TTRm. The minute virus of mouse intron (MVM), the Simian Virus 40 (SV40) polyadenylation site (SV40pA) or a synthetic polyadenylation site (Synt.pA), and the inverted terminal repeats (ITR) are indicated. The sequences flanking/linking the different elements are indicated. The indicated vector sizes include both ITR's.

[0066] FIG. 3: FIX protein levels upon transduction of the described SC-vectors. FIG. 3A: the protein expression levels achieved upon transduction with 1.times.10e9 vg/mouse (Low Dose) for the different vector systems described in FIG. 1. FIG. 3B: the protein expression levels achieved upon transduction with 5.times.10e9 vg/mouse (High Dose) for the different vector systems described in FIG. 1.

[0067] FIG. 4: FVIII protein levels upon transduction of the described SS-vectors. FIG. 4A: the protein expression levels achieved upon transduction with 1.times.10e9 vg/mouse (Low Dose) for the different vector systems described in FIG. 2. FIG. 4B: the protein expression levels achieved upon transduction with 5.times.10e9 vg/mouse (High Dose) for the different vector systems described in FIG. 2.

[0068] FIG. 5: Plasmid map of the pAAVsc-SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA vector

[0069] FIG. 6: Plasmid map of the pAAVsc-3.times.SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA vector

[0070] FIG. 7: Plasmid map of the pAAVsc-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA vector

[0071] FIG. 8: Plasmid map of the pAAVsc-3.times.SerpEnh-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA vector

[0072] FIG. 9: Plasmid map of the pAAVss-TTRm-MVM-coFVIIIdeltaB-Sv40pA vector

[0073] FIG. 10: Plasmid map of the pAAVss-SerpEnh-TTRm-MVM-coFVIIIdeltaB-Sv40pA vector

[0074] FIG. 11: Plasmid map of the pAAVss-3.times.SerpEnh-TTRm-MVM-coFVIIIdeltaB-Sv40pA vector

[0075] FIG. 12: Plasmid map of the pAAVss-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA vector

[0076] FIG. 13: Nucleotide sequence of pAAVsc-SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:1); pAAVsc-3.times.SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:2); pAAVsc-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:3); pAAVsc-3.times.SerpEnh-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:4); the Serpin enhancer (SerpEnh) (SEQ ID NO:5); the minimal transthyretin (TTRm) promoter (SEQ ID NO:6); the 5' untranslated region (UTR) of TTRm (TTRm5'UTR) (SEQ ID NO:7); the Minute Virus of Mouse (MVM) intron (SEQ ID NO:8); the codon-optimized transgene encoding human FIX Padua mutant (Co-FIX-R338L) (SEQ ID NO:9); the Bovine Growth Hormone polyadenylation signal (BGHpA) (SEQ ID NO:10); triple tandem repeat of the Serpin enhancer (3.times.SERP) (SEQ ID NO:11, the nucleotide linking the repeats is indicated in bold); the transthyretin enhancer (TTRe) (SEQ ID NO:12); a triple tandem repeat of the Serpin enhancer (underlined) linked to the transthyretin enhancer (italics) (3.times.SERP-Flank-TTRe) (SEQ ID NO:13); pAAVsc-3.times.SerpEnh-TTREnh-TTRm-MVM-co-FIX-R338L-Synt.pA (SEQ ID NO:25); Flank-3.times.SERP-Flank-TTRe (SEQ ID NO:26); Flank-3.times.SERP-Flank-TTRe-Flank (SEQ ID NO:27); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm (SEQ ID NO:28); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank (SEQ ID NO:29); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-MVM (SEQ ID NO:30); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank (SEQ ID NO:31); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L (SEQ ID NO:32), Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Fla- nk (SEQ ID NO:33); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Fla- nk-BGHpA (SEQ ID NO:34); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Fla- nk-BGHpA-Flank (SEQ ID NO:35); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Fla- nk-Synt.pA (SEQ ID NO:36); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-MVM-Flank-co-FIX-R338L-Fla- nk-Synt.pA-Flank (SEQ ID NO:37).

[0077] FIG. 14: Nucleotide sequence of pAAVss-TTRm-MVM-coFV1IldeltaB-Sv40pA (SEQ ID NO:14); pAAVss-SerpEnh-TTRm-MVM- coFVIIIdeltaB-Sv40pA (SEQ ID NO:15); pAAVss-3.times.SerpEnh-TTRm-MVM- coFVIIIdeltaB-Sv40pA (SEQ ID NO:16); pAAVss-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:17); codon-optimized transgene encoding B domain deleted human factor VIII (coFVIIIdeltaB) (SEQ ID NO:18); Simian virus 40 polyadenylation signal (SV40polyA) (SEQ ID NO:19); pAAVss-3.times.SerpEnh-TTRm-MVM-coFVIIIdeltaB-Synt.pA (SEQ ID NO:20); pAAVss-3.times.SerpEnh-TTRm-coFV1IldeltaB-Synt.pA (SEQ ID NO:21); pAAVss-3.times.SerpEnh-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:22); pAAVss-3.times.SerpEnh-TTRe-TTRm-coFVIIIdeltaB-Synt.pA (SEQ ID NO:23); pAAVss-TTRe-TTRnn-coFVIIIdeltaB-Synt. pA (SEQ ID NO:24); Flank-3.times.SERP-F/ank (SEQ ID NO:38); Flank-3.times.SERP-Flank-TTRe (SEQ ID NO:39); Flank-3.times.SERP-Flank-TTRe-Flank (SEQ ID NO:40); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm (SEQ ID NO:41); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank (SEQ ID NO:42); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB (SEQ ID NO:43); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flan- k (SEQ ID NO:44); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flank-SV40pA (SEQ ID NO:45); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flank-SV40pA- -Flank (SEQ ID NO:46); Flank-3.times.SERP-Flank- TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flank-Synt.pA (SEQ ID NO:47); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-Flank-coFVIIIdeltaB-Flank-SyntpA- -Flank (SEQ ID NO:48); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-MVM-Flank (SEQ ID NO:49); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB (SEQ ID NO:50); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-- Flank (SEQ ID NO:51); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-Flank-SV- 40pA (SEQ ID NO:52); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-Flank-SV- 40pA-Flank (SEQ ID NO:53); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-Flank-Sy- nt.pA (SEQ ID NO:54); Flank-3.times.SERP-Flank-TTRe-Flank-TTRm-MVM-Flank-coFVIIIdeltaB-Flank-Sy- nt.pA-Flank (SEQ ID NO:55); synthetic polyadenylation signal (Synt.pA) (SEQ ID NO:56); 3.times.SERP-Flank-TTRe-Flank (SEQ ID NO:57); 3.times.SERP-Flank-TTRe-Flank-TTRm (SEQ ID NO:58).

[0078] FIG. 15: FVIII protein levels in C57BL6 mice injected with the pAAVss-3.times.SerpEnh-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pA and pAAVss-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pA plasmids as described in FIG. 2. C57BL6 mice were injected with 300 ng of the respective plasmids. FVIII protein levels were measured by ELISA in plasma samples collected on day 1 (black bar) and day 2 (white bar) post injection.

[0079] FIG. 16: Plasmid map of the pAAVsc-3.times.SerpEnh-TTRe-TTRm-MVM-co-FIX-R338L-Synt.pA vector

[0080] FIG. 17: Plasmid map of the pAAVss-3.times.SerpEnh-TTRm-MVM-coFVIIIdeltaB-Synt.pA vector

[0081] FIG. 18: Plasmid map of the pAAVss-3.times.SerpEnh-TTRm-coFVIIIdeltaB-Synt.pA vector

[0082] FIG. 19: Plasmid map of the pAAVss-3.times.SerpEnh-TTRe-TTRm-MVM-coFV1IldeltaB-Sv40pA vector

[0083] FIG. 20: Plasmid map of the pAAVss-3.times.SerpEnh-TTRe-TTRm-coFVIIIdeltaB-Synt.pA vector

[0084] FIG. 21: Plasmid map of the pAAVss-TTRe-TTRm-coFVIIIdeltaB-Synt.pA vector.

[0085] FIG. 22: Design of comparative example of self-complementary (sc), double-stranded adeno-associated viral (AAV) vectors expressing co-FVIII delta-B. The following vectors were compared for FVIII expression levels: pAAVss-TTRm-MVM-coFV1IldeltaB-Sv40pA (SEQ ID NO. 14); pAAVss-3.times.SerpEnh-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:16); pAAVss-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:17) and pAAVss-3.times.SerpEnh-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA (SEQ ID NO:22).

[0086] FIG. 23: Design of comparative example of self-complementary (sc), double-stranded adeno-associated viral (AAV) vectors expressing co-FIX. The following vectors were compared for FIX expression: pAAVss-3.times.SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:65); pAAVss-3.times.SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:66); pAAVss-3.times.SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:67); and pAAVss-3.times.SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:68).

[0087] FIG. 24: Nucleotide sequence of AAT promotor (SEQ ID 64); pAAVss-3.times.SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:65); pAAVss-3.times.SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:66); pAAVss-3.times.SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:67); and pAAVss-3.times.SerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA (SEQ ID NO:68).

[0088] FIG. 25: FIG. 25A: Graph depicting the effect of the 3.times.Serp enhancer on the TTRenh-TTRm promoter in regulating the FVIII expression in C57BL6 mice injected with 300 ng of respective plasmids. The FVIII expression profile was tested using the ELISA, in the plasma samples collected Day 1. The presence of 3.times. Serp enhancer seems to elevate the FVIII expression by 8 fold. FIG. 25B: Graph comparing different constructs tested on FVIII expression as outlined in example 2.

DETAILED DESCRIPTION OF THE INVENTION

[0089] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

[0090] Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. The term "comprising" also encompasses the more specific embodiments defined as "consisting of" and "consisting essentially of".

[0091] Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order.

[0092] It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0093] The following terms or definitions are provided to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art.

[0094] The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0095] The invention relates to nucleic acid expression cassettes and expression vectors comprising said nucleic acid expression cassettes for enhancing liver-specific expression of a (trans)gene.

[0096] As used herein, the term "nucleic acid expression cassette" refers to nucleic acid molecules that include one or more transcriptional control elements (such as, but not limited to promoters, enhancers and/or regulatory elements, polyadenylation sequences, and introns) that direct (trans)gene expression in one or more desired cell types, tissues or organs. Typically, the nucleic acid expression cassettes described herein will contain the FIX transgene or the FVIII transgene as defined herein.

[0097] The invention provides nucleic acid expression cassettes comprising one or more liver-specific nucleic acid regulatory elements, operably linked to a promoter and a transgene.

[0098] The term "operably linked" as used herein refers to the arrangement of various nucleic acid molecule elements relative to each such that the elements are functionally connected and are able to interact with each other. Such elements may include, without limitation, a promoter, an enhancer and/or a regulatory element, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a gene of interest to be expressed (i.e., the transgene). The nucleic acid sequence elements, when properly oriented or operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression of the transgene. By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements may be expressed in terms of the 5' terminus and the 3' terminus of each element, and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements.

[0099] A "regulatory element" as used herein refers to transcriptional control elements, in particular non-coding cis-acting transcriptional control elements, capable of regulating and/or controlling transcription of a gene, in particular tissue-specific transcription of a gene. Regulatory elements comprise at least one transcription factor binding site (TFBS), more in particular at least one binding site for a tissue-specific transcription factor, most particularly at least one binding site for a liver-specific transcription factor. Typically, regulatory elements as used herein increase or enhance promoter-driven gene expression when compared to the transcription of the gene from the promoter alone, without the regulatory elements. Thus, regulatory elements particularly comprise enhancer sequences, although it is to be understood that the regulatory elements enhancing transcription are not limited to typical far upstream enhancer sequences, but may occur at any distance of the gene they regulate. Indeed, it is known in the art that sequences regulating transcription may be situated either upstream (e.g. in the promoter region) or downstream (e.g. in the 3'UTR) of the gene they regulate in vivo, and may be located in the immediate vicinity of the gene or further away. Of note, although regulatory elements as disclosed herein typically are naturally occurring sequences, combinations of (parts of) such regulatory elements or several copies of a regulatory element, i.e. non-naturally occurring sequences, are themselves also envisaged as regulatory element. Regulatory elements as used herein may be part of a larger sequence involved in transcriptional control, e.g. part of a promoter sequence. However, regulatory elements alone are typically not sufficient to initiate transcription, but require a promoter to this end.

[0100] The one or more regulatory elements contained in the nucleic acid expression cassettes and vectors disclosed herein are preferably liver-specific. Non-limiting examples of liver-specific regulatory elements are disclosed in WO 2009/130208, which is specifically incorporated by reference herein. Another example of a liver-specific regulatory element is a regulatory element derived from the transthyretin (TTR) gene, such as the regulatory element defined by SEQ ID NO:12, also referred to herein as "TTRe" or "TTREnh"(Wu et al., 2008). `Liver-specific expression`, as used in the application, refers to the preferential or predominant expression of a (trans)gene (as RNA and/or polypeptide) in the liver as compared to other tissues. According to particular embodiments, at least 50% of the (trans)gene expression occurs within the liver. According to more particular embodiments, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% of the (trans)gene expression occurs within the liver. According to a particular embodiment, liver-specific expression entails that there is no `leakage` of expressed gene product to other organs, such as spleen, muscle, heart and/or lung. The same applies mutatis mutandis for hepatocyte-specific expression, which may be considered as a particular form of liver-specific expression. Throughout the application, where liver-specific is mentioned in the context of expression, hepatocyte-specific expression is also explicitly envisaged. Similarly, where tissue-specific expression is used in the application, cell-type specific expression of the cell type(s) predominantly making up the tissue is also envisaged.

[0101] Preferably, the one or more regulatory element in the nucleic acid expression cassettes and vectors disclosed herein is fully functional while being only of limited length. This allows its use in vectors or nucleic acid expression cassettes without unduly restricting their payload capacity. Accordingly, in embodiments, the one or more regulatory element in the expression cassettes and vectors disclosed herein is a nucleic acid of 1000 nucleotides or less, 800 nucleotides or less, or 600 nucleotides or less, preferably 400 nucleotides or less, such as 300 nucleotides or less, 200 nucleotides or less, 150 nucleotides or less, or 100 nucleotides or less (i.e. the nucleic acid regulatory element has a maximal length of 1000 nucleotides, 800 nucleotides, 600 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 150 nucleotides, or 100 nucleotides).

[0102] However, it is to be understood that the disclosed nucleic acid regulatory elements retain regulatory activity (i.e. with regard to specificity and/or activity of transcription) and thus they particularly have a minimum length of 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, or 70 nucleotides. In preferred embodiments, the one or more regulatory element in the nucleic acid expression cassettes and vectors disclosed herein comprises a sequence from SERPINA1 regulatory elements, i.e. regulatory elements that control expression of the SERPINA1 gene in vivo. Said regulatory element preferably comprises, consists essentially of or consists of the sequence as defined in SEQ ID NO:5, a sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as 96%, 97%, 98% or 99%, identity to said sequence, or a functional fragment thereof. Also preferably, said regulatory element has a maximal length of 150 nucleotides or less, preferably 100 nucleotides or less, and comprises, consists essentially of or consists of the sequence as defined in SEQ ID NO:5, a sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as 96%, 97%, 98% or 99%, identity to said sequence, or a functional fragment thereof. The liver-specific nucleic acid regulatory element consisting of SEQ ID NO:5 is herein referred to as "the Serpin enhancer", "SerpEnh", or "Serp".

[0103] In particularly preferred embodiments, the nucleic acid expression cassettes and vectors disclosed herein comprise two or more, such as two, three, four, five or six, preferably three, (tandem) repeats of a liver-specific regulatory element comprising, consisting essentially of or consisting of SEQ ID NO:5, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as 96%, 97%, 98% or 99%, identity to said sequence, more preferably a liver-specific regulatory element of 150 nucleotides or less, preferably 100 nucleotides or less, more preferably 80 nucleotides or less, comprising, consisting essentially of or consisting of SEQ ID NO:5, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as 96%, 97%, 98% or 99%, identity to said sequence. A preferred nucleic acid regulatory element comprising three tandem repeats of SEQ ID NO:5 is herein referred to as "3.times.Serp" and is defined by SEQ ID NO:11.

[0104] In further embodiments, the nucleic acid expression cassettes and vectors disclosed herein comprise two or more, such as two, three, four, five or six, more preferably three, (tandem) repeats of a liver-specific regulatory element comprising, consisting essentially of or consisting of SEQ ID NO:5, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as 96%, 97%, 98% or 99%, identity to said sequence, preferably a liver-specific regulatory element of 150 nucleotides or less, preferably 100 nucleotides or less, more preferably 80 nucleotides or less, comprising, consisting essentially of or consisting of SEQ ID NO:5, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as 96%, 97%, 98% or 99%, identity to said sequence; and a regulatory element comprising, consisting essentially of or consisting of SEQ ID NO:12, preferably a regulatory element of 150 nucleotides or less, preferably 120 nucleotides or less, comprising, consisting essentially of or consisting of SEQ ID NO:12. A preferred liver-specific nucleic acid regulatory element comprising three tandem repeats of SEQ ID NO:5, and SEQ ID NO:12, is herein referred to as "3.times.Serp-flank-TTRe" and is defined by SEQ ID NO:13 A further preferred liver-specific nucleic acid regulatory element comprising three tandem repeats of SEQ ID NO:5, and SEQ ID NO:12, is herein referred to as "3.times.Serp-flank-TTRe-flank" and is defined by SEQ

[0105] ID NO:57. Preferably, the liver-specific nucleic acid regulatory element in the nucleic acid expression cassettes and vectors disclosed herein comprises three tandem repeats of SEQ ID NO:5, and SEQ ID NO:12; more preferably SEQ ID NO:13; even more preferably SEQ ID NO:57, such as SEQ ID NO:27 or SEQ ID NO:39. It has been shown herein that said specific combinations of liver-specific regulatory elements resulted in unexpectedly enhanced liver-specific expression of the transgene, in particular the FIX transgene or FVIII transgene described herein, operably linked thereto.

[0106] In further embodiments, said nucleic acid expression cassettes and vectors disclosed herein comprise three tandem repeats of SEQ ID NO:5 (such as SEQ ID NO.13), and a further enhancer element TTRe defined by SEQ ID NO:12, for example as defined by SEQ ID NO:13, in combination with a liver-specific promotor. In one particular example, said promotor is the TTRm minimal promoter as defined by SEQ ID NO.6. In an alternative embodiment, said liver-specific promoter is the AAT promoter, such as the promoter defined by SEQ ID NO.64. It has been shown herein that said specific combinations of liver-specific regulatory elements resulted in unexpectedly enhanced liver-specific expression of the transgene, in particular the FIX transgene or FVIII transgene described herein, operably linked thereto

[0107] As used herein, the terms "identity" and "identical" and the like refer to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules. Sequence alignments and determination of sequence identity can be done, e.g., using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences" algorithm described by

[0108] Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250). Typically, the percentage sequence identity is calculated over the entire length of the sequence. As used herein, the term "substantially identical" denotes at least 90%, preferably at least 95%, such as 95%, 96%, 97%, 98% or 99%, sequence identity.

[0109] The term "functional fragment" as used in the application refers to fragments of the sequences disclosed herein that retain the capability of regulating liver-specific expression, i.e. they still confer tissue specificity and they are capable of regulating expression of a (trans)gene in the same way (although possibly not to the same extent) as the sequence from which they are derived. Fragments comprise at least 10 contiguous nucleotides from the sequence from which they are derived. In further particular embodiments, fragments comprise at least 15, at least 20, at least 25, at least 30, at least 35 or at least 40 contiguous nucleotides from the sequence from which they are derived. Also preferably, functional fragments may comprise at least 1, more preferably at least 2, at least 3, or at least 4, even more preferably at least 5, at least 10, or at least 15, of the transcription factor binding sites (TFBS) that are present in the sequence from which they are derived.

[0110] As used in the application, the term "promoter" refers to nucleic acid sequences that regulate, either directly or indirectly, the transcription of corresponding nucleic acid coding sequences to which they are operably linked (e.g. a transgene or endogenous gene). A promoter may function alone to regulate transcription or may act in concert with one or more other regulatory sequences (e.g. enhancers or silencers). In the context of the present application, a promoter is typically operably linked to regulatory elements to regulate transcription of a transgene.

[0111] When a regulatory element as described herein is operably linked to both a promoter and a transgene, the regulatory element can (1) confer a significant degree of liver specific expression in vivo (and/or in hepatocytes/hepatic cell lines in vitro) of the transgene, and/or (2) can increase the level of expression of the transgene in the liver (and/or in hepatocytes/hepatocyte cell lines in vitro).

[0112] According to a particular embodiment, the promoter contained in the nucleic acid expression cassettes and vectors disclosed herein is a liver-specific promoter. This is to increase liver specificity and/or avoid leakage of expression in other tissues. According to a further particular embodiment, the liver-specific promoter is from the transthyretin (TTR) gene or from the Alpha-1-antitrypsin (AAT) gene. According to yet a further particular embodiment, the TTR promoter is a minimal promoter (also referred to as TTRm or TRRmin herein), most particularly the minimal TTR promoter as defined in SEQ ID NO: 6. According to yet a further particular embodiment, the AAT promoter is as defined in SEQ ID NO: 64.

[0113] According to particular embodiments, the promoter in the nucleic acid expression cassettes and vectors disclosed herein is a minimal promoter.

[0114] A `minimal promoter` as used herein is part of a full-size promoter still capable of driving expression, but lacking at least part of the sequence that contributes to regulating (e.g. tissue-specific) expression. This definition covers both promoters from which (tissue-specific) regulatory elements have been deleted- that are capable of driving expression of a gene but have lost their ability to express that gene in a tissue-specific fashion and promoters from which (tissue-specific) regulatory elements have been deleted that are capable of driving (possibly decreased) expression of a gene but have not necessarily lost their ability to express that gene in a tissue-specific fashion. Minimal promoters have been extensively documented in the art, a non-limiting list of minimal promoters is provided in the specification.

[0115] The term "liver-specific promoter" encompasses any promoter that confers liver-specific expression to a (trans)gene. Non-limiting examples of liver-specific promoters are provided on the Liver Specific Gene Promoter Database (LSPD, http://rulai.cshl.edu/LSPD/), and include, for example, the transthyretin (TTR) promoter or TTR-minimal promoter (TTRm), the alpha 1-antitrypsin (AAT) promoter, the albumin (ALB) promotor or minimal promoter, the apolipoprotein A1 (APOA1) promoter or minimal promoter, the complement factor B (CFB) promoter, the ketohexokinase (KHK) promoter, the hemopexin (HPX) promoter or minimal promoter, the nicotinamide N-methyltransferase (NNMT) promoter or minimal promoter, the (liver) carboxylesterase 1 (CES1) promoter or minimal promoter, the protein C (PROC) promoter or minimal promoter, the apolipoprotein C3 (APOC3) promoter or minimal promoter, the mannan-binding lectin serine protease 2 (MASP2) promoter or minimal promoter, the hepcidin antimicrobial peptide (HAMP) promoter or minimal promoter, and the serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) promoter or minimal promoter.

[0116] In particularly preferred embodiments, the promoter is a mammalian liver-specific promoter, in particular a murine or human liver-specific promoter. More preferably, said promoters are the respective minimal promoters

[0117] The term "liver-specific expression" as used in the application, refers to the preferential or predominant expression of a (trans)gene (as RNA and/or polypeptide) in the liver, in liver tissue or in liver cells, as compared to other (i.e. non-liver) tissues or cells. According to particular embodiments, at least 50%, more particularly at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% of the (trans)gene expression occurs within liver tissue or liver cells. According to a particular embodiment, liver-specific expression entails that there is no `leakage` of expressed gene product to other organs or tissue than liver, such as lung, muscle, brain, kidney and/or spleen. The same applies mutatis mutandis for hepatocyte-specific expression and hepatoblast-specific expression, which may be considered as particular forms of liver-specific expression. Throughout the application, where liver-specific is mentioned in the context of expression, hepatocyte-specific expression and hepatoblast-specific expression are also explicitly envisaged.

[0118] As used herein, the term "liver cells" encompasses the cells predominantly populating the liver and encompasses mainly hepatocytes, oval cells, liver sinusoidal endothelial cells (LSEC) and cholangiocytes (epithelial cells forming the bile ducts).

[0119] The term "hepatocyte," as used herein, refers to a cell that has been differentiated from a progenitor hepatoblast such that it is capable of expressing liver-specific phenotype under appropriate conditions. The term "hepatocyte" also refers to hepatocytes that are de-differentiated. The term includes cells in vivo and cells cultured ex vivo regardless of whether such cells are primary or passaged.

[0120] The term "hepatoblast" as used herein, refers to an embryonic cell in the mesoderm that differentiates to give rise to a hepatocyte, an oval cell, or a cholangiocyte. The term includes cells in vivo and cells cultured ex vivo regardless of whether such cells are primary or passaged.

[0121] The term "transgene" or "(trans)gene" as used herein refers to particular nucleic acid sequences encoding a polypeptide or a portion of a polypeptide to be expressed in a cell into which the nucleic acid sequence is inserted. However, it is also possible that transgenes are expressed as RNA, typically to lower the amount of a particular polypeptide in a cell into which the nucleic acid sequence is inserted. These RNA molecules include but are not limited to molecules that exert their function through RNA interference (shRNA, RNAi), micro-RNA regulation (miRNA), catalytic RNA, antisense RNA, RNA aptamers, etc. How the nucleic acid sequence is introduced into a cell is not essential to the invention, it may for instance be through integration in the genome or as an episomal plasmid. Of note, expression of the transgene may be restricted to a subset of the cells into which the nucleic acid sequence is inserted. The term `transgene` is meant to include (1) a nucleic acid sequence that is not naturally found in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence naturally found in the cell into which it has been introduced ; (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or a similar nucleic acid sequence naturally occurring in the cell into which it has been introduced ; or (4) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been introduced. By `mutant form` is meant a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or naturally occurring sequence, i.e., the mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and/or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence such that the transgene product will be secreted from the cell.

[0122] Typically, the transgenes in the expression cassettes and vectors described herein encode coagulation factor IX or coagulation factor VIII.

[0123] The term "coagulation factor IX" has the meaning as known in the art. Synonyms of coagulation factor IX are "FIX" or "Christmas factor" or "F9" and can be used interchangeably.

[0124] In particular, the term "coagulation factor IX" encompasses the human protein encoded by the mRNA sequence as defined in Genbank accession number NM_000133.

[0125] Preferably, said FIX is a mutated FIX, which is hyperactive or hyper-functional as compared to the wild type FIX. Modifying functional activity of human coagulation factor can be done by bioengineering e.g. by introduction of point mutations. By this approach a hyperactive R338A variant was reported, which showed a 3 fold increased clotting activity compared to the wild type human FIX in an in vitro activated partial thromboplastin time assay (APPT) (Chang et al., 1998) and a 2 to 6-fold higher specific activity in hemophilia B mice transduced with the mutant FIX gene (Schuettrumpf et al., 2005). Further exemplary FIX point-mutants or domain exchange mutants with even higher clotting activities have been described: FIX, with the EGF-1 domain replaced with the EGF-1 domain from FVII, alone or in combination with a R338A point mutation (Brunetti-Pierri et al., 2009), the V86A/E277A/R338A triple mutant (Lin et al., 2010), the Y259F, K265T, and/or Y345T single, double or triple mutants (Milanov, et al., 2012), and the G190V point mutant (Kao et al., 2010), all incorporated herein by reference. In a particularly preferred embodiment, the FIX mutant is the one described by Simioni et al., in 2009 and denominated as the "factor IX Padua" mutant, causing X-linked thrombophilia. Said mutant factor IX is hyperactive and carries an R338L amino acid substitution. In a preferred embodiment of the present invention, the FIX transgene used in nucleic acid expression cassettes and expression vectors described herein encodes the human FIX protein, most preferably the FIX transgene encodes for the Padua mutant of the human FIX protein.

[0126] Accordingly, in a particularly preferred embodiment of the present invention, the transgene has SEQ ID NO:9 (i.e. codon-optimized transgene encoding for the Padua mutant of the human FIX protein).

[0127] The term "coagulation factor VIII" has the meaning as known in the art. Synonyms of coagulation factor VIII are "FVIII" or "anti-hemophilic factor" or "AHF" and can be used interchangeably herein. The term "coagulation factor VIII" encompasses, for example, the human protein having the amino acid sequence as defined in Uniprot accession number P00451.

[0128] In embodiments, said FVIII is a FVIII wherein the B domain is deleted (i.e. B domain deleted FVIII, also referred to as BDD FVIII or FVIIIAB or FVIIIdeltaB herein). The term "B domain deleted FVIII" encompasses for example, but without limitation, FVIII mutants wherein whole or a part of the B domain is deleted and FVIII mutants wherein the B domain is replaced by a linker. Non-limiting examples of B domain deleted FVIII are described in Ward et al. (2011) and WO 2011/005968, which are specifically incorporated by reference herein.

[0129] In preferred embodiments, said FVIII is B domain deleted FVIII wherein the B domain is replaced by a linker having the following sequence: SFSQNPPVLTRHQR (SEQ ID NO: 59) (i.e. SQ FVIII as defined in Ward et al. (2011)). In particularly preferred embodiments, said transgene encoding FVIII has SEQ ID NO:18 (i.e. codon-optimized transgene encoding B domain deleted human FVIII, also referred to herein as (h)FVIIIcopt or co(h)FVIIIdeltaB or co(h)FVIIIdeltaB transgene), as disclosed also in WO 2011/005968.

[0130] Other sequences may be incorporated in the nucleic acid expression cassette disclosed herein as well, typically to further increase or stabilize the expression of the transgene product (e.g. introns and/or polyadenylation sequences).

[0131] Any intron can be utilized in the expression cassettes described herein. The term "intron" encompasses any portion of a whole intron that is large enough to be recognized and spliced by the nuclear splicing apparatus. Typically, short, functional, intron sequences are preferred in order to keep the size of the expression cassette as small as possible which facilitates the construction and manipulation of the expression cassette. In some embodiments, the intron is obtained from a gene that encodes the protein that is encoded by the coding sequence within the expression cassette. The intron can be located 5' to the coding sequence, 3' to the coding sequence, or within the coding sequence. An advantage of locating the intron 5' to the coding sequence is to minimize the chance of the intron interfering with the function of the polyadenylation signal. In embodiments, the nucleic acid expression cassette disclosed herein further comprises an intron. Non-limiting examples of suitable introns are Minute Virus of Mice (MVM) intron, beta-globin intron (betalVS-II), factor IX (FIX) intron A, Simian virus 40 (SV40) small-t intron, and beta-actin intron. Preferably, the intron is an MVM intron, more preferably the MVM mini-intron as defined by SEQ ID NO: 8. The cloning of the MVM intron into a nucleic acid expression cassette described herein was shown to result in unexpectedly high expression levels of the transgene operably linked thereto.

[0132] Any polyadenylation signal that directs the synthesis of a polyA tail is useful in the expression cassettes described herein, examples of those are well known to one of skill in the art. Exemplary polyadenylation signals include, but are not limited to, polyA sequences derived from the Simian virus 40 (SV40) late gene, the bovine growth hormone (BGH) polyadenylation signal, the minimal rabbit f3-globin (mRBG) gene, and the synthetic polyA (SPA) site as described in Levitt et al. (1989, Genes Dev 3:1019-1025) (SEQ ID NO:56). Preferably, the polyadenylation signal is the bovine growth hormone (BGH) polyadenylation signal (SEQ ID NO:10) or the Simian virus 40 (SV40) polyadenylation signal (SEQ ID NO:19).

[0133] Typically, the nucleic acid expression cassette according to the invention comprises a promotor, an enhancer, a (trans)gene, and a transcription terminator.

[0134] In a typical embodiment of the present invention, a nucleic acid expression cassette is disclosed and comprises:

[0135] a liver-specific nucleic acid regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQ ID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12,

[0136] a liver-specific promoter, and

[0137] a transgene.

[0138] In a typical embodiment of the present invention, a nucleic acid expression cassette is disclosed and comprises:

[0139] a liver-specific nucleic acid regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQ ID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12,

[0140] the liver-specific TTRm promoter (e.g. defined by SEQ ID NO.6, and

[0141] a transgene, preferably wherein the combination of the TTRe and TTRm nucleic acids is defined by SEQ ID NO.69.

[0142] In a typical embodiment of the present invention, a nucleic acid expression cassette is disclosed and comprises:

[0143] a liver-specific nucleic acid regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQ ID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12,

[0144] the liver-specific TTRm promoter, e.g. as defined by SEQ ID NO.6,

[0145] an intron, preferably the MVM intron, e.g. as defined by SEQ ID NO.8, and

[0146] a transgene preferably wherein the combination of the TTRe and TTRm nucleic acids is defined by SEQ ID NO.69.

[0147] In a typical embodiment of the present invention, a nucleic acid expression cassette is disclosed and comprises:

[0148] a liver-specific nucleic acid regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQ ID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12,

[0149] the liver-specific TTRm promoter, e.g. as defined by SEQ ID NO.6,

[0150] an intron, preferably the MVM intron, e.g. as defined by SEQ ID NO.8, and

[0151] a transgene, preferably the FIX or FVIII transgene as defined herein elsewhere and optionally a transcription terminator as defined herein elsewhere, preferably wherein the combination of the TTRe and TTRm nucleic acids is defined by SEQ ID NO.69.

[0152] In another preferred embodiment of the present invention, a nucleic acid expression cassette is disclosed and comprises:

[0153] a liver-specific nucleic acid regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQ ID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12,

[0154] the liver-specific AAT promoter, e.g. as defined by SEQ ID NO.64 , and

[0155] a transgene.

[0156] In another preferred embodiment of the present invention, a nucleic acid expression cassette is disclosed and comprises:

[0157] a liver-specific nucleic acid regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQ ID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12,

[0158] the liver-specific AAT promoter, e.g. as defined by SEQ ID NO.64 , and

[0159] an intron, preferably the MVM intron, e.g. as defined by SEQ ID NO.8, and

[0160] a transgene.

[0161] In another preferred embodiment of the present invention, a nucleic acid expression cassette is disclosed and comprises:

[0162] a liver-specific nucleic acid regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQ ID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12,

[0163] the liver-specific AAT promoter, e.g. as defined by SEQ ID NO.64, and

[0164] an intron, preferably the MVM intron, e.g. as defined by SEQ ID NO.8, and

[0165] a transgene, preferably the FIX or FVIII transgene as defined herein elsewhere and optionally a transcription terminator as defined herein elsewhere.

[0166] In a typical embodiment of the invention, said nucleic acid expression cassette disclosed herein comprises:

[0167] a liver-specific regulatory element, preferably three tandem repeats of the Serpin enhancer (SEQ ID NO.5), e.g. a regulatory element as defined by SEQ ID NO:11,

[0168] a promoter, preferably the minimal TTR promoter,

[0169] an intron, preferably the MVM intron

[0170] a transgene, preferably codon-optimized factor IX cDNA, even more preferably codon-optimized factor IX Padua cDNA

[0171] a transcription terminator, preferably a polyadenylation signal such as the bovine growth hormone polyadenylation signal (BGHpA) e.g. as defined by SEQ ID NO.10, or the synthetic polyA site as defined by SEQ ID NO:56.

[0172] In a further typical embodiment of the present invention, said nucleic acid expression cassette disclosed herein comprises:

[0173] a liver-specific regulatory element, preferably three tandem repeats of the Serpin enhancer and the transthyretin enhancer (TTRe) (e.g. a regulatory element comprising SEQ ID NO:13, preferably comprising SEQ ID NO:57),

[0174] a promoter, preferably the minimal TTR promoter,

[0175] an intron, preferably the MVM intron,

[0176] a (trans)gene, preferably codon-optimized factor IX cDNA, even more preferably codon-optimized factor IX Padua cDNA,

[0177] a transcription terminator, preferably a polyadenylation signal such as the bovine growth hormone polyadenylation signal (BGHpA) or the synthetic polyA site as defined by SEQ ID NO:56.

[0178] In another typical embodiment of the present invention, said nucleic acid expression cassette disclosed herein comprises:

[0179] a liver-specific regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. a regulatory element comprising SEQ ID NO:11), more preferably three tandem repeats of the Serpin enhancer and the transthyretin enhancer (TTRe) (e.g. a regulatory element comprising SEQ ID NO:13, preferably comprising SEQ ID NO:57),

[0180] a promoter, preferably the minimal TTR promoter,

[0181] an intron, preferably the MVM intron,

[0182] a (trans)gene, preferably codon-optimized factor VIII cDNA, even more preferably codon-optimized B domain deleted factor VIII cDNA,

[0183] a transcription terminator, preferably a polyadenylation signal such as the Simian vacuolating virus 40 or Simian virus 40 (SV40) polyadenylation signal or the synthetic polyA site as defined by SEQ ID NO:56.

[0184] In another typical embodiment of the present invention, said nucleic acid expression cassette disclosed herein comprises:

[0185] a liver-specific regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQ ID NO:11 and the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12, such as the regulatory element comprising SEQ ID NO:13, preferably comprising SEQ ID NO:57,

[0186] a promoter, preferably the AAT promoter such as the promoter as defined by SEQ ID NO. 64,

[0187] an intron, preferably the MVM intron such as the MVM intron as defined by SEQ ID NO.8, a (trans)gene, preferably codon-optimized factor IX cDNA, even more preferably codon-optimized factor IX Padua cDNA,

[0188] a transcription terminator, preferably a polyadenylation signal such as the bovine growth hormone polyadenylation signal (BGHpA) as defined by SEQ ID NO.10, or the synthetic polyA site as defined by SEQ ID NO:56. As a non-limiting example, such a vector is defined by SEQ ID NO. 65 (cf. FIG. 25).

[0189] In another typical embodiment of the present invention, said nucleic acid expression cassette disclosed herein comprises:

[0190] a liver-specific regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), such as a regulatory element comprising SEQ ID NO:11,

[0191] a promoter, preferably the AAT promoter such as the promoter as defined by SEQ ID NO. 64,

[0192] an intron, preferably the MVM intron such as the MVM intron as defined by SEQ ID NO.8 , a (trans)gene, preferably codon-optimized factor IX cDNA, even more preferably codon-optimized factor IX Padua cDNA,

[0193] a transcription terminator, preferably a polyadenylation signal such as the bovine growth hormone polyadenylation signal (BGHpA) as defined by SEQ ID NO.10, or the synthetic polyA site as defined by SEQ ID NO:56. As a non-limiting example, such a vector is defined by SEQ ID NO. 66 (cf. FIG. 25).

[0194] In another typical embodiment of the present invention, said nucleic acid expression cassette disclosed herein comprises:

[0195] a liver-specific regulatory element, preferably a regulatory element comprising the transthyretin enhancer (TTRe) as defined by SEQ ID NO.12, operably linked to three tandem repeats of the Serpin enhancer (e.g. SEQ ID NO.5), more preferably as defined by SEQ ID NO:11,

[0196] a promoter, preferably the AAT promoter such as the promoter as defined by SEQ ID NO. 64,

[0197] an intron, preferably the MVM intron such as the MVM intron as defined by SEQ ID NO.8, a (trans)gene, preferably codon-optimized factor IX cDNA, even more preferably codon-optimized factor IX Padua cDNA,

[0198] a transcription terminator, preferably a polyadenylation signal such as the bovine growth hormone polyadenylation signal (BGHpA) as defined by SEQ ID NO.10, or the synthetic polyA site as defined by SEQ ID NO:56. As a non-limiting example, such a vector is defined by SEQ ID NO.68 (cf. FIG. 25).

[0199] The expression cassettes disclosed herein may be used as such, or typically, they may be part of a nucleic acid vector. Accordingly, a further aspect relates to the use of a nucleic acid expression cassette as described herein in a vector, in particular a nucleic acid vector.

[0200] In an aspect, the invention also provides a vector comprising a nucleic acid expression cassette as disclosed herein.

[0201] The term `vector` as used in the application refers to nucleic acid molecules, usually double-stranded DNA, which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a cDNA molecule. The vector is used to transport the insert nucleic acid molecule into a suitable host cell. A vector may contain the necessary elements that permit transcribing the insert nucleic acid molecule, and, optionally, translating the transcript into a polypeptide. The insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated.

[0202] The term "vector" may thus also be defined as a gene delivery vehicle that facilitates gene transfer into a target cell. This definition includes both non-viral and viral vectors. Non-viral vectors include but are not limited to cationic lipids, liposomes, nanoparticles, PEG, PEI, etc. Viral vectors are derived from viruses including but not limited to: retrovirus, lentivirus, adeno-associated virus, adenovirus, herpesvirus, hepatitis virus or the like. Alternatively, gene delivery systems can be used to combine viral and non-viral components, such as nanoparticles or virosomes (Yamada et al., 2003).

[0203] Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. Preferred vectors are derived from adeno-associated virus, adenovirus, retroviruses and Antiviruses.

[0204] Retroviruses and Antiviruses are RNA viruses that have the ability to insert their genes into host cell chromosomes after infection. Retroviral and lentiviral vectors have been developed that lack the genes encoding viral proteins, but retain the ability to infect cells and insert their genes into the chromosomes of the target cell (Miller, 1990; Naldini et al., 1996, VandenDriessche et al., 1999). The difference between a lentiviral and a classical Moloney-murine leukemia-virus (MLV) based retroviral vector is that lentiviral vectors can transduce both dividing and non-dividing cells whereas MLV-based retroviral vectors can only transduce dividing cells.

[0205] Adenoviral vectors are designed to be administered directly to a living subject. Unlike retroviral vectors, most of the adenoviral vector genomes do not integrate into the chromosome of the host cell. Instead, genes introduced into cells using adenoviral vectors are maintained in the nucleus as an extrachromosomal element (episome) that persists for an extended period of time. Adenoviral vectors will transduce dividing and nondividing cells in many different tissues in vivo including airway epithelial cells, endothelial cells, hepatocytes and various tumors (Trapnell, 1993; Chuah et al., 2003). Another viral vector is derived from the herpes simplex virus, a large, double-stranded DNA virus. Recombinant forms of the vaccinia virus, another dsDNA virus, can accommodate large inserts and are generated by homologous recombination.

[0206] Adeno-associated virus (AAV) is a small ssDNA virus which infects humans and some other primate species, not known to cause disease and consequently causing only a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy, although the cloning capacity of the vector is relatively limited. Accordingly, in preferred embodiments of the invention, the vector used is derived from adeno-associated virus (i.e. AAV vector).

[0207] Different serotypes of AAVs have been isolated and characterized, such as, for example AAV serotype 2, AAV serotype 5, AAV serotype 8, and AAV serotype 9, and all AAV serotypes are contemplated herein. In particular, AAV vectors that comprise a FIX transgene as disclosed herein are preferably AAV serotype 9 vectors, and AAV vectors that comprise a FVIII transgene as disclosed herein are preferably AAV serotype 8 vectors.

[0208] The AAV vectors disclosed herein may be single-stranded (i.e. ssAAV vectors) or self-complementary (i.e. scAAV vectors). In particular, AAV vectors that comprise a FIX transgene as disclosed herein are preferably self-complementary, and AAV vectors that comprise a FVIII transgene as disclosed herein are preferably single-stranded. With the term "self-complementary AAV" is meant herein a recombinant AAV-derived vector wherein the coding region has been designed to form an intra-molecular double-stranded DNA template.

[0209] Gene therapy with adeno-associated viral vectors disclosed herein was shown to induce immune tolerance towards the transgene comprised in the vector.

[0210] In embodiments, the vector according to the invention comprises the following elements (cfr. FIG. 6):

[0211] an Inverted Terminal Repeat sequence (ITR), optionally mutated,

[0212] a liver-specific regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer ("Serp" or "SerpEnh") (e.g. a regulatory element comprising the nucleic acid fragment defined by SEQ ID NO:11),

[0213] a promoter, preferably the minimal TTR promoter (TTRm),

[0214] an intron, preferably the MVM intron,

[0215] a (trans)gene, preferably codon-optimized factor IX cDNA, even more preferably codon-optimized factor IX Padua cDNA,

[0216] a transcription terminator, preferably a polyadenylation signal such as the BGHpA or the synthetic polyA site as defined by SEQ ID NO:56,

[0217] an Inverted Terminal Repeat sequence (ITR).

[0218] Preferably, the vector is an adeno-associated virus-derived vector, more preferably a self-complementary AAV vector, even more preferably a self-complementary AAV serotype 9 vector, such as the vector as defined by SEQ ID NO:2.

[0219] In a further typical embodiment of the present invention, said vector comprises the following elements (cf. FIG. 8 and FIG. 16):

[0220] an Inverted Terminal Repeat sequence (ITR), optionally mutated,

[0221] a liver-specific regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer ("Serp" or "SerpEnh") and the transthyretin enhancer (TTRe) (e.g. a regulatory element comprising SEQ ID NO:13, preferably comprising SEQ ID NO:57),

[0222] a promoter, preferably the minimal TTR promoter (TTRm),

[0223] an intron, preferably the MVM intron,

[0224] a (trans)gene, preferably codon-optimized factor IX cDNA, even more preferably codon-optimized factor IX Padua cDNA,

[0225] a transcription terminator, preferably a polyadenylation signal such as the BGHpA or the synthetic polyA site as defined by SEQ ID NO:56, and

[0226] an Inverted Terminal Repeat sequence (ITR).

[0227] The combination of said elements resulted in an unexpectedly high expression level of FIX, and in particular of the Padua mutant thereof, in the liver of subjects. Preferably, the vector is an adeno-associated virus-derived vector, more preferably a self-complementary AAV vector, even more preferably a self-complementary AAV serotype 9 vector, such as the vector as defined by SEQ ID NO:4 or SEQ ID NO:25.

[0228] In another typical embodiment of the present invention, said vector comprises the following elements (cfr. FIG. 11, 17 or 18):

[0229] an Inverted Terminal Repeat sequence (ITR), optionally mutated,

[0230] a liver-specific regulatory element, preferably a regulatory element comprising three tandem repeats of the Serpin enhancer (e.g. a regulatory element comprising SEQ ID NO:11),

[0231] a promoter, preferably the minimal TTR promoter,

[0232] an intron, preferably the MVM intron,

[0233] a (trans)gene, preferably codon-optimized factor VIII cDNA, even more preferably codon-optimized B domain deleted factor VIII cDNA,

[0234] a transcription terminator, preferably a polyadenylation signal such as the Simian vacuolating virus 40 or Simian virus 40 (SV40) polyadenylation signal or the synthetic polyA site as defined by SEQ ID NO:56, and

[0235] an Inverted Terminal Repeat sequence (ITR).

[0236] The combination of said elements resulted in an unexpectedly high expression level of FVIII specifically in the liver of subjects. Preferably, the vector is an adeno-associated virus(AAV)-derived vector, more preferably a single-stranded AAV vector, even more preferably a single-stranded AAV serotype 8 vector, such as the vector as defined by SEQ ID NO:16, SEQ ID NO:20 or SEQ ID NO:21.

[0237] In a further embodiment, said vector comprises the following elements (cfr. FIG. 19 or 20):

[0238] an Inverted Terminal Repeat sequence (ITR), optionally mutated,

[0239] a liver-specific regulatory element, preferably preferably a regulatory element comprising three tandem repeats of the Serpin enhancer ("Serp" or "SerpEnh") and the transthyretin enhancer (TTRe) (e.g. a regulatory element comprising SEQ ID NO:13, preferably comprising SEQ ID NO:57),

[0240] a promoter, preferably the minimal TTR promoter,

[0241] an intron, preferably the MVM intron,

[0242] a (trans)gene, preferably codon-optimized factor VIII cDNA, even more preferably codon-optimized B domain deleted factor VIII cDNA,

[0243] a transcription terminator, preferably a polyadenylation signal such as the Simian vacuolating virus 40 or Simian virus 40 (SV40) polyadenylation signal or the synthetic polyA site as defined by SEQ ID NO:56, and

[0244] an Inverted Terminal Repeat sequence (ITR).

[0245] The combination of said elements resulted in an unexpectedly high expression level of FVIII specifically in the liver of subjects. Preferably, the vector is an adeno-associated virus(AAV)-derived vector, more preferably a single-stranded AAV vector, even more preferably a single-stranded AAV serotype 8 vector, such as the vector as defined by SEQ ID NO:22 or SEQ ID NO:23.

[0246] The combination of the triple repeat of the Serpin enhancer defined by SEQ ID NO. 5 and the transthyretin enhancer defined by SEQ ID NO:12 including a specific spacer fragment has been shown to be unexpectedly potent in increasing expression of a transgene operably linked to it. Said regulatory element is defined by SEQ ID NO:13. Said regulatory element can further be combined with the transthyretin minimal promotor as defined by SEQ ID NO.6. This creates a combination of 3.times. the SerpEnh (3.times. SEQ ID NO.5, e.g. such as in SEQ ID NO.11) with the TTRe and TTRm nucleic acid sequence e.g. as defined by SEQ ID NO.69. For example, such a construct results in a regulatory element as defined by SEQ ID NO:58, which has been tested to increase the expression of both FVIII and FIX transgenes as shown herein.

[0247] In specific embodiments the following plamids/vectors are provided: pAAVsc-3.times.CRM8-TTRe-TTRm-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-TTRm-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-TTRm-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-TTRm-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-TTRm-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-TTRm-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-AAT-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-AAT-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-AAT-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-AAT-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-AAT-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-AAT-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-ALBp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-ALBp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-ALBp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-ALBp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-ALBp-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-ALBp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-APOA1p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-APOA1p-MVM-FVIIIcodeltaB-sv40pA pAAVsc-TTRe-3.times.CRM8-APOA1p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-APOA1p-MVM-FVIIIcodeltaB-sv40pA pAAVsc-CRM8-TTRe-APOA1p-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-APOA1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-CFBp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-CFBp-MVM-FVI I IcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-CFBp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-CFBp-MVM-FVI I IcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-CFBp-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-CFBp-MVM-FVI I IcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-KHKp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-KHKp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-KHKp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-KHKp-MVM-FVI I IcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-KHKp-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-KHKp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-HPXp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-HPXp-MVM-FVI I IcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-HPXp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-HPXp-MVM-FVI I IcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-HPXp-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-HPXp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-NNMTp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-NNMTp-MVM-FVI I IcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-NNMTp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-NNMTp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-NNMTp-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-NNMTp-MVM-FVI I IcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-CES1p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-CES1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-CES1p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-CES1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-CES1p-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-CES1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-PROCp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-PROCp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-PROCp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-PROCp-MVM-FVI I IcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-PROCp-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-PROCp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-APOC3p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-APOC3p-MVM-FVII IcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-APOC3p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-APOC3p-MVM-FVII IcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-APOC3p-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-APOC3p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-MASP2p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-MASP2p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-MASP2p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-MASP2p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-MASP2p-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-MASP2p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-SERPINC1p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-SERPINC1p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-SERPINC1p-MVM-FIXcoR338L-BGHpA, pAAVss-CRM8-TTRe-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-3.times.CRM8-TTRe-HAMPp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-HAMPp-MVM-FVIIIcodeltaB-sv40pA pAAVsc-TTRe-3.times.CRM8-HAMPp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-HAMPp-MVM-FVIIIcodeltaB-sv40pA pAAVsc-CRM8-TTRe-HAMPp-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-HAMPp-MVM-FVIIIcodeltaB-sv40pA.

[0248] In any one of said plasmids the poly adenylation signal can be replaced by any other poly adenylation signal such as e.g. the synthetic polyA site as defined by SEQ ID NO:56 (Synth.pA).

[0249] In other embodiments, the vector is a non-viral vector, such as a transposon-based vector. Preferably, said transposon-based vectors are derived from Sleeping Beauty (SB) or PiggyBac (PB). A preferred SB transposon has been described in Ivics et al. (1997) and its hyperactive versions, including SB100X, as described in Mates et al. (2009). PiggyBac-based transposons are safe vectors in that they do no enhance the tumorigenic risk. Furthermore, liver-directed gene therapy with these vectors was shown to induce immune tolerance towards the transgene, in particular the hFIX or hFVIII transgene, comprised in the vector.

[0250] The transposon-based vectors are preferably administered in combination with a vector encoding a transposase for gene therapy. For example, the PiggyBac-derived transposon-based vector can be administered with wild-type PiggyBac transposase (Pbase) or mouse codon-optimized PiggyBac transposase (mPBase) Preferably, said transposases are hyperactive transposases, such as, for example, hyperactive PB (hyPB) transposase containing seven amino acid substitutions (I30V, S103P, G165S, M282V, S509G, N538K, N570S) as described in Yusa et al. (2011), which is specifically incorporated by reference herein.

[0251] Transposon/transposase constructs can be delivered by hydrodynamic injection or using non-viral nanoparticles to transfect hepatocytes.

[0252] In a further aspect, the nucleic acid regulatory elements, the nucleic acid expression cassettes and the vectors described herein can be used in gene therapy. Gene therapy protocols, intended to achieve therapeutic gene product expression in target cells, in vitro, but also particularly in vivo, have been extensively described in the art. These include, but are not limited to, intramuscular injection of plasmid DNA (naked or in liposomes), interstitial injection, instillation in airways, application to endothelium, intra-hepatic parenchyme, and intravenous or intra-arterial administration (e.g. intra-hepatic artery, intra-hepatic vein). Various devices have been developed for enhancing the availability of DNA to the target cell. A simple approach is to contact the target cell physically with catheters or implantable materials containing DNA. Another approach is to utilize needle-free, jet injection devices which project a column of liquid directly into the target tissue under high pressure. These delivery paradigms can also be used to deliver viral vectors. Another approach to targeted gene delivery is the use of molecular conjugates, which consist of protein or synthetic ligands to which a nucleic acid-or DNA-binding agent has been attached for the specific targeting of nucleic acids to cells (Cristiano et al., 1993).

[0253] According to particular embodiments, the use of the nucleic acid expression cassettes and vectors as described herein is envisaged for gene therapy of liver cells (i.e. liver-directed gene therapy). According to a further particular embodiment, the use of the regulatory elements, expression cassettes or vectors is for gene therapy, in particular liver-directed gene therapy, in vivo. According to yet a further particular embodiment, the use is for a method of gene therapy, in particular liver-directed gene therapy, to treat hemophilia, in particular to treat hemophilia B or hemophilia A.

[0254] Gene transfer into mammalian hepatocytes has been performed using both ex vivo and in vivo procedures. The ex vivo approach requires harvesting of the liver cells, in vitro transduction with long-term expression vectors, and reintroduction of the transduced hepatocytes into the portal circulation (Kay et al., 1992; Chowdhury et al., 1991). In vivo targeting has been done by injecting DNA or viral vectors into the liver parenchyma, hepatic artery, or portal vein, as well as via transcriptional targeting (Kuriyama et al., 1991; Kistner et al., 1996). Recent methods also include intraportal delivery of naked DNA (Budker et al., 1996) and hydrodynamic tail vein transfection (Liu et al., 1999; Zhang et al., 1999). According to a further aspect, methods for expressing a protein in liver cells are provided, comprising the steps of introducing in liver cells the nucleic acid expression cassette or a vector as described herein and expressing the transgene protein product in the liver cells. These methods may be performed both in vitro and in vivo.

[0255] Methods of gene therapy for a subject in need thereof are also provided, comprising the steps of introducing in the liver of the subject a nucleic acid expression cassette containing a transgene encoding a therapeutic protein, and expressing a therapeutic amount of the therapeutic protein in the liver. According to a further embodiment, the method comprises the steps of introducing in the liver of the subject a vector comprising the nucleic acid expression cassette containing a transgene encoding a therapeutic protein, and expressing a therapeutic amount of the therapeutic protein in the liver.

[0256] According to a very specific embodiment, the therapeutic protein encoded by the transgene in the nucleic acid expression cassette or the vector is factor IX, and the method is a method for treating hemophilia B. By expressing factor IX in the liver via gene therapy, hemophilia B can be treated (Snyder et al., 1999). According to another very specific embodiment, the therapeutic protein encoded by the transgene in the nucleic acid expression cassette or the vector is factor VIII, and the method is a method for treating hemophilia A.

[0257] Except when noted differently, the terms "subject" or "patient" are used interchangeably and refer to animals, preferably vertebrates, more preferably mammals, and specifically includes human patients and non-human mammals, such as e.g. mice. Preferred patients or subjects are human subjects.

[0258] As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of proliferative disease, e.g., cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0259] As used herein, a phrase such as "a subject in need of treatment" includes subjects, such as mammalian subjects, that would benefit from treatment of a given condition, such as, hemophilia B or hemophilia A. Such subjects will typically include, without limitation, those that have been diagnosed with the condition, those prone to have or develop the said condition and/or those in whom the condition is to be prevented.

[0260] The term "therapeutically effective amount" refers to an amount of a compound or pharmaceutical composition effective to treat a disease or disorder in a subject, i.e., to obtain a desired local or systemic effect and performance. In a particular embodiment, the term implies that levels of factor IX in plasma equal to or higher than the therapeutic threshold concentration of 10 mU/ml (milli-units per milliliter) plasma, 50 mU/ml plasma, 100 mU/ml plasma, 150 mU/ml or 200 mU/ml plasma in a subject can be obtained by transduction or transfection of the vector according to any one the embodiments described herein into a subject. Due to the very high efficiency of the vectors and nucleic acid expression cassettes of the present invention, this high physiological level of factor IX in the subject can be obtained even by administering relatively low doses of vector. In another particular embodiment, the term implies that levels of factor VIII in plasma equal to or higher than the therapeutic threshold concentration of 10 mU/ml (milli-units per milliliter) plasma, 50 mU/ml plasma, 100 mU/ml plasma, 150 mU/ml plasma, 200 mU/ml plasma or higher can be obtained by transduction or transfection of any of the vectors disclosed herein into a subject. Due to the very high efficiency of the vectors and nucleic acid expression cassettes disclosed herein, these high physiological levels of factor VIII in the subject can be obtained even by administering relatively low doses of vector. The term thus refers to the quantity of compound or pharmaceutical composition that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the hemophilia being treated. In particular, these terms refer to the quantity of compound or pharmaceutical composition according to the invention which is necessary to prevent, cure, ameliorate, or at least minimize the clinical impairment, symptoms, or complications associated with hemophilia, in particular hemophilia B or hemophilia A, in either a single or multiple dose.

[0261] In particular, the transduction of the vector according to any one of the embodiments defined herein into the subject can be done at a dose lower than 2.times.10.sup.11 vg/kg (viral genomes per kilogram) to obtain a therapeutic factor IX level of 10 mU/ml plasma or of 50 mU/ml plasma in a subject.

[0262] Alternatively, if a level of factor IX of 100 mU/ml plasma needs to be reached in a subject, the transduction of the vector according to any one of the embodiments defined herein into the subject can be done at a dose lower than or equal to 6.times.10.sup.11 vg/kg.

[0263] Further, if a level of factor IX equal to 150 mU/ml plasma or higher needs to be reached, the transduction of the vector according to any one of the embodiments defined herein into the subject can be done ata dose lower than or equal than 2.times.10.sup.12 vg/kg.

[0264] In a preferred embodiment, a level of factor IX of 200 mU/ml plasma or higher can be reached in a subject, when the transduction of the vector according to any one of the embodiments defined herein into the subject is done at a dose lower than or equal to 2.times.10.sup.12 vg/kg.

[0265] In particular, the transduction of the vector according to any one of the embodiments defined herein into the subject can be done at a dose lower than or equal to 2.times.10.sup.12 vg/kg (viral genomes per kilogram), such as lower than or equal to 1.times.10.sup.12 vg/kg, 5.times.10.sup.11 vg/kg, 2.5.times.10.sup.11 vg/kg, 1.times.10.sup.11 vg/kg, 5.times.10.sup.1.degree. vg/kg, 1.times.10.sup.1.degree. vg/kg, 5.times.10.sup.9 vg/kg, or 1.times.10.sup.9 vg/kg preferably at a dose lower than or equal to 2.5.times.10.sup.11 vg/kg, to obtain a therapeutic factor VIII level of 10 mU/ml plasma, 50 mU/ml plasma, 100 mU/ml plasma, 150 mU/ml plasma, 200 mU/ml plasma, or higher in a subject.

[0266] For hemophilia therapy, efficacy of the treatment can, for example, be measured by assessing the hemophilia-caused bleeding in the subject. In vitro tests such as, but not limited to the in vitro activated partial thromboplastin time assay (APPT), test factor IX chromogenic activity assays, blood clotting times, factor IX or human factor VIII-specific ELISAs are also available. Any other tests for assessing the efficacy of the treatment known in the art can of course be used.

[0267] The nucleic acid expression cassette, the vector or the pharmaceutical composition of the invention may be used alone or in combination with any of the know hemophilia therapies, such as the administration of recombinant or purified clotting factors. The nucleic acid expression cassette, the vector or the pharmaceutical composition of the invention can thus be administered alone or in combination with one or more active compounds. The latter can be administered before, after or simultaneously with the administration of the said agent(s).

[0268] A further object of the invention are pharmaceutical preparations which comprise a therapeutically effective amount of the nucleic acid expression cassette or the expression vector as defined herein, and a pharmaceutically acceptable carrier, i.e., one or more pharmaceutically acceptable carrier substances and/or additives, e.g., buffers, carriers, excipients, stabilisers, etc. The term "pharmaceutically acceptable" as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof. The term "pharmaceutically acceptable salts" as used herein means an inorganic acid addition salt such as hydrochloride, sulfate, and phosphate, or an organic acid addition salt such as acetate, maleate, fumarate, tartrate, and citrate. Examples of pharmaceutically acceptable metal salts are alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of pharmaceutically acceptable ammonium salts are ammonium salt and tetramethylammonium salt. Examples of pharmaceutically acceptable organic amine addition salts are salts with morpholine and piperidine. Examples of pharmaceutically acceptable amino acid addition salts are salts with lysine, glycine, and phenylalanine. The pharmaceutical composition according to the invention can be administered orally, for example in the form of pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions, or rectally, for example in the form of suppositories.

[0269] Administration can also be carried out parenterally, for example subcutaneously, intramuscularly or intravenously in the form of solutions for injection or infusion. Other suitable administration forms are, for example, percutaneous or topical administration, for example in the form of ointments, tinctures, sprays or transdermal therapeutic systems, or the inhalative administration in the form of nasal sprays or aerosol mixtures, or, for example, microcapsules, implants or rods. The pharmaceutical composition can be prepared in a manner known per se to one of skill in the art. For this purpose, the nucleic acid expression cassette or the expression vector as defined herein, one or more solid or liquid pharmaceutically acceptable excipients and, if desired, in combination with other pharmaceutical active compounds, are brought into a suitable administration form or dosage form which can then be used as a pharmaceutical in human medicine or veterinary medicine.

[0270] According to another aspect, a pharmaceutical composition is provided comprising a nucleic acid expression cassette containing a transgene encoding a therapeutic protein, and a pharmaceutically acceptable carrier. According to another embodiment, the pharmaceutical composition comprises a vector containing the nucleic acid expression cassette containing a transgene encoding a therapeutic protein, and a pharmaceutically acceptable carrier. According to further particular embodiments, the transgene encodes factor IX and the pharmaceutical composition is for treating hemophilia B or the transgene encodes factor VIII and the pharmaceutical composition is for treating hemophilia A.

[0271] The use of the nucleic acid expression cassette, its regulatory elements and the vector components as disclosed herein for the manufacture of these pharmaceutical compositions for use in treating hemophilia, preferably hemophilia B or hemophilia A, is also envisaged.

[0272] It is to be understood that although particular embodiments, specific constructions and configurations, as well as materials, have been discussed herein for methods and applications according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention.

[0273] The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

EXAMPLES

[0274] Example 1

Design of the AAV Vectors Used

[0275] The design of the self-complementary (sc), double-stranded adeno-associated viral (AAV) vectors is depicted in FIG. 1. The minimal transthyretin promoter (TTRm) is driving the expression of the human codon-optimized factor IX (co-FIX-R338L) containing the hyper-activating, thrombophilic FIX mutation (R338L). The minimal TTR promoter (TTRm) is used in conjunction with either the Serpin enhancer (SERP), a triple repeat of the Serpin enhancer (3.times.SERP), the native TTR enhancer (TTRe), or a combination of a triple repeat of the Serpin enhancer and the native TTR enhancer (3.times.SERP-TTRe). The minute virus of mouse intron (MVM), the bovine growth hormone polyadenylation site (BGHpA) or a synthetic polyadenylation site (Synt.pA), and the inverted terminal repeats (ITR) are indicated; 3.times.SerpEnh means that this element was cloned as a triplet repeat upstream of the TTRm. The vector size (including both ITR's) is indicated.

[0276] FIG. 2 shows the design of the single-stranded (ss) adeno-associated viral (AAV) vectors. The minimal transthyretin promoter (TTRm) is driving the expression of the human codon-optimized B-domain deleted factor VIII (cohFVIIIdeltaB). Optionally, the Serpin enhancer (SERP), a triple repeat of the Serpin enhancer (3.times.SERP), the native TTR enhancer (TTRe), or a combination of a triple repeat of the Serpin enhancer and the native TTR enhancer (3.times.SERP-TTRe) are cloned upstream of the TTRm. The minute virus of mouse intron (MVM), the Simian Virus 40 (SV40) polyadenylation site (Sv40pA) or a synthetic polyadenylation site (Synt.pA), and the inverted terminal repeats (ITR) are indicated. The vector size (including both ITR's) is indicated.

Materials and Methods:

Cloning Strategy

[0277] FIX constructs: The basic AAVsc-SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA plasmid (Nair et al. Blood, 2014) was used to create the other AAV-FIX constructs. This plasmid contains the Serp enhancer (SerpEnh), a liver-specific transthyretin (TTRm) promoter, a minute virus of mouse (MVM) small intron, a codon optimized human FIX transgene containing a R338L mutation, and a bovine growth hormone poly A (BGHpA). The 3.times.SerpEnh-TTRm-MVM sequence was synthesized by GeneArt (Life Technologies, Regensburg, Germany) and cloned into the basic construct using Ascl and Nhel, thereby replacing SerpEnh-TTRm-MVM, creating pAAVsc-3.times.SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA. Into this plasmid, the 3.times.SerpEnh sequence was replaced by the TTR enhancer (TTREnh) or 3.times.SerpEnh-TTREnh (constructed by GeneArt), thereby respectively creating pAAVsc-TTREnh-TTRm-MVM-co-FIX-R338L-BGHpA and pAAVsc-3.times.SerpEnh-TTREnh-TTRm-MVM-co-FIX-R338L-BGHpA respectively.

[0278] FVIII constructs: The basic AAVss-SerpEnh-TTRm-MVM-FVIIIcopt-Sv40pA plasmid was used to create the other AAV-FVIII constructs. This plasmid contains the Serp enhancer (SerpEnh), a liver-specific transthyretin (TTRm) promoter, an MVM intron, a codon optimized human FVIII transgene (Di Matteo et al., 2014), and an SV40 poly A. The SerpEnh was removed using Kpnl, followed by re-ligation of the backbone, thereby creating pAAVss-TTRm-MVM-FVIIIcopt-Sv40pA. To create pAAVsc-3.times.SerpEnh-TTRm-MVM-FVIIIcopt-Sv40pA, the 3.times.SerpEnh was first cloned into a FVIIIcopt plasmid with a different backbone, after which the entire expression cassette (3.times.SerpEnh-TTRm-MVM-FVIIIcopt-Sv40pA) was cloned into the AAVss backbone using Notl and Xhol. From this final plasmid, 3.times.SerpEnh was removed and replaced with the TTREnh (constructed by GeneArt), thereby creating pAAVss-TTREnh-TTRm-MVM-FVIIIcopt-Sv40pA. In order to generate pAAVss-3.times. SerpEnh-TTREnh-TTRm-MVM-cohFVIIIdeltaB-SV40pA (5493bp), pAAVss-TTREnh-TTRm-MVM-cohFVIIIdeltaB-SV40pA (the vector) was restricted with Notl-Acc651. A fragment with 3.times.Serp flanked by Notl/Acc651 was synthesized by GenArt and ligated into the restricted vector.

[0279] AAV Vector Production, Purification and Titration

[0280] AAV vectors were produced by calcium phosphate (Invitrogen Corp, Carlsbad, Calif.) co-transfection of AAV-293 human embryonic kidney carcinoma cells (Stratagene, Carlsbad, Calif., catalog No 240073; with the pAAV plasmid of interest, an adenoviral helper plasmid and a chimeric packaging constructs that delivers the AAV2 Rep gene together with the AAV8 or AAV9 Cap gene, as described previously (VandenDriessche et al, 2007, VandenDriessche et al; 2007, J. Thromb. Haemost. JTH 5:16-24). For the AAV-FIX vectors, the AAV9 serotype was used and for the AAV-FVIII vectors the AAV8 serotype. The AAV-293 cells are free of microbial contamination as determined by PCR for detection of mycoplasma. Briefly, two days post transfection, cells were harvested and vector particles were purified using isopycnic centrifugation methods. Harvested cells were lysed by successive freeze/thaw cycles and sonication, treated with benzonase (Novagen, Madison, Wis.) and deoxycholic acid (Sigma-Aldrich, St. Louis, Mo.) and subsequently subjected to 2 successive rounds of cesium chloride (Invitrogen Corp, Carlsbad, Calif.) density gradient ultracentrifugation. Fractions containing the AAV vector were collected, concentrated in 1mM MgCl.sub.2 in Dulbecco's phosphate buffered saline (PBS) (Gibco, BRL) and stored at -80.degree. C. The vector titers (in viral genomes (vg)/ml) were determined by quantitative real-time PCR using specific primers. For the FIX vectors, primers specific for the bovine growth hormone poly A sequence were used. The forward and reverse primers used were 5'-GCCTTCTAGTTGCCAGCCAT-3' (SEQ ID NO:60) and 5'-GGCACCTTCCAGGGTCAAG-3' (SEQ ID NO:61), respectively. For the FVIII vectors, primers specific for the FVIII gene sequence were used. The forward and reverse primers used were 5'-AACGGCTACGTGAACAGAAG -3' (SEQ ID NO:62) and 5'-GATAGGGCTGATTTCCAGGC-3' (SEQ ID NO:63), respectively. Reactions were performed in SybrGreen PCR Master Mix (Applied Biosystems, Foster City, Calif., USA), on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City,CA,USA). Known copy numbers (102-107) of the respective vector plasmids used to generate the corresponding AAV vectors, carrying the appropriate cDNAs were used to generate the standard curves.

[0281] Animal Study and Blood Collection

[0282] AAV vector administration is carried out by tail vein injection on adult C57B6 mice at two different doses: 1.times.10.sup.9 (low dose) and 5.times.10.sup.9 (high dose) vector genomes (vg) per mouse as detailed below. Mice were bled at different time points after gene transfer in order to evaluate the FIX/FVIII gene expression. The pAAVss-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pA and pAAVss-3.times.SerpEnh-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pA plasmid constructs were administered by hydrodynamic delivery into C57BL/6 mice at a dose of 300 ng per mouse. Animals were anesthetized using isoflurane and blood samples were taken by retro-orbital bleeding on trisodium citrate. Plasma was prepared immediately after blood collection by centrifugation at 14000 rpm for 3 minutes at 4.degree. C. Plasma was immediately stored at -80.degree. C. for further analysis.

[0283] FIX ELISA

[0284] The concentration of hFIX antigens in citrated plasma was measured by enzyme-linked immunosorbent assay (ELISA) using manufacturer's protocol (Diagnostica Stago, France). The hFIX standards (available in the kit) were serially diluted using the dilution buffer and used for calibration. Here the 100% of the standard corresponds to 5000 ng of FIX protein. The aliquots of the plasma samples were thawed and diluted in order to make their reading fall in the linear range of standards. Standards and samples were then added to a 96 well plate pre-coated with primary anti-human FIX antibodies. After an incubation of 1 hour at room temperature, the plate was washed and a solution containing secondary antibodies coupled to peroxidase were added followed by another incubation of 1 hour at room temperature. After incubation, the peroxidase substrate TMB (chromogenic solution) was added which resulted in color development. After exact 5 minutes of incubation 1M H2SO4 was added to all wells to stop the reaction. After an incubation of 15minutes the absorbance was measured at 450 nm using the microplate reader. Using the obtained standard curve, FIX levels were determined.

[0285] FVIII ELISA

[0286] The concentration of hFVIII antigens in citrated plasma was measured by enzyme-linked immunosorbent assay (ELISA) using manufacturer's protocol (Diagnostica Stago, France).

[0287] The hFVIII standards (available in the kit) were serially diluted using the dilution buffer and used for calibration. Here the 100% of the standard corresponds to 200 ng of FVIII protein. The aliquots of the plasma samples were thawed and diluted in order to make their reading fall in the linear range of standards. Standards and samples were then added to a 96 well plate pre-coated with primary anti-human FVIII antibodies. After an incubation of 2 hour at room temperature, the plate was washed and a solution containing secondary antibodies coupled to peroxidase was added followed by another incubation of 2 hour at room temperature. After incubation, the peroxidase substrate TMB (chromogenic solution) was added which resulted in color development. After exact 5 minutes of incubation 1M H2504 was added to all wells to stop the reaction. After an incubation of 15 minutes the absorbance was measured at 450 nm using the microplate reader. Using the obtained standard curve, FIX levels were determined.

[0288] STATISTICS

[0289] Data were analyzed using Microsoft Excel Statistics package. Values shown in FIG. 3 and FIG. 4 are the mean +SEM. Significance values were obtained by comparison using t-test.

Viral Administration

[0290] Male adult C57BL/6 mice (18-20 grams) were administrated with AAV9 FIX vectors (see the table below) by tail vain injection at doses of 1.times.10.sup.9 vg/mouse and 5.times.10.sup.9 vg/mouse.

TABLE-US-00001 Construct Size Dose Mice AAV9sc-SerpEnh-TTRm-MVM-co-FIX- 2510 bp 1 .times. 10.sup.9 4 R338L-BGHpolyA 5 .times. 10.sup.9 4 AAV9sc-3xSerpEnh-TTRm-MVM-co-FIX- 2682 bp 1 .times. 10.sup.9 4 R338L-BGHpolyA 5 .times. 10.sup.9 4 AAV9sc-TTREnh-TTRm-MVM-co-FIX- 2540 bp 1 .times. 10.sup.9 4 R338L-BGHpolyA 5 .times. 10.sup.9 4 AAV9sc-3xSerpEnh-TTREnh-TTRm-MVM- 2760 bp 1 .times. 10.sup.9 4 co-FIX-R338L-BGHpolyA 5 .times. 10.sup.9 4

[0291] Male adult CB17SCID mice (18-20 grams) were administrated with AAV8 XVIII vectors (see the table below) by tail vain injection at doses of 1.times.10.sup.9 vg/mouse and 5.times.10.sup.9 vg/mouse.

TABLE-US-00002 Construct/condition Size Dose Mice AAV8ss-TTRm-MVM-FVIIIcopt-sv40pA 5160 bp 1 .times. 10.sup.9 4 5 .times. 10.sup.9 4 AAV8ss-SerpEnh-TTRm-MVM-FVIIIcopt- 5252 bp 1 .times. 10.sup.9 4 sv40pA 5 .times. 10.sup.9 4 AAV8ss-3xSerpEnh-TTRm-MVM-FVIIIcopt- 5410 bp 1 .times. 10.sup.9 4 sv40pA 5 .times. 10.sup.9 4 AAV8ss-TTREnh-TTRm-MVM-FVIIIcopt- 5272 bp 1 .times. 10.sup.9 4 sv40pA 5 .times. 10.sup.9 4

[0292] Plasmid Administration

[0293] Male adult C57BL/6 mice (22-24 grams) were administrated with respective plasmids (see the table below) by hydrodynamic tail vein injection at doses of 300 ng.

TABLE-US-00003 Construct/condition Size Dose Mice pAAVss-TTREnh-TTRm-MVM-co-hFVIII- 5272 p 300 ng 2 deltaB-SV40pA pAAVss-3XSerpEnh-TTREnh-TTRm-MVM- 5493 bp 300 ng 2 co-hFVIII-deltaB-SV40pA

[0294] Results

[0295] Factor IX:

[0296] The AAV vector containing 3.times.SerpEnh-TTREnh-TTRm, with 3 copies of the Serp enhancer combined with the natural TTRe enhancer, and in combination with the TTRm promoter, led to the highest FIX levels compared to any of the other expression cassettes, over 4 time points until at least day 39 after AAV vector injection. The FIX expression showed a continuous increase over time.

[0297] The AAV vector containing the most robust 3.times.SerpEnh-TTREnh-TTRm regulatory elements showed about 11 to 6 fold (at low and high vector dose, respectively) higher FIX expression when compared to a control AAV vector that expressed FIX from the TTREnh-TTRm enhancer/promoter (i.e. lacking the SerpEnh enhancer).

[0298] The AAV vector containing the most robust 3.times.SerpEnh-TTREnh-TTRm regulatory elements showed about 4 to 3 fold (low and high vector dose, respectively) higher FIX expression when compared to a vector that expressed FIX from the SerpEnh-TTRm enhancer/promoter, which contains only one instead of 3 copies of the Serp enhancer, and no TTRe enhancer.

[0299] The AAV vector containing the most robust 3.times.SerpEnh-TTREnh-TTRm showed about 3 to 2 fold (low and high vector dose, respectively) higher expression when compared to a vector that expressed FIX from the 3.times.SerpEnh-TTRm enhancer/promoter which also 3 copies of the Serp enhancer, but is devoid of the TTR enhancer.

[0300] Factor VIII

[0301] The AAV vector containing 3.times.SerpEnh-TTRm, with 3 copies of the Serp enhancer, led to the highest FVIII levels compared to any of the other expression cassettes, until at least day 40 after AAV vector injection. The FVIII expression showed a continuous increase over time.

[0302] The AAV vector containing the 3.times.SerpEnh-TTRm regulatory elements showed about 30 to 12 fold (low and high vector dose, respectively) higher FVIII expression when compared to a reference control cassette (TTRm) which consist of only the TTR minimal promoter and that do not contain any enhancer.

[0303] The AAV vector containing the 3.times.SerpEnh-TTRm regulatory elements showed about 10 to 1.5-fold (low and high vector dose, respectively) higher FVIII expression when compared to an AAV vector containing SerpEnh-TTRm with only one copy of the Serp enhancer.

[0304] The AAV vector containing the 3.times.SerpEnh-TTRm regulatory elements showed about 5 to 2 fold (low and high vector dose, respectively) higher expression when compared to an AAV vector containing the TTREnh-TTRm enhancer/promoter. The 3.times.SerpEnh regulatory element could further increase FVIII expression from the TTRenh-TTRm enhancer/promoter (FIG. 15). FVIII protein levels were about 8 to 5 fold higher in mice injected with the pAAVss-3.times.SerpEnh-TTREnh-TTRm-MVM-co-hFVIII-deltaB-SV40pA plasmid as compared to the pAAVss-TTREnh-TTRm-MVM-co-hFV111-deltaB-SV40pA plasmid.

Example 2

Studying the In Vivo Effect of 3.times.CRM8 and TTRenhancer on FVIII Expression in Various Constructs by Hydrodynamic Injection (2 ml) into CB17-SCID Mice

[0305] The aim of this example was to study the in vivo effect of 3.times.CRM8 and TTRenhancer on FVIII expression in various constructs by hydrodynamic injection (2m1) into CB17-SCID mice.

[0306] Constructs: The following vectors were compared for FVIII expression levels (cf. FIG. 22):

TABLE-US-00004 (SEQ ID NO. 14) pAAVss-TTRm-MVM-coFVIIIdeltaB-Sv40pA; (SEQ ID NO: 16) pAAVss-3xSerpEnh-TTRm-MVM-coFVIIIdeltaB-Sv40pA; (SEQ ID NO: 17) pAAVss-TTRe-TTRm-MVM-coFVIIIdeltaB-Sv40pA; and (SEQ ID NO: 22) pAAVss-3xSerpEnh-TTRe-TTRm-MVM-coFVIIIdeltaB- Sv40pA.

[0307] In a first experiment, the following conditions were used:

[0308] Mouse model: CB17-SCID, male, adult mice of 18-20 grams

[0309] Doses: 150 ng plasmid/mouse

[0310] Analysis points: Blood collection at Dayl

[0311] No of mice: 3 per condition

[0312] Design: See the table below

TABLE-US-00005 Construct/condition Dose Mice PBS control -- 3 1. pAAVss-TTRe-TTRm-MVM-FVIIIco.DELTA.B-sv40pA 300 ng 3 2. pAAVss-3xCRM8-TTRe-TTRm-MVM-FVIIIco.DELTA.B- 300 ng 3 sv40pA

[0313] Results: The expression level of FVIII was analysed and compared. FIG. 25A example shows a graph depicting the effect of the 3.times.Serp enhancer on the TTRenh-TTRm promoter in regulating the FVIII expression in C57BL6 mice injected with 300ng of respective plasmids. The FVIII expression profile was tested using the ELISA, in the plasma samples collected Day 1. The presence of 3.times.Serp enhancer seems to elevate the FVIII expression by 8 fold.

[0314] In a second experiment the following conditions were used:

[0315] Mouse model: CB17-SCID, male, adult mice of 18-20 grams

[0316] Doses: 150 ng plasmid/mouse

[0317] Analysis points: Blood collection at Dayl

[0318] No of mice: 3 per condition

[0319] Design: See the table below

TABLE-US-00006 Construct/condition Dose Mice PBS control -- 3 1. pAAVss-TTRm-MVM-FVIIIco.DELTA.B-sv40pA 150 ng 3 2. pAAVss-3xCRM8-TTRm-MVM-FVIIIco.DELTA.B -sv40pA 150 ng 3 3. pAAVss-TTRe-TTRm-MVM-FVIIIco.DELTA.B-sv40pA 150 ng 3 4. pAAVss-3xCRM8-TTRe-TTRm-MVM-FVIIIco.DELTA.B- 150 ng 3 sv40pA

[0320] Results: FIG. 25B shows the compiled data from comparing the 4 constructs (see table below for actual FVIII expression values). Addition of 3.times.SERP to the TTRm construct enhances FVIII expression 17 fold. Addition of 3.times.SERP to the TTRe-TTRm construct enhances FVIII expression 13 fold. Addition of TTRe to the 3.times.SERP-TTRm construct still enhances FVIII expression 4 fold.

TABLE-US-00007 Vectors FVIII level (ng/ml) TTRm 0.68 3xCRM8-TTRm 11.74 TTRe-TTRm 3.52 3xCRM8-TTRe-TTRm 46.67

Example 3

Studying the In Vivo Effect of 3.times.CRM8 and TTRenhancer in Combination with an AAT Promoter on FIX Expression in Various Constructs by Hydrodynamic Injection (2 ml) into C57131/6 Mice

[0321] In this experiment, the TTRm minimal promotor is replaced by another liver-specific promotor to show the versatility of the regulator element.

[0322] As a first example, the AAT liver-specific promoter (AAT) is tested.

[0323] Constructs: Four different constructs were prepared as depicted in FIG. 23 (the sequences are depicted in FIG. 24):

TABLE-US-00008 (SEQ ID NO: 65) pAAVss-3xSerpEnh-TTRe-AAT-MVM-co-FIX-R338L-BGHpA; (SEQ ID NO: 66) pAAVss-3xSerpEnh-AAT-MVM-co-FIX-R338L-BGHpA; (SEQ ID NO: 67) pAAVss-TTRe-AAT-MVM-co-FIX-R338L-BGHpA; and (SEQ ID NO: 68) pAAVss-TTRe-3xSerpEnh-AAT-MVM-co-FIX-R338L-BGHpA;

[0324] All constructs tested make use of the AAT promoter (SEQ ID NO.64).

[0325] Mouse model: C57BL/6, male, adult mice of 18-20 grams

[0326] Doses: 1 .mu.g and 2 .mu.g plasmid/mouse

[0327] Analysis points: Blood collection at D1, D2

[0328] No of mice: 3 per condition

[0329] Design: See the table below

TABLE-US-00009 Construct/condition Dose Mice PBS control -- 3 1. pAAVsc-3xCRM8-TTRe-AAT-MVM-FIXcoR338L- 1 .mu.g 3 BGHpA 2 .mu.g 3 2. pAAVsc-3xCRM8-AAT-MVM-FIXcoR338L-BGHpA 1 .mu.g 3 2 .mu.g 3 3. pAAVsc-TTRe-AAT-MVM-FIXcoR338L-BGHpA 1 .mu.g 3 2 .mu.g 3 4. pAAVsc-TTRe-3xCRM8-AAT-MVM-FIXcoR338L- 1 .mu.g 3 BGHpA 2 .mu.g 3

[0330] Also the corresponding FVIII constructs will be tested for FVIII expression with the AAT promotor, hence encompassing the following constructs: pAAVss-CRM8-TTRe-AAT-MVM-FVIIIcodeltaB-sv40pA. pAAVss-3.times.CRM8-AAT-MVM-FVIIIcodeltaB-sv40pA. pAAVss-3.times.CRM8-TTRe-AAT-MVM-FVIIIcodeltaB-sv40pA and pAAVss-TTRe-3.times.CRM8-AAT-MVM-FVIIIcodeltaB-sv40pA.

[0331] In analogy, the following examples follow the outline of the example with the AAT promoter above, but each time with a different liver-specific promoter or minimal promoter.

[0332] In further examples, the albumin promotor (ALBp) is used to replace the TTRm promotor in said constructs, hence encompassing the following constructs: pAAVsc-3.times.CRM8-TTRe-ALBp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-ALBp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-ALBp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-ALBp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-ALBp-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-ALBp-MVM-FVIIIcodeltaB-sv40pA.

[0333] In further examples, the apolipoprotein Al promotor (APOA1p) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct:

[0334] pAAVsc-3.times.CRM8-TTRe-APOA1p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-APOA1p-MVM-FVIIIcodeltaB-sv40pA pAAVsc-TTRe-3.times.CRM8-APOA1p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-APOA1p-MVM-FVIIIcodeltaB-sv40pA pAAVsc-CRM8-TTRe-APOA1p-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-APOA1p-MVM-FVIIIcodeltaB-sv40pA.

[0335] In further examples, the complement factor B promoter (CFBp) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct:

[0336] pAAVsc-3.times.CRM8-TTRe-CFBp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-CFBp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-CFBp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-CFBp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-CFBp-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-CFBp-MVM-FVIIIcodeltaB-sv40pA.

[0337] In further examples, the ketohexokinase promoter (KHKp) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct: pAAVsc-3.times.CRM8-TTRe-KHKp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-KHKp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-KHKp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-KHKp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-KHKp-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-KHKp-MVM-FVIIIcodeltaB-sv40pA.

[0338] In further examples, the hemopexin promoter (HPXp) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct:

[0339] pAAVsc-3.times.CRM8-TTRe-HPXp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-HPXp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-HPXp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-HPXp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-HPXp-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-HPXp-MVM-FVIIIcodeltaB-sv40pA.

[0340] In further examples, the nicotinamide N-methyltransferase promoter (NNMTp) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct: pAAVsc-3.times.CRM8-TTRe-NNMTp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-NNMTp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-NNMTp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-NNMTp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-NNMTp-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-NNMTp-MVM-FVIIIcodeltaB-sv40pA.

[0341] In further examples, the (liver) carboxylesterase 1 promoter (CES1p) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct: pAAVsc-3.times.CRM8-TTRe-CES1p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-CES1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-CES1p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-CES1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-CES1p-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-CES1p-MVM-FVIIIcodeltaB-sv40pA.

[0342] In further examples, the protein C promoter (PROCp) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct: pAAVsc-3.times.CRM8-TTRe-PROCp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-PROCp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-PROCp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-PROCp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-PROCp-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-PROCp-MVM-FVIIIcodeltaB-sv40pA.

[0343] In further examples, the apolipoprotein C3 promoter (APOC3p) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct: pAAVsc-3.times.CRM8-TTRe-APOC3p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-APOC3p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-APOC3p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-APOC3p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-APOC3p-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-APOC3p-MVM-FVIIIcodeltaB-sv40pA.

[0344] In further examples, the mannan-binding lectin serine protease 2 (MASP2p) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct: pAAVsc-3.times.CRM8-TTRe-MASP2p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-MASP2p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-MASP2p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-MASP2p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-MASP2p-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-MASP2p-MVM-FVIIIcodeltaB-sv40pA. In further examples, the serpin peptidase inhibitor, clade C (antithrombin) promoter (SERPINC1p) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct: pAAVsc-3.times.CRM8-TTRe-SERPINC1p-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-TTRe-3.times.CRM8-SERPINC1p-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-SERPINC1p-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-SERPINC1p-MVM-FVIIIcodeltaB-sv40pA.

[0345] In further examples, the serpin peptidase inhibitor promoter (HAMPp) is used to replace the TTRm promotor in said constructs, hence encompassing the following construct: pAAVsc-3.times.CRM8-TTRe-HAMPp-MVM-FIXcoR338L-BGHpA, pAAVss-3.times.CRM8-TTRe-HAMPp-MVM-FVIIIcodeltaB-sv40pA pAAVsc-TTRe-3.times.CRM8-HAMPp-MVM-FIXcoR338L-BGHpA, pAAVss-TTRe-3.times.CRM8-HAMPp-MVM-FVIIIcodeltaB-sv40pA, pAAVsc-CRM8-TTRe-HAMPp-MVM-FIXcoR338L-BGHpA, or pAAVss-CRM8-TTRe-HAMPp-MVM-FVIIIcodeltaB-sv40pA.

[0346] Results: The expression level of FIX or FVIII will be analysed and compared as explained in Example 2.

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[0429] WU Z, SUN J, ZHANG T, YIN C, YIN F, VAN DYKE T, SAMULSKI R J, and MONAHAN P E. (2008). Optimization of self-complementary AAV vectors for liver-directed expression results in sustained correction of hemophilia B at low vector dose. Mol Ther. 16, 280-9.

[0430] XU L, GAO C, and SANDS M S. (2003). Neonatal or hepatocyte growth factor-potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B. Blood 101, 3924-3932.

[0431] XU L, NICHOLS T C, SARKAR R, Mc CORQUODALE S, BELLINGER D A, PONDER K P. (2005). Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy. Proc Natl Acad Sci USA 102, 6080-6085.

[0432] YAMADA T, IWASAKI Y, TADA H, IWABUKI H, CHUAH M K, VANDENDRIESSCHE T, FUKUDA H, KONDO A, UEDA M, SENO M, TANIZAWA K, KURODA S. (2003) Nanoparticles for the delivery of genes and drugs to human hepatocytes. Nat Biotechnol. August; 21 (8):885-90.

[0433] YANT S R, MEUSE L, CHIU W, IVICS Z, IZSVAK Z, and KAY M A. (2000). Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet. 25, 35-41. Yusa et al. A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci USA. 2011;108(4):1531-6.

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Sequence CWU 1

1

8516198DNAArtificial SequencepAAVsc-SerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA 1agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020gcagagaggg agtggaattc acgcgtggat ctgaattcaa ttcacgcgtg gtacggccgc 1080ggtaccggcg cgccggggga ggctgctggt gaatattaac caaggtcacc ccagttatcg 1140gaggagcaaa caggggctaa gtccacacgc gtggtaccgt ctgtctgcac atttcgtaga 1200gcgagtgttc cgatactcta atctccctag gcaaggttca tatttgtgta ggttacttat 1260tctccttttg ttgactaagt caataatcag aatcagcagg tttggagtca gcttggcagg 1320gatcagcagc ctgggttgga aggagggggt ataaaagccc cttcaccagg agaagccgtc 1380acacagatcc acaagctcct gaagaggtaa gggtttaagg gatggttggt tggtggggta 1440ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt tggctagccc 1500accatgcagc gcgtgaacat gatcatggcc gagagccccg gcctgatcac catctgcctg 1560ctgggctacc tgctgagcgc cgagtgcacc gtgttcctgg accacgagaa cgccaacaag 1620atcctgaacc gccccaagcg ctacaacagc ggcaagctgg aggagttcgt gcagggcaac 1680ctggagcgcg agtgcatgga ggagaagtgc agcttcgagg aggcccgcga ggtgttcgag 1740aacaccgagc gcaccaccga gttctggaag cagtacgtgg acggcgacca gtgcgagagc 1800aacccctgcc tgaacggcgg cagctgcaag gacgacatca acagctacga gtgctggtgc 1860cccttcggct tcgagggcaa gaactgcgag ctggacgtga cctgcaacat caagaacggc 1920cgctgcgagc agttctgcaa gaacagcgcc gacaacaagg tggtgtgcag ctgcaccgag 1980ggctaccgcc tggccgagaa ccagaagagc tgcgagcccg ccgtgccctt cccctgcggc 2040cgcgtgagcg tgagccagac cagcaagctg acccgcgccg aggccgtgtt ccccgacgtg 2100gactacgtga acagcaccga ggccgagacc atcctggaca acatcaccca gagcacccag 2160agcttcaacg acttcacccg cgtggtgggc ggcgaggacg ccaagcccgg ccagttcccc 2220tggcaggtgg tgctgaacgg caaggtggac gccttctgcg gcggcagcat cgtgaacgag 2280aagtggatcg tgaccgccgc ccactgcgtg gagaccggcg tgaagatcac cgtggtggcc 2340ggcgagcaca acatcgagga gaccgagcac accgagcaga agcgcaacgt gatccgcatc 2400atcccccacc acaactacaa cgccgccatc aacaagtaca accacgacat cgccctgctg 2460gagctggacg agcccctggt gctgaacagc tacgtgaccc ccatctgcat cgccgacaag 2520gagtacacca acatcttcct gaagttcggc agcggctacg tgagcggctg gggccgcgtg 2580ttccacaagg gccgcagcgc cctggtgctg cagtacctgc gcgtgcccct ggtggaccgc 2640gccacctgcc tgctgagcac caagttcacc atctacaaca acatgttctg cgccggcttc 2700cacgagggcg gccgcgacag ctgccagggc gacagcggcg gcccccacgt gaccgaggtg 2760gagggcacca gcttcctgac cggcatcatc agctggggcg aggagtgcgc catgaagggc 2820aagtacggca tctacaccaa ggtgagccgc tacgtgaact ggatcaagga gaagaccaag 2880ctgacctaat gaaagatgga tttccaaggt taattcattg gaattgaaaa ttaacagccc 2940cccccccccc ccccctgcag atctgagccg aattcctgca gcccggggga tcagcctcga 3000ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 3060tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 3120tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 3180gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 3240gaaccagctg gggaccggtg gatctcgata gcaggcatgc tggggagaga tcgatctgag 3300gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc 3360gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga 3420gcgcgcagag agggagtggc caaccccccc cccccccccc cccggcgatt ctcttgtttg 3480ctccagactc tcaggcaatg acctgatagc ctttgtagag acctctcaaa aatagctacc 3540ctctccggca tgaatttatc agctagaacg gttgaatatc atattgatgg tgatttgact 3600gtctccggcc tttctcaccc gtttgaatct ttacctacac attactcagg cattgcattt 3660aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc ttctcccgca 3720aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg ctctgaggct 3780ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga tgttggaatc 3840gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 3900ctctcagtac aatctgctct gatgccgcat agttatatgg tgcactctca gtacaatctg 3960ctctgatgcc gcatagttaa gccagccccg acacccgcca acacagccag ccccgacacc 4020cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 4080aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 4140gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 4200tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 4260tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 4320tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 4380cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 4440agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 4500taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 4560tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 4620catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 4680ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 4740ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 4800catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 4860aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 4920aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 4980taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 5040atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 5100gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 5160tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 5220ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 5280gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 5340agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 5400aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 5460agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 5520tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 5580atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 5640taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 5700gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 5760gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 5820aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 5880tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 5940gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 6000cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 6060ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 6120cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg 6180ttggccgatt cattaatg 619826370DNAArtificial SequencepAAVsc-3xSerpEnh-TTRm-MVM-co-FIX-R338L-BGHpA 2agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020gcagagaggg agtggaattc acgcgtggat ctgaattcaa ttcacgcgtg gtacggccgc 1080ggtaccggcg cgcccgtacg cgggggaggc tgctggtgaa tattaaccaa ggtcacccca 1140gttatcggag gagcaaacag gggctaagtc caccggggga ggctgctggt gaatattaac 1200caaggtcacc ccagttatcg gaggagcaaa caggggctaa gtccaccggg ggaggctgct 1260ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccact 1320gtacaacgcg tgaattcgct agcgtctgtc tgcacatttc gtagagcgag tgttccgata 1380ctctaatctc cctaggcaag gttcatattt gtgtaggtta cttattctcc ttttgttgac 1440taagtcaata atcagaatca gcaggtttgg agtcagcttg gcagggatca gcagcctggg 1500ttggaaggag ggggtataaa agccccttca ccaggagaag ccgtcacaca gatccacaag 1560ctcctgtcta gaaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt 1620aattacctgg agcacctgcc tgaaatcact ttttttcagg ttgggctagc ccaccatgca 1680gcgcgtgaac atgatcatgg ccgagagccc cggcctgatc accatctgcc tgctgggcta 1740cctgctgagc gccgagtgca ccgtgttcct ggaccacgag aacgccaaca agatcctgaa 1800ccgccccaag cgctacaaca gcggcaagct ggaggagttc gtgcagggca acctggagcg 1860cgagtgcatg gaggagaagt gcagcttcga ggaggcccgc gaggtgttcg agaacaccga 1920gcgcaccacc gagttctgga agcagtacgt ggacggcgac cagtgcgaga gcaacccctg 1980cctgaacggc ggcagctgca aggacgacat caacagctac gagtgctggt gccccttcgg 2040cttcgagggc aagaactgcg agctggacgt gacctgcaac atcaagaacg gccgctgcga 2100gcagttctgc aagaacagcg ccgacaacaa ggtggtgtgc agctgcaccg agggctaccg 2160cctggccgag aaccagaaga gctgcgagcc cgccgtgccc ttcccctgcg gccgcgtgag 2220cgtgagccag accagcaagc tgacccgcgc cgaggccgtg ttccccgacg tggactacgt 2280gaacagcacc gaggccgaga ccatcctgga caacatcacc cagagcaccc agagcttcaa 2340cgacttcacc cgcgtggtgg gcggcgagga cgccaagccc ggccagttcc cctggcaggt 2400ggtgctgaac ggcaaggtgg acgccttctg cggcggcagc atcgtgaacg agaagtggat 2460cgtgaccgcc gcccactgcg tggagaccgg cgtgaagatc accgtggtgg ccggcgagca 2520caacatcgag gagaccgagc acaccgagca gaagcgcaac gtgatccgca tcatccccca 2580ccacaactac aacgccgcca tcaacaagta caaccacgac atcgccctgc tggagctgga 2640cgagcccctg gtgctgaaca gctacgtgac ccccatctgc atcgccgaca aggagtacac 2700caacatcttc ctgaagttcg gcagcggcta cgtgagcggc tggggccgcg tgttccacaa 2760gggccgcagc gccctggtgc tgcagtacct gcgcgtgccc ctggtggacc gcgccacctg 2820cctgctgagc accaagttca ccatctacaa caacatgttc tgcgccggct tccacgaggg 2880cggccgcgac agctgccagg gcgacagcgg cggcccccac gtgaccgagg tggagggcac 2940cagcttcctg accggcatca tcagctgggg cgaggagtgc gccatgaagg gcaagtacgg 3000catctacacc aaggtgagcc gctacgtgaa ctggatcaag gagaagacca agctgaccta 3060atgaaagatg gatttccaag gttaattcat tggaattgaa aattaacagc cccccccccc 3120ccccccctgc agatctgagc cgaattcctg cagcccgggg gatcagcctc gactgtgcct 3180tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 3240gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 3300tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac 3360aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc 3420tggggaccgg tggatctcga tagcaggcat gctggggaga gatcgatctg aggaacccct 3480agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc 3540aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag 3600agagggagtg gccaaccccc cccccccccc cccccggcga ttctcttgtt tgctccagac 3660tctcaggcaa tgacctgata gcctttgtag agacctctca aaaatagcta ccctctccgg 3720catgaattta tcagctagaa cggttgaata tcatattgat ggtgatttga ctgtctccgg 3780cctttctcac ccgtttgaat ctttacctac acattactca ggcattgcat ttaaaatata 3840tgagggttct aaaaattttt atccttgcgt tgaaataaag gcttctcccg caaaagtatt 3900acagggtcat aatgtttttg gtacaaccga tttagcttta tgctctgagg ctttattgct 3960taattttgct aattctttgc cttgcctgta tgatttattg gatgttggaa tcgcctgatg 4020cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt 4080acaatctgct ctgatgccgc atagttatat ggtgcactct cagtacaatc tgctctgatg 4140ccgcatagtt aagccagccc cgacacccgc caacacagcc agccccgaca cccgccaaca 4200cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 4260accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 4320cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 4380tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 4440taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gctcaataat 4500attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 4560cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 4620aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 4680ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 4740gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 4800attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 4860tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 4920tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 4980atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 5040agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 5100aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 5160caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 5220ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 5280gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 5340tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 5400atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 5460tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 5520accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 5580gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 5640caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 5700tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 5760ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 5820tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 5880gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 5940tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 6000gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 6060gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 6120ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 6180ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 6240ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 6300tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga 6360ttcattaatg 637036228DNAArtificial SequencepAAVsc-TTRe-TTRm-MVM-co-FIX-R338L-BGHpA 3agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020gcagagaggg agtggaattc acgcgtggat ctgaattcaa ttcacgcgtg gtacggccgc 1080ggtacccact gggaggatgt tgagtaagat ggaaaactac tgatgaccct tgcagagaca 1140gagtattagg acatgtttga acaggggccg ggcgatcagc aggtagctct agaggatccc 1200cgtctgtctg cacatttcgt agagcgagtg ttccgatact ctaatctccc taggcaaggt 1260tcatatttgt gtaggttact tattctcctt ttgttgacta agtcaataat cagaatcagc 1320aggtttggag tcagcttggc agggatcagc agcctgggtt ggaaggaggg ggtataaaag 1380ccccttcacc aggagaagcc gtcacacaga tccacaagct cctggctaga aagaggtaag 1440ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag cacctgcctg 1500aaatcacttt ttttcaggtt gggctagccc accatgcagc gcgtgaacat gatcatggcc 1560gagagccccg gcctgatcac catctgcctg ctgggctacc tgctgagcgc cgagtgcacc 1620gtgttcctgg accacgagaa cgccaacaag atcctgaacc gccccaagcg ctacaacagc 1680ggcaagctgg aggagttcgt gcagggcaac ctggagcgcg agtgcatgga ggagaagtgc 1740agcttcgagg aggcccgcga ggtgttcgag aacaccgagc gcaccaccga gttctggaag 1800cagtacgtgg acggcgacca gtgcgagagc aacccctgcc tgaacggcgg cagctgcaag 1860gacgacatca acagctacga gtgctggtgc cccttcggct tcgagggcaa gaactgcgag 1920ctggacgtga cctgcaacat caagaacggc cgctgcgagc agttctgcaa gaacagcgcc 1980gacaacaagg tggtgtgcag ctgcaccgag ggctaccgcc tggccgagaa ccagaagagc 2040tgcgagcccg ccgtgccctt cccctgcggc cgcgtgagcg tgagccagac cagcaagctg 2100acccgcgccg aggccgtgtt ccccgacgtg gactacgtga acagcaccga ggccgagacc

2160atcctggaca acatcaccca gagcacccag agcttcaacg acttcacccg cgtggtgggc 2220ggcgaggacg ccaagcccgg ccagttcccc tggcaggtgg tgctgaacgg caaggtggac 2280gccttctgcg gcggcagcat cgtgaacgag aagtggatcg tgaccgccgc ccactgcgtg 2340gagaccggcg tgaagatcac cgtggtggcc ggcgagcaca acatcgagga gaccgagcac 2400accgagcaga agcgcaacgt gatccgcatc atcccccacc acaactacaa cgccgccatc 2460aacaagtaca accacgacat cgccctgctg gagctggacg agcccctggt gctgaacagc 2520tacgtgaccc ccatctgcat cgccgacaag gagtacacca acatcttcct gaagttcggc 2580agcggctacg tgagcggctg gggccgcgtg ttccacaagg gccgcagcgc cctggtgctg 2640cagtacctgc gcgtgcccct ggtggaccgc gccacctgcc tgctgagcac caagttcacc 2700atctacaaca acatgttctg cgccggcttc cacgagggcg gccgcgacag ctgccagggc 2760gacagcggcg gcccccacgt gaccgaggtg gagggcacca gcttcctgac cggcatcatc 2820agctggggcg aggagtgcgc catgaagggc aagtacggca tctacaccaa ggtgagccgc 2880tacgtgaact ggatcaagga gaagaccaag ctgacctaat gaaagatgga tttccaaggt 2940taattcattg gaattgaaaa ttaacagccc cccccccccc ccccctgcag atctgagccg 3000aattcctgca gcccggggga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 3060tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 3120aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 3180gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 3240cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggaccggtg gatctcgata 3300gcaggcatgc tggggagaga tcgatctgag gaacccctag tgatggagtt ggccactccc 3360tctctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc 3420tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caaccccccc 3480cccccccccc cccggcgatt ctcttgtttg ctccagactc tcaggcaatg acctgatagc 3540ctttgtagag acctctcaaa aatagctacc ctctccggca tgaatttatc agctagaacg 3600gttgaatatc atattgatgg tgatttgact gtctccggcc tttctcaccc gtttgaatct 3660ttacctacac attactcagg cattgcattt aaaatatatg agggttctaa aaatttttat 3720ccttgcgttg aaataaaggc ttctcccgca aaagtattac agggtcataa tgtttttggt 3780acaaccgatt tagctttatg ctctgaggct ttattgctta attttgctaa ttctttgcct 3840tgcctgtatg atttattgga tgttggaatc gcctgatgcg gtattttctc cttacgcatc 3900tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 3960agttatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg 4020acacccgcca acacagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 4080cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 4140gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 4200tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 4260ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 4320cgctcatgag acaataaccc tgataaatgc tcaataatat tgaaaaagga agagtatgag 4380tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4440tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4500gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4560acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 4620tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4680gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 4740tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 4800accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 4860ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 4920agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 4980gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5040ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5100tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5160ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5220gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5280acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5340aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5400atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5460gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5520tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5580ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5640ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 5700ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 5760aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 5820cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 5880gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 5940ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6000cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6060tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6120cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6180cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatg 622846448DNAArtificial SequencepAAVsc-3xSerpEnh-TTRe-TTRm-MVM-co-FIX-R338L- BGHpA 4agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020gcagagaggg agtggaattc acgcgtggat ctgaattcaa ttcacgcgtg gtacggccgc 1080gggggggagg ctgctggtga atattaacca aggtcacccc agttatcgga ggagcaaaca 1140ggggctaagt ccaccggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 1200ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 1260caccccagtt atcggaggag caaacagggg ctaagtccac ggtacccact gggaggatgt 1320tgagtaagat ggaaaactac tgatgaccct tgcagagaca gagtattagg acatgtttga 1380acaggggccg ggcgatcagc aggtagctct agaggatccc cgtctgtctg cacatttcgt 1440agagcgagtg ttccgatact ctaatctccc taggcaaggt tcatatttgt gtaggttact 1500tattctcctt ttgttgacta agtcaataat cagaatcagc aggtttggag tcagcttggc 1560agggatcagc agcctgggtt ggaaggaggg ggtataaaag ccccttcacc aggagaagcc 1620gtcacacaga tccacaagct cctggctaga aagaggtaag ggtttaaggg atggttggtt 1680ggtggggtat taatgtttaa ttacctggag cacctgcctg aaatcacttt ttttcaggtt 1740gggctagccc accatgcagc gcgtgaacat gatcatggcc gagagccccg gcctgatcac 1800catctgcctg ctgggctacc tgctgagcgc cgagtgcacc gtgttcctgg accacgagaa 1860cgccaacaag atcctgaacc gccccaagcg ctacaacagc ggcaagctgg aggagttcgt 1920gcagggcaac ctggagcgcg agtgcatgga ggagaagtgc agcttcgagg aggcccgcga 1980ggtgttcgag aacaccgagc gcaccaccga gttctggaag cagtacgtgg acggcgacca 2040gtgcgagagc aacccctgcc tgaacggcgg cagctgcaag gacgacatca acagctacga 2100gtgctggtgc cccttcggct tcgagggcaa gaactgcgag ctggacgtga cctgcaacat 2160caagaacggc cgctgcgagc agttctgcaa gaacagcgcc gacaacaagg tggtgtgcag 2220ctgcaccgag ggctaccgcc tggccgagaa ccagaagagc tgcgagcccg ccgtgccctt 2280cccctgcggc cgcgtgagcg tgagccagac cagcaagctg acccgcgccg aggccgtgtt 2340ccccgacgtg gactacgtga acagcaccga ggccgagacc atcctggaca acatcaccca 2400gagcacccag agcttcaacg acttcacccg cgtggtgggc ggcgaggacg ccaagcccgg 2460ccagttcccc tggcaggtgg tgctgaacgg caaggtggac gccttctgcg gcggcagcat 2520cgtgaacgag aagtggatcg tgaccgccgc ccactgcgtg gagaccggcg tgaagatcac 2580cgtggtggcc ggcgagcaca acatcgagga gaccgagcac accgagcaga agcgcaacgt 2640gatccgcatc atcccccacc acaactacaa cgccgccatc aacaagtaca accacgacat 2700cgccctgctg gagctggacg agcccctggt gctgaacagc tacgtgaccc ccatctgcat 2760cgccgacaag gagtacacca acatcttcct gaagttcggc agcggctacg tgagcggctg 2820gggccgcgtg ttccacaagg gccgcagcgc cctggtgctg cagtacctgc gcgtgcccct 2880ggtggaccgc gccacctgcc tgctgagcac caagttcacc atctacaaca acatgttctg 2940cgccggcttc cacgagggcg gccgcgacag ctgccagggc gacagcggcg gcccccacgt 3000gaccgaggtg gagggcacca gcttcctgac cggcatcatc agctggggcg aggagtgcgc 3060catgaagggc aagtacggca tctacaccaa ggtgagccgc tacgtgaact ggatcaagga 3120gaagaccaag ctgacctaat gaaagatgga tttccaaggt taattcattg gaattgaaaa 3180ttaacagccc cccccccccc ccccctgcag atctgagccg aattcctgca gcccggggga 3240tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 3300tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 3360tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 3420ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct 3480gaggcggaaa gaaccagctg gggaccggtg gatctcgata gcaggcatgc tggggagaga 3540tcgatctgag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 3600cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt 3660gagcgagcga gcgcgcagag agggagtggc caaccccccc cccccccccc cccggcgatt 3720ctcttgtttg ctccagactc tcaggcaatg acctgatagc ctttgtagag acctctcaaa 3780aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc atattgatgg 3840tgatttgact gtctccggcc tttctcaccc gtttgaatct ttacctacac attactcagg 3900cattgcattt aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc 3960ttctcccgca aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg 4020ctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga 4080tgttggaatc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 4140atatggtgca ctctcagtac aatctgctct gatgccgcat agttatatgg tgcactctca 4200gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacagccag 4260ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 4320gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 4380tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc 4440atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc 4500cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 4560tgataaatgc tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 4620cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 4680tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 4740tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 4800cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 4860tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 4920agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 4980ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 5040ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 5100aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 5160gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 5220tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 5280ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 5340cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 5400atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 5460cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 5520ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 5580cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 5640ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 5700tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 5760taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 5820caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 5880agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 5940gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 6000gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 6060ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 6120acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 6180tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 6240ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 6300ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 6360ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc 6420tccccgcgcg ttggccgatt cattaatg 6448572DNAArtificial SequenceSerpEnh 5gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60ggctaagtcc ac 726202DNAArtificial SequenceTTRm 6gtctgtctgc acatttcgta gagcgagtgt tccgatactc taatctccct aggcaaggtt 60catatttgtg taggttactt attctccttt tgttgactaa gtcaataatc agaatcagca 120ggtttggagt cagcttggca gggatcagca gcctgggttg gaaggagggg gtataaaagc 180cccttcacca ggagaagccg tc 202721DNAArtificial SequenceTTRm5'UTR 7acacagatcc acaagctcct g 21892DNAArtificial SequenceMVM intron 8aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag 60cacctgcctg aaatcacttt ttttcaggtt gg 9291389DNAArtificial SequenceCo-FIX-R338L 9atgcagcgcg tgaacatgat catggccgag agccccggcc tgatcaccat ctgcctgctg 60ggctacctgc tgagcgccga gtgcaccgtg ttcctggacc acgagaacgc caacaagatc 120ctgaaccgcc ccaagcgcta caacagcggc aagctggagg agttcgtgca gggcaacctg 180gagcgcgagt gcatggagga gaagtgcagc ttcgaggagg cccgcgaggt gttcgagaac 240accgagcgca ccaccgagtt ctggaagcag tacgtggacg gcgaccagtg cgagagcaac 300ccctgcctga acggcggcag ctgcaaggac gacatcaaca gctacgagtg ctggtgcccc 360ttcggcttcg agggcaagaa ctgcgagctg gacgtgacct gcaacatcaa gaacggccgc 420tgcgagcagt tctgcaagaa cagcgccgac aacaaggtgg tgtgcagctg caccgagggc 480taccgcctgg ccgagaacca gaagagctgc gagcccgccg tgcccttccc ctgcggccgc 540gtgagcgtga gccagaccag caagctgacc cgcgccgagg ccgtgttccc cgacgtggac 600tacgtgaaca gcaccgaggc cgagaccatc ctggacaaca tcacccagag cacccagagc 660ttcaacgact tcacccgcgt ggtgggcggc gaggacgcca agcccggcca gttcccctgg 720caggtggtgc tgaacggcaa ggtggacgcc ttctgcggcg gcagcatcgt gaacgagaag 780tggatcgtga ccgccgccca ctgcgtggag accggcgtga agatcaccgt ggtggccggc 840gagcacaaca tcgaggagac cgagcacacc gagcagaagc gcaacgtgat ccgcatcatc 900ccccaccaca actacaacgc cgccatcaac aagtacaacc acgacatcgc cctgctggag 960ctggacgagc ccctggtgct gaacagctac gtgaccccca tctgcatcgc cgacaaggag 1020tacaccaaca tcttcctgaa gttcggcagc ggctacgtga gcggctgggg ccgcgtgttc 1080cacaagggcc gcagcgccct ggtgctgcag tacctgcgcg tgcccctggt ggaccgcgcc 1140acctgcctgc tgagcaccaa gttcaccatc tacaacaaca tgttctgcgc cggcttccac 1200gagggcggcc gcgacagctg ccagggcgac agcggcggcc cccacgtgac cgaggtggag 1260ggcaccagct tcctgaccgg catcatcagc tggggcgagg agtgcgccat gaagggcaag 1320tacggcatct acaccaaggt gagccgctac gtgaactgga tcaaggagaa gaccaagctg 1380acctaatga 138910295DNAArtificial SequenceBGHpolyA 10gatctgagcc gaattcctgc agcccggggg atcagcctcg actgtgcctt ctagttgcca 60gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 120tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 180tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 240tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct gggga 29511218DNAArtificial Sequence3xSERP 11gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 120aggagcaaac aggggctaag tccaccgggg gaggctgctg gtgaatatta accaaggtca 180ccccagttat cggaggagca aacaggggct aagtccac 21812100DNAArtificial SequenceTTRe 12cactgggagg atgttgagta agatggaaaa ctactgatga cccttgcaga gacagagtat 60taggacatgt ttgaacaggg gccgggcgat cagcaggtag 10013324DNAArtificial Sequence3xSERP-Flank-TTRe 13gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 120aggagcaaac aggggctaag tccaccgggg gaggctgctg gtgaatatta accaaggtca 180ccccagttat cggaggagca aacaggggct aagtccacgg tacccactgg gaggatgttg 240agtaagatgg aaaactactg atgacccttg cagagacaga gtattaggac atgtttgaac 300aggggccggg cgatcagcag gtag 324147757DNAArtificial SequencepAAVss-TTRm-MVM-CoFVIIIdeltaB-Sv40pA 14cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggccgcg gtaccgtctg tctgcacatt tcgtagagcg 180agtgttccga tactctaatc tccctaggca aggttcatat ttgtgtaggt tacttattct 240ccttttgttg actaagtcaa taatcagaat cagcaggttt ggagtcagct tggcagggat 300cagcagcctg ggttggaagg agggggtata aaagcccctt caccaggaga agccgtcaca 360cagatccaca agctcctgaa gaggtaaggg tttaagggat ggttggttgg tggggtatta 420atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg ctagtatgca 480gatcgagctg tccacctgct tttttctgtg cctgctgcgg ttctgcttca gcgccacccg 540gcggtactac ctgggcgccg tggagctgtc ctgggactac atgcagagcg acctgggcga 600gctgcccgtg gacgcccggt tcccccccag agtgcccaag agcttcccct tcaacaccag 660cgtggtgtac aagaaaaccc tgttcgtgga gttcaccgac cacctgttca atatcgccaa 720gcccaggccc ccctggatgg gcctgctggg ccccaccatc caggccgagg tgtacgacac 780cgtggtgatc accctgaaga acatggccag ccaccccgtg agcctgcacg ccgtgggcgt 840gagctactgg aaggccagcg agggcgccga gtacgacgac cagaccagcc agcgggagaa 900agaagatgac aaggtgttcc ctggcggcag ccacacctac gtgtggcagg tgctgaaaga 960aaacggcccc atggcctccg

accccctgtg cctgacctac agctacctga gccacgtgga 1020cctggtgaag gacctgaaca gcggcctgat cggcgctctg ctcgtctgcc gggagggcag 1080cctggccaaa gagaaaaccc agaccctgca caagttcatc ctgctgttcg ccgtgttcga 1140cgagggcaag agctggcaca gcgagacaaa gaacagcctg atgcaggacc gggacgccgc 1200ctctgccaga gcctggccca agatgcacac cgtgaacggc tacgtgaaca gaagcctgcc 1260cggcctgatt ggctgccacc ggaagagcgt gtactggcac gtgatcggca tgggcaccac 1320acccgaggtg cacagcatct ttctggaagg gcacaccttt ctggtccgga accaccggca 1380ggccagcctg gaaatcagcc ctatcacctt cctgaccgcc cagacactgc tgatggacct 1440gggccagttc ctgctgtttt gccacatcag ctctcaccag cacgacggca tggaagccta 1500cgtgaaggtg gactcttgcc ccgaggaacc ccagctgcgg atgaagaaca acgaggaagc 1560cgaggactac gacgacgacc tgaccgacag cgagatggac gtggtgcggt tcgacgacga 1620caacagcccc agcttcatcc agatcagaag cgtggccaag aagcacccca agacctgggt 1680gcactatatc gccgccgagg aagaggactg ggactacgcc cccctggtgc tggcccccga 1740cgacagaagc tacaagagcc agtacctgaa caatggcccc cagcggatcg gccggaagta 1800caagaaagtg cggttcatgg cctacaccga cgagacattc aagacccggg aggccatcca 1860gcacgagagc ggcatcctgg gccccctgct gtacggcgaa gtgggcgaca cactgctgat 1920catcttcaag aaccaggcta gccggcccta caacatctac ccccacggca tcaccgacgt 1980gcggcccctg tacagcaggc ggctgcccaa gggcgtgaag cacctgaagg acttccccat 2040cctgcccggc gagatcttca agtacaagtg gaccgtgacc gtggaggacg gccccaccaa 2100gagcgacccc agatgcctga cccggtacta cagcagcttc gtgaacatgg aacgggacct 2160ggcctccggg ctgatcggac ctctgctgat ctgctacaaa gaaagcgtgg accagcgggg 2220caaccagatc atgagcgaca agcggaacgt gatcctgttc agcgtgttcg atgagaaccg 2280gtcctggtat ctgaccgaga acatccagcg gtttctgccc aaccctgccg gcgtgcagct 2340ggaagatccc gagttccagg ccagcaacat catgcactcc atcaatggct acgtgttcga 2400ctctctgcag ctctccgtgt gtctgcacga ggtggcctac tggtacatcc tgagcatcgg 2460cgcccagacc gacttcctga gcgtgttctt cagcggctac accttcaagc acaagatggt 2520gtacgaggac accctgaccc tgttcccttt cagcggcgag acagtgttca tgagcatgga 2580aaaccccggc ctgtggattc tgggctgcca caacagcgac ttccggaacc ggggcatgac 2640cgccctgctg aaggtgtcca gctgcgacaa gaacaccggc gactactacg aggacagcta 2700cgaggatatc agcgcctacc tgctgtccaa gaacaacgcc atcgaacccc ggagcttcag 2760ccagaacccc cccgtgctga cgcgtcacca gcgggagatc acccggacaa ccctgcagtc 2820cgaccaggaa gagatcgatt acgacgacac catcagcgtg gagatgaaga aagaggattt 2880cgatatctac gacgaggacg agaaccagag ccccagaagc ttccagaaga aaacccggca 2940ctacttcatt gccgccgtgg agaggctgtg ggactacggc atgagttcta gcccccacgt 3000gctgcggaac cgggcccaga gcggcagcgt gccccagttc aagaaagtgg tgttccagga 3060attcacagac ggcagcttca cccagcctct gtatagaggc gagctgaacg agcacctggg 3120gctgctgggg ccctacatca gggccgaagt ggaggacaac atcatggtga ccttccggaa 3180tcaggccagc agaccctact ccttctacag cagcctgatc agctacgaag aggaccagcg 3240gcagggcgcc gaaccccgga agaacttcgt gaagcccaac gaaaccaaga cctacttctg 3300gaaagtgcag caccacatgg cccccaccaa ggacgagttc gactgcaagg cctgggccta 3360cttcagcgac gtggatctgg aaaaggacgt gcactctgga ctgattggcc cactcctggt 3420ctgccacact aacaccctca accccgccca cggccgccag gtgaccgtgc aggaattcgc 3480cctgttcttc accatcttcg acgagacaaa gtcctggtac ttcaccgaga atatggaacg 3540gaactgcaga gccccctgca acatccagat ggaagatcct accttcaaag agaactaccg 3600gttccacgcc atcaacggct acatcatgga caccctgcct ggcctggtga tggcccagga 3660ccagagaatc cggtggtatc tgctgtccat gggcagcaac gagaatatcc acagcatcca 3720cttcagcggc cacgtgttca ccgtgcggaa gaaagaagag tacaagatgg ccctgtacaa 3780cctgtacccc ggcgtgttcg agacagtgga gatgctgccc agcaaggccg gcatctggcg 3840ggtggagtgt ctgatcggcg agcacctgca cgctggcatg agcaccctgt ttctggtgta 3900cagcaacaag tgccagaccc cactgggcat ggcctctggc cacatccggg acttccagat 3960caccgcctcc ggccagtacg gccagtgggc ccccaagctg gccagactgc actacagcgg 4020cagcatcaac gcctggtcca ccaaagagcc cttcagctgg atcaaggtgg acctgctggc 4080ccctatgatc atccacggca ttaagaccca gggcgccagg cagaagttca gcagcctgta 4140catcagccag ttcatcatca tgtacagcct ggacggcaag aagtggcaga cctaccgggg 4200caacagcacc ggcaccctga tggtgttctt cggcaatgtg gacagcagcg gcatcaagca 4260caacatcttc aaccccccca tcattgcccg gtacatccgg ctgcacccca cccactacag 4320cattagatcc acactgagaa tggaactgat gggctgcgac ctgaactcct gcagcatgcc 4380tctgggcatg gaaagcaagg ccatcagcga cgcccagatc acagccagca gctacttcac 4440caacatgttc gccacctggt ccccctccaa ggccaggctg cacctgcagg gccggtccaa 4500cgcctggcgg cctcaggtca acaaccccaa agaatggctg caggtggact ttcagaaaac 4560catgaaggtg accggcgtga ccacccaggg cgtgaaaagc ctgctgacca gcatgtacgt 4620gaaagagttt ctgatcagca gctctcagga tggccaccag tggaccctgt tctttcagaa 4680cggcaaggtg aaagtgttcc agggcaacca ggactccttc acccccgtgg tgaactccct 4740ggaccccccc ctgctgaccc gctacctgag aatccacccc cagtcttggg tgcaccagat 4800cgccctcagg atggaagtcc tgggatgtga ggcccaggat ctgtactgat gaggatctag 4860gctcgacatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa 4920gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg 4980aggtgtggga ggttttttaa actcgagatc cacggccgca ggaaccccta gtgatggagt 5040tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc 5100gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg 5160ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc 5220aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 5280gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 5340ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 5400agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg 5460ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 5520gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta 5580ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 5640ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac 5700tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 5760cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 5820cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg 5880aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 5940gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 6000aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 6060ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 6120ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 6180agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 6240tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 6300tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 6360ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 6420gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 6480acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 6540tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 6600gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 6660actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 6720aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 6780cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 6840tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 6900cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 6960tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 7020ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 7080ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 7140cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 7200aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 7260agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 7320tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 7380ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 7440cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 7500atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 7560ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 7620tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 7680gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 7740gccttttgct cacatgt 7757157849DNAArtificial SequencepAAVss-SerpEnh-TTRm-MVM-CoFVIIIdeltaB-Sv40pA 15cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggccgcg gtaccggcgc gccgggggag gctgctggtg 180aatattaacc aaggtcaccc cagttatcgg aggagcaaac aggggctaag tccacacgcg 240tggtaccgtc tgtctgcaca tttcgtagag cgagtgttcc gatactctaa tctccctagg 300caaggttcat atttgtgtag gttacttatt ctccttttgt tgactaagtc aataatcaga 360atcagcaggt ttggagtcag cttggcaggg atcagcagcc tgggttggaa ggagggggta 420taaaagcccc ttcaccagga gaagccgtca cacagatcca caagctcctg aagaggtaag 480ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag cacctgcctg 540aaatcacttt ttttcaggtt ggctagtatg cagatcgagc tgtccacctg cttttttctg 600tgcctgctgc ggttctgctt cagcgccacc cggcggtact acctgggcgc cgtggagctg 660tcctgggact acatgcagag cgacctgggc gagctgcccg tggacgcccg gttccccccc 720agagtgccca agagcttccc cttcaacacc agcgtggtgt acaagaaaac cctgttcgtg 780gagttcaccg accacctgtt caatatcgcc aagcccaggc ccccctggat gggcctgctg 840ggccccacca tccaggccga ggtgtacgac accgtggtga tcaccctgaa gaacatggcc 900agccaccccg tgagcctgca cgccgtgggc gtgagctact ggaaggccag cgagggcgcc 960gagtacgacg accagaccag ccagcgggag aaagaagatg acaaggtgtt ccctggcggc 1020agccacacct acgtgtggca ggtgctgaaa gaaaacggcc ccatggcctc cgaccccctg 1080tgcctgacct acagctacct gagccacgtg gacctggtga aggacctgaa cagcggcctg 1140atcggcgctc tgctcgtctg ccgggagggc agcctggcca aagagaaaac ccagaccctg 1200cacaagttca tcctgctgtt cgccgtgttc gacgagggca agagctggca cagcgagaca 1260aagaacagcc tgatgcagga ccgggacgcc gcctctgcca gagcctggcc caagatgcac 1320accgtgaacg gctacgtgaa cagaagcctg cccggcctga ttggctgcca ccggaagagc 1380gtgtactggc acgtgatcgg catgggcacc acacccgagg tgcacagcat ctttctggaa 1440gggcacacct ttctggtccg gaaccaccgg caggccagcc tggaaatcag ccctatcacc 1500ttcctgaccg cccagacact gctgatggac ctgggccagt tcctgctgtt ttgccacatc 1560agctctcacc agcacgacgg catggaagcc tacgtgaagg tggactcttg ccccgaggaa 1620ccccagctgc ggatgaagaa caacgaggaa gccgaggact acgacgacga cctgaccgac 1680agcgagatgg acgtggtgcg gttcgacgac gacaacagcc ccagcttcat ccagatcaga 1740agcgtggcca agaagcaccc caagacctgg gtgcactata tcgccgccga ggaagaggac 1800tgggactacg cccccctggt gctggccccc gacgacagaa gctacaagag ccagtacctg 1860aacaatggcc cccagcggat cggccggaag tacaagaaag tgcggttcat ggcctacacc 1920gacgagacat tcaagacccg ggaggccatc cagcacgaga gcggcatcct gggccccctg 1980ctgtacggcg aagtgggcga cacactgctg atcatcttca agaaccaggc tagccggccc 2040tacaacatct acccccacgg catcaccgac gtgcggcccc tgtacagcag gcggctgccc 2100aagggcgtga agcacctgaa ggacttcccc atcctgcccg gcgagatctt caagtacaag 2160tggaccgtga ccgtggagga cggccccacc aagagcgacc ccagatgcct gacccggtac 2220tacagcagct tcgtgaacat ggaacgggac ctggcctccg ggctgatcgg acctctgctg 2280atctgctaca aagaaagcgt ggaccagcgg ggcaaccaga tcatgagcga caagcggaac 2340gtgatcctgt tcagcgtgtt cgatgagaac cggtcctggt atctgaccga gaacatccag 2400cggtttctgc ccaaccctgc cggcgtgcag ctggaagatc ccgagttcca ggccagcaac 2460atcatgcact ccatcaatgg ctacgtgttc gactctctgc agctctccgt gtgtctgcac 2520gaggtggcct actggtacat cctgagcatc ggcgcccaga ccgacttcct gagcgtgttc 2580ttcagcggct acaccttcaa gcacaagatg gtgtacgagg acaccctgac cctgttccct 2640ttcagcggcg agacagtgtt catgagcatg gaaaaccccg gcctgtggat tctgggctgc 2700cacaacagcg acttccggaa ccggggcatg accgccctgc tgaaggtgtc cagctgcgac 2760aagaacaccg gcgactacta cgaggacagc tacgaggata tcagcgccta cctgctgtcc 2820aagaacaacg ccatcgaacc ccggagcttc agccagaacc cccccgtgct gacgcgtcac 2880cagcgggaga tcacccggac aaccctgcag tccgaccagg aagagatcga ttacgacgac 2940accatcagcg tggagatgaa gaaagaggat ttcgatatct acgacgagga cgagaaccag 3000agccccagaa gcttccagaa gaaaacccgg cactacttca ttgccgccgt ggagaggctg 3060tgggactacg gcatgagttc tagcccccac gtgctgcgga accgggccca gagcggcagc 3120gtgccccagt tcaagaaagt ggtgttccag gaattcacag acggcagctt cacccagcct 3180ctgtatagag gcgagctgaa cgagcacctg gggctgctgg ggccctacat cagggccgaa 3240gtggaggaca acatcatggt gaccttccgg aatcaggcca gcagacccta ctccttctac 3300agcagcctga tcagctacga agaggaccag cggcagggcg ccgaaccccg gaagaacttc 3360gtgaagccca acgaaaccaa gacctacttc tggaaagtgc agcaccacat ggcccccacc 3420aaggacgagt tcgactgcaa ggcctgggcc tacttcagcg acgtggatct ggaaaaggac 3480gtgcactctg gactgattgg cccactcctg gtctgccaca ctaacaccct caaccccgcc 3540cacggccgcc aggtgaccgt gcaggaattc gccctgttct tcaccatctt cgacgagaca 3600aagtcctggt acttcaccga gaatatggaa cggaactgca gagccccctg caacatccag 3660atggaagatc ctaccttcaa agagaactac cggttccacg ccatcaacgg ctacatcatg 3720gacaccctgc ctggcctggt gatggcccag gaccagagaa tccggtggta tctgctgtcc 3780atgggcagca acgagaatat ccacagcatc cacttcagcg gccacgtgtt caccgtgcgg 3840aagaaagaag agtacaagat ggccctgtac aacctgtacc ccggcgtgtt cgagacagtg 3900gagatgctgc ccagcaaggc cggcatctgg cgggtggagt gtctgatcgg cgagcacctg 3960cacgctggca tgagcaccct gtttctggtg tacagcaaca agtgccagac cccactgggc 4020atggcctctg gccacatccg ggacttccag atcaccgcct ccggccagta cggccagtgg 4080gcccccaagc tggccagact gcactacagc ggcagcatca acgcctggtc caccaaagag 4140cccttcagct ggatcaaggt ggacctgctg gcccctatga tcatccacgg cattaagacc 4200cagggcgcca ggcagaagtt cagcagcctg tacatcagcc agttcatcat catgtacagc 4260ctggacggca agaagtggca gacctaccgg ggcaacagca ccggcaccct gatggtgttc 4320ttcggcaatg tggacagcag cggcatcaag cacaacatct tcaacccccc catcattgcc 4380cggtacatcc ggctgcaccc cacccactac agcattagat ccacactgag aatggaactg 4440atgggctgcg acctgaactc ctgcagcatg cctctgggca tggaaagcaa ggccatcagc 4500gacgcccaga tcacagccag cagctacttc accaacatgt tcgccacctg gtccccctcc 4560aaggccaggc tgcacctgca gggccggtcc aacgcctggc ggcctcaggt caacaacccc 4620aaagaatggc tgcaggtgga ctttcagaaa accatgaagg tgaccggcgt gaccacccag 4680ggcgtgaaaa gcctgctgac cagcatgtac gtgaaagagt ttctgatcag cagctctcag 4740gatggccacc agtggaccct gttctttcag aacggcaagg tgaaagtgtt ccagggcaac 4800caggactcct tcacccccgt ggtgaactcc ctggaccccc ccctgctgac ccgctacctg 4860agaatccacc cccagtcttg ggtgcaccag atcgccctca ggatggaagt cctgggatgt 4920gaggcccagg atctgtactg atgaggatct aggctcgaca tgctttattt gtgaaatttg 4980tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa 5040ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaactcgaga 5100tccacggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 5160gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 5220agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac 5280gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 5340ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 5400gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 5460ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 5520ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 5580acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 5640actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg 5700atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 5760aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 5820tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 5880ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 5940ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 6000taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 6060gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 6120agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 6180catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 6240ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 6300atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 6360ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 6420gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 6480ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 6540ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 6600gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 6660ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 6720gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 6780ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 6840gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 6900gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 6960caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 7020cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 7080ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 7140taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 7200tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 7260gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 7320agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 7380aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 7440gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 7500gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 7560tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 7620agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 7680cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 7740gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 7800gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt 7849168007DNAArtificial SequencepAAVss-3xSerpEnh-TTRm-MVM-CoFVIIIdeltaB-Sv40pA 16cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggccgcg gtacgcgggg gaggctgctg gtgaatatta 180accaaggtca

ccccagttat cggaggagca aacaggggct aagtccaccg ggggaggctg 240ctggtgaata ttaaccaagg tcaccccagt tatcggagga gcaaacaggg gctaagtcca 300ccgggggagg ctgctggtga atattaacca aggtcacccc agttatcgga ggagcaaaca 360ggggctaagt ccactgtaca acgcgtgaat tcgctagcgt ctgtctgcac atttcgtaga 420gcgagtgttc cgatactcta atctccctag gcaaggttca tatttgtgta ggttacttat 480tctccttttg ttgactaagt caataatcag aatcagcagg tttggagtca gcttggcagg 540gatcagcagc ctgggttgga aggagggggt ataaaagccc cttcaccagg agaagccgtc 600acacagatcc acaagctcct gtctagaaag aggtaagggt ttaagggatg gttggttggt 660ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttggg 720ctagcatgca gatcgagctg tccacctgct tttttctgtg cctgctgcgg ttctgcttca 780gcgccacccg gcggtactac ctgggcgccg tggagctgtc ctgggactac atgcagagcg 840acctgggcga gctgcccgtg gacgcccggt tcccccccag agtgcccaag agcttcccct 900tcaacaccag cgtggtgtac aagaaaaccc tgttcgtgga gttcaccgac cacctgttca 960atatcgccaa gcccaggccc ccctggatgg gcctgctggg ccccaccatc caggccgagg 1020tgtacgacac cgtggtgatc accctgaaga acatggccag ccaccccgtg agcctgcacg 1080ccgtgggcgt gagctactgg aaggccagcg agggcgccga gtacgacgac cagaccagcc 1140agcgggagaa agaagatgac aaggtgttcc ctggcggcag ccacacctac gtgtggcagg 1200tgctgaaaga aaacggcccc atggcctccg accccctgtg cctgacctac agctacctga 1260gccacgtgga cctggtgaag gacctgaaca gcggcctgat cggcgctctg ctcgtctgcc 1320gggagggcag cctggccaaa gagaaaaccc agaccctgca caagttcatc ctgctgttcg 1380ccgtgttcga cgagggcaag agctggcaca gcgagacaaa gaacagcctg atgcaggacc 1440gggacgccgc ctctgccaga gcctggccca agatgcacac cgtgaacggc tacgtgaaca 1500gaagcctgcc cggcctgatt ggctgccacc ggaagagcgt gtactggcac gtgatcggca 1560tgggcaccac acccgaggtg cacagcatct ttctggaagg gcacaccttt ctggtccgga 1620accaccggca ggccagcctg gaaatcagcc ctatcacctt cctgaccgcc cagacactgc 1680tgatggacct gggccagttc ctgctgtttt gccacatcag ctctcaccag cacgacggca 1740tggaagccta cgtgaaggtg gactcttgcc ccgaggaacc ccagctgcgg atgaagaaca 1800acgaggaagc cgaggactac gacgacgacc tgaccgacag cgagatggac gtggtgcggt 1860tcgacgacga caacagcccc agcttcatcc agatcagaag cgtggccaag aagcacccca 1920agacctgggt gcactatatc gccgccgagg aagaggactg ggactacgcc cccctggtgc 1980tggcccccga cgacagaagc tacaagagcc agtacctgaa caatggcccc cagcggatcg 2040gccggaagta caagaaagtg cggttcatgg cctacaccga cgagacattc aagacccggg 2100aggccatcca gcacgagagc ggcatcctgg gccccctgct gtacggcgaa gtgggcgaca 2160cactgctgat catcttcaag aaccaggcta gccggcccta caacatctac ccccacggca 2220tcaccgacgt gcggcccctg tacagcaggc ggctgcccaa gggcgtgaag cacctgaagg 2280acttccccat cctgcccggc gagatcttca agtacaagtg gaccgtgacc gtggaggacg 2340gccccaccaa gagcgacccc agatgcctga cccggtacta cagcagcttc gtgaacatgg 2400aacgggacct ggcctccggg ctgatcggac ctctgctgat ctgctacaaa gaaagcgtgg 2460accagcgggg caaccagatc atgagcgaca agcggaacgt gatcctgttc agcgtgttcg 2520atgagaaccg gtcctggtat ctgaccgaga acatccagcg gtttctgccc aaccctgccg 2580gcgtgcagct ggaagatccc gagttccagg ccagcaacat catgcactcc atcaatggct 2640acgtgttcga ctctctgcag ctctccgtgt gtctgcacga ggtggcctac tggtacatcc 2700tgagcatcgg cgcccagacc gacttcctga gcgtgttctt cagcggctac accttcaagc 2760acaagatggt gtacgaggac accctgaccc tgttcccttt cagcggcgag acagtgttca 2820tgagcatgga aaaccccggc ctgtggattc tgggctgcca caacagcgac ttccggaacc 2880ggggcatgac cgccctgctg aaggtgtcca gctgcgacaa gaacaccggc gactactacg 2940aggacagcta cgaggatatc agcgcctacc tgctgtccaa gaacaacgcc atcgaacccc 3000ggagcttcag ccagaacccc cccgtgctga cgcgtcacca gcgggagatc acccggacaa 3060ccctgcagtc cgaccaggaa gagatcgatt acgacgacac catcagcgtg gagatgaaga 3120aagaggattt cgatatctac gacgaggacg agaaccagag ccccagaagc ttccagaaga 3180aaacccggca ctacttcatt gccgccgtgg agaggctgtg ggactacggc atgagttcta 3240gcccccacgt gctgcggaac cgggcccaga gcggcagcgt gccccagttc aagaaagtgg 3300tgttccagga attcacagac ggcagcttca cccagcctct gtatagaggc gagctgaacg 3360agcacctggg gctgctgggg ccctacatca gggccgaagt ggaggacaac atcatggtga 3420ccttccggaa tcaggccagc agaccctact ccttctacag cagcctgatc agctacgaag 3480aggaccagcg gcagggcgcc gaaccccgga agaacttcgt gaagcccaac gaaaccaaga 3540cctacttctg gaaagtgcag caccacatgg cccccaccaa ggacgagttc gactgcaagg 3600cctgggccta cttcagcgac gtggatctgg aaaaggacgt gcactctgga ctgattggcc 3660cactcctggt ctgccacact aacaccctca accccgccca cggccgccag gtgaccgtgc 3720aggaattcgc cctgttcttc accatcttcg acgagacaaa gtcctggtac ttcaccgaga 3780atatggaacg gaactgcaga gccccctgca acatccagat ggaagatcct accttcaaag 3840agaactaccg gttccacgcc atcaacggct acatcatgga caccctgcct ggcctggtga 3900tggcccagga ccagagaatc cggtggtatc tgctgtccat gggcagcaac gagaatatcc 3960acagcatcca cttcagcggc cacgtgttca ccgtgcggaa gaaagaagag tacaagatgg 4020ccctgtacaa cctgtacccc ggcgtgttcg agacagtgga gatgctgccc agcaaggccg 4080gcatctggcg ggtggagtgt ctgatcggcg agcacctgca cgctggcatg agcaccctgt 4140ttctggtgta cagcaacaag tgccagaccc cactgggcat ggcctctggc cacatccggg 4200acttccagat caccgcctcc ggccagtacg gccagtgggc ccccaagctg gccagactgc 4260actacagcgg cagcatcaac gcctggtcca ccaaagagcc cttcagctgg atcaaggtgg 4320acctgctggc ccctatgatc atccacggca ttaagaccca gggcgccagg cagaagttca 4380gcagcctgta catcagccag ttcatcatca tgtacagcct ggacggcaag aagtggcaga 4440cctaccgggg caacagcacc ggcaccctga tggtgttctt cggcaatgtg gacagcagcg 4500gcatcaagca caacatcttc aaccccccca tcattgcccg gtacatccgg ctgcacccca 4560cccactacag cattagatcc acactgagaa tggaactgat gggctgcgac ctgaactcct 4620gcagcatgcc tctgggcatg gaaagcaagg ccatcagcga cgcccagatc acagccagca 4680gctacttcac caacatgttc gccacctggt ccccctccaa ggccaggctg cacctgcagg 4740gccggtccaa cgcctggcgg cctcaggtca acaaccccaa agaatggctg caggtggact 4800ttcagaaaac catgaaggtg accggcgtga ccacccaggg cgtgaaaagc ctgctgacca 4860gcatgtacgt gaaagagttt ctgatcagca gctctcagga tggccaccag tggaccctgt 4920tctttcagaa cggcaaggtg aaagtgttcc agggcaacca ggactccttc acccccgtgg 4980tgaactccct ggaccccccc ctgctgaccc gctacctgag aatccacccc cagtcttggg 5040tgcaccagat cgccctcagg atggaagtcc tgggatgtga ggcccaggat ctgtactgat 5100gaggatctag gctcgacatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 5160accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag 5220gttcaggggg aggtgtggga ggttttttaa actcgagatc cacggccgca ggaaccccta 5280gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca 5340aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc 5400tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 5460cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg 5520tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 5580ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 5640ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 5700tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 5760tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 5820tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 5880atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 5940tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 6000cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 6060aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 6120gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 6180tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 6240tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 6300ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 6360ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 6420aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 6480gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 6540ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 6600gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 6660cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 6720cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 6780acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 6840caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 6900taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 6960ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 7020aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 7080agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 7140atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 7200tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 7260tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 7320gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 7380taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 7440aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 7500ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 7560catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 7620ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 7680ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 7740agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 7800taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 7860atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 7920cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 7980ccttttgctg gccttttgct cacatgt 8007177872DNAArtificial SequencepAAVss-TTRe-TTRm-MVM-CoFVIIIdeltaB-Sv40pA 17cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggccgcg gtacccactg ggaggatgtt gagtaagatg 180gaaaactact gatgaccctt gcagagacag agtattagga catgtttgaa caggggccgg 240gcgatcagca ggtagctcta gaggatcccc gtctgtctgc acatttcgta gagcgagtgt 300tccgatactc taatctccct aggcaaggtt catatttgtg taggttactt attctccttt 360tgttgactaa gtcaataatc agaatcagca ggtttggagt cagcttggca gggatcagca 420gcctgggttg gaaggagggg gtataaaagc cccttcacca ggagaagccg tcacacagat 480ccacaagctc ctgaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt 540taattacctg gagcacctgc ctgaaatcac tttttttcag gttggctagt atgcagatcg 600agctgtccac ctgctttttt ctgtgcctgc tgcggttctg cttcagcgcc acccggcggt 660actacctggg cgccgtggag ctgtcctggg actacatgca gagcgacctg ggcgagctgc 720ccgtggacgc ccggttcccc cccagagtgc ccaagagctt ccccttcaac accagcgtgg 780tgtacaagaa aaccctgttc gtggagttca ccgaccacct gttcaatatc gccaagccca 840ggcccccctg gatgggcctg ctgggcccca ccatccaggc cgaggtgtac gacaccgtgg 900tgatcaccct gaagaacatg gccagccacc ccgtgagcct gcacgccgtg ggcgtgagct 960actggaaggc cagcgagggc gccgagtacg acgaccagac cagccagcgg gagaaagaag 1020atgacaaggt gttccctggc ggcagccaca cctacgtgtg gcaggtgctg aaagaaaacg 1080gccccatggc ctccgacccc ctgtgcctga cctacagcta cctgagccac gtggacctgg 1140tgaaggacct gaacagcggc ctgatcggcg ctctgctcgt ctgccgggag ggcagcctgg 1200ccaaagagaa aacccagacc ctgcacaagt tcatcctgct gttcgccgtg ttcgacgagg 1260gcaagagctg gcacagcgag acaaagaaca gcctgatgca ggaccgggac gccgcctctg 1320ccagagcctg gcccaagatg cacaccgtga acggctacgt gaacagaagc ctgcccggcc 1380tgattggctg ccaccggaag agcgtgtact ggcacgtgat cggcatgggc accacacccg 1440aggtgcacag catctttctg gaagggcaca cctttctggt ccggaaccac cggcaggcca 1500gcctggaaat cagccctatc accttcctga ccgcccagac actgctgatg gacctgggcc 1560agttcctgct gttttgccac atcagctctc accagcacga cggcatggaa gcctacgtga 1620aggtggactc ttgccccgag gaaccccagc tgcggatgaa gaacaacgag gaagccgagg 1680actacgacga cgacctgacc gacagcgaga tggacgtggt gcggttcgac gacgacaaca 1740gccccagctt catccagatc agaagcgtgg ccaagaagca ccccaagacc tgggtgcact 1800atatcgccgc cgaggaagag gactgggact acgcccccct ggtgctggcc cccgacgaca 1860gaagctacaa gagccagtac ctgaacaatg gcccccagcg gatcggccgg aagtacaaga 1920aagtgcggtt catggcctac accgacgaga cattcaagac ccgggaggcc atccagcacg 1980agagcggcat cctgggcccc ctgctgtacg gcgaagtggg cgacacactg ctgatcatct 2040tcaagaacca ggctagccgg ccctacaaca tctaccccca cggcatcacc gacgtgcggc 2100ccctgtacag caggcggctg cccaagggcg tgaagcacct gaaggacttc cccatcctgc 2160ccggcgagat cttcaagtac aagtggaccg tgaccgtgga ggacggcccc accaagagcg 2220accccagatg cctgacccgg tactacagca gcttcgtgaa catggaacgg gacctggcct 2280ccgggctgat cggacctctg ctgatctgct acaaagaaag cgtggaccag cggggcaacc 2340agatcatgag cgacaagcgg aacgtgatcc tgttcagcgt gttcgatgag aaccggtcct 2400ggtatctgac cgagaacatc cagcggtttc tgcccaaccc tgccggcgtg cagctggaag 2460atcccgagtt ccaggccagc aacatcatgc actccatcaa tggctacgtg ttcgactctc 2520tgcagctctc cgtgtgtctg cacgaggtgg cctactggta catcctgagc atcggcgccc 2580agaccgactt cctgagcgtg ttcttcagcg gctacacctt caagcacaag atggtgtacg 2640aggacaccct gaccctgttc cctttcagcg gcgagacagt gttcatgagc atggaaaacc 2700ccggcctgtg gattctgggc tgccacaaca gcgacttccg gaaccggggc atgaccgccc 2760tgctgaaggt gtccagctgc gacaagaaca ccggcgacta ctacgaggac agctacgagg 2820atatcagcgc ctacctgctg tccaagaaca acgccatcga accccggagc ttcagccaga 2880acccccccgt gctgacgcgt caccagcggg agatcacccg gacaaccctg cagtccgacc 2940aggaagagat cgattacgac gacaccatca gcgtggagat gaagaaagag gatttcgata 3000tctacgacga ggacgagaac cagagcccca gaagcttcca gaagaaaacc cggcactact 3060tcattgccgc cgtggagagg ctgtgggact acggcatgag ttctagcccc cacgtgctgc 3120ggaaccgggc ccagagcggc agcgtgcccc agttcaagaa agtggtgttc caggaattca 3180cagacggcag cttcacccag cctctgtata gaggcgagct gaacgagcac ctggggctgc 3240tggggcccta catcagggcc gaagtggagg acaacatcat ggtgaccttc cggaatcagg 3300ccagcagacc ctactccttc tacagcagcc tgatcagcta cgaagaggac cagcggcagg 3360gcgccgaacc ccggaagaac ttcgtgaagc ccaacgaaac caagacctac ttctggaaag 3420tgcagcacca catggccccc accaaggacg agttcgactg caaggcctgg gcctacttca 3480gcgacgtgga tctggaaaag gacgtgcact ctggactgat tggcccactc ctggtctgcc 3540acactaacac cctcaacccc gcccacggcc gccaggtgac cgtgcaggaa ttcgccctgt 3600tcttcaccat cttcgacgag acaaagtcct ggtacttcac cgagaatatg gaacggaact 3660gcagagcccc ctgcaacatc cagatggaag atcctacctt caaagagaac taccggttcc 3720acgccatcaa cggctacatc atggacaccc tgcctggcct ggtgatggcc caggaccaga 3780gaatccggtg gtatctgctg tccatgggca gcaacgagaa tatccacagc atccacttca 3840gcggccacgt gttcaccgtg cggaagaaag aagagtacaa gatggccctg tacaacctgt 3900accccggcgt gttcgagaca gtggagatgc tgcccagcaa ggccggcatc tggcgggtgg 3960agtgtctgat cggcgagcac ctgcacgctg gcatgagcac cctgtttctg gtgtacagca 4020acaagtgcca gaccccactg ggcatggcct ctggccacat ccgggacttc cagatcaccg 4080cctccggcca gtacggccag tgggccccca agctggccag actgcactac agcggcagca 4140tcaacgcctg gtccaccaaa gagcccttca gctggatcaa ggtggacctg ctggccccta 4200tgatcatcca cggcattaag acccagggcg ccaggcagaa gttcagcagc ctgtacatca 4260gccagttcat catcatgtac agcctggacg gcaagaagtg gcagacctac cggggcaaca 4320gcaccggcac cctgatggtg ttcttcggca atgtggacag cagcggcatc aagcacaaca 4380tcttcaaccc ccccatcatt gcccggtaca tccggctgca ccccacccac tacagcatta 4440gatccacact gagaatggaa ctgatgggct gcgacctgaa ctcctgcagc atgcctctgg 4500gcatggaaag caaggccatc agcgacgccc agatcacagc cagcagctac ttcaccaaca 4560tgttcgccac ctggtccccc tccaaggcca ggctgcacct gcagggccgg tccaacgcct 4620ggcggcctca ggtcaacaac cccaaagaat ggctgcaggt ggactttcag aaaaccatga 4680aggtgaccgg cgtgaccacc cagggcgtga aaagcctgct gaccagcatg tacgtgaaag 4740agtttctgat cagcagctct caggatggcc accagtggac cctgttcttt cagaacggca 4800aggtgaaagt gttccagggc aaccaggact ccttcacccc cgtggtgaac tccctggacc 4860cccccctgct gacccgctac ctgagaatcc acccccagtc ttgggtgcac cagatcgccc 4920tcaggatgga agtcctggga tgtgaggccc aggatctgta ctgatgagga tctaggctcg 4980acatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc 5040aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg 5100tgggaggttt tttaaactcg agatccacgg ccgcaggaac ccctagtgat ggagttggcc 5160actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc 5220ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc 5280ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc 5340aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 5400gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 5460ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 5520tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac 5580gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 5640ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt 5700ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 5760aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca 5820gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 5880acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 5940ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 6000gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt 6060caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 6120attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 6180aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 6240tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 6300agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 6360gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 6420cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 6480agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 6540taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 6600tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 6660taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 6720acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 6780ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 6840cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 6900agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 6960tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 7020agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 7080tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg

7140ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 7200tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 7260aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 7320tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 7380agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 7440taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 7500caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 7560agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 7620aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 7680gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 7740tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 7800gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 7860ttgctcacat gt 7872184377DNAArtificial SequenceCoFVIIIdeltaB 18atgcagatcg agctgtccac ctgctttttt ctgtgcctgc tgcggttctg cttcagcgcc 60acccggcggt actacctggg cgccgtggag ctgtcctggg actacatgca gagcgacctg 120ggcgagctgc ccgtggacgc ccggttcccc cccagagtgc ccaagagctt ccccttcaac 180accagcgtgg tgtacaagaa aaccctgttc gtggagttca ccgaccacct gttcaatatc 240gccaagccca ggcccccctg gatgggcctg ctgggcccca ccatccaggc cgaggtgtac 300gacaccgtgg tgatcaccct gaagaacatg gccagccacc ccgtgagcct gcacgccgtg 360ggcgtgagct actggaaggc cagcgagggc gccgagtacg acgaccagac cagccagcgg 420gagaaagaag atgacaaggt gttccctggc ggcagccaca cctacgtgtg gcaggtgctg 480aaagaaaacg gccccatggc ctccgacccc ctgtgcctga cctacagcta cctgagccac 540gtggacctgg tgaaggacct gaacagcggc ctgatcggcg ctctgctcgt ctgccgggag 600ggcagcctgg ccaaagagaa aacccagacc ctgcacaagt tcatcctgct gttcgccgtg 660ttcgacgagg gcaagagctg gcacagcgag acaaagaaca gcctgatgca ggaccgggac 720gccgcctctg ccagagcctg gcccaagatg cacaccgtga acggctacgt gaacagaagc 780ctgcccggcc tgattggctg ccaccggaag agcgtgtact ggcacgtgat cggcatgggc 840accacacccg aggtgcacag catctttctg gaagggcaca cctttctggt ccggaaccac 900cggcaggcca gcctggaaat cagccctatc accttcctga ccgcccagac actgctgatg 960gacctgggcc agttcctgct gttttgccac atcagctctc accagcacga cggcatggaa 1020gcctacgtga aggtggactc ttgccccgag gaaccccagc tgcggatgaa gaacaacgag 1080gaagccgagg actacgacga cgacctgacc gacagcgaga tggacgtggt gcggttcgac 1140gacgacaaca gccccagctt catccagatc agaagcgtgg ccaagaagca ccccaagacc 1200tgggtgcact atatcgccgc cgaggaagag gactgggact acgcccccct ggtgctggcc 1260cccgacgaca gaagctacaa gagccagtac ctgaacaatg gcccccagcg gatcggccgg 1320aagtacaaga aagtgcggtt catggcctac accgacgaga cattcaagac ccgggaggcc 1380atccagcacg agagcggcat cctgggcccc ctgctgtacg gcgaagtggg cgacacactg 1440ctgatcatct tcaagaacca ggctagccgg ccctacaaca tctaccccca cggcatcacc 1500gacgtgcggc ccctgtacag caggcggctg cccaagggcg tgaagcacct gaaggacttc 1560cccatcctgc ccggcgagat cttcaagtac aagtggaccg tgaccgtgga ggacggcccc 1620accaagagcg accccagatg cctgacccgg tactacagca gcttcgtgaa catggaacgg 1680gacctggcct ccgggctgat cggacctctg ctgatctgct acaaagaaag cgtggaccag 1740cggggcaacc agatcatgag cgacaagcgg aacgtgatcc tgttcagcgt gttcgatgag 1800aaccggtcct ggtatctgac cgagaacatc cagcggtttc tgcccaaccc tgccggcgtg 1860cagctggaag atcccgagtt ccaggccagc aacatcatgc actccatcaa tggctacgtg 1920ttcgactctc tgcagctctc cgtgtgtctg cacgaggtgg cctactggta catcctgagc 1980atcggcgccc agaccgactt cctgagcgtg ttcttcagcg gctacacctt caagcacaag 2040atggtgtacg aggacaccct gaccctgttc cctttcagcg gcgagacagt gttcatgagc 2100atggaaaacc ccggcctgtg gattctgggc tgccacaaca gcgacttccg gaaccggggc 2160atgaccgccc tgctgaaggt gtccagctgc gacaagaaca ccggcgacta ctacgaggac 2220agctacgagg atatcagcgc ctacctgctg tccaagaaca acgccatcga accccggagc 2280ttcagccaga acccccccgt gctgacgcgt caccagcggg agatcacccg gacaaccctg 2340cagtccgacc aggaagagat cgattacgac gacaccatca gcgtggagat gaagaaagag 2400gatttcgata tctacgacga ggacgagaac cagagcccca gaagcttcca gaagaaaacc 2460cggcactact tcattgccgc cgtggagagg ctgtgggact acggcatgag ttctagcccc 2520cacgtgctgc ggaaccgggc ccagagcggc agcgtgcccc agttcaagaa agtggtgttc 2580caggaattca cagacggcag cttcacccag cctctgtata gaggcgagct gaacgagcac 2640ctggggctgc tggggcccta catcagggcc gaagtggagg acaacatcat ggtgaccttc 2700cggaatcagg ccagcagacc ctactccttc tacagcagcc tgatcagcta cgaagaggac 2760cagcggcagg gcgccgaacc ccggaagaac ttcgtgaagc ccaacgaaac caagacctac 2820ttctggaaag tgcagcacca catggccccc accaaggacg agttcgactg caaggcctgg 2880gcctacttca gcgacgtgga tctggaaaag gacgtgcact ctggactgat tggcccactc 2940ctggtctgcc acactaacac cctcaacccc gcccacggcc gccaggtgac cgtgcaggaa 3000ttcgccctgt tcttcaccat cttcgacgag acaaagtcct ggtacttcac cgagaatatg 3060gaacggaact gcagagcccc ctgcaacatc cagatggaag atcctacctt caaagagaac 3120taccggttcc acgccatcaa cggctacatc atggacaccc tgcctggcct ggtgatggcc 3180caggaccaga gaatccggtg gtatctgctg tccatgggca gcaacgagaa tatccacagc 3240atccacttca gcggccacgt gttcaccgtg cggaagaaag aagagtacaa gatggccctg 3300tacaacctgt accccggcgt gttcgagaca gtggagatgc tgcccagcaa ggccggcatc 3360tggcgggtgg agtgtctgat cggcgagcac ctgcacgctg gcatgagcac cctgtttctg 3420gtgtacagca acaagtgcca gaccccactg ggcatggcct ctggccacat ccgggacttc 3480cagatcaccg cctccggcca gtacggccag tgggccccca agctggccag actgcactac 3540agcggcagca tcaacgcctg gtccaccaaa gagcccttca gctggatcaa ggtggacctg 3600ctggccccta tgatcatcca cggcattaag acccagggcg ccaggcagaa gttcagcagc 3660ctgtacatca gccagttcat catcatgtac agcctggacg gcaagaagtg gcagacctac 3720cggggcaaca gcaccggcac cctgatggtg ttcttcggca atgtggacag cagcggcatc 3780aagcacaaca tcttcaaccc ccccatcatt gcccggtaca tccggctgca ccccacccac 3840tacagcatta gatccacact gagaatggaa ctgatgggct gcgacctgaa ctcctgcagc 3900atgcctctgg gcatggaaag caaggccatc agcgacgccc agatcacagc cagcagctac 3960ttcaccaaca tgttcgccac ctggtccccc tccaaggcca ggctgcacct gcagggccgg 4020tccaacgcct ggcggcctca ggtcaacaac cccaaagaat ggctgcaggt ggactttcag 4080aaaaccatga aggtgaccgg cgtgaccacc cagggcgtga aaagcctgct gaccagcatg 4140tacgtgaaag agtttctgat cagcagctct caggatggcc accagtggac cctgttcttt 4200cagaacggca aggtgaaagt gttccagggc aaccaggact ccttcacccc cgtggtgaac 4260tccctggacc cccccctgct gacccgctac ctgagaatcc acccccagtc ttgggtgcac 4320cagatcgccc tcaggatgga agtcctggga tgtgaggccc aggatctgta ctgatga 437719134DNAArtificial SequenceSV40polyA 19atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 60taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg 120ggaggttttt taaa 134207922DNAArtificial SequencepAAVss-3xSerpEnh-TTRm-MVM-coFVIIIdeltaB-Synt-pA 20cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggccgcg gtacgcgggg gaggctgctg gtgaatatta 180accaaggtca ccccagttat cggaggagca aacaggggct aagtccaccg ggggaggctg 240ctggtgaata ttaaccaagg tcaccccagt tatcggagga gcaaacaggg gctaagtcca 300ccgggggagg ctgctggtga atattaacca aggtcacccc agttatcgga ggagcaaaca 360ggggctaagt ccactgtaca acgcgtgaat tcgctagcgt ctgtctgcac atttcgtaga 420gcgagtgttc cgatactcta atctccctag gcaaggttca tatttgtgta ggttacttat 480tctccttttg ttgactaagt caataatcag aatcagcagg tttggagtca gcttggcagg 540gatcagcagc ctgggttgga aggagggggt ataaaagccc cttcaccagg agaagccgtc 600acacagatcc acaagctcct gtctagaaag aggtaagggt ttaagggatg gttggttggt 660ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttggg 720ctagcatgca gatcgagctg tccacctgct tttttctgtg cctgctgcgg ttctgcttca 780gcgccacccg gcggtactac ctgggcgccg tggagctgtc ctgggactac atgcagagcg 840acctgggcga gctgcccgtg gacgcccggt tcccccccag agtgcccaag agcttcccct 900tcaacaccag cgtggtgtac aagaaaaccc tgttcgtgga gttcaccgac cacctgttca 960atatcgccaa gcccaggccc ccctggatgg gcctgctggg ccccaccatc caggccgagg 1020tgtacgacac cgtggtgatc accctgaaga acatggccag ccaccccgtg agcctgcacg 1080ccgtgggcgt gagctactgg aaggccagcg agggcgccga gtacgacgac cagaccagcc 1140agcgggagaa agaagatgac aaggtgttcc ctggcggcag ccacacctac gtgtggcagg 1200tgctgaaaga aaacggcccc atggcctccg accccctgtg cctgacctac agctacctga 1260gccacgtgga cctggtgaag gacctgaaca gcggcctgat cggcgctctg ctcgtctgcc 1320gggagggcag cctggccaaa gagaaaaccc agaccctgca caagttcatc ctgctgttcg 1380ccgtgttcga cgagggcaag agctggcaca gcgagacaaa gaacagcctg atgcaggacc 1440gggacgccgc ctctgccaga gcctggccca agatgcacac cgtgaacggc tacgtgaaca 1500gaagcctgcc cggcctgatt ggctgccacc ggaagagcgt gtactggcac gtgatcggca 1560tgggcaccac acccgaggtg cacagcatct ttctggaagg gcacaccttt ctggtccgga 1620accaccggca ggccagcctg gaaatcagcc ctatcacctt cctgaccgcc cagacactgc 1680tgatggacct gggccagttc ctgctgtttt gccacatcag ctctcaccag cacgacggca 1740tggaagccta cgtgaaggtg gactcttgcc ccgaggaacc ccagctgcgg atgaagaaca 1800acgaggaagc cgaggactac gacgacgacc tgaccgacag cgagatggac gtggtgcggt 1860tcgacgacga caacagcccc agcttcatcc agatcagaag cgtggccaag aagcacccca 1920agacctgggt gcactatatc gccgccgagg aagaggactg ggactacgcc cccctggtgc 1980tggcccccga cgacagaagc tacaagagcc agtacctgaa caatggcccc cagcggatcg 2040gccggaagta caagaaagtg cggttcatgg cctacaccga cgagacattc aagacccggg 2100aggccatcca gcacgagagc ggcatcctgg gccccctgct gtacggcgaa gtgggcgaca 2160cactgctgat catcttcaag aaccaggcta gccggcccta caacatctac ccccacggca 2220tcaccgacgt gcggcccctg tacagcaggc ggctgcccaa gggcgtgaag cacctgaagg 2280acttccccat cctgcccggc gagatcttca agtacaagtg gaccgtgacc gtggaggacg 2340gccccaccaa gagcgacccc agatgcctga cccggtacta cagcagcttc gtgaacatgg 2400aacgggacct ggcctccggg ctgatcggac ctctgctgat ctgctacaaa gaaagcgtgg 2460accagcgggg caaccagatc atgagcgaca agcggaacgt gatcctgttc agcgtgttcg 2520atgagaaccg gtcctggtat ctgaccgaga acatccagcg gtttctgccc aaccctgccg 2580gcgtgcagct ggaagatccc gagttccagg ccagcaacat catgcactcc atcaatggct 2640acgtgttcga ctctctgcag ctctccgtgt gtctgcacga ggtggcctac tggtacatcc 2700tgagcatcgg cgcccagacc gacttcctga gcgtgttctt cagcggctac accttcaagc 2760acaagatggt gtacgaggac accctgaccc tgttcccttt cagcggcgag acagtgttca 2820tgagcatgga aaaccccggc ctgtggattc tgggctgcca caacagcgac ttccggaacc 2880ggggcatgac cgccctgctg aaggtgtcca gctgcgacaa gaacaccggc gactactacg 2940aggacagcta cgaggatatc agcgcctacc tgctgtccaa gaacaacgcc atcgaacccc 3000ggagcttcag ccagaacccc cccgtgctga cgcgtcacca gcgggagatc acccggacaa 3060ccctgcagtc cgaccaggaa gagatcgatt acgacgacac catcagcgtg gagatgaaga 3120aagaggattt cgatatctac gacgaggacg agaaccagag ccccagaagc ttccagaaga 3180aaacccggca ctacttcatt gccgccgtgg agaggctgtg ggactacggc atgagttcta 3240gcccccacgt gctgcggaac cgggcccaga gcggcagcgt gccccagttc aagaaagtgg 3300tgttccagga attcacagac ggcagcttca cccagcctct gtatagaggc gagctgaacg 3360agcacctggg gctgctgggg ccctacatca gggccgaagt ggaggacaac atcatggtga 3420ccttccggaa tcaggccagc agaccctact ccttctacag cagcctgatc agctacgaag 3480aggaccagcg gcagggcgcc gaaccccgga agaacttcgt gaagcccaac gaaaccaaga 3540cctacttctg gaaagtgcag caccacatgg cccccaccaa ggacgagttc gactgcaagg 3600cctgggccta cttcagcgac gtggatctgg aaaaggacgt gcactctgga ctgattggcc 3660cactcctggt ctgccacact aacaccctca accccgccca cggccgccag gtgaccgtgc 3720aggaattcgc cctgttcttc accatcttcg acgagacaaa gtcctggtac ttcaccgaga 3780atatggaacg gaactgcaga gccccctgca acatccagat ggaagatcct accttcaaag 3840agaactaccg gttccacgcc atcaacggct acatcatgga caccctgcct ggcctggtga 3900tggcccagga ccagagaatc cggtggtatc tgctgtccat gggcagcaac gagaatatcc 3960acagcatcca cttcagcggc cacgtgttca ccgtgcggaa gaaagaagag tacaagatgg 4020ccctgtacaa cctgtacccc ggcgtgttcg agacagtgga gatgctgccc agcaaggccg 4080gcatctggcg ggtggagtgt ctgatcggcg agcacctgca cgctggcatg agcaccctgt 4140ttctggtgta cagcaacaag tgccagaccc cactgggcat ggcctctggc cacatccggg 4200acttccagat caccgcctcc ggccagtacg gccagtgggc ccccaagctg gccagactgc 4260actacagcgg cagcatcaac gcctggtcca ccaaagagcc cttcagctgg atcaaggtgg 4320acctgctggc ccctatgatc atccacggca ttaagaccca gggcgccagg cagaagttca 4380gcagcctgta catcagccag ttcatcatca tgtacagcct ggacggcaag aagtggcaga 4440cctaccgggg caacagcacc ggcaccctga tggtgttctt cggcaatgtg gacagcagcg 4500gcatcaagca caacatcttc aaccccccca tcattgcccg gtacatccgg ctgcacccca 4560cccactacag cattagatcc acactgagaa tggaactgat gggctgcgac ctgaactcct 4620gcagcatgcc tctgggcatg gaaagcaagg ccatcagcga cgcccagatc acagccagca 4680gctacttcac caacatgttc gccacctggt ccccctccaa ggccaggctg cacctgcagg 4740gccggtccaa cgcctggcgg cctcaggtca acaaccccaa agaatggctg caggtggact 4800ttcagaaaac catgaaggtg accggcgtga ccacccaggg cgtgaaaagc ctgctgacca 4860gcatgtacgt gaaagagttt ctgatcagca gctctcagga tggccaccag tggaccctgt 4920tctttcagaa cggcaaggtg aaagtgttcc agggcaacca ggactccttc acccccgtgg 4980tgaactccct ggaccccccc ctgctgaccc gctacctgag aatccacccc cagtcttggg 5040tgcaccagat cgccctcagg atggaagtcc tgggatgtga ggcccaggat ctgtactgat 5100gaggatctag gctcgacaat aaaagatctt tattttcatt agatctgtgt gttggttttt 5160tgtgtgctcg agatccacgg ccgcaggaac ccctagtgat ggagttggcc actccctctc 5220tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 5280cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt 5340attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta 5400cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 5460tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 5520gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 5580tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc 5640atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 5700actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata 5760agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 5820cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg 5880ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg 5940acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 6000catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 6060acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 6120ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 6180gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 6240tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 6300tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 6360acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 6420cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 6480ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 6540ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 6600atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 6660cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 6720tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 6780gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 6840ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 6900ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 6960tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 7020cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 7080ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 7140tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 7200gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 7260caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 7320accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 7380ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt 7440aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 7500accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 7560gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 7620ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 7680gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 7740gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 7800ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 7860aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 7920gt 7922217814DNAArtificial SequencepAAVss-3xSerpEnh-TTRm-coFVIIIdeltaB-Synt-pA 21cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggccgcg gtacgcgggg gaggctgctg gtgaatatta 180accaaggtca ccccagttat cggaggagca aacaggggct aagtccaccg ggggaggctg 240ctggtgaata ttaaccaagg tcaccccagt tatcggagga gcaaacaggg gctaagtcca 300ccgggggagg ctgctggtga atattaacca aggtcacccc agttatcgga ggagcaaaca 360ggggctaagt ccactgtaca acgcgtgaat tcgctagcgt ctgtctgcac atttcgtaga 420gcgagtgttc cgatactcta atctccctag gcaaggttca tatttgtgta ggttacttat 480tctccttttg ttgactaagt caataatcag aatcagcagg tttggagtca gcttggcagg 540gatcagcagc ctgggttgga aggagggggt ataaaagccc cttcaccagg agaagccgtc 600acacagatcc acaagctcct gctagtatgc agatcgagct gtccacctgc ttttttctgt 660gcctgctgcg gttctgcttc agcgccaccc ggcggtacta cctgggcgcc gtggagctgt 720cctgggacta catgcagagc gacctgggcg agctgcccgt ggacgcccgg ttccccccca 780gagtgcccaa gagcttcccc ttcaacacca gcgtggtgta caagaaaacc ctgttcgtgg 840agttcaccga ccacctgttc aatatcgcca agcccaggcc cccctggatg ggcctgctgg 900gccccaccat ccaggccgag gtgtacgaca ccgtggtgat caccctgaag aacatggcca 960gccaccccgt gagcctgcac gccgtgggcg tgagctactg gaaggccagc gagggcgccg 1020agtacgacga ccagaccagc cagcgggaga aagaagatga caaggtgttc cctggcggca 1080gccacaccta cgtgtggcag gtgctgaaag aaaacggccc catggcctcc gaccccctgt 1140gcctgaccta cagctacctg agccacgtgg acctggtgaa ggacctgaac agcggcctga 1200tcggcgctct gctcgtctgc cgggagggca gcctggccaa agagaaaacc cagaccctgc 1260acaagttcat cctgctgttc gccgtgttcg acgagggcaa gagctggcac agcgagacaa 1320agaacagcct gatgcaggac cgggacgccg cctctgccag agcctggccc aagatgcaca 1380ccgtgaacgg ctacgtgaac agaagcctgc ccggcctgat tggctgccac cggaagagcg 1440tgtactggca

cgtgatcggc atgggcacca cacccgaggt gcacagcatc tttctggaag 1500ggcacacctt tctggtccgg aaccaccggc aggccagcct ggaaatcagc cctatcacct 1560tcctgaccgc ccagacactg ctgatggacc tgggccagtt cctgctgttt tgccacatca 1620gctctcacca gcacgacggc atggaagcct acgtgaaggt ggactcttgc cccgaggaac 1680cccagctgcg gatgaagaac aacgaggaag ccgaggacta cgacgacgac ctgaccgaca 1740gcgagatgga cgtggtgcgg ttcgacgacg acaacagccc cagcttcatc cagatcagaa 1800gcgtggccaa gaagcacccc aagacctggg tgcactatat cgccgccgag gaagaggact 1860gggactacgc ccccctggtg ctggcccccg acgacagaag ctacaagagc cagtacctga 1920acaatggccc ccagcggatc ggccggaagt acaagaaagt gcggttcatg gcctacaccg 1980acgagacatt caagacccgg gaggccatcc agcacgagag cggcatcctg ggccccctgc 2040tgtacggcga agtgggcgac acactgctga tcatcttcaa gaaccaggct agccggccct 2100acaacatcta cccccacggc atcaccgacg tgcggcccct gtacagcagg cggctgccca 2160agggcgtgaa gcacctgaag gacttcccca tcctgcccgg cgagatcttc aagtacaagt 2220ggaccgtgac cgtggaggac ggccccacca agagcgaccc cagatgcctg acccggtact 2280acagcagctt cgtgaacatg gaacgggacc tggcctccgg gctgatcgga cctctgctga 2340tctgctacaa agaaagcgtg gaccagcggg gcaaccagat catgagcgac aagcggaacg 2400tgatcctgtt cagcgtgttc gatgagaacc ggtcctggta tctgaccgag aacatccagc 2460ggtttctgcc caaccctgcc ggcgtgcagc tggaagatcc cgagttccag gccagcaaca 2520tcatgcactc catcaatggc tacgtgttcg actctctgca gctctccgtg tgtctgcacg 2580aggtggccta ctggtacatc ctgagcatcg gcgcccagac cgacttcctg agcgtgttct 2640tcagcggcta caccttcaag cacaagatgg tgtacgagga caccctgacc ctgttccctt 2700tcagcggcga gacagtgttc atgagcatgg aaaaccccgg cctgtggatt ctgggctgcc 2760acaacagcga cttccggaac cggggcatga ccgccctgct gaaggtgtcc agctgcgaca 2820agaacaccgg cgactactac gaggacagct acgaggatat cagcgcctac ctgctgtcca 2880agaacaacgc catcgaaccc cggagcttca gccagaaccc ccccgtgctg acgcgtcacc 2940agcgggagat cacccggaca accctgcagt ccgaccagga agagatcgat tacgacgaca 3000ccatcagcgt ggagatgaag aaagaggatt tcgatatcta cgacgaggac gagaaccaga 3060gccccagaag cttccagaag aaaacccggc actacttcat tgccgccgtg gagaggctgt 3120gggactacgg catgagttct agcccccacg tgctgcggaa ccgggcccag agcggcagcg 3180tgccccagtt caagaaagtg gtgttccagg aattcacaga cggcagcttc acccagcctc 3240tgtatagagg cgagctgaac gagcacctgg ggctgctggg gccctacatc agggccgaag 3300tggaggacaa catcatggtg accttccgga atcaggccag cagaccctac tccttctaca 3360gcagcctgat cagctacgaa gaggaccagc ggcagggcgc cgaaccccgg aagaacttcg 3420tgaagcccaa cgaaaccaag acctacttct ggaaagtgca gcaccacatg gcccccacca 3480aggacgagtt cgactgcaag gcctgggcct acttcagcga cgtggatctg gaaaaggacg 3540tgcactctgg actgattggc ccactcctgg tctgccacac taacaccctc aaccccgccc 3600acggccgcca ggtgaccgtg caggaattcg ccctgttctt caccatcttc gacgagacaa 3660agtcctggta cttcaccgag aatatggaac ggaactgcag agccccctgc aacatccaga 3720tggaagatcc taccttcaaa gagaactacc ggttccacgc catcaacggc tacatcatgg 3780acaccctgcc tggcctggtg atggcccagg accagagaat ccggtggtat ctgctgtcca 3840tgggcagcaa cgagaatatc cacagcatcc acttcagcgg ccacgtgttc accgtgcgga 3900agaaagaaga gtacaagatg gccctgtaca acctgtaccc cggcgtgttc gagacagtgg 3960agatgctgcc cagcaaggcc ggcatctggc gggtggagtg tctgatcggc gagcacctgc 4020acgctggcat gagcaccctg tttctggtgt acagcaacaa gtgccagacc ccactgggca 4080tggcctctgg ccacatccgg gacttccaga tcaccgcctc cggccagtac ggccagtggg 4140cccccaagct ggccagactg cactacagcg gcagcatcaa cgcctggtcc accaaagagc 4200ccttcagctg gatcaaggtg gacctgctgg cccctatgat catccacggc attaagaccc 4260agggcgccag gcagaagttc agcagcctgt acatcagcca gttcatcatc atgtacagcc 4320tggacggcaa gaagtggcag acctaccggg gcaacagcac cggcaccctg atggtgttct 4380tcggcaatgt ggacagcagc ggcatcaagc acaacatctt caaccccccc atcattgccc 4440ggtacatccg gctgcacccc acccactaca gcattagatc cacactgaga atggaactga 4500tgggctgcga cctgaactcc tgcagcatgc ctctgggcat ggaaagcaag gccatcagcg 4560acgcccagat cacagccagc agctacttca ccaacatgtt cgccacctgg tccccctcca 4620aggccaggct gcacctgcag ggccggtcca acgcctggcg gcctcaggtc aacaacccca 4680aagaatggct gcaggtggac tttcagaaaa ccatgaaggt gaccggcgtg accacccagg 4740gcgtgaaaag cctgctgacc agcatgtacg tgaaagagtt tctgatcagc agctctcagg 4800atggccacca gtggaccctg ttctttcaga acggcaaggt gaaagtgttc cagggcaacc 4860aggactcctt cacccccgtg gtgaactccc tggacccccc cctgctgacc cgctacctga 4920gaatccaccc ccagtcttgg gtgcaccaga tcgccctcag gatggaagtc ctgggatgtg 4980aggcccagga tctgtactga tgaggatcca ataaaagatc tttattttca ttagatctgt 5040gtgttggttt tttgtgtgct cgagatccac ggccgcagga acccctagtg atggagttgg 5100ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 5160gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc 5220gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa 5280gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 5340cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 5400ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 5460gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc 5520acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 5580ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc 5640ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 5700acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct 5760cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 5820tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 5880ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 5940gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 6000gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 6060acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 6120aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 6180attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 6240tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 6300gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 6360cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 6420tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 6480agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 6540tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 6600tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 6660tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 6720acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 6780accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 6840tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 6900cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 6960tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 7020actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 7080tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 7140cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 7200gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 7260tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 7320gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 7380gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 7440ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 7500acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 7560agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 7620cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 7680tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 7740gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 7800ttttgctcac atgt 7814228090DNAArtificial SequencepAAVss-3xSerpEnh-TTRe-TTRm-MVM-coFVIIIdeltaB- Sv40pA 22cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggccgcg ggggaggctg ctggtgaata ttaaccaagg 180tcaccccagt tatcggagga gcaaacaggg gctaagtcca ccgggggagg ctgctggtga 240atattaacca aggtcacccc agttatcgga ggagcaaaca ggggctaagt ccaccggggg 300aggctgctgg tgaatattaa ccaaggtcac cccagttatc ggaggagcaa acaggggcta 360agtccacggt acccactggg aggatgttga gtaagatgga aaactactga tgacccttgc 420agagacagag tattaggaca tgtttgaaca ggggccgggc gatcagcagg tagctctaga 480ggatccccgt ctgtctgcac atttcgtaga gcgagtgttc cgatactcta atctccctag 540gcaaggttca tatttgtgta ggttacttat tctccttttg ttgactaagt caataatcag 600aatcagcagg tttggagtca gcttggcagg gatcagcagc ctgggttgga aggagggggt 660ataaaagccc cttcaccagg agaagccgtc acacagatcc acaagctcct gaagaggtaa 720gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct 780gaaatcactt tttttcaggt tggctagtat gcagatcgag ctgtccacct gcttttttct 840gtgcctgctg cggttctgct tcagcgccac ccggcggtac tacctgggcg ccgtggagct 900gtcctgggac tacatgcaga gcgacctggg cgagctgccc gtggacgccc ggttcccccc 960cagagtgccc aagagcttcc ccttcaacac cagcgtggtg tacaagaaaa ccctgttcgt 1020ggagttcacc gaccacctgt tcaatatcgc caagcccagg cccccctgga tgggcctgct 1080gggccccacc atccaggccg aggtgtacga caccgtggtg atcaccctga agaacatggc 1140cagccacccc gtgagcctgc acgccgtggg cgtgagctac tggaaggcca gcgagggcgc 1200cgagtacgac gaccagacca gccagcggga gaaagaagat gacaaggtgt tccctggcgg 1260cagccacacc tacgtgtggc aggtgctgaa agaaaacggc cccatggcct ccgaccccct 1320gtgcctgacc tacagctacc tgagccacgt ggacctggtg aaggacctga acagcggcct 1380gatcggcgct ctgctcgtct gccgggaggg cagcctggcc aaagagaaaa cccagaccct 1440gcacaagttc atcctgctgt tcgccgtgtt cgacgagggc aagagctggc acagcgagac 1500aaagaacagc ctgatgcagg accgggacgc cgcctctgcc agagcctggc ccaagatgca 1560caccgtgaac ggctacgtga acagaagcct gcccggcctg attggctgcc accggaagag 1620cgtgtactgg cacgtgatcg gcatgggcac cacacccgag gtgcacagca tctttctgga 1680agggcacacc tttctggtcc ggaaccaccg gcaggccagc ctggaaatca gccctatcac 1740cttcctgacc gcccagacac tgctgatgga cctgggccag ttcctgctgt tttgccacat 1800cagctctcac cagcacgacg gcatggaagc ctacgtgaag gtggactctt gccccgagga 1860accccagctg cggatgaaga acaacgagga agccgaggac tacgacgacg acctgaccga 1920cagcgagatg gacgtggtgc ggttcgacga cgacaacagc cccagcttca tccagatcag 1980aagcgtggcc aagaagcacc ccaagacctg ggtgcactat atcgccgccg aggaagagga 2040ctgggactac gcccccctgg tgctggcccc cgacgacaga agctacaaga gccagtacct 2100gaacaatggc ccccagcgga tcggccggaa gtacaagaaa gtgcggttca tggcctacac 2160cgacgagaca ttcaagaccc gggaggccat ccagcacgag agcggcatcc tgggccccct 2220gctgtacggc gaagtgggcg acacactgct gatcatcttc aagaaccagg ctagccggcc 2280ctacaacatc tacccccacg gcatcaccga cgtgcggccc ctgtacagca ggcggctgcc 2340caagggcgtg aagcacctga aggacttccc catcctgccc ggcgagatct tcaagtacaa 2400gtggaccgtg accgtggagg acggccccac caagagcgac cccagatgcc tgacccggta 2460ctacagcagc ttcgtgaaca tggaacggga cctggcctcc gggctgatcg gacctctgct 2520gatctgctac aaagaaagcg tggaccagcg gggcaaccag atcatgagcg acaagcggaa 2580cgtgatcctg ttcagcgtgt tcgatgagaa ccggtcctgg tatctgaccg agaacatcca 2640gcggtttctg cccaaccctg ccggcgtgca gctggaagat cccgagttcc aggccagcaa 2700catcatgcac tccatcaatg gctacgtgtt cgactctctg cagctctccg tgtgtctgca 2760cgaggtggcc tactggtaca tcctgagcat cggcgcccag accgacttcc tgagcgtgtt 2820cttcagcggc tacaccttca agcacaagat ggtgtacgag gacaccctga ccctgttccc 2880tttcagcggc gagacagtgt tcatgagcat ggaaaacccc ggcctgtgga ttctgggctg 2940ccacaacagc gacttccgga accggggcat gaccgccctg ctgaaggtgt ccagctgcga 3000caagaacacc ggcgactact acgaggacag ctacgaggat atcagcgcct acctgctgtc 3060caagaacaac gccatcgaac cccggagctt cagccagaac ccccccgtgc tgacgcgtca 3120ccagcgggag atcacccgga caaccctgca gtccgaccag gaagagatcg attacgacga 3180caccatcagc gtggagatga agaaagagga tttcgatatc tacgacgagg acgagaacca 3240gagccccaga agcttccaga agaaaacccg gcactacttc attgccgccg tggagaggct 3300gtgggactac ggcatgagtt ctagccccca cgtgctgcgg aaccgggccc agagcggcag 3360cgtgccccag ttcaagaaag tggtgttcca ggaattcaca gacggcagct tcacccagcc 3420tctgtataga ggcgagctga acgagcacct ggggctgctg gggccctaca tcagggccga 3480agtggaggac aacatcatgg tgaccttccg gaatcaggcc agcagaccct actccttcta 3540cagcagcctg atcagctacg aagaggacca gcggcagggc gccgaacccc ggaagaactt 3600cgtgaagccc aacgaaacca agacctactt ctggaaagtg cagcaccaca tggcccccac 3660caaggacgag ttcgactgca aggcctgggc ctacttcagc gacgtggatc tggaaaagga 3720cgtgcactct ggactgattg gcccactcct ggtctgccac actaacaccc tcaaccccgc 3780ccacggccgc caggtgaccg tgcaggaatt cgccctgttc ttcaccatct tcgacgagac 3840aaagtcctgg tacttcaccg agaatatgga acggaactgc agagccccct gcaacatcca 3900gatggaagat cctaccttca aagagaacta ccggttccac gccatcaacg gctacatcat 3960ggacaccctg cctggcctgg tgatggccca ggaccagaga atccggtggt atctgctgtc 4020catgggcagc aacgagaata tccacagcat ccacttcagc ggccacgtgt tcaccgtgcg 4080gaagaaagaa gagtacaaga tggccctgta caacctgtac cccggcgtgt tcgagacagt 4140ggagatgctg cccagcaagg ccggcatctg gcgggtggag tgtctgatcg gcgagcacct 4200gcacgctggc atgagcaccc tgtttctggt gtacagcaac aagtgccaga ccccactggg 4260catggcctct ggccacatcc gggacttcca gatcaccgcc tccggccagt acggccagtg 4320ggcccccaag ctggccagac tgcactacag cggcagcatc aacgcctggt ccaccaaaga 4380gcccttcagc tggatcaagg tggacctgct ggcccctatg atcatccacg gcattaagac 4440ccagggcgcc aggcagaagt tcagcagcct gtacatcagc cagttcatca tcatgtacag 4500cctggacggc aagaagtggc agacctaccg gggcaacagc accggcaccc tgatggtgtt 4560cttcggcaat gtggacagca gcggcatcaa gcacaacatc ttcaaccccc ccatcattgc 4620ccggtacatc cggctgcacc ccacccacta cagcattaga tccacactga gaatggaact 4680gatgggctgc gacctgaact cctgcagcat gcctctgggc atggaaagca aggccatcag 4740cgacgcccag atcacagcca gcagctactt caccaacatg ttcgccacct ggtccccctc 4800caaggccagg ctgcacctgc agggccggtc caacgcctgg cggcctcagg tcaacaaccc 4860caaagaatgg ctgcaggtgg actttcagaa aaccatgaag gtgaccggcg tgaccaccca 4920gggcgtgaaa agcctgctga ccagcatgta cgtgaaagag tttctgatca gcagctctca 4980ggatggccac cagtggaccc tgttctttca gaacggcaag gtgaaagtgt tccagggcaa 5040ccaggactcc ttcacccccg tggtgaactc cctggacccc cccctgctga cccgctacct 5100gagaatccac ccccagtctt gggtgcacca gatcgccctc aggatggaag tcctgggatg 5160tgaggcccag gatctgtact gatgaggatc taggctcgac atgctttatt tgtgaaattt 5220gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca 5280attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaactcgag 5340atccacggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 5400cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 5460cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta 5520cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag 5580cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 5640cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 5700tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 5760cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata 5820gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 5880aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc 5940gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 6000caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc 6060atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 6120gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 6180gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 6240ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 6300tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 6360gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 6420acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 6480cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 6540catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 6600tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 6660cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 6720accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 6780cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 6840ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 6900accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 6960ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 7020attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 7080ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 7140tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 7200tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 7260gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 7320tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 7380ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 7440ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 7500agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 7560cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 7620caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 7680tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 7740ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 7800ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 7860gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 7920gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 7980tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 8040cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 8090237904DNAArtificial SequencepAAVss-3xSerpEnh-TTRe-TTRm-coFVIIIdeltaB-Synt- pA 23cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggccgcg ggggaggctg ctggtgaata ttaaccaagg 180tcaccccagt tatcggagga gcaaacaggg gctaagtcca ccgggggagg ctgctggtga 240atattaacca aggtcacccc agttatcgga ggagcaaaca ggggctaagt ccaccggggg

300aggctgctgg tgaatattaa ccaaggtcac cccagttatc ggaggagcaa acaggggcta 360agtccacggt acccactggg aggatgttga gtaagatgga aaactactga tgacccttgc 420agagacagag tattaggaca tgtttgaaca ggggccgggc gatcagcagg tagctctaga 480ggatccccgt ctgtctgcac atttcgtaga gcgagtgttc cgatactcta atctccctag 540gcaaggttca tatttgtgta ggttacttat tctccttttg ttgactaagt caataatcag 600aatcagcagg tttggagtca gcttggcagg gatcagcagc ctgggttgga aggagggggt 660ataaaagccc cttcaccagg agaagccgtc acacagatcc acaagctcct gctagtatgc 720agatcgagct gtccacctgc ttttttctgt gcctgctgcg gttctgcttc agcgccaccc 780ggcggtacta cctgggcgcc gtggagctgt cctgggacta catgcagagc gacctgggcg 840agctgcccgt ggacgcccgg ttccccccca gagtgcccaa gagcttcccc ttcaacacca 900gcgtggtgta caagaaaacc ctgttcgtgg agttcaccga ccacctgttc aatatcgcca 960agcccaggcc cccctggatg ggcctgctgg gccccaccat ccaggccgag gtgtacgaca 1020ccgtggtgat caccctgaag aacatggcca gccaccccgt gagcctgcac gccgtgggcg 1080tgagctactg gaaggccagc gagggcgccg agtacgacga ccagaccagc cagcgggaga 1140aagaagatga caaggtgttc cctggcggca gccacaccta cgtgtggcag gtgctgaaag 1200aaaacggccc catggcctcc gaccccctgt gcctgaccta cagctacctg agccacgtgg 1260acctggtgaa ggacctgaac agcggcctga tcggcgctct gctcgtctgc cgggagggca 1320gcctggccaa agagaaaacc cagaccctgc acaagttcat cctgctgttc gccgtgttcg 1380acgagggcaa gagctggcac agcgagacaa agaacagcct gatgcaggac cgggacgccg 1440cctctgccag agcctggccc aagatgcaca ccgtgaacgg ctacgtgaac agaagcctgc 1500ccggcctgat tggctgccac cggaagagcg tgtactggca cgtgatcggc atgggcacca 1560cacccgaggt gcacagcatc tttctggaag ggcacacctt tctggtccgg aaccaccggc 1620aggccagcct ggaaatcagc cctatcacct tcctgaccgc ccagacactg ctgatggacc 1680tgggccagtt cctgctgttt tgccacatca gctctcacca gcacgacggc atggaagcct 1740acgtgaaggt ggactcttgc cccgaggaac cccagctgcg gatgaagaac aacgaggaag 1800ccgaggacta cgacgacgac ctgaccgaca gcgagatgga cgtggtgcgg ttcgacgacg 1860acaacagccc cagcttcatc cagatcagaa gcgtggccaa gaagcacccc aagacctggg 1920tgcactatat cgccgccgag gaagaggact gggactacgc ccccctggtg ctggcccccg 1980acgacagaag ctacaagagc cagtacctga acaatggccc ccagcggatc ggccggaagt 2040acaagaaagt gcggttcatg gcctacaccg acgagacatt caagacccgg gaggccatcc 2100agcacgagag cggcatcctg ggccccctgc tgtacggcga agtgggcgac acactgctga 2160tcatcttcaa gaaccaggct agccggccct acaacatcta cccccacggc atcaccgacg 2220tgcggcccct gtacagcagg cggctgccca agggcgtgaa gcacctgaag gacttcccca 2280tcctgcccgg cgagatcttc aagtacaagt ggaccgtgac cgtggaggac ggccccacca 2340agagcgaccc cagatgcctg acccggtact acagcagctt cgtgaacatg gaacgggacc 2400tggcctccgg gctgatcgga cctctgctga tctgctacaa agaaagcgtg gaccagcggg 2460gcaaccagat catgagcgac aagcggaacg tgatcctgtt cagcgtgttc gatgagaacc 2520ggtcctggta tctgaccgag aacatccagc ggtttctgcc caaccctgcc ggcgtgcagc 2580tggaagatcc cgagttccag gccagcaaca tcatgcactc catcaatggc tacgtgttcg 2640actctctgca gctctccgtg tgtctgcacg aggtggccta ctggtacatc ctgagcatcg 2700gcgcccagac cgacttcctg agcgtgttct tcagcggcta caccttcaag cacaagatgg 2760tgtacgagga caccctgacc ctgttccctt tcagcggcga gacagtgttc atgagcatgg 2820aaaaccccgg cctgtggatt ctgggctgcc acaacagcga cttccggaac cggggcatga 2880ccgccctgct gaaggtgtcc agctgcgaca agaacaccgg cgactactac gaggacagct 2940acgaggatat cagcgcctac ctgctgtcca agaacaacgc catcgaaccc cggagcttca 3000gccagaaccc ccccgtgctg acgcgtcacc agcgggagat cacccggaca accctgcagt 3060ccgaccagga agagatcgat tacgacgaca ccatcagcgt ggagatgaag aaagaggatt 3120tcgatatcta cgacgaggac gagaaccaga gccccagaag cttccagaag aaaacccggc 3180actacttcat tgccgccgtg gagaggctgt gggactacgg catgagttct agcccccacg 3240tgctgcggaa ccgggcccag agcggcagcg tgccccagtt caagaaagtg gtgttccagg 3300aattcacaga cggcagcttc acccagcctc tgtatagagg cgagctgaac gagcacctgg 3360ggctgctggg gccctacatc agggccgaag tggaggacaa catcatggtg accttccgga 3420atcaggccag cagaccctac tccttctaca gcagcctgat cagctacgaa gaggaccagc 3480ggcagggcgc cgaaccccgg aagaacttcg tgaagcccaa cgaaaccaag acctacttct 3540ggaaagtgca gcaccacatg gcccccacca aggacgagtt cgactgcaag gcctgggcct 3600acttcagcga cgtggatctg gaaaaggacg tgcactctgg actgattggc ccactcctgg 3660tctgccacac taacaccctc aaccccgccc acggccgcca ggtgaccgtg caggaattcg 3720ccctgttctt caccatcttc gacgagacaa agtcctggta cttcaccgag aatatggaac 3780ggaactgcag agccccctgc aacatccaga tggaagatcc taccttcaaa gagaactacc 3840ggttccacgc catcaacggc tacatcatgg acaccctgcc tggcctggtg atggcccagg 3900accagagaat ccggtggtat ctgctgtcca tgggcagcaa cgagaatatc cacagcatcc 3960acttcagcgg ccacgtgttc accgtgcgga agaaagaaga gtacaagatg gccctgtaca 4020acctgtaccc cggcgtgttc gagacagtgg agatgctgcc cagcaaggcc ggcatctggc 4080gggtggagtg tctgatcggc gagcacctgc acgctggcat gagcaccctg tttctggtgt 4140acagcaacaa gtgccagacc ccactgggca tggcctctgg ccacatccgg gacttccaga 4200tcaccgcctc cggccagtac ggccagtggg cccccaagct ggccagactg cactacagcg 4260gcagcatcaa cgcctggtcc accaaagagc ccttcagctg gatcaaggtg gacctgctgg 4320cccctatgat catccacggc attaagaccc agggcgccag gcagaagttc agcagcctgt 4380acatcagcca gttcatcatc atgtacagcc tggacggcaa gaagtggcag acctaccggg 4440gcaacagcac cggcaccctg atggtgttct tcggcaatgt ggacagcagc ggcatcaagc 4500acaacatctt caaccccccc atcattgccc ggtacatccg gctgcacccc acccactaca 4560gcattagatc cacactgaga atggaactga tgggctgcga cctgaactcc tgcagcatgc 4620ctctgggcat ggaaagcaag gccatcagcg acgcccagat cacagccagc agctacttca 4680ccaacatgtt cgccacctgg tccccctcca aggccaggct gcacctgcag ggccggtcca 4740acgcctggcg gcctcaggtc aacaacccca aagaatggct gcaggtggac tttcagaaaa 4800ccatgaaggt gaccggcgtg accacccagg gcgtgaaaag cctgctgacc agcatgtacg 4860tgaaagagtt tctgatcagc agctctcagg atggccacca gtggaccctg ttctttcaga 4920acggcaaggt gaaagtgttc cagggcaacc aggactcctt cacccccgtg gtgaactccc 4980tggacccccc cctgctgacc cgctacctga gaatccaccc ccagtcttgg gtgcaccaga 5040tcgccctcag gatggaagtc ctgggatgtg aggcccagga tctgtactga tgaggatcca 5100ataaaagatc tttattttca ttagatctgt gtgttggttt tttgtgtgct cgagatccac 5160ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 5220ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 5280gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc 5340tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc 5400attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 5460agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 5520tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 5580ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt 5640ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 5700aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc 5760ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 5820attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 5880aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 5940ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 6000accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 6060taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 6120cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 6180ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 6240ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 6300aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 6360actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 6420gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 6480agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 6540cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 6600catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 6660aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 6720gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 6780aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 6840agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 6900ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 6960actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 7020aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 7080gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 7140atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 7200tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 7260tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 7320ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 7380agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 7440ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 7500tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 7560gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 7620cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 7680ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 7740agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 7800tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 7860ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt 7904247683DNAArtificial SequencepAAVss-TTRe-TTRm-coFVIIIdeltaB-Synt-pA 24cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggccgta cccactggga ggatgttgag taagatggaa 180aactactgat gacccttgca gagacagagt attaggacat gtttgaacag gggccgggcg 240atcagcaggt agctctagag gatccccgtc tgtctgcaca tttcgtagag cgagtgttcc 300gatactctaa tctccctagg caaggttcat atttgtgtag gttacttatt ctccttttgt 360tgactaagtc aataatcaga atcagcaggt ttggagtcag cttggcaggg atcagcagcc 420tgggttggaa ggagggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca 480caagctcctg ctagtatgca gatcgagctg tccacctgct tttttctgtg cctgctgcgg 540ttctgcttca gcgccacccg gcggtactac ctgggcgccg tggagctgtc ctgggactac 600atgcagagcg acctgggcga gctgcccgtg gacgcccggt tcccccccag agtgcccaag 660agcttcccct tcaacaccag cgtggtgtac aagaaaaccc tgttcgtgga gttcaccgac 720cacctgttca atatcgccaa gcccaggccc ccctggatgg gcctgctggg ccccaccatc 780caggccgagg tgtacgacac cgtggtgatc accctgaaga acatggccag ccaccccgtg 840agcctgcacg ccgtgggcgt gagctactgg aaggccagcg agggcgccga gtacgacgac 900cagaccagcc agcgggagaa agaagatgac aaggtgttcc ctggcggcag ccacacctac 960gtgtggcagg tgctgaaaga aaacggcccc atggcctccg accccctgtg cctgacctac 1020agctacctga gccacgtgga cctggtgaag gacctgaaca gcggcctgat cggcgctctg 1080ctcgtctgcc gggagggcag cctggccaaa gagaaaaccc agaccctgca caagttcatc 1140ctgctgttcg ccgtgttcga cgagggcaag agctggcaca gcgagacaaa gaacagcctg 1200atgcaggacc gggacgccgc ctctgccaga gcctggccca agatgcacac cgtgaacggc 1260tacgtgaaca gaagcctgcc cggcctgatt ggctgccacc ggaagagcgt gtactggcac 1320gtgatcggca tgggcaccac acccgaggtg cacagcatct ttctggaagg gcacaccttt 1380ctggtccgga accaccggca ggccagcctg gaaatcagcc ctatcacctt cctgaccgcc 1440cagacactgc tgatggacct gggccagttc ctgctgtttt gccacatcag ctctcaccag 1500cacgacggca tggaagccta cgtgaaggtg gactcttgcc ccgaggaacc ccagctgcgg 1560atgaagaaca acgaggaagc cgaggactac gacgacgacc tgaccgacag cgagatggac 1620gtggtgcggt tcgacgacga caacagcccc agcttcatcc agatcagaag cgtggccaag 1680aagcacccca agacctgggt gcactatatc gccgccgagg aagaggactg ggactacgcc 1740cccctggtgc tggcccccga cgacagaagc tacaagagcc agtacctgaa caatggcccc 1800cagcggatcg gccggaagta caagaaagtg cggttcatgg cctacaccga cgagacattc 1860aagacccggg aggccatcca gcacgagagc ggcatcctgg gccccctgct gtacggcgaa 1920gtgggcgaca cactgctgat catcttcaag aaccaggcta gccggcccta caacatctac 1980ccccacggca tcaccgacgt gcggcccctg tacagcaggc ggctgcccaa gggcgtgaag 2040cacctgaagg acttccccat cctgcccggc gagatcttca agtacaagtg gaccgtgacc 2100gtggaggacg gccccaccaa gagcgacccc agatgcctga cccggtacta cagcagcttc 2160gtgaacatgg aacgggacct ggcctccggg ctgatcggac ctctgctgat ctgctacaaa 2220gaaagcgtgg accagcgggg caaccagatc atgagcgaca agcggaacgt gatcctgttc 2280agcgtgttcg atgagaaccg gtcctggtat ctgaccgaga acatccagcg gtttctgccc 2340aaccctgccg gcgtgcagct ggaagatccc gagttccagg ccagcaacat catgcactcc 2400atcaatggct acgtgttcga ctctctgcag ctctccgtgt gtctgcacga ggtggcctac 2460tggtacatcc tgagcatcgg cgcccagacc gacttcctga gcgtgttctt cagcggctac 2520accttcaagc acaagatggt gtacgaggac accctgaccc tgttcccttt cagcggcgag 2580acagtgttca tgagcatgga aaaccccggc ctgtggattc tgggctgcca caacagcgac 2640ttccggaacc ggggcatgac cgccctgctg aaggtgtcca gctgcgacaa gaacaccggc 2700gactactacg aggacagcta cgaggatatc agcgcctacc tgctgtccaa gaacaacgcc 2760atcgaacccc ggagcttcag ccagaacccc cccgtgctga cgcgtcacca gcgggagatc 2820acccggacaa ccctgcagtc cgaccaggaa gagatcgatt acgacgacac catcagcgtg 2880gagatgaaga aagaggattt cgatatctac gacgaggacg agaaccagag ccccagaagc 2940ttccagaaga aaacccggca ctacttcatt gccgccgtgg agaggctgtg ggactacggc 3000atgagttcta gcccccacgt gctgcggaac cgggcccaga gcggcagcgt gccccagttc 3060aagaaagtgg tgttccagga attcacagac ggcagcttca cccagcctct gtatagaggc 3120gagctgaacg agcacctggg gctgctgggg ccctacatca gggccgaagt ggaggacaac 3180atcatggtga ccttccggaa tcaggccagc agaccctact ccttctacag cagcctgatc 3240agctacgaag aggaccagcg gcagggcgcc gaaccccgga agaacttcgt gaagcccaac 3300gaaaccaaga cctacttctg gaaagtgcag caccacatgg cccccaccaa ggacgagttc 3360gactgcaagg cctgggccta cttcagcgac gtggatctgg aaaaggacgt gcactctgga 3420ctgattggcc cactcctggt ctgccacact aacaccctca accccgccca cggccgccag 3480gtgaccgtgc aggaattcgc cctgttcttc accatcttcg acgagacaaa gtcctggtac 3540ttcaccgaga atatggaacg gaactgcaga gccccctgca acatccagat ggaagatcct 3600accttcaaag agaactaccg gttccacgcc atcaacggct acatcatgga caccctgcct 3660ggcctggtga tggcccagga ccagagaatc cggtggtatc tgctgtccat gggcagcaac 3720gagaatatcc acagcatcca cttcagcggc cacgtgttca ccgtgcggaa gaaagaagag 3780tacaagatgg ccctgtacaa cctgtacccc ggcgtgttcg agacagtgga gatgctgccc 3840agcaaggccg gcatctggcg ggtggagtgt ctgatcggcg agcacctgca cgctggcatg 3900agcaccctgt ttctggtgta cagcaacaag tgccagaccc cactgggcat ggcctctggc 3960cacatccggg acttccagat caccgcctcc ggccagtacg gccagtgggc ccccaagctg 4020gccagactgc actacagcgg cagcatcaac gcctggtcca ccaaagagcc cttcagctgg 4080atcaaggtgg acctgctggc ccctatgatc atccacggca ttaagaccca gggcgccagg 4140cagaagttca gcagcctgta catcagccag ttcatcatca tgtacagcct ggacggcaag 4200aagtggcaga cctaccgggg caacagcacc ggcaccctga tggtgttctt cggcaatgtg 4260gacagcagcg gcatcaagca caacatcttc aaccccccca tcattgcccg gtacatccgg 4320ctgcacccca cccactacag cattagatcc acactgagaa tggaactgat gggctgcgac 4380ctgaactcct gcagcatgcc tctgggcatg gaaagcaagg ccatcagcga cgcccagatc 4440acagccagca gctacttcac caacatgttc gccacctggt ccccctccaa ggccaggctg 4500cacctgcagg gccggtccaa cgcctggcgg cctcaggtca acaaccccaa agaatggctg 4560caggtggact ttcagaaaac catgaaggtg accggcgtga ccacccaggg cgtgaaaagc 4620ctgctgacca gcatgtacgt gaaagagttt ctgatcagca gctctcagga tggccaccag 4680tggaccctgt tctttcagaa cggcaaggtg aaagtgttcc agggcaacca ggactccttc 4740acccccgtgg tgaactccct ggaccccccc ctgctgaccc gctacctgag aatccacccc 4800cagtcttggg tgcaccagat cgccctcagg atggaagtcc tgggatgtga ggcccaggat 4860ctgtactgat gaggatccaa taaaagatct ttattttcat tagatctgtg tgttggtttt 4920ttgtgtgctc gagatccacg gccgcaggaa cccctagtga tggagttggc cactccctct 4980ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt 5040gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg 5100tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt 5160acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 5220ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 5280cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 5340gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc 5400catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 5460gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat 5520aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 5580acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct 5640gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct 5700gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 5760gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga 5820tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca 5880cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 5940tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 6000gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 6060ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 6120cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 6180ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 6240cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 6300tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 6360tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 6420tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 6480ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 6540tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 6600cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 6660gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 6720ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 6780acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 6840cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 6900atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 6960tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 7020tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 7080aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 7140aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 7200taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 7260taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 7320agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct

7380tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 7440cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 7500agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 7560gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 7620aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 7680tgt 7683256207DNAArtificial SequencepAAVsc-3xSerpEnh-TTREnh-TTRm-MVM-co-FIX-R338L- Synt-pA 25agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020gcagagaggg agtggaattc acgcgtggat ctgaattcaa ttcacgcgtg gtacggccgc 1080gggggggagg ctgctggtga atattaacca aggtcacccc agttatcgga ggagcaaaca 1140ggggctaagt ccaccggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 1200ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 1260caccccagtt atcggaggag caaacagggg ctaagtccac ggtacccact gggaggatgt 1320tgagtaagat ggaaaactac tgatgaccct tgcagagaca gagtattagg acatgtttga 1380acaggggccg ggcgatcagc aggtagctct agaggatccc cgtctgtctg cacatttcgt 1440agagcgagtg ttccgatact ctaatctccc taggcaaggt tcatatttgt gtaggttact 1500tattctcctt ttgttgacta agtcaataat cagaatcagc aggtttggag tcagcttggc 1560agggatcagc agcctgggtt ggaaggaggg ggtataaaag ccccttcacc aggagaagcc 1620gtcacacaga tccacaagct cctggctaga aagaggtaag ggtttaaggg atggttggtt 1680ggtggggtat taatgtttaa ttacctggag cacctgcctg aaatcacttt ttttcaggtt 1740gggctagccc accatgcagc gcgtgaacat gatcatggcc gagagccccg gcctgatcac 1800catctgcctg ctgggctacc tgctgagcgc cgagtgcacc gtgttcctgg accacgagaa 1860cgccaacaag atcctgaacc gccccaagcg ctacaacagc ggcaagctgg aggagttcgt 1920gcagggcaac ctggagcgcg agtgcatgga ggagaagtgc agcttcgagg aggcccgcga 1980ggtgttcgag aacaccgagc gcaccaccga gttctggaag cagtacgtgg acggcgacca 2040gtgcgagagc aacccctgcc tgaacggcgg cagctgcaag gacgacatca acagctacga 2100gtgctggtgc cccttcggct tcgagggcaa gaactgcgag ctggacgtga cctgcaacat 2160caagaacggc cgctgcgagc agttctgcaa gaacagcgcc gacaacaagg tggtgtgcag 2220ctgcaccgag ggctaccgcc tggccgagaa ccagaagagc tgcgagcccg ccgtgccctt 2280cccctgcggc cgcgtgagcg tgagccagac cagcaagctg acccgcgccg aggccgtgtt 2340ccccgacgtg gactacgtga acagcaccga ggccgagacc atcctggaca acatcaccca 2400gagcacccag agcttcaacg acttcacccg cgtggtgggc ggcgaggacg ccaagcccgg 2460ccagttcccc tggcaggtgg tgctgaacgg caaggtggac gccttctgcg gcggcagcat 2520cgtgaacgag aagtggatcg tgaccgccgc ccactgcgtg gagaccggcg tgaagatcac 2580cgtggtggcc ggcgagcaca acatcgagga gaccgagcac accgagcaga agcgcaacgt 2640gatccgcatc atcccccacc acaactacaa cgccgccatc aacaagtaca accacgacat 2700cgccctgctg gagctggacg agcccctggt gctgaacagc tacgtgaccc ccatctgcat 2760cgccgacaag gagtacacca acatcttcct gaagttcggc agcggctacg tgagcggctg 2820gggccgcgtg ttccacaagg gccgcagcgc cctggtgctg cagtacctgc gcgtgcccct 2880ggtggaccgc gccacctgcc tgctgagcac caagttcacc atctacaaca acatgttctg 2940cgccggcttc cacgagggcg gccgcgacag ctgccagggc gacagcggcg gcccccacgt 3000gaccgaggtg gagggcacca gcttcctgac cggcatcatc agctggggcg aggagtgcgc 3060catgaagggc aagtacggca tctacaccaa ggtgagccgc tacgtgaact ggatcaagga 3120gaagaccaag ctgacctaat gaaagatgga tttccaaggt taattcattg gaattgaaaa 3180ttaacagccc cccccccccc ccccctgcag atctaataaa agatctttat tttcattaga 3240tctgtgtgtt ggttttttgt gtgccggtgg atctcgatag caggcatgct ggggagagat 3300cgatctgagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 3360actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg 3420agcgagcgag cgcgcagaga gggagtggcc aacccccccc cccccccccc ccggcgattc 3480tcttgtttgc tccagactct caggcaatga cctgatagcc tttgtagaga cctctcaaaa 3540atagctaccc tctccggcat gaatttatca gctagaacgg ttgaatatca tattgatggt 3600gatttgactg tctccggcct ttctcacccg tttgaatctt tacctacaca ttactcaggc 3660attgcattta aaatatatga gggttctaaa aatttttatc cttgcgttga aataaaggct 3720tctcccgcaa aagtattaca gggtcataat gtttttggta caaccgattt agctttatgc 3780tctgaggctt tattgcttaa ttttgctaat tctttgcctt gcctgtatga tttattggat 3840gttggaatcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 3900tatggtgcac tctcagtaca atctgctctg atgccgcata gttatatggt gcactctcag 3960tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacagccagc 4020cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 4080cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 4140caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 4200tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 4260ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 4320gataaatgct caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 4380ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 4440gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 4500caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 4560ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 4620cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 4680gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 4740taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 4800tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 4860agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 4920caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 4980ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 5040tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 5100agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 5160tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 5220agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 5280gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 5340gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 5400tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 5460gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 5520accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 5580accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 5640gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 5700ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 5760atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 5820gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 5880cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 5940gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 6000gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 6060tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 6120cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 6180ccccgcgcgt tggccgattc attaatg 620726371DNAArtificial SequenceFlank-3xSERP-Flank-TTRe 26aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta g 37127386DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank 27aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atcccc 38628588DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm 28aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtc 58829615DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank 29aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac 600aagctcctgg ctaga 61530707DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank-MVM 30aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac 600aagctcctgg ctagaaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 660tttaattacc tggagcacct gcctgaaatc actttttttc aggttgg 70731718DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank-MVM- Flank 31aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac 600aagctcctgg ctagaaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 660tttaattacc tggagcacct gcctgaaatc actttttttc aggttgggct agcccacc 718322107DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank-MVM- Flank-co-FIX-R338L 32aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac 600aagctcctgg ctagaaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 660tttaattacc tggagcacct gcctgaaatc actttttttc aggttgggct agcccaccat 720gcagcgcgtg aacatgatca tggccgagag ccccggcctg atcaccatct gcctgctggg 780ctacctgctg agcgccgagt gcaccgtgtt cctggaccac gagaacgcca acaagatcct 840gaaccgcccc aagcgctaca acagcggcaa gctggaggag ttcgtgcagg gcaacctgga 900gcgcgagtgc atggaggaga agtgcagctt cgaggaggcc cgcgaggtgt tcgagaacac 960cgagcgcacc accgagttct ggaagcagta cgtggacggc gaccagtgcg agagcaaccc 1020ctgcctgaac ggcggcagct gcaaggacga catcaacagc tacgagtgct ggtgcccctt 1080cggcttcgag ggcaagaact gcgagctgga cgtgacctgc aacatcaaga acggccgctg 1140cgagcagttc tgcaagaaca gcgccgacaa caaggtggtg tgcagctgca ccgagggcta 1200ccgcctggcc gagaaccaga agagctgcga gcccgccgtg cccttcccct gcggccgcgt 1260gagcgtgagc cagaccagca agctgacccg cgccgaggcc gtgttccccg acgtggacta 1320cgtgaacagc accgaggccg agaccatcct ggacaacatc acccagagca cccagagctt 1380caacgacttc acccgcgtgg tgggcggcga ggacgccaag cccggccagt tcccctggca 1440ggtggtgctg aacggcaagg tggacgcctt ctgcggcggc agcatcgtga acgagaagtg 1500gatcgtgacc gccgcccact gcgtggagac cggcgtgaag atcaccgtgg tggccggcga 1560gcacaacatc gaggagaccg agcacaccga gcagaagcgc aacgtgatcc gcatcatccc 1620ccaccacaac tacaacgccg ccatcaacaa gtacaaccac gacatcgccc tgctggagct 1680ggacgagccc ctggtgctga acagctacgt gacccccatc tgcatcgccg acaaggagta 1740caccaacatc ttcctgaagt tcggcagcgg ctacgtgagc ggctggggcc gcgtgttcca 1800caagggccgc agcgccctgg tgctgcagta cctgcgcgtg cccctggtgg accgcgccac 1860ctgcctgctg agcaccaagt tcaccatcta caacaacatg ttctgcgccg gcttccacga 1920gggcggccgc gacagctgcc agggcgacag cggcggcccc cacgtgaccg aggtggaggg 1980caccagcttc ctgaccggca tcatcagctg gggcgaggag tgcgccatga agggcaagta 2040cggcatctac accaaggtga gccgctacgt gaactggatc aaggagaaga ccaagctgac 2100ctaatga 2107332174DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank-MVM- Flank-co-FIX-R338L-Flank 33aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac 600aagctcctgg ctagaaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 660tttaattacc tggagcacct gcctgaaatc actttttttc aggttgggct agcccaccat 720gcagcgcgtg aacatgatca tggccgagag ccccggcctg atcaccatct gcctgctggg 780ctacctgctg agcgccgagt gcaccgtgtt cctggaccac gagaacgcca acaagatcct 840gaaccgcccc aagcgctaca acagcggcaa gctggaggag ttcgtgcagg gcaacctgga 900gcgcgagtgc atggaggaga agtgcagctt cgaggaggcc cgcgaggtgt tcgagaacac 960cgagcgcacc accgagttct ggaagcagta cgtggacggc gaccagtgcg agagcaaccc 1020ctgcctgaac ggcggcagct gcaaggacga catcaacagc tacgagtgct ggtgcccctt 1080cggcttcgag ggcaagaact gcgagctgga cgtgacctgc aacatcaaga acggccgctg 1140cgagcagttc tgcaagaaca gcgccgacaa caaggtggtg tgcagctgca ccgagggcta 1200ccgcctggcc gagaaccaga agagctgcga gcccgccgtg cccttcccct gcggccgcgt 1260gagcgtgagc cagaccagca agctgacccg cgccgaggcc gtgttccccg acgtggacta 1320cgtgaacagc accgaggccg agaccatcct ggacaacatc acccagagca cccagagctt 1380caacgacttc acccgcgtgg tgggcggcga ggacgccaag cccggccagt tcccctggca 1440ggtggtgctg aacggcaagg tggacgcctt ctgcggcggc agcatcgtga acgagaagtg 1500gatcgtgacc gccgcccact gcgtggagac cggcgtgaag atcaccgtgg tggccggcga 1560gcacaacatc gaggagaccg agcacaccga gcagaagcgc aacgtgatcc gcatcatccc 1620ccaccacaac tacaacgccg ccatcaacaa gtacaaccac gacatcgccc tgctggagct 1680ggacgagccc ctggtgctga acagctacgt gacccccatc tgcatcgccg acaaggagta 1740caccaacatc ttcctgaagt tcggcagcgg ctacgtgagc ggctggggcc gcgtgttcca 1800caagggccgc agcgccctgg tgctgcagta cctgcgcgtg cccctggtgg accgcgccac 1860ctgcctgctg agcaccaagt tcaccatcta caacaacatg

ttctgcgccg gcttccacga 1920gggcggccgc gacagctgcc agggcgacag cggcggcccc cacgtgaccg aggtggaggg 1980caccagcttc ctgaccggca tcatcagctg gggcgaggag tgcgccatga agggcaagta 2040cggcatctac accaaggtga gccgctacgt gaactggatc aaggagaaga ccaagctgac 2100ctaatgaaag atggatttcc aaggttaatt cattggaatt gaaaattaac agcccccccc 2160cccccccccc tgca 2174342469DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank-MVM- Flank-co-FIX-R338L-Flank-BGHpA 34aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac 600aagctcctgg ctagaaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 660tttaattacc tggagcacct gcctgaaatc actttttttc aggttgggct agcccaccat 720gcagcgcgtg aacatgatca tggccgagag ccccggcctg atcaccatct gcctgctggg 780ctacctgctg agcgccgagt gcaccgtgtt cctggaccac gagaacgcca acaagatcct 840gaaccgcccc aagcgctaca acagcggcaa gctggaggag ttcgtgcagg gcaacctgga 900gcgcgagtgc atggaggaga agtgcagctt cgaggaggcc cgcgaggtgt tcgagaacac 960cgagcgcacc accgagttct ggaagcagta cgtggacggc gaccagtgcg agagcaaccc 1020ctgcctgaac ggcggcagct gcaaggacga catcaacagc tacgagtgct ggtgcccctt 1080cggcttcgag ggcaagaact gcgagctgga cgtgacctgc aacatcaaga acggccgctg 1140cgagcagttc tgcaagaaca gcgccgacaa caaggtggtg tgcagctgca ccgagggcta 1200ccgcctggcc gagaaccaga agagctgcga gcccgccgtg cccttcccct gcggccgcgt 1260gagcgtgagc cagaccagca agctgacccg cgccgaggcc gtgttccccg acgtggacta 1320cgtgaacagc accgaggccg agaccatcct ggacaacatc acccagagca cccagagctt 1380caacgacttc acccgcgtgg tgggcggcga ggacgccaag cccggccagt tcccctggca 1440ggtggtgctg aacggcaagg tggacgcctt ctgcggcggc agcatcgtga acgagaagtg 1500gatcgtgacc gccgcccact gcgtggagac cggcgtgaag atcaccgtgg tggccggcga 1560gcacaacatc gaggagaccg agcacaccga gcagaagcgc aacgtgatcc gcatcatccc 1620ccaccacaac tacaacgccg ccatcaacaa gtacaaccac gacatcgccc tgctggagct 1680ggacgagccc ctggtgctga acagctacgt gacccccatc tgcatcgccg acaaggagta 1740caccaacatc ttcctgaagt tcggcagcgg ctacgtgagc ggctggggcc gcgtgttcca 1800caagggccgc agcgccctgg tgctgcagta cctgcgcgtg cccctggtgg accgcgccac 1860ctgcctgctg agcaccaagt tcaccatcta caacaacatg ttctgcgccg gcttccacga 1920gggcggccgc gacagctgcc agggcgacag cggcggcccc cacgtgaccg aggtggaggg 1980caccagcttc ctgaccggca tcatcagctg gggcgaggag tgcgccatga agggcaagta 2040cggcatctac accaaggtga gccgctacgt gaactggatc aaggagaaga ccaagctgac 2100ctaatgaaag atggatttcc aaggttaatt cattggaatt gaaaattaac agcccccccc 2160cccccccccc tgcagatctg agccgaattc ctgcagcccg ggggatcagc ctcgactgtg 2220ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 2280ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 2340aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 2400gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 2460agctgggga 2469352508DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank-MVM- Flank-co-FIX-R338L-Flank-BGHpA-Flank 35aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac 600aagctcctgg ctagaaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 660tttaattacc tggagcacct gcctgaaatc actttttttc aggttgggct agcccaccat 720gcagcgcgtg aacatgatca tggccgagag ccccggcctg atcaccatct gcctgctggg 780ctacctgctg agcgccgagt gcaccgtgtt cctggaccac gagaacgcca acaagatcct 840gaaccgcccc aagcgctaca acagcggcaa gctggaggag ttcgtgcagg gcaacctgga 900gcgcgagtgc atggaggaga agtgcagctt cgaggaggcc cgcgaggtgt tcgagaacac 960cgagcgcacc accgagttct ggaagcagta cgtggacggc gaccagtgcg agagcaaccc 1020ctgcctgaac ggcggcagct gcaaggacga catcaacagc tacgagtgct ggtgcccctt 1080cggcttcgag ggcaagaact gcgagctgga cgtgacctgc aacatcaaga acggccgctg 1140cgagcagttc tgcaagaaca gcgccgacaa caaggtggtg tgcagctgca ccgagggcta 1200ccgcctggcc gagaaccaga agagctgcga gcccgccgtg cccttcccct gcggccgcgt 1260gagcgtgagc cagaccagca agctgacccg cgccgaggcc gtgttccccg acgtggacta 1320cgtgaacagc accgaggccg agaccatcct ggacaacatc acccagagca cccagagctt 1380caacgacttc acccgcgtgg tgggcggcga ggacgccaag cccggccagt tcccctggca 1440ggtggtgctg aacggcaagg tggacgcctt ctgcggcggc agcatcgtga acgagaagtg 1500gatcgtgacc gccgcccact gcgtggagac cggcgtgaag atcaccgtgg tggccggcga 1560gcacaacatc gaggagaccg agcacaccga gcagaagcgc aacgtgatcc gcatcatccc 1620ccaccacaac tacaacgccg ccatcaacaa gtacaaccac gacatcgccc tgctggagct 1680ggacgagccc ctggtgctga acagctacgt gacccccatc tgcatcgccg acaaggagta 1740caccaacatc ttcctgaagt tcggcagcgg ctacgtgagc ggctggggcc gcgtgttcca 1800caagggccgc agcgccctgg tgctgcagta cctgcgcgtg cccctggtgg accgcgccac 1860ctgcctgctg agcaccaagt tcaccatcta caacaacatg ttctgcgccg gcttccacga 1920gggcggccgc gacagctgcc agggcgacag cggcggcccc cacgtgaccg aggtggaggg 1980caccagcttc ctgaccggca tcatcagctg gggcgaggag tgcgccatga agggcaagta 2040cggcatctac accaaggtga gccgctacgt gaactggatc aaggagaaga ccaagctgac 2100ctaatgaaag atggatttcc aaggttaatt cattggaatt gaaaattaac agcccccccc 2160cccccccccc tgcagatctg agccgaattc ctgcagcccg ggggatcagc ctcgactgtg 2220ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 2280ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 2340aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 2400gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 2460agctggggac cggtggatct cgatagcagg catgctgggg agagatcg 2508362223DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank-MVM- Flank-co-FIX-R338L-Flank-Synt-pA 36aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac 600aagctcctgg ctagaaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 660tttaattacc tggagcacct gcctgaaatc actttttttc aggttgggct agcccaccat 720gcagcgcgtg aacatgatca tggccgagag ccccggcctg atcaccatct gcctgctggg 780ctacctgctg agcgccgagt gcaccgtgtt cctggaccac gagaacgcca acaagatcct 840gaaccgcccc aagcgctaca acagcggcaa gctggaggag ttcgtgcagg gcaacctgga 900gcgcgagtgc atggaggaga agtgcagctt cgaggaggcc cgcgaggtgt tcgagaacac 960cgagcgcacc accgagttct ggaagcagta cgtggacggc gaccagtgcg agagcaaccc 1020ctgcctgaac ggcggcagct gcaaggacga catcaacagc tacgagtgct ggtgcccctt 1080cggcttcgag ggcaagaact gcgagctgga cgtgacctgc aacatcaaga acggccgctg 1140cgagcagttc tgcaagaaca gcgccgacaa caaggtggtg tgcagctgca ccgagggcta 1200ccgcctggcc gagaaccaga agagctgcga gcccgccgtg cccttcccct gcggccgcgt 1260gagcgtgagc cagaccagca agctgacccg cgccgaggcc gtgttccccg acgtggacta 1320cgtgaacagc accgaggccg agaccatcct ggacaacatc acccagagca cccagagctt 1380caacgacttc acccgcgtgg tgggcggcga ggacgccaag cccggccagt tcccctggca 1440ggtggtgctg aacggcaagg tggacgcctt ctgcggcggc agcatcgtga acgagaagtg 1500gatcgtgacc gccgcccact gcgtggagac cggcgtgaag atcaccgtgg tggccggcga 1560gcacaacatc gaggagaccg agcacaccga gcagaagcgc aacgtgatcc gcatcatccc 1620ccaccacaac tacaacgccg ccatcaacaa gtacaaccac gacatcgccc tgctggagct 1680ggacgagccc ctggtgctga acagctacgt gacccccatc tgcatcgccg acaaggagta 1740caccaacatc ttcctgaagt tcggcagcgg ctacgtgagc ggctggggcc gcgtgttcca 1800caagggccgc agcgccctgg tgctgcagta cctgcgcgtg cccctggtgg accgcgccac 1860ctgcctgctg agcaccaagt tcaccatcta caacaacatg ttctgcgccg gcttccacga 1920gggcggccgc gacagctgcc agggcgacag cggcggcccc cacgtgaccg aggtggaggg 1980caccagcttc ctgaccggca tcatcagctg gggcgaggag tgcgccatga agggcaagta 2040cggcatctac accaaggtga gccgctacgt gaactggatc aaggagaaga ccaagctgac 2100ctaatgaaag atggatttcc aaggttaatt cattggaatt gaaaattaac agcccccccc 2160cccccccccc tgcaaataaa agatctttat tttcattaga tctgtgtgtt ggttttttgt 2220gtg 2223372262DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank-MVM- Flank-co-FIX-R338L-Flank-Synt.pA-Flank 37aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggggg ggaggctgct 60ggtgaatatt aaccaaggtc accccagtta tcggaggagc aaacaggggc taagtccacc 120gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 180ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 240aggagcaaac aggggctaag tccacggtac ccactgggag gatgttgagt aagatggaaa 300actactgatg acccttgcag agacagagta ttaggacatg tttgaacagg ggccgggcga 360tcagcaggta gctctagagg atccccgtct gtctgcacat ttcgtagagc gagtgttccg 420atactctaat ctccctaggc aaggttcata tttgtgtagg ttacttattc tccttttgtt 480gactaagtca ataatcagaa tcagcaggtt tggagtcagc ttggcaggga tcagcagcct 540gggttggaag gagggggtat aaaagcccct tcaccaggag aagccgtcac acagatccac 600aagctcctgg ctagaaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 660tttaattacc tggagcacct gcctgaaatc actttttttc aggttgggct agcccaccat 720gcagcgcgtg aacatgatca tggccgagag ccccggcctg atcaccatct gcctgctggg 780ctacctgctg agcgccgagt gcaccgtgtt cctggaccac gagaacgcca acaagatcct 840gaaccgcccc aagcgctaca acagcggcaa gctggaggag ttcgtgcagg gcaacctgga 900gcgcgagtgc atggaggaga agtgcagctt cgaggaggcc cgcgaggtgt tcgagaacac 960cgagcgcacc accgagttct ggaagcagta cgtggacggc gaccagtgcg agagcaaccc 1020ctgcctgaac ggcggcagct gcaaggacga catcaacagc tacgagtgct ggtgcccctt 1080cggcttcgag ggcaagaact gcgagctgga cgtgacctgc aacatcaaga acggccgctg 1140cgagcagttc tgcaagaaca gcgccgacaa caaggtggtg tgcagctgca ccgagggcta 1200ccgcctggcc gagaaccaga agagctgcga gcccgccgtg cccttcccct gcggccgcgt 1260gagcgtgagc cagaccagca agctgacccg cgccgaggcc gtgttccccg acgtggacta 1320cgtgaacagc accgaggccg agaccatcct ggacaacatc acccagagca cccagagctt 1380caacgacttc acccgcgtgg tgggcggcga ggacgccaag cccggccagt tcccctggca 1440ggtggtgctg aacggcaagg tggacgcctt ctgcggcggc agcatcgtga acgagaagtg 1500gatcgtgacc gccgcccact gcgtggagac cggcgtgaag atcaccgtgg tggccggcga 1560gcacaacatc gaggagaccg agcacaccga gcagaagcgc aacgtgatcc gcatcatccc 1620ccaccacaac tacaacgccg ccatcaacaa gtacaaccac gacatcgccc tgctggagct 1680ggacgagccc ctggtgctga acagctacgt gacccccatc tgcatcgccg acaaggagta 1740caccaacatc ttcctgaagt tcggcagcgg ctacgtgagc ggctggggcc gcgtgttcca 1800caagggccgc agcgccctgg tgctgcagta cctgcgcgtg cccctggtgg accgcgccac 1860ctgcctgctg agcaccaagt tcaccatcta caacaacatg ttctgcgccg gcttccacga 1920gggcggccgc gacagctgcc agggcgacag cggcggcccc cacgtgaccg aggtggaggg 1980caccagcttc ctgaccggca tcatcagctg gggcgaggag tgcgccatga agggcaagta 2040cggcatctac accaaggtga gccgctacgt gaactggatc aaggagaaga ccaagctgac 2100ctaatgaaag atggatttcc aaggttaatt cattggaatt gaaaattaac agcccccccc 2160cccccccccc tgcaaataaa agatctttat tttcattaga tctgtgtgtt ggttttttgt 2220gtgccggtgg atctcgatag caggcatgct ggggagagat cg 226238239DNAArtificial SequenceFlank-3xSERP-Flank 38gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtacc 23939339DNAArtificial SequenceFlank-3xSERP-Flank-TTRe 39gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtag 33940354DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank 40gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat cccc 35441556DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm 41gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtc 55642561DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank 42gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcctag t 561434938DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank- coFVIIIdeltaB 43gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcctag tatgcagatc gagctgtcca cctgcttttt tctgtgcctg 600ctgcggttct gcttcagcgc cacccggcgg tactacctgg gcgccgtgga gctgtcctgg 660gactacatgc agagcgacct gggcgagctg cccgtggacg cccggttccc ccccagagtg 720cccaagagct tccccttcaa caccagcgtg gtgtacaaga aaaccctgtt cgtggagttc 780accgaccacc tgttcaatat cgccaagccc aggcccccct ggatgggcct gctgggcccc 840accatccagg ccgaggtgta cgacaccgtg gtgatcaccc tgaagaacat ggccagccac 900cccgtgagcc tgcacgccgt gggcgtgagc tactggaagg ccagcgaggg cgccgagtac 960gacgaccaga ccagccagcg ggagaaagaa gatgacaagg tgttccctgg cggcagccac 1020acctacgtgt ggcaggtgct gaaagaaaac ggccccatgg cctccgaccc cctgtgcctg 1080acctacagct acctgagcca cgtggacctg gtgaaggacc tgaacagcgg cctgatcggc 1140gctctgctcg tctgccggga gggcagcctg gccaaagaga aaacccagac cctgcacaag 1200ttcatcctgc tgttcgccgt gttcgacgag ggcaagagct ggcacagcga gacaaagaac 1260agcctgatgc aggaccggga cgccgcctct gccagagcct ggcccaagat gcacaccgtg 1320aacggctacg tgaacagaag cctgcccggc ctgattggct gccaccggaa gagcgtgtac 1380tggcacgtga tcggcatggg caccacaccc gaggtgcaca gcatctttct ggaagggcac 1440acctttctgg tccggaacca ccggcaggcc agcctggaaa tcagccctat caccttcctg 1500accgcccaga cactgctgat ggacctgggc cagttcctgc tgttttgcca catcagctct 1560caccagcacg acggcatgga agcctacgtg aaggtggact cttgccccga ggaaccccag 1620ctgcggatga agaacaacga ggaagccgag gactacgacg acgacctgac cgacagcgag 1680atggacgtgg tgcggttcga cgacgacaac agccccagct tcatccagat cagaagcgtg 1740gccaagaagc accccaagac ctgggtgcac tatatcgccg ccgaggaaga ggactgggac 1800tacgcccccc tggtgctggc ccccgacgac agaagctaca agagccagta cctgaacaat 1860ggcccccagc ggatcggccg gaagtacaag aaagtgcggt tcatggccta caccgacgag 1920acattcaaga cccgggaggc catccagcac gagagcggca tcctgggccc cctgctgtac 1980ggcgaagtgg gcgacacact gctgatcatc ttcaagaacc aggctagccg gccctacaac 2040atctaccccc acggcatcac cgacgtgcgg cccctgtaca gcaggcggct gcccaagggc

2100gtgaagcacc tgaaggactt ccccatcctg cccggcgaga tcttcaagta caagtggacc 2160gtgaccgtgg aggacggccc caccaagagc gaccccagat gcctgacccg gtactacagc 2220agcttcgtga acatggaacg ggacctggcc tccgggctga tcggacctct gctgatctgc 2280tacaaagaaa gcgtggacca gcggggcaac cagatcatga gcgacaagcg gaacgtgatc 2340ctgttcagcg tgttcgatga gaaccggtcc tggtatctga ccgagaacat ccagcggttt 2400ctgcccaacc ctgccggcgt gcagctggaa gatcccgagt tccaggccag caacatcatg 2460cactccatca atggctacgt gttcgactct ctgcagctct ccgtgtgtct gcacgaggtg 2520gcctactggt acatcctgag catcggcgcc cagaccgact tcctgagcgt gttcttcagc 2580ggctacacct tcaagcacaa gatggtgtac gaggacaccc tgaccctgtt ccctttcagc 2640ggcgagacag tgttcatgag catggaaaac cccggcctgt ggattctggg ctgccacaac 2700agcgacttcc ggaaccgggg catgaccgcc ctgctgaagg tgtccagctg cgacaagaac 2760accggcgact actacgagga cagctacgag gatatcagcg cctacctgct gtccaagaac 2820aacgccatcg aaccccggag cttcagccag aacccccccg tgctgacgcg tcaccagcgg 2880gagatcaccc ggacaaccct gcagtccgac caggaagaga tcgattacga cgacaccatc 2940agcgtggaga tgaagaaaga ggatttcgat atctacgacg aggacgagaa ccagagcccc 3000agaagcttcc agaagaaaac ccggcactac ttcattgccg ccgtggagag gctgtgggac 3060tacggcatga gttctagccc ccacgtgctg cggaaccggg cccagagcgg cagcgtgccc 3120cagttcaaga aagtggtgtt ccaggaattc acagacggca gcttcaccca gcctctgtat 3180agaggcgagc tgaacgagca cctggggctg ctggggccct acatcagggc cgaagtggag 3240gacaacatca tggtgacctt ccggaatcag gccagcagac cctactcctt ctacagcagc 3300ctgatcagct acgaagagga ccagcggcag ggcgccgaac cccggaagaa cttcgtgaag 3360cccaacgaaa ccaagaccta cttctggaaa gtgcagcacc acatggcccc caccaaggac 3420gagttcgact gcaaggcctg ggcctacttc agcgacgtgg atctggaaaa ggacgtgcac 3480tctggactga ttggcccact cctggtctgc cacactaaca ccctcaaccc cgcccacggc 3540cgccaggtga ccgtgcagga attcgccctg ttcttcacca tcttcgacga gacaaagtcc 3600tggtacttca ccgagaatat ggaacggaac tgcagagccc cctgcaacat ccagatggaa 3660gatcctacct tcaaagagaa ctaccggttc cacgccatca acggctacat catggacacc 3720ctgcctggcc tggtgatggc ccaggaccag agaatccggt ggtatctgct gtccatgggc 3780agcaacgaga atatccacag catccacttc agcggccacg tgttcaccgt gcggaagaaa 3840gaagagtaca agatggccct gtacaacctg taccccggcg tgttcgagac agtggagatg 3900ctgcccagca aggccggcat ctggcgggtg gagtgtctga tcggcgagca cctgcacgct 3960ggcatgagca ccctgtttct ggtgtacagc aacaagtgcc agaccccact gggcatggcc 4020tctggccaca tccgggactt ccagatcacc gcctccggcc agtacggcca gtgggccccc 4080aagctggcca gactgcacta cagcggcagc atcaacgcct ggtccaccaa agagcccttc 4140agctggatca aggtggacct gctggcccct atgatcatcc acggcattaa gacccagggc 4200gccaggcaga agttcagcag cctgtacatc agccagttca tcatcatgta cagcctggac 4260ggcaagaagt ggcagaccta ccggggcaac agcaccggca ccctgatggt gttcttcggc 4320aatgtggaca gcagcggcat caagcacaac atcttcaacc cccccatcat tgcccggtac 4380atccggctgc accccaccca ctacagcatt agatccacac tgagaatgga actgatgggc 4440tgcgacctga actcctgcag catgcctctg ggcatggaaa gcaaggccat cagcgacgcc 4500cagatcacag ccagcagcta cttcaccaac atgttcgcca cctggtcccc ctccaaggcc 4560aggctgcacc tgcagggccg gtccaacgcc tggcggcctc aggtcaacaa ccccaaagaa 4620tggctgcagg tggactttca gaaaaccatg aaggtgaccg gcgtgaccac ccagggcgtg 4680aaaagcctgc tgaccagcat gtacgtgaaa gagtttctga tcagcagctc tcaggatggc 4740caccagtgga ccctgttctt tcagaacggc aaggtgaaag tgttccaggg caaccaggac 4800tccttcaccc ccgtggtgaa ctccctggac ccccccctgc tgacccgcta cctgagaatc 4860cacccccagt cttgggtgca ccagatcgcc ctcaggatgg aagtcctggg atgtgaggcc 4920caggatctgt actgatga 4938444944DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank- coFVIIIdeltaB-Flank 44gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcctag tatgcagatc gagctgtcca cctgcttttt tctgtgcctg 600ctgcggttct gcttcagcgc cacccggcgg tactacctgg gcgccgtgga gctgtcctgg 660gactacatgc agagcgacct gggcgagctg cccgtggacg cccggttccc ccccagagtg 720cccaagagct tccccttcaa caccagcgtg gtgtacaaga aaaccctgtt cgtggagttc 780accgaccacc tgttcaatat cgccaagccc aggcccccct ggatgggcct gctgggcccc 840accatccagg ccgaggtgta cgacaccgtg gtgatcaccc tgaagaacat ggccagccac 900cccgtgagcc tgcacgccgt gggcgtgagc tactggaagg ccagcgaggg cgccgagtac 960gacgaccaga ccagccagcg ggagaaagaa gatgacaagg tgttccctgg cggcagccac 1020acctacgtgt ggcaggtgct gaaagaaaac ggccccatgg cctccgaccc cctgtgcctg 1080acctacagct acctgagcca cgtggacctg gtgaaggacc tgaacagcgg cctgatcggc 1140gctctgctcg tctgccggga gggcagcctg gccaaagaga aaacccagac cctgcacaag 1200ttcatcctgc tgttcgccgt gttcgacgag ggcaagagct ggcacagcga gacaaagaac 1260agcctgatgc aggaccggga cgccgcctct gccagagcct ggcccaagat gcacaccgtg 1320aacggctacg tgaacagaag cctgcccggc ctgattggct gccaccggaa gagcgtgtac 1380tggcacgtga tcggcatggg caccacaccc gaggtgcaca gcatctttct ggaagggcac 1440acctttctgg tccggaacca ccggcaggcc agcctggaaa tcagccctat caccttcctg 1500accgcccaga cactgctgat ggacctgggc cagttcctgc tgttttgcca catcagctct 1560caccagcacg acggcatgga agcctacgtg aaggtggact cttgccccga ggaaccccag 1620ctgcggatga agaacaacga ggaagccgag gactacgacg acgacctgac cgacagcgag 1680atggacgtgg tgcggttcga cgacgacaac agccccagct tcatccagat cagaagcgtg 1740gccaagaagc accccaagac ctgggtgcac tatatcgccg ccgaggaaga ggactgggac 1800tacgcccccc tggtgctggc ccccgacgac agaagctaca agagccagta cctgaacaat 1860ggcccccagc ggatcggccg gaagtacaag aaagtgcggt tcatggccta caccgacgag 1920acattcaaga cccgggaggc catccagcac gagagcggca tcctgggccc cctgctgtac 1980ggcgaagtgg gcgacacact gctgatcatc ttcaagaacc aggctagccg gccctacaac 2040atctaccccc acggcatcac cgacgtgcgg cccctgtaca gcaggcggct gcccaagggc 2100gtgaagcacc tgaaggactt ccccatcctg cccggcgaga tcttcaagta caagtggacc 2160gtgaccgtgg aggacggccc caccaagagc gaccccagat gcctgacccg gtactacagc 2220agcttcgtga acatggaacg ggacctggcc tccgggctga tcggacctct gctgatctgc 2280tacaaagaaa gcgtggacca gcggggcaac cagatcatga gcgacaagcg gaacgtgatc 2340ctgttcagcg tgttcgatga gaaccggtcc tggtatctga ccgagaacat ccagcggttt 2400ctgcccaacc ctgccggcgt gcagctggaa gatcccgagt tccaggccag caacatcatg 2460cactccatca atggctacgt gttcgactct ctgcagctct ccgtgtgtct gcacgaggtg 2520gcctactggt acatcctgag catcggcgcc cagaccgact tcctgagcgt gttcttcagc 2580ggctacacct tcaagcacaa gatggtgtac gaggacaccc tgaccctgtt ccctttcagc 2640ggcgagacag tgttcatgag catggaaaac cccggcctgt ggattctggg ctgccacaac 2700agcgacttcc ggaaccgggg catgaccgcc ctgctgaagg tgtccagctg cgacaagaac 2760accggcgact actacgagga cagctacgag gatatcagcg cctacctgct gtccaagaac 2820aacgccatcg aaccccggag cttcagccag aacccccccg tgctgacgcg tcaccagcgg 2880gagatcaccc ggacaaccct gcagtccgac caggaagaga tcgattacga cgacaccatc 2940agcgtggaga tgaagaaaga ggatttcgat atctacgacg aggacgagaa ccagagcccc 3000agaagcttcc agaagaaaac ccggcactac ttcattgccg ccgtggagag gctgtgggac 3060tacggcatga gttctagccc ccacgtgctg cggaaccggg cccagagcgg cagcgtgccc 3120cagttcaaga aagtggtgtt ccaggaattc acagacggca gcttcaccca gcctctgtat 3180agaggcgagc tgaacgagca cctggggctg ctggggccct acatcagggc cgaagtggag 3240gacaacatca tggtgacctt ccggaatcag gccagcagac cctactcctt ctacagcagc 3300ctgatcagct acgaagagga ccagcggcag ggcgccgaac cccggaagaa cttcgtgaag 3360cccaacgaaa ccaagaccta cttctggaaa gtgcagcacc acatggcccc caccaaggac 3420gagttcgact gcaaggcctg ggcctacttc agcgacgtgg atctggaaaa ggacgtgcac 3480tctggactga ttggcccact cctggtctgc cacactaaca ccctcaaccc cgcccacggc 3540cgccaggtga ccgtgcagga attcgccctg ttcttcacca tcttcgacga gacaaagtcc 3600tggtacttca ccgagaatat ggaacggaac tgcagagccc cctgcaacat ccagatggaa 3660gatcctacct tcaaagagaa ctaccggttc cacgccatca acggctacat catggacacc 3720ctgcctggcc tggtgatggc ccaggaccag agaatccggt ggtatctgct gtccatgggc 3780agcaacgaga atatccacag catccacttc agcggccacg tgttcaccgt gcggaagaaa 3840gaagagtaca agatggccct gtacaacctg taccccggcg tgttcgagac agtggagatg 3900ctgcccagca aggccggcat ctggcgggtg gagtgtctga tcggcgagca cctgcacgct 3960ggcatgagca ccctgtttct ggtgtacagc aacaagtgcc agaccccact gggcatggcc 4020tctggccaca tccgggactt ccagatcacc gcctccggcc agtacggcca gtgggccccc 4080aagctggcca gactgcacta cagcggcagc atcaacgcct ggtccaccaa agagcccttc 4140agctggatca aggtggacct gctggcccct atgatcatcc acggcattaa gacccagggc 4200gccaggcaga agttcagcag cctgtacatc agccagttca tcatcatgta cagcctggac 4260ggcaagaagt ggcagaccta ccggggcaac agcaccggca ccctgatggt gttcttcggc 4320aatgtggaca gcagcggcat caagcacaac atcttcaacc cccccatcat tgcccggtac 4380atccggctgc accccaccca ctacagcatt agatccacac tgagaatgga actgatgggc 4440tgcgacctga actcctgcag catgcctctg ggcatggaaa gcaaggccat cagcgacgcc 4500cagatcacag ccagcagcta cttcaccaac atgttcgcca cctggtcccc ctccaaggcc 4560aggctgcacc tgcagggccg gtccaacgcc tggcggcctc aggtcaacaa ccccaaagaa 4620tggctgcagg tggactttca gaaaaccatg aaggtgaccg gcgtgaccac ccagggcgtg 4680aaaagcctgc tgaccagcat gtacgtgaaa gagtttctga tcagcagctc tcaggatggc 4740caccagtgga ccctgttctt tcagaacggc aaggtgaaag tgttccaggg caaccaggac 4800tccttcaccc ccgtggtgaa ctccctggac ccccccctgc tgacccgcta cctgagaatc 4860cacccccagt cttgggtgca ccagatcgcc ctcaggatgg aagtcctggg atgtgaggcc 4920caggatctgt actgatgagg atcc 4944455078DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank- coFVIIIdeltaB-Flank-SV40pA 45gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcctag tatgcagatc gagctgtcca cctgcttttt tctgtgcctg 600ctgcggttct gcttcagcgc cacccggcgg tactacctgg gcgccgtgga gctgtcctgg 660gactacatgc agagcgacct gggcgagctg cccgtggacg cccggttccc ccccagagtg 720cccaagagct tccccttcaa caccagcgtg gtgtacaaga aaaccctgtt cgtggagttc 780accgaccacc tgttcaatat cgccaagccc aggcccccct ggatgggcct gctgggcccc 840accatccagg ccgaggtgta cgacaccgtg gtgatcaccc tgaagaacat ggccagccac 900cccgtgagcc tgcacgccgt gggcgtgagc tactggaagg ccagcgaggg cgccgagtac 960gacgaccaga ccagccagcg ggagaaagaa gatgacaagg tgttccctgg cggcagccac 1020acctacgtgt ggcaggtgct gaaagaaaac ggccccatgg cctccgaccc cctgtgcctg 1080acctacagct acctgagcca cgtggacctg gtgaaggacc tgaacagcgg cctgatcggc 1140gctctgctcg tctgccggga gggcagcctg gccaaagaga aaacccagac cctgcacaag 1200ttcatcctgc tgttcgccgt gttcgacgag ggcaagagct ggcacagcga gacaaagaac 1260agcctgatgc aggaccggga cgccgcctct gccagagcct ggcccaagat gcacaccgtg 1320aacggctacg tgaacagaag cctgcccggc ctgattggct gccaccggaa gagcgtgtac 1380tggcacgtga tcggcatggg caccacaccc gaggtgcaca gcatctttct ggaagggcac 1440acctttctgg tccggaacca ccggcaggcc agcctggaaa tcagccctat caccttcctg 1500accgcccaga cactgctgat ggacctgggc cagttcctgc tgttttgcca catcagctct 1560caccagcacg acggcatgga agcctacgtg aaggtggact cttgccccga ggaaccccag 1620ctgcggatga agaacaacga ggaagccgag gactacgacg acgacctgac cgacagcgag 1680atggacgtgg tgcggttcga cgacgacaac agccccagct tcatccagat cagaagcgtg 1740gccaagaagc accccaagac ctgggtgcac tatatcgccg ccgaggaaga ggactgggac 1800tacgcccccc tggtgctggc ccccgacgac agaagctaca agagccagta cctgaacaat 1860ggcccccagc ggatcggccg gaagtacaag aaagtgcggt tcatggccta caccgacgag 1920acattcaaga cccgggaggc catccagcac gagagcggca tcctgggccc cctgctgtac 1980ggcgaagtgg gcgacacact gctgatcatc ttcaagaacc aggctagccg gccctacaac 2040atctaccccc acggcatcac cgacgtgcgg cccctgtaca gcaggcggct gcccaagggc 2100gtgaagcacc tgaaggactt ccccatcctg cccggcgaga tcttcaagta caagtggacc 2160gtgaccgtgg aggacggccc caccaagagc gaccccagat gcctgacccg gtactacagc 2220agcttcgtga acatggaacg ggacctggcc tccgggctga tcggacctct gctgatctgc 2280tacaaagaaa gcgtggacca gcggggcaac cagatcatga gcgacaagcg gaacgtgatc 2340ctgttcagcg tgttcgatga gaaccggtcc tggtatctga ccgagaacat ccagcggttt 2400ctgcccaacc ctgccggcgt gcagctggaa gatcccgagt tccaggccag caacatcatg 2460cactccatca atggctacgt gttcgactct ctgcagctct ccgtgtgtct gcacgaggtg 2520gcctactggt acatcctgag catcggcgcc cagaccgact tcctgagcgt gttcttcagc 2580ggctacacct tcaagcacaa gatggtgtac gaggacaccc tgaccctgtt ccctttcagc 2640ggcgagacag tgttcatgag catggaaaac cccggcctgt ggattctggg ctgccacaac 2700agcgacttcc ggaaccgggg catgaccgcc ctgctgaagg tgtccagctg cgacaagaac 2760accggcgact actacgagga cagctacgag gatatcagcg cctacctgct gtccaagaac 2820aacgccatcg aaccccggag cttcagccag aacccccccg tgctgacgcg tcaccagcgg 2880gagatcaccc ggacaaccct gcagtccgac caggaagaga tcgattacga cgacaccatc 2940agcgtggaga tgaagaaaga ggatttcgat atctacgacg aggacgagaa ccagagcccc 3000agaagcttcc agaagaaaac ccggcactac ttcattgccg ccgtggagag gctgtgggac 3060tacggcatga gttctagccc ccacgtgctg cggaaccggg cccagagcgg cagcgtgccc 3120cagttcaaga aagtggtgtt ccaggaattc acagacggca gcttcaccca gcctctgtat 3180agaggcgagc tgaacgagca cctggggctg ctggggccct acatcagggc cgaagtggag 3240gacaacatca tggtgacctt ccggaatcag gccagcagac cctactcctt ctacagcagc 3300ctgatcagct acgaagagga ccagcggcag ggcgccgaac cccggaagaa cttcgtgaag 3360cccaacgaaa ccaagaccta cttctggaaa gtgcagcacc acatggcccc caccaaggac 3420gagttcgact gcaaggcctg ggcctacttc agcgacgtgg atctggaaaa ggacgtgcac 3480tctggactga ttggcccact cctggtctgc cacactaaca ccctcaaccc cgcccacggc 3540cgccaggtga ccgtgcagga attcgccctg ttcttcacca tcttcgacga gacaaagtcc 3600tggtacttca ccgagaatat ggaacggaac tgcagagccc cctgcaacat ccagatggaa 3660gatcctacct tcaaagagaa ctaccggttc cacgccatca acggctacat catggacacc 3720ctgcctggcc tggtgatggc ccaggaccag agaatccggt ggtatctgct gtccatgggc 3780agcaacgaga atatccacag catccacttc agcggccacg tgttcaccgt gcggaagaaa 3840gaagagtaca agatggccct gtacaacctg taccccggcg tgttcgagac agtggagatg 3900ctgcccagca aggccggcat ctggcgggtg gagtgtctga tcggcgagca cctgcacgct 3960ggcatgagca ccctgtttct ggtgtacagc aacaagtgcc agaccccact gggcatggcc 4020tctggccaca tccgggactt ccagatcacc gcctccggcc agtacggcca gtgggccccc 4080aagctggcca gactgcacta cagcggcagc atcaacgcct ggtccaccaa agagcccttc 4140agctggatca aggtggacct gctggcccct atgatcatcc acggcattaa gacccagggc 4200gccaggcaga agttcagcag cctgtacatc agccagttca tcatcatgta cagcctggac 4260ggcaagaagt ggcagaccta ccggggcaac agcaccggca ccctgatggt gttcttcggc 4320aatgtggaca gcagcggcat caagcacaac atcttcaacc cccccatcat tgcccggtac 4380atccggctgc accccaccca ctacagcatt agatccacac tgagaatgga actgatgggc 4440tgcgacctga actcctgcag catgcctctg ggcatggaaa gcaaggccat cagcgacgcc 4500cagatcacag ccagcagcta cttcaccaac atgttcgcca cctggtcccc ctccaaggcc 4560aggctgcacc tgcagggccg gtccaacgcc tggcggcctc aggtcaacaa ccccaaagaa 4620tggctgcagg tggactttca gaaaaccatg aaggtgaccg gcgtgaccac ccagggcgtg 4680aaaagcctgc tgaccagcat gtacgtgaaa gagtttctga tcagcagctc tcaggatggc 4740caccagtgga ccctgttctt tcagaacggc aaggtgaaag tgttccaggg caaccaggac 4800tccttcaccc ccgtggtgaa ctccctggac ccccccctgc tgacccgcta cctgagaatc 4860cacccccagt cttgggtgca ccagatcgcc ctcaggatgg aagtcctggg atgtgaggcc 4920caggatctgt actgatgagg atccatgctt tatttgtgaa atttgtgatg ctattgcttt 4980atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 5040gtttcaggtt cagggggagg tgtgggaggt tttttaaa 5078465096DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank- coFVIIIdeltaB-Flank-SV40pA-Flank 46gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcctag tatgcagatc gagctgtcca cctgcttttt tctgtgcctg 600ctgcggttct gcttcagcgc cacccggcgg tactacctgg gcgccgtgga gctgtcctgg 660gactacatgc agagcgacct gggcgagctg cccgtggacg cccggttccc ccccagagtg 720cccaagagct tccccttcaa caccagcgtg gtgtacaaga aaaccctgtt cgtggagttc 780accgaccacc tgttcaatat cgccaagccc aggcccccct ggatgggcct gctgggcccc 840accatccagg ccgaggtgta cgacaccgtg gtgatcaccc tgaagaacat ggccagccac 900cccgtgagcc tgcacgccgt gggcgtgagc tactggaagg ccagcgaggg cgccgagtac 960gacgaccaga ccagccagcg ggagaaagaa gatgacaagg tgttccctgg cggcagccac 1020acctacgtgt ggcaggtgct gaaagaaaac ggccccatgg cctccgaccc cctgtgcctg 1080acctacagct acctgagcca cgtggacctg gtgaaggacc tgaacagcgg cctgatcggc 1140gctctgctcg tctgccggga gggcagcctg gccaaagaga aaacccagac cctgcacaag 1200ttcatcctgc tgttcgccgt gttcgacgag ggcaagagct ggcacagcga gacaaagaac 1260agcctgatgc aggaccggga cgccgcctct gccagagcct ggcccaagat gcacaccgtg 1320aacggctacg tgaacagaag cctgcccggc ctgattggct gccaccggaa gagcgtgtac 1380tggcacgtga tcggcatggg caccacaccc gaggtgcaca gcatctttct ggaagggcac 1440acctttctgg tccggaacca ccggcaggcc agcctggaaa tcagccctat caccttcctg 1500accgcccaga cactgctgat ggacctgggc cagttcctgc tgttttgcca catcagctct 1560caccagcacg acggcatgga agcctacgtg aaggtggact cttgccccga ggaaccccag 1620ctgcggatga agaacaacga ggaagccgag gactacgacg acgacctgac cgacagcgag 1680atggacgtgg tgcggttcga cgacgacaac agccccagct tcatccagat cagaagcgtg 1740gccaagaagc accccaagac ctgggtgcac

tatatcgccg ccgaggaaga ggactgggac 1800tacgcccccc tggtgctggc ccccgacgac agaagctaca agagccagta cctgaacaat 1860ggcccccagc ggatcggccg gaagtacaag aaagtgcggt tcatggccta caccgacgag 1920acattcaaga cccgggaggc catccagcac gagagcggca tcctgggccc cctgctgtac 1980ggcgaagtgg gcgacacact gctgatcatc ttcaagaacc aggctagccg gccctacaac 2040atctaccccc acggcatcac cgacgtgcgg cccctgtaca gcaggcggct gcccaagggc 2100gtgaagcacc tgaaggactt ccccatcctg cccggcgaga tcttcaagta caagtggacc 2160gtgaccgtgg aggacggccc caccaagagc gaccccagat gcctgacccg gtactacagc 2220agcttcgtga acatggaacg ggacctggcc tccgggctga tcggacctct gctgatctgc 2280tacaaagaaa gcgtggacca gcggggcaac cagatcatga gcgacaagcg gaacgtgatc 2340ctgttcagcg tgttcgatga gaaccggtcc tggtatctga ccgagaacat ccagcggttt 2400ctgcccaacc ctgccggcgt gcagctggaa gatcccgagt tccaggccag caacatcatg 2460cactccatca atggctacgt gttcgactct ctgcagctct ccgtgtgtct gcacgaggtg 2520gcctactggt acatcctgag catcggcgcc cagaccgact tcctgagcgt gttcttcagc 2580ggctacacct tcaagcacaa gatggtgtac gaggacaccc tgaccctgtt ccctttcagc 2640ggcgagacag tgttcatgag catggaaaac cccggcctgt ggattctggg ctgccacaac 2700agcgacttcc ggaaccgggg catgaccgcc ctgctgaagg tgtccagctg cgacaagaac 2760accggcgact actacgagga cagctacgag gatatcagcg cctacctgct gtccaagaac 2820aacgccatcg aaccccggag cttcagccag aacccccccg tgctgacgcg tcaccagcgg 2880gagatcaccc ggacaaccct gcagtccgac caggaagaga tcgattacga cgacaccatc 2940agcgtggaga tgaagaaaga ggatttcgat atctacgacg aggacgagaa ccagagcccc 3000agaagcttcc agaagaaaac ccggcactac ttcattgccg ccgtggagag gctgtgggac 3060tacggcatga gttctagccc ccacgtgctg cggaaccggg cccagagcgg cagcgtgccc 3120cagttcaaga aagtggtgtt ccaggaattc acagacggca gcttcaccca gcctctgtat 3180agaggcgagc tgaacgagca cctggggctg ctggggccct acatcagggc cgaagtggag 3240gacaacatca tggtgacctt ccggaatcag gccagcagac cctactcctt ctacagcagc 3300ctgatcagct acgaagagga ccagcggcag ggcgccgaac cccggaagaa cttcgtgaag 3360cccaacgaaa ccaagaccta cttctggaaa gtgcagcacc acatggcccc caccaaggac 3420gagttcgact gcaaggcctg ggcctacttc agcgacgtgg atctggaaaa ggacgtgcac 3480tctggactga ttggcccact cctggtctgc cacactaaca ccctcaaccc cgcccacggc 3540cgccaggtga ccgtgcagga attcgccctg ttcttcacca tcttcgacga gacaaagtcc 3600tggtacttca ccgagaatat ggaacggaac tgcagagccc cctgcaacat ccagatggaa 3660gatcctacct tcaaagagaa ctaccggttc cacgccatca acggctacat catggacacc 3720ctgcctggcc tggtgatggc ccaggaccag agaatccggt ggtatctgct gtccatgggc 3780agcaacgaga atatccacag catccacttc agcggccacg tgttcaccgt gcggaagaaa 3840gaagagtaca agatggccct gtacaacctg taccccggcg tgttcgagac agtggagatg 3900ctgcccagca aggccggcat ctggcgggtg gagtgtctga tcggcgagca cctgcacgct 3960ggcatgagca ccctgtttct ggtgtacagc aacaagtgcc agaccccact gggcatggcc 4020tctggccaca tccgggactt ccagatcacc gcctccggcc agtacggcca gtgggccccc 4080aagctggcca gactgcacta cagcggcagc atcaacgcct ggtccaccaa agagcccttc 4140agctggatca aggtggacct gctggcccct atgatcatcc acggcattaa gacccagggc 4200gccaggcaga agttcagcag cctgtacatc agccagttca tcatcatgta cagcctggac 4260ggcaagaagt ggcagaccta ccggggcaac agcaccggca ccctgatggt gttcttcggc 4320aatgtggaca gcagcggcat caagcacaac atcttcaacc cccccatcat tgcccggtac 4380atccggctgc accccaccca ctacagcatt agatccacac tgagaatgga actgatgggc 4440tgcgacctga actcctgcag catgcctctg ggcatggaaa gcaaggccat cagcgacgcc 4500cagatcacag ccagcagcta cttcaccaac atgttcgcca cctggtcccc ctccaaggcc 4560aggctgcacc tgcagggccg gtccaacgcc tggcggcctc aggtcaacaa ccccaaagaa 4620tggctgcagg tggactttca gaaaaccatg aaggtgaccg gcgtgaccac ccagggcgtg 4680aaaagcctgc tgaccagcat gtacgtgaaa gagtttctga tcagcagctc tcaggatggc 4740caccagtgga ccctgttctt tcagaacggc aaggtgaaag tgttccaggg caaccaggac 4800tccttcaccc ccgtggtgaa ctccctggac ccccccctgc tgacccgcta cctgagaatc 4860cacccccagt cttgggtgca ccagatcgcc ctcaggatgg aagtcctggg atgtgaggcc 4920caggatctgt actgatgagg atccatgctt tatttgtgaa atttgtgatg ctattgcttt 4980atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 5040gtttcaggtt cagggggagg tgtgggaggt tttttaaact cgagatccac ggccgc 5096474993DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank- coFVIIIdeltaB-Flank-Synt.pA 47gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcctag tatgcagatc gagctgtcca cctgcttttt tctgtgcctg 600ctgcggttct gcttcagcgc cacccggcgg tactacctgg gcgccgtgga gctgtcctgg 660gactacatgc agagcgacct gggcgagctg cccgtggacg cccggttccc ccccagagtg 720cccaagagct tccccttcaa caccagcgtg gtgtacaaga aaaccctgtt cgtggagttc 780accgaccacc tgttcaatat cgccaagccc aggcccccct ggatgggcct gctgggcccc 840accatccagg ccgaggtgta cgacaccgtg gtgatcaccc tgaagaacat ggccagccac 900cccgtgagcc tgcacgccgt gggcgtgagc tactggaagg ccagcgaggg cgccgagtac 960gacgaccaga ccagccagcg ggagaaagaa gatgacaagg tgttccctgg cggcagccac 1020acctacgtgt ggcaggtgct gaaagaaaac ggccccatgg cctccgaccc cctgtgcctg 1080acctacagct acctgagcca cgtggacctg gtgaaggacc tgaacagcgg cctgatcggc 1140gctctgctcg tctgccggga gggcagcctg gccaaagaga aaacccagac cctgcacaag 1200ttcatcctgc tgttcgccgt gttcgacgag ggcaagagct ggcacagcga gacaaagaac 1260agcctgatgc aggaccggga cgccgcctct gccagagcct ggcccaagat gcacaccgtg 1320aacggctacg tgaacagaag cctgcccggc ctgattggct gccaccggaa gagcgtgtac 1380tggcacgtga tcggcatggg caccacaccc gaggtgcaca gcatctttct ggaagggcac 1440acctttctgg tccggaacca ccggcaggcc agcctggaaa tcagccctat caccttcctg 1500accgcccaga cactgctgat ggacctgggc cagttcctgc tgttttgcca catcagctct 1560caccagcacg acggcatgga agcctacgtg aaggtggact cttgccccga ggaaccccag 1620ctgcggatga agaacaacga ggaagccgag gactacgacg acgacctgac cgacagcgag 1680atggacgtgg tgcggttcga cgacgacaac agccccagct tcatccagat cagaagcgtg 1740gccaagaagc accccaagac ctgggtgcac tatatcgccg ccgaggaaga ggactgggac 1800tacgcccccc tggtgctggc ccccgacgac agaagctaca agagccagta cctgaacaat 1860ggcccccagc ggatcggccg gaagtacaag aaagtgcggt tcatggccta caccgacgag 1920acattcaaga cccgggaggc catccagcac gagagcggca tcctgggccc cctgctgtac 1980ggcgaagtgg gcgacacact gctgatcatc ttcaagaacc aggctagccg gccctacaac 2040atctaccccc acggcatcac cgacgtgcgg cccctgtaca gcaggcggct gcccaagggc 2100gtgaagcacc tgaaggactt ccccatcctg cccggcgaga tcttcaagta caagtggacc 2160gtgaccgtgg aggacggccc caccaagagc gaccccagat gcctgacccg gtactacagc 2220agcttcgtga acatggaacg ggacctggcc tccgggctga tcggacctct gctgatctgc 2280tacaaagaaa gcgtggacca gcggggcaac cagatcatga gcgacaagcg gaacgtgatc 2340ctgttcagcg tgttcgatga gaaccggtcc tggtatctga ccgagaacat ccagcggttt 2400ctgcccaacc ctgccggcgt gcagctggaa gatcccgagt tccaggccag caacatcatg 2460cactccatca atggctacgt gttcgactct ctgcagctct ccgtgtgtct gcacgaggtg 2520gcctactggt acatcctgag catcggcgcc cagaccgact tcctgagcgt gttcttcagc 2580ggctacacct tcaagcacaa gatggtgtac gaggacaccc tgaccctgtt ccctttcagc 2640ggcgagacag tgttcatgag catggaaaac cccggcctgt ggattctggg ctgccacaac 2700agcgacttcc ggaaccgggg catgaccgcc ctgctgaagg tgtccagctg cgacaagaac 2760accggcgact actacgagga cagctacgag gatatcagcg cctacctgct gtccaagaac 2820aacgccatcg aaccccggag cttcagccag aacccccccg tgctgacgcg tcaccagcgg 2880gagatcaccc ggacaaccct gcagtccgac caggaagaga tcgattacga cgacaccatc 2940agcgtggaga tgaagaaaga ggatttcgat atctacgacg aggacgagaa ccagagcccc 3000agaagcttcc agaagaaaac ccggcactac ttcattgccg ccgtggagag gctgtgggac 3060tacggcatga gttctagccc ccacgtgctg cggaaccggg cccagagcgg cagcgtgccc 3120cagttcaaga aagtggtgtt ccaggaattc acagacggca gcttcaccca gcctctgtat 3180agaggcgagc tgaacgagca cctggggctg ctggggccct acatcagggc cgaagtggag 3240gacaacatca tggtgacctt ccggaatcag gccagcagac cctactcctt ctacagcagc 3300ctgatcagct acgaagagga ccagcggcag ggcgccgaac cccggaagaa cttcgtgaag 3360cccaacgaaa ccaagaccta cttctggaaa gtgcagcacc acatggcccc caccaaggac 3420gagttcgact gcaaggcctg ggcctacttc agcgacgtgg atctggaaaa ggacgtgcac 3480tctggactga ttggcccact cctggtctgc cacactaaca ccctcaaccc cgcccacggc 3540cgccaggtga ccgtgcagga attcgccctg ttcttcacca tcttcgacga gacaaagtcc 3600tggtacttca ccgagaatat ggaacggaac tgcagagccc cctgcaacat ccagatggaa 3660gatcctacct tcaaagagaa ctaccggttc cacgccatca acggctacat catggacacc 3720ctgcctggcc tggtgatggc ccaggaccag agaatccggt ggtatctgct gtccatgggc 3780agcaacgaga atatccacag catccacttc agcggccacg tgttcaccgt gcggaagaaa 3840gaagagtaca agatggccct gtacaacctg taccccggcg tgttcgagac agtggagatg 3900ctgcccagca aggccggcat ctggcgggtg gagtgtctga tcggcgagca cctgcacgct 3960ggcatgagca ccctgtttct ggtgtacagc aacaagtgcc agaccccact gggcatggcc 4020tctggccaca tccgggactt ccagatcacc gcctccggcc agtacggcca gtgggccccc 4080aagctggcca gactgcacta cagcggcagc atcaacgcct ggtccaccaa agagcccttc 4140agctggatca aggtggacct gctggcccct atgatcatcc acggcattaa gacccagggc 4200gccaggcaga agttcagcag cctgtacatc agccagttca tcatcatgta cagcctggac 4260ggcaagaagt ggcagaccta ccggggcaac agcaccggca ccctgatggt gttcttcggc 4320aatgtggaca gcagcggcat caagcacaac atcttcaacc cccccatcat tgcccggtac 4380atccggctgc accccaccca ctacagcatt agatccacac tgagaatgga actgatgggc 4440tgcgacctga actcctgcag catgcctctg ggcatggaaa gcaaggccat cagcgacgcc 4500cagatcacag ccagcagcta cttcaccaac atgttcgcca cctggtcccc ctccaaggcc 4560aggctgcacc tgcagggccg gtccaacgcc tggcggcctc aggtcaacaa ccccaaagaa 4620tggctgcagg tggactttca gaaaaccatg aaggtgaccg gcgtgaccac ccagggcgtg 4680aaaagcctgc tgaccagcat gtacgtgaaa gagtttctga tcagcagctc tcaggatggc 4740caccagtgga ccctgttctt tcagaacggc aaggtgaaag tgttccaggg caaccaggac 4800tccttcaccc ccgtggtgaa ctccctggac ccccccctgc tgacccgcta cctgagaatc 4860cacccccagt cttgggtgca ccagatcgcc ctcaggatgg aagtcctggg atgtgaggcc 4920caggatctgt actgatgagg atccaataaa agatctttat tttcattaga tctgtgtgtt 4980ggttttttgt gtg 4993485011DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-Flank- coFVIIIdeltaB-Flank-SyntpA-Flank 48gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcctag tatgcagatc gagctgtcca cctgcttttt tctgtgcctg 600ctgcggttct gcttcagcgc cacccggcgg tactacctgg gcgccgtgga gctgtcctgg 660gactacatgc agagcgacct gggcgagctg cccgtggacg cccggttccc ccccagagtg 720cccaagagct tccccttcaa caccagcgtg gtgtacaaga aaaccctgtt cgtggagttc 780accgaccacc tgttcaatat cgccaagccc aggcccccct ggatgggcct gctgggcccc 840accatccagg ccgaggtgta cgacaccgtg gtgatcaccc tgaagaacat ggccagccac 900cccgtgagcc tgcacgccgt gggcgtgagc tactggaagg ccagcgaggg cgccgagtac 960gacgaccaga ccagccagcg ggagaaagaa gatgacaagg tgttccctgg cggcagccac 1020acctacgtgt ggcaggtgct gaaagaaaac ggccccatgg cctccgaccc cctgtgcctg 1080acctacagct acctgagcca cgtggacctg gtgaaggacc tgaacagcgg cctgatcggc 1140gctctgctcg tctgccggga gggcagcctg gccaaagaga aaacccagac cctgcacaag 1200ttcatcctgc tgttcgccgt gttcgacgag ggcaagagct ggcacagcga gacaaagaac 1260agcctgatgc aggaccggga cgccgcctct gccagagcct ggcccaagat gcacaccgtg 1320aacggctacg tgaacagaag cctgcccggc ctgattggct gccaccggaa gagcgtgtac 1380tggcacgtga tcggcatggg caccacaccc gaggtgcaca gcatctttct ggaagggcac 1440acctttctgg tccggaacca ccggcaggcc agcctggaaa tcagccctat caccttcctg 1500accgcccaga cactgctgat ggacctgggc cagttcctgc tgttttgcca catcagctct 1560caccagcacg acggcatgga agcctacgtg aaggtggact cttgccccga ggaaccccag 1620ctgcggatga agaacaacga ggaagccgag gactacgacg acgacctgac cgacagcgag 1680atggacgtgg tgcggttcga cgacgacaac agccccagct tcatccagat cagaagcgtg 1740gccaagaagc accccaagac ctgggtgcac tatatcgccg ccgaggaaga ggactgggac 1800tacgcccccc tggtgctggc ccccgacgac agaagctaca agagccagta cctgaacaat 1860ggcccccagc ggatcggccg gaagtacaag aaagtgcggt tcatggccta caccgacgag 1920acattcaaga cccgggaggc catccagcac gagagcggca tcctgggccc cctgctgtac 1980ggcgaagtgg gcgacacact gctgatcatc ttcaagaacc aggctagccg gccctacaac 2040atctaccccc acggcatcac cgacgtgcgg cccctgtaca gcaggcggct gcccaagggc 2100gtgaagcacc tgaaggactt ccccatcctg cccggcgaga tcttcaagta caagtggacc 2160gtgaccgtgg aggacggccc caccaagagc gaccccagat gcctgacccg gtactacagc 2220agcttcgtga acatggaacg ggacctggcc tccgggctga tcggacctct gctgatctgc 2280tacaaagaaa gcgtggacca gcggggcaac cagatcatga gcgacaagcg gaacgtgatc 2340ctgttcagcg tgttcgatga gaaccggtcc tggtatctga ccgagaacat ccagcggttt 2400ctgcccaacc ctgccggcgt gcagctggaa gatcccgagt tccaggccag caacatcatg 2460cactccatca atggctacgt gttcgactct ctgcagctct ccgtgtgtct gcacgaggtg 2520gcctactggt acatcctgag catcggcgcc cagaccgact tcctgagcgt gttcttcagc 2580ggctacacct tcaagcacaa gatggtgtac gaggacaccc tgaccctgtt ccctttcagc 2640ggcgagacag tgttcatgag catggaaaac cccggcctgt ggattctggg ctgccacaac 2700agcgacttcc ggaaccgggg catgaccgcc ctgctgaagg tgtccagctg cgacaagaac 2760accggcgact actacgagga cagctacgag gatatcagcg cctacctgct gtccaagaac 2820aacgccatcg aaccccggag cttcagccag aacccccccg tgctgacgcg tcaccagcgg 2880gagatcaccc ggacaaccct gcagtccgac caggaagaga tcgattacga cgacaccatc 2940agcgtggaga tgaagaaaga ggatttcgat atctacgacg aggacgagaa ccagagcccc 3000agaagcttcc agaagaaaac ccggcactac ttcattgccg ccgtggagag gctgtgggac 3060tacggcatga gttctagccc ccacgtgctg cggaaccggg cccagagcgg cagcgtgccc 3120cagttcaaga aagtggtgtt ccaggaattc acagacggca gcttcaccca gcctctgtat 3180agaggcgagc tgaacgagca cctggggctg ctggggccct acatcagggc cgaagtggag 3240gacaacatca tggtgacctt ccggaatcag gccagcagac cctactcctt ctacagcagc 3300ctgatcagct acgaagagga ccagcggcag ggcgccgaac cccggaagaa cttcgtgaag 3360cccaacgaaa ccaagaccta cttctggaaa gtgcagcacc acatggcccc caccaaggac 3420gagttcgact gcaaggcctg ggcctacttc agcgacgtgg atctggaaaa ggacgtgcac 3480tctggactga ttggcccact cctggtctgc cacactaaca ccctcaaccc cgcccacggc 3540cgccaggtga ccgtgcagga attcgccctg ttcttcacca tcttcgacga gacaaagtcc 3600tggtacttca ccgagaatat ggaacggaac tgcagagccc cctgcaacat ccagatggaa 3660gatcctacct tcaaagagaa ctaccggttc cacgccatca acggctacat catggacacc 3720ctgcctggcc tggtgatggc ccaggaccag agaatccggt ggtatctgct gtccatgggc 3780agcaacgaga atatccacag catccacttc agcggccacg tgttcaccgt gcggaagaaa 3840gaagagtaca agatggccct gtacaacctg taccccggcg tgttcgagac agtggagatg 3900ctgcccagca aggccggcat ctggcgggtg gagtgtctga tcggcgagca cctgcacgct 3960ggcatgagca ccctgtttct ggtgtacagc aacaagtgcc agaccccact gggcatggcc 4020tctggccaca tccgggactt ccagatcacc gcctccggcc agtacggcca gtgggccccc 4080aagctggcca gactgcacta cagcggcagc atcaacgcct ggtccaccaa agagcccttc 4140agctggatca aggtggacct gctggcccct atgatcatcc acggcattaa gacccagggc 4200gccaggcaga agttcagcag cctgtacatc agccagttca tcatcatgta cagcctggac 4260ggcaagaagt ggcagaccta ccggggcaac agcaccggca ccctgatggt gttcttcggc 4320aatgtggaca gcagcggcat caagcacaac atcttcaacc cccccatcat tgcccggtac 4380atccggctgc accccaccca ctacagcatt agatccacac tgagaatgga actgatgggc 4440tgcgacctga actcctgcag catgcctctg ggcatggaaa gcaaggccat cagcgacgcc 4500cagatcacag ccagcagcta cttcaccaac atgttcgcca cctggtcccc ctccaaggcc 4560aggctgcacc tgcagggccg gtccaacgcc tggcggcctc aggtcaacaa ccccaaagaa 4620tggctgcagg tggactttca gaaaaccatg aaggtgaccg gcgtgaccac ccagggcgtg 4680aaaagcctgc tgaccagcat gtacgtgaaa gagtttctga tcagcagctc tcaggatggc 4740caccagtgga ccctgttctt tcagaacggc aaggtgaaag tgttccaggg caaccaggac 4800tccttcaccc ccgtggtgaa ctccctggac ccccccctgc tgacccgcta cctgagaatc 4860cacccccagt cttgggtgca ccagatcgcc ctcaggatgg aagtcctggg atgtgaggcc 4920caggatctgt actgatgagg atccaataaa agatctttat tttcattaga tctgtgtgtt 4980ggttttttgt gtgctcgaga tccacggccg c 501149653DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-MVM-Flank 49gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat 600gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggct agt 653505030DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-MVM-Flank- coFVIIIdeltaB 50gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc

540accaggagaa gccgtcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat 600gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggct agtatgcaga 660tcgagctgtc cacctgcttt tttctgtgcc tgctgcggtt ctgcttcagc gccacccggc 720ggtactacct gggcgccgtg gagctgtcct gggactacat gcagagcgac ctgggcgagc 780tgcccgtgga cgcccggttc ccccccagag tgcccaagag cttccccttc aacaccagcg 840tggtgtacaa gaaaaccctg ttcgtggagt tcaccgacca cctgttcaat atcgccaagc 900ccaggccccc ctggatgggc ctgctgggcc ccaccatcca ggccgaggtg tacgacaccg 960tggtgatcac cctgaagaac atggccagcc accccgtgag cctgcacgcc gtgggcgtga 1020gctactggaa ggccagcgag ggcgccgagt acgacgacca gaccagccag cgggagaaag 1080aagatgacaa ggtgttccct ggcggcagcc acacctacgt gtggcaggtg ctgaaagaaa 1140acggccccat ggcctccgac cccctgtgcc tgacctacag ctacctgagc cacgtggacc 1200tggtgaagga cctgaacagc ggcctgatcg gcgctctgct cgtctgccgg gagggcagcc 1260tggccaaaga gaaaacccag accctgcaca agttcatcct gctgttcgcc gtgttcgacg 1320agggcaagag ctggcacagc gagacaaaga acagcctgat gcaggaccgg gacgccgcct 1380ctgccagagc ctggcccaag atgcacaccg tgaacggcta cgtgaacaga agcctgcccg 1440gcctgattgg ctgccaccgg aagagcgtgt actggcacgt gatcggcatg ggcaccacac 1500ccgaggtgca cagcatcttt ctggaagggc acacctttct ggtccggaac caccggcagg 1560ccagcctgga aatcagccct atcaccttcc tgaccgccca gacactgctg atggacctgg 1620gccagttcct gctgttttgc cacatcagct ctcaccagca cgacggcatg gaagcctacg 1680tgaaggtgga ctcttgcccc gaggaacccc agctgcggat gaagaacaac gaggaagccg 1740aggactacga cgacgacctg accgacagcg agatggacgt ggtgcggttc gacgacgaca 1800acagccccag cttcatccag atcagaagcg tggccaagaa gcaccccaag acctgggtgc 1860actatatcgc cgccgaggaa gaggactggg actacgcccc cctggtgctg gcccccgacg 1920acagaagcta caagagccag tacctgaaca atggccccca gcggatcggc cggaagtaca 1980agaaagtgcg gttcatggcc tacaccgacg agacattcaa gacccgggag gccatccagc 2040acgagagcgg catcctgggc cccctgctgt acggcgaagt gggcgacaca ctgctgatca 2100tcttcaagaa ccaggctagc cggccctaca acatctaccc ccacggcatc accgacgtgc 2160ggcccctgta cagcaggcgg ctgcccaagg gcgtgaagca cctgaaggac ttccccatcc 2220tgcccggcga gatcttcaag tacaagtgga ccgtgaccgt ggaggacggc cccaccaaga 2280gcgaccccag atgcctgacc cggtactaca gcagcttcgt gaacatggaa cgggacctgg 2340cctccgggct gatcggacct ctgctgatct gctacaaaga aagcgtggac cagcggggca 2400accagatcat gagcgacaag cggaacgtga tcctgttcag cgtgttcgat gagaaccggt 2460cctggtatct gaccgagaac atccagcggt ttctgcccaa ccctgccggc gtgcagctgg 2520aagatcccga gttccaggcc agcaacatca tgcactccat caatggctac gtgttcgact 2580ctctgcagct ctccgtgtgt ctgcacgagg tggcctactg gtacatcctg agcatcggcg 2640cccagaccga cttcctgagc gtgttcttca gcggctacac cttcaagcac aagatggtgt 2700acgaggacac cctgaccctg ttccctttca gcggcgagac agtgttcatg agcatggaaa 2760accccggcct gtggattctg ggctgccaca acagcgactt ccggaaccgg ggcatgaccg 2820ccctgctgaa ggtgtccagc tgcgacaaga acaccggcga ctactacgag gacagctacg 2880aggatatcag cgcctacctg ctgtccaaga acaacgccat cgaaccccgg agcttcagcc 2940agaacccccc cgtgctgacg cgtcaccagc gggagatcac ccggacaacc ctgcagtccg 3000accaggaaga gatcgattac gacgacacca tcagcgtgga gatgaagaaa gaggatttcg 3060atatctacga cgaggacgag aaccagagcc ccagaagctt ccagaagaaa acccggcact 3120acttcattgc cgccgtggag aggctgtggg actacggcat gagttctagc ccccacgtgc 3180tgcggaaccg ggcccagagc ggcagcgtgc cccagttcaa gaaagtggtg ttccaggaat 3240tcacagacgg cagcttcacc cagcctctgt atagaggcga gctgaacgag cacctggggc 3300tgctggggcc ctacatcagg gccgaagtgg aggacaacat catggtgacc ttccggaatc 3360aggccagcag accctactcc ttctacagca gcctgatcag ctacgaagag gaccagcggc 3420agggcgccga accccggaag aacttcgtga agcccaacga aaccaagacc tacttctgga 3480aagtgcagca ccacatggcc cccaccaagg acgagttcga ctgcaaggcc tgggcctact 3540tcagcgacgt ggatctggaa aaggacgtgc actctggact gattggccca ctcctggtct 3600gccacactaa caccctcaac cccgcccacg gccgccaggt gaccgtgcag gaattcgccc 3660tgttcttcac catcttcgac gagacaaagt cctggtactt caccgagaat atggaacgga 3720actgcagagc cccctgcaac atccagatgg aagatcctac cttcaaagag aactaccggt 3780tccacgccat caacggctac atcatggaca ccctgcctgg cctggtgatg gcccaggacc 3840agagaatccg gtggtatctg ctgtccatgg gcagcaacga gaatatccac agcatccact 3900tcagcggcca cgtgttcacc gtgcggaaga aagaagagta caagatggcc ctgtacaacc 3960tgtaccccgg cgtgttcgag acagtggaga tgctgcccag caaggccggc atctggcggg 4020tggagtgtct gatcggcgag cacctgcacg ctggcatgag caccctgttt ctggtgtaca 4080gcaacaagtg ccagacccca ctgggcatgg cctctggcca catccgggac ttccagatca 4140ccgcctccgg ccagtacggc cagtgggccc ccaagctggc cagactgcac tacagcggca 4200gcatcaacgc ctggtccacc aaagagccct tcagctggat caaggtggac ctgctggccc 4260ctatgatcat ccacggcatt aagacccagg gcgccaggca gaagttcagc agcctgtaca 4320tcagccagtt catcatcatg tacagcctgg acggcaagaa gtggcagacc taccggggca 4380acagcaccgg caccctgatg gtgttcttcg gcaatgtgga cagcagcggc atcaagcaca 4440acatcttcaa cccccccatc attgcccggt acatccggct gcaccccacc cactacagca 4500ttagatccac actgagaatg gaactgatgg gctgcgacct gaactcctgc agcatgcctc 4560tgggcatgga aagcaaggcc atcagcgacg cccagatcac agccagcagc tacttcacca 4620acatgttcgc cacctggtcc ccctccaagg ccaggctgca cctgcagggc cggtccaacg 4680cctggcggcc tcaggtcaac aaccccaaag aatggctgca ggtggacttt cagaaaacca 4740tgaaggtgac cggcgtgacc acccagggcg tgaaaagcct gctgaccagc atgtacgtga 4800aagagtttct gatcagcagc tctcaggatg gccaccagtg gaccctgttc tttcagaacg 4860gcaaggtgaa agtgttccag ggcaaccagg actccttcac ccccgtggtg aactccctgg 4920acccccccct gctgacccgc tacctgagaa tccaccccca gtcttgggtg caccagatcg 4980ccctcaggat ggaagtcctg ggatgtgagg cccaggatct gtactgatga 5030515036DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-MVM-Flank- coFVIIIdeltaB-Flank 51gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat 600gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggct agtatgcaga 660tcgagctgtc cacctgcttt tttctgtgcc tgctgcggtt ctgcttcagc gccacccggc 720ggtactacct gggcgccgtg gagctgtcct gggactacat gcagagcgac ctgggcgagc 780tgcccgtgga cgcccggttc ccccccagag tgcccaagag cttccccttc aacaccagcg 840tggtgtacaa gaaaaccctg ttcgtggagt tcaccgacca cctgttcaat atcgccaagc 900ccaggccccc ctggatgggc ctgctgggcc ccaccatcca ggccgaggtg tacgacaccg 960tggtgatcac cctgaagaac atggccagcc accccgtgag cctgcacgcc gtgggcgtga 1020gctactggaa ggccagcgag ggcgccgagt acgacgacca gaccagccag cgggagaaag 1080aagatgacaa ggtgttccct ggcggcagcc acacctacgt gtggcaggtg ctgaaagaaa 1140acggccccat ggcctccgac cccctgtgcc tgacctacag ctacctgagc cacgtggacc 1200tggtgaagga cctgaacagc ggcctgatcg gcgctctgct cgtctgccgg gagggcagcc 1260tggccaaaga gaaaacccag accctgcaca agttcatcct gctgttcgcc gtgttcgacg 1320agggcaagag ctggcacagc gagacaaaga acagcctgat gcaggaccgg gacgccgcct 1380ctgccagagc ctggcccaag atgcacaccg tgaacggcta cgtgaacaga agcctgcccg 1440gcctgattgg ctgccaccgg aagagcgtgt actggcacgt gatcggcatg ggcaccacac 1500ccgaggtgca cagcatcttt ctggaagggc acacctttct ggtccggaac caccggcagg 1560ccagcctgga aatcagccct atcaccttcc tgaccgccca gacactgctg atggacctgg 1620gccagttcct gctgttttgc cacatcagct ctcaccagca cgacggcatg gaagcctacg 1680tgaaggtgga ctcttgcccc gaggaacccc agctgcggat gaagaacaac gaggaagccg 1740aggactacga cgacgacctg accgacagcg agatggacgt ggtgcggttc gacgacgaca 1800acagccccag cttcatccag atcagaagcg tggccaagaa gcaccccaag acctgggtgc 1860actatatcgc cgccgaggaa gaggactggg actacgcccc cctggtgctg gcccccgacg 1920acagaagcta caagagccag tacctgaaca atggccccca gcggatcggc cggaagtaca 1980agaaagtgcg gttcatggcc tacaccgacg agacattcaa gacccgggag gccatccagc 2040acgagagcgg catcctgggc cccctgctgt acggcgaagt gggcgacaca ctgctgatca 2100tcttcaagaa ccaggctagc cggccctaca acatctaccc ccacggcatc accgacgtgc 2160ggcccctgta cagcaggcgg ctgcccaagg gcgtgaagca cctgaaggac ttccccatcc 2220tgcccggcga gatcttcaag tacaagtgga ccgtgaccgt ggaggacggc cccaccaaga 2280gcgaccccag atgcctgacc cggtactaca gcagcttcgt gaacatggaa cgggacctgg 2340cctccgggct gatcggacct ctgctgatct gctacaaaga aagcgtggac cagcggggca 2400accagatcat gagcgacaag cggaacgtga tcctgttcag cgtgttcgat gagaaccggt 2460cctggtatct gaccgagaac atccagcggt ttctgcccaa ccctgccggc gtgcagctgg 2520aagatcccga gttccaggcc agcaacatca tgcactccat caatggctac gtgttcgact 2580ctctgcagct ctccgtgtgt ctgcacgagg tggcctactg gtacatcctg agcatcggcg 2640cccagaccga cttcctgagc gtgttcttca gcggctacac cttcaagcac aagatggtgt 2700acgaggacac cctgaccctg ttccctttca gcggcgagac agtgttcatg agcatggaaa 2760accccggcct gtggattctg ggctgccaca acagcgactt ccggaaccgg ggcatgaccg 2820ccctgctgaa ggtgtccagc tgcgacaaga acaccggcga ctactacgag gacagctacg 2880aggatatcag cgcctacctg ctgtccaaga acaacgccat cgaaccccgg agcttcagcc 2940agaacccccc cgtgctgacg cgtcaccagc gggagatcac ccggacaacc ctgcagtccg 3000accaggaaga gatcgattac gacgacacca tcagcgtgga gatgaagaaa gaggatttcg 3060atatctacga cgaggacgag aaccagagcc ccagaagctt ccagaagaaa acccggcact 3120acttcattgc cgccgtggag aggctgtggg actacggcat gagttctagc ccccacgtgc 3180tgcggaaccg ggcccagagc ggcagcgtgc cccagttcaa gaaagtggtg ttccaggaat 3240tcacagacgg cagcttcacc cagcctctgt atagaggcga gctgaacgag cacctggggc 3300tgctggggcc ctacatcagg gccgaagtgg aggacaacat catggtgacc ttccggaatc 3360aggccagcag accctactcc ttctacagca gcctgatcag ctacgaagag gaccagcggc 3420agggcgccga accccggaag aacttcgtga agcccaacga aaccaagacc tacttctgga 3480aagtgcagca ccacatggcc cccaccaagg acgagttcga ctgcaaggcc tgggcctact 3540tcagcgacgt ggatctggaa aaggacgtgc actctggact gattggccca ctcctggtct 3600gccacactaa caccctcaac cccgcccacg gccgccaggt gaccgtgcag gaattcgccc 3660tgttcttcac catcttcgac gagacaaagt cctggtactt caccgagaat atggaacgga 3720actgcagagc cccctgcaac atccagatgg aagatcctac cttcaaagag aactaccggt 3780tccacgccat caacggctac atcatggaca ccctgcctgg cctggtgatg gcccaggacc 3840agagaatccg gtggtatctg ctgtccatgg gcagcaacga gaatatccac agcatccact 3900tcagcggcca cgtgttcacc gtgcggaaga aagaagagta caagatggcc ctgtacaacc 3960tgtaccccgg cgtgttcgag acagtggaga tgctgcccag caaggccggc atctggcggg 4020tggagtgtct gatcggcgag cacctgcacg ctggcatgag caccctgttt ctggtgtaca 4080gcaacaagtg ccagacccca ctgggcatgg cctctggcca catccgggac ttccagatca 4140ccgcctccgg ccagtacggc cagtgggccc ccaagctggc cagactgcac tacagcggca 4200gcatcaacgc ctggtccacc aaagagccct tcagctggat caaggtggac ctgctggccc 4260ctatgatcat ccacggcatt aagacccagg gcgccaggca gaagttcagc agcctgtaca 4320tcagccagtt catcatcatg tacagcctgg acggcaagaa gtggcagacc taccggggca 4380acagcaccgg caccctgatg gtgttcttcg gcaatgtgga cagcagcggc atcaagcaca 4440acatcttcaa cccccccatc attgcccggt acatccggct gcaccccacc cactacagca 4500ttagatccac actgagaatg gaactgatgg gctgcgacct gaactcctgc agcatgcctc 4560tgggcatgga aagcaaggcc atcagcgacg cccagatcac agccagcagc tacttcacca 4620acatgttcgc cacctggtcc ccctccaagg ccaggctgca cctgcagggc cggtccaacg 4680cctggcggcc tcaggtcaac aaccccaaag aatggctgca ggtggacttt cagaaaacca 4740tgaaggtgac cggcgtgacc acccagggcg tgaaaagcct gctgaccagc atgtacgtga 4800aagagtttct gatcagcagc tctcaggatg gccaccagtg gaccctgttc tttcagaacg 4860gcaaggtgaa agtgttccag ggcaaccagg actccttcac ccccgtggtg aactccctgg 4920acccccccct gctgacccgc tacctgagaa tccaccccca gtcttgggtg caccagatcg 4980ccctcaggat ggaagtcctg ggatgtgagg cccaggatct gtactgatga ggatcc 5036525170DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-MVM-Flank- coFVIIIdeltaB-Flank-SV40pA 52gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat 600gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggct agtatgcaga 660tcgagctgtc cacctgcttt tttctgtgcc tgctgcggtt ctgcttcagc gccacccggc 720ggtactacct gggcgccgtg gagctgtcct gggactacat gcagagcgac ctgggcgagc 780tgcccgtgga cgcccggttc ccccccagag tgcccaagag cttccccttc aacaccagcg 840tggtgtacaa gaaaaccctg ttcgtggagt tcaccgacca cctgttcaat atcgccaagc 900ccaggccccc ctggatgggc ctgctgggcc ccaccatcca ggccgaggtg tacgacaccg 960tggtgatcac cctgaagaac atggccagcc accccgtgag cctgcacgcc gtgggcgtga 1020gctactggaa ggccagcgag ggcgccgagt acgacgacca gaccagccag cgggagaaag 1080aagatgacaa ggtgttccct ggcggcagcc acacctacgt gtggcaggtg ctgaaagaaa 1140acggccccat ggcctccgac cccctgtgcc tgacctacag ctacctgagc cacgtggacc 1200tggtgaagga cctgaacagc ggcctgatcg gcgctctgct cgtctgccgg gagggcagcc 1260tggccaaaga gaaaacccag accctgcaca agttcatcct gctgttcgcc gtgttcgacg 1320agggcaagag ctggcacagc gagacaaaga acagcctgat gcaggaccgg gacgccgcct 1380ctgccagagc ctggcccaag atgcacaccg tgaacggcta cgtgaacaga agcctgcccg 1440gcctgattgg ctgccaccgg aagagcgtgt actggcacgt gatcggcatg ggcaccacac 1500ccgaggtgca cagcatcttt ctggaagggc acacctttct ggtccggaac caccggcagg 1560ccagcctgga aatcagccct atcaccttcc tgaccgccca gacactgctg atggacctgg 1620gccagttcct gctgttttgc cacatcagct ctcaccagca cgacggcatg gaagcctacg 1680tgaaggtgga ctcttgcccc gaggaacccc agctgcggat gaagaacaac gaggaagccg 1740aggactacga cgacgacctg accgacagcg agatggacgt ggtgcggttc gacgacgaca 1800acagccccag cttcatccag atcagaagcg tggccaagaa gcaccccaag acctgggtgc 1860actatatcgc cgccgaggaa gaggactggg actacgcccc cctggtgctg gcccccgacg 1920acagaagcta caagagccag tacctgaaca atggccccca gcggatcggc cggaagtaca 1980agaaagtgcg gttcatggcc tacaccgacg agacattcaa gacccgggag gccatccagc 2040acgagagcgg catcctgggc cccctgctgt acggcgaagt gggcgacaca ctgctgatca 2100tcttcaagaa ccaggctagc cggccctaca acatctaccc ccacggcatc accgacgtgc 2160ggcccctgta cagcaggcgg ctgcccaagg gcgtgaagca cctgaaggac ttccccatcc 2220tgcccggcga gatcttcaag tacaagtgga ccgtgaccgt ggaggacggc cccaccaaga 2280gcgaccccag atgcctgacc cggtactaca gcagcttcgt gaacatggaa cgggacctgg 2340cctccgggct gatcggacct ctgctgatct gctacaaaga aagcgtggac cagcggggca 2400accagatcat gagcgacaag cggaacgtga tcctgttcag cgtgttcgat gagaaccggt 2460cctggtatct gaccgagaac atccagcggt ttctgcccaa ccctgccggc gtgcagctgg 2520aagatcccga gttccaggcc agcaacatca tgcactccat caatggctac gtgttcgact 2580ctctgcagct ctccgtgtgt ctgcacgagg tggcctactg gtacatcctg agcatcggcg 2640cccagaccga cttcctgagc gtgttcttca gcggctacac cttcaagcac aagatggtgt 2700acgaggacac cctgaccctg ttccctttca gcggcgagac agtgttcatg agcatggaaa 2760accccggcct gtggattctg ggctgccaca acagcgactt ccggaaccgg ggcatgaccg 2820ccctgctgaa ggtgtccagc tgcgacaaga acaccggcga ctactacgag gacagctacg 2880aggatatcag cgcctacctg ctgtccaaga acaacgccat cgaaccccgg agcttcagcc 2940agaacccccc cgtgctgacg cgtcaccagc gggagatcac ccggacaacc ctgcagtccg 3000accaggaaga gatcgattac gacgacacca tcagcgtgga gatgaagaaa gaggatttcg 3060atatctacga cgaggacgag aaccagagcc ccagaagctt ccagaagaaa acccggcact 3120acttcattgc cgccgtggag aggctgtggg actacggcat gagttctagc ccccacgtgc 3180tgcggaaccg ggcccagagc ggcagcgtgc cccagttcaa gaaagtggtg ttccaggaat 3240tcacagacgg cagcttcacc cagcctctgt atagaggcga gctgaacgag cacctggggc 3300tgctggggcc ctacatcagg gccgaagtgg aggacaacat catggtgacc ttccggaatc 3360aggccagcag accctactcc ttctacagca gcctgatcag ctacgaagag gaccagcggc 3420agggcgccga accccggaag aacttcgtga agcccaacga aaccaagacc tacttctgga 3480aagtgcagca ccacatggcc cccaccaagg acgagttcga ctgcaaggcc tgggcctact 3540tcagcgacgt ggatctggaa aaggacgtgc actctggact gattggccca ctcctggtct 3600gccacactaa caccctcaac cccgcccacg gccgccaggt gaccgtgcag gaattcgccc 3660tgttcttcac catcttcgac gagacaaagt cctggtactt caccgagaat atggaacgga 3720actgcagagc cccctgcaac atccagatgg aagatcctac cttcaaagag aactaccggt 3780tccacgccat caacggctac atcatggaca ccctgcctgg cctggtgatg gcccaggacc 3840agagaatccg gtggtatctg ctgtccatgg gcagcaacga gaatatccac agcatccact 3900tcagcggcca cgtgttcacc gtgcggaaga aagaagagta caagatggcc ctgtacaacc 3960tgtaccccgg cgtgttcgag acagtggaga tgctgcccag caaggccggc atctggcggg 4020tggagtgtct gatcggcgag cacctgcacg ctggcatgag caccctgttt ctggtgtaca 4080gcaacaagtg ccagacccca ctgggcatgg cctctggcca catccgggac ttccagatca 4140ccgcctccgg ccagtacggc cagtgggccc ccaagctggc cagactgcac tacagcggca 4200gcatcaacgc ctggtccacc aaagagccct tcagctggat caaggtggac ctgctggccc 4260ctatgatcat ccacggcatt aagacccagg gcgccaggca gaagttcagc agcctgtaca 4320tcagccagtt catcatcatg tacagcctgg acggcaagaa gtggcagacc taccggggca 4380acagcaccgg caccctgatg gtgttcttcg gcaatgtgga cagcagcggc atcaagcaca 4440acatcttcaa cccccccatc attgcccggt acatccggct gcaccccacc cactacagca 4500ttagatccac actgagaatg gaactgatgg gctgcgacct gaactcctgc agcatgcctc 4560tgggcatgga aagcaaggcc atcagcgacg cccagatcac agccagcagc tacttcacca 4620acatgttcgc cacctggtcc ccctccaagg ccaggctgca cctgcagggc cggtccaacg 4680cctggcggcc tcaggtcaac aaccccaaag aatggctgca ggtggacttt cagaaaacca 4740tgaaggtgac cggcgtgacc acccagggcg tgaaaagcct gctgaccagc atgtacgtga 4800aagagtttct gatcagcagc tctcaggatg gccaccagtg gaccctgttc tttcagaacg 4860gcaaggtgaa agtgttccag ggcaaccagg actccttcac ccccgtggtg aactccctgg 4920acccccccct gctgacccgc tacctgagaa tccaccccca gtcttgggtg caccagatcg 4980ccctcaggat ggaagtcctg ggatgtgagg cccaggatct gtactgatga ggatccatgc 5040tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 5100caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag 5160gttttttaaa 5170535188DNAArtificial

SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-MVM-Flank- coFVIIIdeltaB-Flank-SV40pA-Flank 53gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat 600gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggct agtatgcaga 660tcgagctgtc cacctgcttt tttctgtgcc tgctgcggtt ctgcttcagc gccacccggc 720ggtactacct gggcgccgtg gagctgtcct gggactacat gcagagcgac ctgggcgagc 780tgcccgtgga cgcccggttc ccccccagag tgcccaagag cttccccttc aacaccagcg 840tggtgtacaa gaaaaccctg ttcgtggagt tcaccgacca cctgttcaat atcgccaagc 900ccaggccccc ctggatgggc ctgctgggcc ccaccatcca ggccgaggtg tacgacaccg 960tggtgatcac cctgaagaac atggccagcc accccgtgag cctgcacgcc gtgggcgtga 1020gctactggaa ggccagcgag ggcgccgagt acgacgacca gaccagccag cgggagaaag 1080aagatgacaa ggtgttccct ggcggcagcc acacctacgt gtggcaggtg ctgaaagaaa 1140acggccccat ggcctccgac cccctgtgcc tgacctacag ctacctgagc cacgtggacc 1200tggtgaagga cctgaacagc ggcctgatcg gcgctctgct cgtctgccgg gagggcagcc 1260tggccaaaga gaaaacccag accctgcaca agttcatcct gctgttcgcc gtgttcgacg 1320agggcaagag ctggcacagc gagacaaaga acagcctgat gcaggaccgg gacgccgcct 1380ctgccagagc ctggcccaag atgcacaccg tgaacggcta cgtgaacaga agcctgcccg 1440gcctgattgg ctgccaccgg aagagcgtgt actggcacgt gatcggcatg ggcaccacac 1500ccgaggtgca cagcatcttt ctggaagggc acacctttct ggtccggaac caccggcagg 1560ccagcctgga aatcagccct atcaccttcc tgaccgccca gacactgctg atggacctgg 1620gccagttcct gctgttttgc cacatcagct ctcaccagca cgacggcatg gaagcctacg 1680tgaaggtgga ctcttgcccc gaggaacccc agctgcggat gaagaacaac gaggaagccg 1740aggactacga cgacgacctg accgacagcg agatggacgt ggtgcggttc gacgacgaca 1800acagccccag cttcatccag atcagaagcg tggccaagaa gcaccccaag acctgggtgc 1860actatatcgc cgccgaggaa gaggactggg actacgcccc cctggtgctg gcccccgacg 1920acagaagcta caagagccag tacctgaaca atggccccca gcggatcggc cggaagtaca 1980agaaagtgcg gttcatggcc tacaccgacg agacattcaa gacccgggag gccatccagc 2040acgagagcgg catcctgggc cccctgctgt acggcgaagt gggcgacaca ctgctgatca 2100tcttcaagaa ccaggctagc cggccctaca acatctaccc ccacggcatc accgacgtgc 2160ggcccctgta cagcaggcgg ctgcccaagg gcgtgaagca cctgaaggac ttccccatcc 2220tgcccggcga gatcttcaag tacaagtgga ccgtgaccgt ggaggacggc cccaccaaga 2280gcgaccccag atgcctgacc cggtactaca gcagcttcgt gaacatggaa cgggacctgg 2340cctccgggct gatcggacct ctgctgatct gctacaaaga aagcgtggac cagcggggca 2400accagatcat gagcgacaag cggaacgtga tcctgttcag cgtgttcgat gagaaccggt 2460cctggtatct gaccgagaac atccagcggt ttctgcccaa ccctgccggc gtgcagctgg 2520aagatcccga gttccaggcc agcaacatca tgcactccat caatggctac gtgttcgact 2580ctctgcagct ctccgtgtgt ctgcacgagg tggcctactg gtacatcctg agcatcggcg 2640cccagaccga cttcctgagc gtgttcttca gcggctacac cttcaagcac aagatggtgt 2700acgaggacac cctgaccctg ttccctttca gcggcgagac agtgttcatg agcatggaaa 2760accccggcct gtggattctg ggctgccaca acagcgactt ccggaaccgg ggcatgaccg 2820ccctgctgaa ggtgtccagc tgcgacaaga acaccggcga ctactacgag gacagctacg 2880aggatatcag cgcctacctg ctgtccaaga acaacgccat cgaaccccgg agcttcagcc 2940agaacccccc cgtgctgacg cgtcaccagc gggagatcac ccggacaacc ctgcagtccg 3000accaggaaga gatcgattac gacgacacca tcagcgtgga gatgaagaaa gaggatttcg 3060atatctacga cgaggacgag aaccagagcc ccagaagctt ccagaagaaa acccggcact 3120acttcattgc cgccgtggag aggctgtggg actacggcat gagttctagc ccccacgtgc 3180tgcggaaccg ggcccagagc ggcagcgtgc cccagttcaa gaaagtggtg ttccaggaat 3240tcacagacgg cagcttcacc cagcctctgt atagaggcga gctgaacgag cacctggggc 3300tgctggggcc ctacatcagg gccgaagtgg aggacaacat catggtgacc ttccggaatc 3360aggccagcag accctactcc ttctacagca gcctgatcag ctacgaagag gaccagcggc 3420agggcgccga accccggaag aacttcgtga agcccaacga aaccaagacc tacttctgga 3480aagtgcagca ccacatggcc cccaccaagg acgagttcga ctgcaaggcc tgggcctact 3540tcagcgacgt ggatctggaa aaggacgtgc actctggact gattggccca ctcctggtct 3600gccacactaa caccctcaac cccgcccacg gccgccaggt gaccgtgcag gaattcgccc 3660tgttcttcac catcttcgac gagacaaagt cctggtactt caccgagaat atggaacgga 3720actgcagagc cccctgcaac atccagatgg aagatcctac cttcaaagag aactaccggt 3780tccacgccat caacggctac atcatggaca ccctgcctgg cctggtgatg gcccaggacc 3840agagaatccg gtggtatctg ctgtccatgg gcagcaacga gaatatccac agcatccact 3900tcagcggcca cgtgttcacc gtgcggaaga aagaagagta caagatggcc ctgtacaacc 3960tgtaccccgg cgtgttcgag acagtggaga tgctgcccag caaggccggc atctggcggg 4020tggagtgtct gatcggcgag cacctgcacg ctggcatgag caccctgttt ctggtgtaca 4080gcaacaagtg ccagacccca ctgggcatgg cctctggcca catccgggac ttccagatca 4140ccgcctccgg ccagtacggc cagtgggccc ccaagctggc cagactgcac tacagcggca 4200gcatcaacgc ctggtccacc aaagagccct tcagctggat caaggtggac ctgctggccc 4260ctatgatcat ccacggcatt aagacccagg gcgccaggca gaagttcagc agcctgtaca 4320tcagccagtt catcatcatg tacagcctgg acggcaagaa gtggcagacc taccggggca 4380acagcaccgg caccctgatg gtgttcttcg gcaatgtgga cagcagcggc atcaagcaca 4440acatcttcaa cccccccatc attgcccggt acatccggct gcaccccacc cactacagca 4500ttagatccac actgagaatg gaactgatgg gctgcgacct gaactcctgc agcatgcctc 4560tgggcatgga aagcaaggcc atcagcgacg cccagatcac agccagcagc tacttcacca 4620acatgttcgc cacctggtcc ccctccaagg ccaggctgca cctgcagggc cggtccaacg 4680cctggcggcc tcaggtcaac aaccccaaag aatggctgca ggtggacttt cagaaaacca 4740tgaaggtgac cggcgtgacc acccagggcg tgaaaagcct gctgaccagc atgtacgtga 4800aagagtttct gatcagcagc tctcaggatg gccaccagtg gaccctgttc tttcagaacg 4860gcaaggtgaa agtgttccag ggcaaccagg actccttcac ccccgtggtg aactccctgg 4920acccccccct gctgacccgc tacctgagaa tccaccccca gtcttgggtg caccagatcg 4980ccctcaggat ggaagtcctg ggatgtgagg cccaggatct gtactgatga ggatccatgc 5040tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 5100caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag 5160gttttttaaa ctcgagatcc acggccgc 5188545085DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-MVM-Flank- coFVIIIdeltaB-Flank-Synt-pA 54gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat 600gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggct agtatgcaga 660tcgagctgtc cacctgcttt tttctgtgcc tgctgcggtt ctgcttcagc gccacccggc 720ggtactacct gggcgccgtg gagctgtcct gggactacat gcagagcgac ctgggcgagc 780tgcccgtgga cgcccggttc ccccccagag tgcccaagag cttccccttc aacaccagcg 840tggtgtacaa gaaaaccctg ttcgtggagt tcaccgacca cctgttcaat atcgccaagc 900ccaggccccc ctggatgggc ctgctgggcc ccaccatcca ggccgaggtg tacgacaccg 960tggtgatcac cctgaagaac atggccagcc accccgtgag cctgcacgcc gtgggcgtga 1020gctactggaa ggccagcgag ggcgccgagt acgacgacca gaccagccag cgggagaaag 1080aagatgacaa ggtgttccct ggcggcagcc acacctacgt gtggcaggtg ctgaaagaaa 1140acggccccat ggcctccgac cccctgtgcc tgacctacag ctacctgagc cacgtggacc 1200tggtgaagga cctgaacagc ggcctgatcg gcgctctgct cgtctgccgg gagggcagcc 1260tggccaaaga gaaaacccag accctgcaca agttcatcct gctgttcgcc gtgttcgacg 1320agggcaagag ctggcacagc gagacaaaga acagcctgat gcaggaccgg gacgccgcct 1380ctgccagagc ctggcccaag atgcacaccg tgaacggcta cgtgaacaga agcctgcccg 1440gcctgattgg ctgccaccgg aagagcgtgt actggcacgt gatcggcatg ggcaccacac 1500ccgaggtgca cagcatcttt ctggaagggc acacctttct ggtccggaac caccggcagg 1560ccagcctgga aatcagccct atcaccttcc tgaccgccca gacactgctg atggacctgg 1620gccagttcct gctgttttgc cacatcagct ctcaccagca cgacggcatg gaagcctacg 1680tgaaggtgga ctcttgcccc gaggaacccc agctgcggat gaagaacaac gaggaagccg 1740aggactacga cgacgacctg accgacagcg agatggacgt ggtgcggttc gacgacgaca 1800acagccccag cttcatccag atcagaagcg tggccaagaa gcaccccaag acctgggtgc 1860actatatcgc cgccgaggaa gaggactggg actacgcccc cctggtgctg gcccccgacg 1920acagaagcta caagagccag tacctgaaca atggccccca gcggatcggc cggaagtaca 1980agaaagtgcg gttcatggcc tacaccgacg agacattcaa gacccgggag gccatccagc 2040acgagagcgg catcctgggc cccctgctgt acggcgaagt gggcgacaca ctgctgatca 2100tcttcaagaa ccaggctagc cggccctaca acatctaccc ccacggcatc accgacgtgc 2160ggcccctgta cagcaggcgg ctgcccaagg gcgtgaagca cctgaaggac ttccccatcc 2220tgcccggcga gatcttcaag tacaagtgga ccgtgaccgt ggaggacggc cccaccaaga 2280gcgaccccag atgcctgacc cggtactaca gcagcttcgt gaacatggaa cgggacctgg 2340cctccgggct gatcggacct ctgctgatct gctacaaaga aagcgtggac cagcggggca 2400accagatcat gagcgacaag cggaacgtga tcctgttcag cgtgttcgat gagaaccggt 2460cctggtatct gaccgagaac atccagcggt ttctgcccaa ccctgccggc gtgcagctgg 2520aagatcccga gttccaggcc agcaacatca tgcactccat caatggctac gtgttcgact 2580ctctgcagct ctccgtgtgt ctgcacgagg tggcctactg gtacatcctg agcatcggcg 2640cccagaccga cttcctgagc gtgttcttca gcggctacac cttcaagcac aagatggtgt 2700acgaggacac cctgaccctg ttccctttca gcggcgagac agtgttcatg agcatggaaa 2760accccggcct gtggattctg ggctgccaca acagcgactt ccggaaccgg ggcatgaccg 2820ccctgctgaa ggtgtccagc tgcgacaaga acaccggcga ctactacgag gacagctacg 2880aggatatcag cgcctacctg ctgtccaaga acaacgccat cgaaccccgg agcttcagcc 2940agaacccccc cgtgctgacg cgtcaccagc gggagatcac ccggacaacc ctgcagtccg 3000accaggaaga gatcgattac gacgacacca tcagcgtgga gatgaagaaa gaggatttcg 3060atatctacga cgaggacgag aaccagagcc ccagaagctt ccagaagaaa acccggcact 3120acttcattgc cgccgtggag aggctgtggg actacggcat gagttctagc ccccacgtgc 3180tgcggaaccg ggcccagagc ggcagcgtgc cccagttcaa gaaagtggtg ttccaggaat 3240tcacagacgg cagcttcacc cagcctctgt atagaggcga gctgaacgag cacctggggc 3300tgctggggcc ctacatcagg gccgaagtgg aggacaacat catggtgacc ttccggaatc 3360aggccagcag accctactcc ttctacagca gcctgatcag ctacgaagag gaccagcggc 3420agggcgccga accccggaag aacttcgtga agcccaacga aaccaagacc tacttctgga 3480aagtgcagca ccacatggcc cccaccaagg acgagttcga ctgcaaggcc tgggcctact 3540tcagcgacgt ggatctggaa aaggacgtgc actctggact gattggccca ctcctggtct 3600gccacactaa caccctcaac cccgcccacg gccgccaggt gaccgtgcag gaattcgccc 3660tgttcttcac catcttcgac gagacaaagt cctggtactt caccgagaat atggaacgga 3720actgcagagc cccctgcaac atccagatgg aagatcctac cttcaaagag aactaccggt 3780tccacgccat caacggctac atcatggaca ccctgcctgg cctggtgatg gcccaggacc 3840agagaatccg gtggtatctg ctgtccatgg gcagcaacga gaatatccac agcatccact 3900tcagcggcca cgtgttcacc gtgcggaaga aagaagagta caagatggcc ctgtacaacc 3960tgtaccccgg cgtgttcgag acagtggaga tgctgcccag caaggccggc atctggcggg 4020tggagtgtct gatcggcgag cacctgcacg ctggcatgag caccctgttt ctggtgtaca 4080gcaacaagtg ccagacccca ctgggcatgg cctctggcca catccgggac ttccagatca 4140ccgcctccgg ccagtacggc cagtgggccc ccaagctggc cagactgcac tacagcggca 4200gcatcaacgc ctggtccacc aaagagccct tcagctggat caaggtggac ctgctggccc 4260ctatgatcat ccacggcatt aagacccagg gcgccaggca gaagttcagc agcctgtaca 4320tcagccagtt catcatcatg tacagcctgg acggcaagaa gtggcagacc taccggggca 4380acagcaccgg caccctgatg gtgttcttcg gcaatgtgga cagcagcggc atcaagcaca 4440acatcttcaa cccccccatc attgcccggt acatccggct gcaccccacc cactacagca 4500ttagatccac actgagaatg gaactgatgg gctgcgacct gaactcctgc agcatgcctc 4560tgggcatgga aagcaaggcc atcagcgacg cccagatcac agccagcagc tacttcacca 4620acatgttcgc cacctggtcc ccctccaagg ccaggctgca cctgcagggc cggtccaacg 4680cctggcggcc tcaggtcaac aaccccaaag aatggctgca ggtggacttt cagaaaacca 4740tgaaggtgac cggcgtgacc acccagggcg tgaaaagcct gctgaccagc atgtacgtga 4800aagagtttct gatcagcagc tctcaggatg gccaccagtg gaccctgttc tttcagaacg 4860gcaaggtgaa agtgttccag ggcaaccagg actccttcac ccccgtggtg aactccctgg 4920acccccccct gctgacccgc tacctgagaa tccaccccca gtcttgggtg caccagatcg 4980ccctcaggat ggaagtcctg ggatgtgagg cccaggatct gtactgatga ggatccaata 5040aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtg 5085555103DNAArtificial SequenceFlank-3xSERP-Flank-TTRe-Flank-TTRm-MVM-Flank- coFVIIIdeltaB-Flank-Synt.pA-Flank 55gcggccgcgg tacgcggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 60ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 120caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 180tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccc 240actgggagga tgttgagtaa gatggaaaac tactgatgac ccttgcagag acagagtatt 300aggacatgtt tgaacagggg ccgggcgatc agcaggtagc tctagaggat ccccgtctgt 360ctgcacattt cgtagagcga gtgttccgat actctaatct ccctaggcaa ggttcatatt 420tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagaatc agcaggtttg 480gagtcagctt ggcagggatc agcagcctgg gttggaagga gggggtataa aagccccttc 540accaggagaa gccgtcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat 600gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggct agtatgcaga 660tcgagctgtc cacctgcttt tttctgtgcc tgctgcggtt ctgcttcagc gccacccggc 720ggtactacct gggcgccgtg gagctgtcct gggactacat gcagagcgac ctgggcgagc 780tgcccgtgga cgcccggttc ccccccagag tgcccaagag cttccccttc aacaccagcg 840tggtgtacaa gaaaaccctg ttcgtggagt tcaccgacca cctgttcaat atcgccaagc 900ccaggccccc ctggatgggc ctgctgggcc ccaccatcca ggccgaggtg tacgacaccg 960tggtgatcac cctgaagaac atggccagcc accccgtgag cctgcacgcc gtgggcgtga 1020gctactggaa ggccagcgag ggcgccgagt acgacgacca gaccagccag cgggagaaag 1080aagatgacaa ggtgttccct ggcggcagcc acacctacgt gtggcaggtg ctgaaagaaa 1140acggccccat ggcctccgac cccctgtgcc tgacctacag ctacctgagc cacgtggacc 1200tggtgaagga cctgaacagc ggcctgatcg gcgctctgct cgtctgccgg gagggcagcc 1260tggccaaaga gaaaacccag accctgcaca agttcatcct gctgttcgcc gtgttcgacg 1320agggcaagag ctggcacagc gagacaaaga acagcctgat gcaggaccgg gacgccgcct 1380ctgccagagc ctggcccaag atgcacaccg tgaacggcta cgtgaacaga agcctgcccg 1440gcctgattgg ctgccaccgg aagagcgtgt actggcacgt gatcggcatg ggcaccacac 1500ccgaggtgca cagcatcttt ctggaagggc acacctttct ggtccggaac caccggcagg 1560ccagcctgga aatcagccct atcaccttcc tgaccgccca gacactgctg atggacctgg 1620gccagttcct gctgttttgc cacatcagct ctcaccagca cgacggcatg gaagcctacg 1680tgaaggtgga ctcttgcccc gaggaacccc agctgcggat gaagaacaac gaggaagccg 1740aggactacga cgacgacctg accgacagcg agatggacgt ggtgcggttc gacgacgaca 1800acagccccag cttcatccag atcagaagcg tggccaagaa gcaccccaag acctgggtgc 1860actatatcgc cgccgaggaa gaggactggg actacgcccc cctggtgctg gcccccgacg 1920acagaagcta caagagccag tacctgaaca atggccccca gcggatcggc cggaagtaca 1980agaaagtgcg gttcatggcc tacaccgacg agacattcaa gacccgggag gccatccagc 2040acgagagcgg catcctgggc cccctgctgt acggcgaagt gggcgacaca ctgctgatca 2100tcttcaagaa ccaggctagc cggccctaca acatctaccc ccacggcatc accgacgtgc 2160ggcccctgta cagcaggcgg ctgcccaagg gcgtgaagca cctgaaggac ttccccatcc 2220tgcccggcga gatcttcaag tacaagtgga ccgtgaccgt ggaggacggc cccaccaaga 2280gcgaccccag atgcctgacc cggtactaca gcagcttcgt gaacatggaa cgggacctgg 2340cctccgggct gatcggacct ctgctgatct gctacaaaga aagcgtggac cagcggggca 2400accagatcat gagcgacaag cggaacgtga tcctgttcag cgtgttcgat gagaaccggt 2460cctggtatct gaccgagaac atccagcggt ttctgcccaa ccctgccggc gtgcagctgg 2520aagatcccga gttccaggcc agcaacatca tgcactccat caatggctac gtgttcgact 2580ctctgcagct ctccgtgtgt ctgcacgagg tggcctactg gtacatcctg agcatcggcg 2640cccagaccga cttcctgagc gtgttcttca gcggctacac cttcaagcac aagatggtgt 2700acgaggacac cctgaccctg ttccctttca gcggcgagac agtgttcatg agcatggaaa 2760accccggcct gtggattctg ggctgccaca acagcgactt ccggaaccgg ggcatgaccg 2820ccctgctgaa ggtgtccagc tgcgacaaga acaccggcga ctactacgag gacagctacg 2880aggatatcag cgcctacctg ctgtccaaga acaacgccat cgaaccccgg agcttcagcc 2940agaacccccc cgtgctgacg cgtcaccagc gggagatcac ccggacaacc ctgcagtccg 3000accaggaaga gatcgattac gacgacacca tcagcgtgga gatgaagaaa gaggatttcg 3060atatctacga cgaggacgag aaccagagcc ccagaagctt ccagaagaaa acccggcact 3120acttcattgc cgccgtggag aggctgtggg actacggcat gagttctagc ccccacgtgc 3180tgcggaaccg ggcccagagc ggcagcgtgc cccagttcaa gaaagtggtg ttccaggaat 3240tcacagacgg cagcttcacc cagcctctgt atagaggcga gctgaacgag cacctggggc 3300tgctggggcc ctacatcagg gccgaagtgg aggacaacat catggtgacc ttccggaatc 3360aggccagcag accctactcc ttctacagca gcctgatcag ctacgaagag gaccagcggc 3420agggcgccga accccggaag aacttcgtga agcccaacga aaccaagacc tacttctgga 3480aagtgcagca ccacatggcc cccaccaagg acgagttcga ctgcaaggcc tgggcctact 3540tcagcgacgt ggatctggaa aaggacgtgc actctggact gattggccca ctcctggtct 3600gccacactaa caccctcaac cccgcccacg gccgccaggt gaccgtgcag gaattcgccc 3660tgttcttcac catcttcgac gagacaaagt cctggtactt caccgagaat atggaacgga 3720actgcagagc cccctgcaac atccagatgg aagatcctac cttcaaagag aactaccggt 3780tccacgccat caacggctac atcatggaca ccctgcctgg cctggtgatg gcccaggacc 3840agagaatccg gtggtatctg ctgtccatgg gcagcaacga gaatatccac agcatccact 3900tcagcggcca cgtgttcacc gtgcggaaga aagaagagta caagatggcc ctgtacaacc 3960tgtaccccgg cgtgttcgag acagtggaga tgctgcccag caaggccggc atctggcggg 4020tggagtgtct gatcggcgag cacctgcacg ctggcatgag caccctgttt ctggtgtaca 4080gcaacaagtg ccagacccca ctgggcatgg cctctggcca catccgggac ttccagatca 4140ccgcctccgg ccagtacggc cagtgggccc ccaagctggc cagactgcac tacagcggca 4200gcatcaacgc ctggtccacc aaagagccct tcagctggat caaggtggac ctgctggccc 4260ctatgatcat ccacggcatt aagacccagg gcgccaggca gaagttcagc agcctgtaca 4320tcagccagtt catcatcatg tacagcctgg acggcaagaa gtggcagacc taccggggca 4380acagcaccgg caccctgatg gtgttcttcg

gcaatgtgga cagcagcggc atcaagcaca 4440acatcttcaa cccccccatc attgcccggt acatccggct gcaccccacc cactacagca 4500ttagatccac actgagaatg gaactgatgg gctgcgacct gaactcctgc agcatgcctc 4560tgggcatgga aagcaaggcc atcagcgacg cccagatcac agccagcagc tacttcacca 4620acatgttcgc cacctggtcc ccctccaagg ccaggctgca cctgcagggc cggtccaacg 4680cctggcggcc tcaggtcaac aaccccaaag aatggctgca ggtggacttt cagaaaacca 4740tgaaggtgac cggcgtgacc acccagggcg tgaaaagcct gctgaccagc atgtacgtga 4800aagagtttct gatcagcagc tctcaggatg gccaccagtg gaccctgttc tttcagaacg 4860gcaaggtgaa agtgttccag ggcaaccagg actccttcac ccccgtggtg aactccctgg 4920acccccccct gctgacccgc tacctgagaa tccaccccca gtcttgggtg caccagatcg 4980ccctcaggat ggaagtcctg ggatgtgagg cccaggatct gtactgatga ggatccaata 5040aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtgctcga gatccacggc 5100cgc 51035649DNAArtificial Sequencesynthetic polyadenylation signal (Synt-pA) 56aataaaagat ctttattttc attagatctg tgtgttggtt ttttgtgtg 4957339DNAArtificial Sequence3xSERP-Flank-TTRe-Flank 57gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 120aggagcaaac aggggctaag tccaccgggg gaggctgctg gtgaatatta accaaggtca 180ccccagttat cggaggagca aacaggggct aagtccacgg tacccactgg gaggatgttg 240agtaagatgg aaaactactg atgacccttg cagagacaga gtattaggac atgtttgaac 300aggggccggg cgatcagcag gtagctctag aggatcccc 33958541DNAArtificial Sequence3xSERP-Flank-TTRe-Flank-TTRm 58gggggaggct gctggtgaat attaaccaag gtcaccccag ttatcggagg agcaaacagg 60ggctaagtcc accgggggag gctgctggtg aatattaacc aaggtcaccc cagttatcgg 120aggagcaaac aggggctaag tccaccgggg gaggctgctg gtgaatatta accaaggtca 180ccccagttat cggaggagca aacaggggct aagtccacgg tacccactgg gaggatgttg 240agtaagatgg aaaactactg atgacccttg cagagacaga gtattaggac atgtttgaac 300aggggccggg cgatcagcag gtagctctag aggatccccg tctgtctgca catttcgtag 360agcgagtgtt ccgatactct aatctcccta ggcaaggttc atatttgtgt aggttactta 420ttctcctttt gttgactaag tcaataatca gaatcagcag gtttggagtc agcttggcag 480ggatcagcag cctgggttgg aaggaggggg tataaaagcc ccttcaccag gagaagccgt 540c 5415914PRTArtificial Sequencelinker 59Ser Phe Ser Gln Asn Pro Pro Val Leu Thr Arg His Gln Arg1 5 106020DNAArtificial Sequenceprimer 60gccttctagt tgccagccat 206119DNAArtificial Sequenceprimer 61ggcaccttcc agggtcaag 196220DNAArtificial Sequenceprimer 62aacggctacg tgaacagaag 206320DNAArtificial Sequenceprimer 63gatagggctg atttccaggc 2064418DNAHomo sapiens 64ggatcttgct accagtggaa cagccactaa ggattctgca gtgagagcag agggccagct 60aagtggtact ctcccagaga ctgtctgact cacgccaccc cctccacctt ggacacagga 120cgctgtggtt tctgagccag gtacaatgac tcctttcggt aagtgcagtg gaagctgtac 180actgcccagg caaagcgtcc gggcagcgta ggcgggcgac tcagatccca gccagtggac 240ttagcccctg tttgctcctc cgataactgg ggtgaccttg gttaatattc accagcagcc 300tcccccgttg cccctctgga tccactgctt aaatacggac gaggacaggg ccctgtctcc 360tcagcttcag gcaccaccac tgacctggga cagtgaatga tccccctgat ctgcggcc 418656601DNAArtificial SequencepAAVsc-3xSerpEnh-TTRe-AAT-FIX-co-R338L-BGHpA 65agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020gcagagaggg agtggaattc acgcgtcggg ggaggctgct ggtgaatatt aaccaaggtc 1080accccagtta tcggaggagc aaacaggggc taagtccacc gggggaggct gctggtgaat 1140attaaccaag gtcaccccag ttatcggagg agcaaacagg ggctaagtcc accgggggag 1200gctgctggtg aatattaacc aaggtcaccc cagttatcgg aggagcaaac aggggctaag 1260tccacggcgc gcccactggg aggatgttga gtaagatgga aaactactga tgacccttgc 1320agagacagag tattaggaca tgtttgaaca ggggccgggc gatcagcagg tagggtaccg 1380gatcttgcta ccagtggaac agccactaag gattctgcag tgagagcaga gggccagcta 1440agtggtactc tcccagagac tgtctgactc acgccacccc ctccaccttg gacacaggac 1500gctgtggttt ctgagccagg tacaatgact cctttcggta agtgcagtgg aagctgtaca 1560ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact 1620tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct 1680cccccgttgc ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct 1740cagcttcagg caccaccact gacctgggac agtgaatgat ccccctgatc tgcggcctct 1800agaaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg 1860gagcacctgc ctgaaatcac tttttttcag gttgggctag cccaccatgc agcgcgtgaa 1920catgatcatg gccgagagcc ccggcctgat caccatctgc ctgctgggct acctgctgag 1980cgccgagtgc accgtgttcc tggaccacga gaacgccaac aagatcctga accgccccaa 2040gcgctacaac agcggcaagc tggaggagtt cgtgcagggc aacctggagc gcgagtgcat 2100ggaggagaag tgcagcttcg aggaggcccg cgaggtgttc gagaacaccg agcgcaccac 2160cgagttctgg aagcagtacg tggacggcga ccagtgcgag agcaacccct gcctgaacgg 2220cggcagctgc aaggacgaca tcaacagcta cgagtgctgg tgccccttcg gcttcgaggg 2280caagaactgc gagctggacg tgacctgcaa catcaagaac ggccgctgcg agcagttctg 2340caagaacagc gccgacaaca aggtggtgtg cagctgcacc gagggctacc gcctggccga 2400gaaccagaag agctgcgagc ccgccgtgcc cttcccctgc ggccgcgtga gcgtgagcca 2460gaccagcaag ctgacccgcg ccgaggccgt gttccccgac gtggactacg tgaacagcac 2520cgaggccgag accatcctgg acaacatcac ccagagcacc cagagcttca acgacttcac 2580ccgcgtggtg ggcggcgagg acgccaagcc cggccagttc ccctggcagg tggtgctgaa 2640cggcaaggtg gacgccttct gcggcggcag catcgtgaac gagaagtgga tcgtgaccgc 2700cgcccactgc gtggagaccg gcgtgaagat caccgtggtg gccggcgagc acaacatcga 2760ggagaccgag cacaccgagc agaagcgcaa cgtgatccgc atcatccccc accacaacta 2820caacgccgcc atcaacaagt acaaccacga catcgccctg ctggagctgg acgagcccct 2880ggtgctgaac agctacgtga cccccatctg catcgccgac aaggagtaca ccaacatctt 2940cctgaagttc ggcagcggct acgtgagcgg ctggggccgc gtgttccaca agggccgcag 3000cgccctggtg ctgcagtacc tgcgcgtgcc cctggtggac cgcgccacct gcctgctgag 3060caccaagttc accatctaca acaacatgtt ctgcgccggc ttccacgagg gcggccgcga 3120cagctgccag ggcgacagcg gcggccccca cgtgaccgag gtggagggca ccagcttcct 3180gaccggcatc atcagctggg gcgaggagtg cgccatgaag ggcaagtacg gcatctacac 3240caaggtgagc cgctacgtga actggatcaa ggagaagacc aagctgacct aatgaaagat 3300ggatttccaa ggttaattca ttggaattga aaattaacag cccccccccc cccccccctg 3360cagatctgag ccgaattcct gcagcccggg ggatcagcct cgactgtgcc ttctagttgc 3420cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 3480actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 3540attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 3600catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggaccg 3660gtggatctcg atagcaggca tgctggggag agatcgatct gaggaacccc tagtgatgga 3720gttggccact ccctctctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg 3780gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt 3840ggccaacccc cccccccccc ccccccggcg attctcttgt ttgctccaga ctctcaggca 3900atgacctgat agcctttgta gagacctctc aaaaatagct accctctccg gcatgaattt 3960atcagctaga acggttgaat atcatattga tggtgatttg actgtctccg gcctttctca 4020cccgtttgaa tctttaccta cacattactc aggcattgca tttaaaatat atgagggttc 4080taaaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat tacagggtca 4140taatgttttt ggtacaaccg atttagcttt atgctctgag gctttattgc ttaattttgc 4200taattctttg ccttgcctgt atgatttatt ggatgttgga atcgcctgat gcggtatttt 4260ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc 4320tctgatgccg catagttata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 4380taagccagcc ccgacacccg ccaacacagc cagccccgac acccgccaac acccgctgac 4440gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 4500gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 4560ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 4620ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 4680tcaaatatgt atccgctcat gagacaataa ccctgataaa tgctcaataa tattgaaaaa 4740ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 4800gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 4860tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 4920ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 4980tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 5040atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 5100gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 5160caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 5220ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 5280ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 5340ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 5400ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 5460gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 5520ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 5580taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 5640agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 5700atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 5760aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 5820caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 5880ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 5940cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 6000tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 6060gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 6120ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 6180gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 6240caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 6300ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 6360tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 6420ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 6480agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 6540aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 6600g 6601666494DNAArtificial SequencepAAVsc-3xSerpEnh-AAT-FIX-co-R338L-BGHpA 66agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020gcagagaggg agtggaattc acgcgcccgg gggaggctgc tggtgaatat taaccaaggt 1080caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 1140tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc caccggggga 1200ggctgctggt gaatattaac caaggtcacc ccagttatcg gaggagcaaa caggggctaa 1260gtccacggta ccggatcttg ctaccagtgg aacagccact aaggattctg cagtgagagc 1320agagggccag ctaagtggta ctctcccaga gactgtctga ctcacgccac cccctccacc 1380ttggacacag gacgctgtgg tttctgagcc aggtacaatg actcctttcg gtaagtgcag 1440tggaagctgt acactgccca ggcaaagcgt ccgggcagcg taggcgggcg actcagatcc 1500cagccagtgg acttagcccc tgtttgctcc tccgataact ggggtgacct tggttaatat 1560tcaccagcag cctcccccgt tgcccctctg gatccactgc ttaaatacgg acgaggacag 1620ggccctgtct cctcagcttc aggcaccacc actgacctgg gacagtgaat gatccccctg 1680atctgcggcc tctagaaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat 1740gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttgggc tagcccacca 1800tgcagcgcgt gaacatgatc atggccgaga gccccggcct gatcaccatc tgcctgctgg 1860gctacctgct gagcgccgag tgcaccgtgt tcctggacca cgagaacgcc aacaagatcc 1920tgaaccgccc caagcgctac aacagcggca agctggagga gttcgtgcag ggcaacctgg 1980agcgcgagtg catggaggag aagtgcagct tcgaggaggc ccgcgaggtg ttcgagaaca 2040ccgagcgcac caccgagttc tggaagcagt acgtggacgg cgaccagtgc gagagcaacc 2100cctgcctgaa cggcggcagc tgcaaggacg acatcaacag ctacgagtgc tggtgcccct 2160tcggcttcga gggcaagaac tgcgagctgg acgtgacctg caacatcaag aacggccgct 2220gcgagcagtt ctgcaagaac agcgccgaca acaaggtggt gtgcagctgc accgagggct 2280accgcctggc cgagaaccag aagagctgcg agcccgccgt gcccttcccc tgcggccgcg 2340tgagcgtgag ccagaccagc aagctgaccc gcgccgaggc cgtgttcccc gacgtggact 2400acgtgaacag caccgaggcc gagaccatcc tggacaacat cacccagagc acccagagct 2460tcaacgactt cacccgcgtg gtgggcggcg aggacgccaa gcccggccag ttcccctggc 2520aggtggtgct gaacggcaag gtggacgcct tctgcggcgg cagcatcgtg aacgagaagt 2580ggatcgtgac cgccgcccac tgcgtggaga ccggcgtgaa gatcaccgtg gtggccggcg 2640agcacaacat cgaggagacc gagcacaccg agcagaagcg caacgtgatc cgcatcatcc 2700cccaccacaa ctacaacgcc gccatcaaca agtacaacca cgacatcgcc ctgctggagc 2760tggacgagcc cctggtgctg aacagctacg tgacccccat ctgcatcgcc gacaaggagt 2820acaccaacat cttcctgaag ttcggcagcg gctacgtgag cggctggggc cgcgtgttcc 2880acaagggccg cagcgccctg gtgctgcagt acctgcgcgt gcccctggtg gaccgcgcca 2940cctgcctgct gagcaccaag ttcaccatct acaacaacat gttctgcgcc ggcttccacg 3000agggcggccg cgacagctgc cagggcgaca gcggcggccc ccacgtgacc gaggtggagg 3060gcaccagctt cctgaccggc atcatcagct ggggcgagga gtgcgccatg aagggcaagt 3120acggcatcta caccaaggtg agccgctacg tgaactggat caaggagaag accaagctga 3180cctaatgaaa gatggatttc caaggttaat tcattggaat tgaaaattaa cagccccccc 3240cccccccccc ctgcagatct gagccgaatt cctgcagccc gggggatcag cctcgactgt 3300gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 3360aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 3420taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 3480agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 3540cagctgggga ccggtggatc tcgatagcag gcatgctggg gagagatcga tctgaggaac 3600ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgccc 3660gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 3720gcagagaggg agtggccaac cccccccccc cccccccccg gcgattctct tgtttgctcc 3780agactctcag gcaatgacct gatagccttt gtagagacct ctcaaaaata gctaccctct 3840ccggcatgaa tttatcagct agaacggttg aatatcatat tgatggtgat ttgactgtct 3900ccggcctttc tcacccgttt gaatctttac ctacacatta ctcaggcatt gcatttaaaa 3960tatatgaggg ttctaaaaat ttttatcctt gcgttgaaat aaaggcttct cccgcaaaag 4020tattacaggg tcataatgtt tttggtacaa ccgatttagc tttatgctct gaggctttat 4080tgcttaattt tgctaattct ttgccttgcc tgtatgattt attggatgtt ggaatcgcct 4140gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 4200cagtacaatc tgctctgatg ccgcatagtt atatggtgca ctctcagtac aatctgctct 4260gatgccgcat agttaagcca gccccgacac ccgccaacac agccagcccc gacacccgcc 4320aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 4380tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 4440gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 4500ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 4560tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgctcaa 4620taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 4680tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 4740gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 4800atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 4860ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 4920cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 4980ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 5040aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 5100ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 5160gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 5220ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 5280gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct

5340ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 5400tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 5460cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 5520tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 5580atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 5640tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 5700tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 5760ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 5820cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 5880ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 5940gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 6000tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 6060gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 6120ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 6180tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 6240ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 6300tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 6360attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 6420tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 6480ccgattcatt aatg 6494676375DNAArtificial SequencepAAVsc-TTRe-AAT-FIX-co-R338L-BGHpA 67agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020gcagagaggg agtggaattc acgcgcccac tgggaggatg ttgagtaaga tggaaaacta 1080ctgatgaccc ttgcagagac agagtattag gacatgtttg aacaggggcc gggcgatcag 1140caggtagggt accggatctt gctaccagtg gaacagccac taaggattct gcagtgagag 1200cagagggcca gctaagtggt actctcccag agactgtctg actcacgcca ccccctccac 1260cttggacaca ggacgctgtg gtttctgagc caggtacaat gactcctttc ggtaagtgca 1320gtggaagctg tacactgccc aggcaaagcg tccgggcagc gtaggcgggc gactcagatc 1380ccagccagtg gacttagccc ctgtttgctc ctccgataac tggggtgacc ttggttaata 1440ttcaccagca gcctcccccg ttgcccctct ggatccactg cttaaatacg gacgaggaca 1500gggccctgtc tcctcagctt caggcaccac cactgacctg ggacagtgaa tgatccccct 1560gatctgcggc ctctagaaag aggtaagggt ttaagggatg gttggttggt ggggtattaa 1620tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttggg ctagcccacc 1680atgcagcgcg tgaacatgat catggccgag agccccggcc tgatcaccat ctgcctgctg 1740ggctacctgc tgagcgccga gtgcaccgtg ttcctggacc acgagaacgc caacaagatc 1800ctgaaccgcc ccaagcgcta caacagcggc aagctggagg agttcgtgca gggcaacctg 1860gagcgcgagt gcatggagga gaagtgcagc ttcgaggagg cccgcgaggt gttcgagaac 1920accgagcgca ccaccgagtt ctggaagcag tacgtggacg gcgaccagtg cgagagcaac 1980ccctgcctga acggcggcag ctgcaaggac gacatcaaca gctacgagtg ctggtgcccc 2040ttcggcttcg agggcaagaa ctgcgagctg gacgtgacct gcaacatcaa gaacggccgc 2100tgcgagcagt tctgcaagaa cagcgccgac aacaaggtgg tgtgcagctg caccgagggc 2160taccgcctgg ccgagaacca gaagagctgc gagcccgccg tgcccttccc ctgcggccgc 2220gtgagcgtga gccagaccag caagctgacc cgcgccgagg ccgtgttccc cgacgtggac 2280tacgtgaaca gcaccgaggc cgagaccatc ctggacaaca tcacccagag cacccagagc 2340ttcaacgact tcacccgcgt ggtgggcggc gaggacgcca agcccggcca gttcccctgg 2400caggtggtgc tgaacggcaa ggtggacgcc ttctgcggcg gcagcatcgt gaacgagaag 2460tggatcgtga ccgccgccca ctgcgtggag accggcgtga agatcaccgt ggtggccggc 2520gagcacaaca tcgaggagac cgagcacacc gagcagaagc gcaacgtgat ccgcatcatc 2580ccccaccaca actacaacgc cgccatcaac aagtacaacc acgacatcgc cctgctggag 2640ctggacgagc ccctggtgct gaacagctac gtgaccccca tctgcatcgc cgacaaggag 2700tacaccaaca tcttcctgaa gttcggcagc ggctacgtga gcggctgggg ccgcgtgttc 2760cacaagggcc gcagcgccct ggtgctgcag tacctgcgcg tgcccctggt ggaccgcgcc 2820acctgcctgc tgagcaccaa gttcaccatc tacaacaaca tgttctgcgc cggcttccac 2880gagggcggcc gcgacagctg ccagggcgac agcggcggcc cccacgtgac cgaggtggag 2940ggcaccagct tcctgaccgg catcatcagc tggggcgagg agtgcgccat gaagggcaag 3000tacggcatct acaccaaggt gagccgctac gtgaactgga tcaaggagaa gaccaagctg 3060acctaatgaa agatggattt ccaaggttaa ttcattggaa ttgaaaatta acagcccccc 3120cccccccccc cctgcagatc tgagccgaat tcctgcagcc cgggggatca gcctcgactg 3180tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 3240aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 3300gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 3360aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 3420ccagctgggg accggtggat ctcgatagca ggcatgctgg ggagagatcg atctgaggaa 3480cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccgcc 3540cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg 3600cgcagagagg gagtggccaa cccccccccc cccccccccc ggcgattctc ttgtttgctc 3660cagactctca ggcaatgacc tgatagcctt tgtagagacc tctcaaaaat agctaccctc 3720tccggcatga atttatcagc tagaacggtt gaatatcata ttgatggtga tttgactgtc 3780tccggccttt ctcacccgtt tgaatcttta cctacacatt actcaggcat tgcatttaaa 3840atatatgagg gttctaaaaa tttttatcct tgcgttgaaa taaaggcttc tcccgcaaaa 3900gtattacagg gtcataatgt ttttggtaca accgatttag ctttatgctc tgaggcttta 3960ttgcttaatt ttgctaattc tttgccttgc ctgtatgatt tattggatgt tggaatcgcc 4020tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 4080tcagtacaat ctgctctgat gccgcatagt tatatggtgc actctcagta caatctgctc 4140tgatgccgca tagttaagcc agccccgaca cccgccaaca cagccagccc cgacacccgc 4200caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 4260ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 4320cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 4380tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 4440ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgctca 4500ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 4560ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 4620tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 4680gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 4740gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 4800acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 4860tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 4920caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 4980gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 5040cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 5100tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 5160agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 5220tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 5280ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 5340acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 5400ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 5460gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 5520gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 5580ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 5640gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 5700ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 5760cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 5820cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 5880ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 5940tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 6000cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 6060ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 6120aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 6180ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 6240tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 6300gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 6360gccgattcat taatg 6375686601DNAArtificial SequencepAAVsc-TTRe-3xSerpEnh-AAT-FIX-co-R338L-BGHpA 68agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 60aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 120caaggccgat agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag 180tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 240ttataaaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 300cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 360agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 420gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 480cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 540ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 600cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 660tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 720cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 780aacaaaaatt taacgcgaac tttaacaaaa tattaacgtt tacaatttaa atatttgctt 840atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 900tgctagtttt acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc 960gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 1020gcagagaggg agtggaattc acgcgtcact gggaggatgt tgagtaagat ggaaaactac 1080tgatgaccct tgcagagaca gagtattagg acatgtttga acaggggccg ggcgatcagc 1140aggtagggcg cgcccggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 1200ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 1260caccccagtt atcggaggag caaacagggg ctaagtccac cgggggaggc tgctggtgaa 1320tattaaccaa ggtcacccca gttatcggag gagcaaacag gggctaagtc cacggtaccg 1380gatcttgcta ccagtggaac agccactaag gattctgcag tgagagcaga gggccagcta 1440agtggtactc tcccagagac tgtctgactc acgccacccc ctccaccttg gacacaggac 1500gctgtggttt ctgagccagg tacaatgact cctttcggta agtgcagtgg aagctgtaca 1560ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact 1620tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct 1680cccccgttgc ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct 1740cagcttcagg caccaccact gacctgggac agtgaatgat ccccctgatc tgcggcctct 1800agaaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg 1860gagcacctgc ctgaaatcac tttttttcag gttgggctag cccaccatgc agcgcgtgaa 1920catgatcatg gccgagagcc ccggcctgat caccatctgc ctgctgggct acctgctgag 1980cgccgagtgc accgtgttcc tggaccacga gaacgccaac aagatcctga accgccccaa 2040gcgctacaac agcggcaagc tggaggagtt cgtgcagggc aacctggagc gcgagtgcat 2100ggaggagaag tgcagcttcg aggaggcccg cgaggtgttc gagaacaccg agcgcaccac 2160cgagttctgg aagcagtacg tggacggcga ccagtgcgag agcaacccct gcctgaacgg 2220cggcagctgc aaggacgaca tcaacagcta cgagtgctgg tgccccttcg gcttcgaggg 2280caagaactgc gagctggacg tgacctgcaa catcaagaac ggccgctgcg agcagttctg 2340caagaacagc gccgacaaca aggtggtgtg cagctgcacc gagggctacc gcctggccga 2400gaaccagaag agctgcgagc ccgccgtgcc cttcccctgc ggccgcgtga gcgtgagcca 2460gaccagcaag ctgacccgcg ccgaggccgt gttccccgac gtggactacg tgaacagcac 2520cgaggccgag accatcctgg acaacatcac ccagagcacc cagagcttca acgacttcac 2580ccgcgtggtg ggcggcgagg acgccaagcc cggccagttc ccctggcagg tggtgctgaa 2640cggcaaggtg gacgccttct gcggcggcag catcgtgaac gagaagtgga tcgtgaccgc 2700cgcccactgc gtggagaccg gcgtgaagat caccgtggtg gccggcgagc acaacatcga 2760ggagaccgag cacaccgagc agaagcgcaa cgtgatccgc atcatccccc accacaacta 2820caacgccgcc atcaacaagt acaaccacga catcgccctg ctggagctgg acgagcccct 2880ggtgctgaac agctacgtga cccccatctg catcgccgac aaggagtaca ccaacatctt 2940cctgaagttc ggcagcggct acgtgagcgg ctggggccgc gtgttccaca agggccgcag 3000cgccctggtg ctgcagtacc tgcgcgtgcc cctggtggac cgcgccacct gcctgctgag 3060caccaagttc accatctaca acaacatgtt ctgcgccggc ttccacgagg gcggccgcga 3120cagctgccag ggcgacagcg gcggccccca cgtgaccgag gtggagggca ccagcttcct 3180gaccggcatc atcagctggg gcgaggagtg cgccatgaag ggcaagtacg gcatctacac 3240caaggtgagc cgctacgtga actggatcaa ggagaagacc aagctgacct aatgaaagat 3300ggatttccaa ggttaattca ttggaattga aaattaacag cccccccccc cccccccctg 3360cagatctgag ccgaattcct gcagcccggg ggatcagcct cgactgtgcc ttctagttgc 3420cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 3480actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 3540attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 3600catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggaccg 3660gtggatctcg atagcaggca tgctggggag agatcgatct gaggaacccc tagtgatgga 3720gttggccact ccctctctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg 3780gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt 3840ggccaacccc cccccccccc ccccccggcg attctcttgt ttgctccaga ctctcaggca 3900atgacctgat agcctttgta gagacctctc aaaaatagct accctctccg gcatgaattt 3960atcagctaga acggttgaat atcatattga tggtgatttg actgtctccg gcctttctca 4020cccgtttgaa tctttaccta cacattactc aggcattgca tttaaaatat atgagggttc 4080taaaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat tacagggtca 4140taatgttttt ggtacaaccg atttagcttt atgctctgag gctttattgc ttaattttgc 4200taattctttg ccttgcctgt atgatttatt ggatgttgga atcgcctgat gcggtatttt 4260ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc 4320tctgatgccg catagttata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 4380taagccagcc ccgacacccg ccaacacagc cagccccgac acccgccaac acccgctgac 4440gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 4500gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 4560ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 4620ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 4680tcaaatatgt atccgctcat gagacaataa ccctgataaa tgctcaataa tattgaaaaa 4740ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 4800gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 4860tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 4920ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 4980tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 5040atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 5100gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 5160caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 5220ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 5280ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 5340ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 5400ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 5460gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 5520ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 5580taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 5640agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 5700atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 5760aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 5820caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 5880ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 5940cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 6000tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 6060gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 6120ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 6180gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 6240caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 6300ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 6360tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 6420ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 6480agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 6540aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 6600g 660169317DNAArtificial SequenceTTRe-TTRm 69cactgggagg atgttgagta agatggaaaa ctactgatga cccttgcaga gacagagtat 60taggacatgt ttgaacaggg gccgggcgat cagcaggtag ctctagagga tccccgtctg 120tctgcacatt tcgtagagcg agtgttccga tactctaatc tccctaggca aggttcatat 180ttgtgtaggt tacttattct ccttttgttg actaagtcaa taatcagaat cagcaggttt 240ggagtcagct tggcagggat cagcagcctg ggttggaagg agggggtata aaagcccctt 300caccaggaga agccgtc 3177059DNAArtificial Sequenceflanking sequence 70aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggtac cggcgcgcc 597112DNAArtificial Sequenceflanking sequence 71acgcgtggta cc

127210DNAArtificial Sequenceflanking sequence 72ctagcccacc 107367DNAArtificial Sequenceflanking sequence 73aagatggatt tccaaggtta attcattgga attgaaaatt aacagccccc cccccccccc 60ccctgca 677439DNAArtificial Sequenceflanking sequence 74ccggtggatc tcgatagcag gcatgctggg gagagatcg 397566DNAArtificial Sequenceflanking sequence 75aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggtac cggcgcgccc 60gtacgc 667624DNAArtificial Sequenceflanking sequence 76tgtacaacgc gtgaattcgc tagc 247751DNAArtificial Sequenceflanking sequence 77aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcggtac c 517815DNAArtificial Sequenceflanking sequence 78ctctagagga tcccc 157947DNAArtificial Sequenceflanking sequence 79aattcacgcg tggatctgaa ttcaattcac gcgtggtacg gccgcgg 478014DNAArtificial Sequenceflanking sequence 80gcggccgcgg tacc 148115DNAArtificial Sequenceflanking sequence 81ggatctaggc tcgac 158218DNAArtificial Sequenceflanking sequence 82ctcgagatcc acggccgc 188322DNAArtificial Sequenceflanking sequence 83gcggccgcgg taccggcgcg cc 228415DNAArtificial Sequenceflanking sequence 84gcggccgcgg tacgc 158511DNAArtificial Sequenceflanking sequence 85gcggccgtac c 11

Claims

1. A nucleic acid expression cassette comprising a triple repeat of a liver-specific nucleic acid regulatory element comprising the nucleic acid fragment defined by SEQ ID NO:5 or a sequence having at least 95% identity to said sequence, wherein the liver-specific nucleic acid regulatory elements are operably linked; and a nucleic acid regulatory element comprising the nucleic acid fragment defined by SEQ ID NO: 12 or a sequence having at least 95% identity to said sequence; operably linked to a promoter and a transgene.

2. The nucleic acid expression cassette according to claim 1, wherein the promoter is a liver-specific promoter selected from the group consisting of the minimal TTR promotor (TTRm), the AAT promoter, the albumin (ALB) promotor or minimal promoter, the apolipoprotein A1 (APOA1) promoter or minimal promoter, the complement factor B (CFB) promoter, the ketohexokinase (KHK) promoter, the hemopexin (HPX) promoter or minimal promoter, the nicotinamide N-methyltransferase (NNMT) promoter or minimal promoter, the (liver) carboxylesterase 1 (CES1) promoter or minimal promoter, the protein C (PROC) promoter or minimal promoter, the apolipoprotein C3 (APOC3) promoter or minimal promoter, the mannan-binding lectin serine protease 2 (MASP2) promoter or minimal promoter, the hepcidin antimicrobial peptide (HAMP) promoter or minimal promoter, and the serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) promoter or minimal promoter.

3. The nucleic acid expression cassette according to claim 1, wherein said promoter is the minimal TTR promotor (TTRm) as defined by SEQ ID NO:6.

4. The nucleic acid expression cassette according to claim 1, wherein said promoter is the AAT promoter as defined by SEQ ID NO:64.

5. The nucleic acid expression cassette according to claim 1, wherein the liver-specific regulatory elements of the triple repeat consist of the the nucleic acid fragment defined by SEQ ID NO:5.

6. The nucleic acid expression cassette according to 1, wherein said transgene is codon-optimized.

7. The nucleic acid expression cassette according to claim 1, wherein said transgene encodes coagulation factor IX (FIX) or wherein said transgene encodes coagulation factor FIX containing a hyper-activating mutation.

8. The nucleic acid expression cassette according to claim 1, wherein said transgene encodes coagulation factor VIII (FVIII) or wherein said transgene encodes coagulation factor VIII having a deletion of the B domain.

9. The nucleic acid expression cassette according to claim 1, wherein the promoter is the transthyretin (TTR) promoter, thereby comprising the combination of the TTRe and TTRm nucleic acids as defined by SEQ ID NO:69.

10. The nucleic acid expression cassette according to claim 1, further comprising a minute virus of mouse (MVM) intron.

11. The nucleic acid expression cassette according to claim 1, further comprising a transcriptional termination signal derived from the bovine growth hormone polyadenylation signal (BGHpA) or derived from the Simian virus 40 polyadenylation signal (SV40pA), or the synthetic polyadenylation signal as defined by SEQ ID NO:56.

12. A vector comprising the nucleic acid expression cassette according to claim 1.

13. A method of treating hemophilia A or hemophilia B comprising transducing or transfecting the vector according to claim 12 into a subject, wherein the vector comprises the FVIII transgene for use in treating hemophilia A or the vector comprises the FIX transgene for use in treating hemophilia B.

14. The method according to claim 13, wherein after transduction or transfection of the vector according to claim 12 into a subject, levels of factor IX or FVIII in plasma are equal to or higher than the therapeutic threshold concentration of 10 mU/ml plasma in the subject are obtained.

15. The method according to claim 14, wherein the transduction of the viral vector into the subject is done at a dose lower than 2.times.10.sup.11 vg/kg.

16. The method according to claim 15, wherein the transduction of the viral vector into the subject is done at a dose lower than or equal to 6.times.10.sup.11 vg/kg, and wherein levels of factor IX or FVIII in plasma equal to or higher than the therapeutic concentration of 100 mU/ml are obtained in said subject; or wherein the transduction of the viral vector into the subject is done at a dose lower than or equal to 6.times.10.sup.11 vg/kg, and wherein levels of factor IX or FVIII in plasma equal to or higher than the therapeutic concentration of 50 mU/ml are obtained in said subject; or wherein the transduction of the viral vector into the subject is done at a dose lower than or equal to 2.times.10.sup.12 vg/kg, and wherein levels of factor IX or FVIII in plasma equal to or higher than the therapeutic concentration of 200 mU/ml are obtained in said subject; or wherein the transduction of the viral vector into the subject is done at a dose lower than or equal to 2.times.10.sup.12 vg/kg, and wherein levels of factor IX or FVIII in plasma equal to or higher than the therapeutic concentration of 150 mU/ml are obtained in said subject.

17. A pharmaceutical composition comprising a vector according to claim 12 and a pharmaceutically acceptable carrier, and optionally further comprising an active ingredient for treating hemophilia A when the transgene in said vector is FVIII, or an active ingredient for treating hemophilia B when the transgene in said vector is FIX.

18. The nucleic acid expression cassette according to claim 7, wherein the hyper-activating mutation corresponds to an R338L amino acid substitution.

19. The nucleic acid expression cassette according to claim 8, wherein the B domain of the FVIII is replaced by a linker defined by SEQ ID NO:59.

20. The vector of claim 12, wherein the vector is a viral vector.