POST-NATAL TRANSPLANTATION OF FACTOR VIII-EXPRESSING CELLS FOR TREATMENT OF HEMOPHILIA

Abstract

Disclosed herein are method of treating hemophilia A in a subject comprising injecting the subject with mesenchymal stromal/stem cells (MSC) modified to express high levels of Factor VIII protein. The MSC are isolated prenatally, at birth, or after the subject's birth. The modified MSC may also express high levels von Willebrand factor protein.

Description

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 62/549,280, filed Aug. 23, 2017, the contents of this application is herein incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

[0003] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named SEQ_WFIRM17-908.txt, created on Aug. 23, 2018, and having a size of 176 KB and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND

[0004] Factor VIII is an essential blood clotting factor. The protein circulates in the bloodstream in an inactive form, bound to another molecule called von Willebrand factor, until an injury that damages blood vessels occurs. In response to injury, coagulation factor VIII is activated and separates from von Willebrand factor. The active protein interacts with another coagulation factor called Factor IX. This interaction sets off a chain of additional chemical reactions that form a blood clot.

[0005] Hemophilia A (HA) is the most common inheritable coagulation deficiency, affecting 1 in 5000 boys, approximately 60% of whom present with the severe form of the disease. Mutations in the Factor VIII gene that result in decreased or defective Factor VIII protein give rise to HA, a recessive X-linked disorder. Individuals with severe HA experience recurrent hematomas of subcutaneous connective tissue/muscle, internal bleeding, and frequent hemarthrosis, leading to chronic debilitating arthropathies. Current treatment is frequent infusions of Factor VIII (plasma-derived or recombinant) to maintain hemostasis, which greatly improves quality of life for many HA patients. While current therapeutic products for HA offer reliable prophylactic and therapeutic efficacy, they are very expensive and do not cure the underlying disease, thus requiring administration for the entire life of the patient. In addition, more than 30% of patients with severe HA develop inhibitory antibodies to the infused Factor VIII therapeutic, placing them in danger of treatment failure. This is a significant and serious complication/challenge in the clinical management/treatment of HA. While protein-based immune tolerance induction (ITI) therapy has been used with some success in this patient group, its cost extends into the millions of dollars per patient, it is only effective in about 60% of patients, and its mechanism of action is largely unknown. These shortcomings with existing therapy for patients who develop inhibitors highlight the need for innovative approaches to surmount this immunological hurdle.

BRIEF SUMMARY

[0006] In one aspect, provided are methods of treating a subject diagnosed with hemophilia A, the method involving the steps of (a) modifying mesenchymal stem/stromal cells (MSC) to express high levels of Factor VIII protein thereby generating modified MSC, the MSC comprising bone-marrow MSC isolated from the subject; (b) generating an expanded modified MSC population by in vitro culturing the modified MSC; and (c) injecting MSC from the expanded modified MSC population into the subject.

[0007] In another aspect, provided are methods of treating a subject prenatally diagnosed as having hemophilia A, the method involving the steps of (a) modifying mesenchymal stem/stromal cells (MSC) to express high levels of Factor VIII protein thereby generating modified MSC, the MSC comprising MSC isolated from at least one of amniotic fluid, placental tissue, or umbilical cord tissue obtained at the time of the subject's birth or prenatally from the subject's mother; (b) generating an expanded modified MSC population by in vitro culturing the modified MSC; and (c) injecting MSC from the expanded modified MSC population into the subject.

[0008] The above described and many other features and attendant advantages of embodiments of the present disclosure will become apparent and further understood by reference to the following detailed description when considered in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] These figures are intended to be illustrative, not limiting. Although the aspects of the disclosure are generally described in the context of these figures, it should be understood that it is not intended to limit the scope of the disclosure to these particular aspects.

[0010] FIG. 1 shows the complete absence of any FVIII antigen/cross-reactive material (CRM) in the hemophilia A sheep according to some aspects of the disclosure.

[0011] FIG. 2 shows assessment of phenotypic markers in MSC isolated from sheep according to some aspects of the disclosure.

[0012] FIG. 3 shows a schematic diagram of an ovine Factor VIII transgene expression vector according to some aspects of the disclosure.

[0013] FIG. 4 shows a schematic diagram of an ovine vWF transgene expression vector according to some aspects of the disclosure.

[0014] FIG. 5 shows endogenous expression of Factor VIII in MSC isolated from amniotic fluid according to some aspects of the disclosure.

[0015] FIG. 6 shows endogenous expression of vWF in MSC isolated from amniotic fluid according to some aspects of the disclosure.

[0016] FIG. 7 shows exogenous expression of Factor VIII in MSC isolated from amniotic fluid and transduced with a lentivector encoding Factor VIII according to some aspects of the disclosure.

[0017] FIG. 8A shows assessment of phenotypic markers in PLCs according to some aspects of the disclosure.

[0018] FIG. 8B shows flow cytometric analysis of PLC constitutively expressed levels of FVIII protein according to some aspects of the disclosure.

[0019] FIG. 8C shows assessment of normalized levels of PLC constitutively expressed levels of FVIII protein according to some aspects of the disclosure.

[0020] FIG. 9A shows assessment of normalized levels of secretion of FVIII protein by PLC engineered/transduced to express high levels of FVIII according to some aspects of the disclosure.

[0021] FIG. 9B shows assessment of secretion of FVIII protein by transduced as compared to non-transduced PLCs according to some aspects of the disclosure.

[0022] FIGS. 10A-10C show assessment of phenotypic markers in transduced as compared to non-transduced PLCs according to some aspects of the disclosure.

[0023] FIG. 11A shows assessment of expression of Toll-like receptors (TLRs) in mcoET3-transduced PLCs as compared to non-transduced PLCs according to some aspects of the disclosure.

[0024] FIG. 11B shows assessment of expression of stress molecules in mcoET3-transduced PLCs as compared to non-transduced PLCs according to some aspects of the disclosure.

[0025] FIG. 12 shows assessment of normalized levels of PLC secretion of FVIII protein by non-transduced-PLC, lcoHSQ-PLC, lcoET3-PLC, ET3-PLC, and mcoET3-PLC according to some aspects of the disclosure.

DETAILED DESCRIPTION

[0026] Provided in this disclosure are methods of treatment for subjects having hemophilia A. The methods are post-natal therapies comprising administering to a subject with hemophilia A autologous mesenchymal stem/stromal cells (MSC) that have been modified to express Factor VIII. Provided methods are effective as first-line therapies for subjects that have been diagnosed prenatally or at an early age and who have not received Factor VIII therapy. Provided methods are also effective as second-line therapies for the treatment of subjects that have been receiving Factor VIII therapy and, in some instances, have developed an immune response to standard infusion therapy of exogenous Factor VIII. The MSC used in the methods are isolated from biological samples obtained prenatally or after the subject's birth. The MSC are modified to express high levels of Factor VIII protein. In some instances, the MSC are modified to express high levels of Factor VIII and high levels of another protein, such as von Willebrand factor. The MSC may be modified by the introduction of a transgene (for example, using a viral vector) or via genome-editing (for example, using the CRISPR/Cas9 system). Administering the modified MSC to the subject results in engraftment of the modified cells. The engrafted cells produce Factor VIII on a continuing basis in the subject and provide long-lasting (ideally lifelong) therapeutic benefit to the subject by promoting blood coagulation.

[0027] In one aspect, provided is a method of treating a subject diagnosed clinically or genetically with hemophilia A comprising: (a) modifying mesenchymal stem/stromal cells (MSC) to express high levels of Factor VIII protein thereby generating modified MSC, the MSC comprising bone marrow MSC isolated from the subject; (b) generating an expanded modified MSC population by in vitro culturing the modified MSC; and (c) injecting MSC from the expanded modified MSC population into the subject. The bone marrow MSC express at least one of Stro-1 or CD146. In some instances, the bone marrow MSC may be isolated based on expression of at least one of Stro-1 or CD146.

[0028] In another aspect, provided is a method of treating a subject prenatally diagnosed as having hemophilia A comprising: (a) modifying mesenchymal stem/stromal cells (MSC) to express high levels of Factor VIII protein thereby generating modified MSC, the MSC comprising MSC isolated from at least one of amniotic fluid, placental tissue, or umbilical cord tissue obtained at the time of the subject's birth or prenatally; (b) generating an expanded modified MSC population by in vitro culturing the modified MSC; and (c) injecting MSC from the expanded modified MSC population into the subject. In some instances, the MSC are amniotic fluid MSC. In some instances, the MSC are placental MSC (PLC). In some instances, the MSC are umbilical cord tissue MSC.

[0029] As used herein the terms treatment, treat, or treating refer to a method of reducing one or more symptoms of a disease or condition. In some instances, treatment results in a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of one or more symptoms of the disease or condition. In some instances, treatment results in at least a 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction in the severity of one or more symptoms of the disease or condition. In some instances, treatment results in a 100% reduction in the severity of one or more symptoms of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 5% reduction in one or more symptoms or signs. As used herein, control refers to the untreated condition. In some instances, the reduction can be a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. In some instances, the reduction can be at least a 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction as compared to native or control levels. In some instances, the reduction can be a 100% reduction. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. As used herein, references to decreasing, reducing, or inhibiting include a change of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include, but do not necessarily include, complete elimination.

[0030] The subject on which the method is performed has been diagnosed with hemophilia A. The subject is mammalian, including humans; non-human primates, such as apes and monkeys; cattle; horses; sheep; rats; dogs; cats; mice; pigs; and goats. In some embodiments, the subject is a human, a dog, a horse, a sheep, a cow, or a cat. The subject may be male or female. The subject may be a juvenile subject or an adult subject. As a recessive X-linked disorder, a male subject will carry an X chromosome that has a mutation in the Factor VIII gene. A female subject that has hemophilia A will either have a mutated Factor VIII allele on both X chromosomes or will have a mutant Factor VIII allele on one X chromosome and have an inactive Factor VIII allele on the other X chromosome. In some instances, the subject may be a female carrier of hemophilia A that has a mutant Factor VIII allele on one X chromosome and a normal Factor VIII gene on the other X chromosome. Subjects may be diagnosed via prenatal genetic testing, particularly in instances where there is a family history of hemophilia. The DNA from biological samples obtained from amniocentesis, chorionic villi sampling, or cell-free fetal DNA present in the maternal peripheral blood may be analyzed for abnormalities on the X chromosome or mutations in the Factor VIII gene. Alternatively, subjects may be diagnosed after birth by assessing the ability of the subject's blood to clot properly. For example, screening tests include activated partial thromboplastin time (APTT) test, prothrombin time (PT) test, and fibrinogen test. Diagnosis of hemophilia A (type and severity) can also be performed with antigen-based tests that assess the amount of Factor VIII protein in the subject's blood. In some instances, the subject may have received therapy with infused Factor VIII protein. Where the subject has received such therapy, in some instances the subject may have developed an immune response to Factor VIII protein (developed inhibitory antibodies that impair the effectiveness of the therapy). In some embodiments, the subject has received prior treatment with exogenous Factor VIII and has developed an inhibitory immune response that diminishes the effectiveness of the exogenous Factor VIII treatment.

[0031] MSC, referred to in the field as mesenchymal stem cells, mesenchymal stromal cells, and, when isolated from bone marrow, also marrow stromal progenitors (MSP), are multipotent stromal cells that can differentiate into a variety of cell types, including: osteoblasts, chondrocytes, myocytes, and adipocytes. MSC do not have the capacity to reconstitute an entire organ. The term encompasses multipotent cells derived from other non-marrow tissues, such as placenta, umbilical cord blood, adipose tissue, or the dental pulp of deciduous baby teeth. MSC are heterogeneous and different subsets of MSC may have different capabilities. Different methods of isolation will result in different populations of MSC. Such different populations may express different protein markers. MSC subpopulations with different marker expression profiles have been found to have different capabilities. See, for example, Thierry, D., et al., Stro-1 Positive and Stro-1 Negative Human Mesenchymal Stem Cells Express Different Levels of Immunosuppression, Blood 104(11): 4964 (2004). The extent to which a MSC population isolated using one method and having a particular marker profile will share properties with a MSC population isolated using a different method and having a different marker profile has not been determined.

[0032] In some instances, the MSC used in the method are bone marrow-derived MSC--that is, the MSC are isolated from bone marrow. Specifically, the bone marrow-derived MSC are isolated from bone marrow obtained from the subject (autologous MSC). In some instances, the bone marrow-derived MSC used in the method express Stro-1, CD146, or both Stro-1 and CD146. Flow cytometry methods may be used to isolate MSC expressing these markers such as described, for example, in Sanada C., et al., Mesenchymal stem cells contribute to endogenous FVIII:c production. J Cell Physiol. 2013; 228(5):1010-1016 and Chamberlain J. L., et al., Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep. Hepatology. 2007; 46(6):1935-1945. Isolating MSC based on Stro-1 and/or CD146 results in a distinct cell population from that isolated using the traditional approach in which bulk unpurified bone marrow or Ficoll-purified bone marrow mononuclear cells are plated directly into plastic cell culture plates or flasks to which the adherent MSC population binds.

[0033] In some instances, the MSC used in the method are MSC isolated from a birth tissue or birth fluid. Specifically, the MSC may be isolated from amniotic fluid, placental tissue, chorionic villi, or umbilical cord tissue. In some instances, the MSC used in the method express c-kit. Methods of isolating such cells are described in U.S. Pat. Nos. 7,968,336 and 8,021,876, which are incorporated herein by reference in their entirety. In some instances, the MSC express at least one of c-kit, CD34, CD90, or CD133. In some instances, the MSC express c-kit and at least one of CD34, CD90, or CD133. In some instances, the MSC are isolated based on expression of c-kit.

[0034] For juvenile patients for whom prenatal biological samples are available, the MSC may be isolated from such samples (such as amniotic fluid, placental, cord tissue). Such samples may be available where the subject is diagnosed with hemophilia prior to birth. In some instances, appropriate biological samples may be obtained at the time of the subject's birth (such as amniotic fluid, placental, cord tissue). For adult patients, or juvenile patients for which prenatal biological samples are not available, the MSC used in the method may be bone marrow derived mesenchymal stem/stromal cells (MSC), also referred to as bone marrow stromal cells.

[0035] The MSC used in the method are modified to express high levels of Factor VIII. In some instances, the MSC may be modified to also express high levels of von Willebrand factor (vWF).

[0036] In some instances, an exogenous gene sequence encoding one or both of these proteins may be introduced into the MSC via one or more vectors. In some instances, the MSC may be modified to express high levels of Factor VIII protein via introduction into the MSC of a vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter. In some instances, the MSC may be modified to express high levels of vWF protein via introduction into the MSC of a vector comprising a vWF gene sequence operatively linked to a constitutively active promoter. In some instances, the MSC may be modified to express high levels of Factor VIII protein and vWF protein via introduction into the MSC of a vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter and a vector encoding a vWF gene sequence operatively linked to a constitutively active promoter. In some instances, the Factor VIII gene sequence and the vWF gene sequence may be operatively linked to the same constitutively active promoter. Alternatively, the Factor VIII gene sequence and the vWF gene sequence may be operatively linked to different constitutively active promoters.

[0037] Exemplary vectors include, for example, plasmids and viral vectors (including but not limited to adenoviral, adeno-associated viral (AAV), or retroviruses such as lentiviruses. In preferred embodiments, the vector is a viral vector. In some instances, the vector may be a vector that integrates into the genome of transduced cells. For example, the vector may be a lentivirus vector. In preferred embodiments, the vector is a lentivirus vector. In some instances, the lentivirus vector contains a 3'-modified long terminal repeat (LTR), resulting in a self-inactivating (SIN) lentivector. A lentivirus vector may integrate into the genome of dividing or non-dividing cells. The lentivirus genome in the form of RNA is reverse-transcribed to DNA when the virus enters the cell, and is then inserted into the genome by the viral integrase enzyme. The lentivirus vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides. In another example, the vector may be an adeno-associated virus (AAV) vector, which, in contrast to wild-type AAV, only rarely integrates into the genome of the cells it transduces. In one example, the vector may be an adenoviral vector. An adenoviral vector does not integrate into the genome. In another instance, the vector may be a murine retrovirus vector. In another example, the vector may be a foamy virus vector, which may have a larger capacity for inserts than lentiviral vectors. In another example, the vector may be Sendai virus vector.

[0038] The exogenous gene sequences are operatively linked to one or more promoter sequences within the vector. The term "promoter sequence" or "promoter element" refers to a nucleotide sequence that assists with controlling expression of a coding sequence. Generally, promoter elements are located 5' of the translation start site of a gene. However, in certain embodiments, a promoter element may be located within an intron sequence, or 3' of the coding sequence. In some embodiments, a promoter useful for a gene therapy vector is derived from the native gene of the target protein (e.g., a Factor VIII promoter). In some embodiments, the promoter is a constitutive promoter, which drives substantially constant expression of protein from the exogenous gene sequence. Non-limiting examples of well-characterized promoter elements include the cytomegalovirus immediate-early promoter (CMV), the .beta.-actin promoter, the methyl CpG binding protein 2 (MeCP2) promoter, the simian virus 40 early (SV40) promoter, human Ubiquitin C promoter (UBC), human elongation factor 1.alpha. promoter (EF1.alpha.), the phosphoglycerate kinase 1 promoter (PGK), or the CMV immediate early enhancer/chicken beta actin (CAG) promoter. The vector will generally also contain one or more of a promoter regulatory region (e.g., one conferring constitutive expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

[0039] In some instances, the Factor VIII transgene is operably linked to a promoter. A number of promoters can be used in the practice of the invention. The promoters can be selected based on desired outcome. The nucleic acids can be combined with constitutive, inducible, tissue-preferred, or other promoters for expression in the organism of interest. See, for example, promoters set forth as SEQ ID NOs: 1-6 as described in Brown et al. (2018) Target-Cell-Directed Bioengineering Approaches for Gene Therapy of Hemophilia A. Mol. Ther. Methods Clin. Dev., 2018. 9:57-69, which is herein incorporated by reference in its entirety for all purposes.

[0040] Where the MSC are modified to express high levels of Factor VIII via transduction with an exogenous Factor VIII gene sequence, the exogenous Factor VIII gene sequence may be human Factor VIII gene sequence, porcine Factor VIII gene sequence, or a hybrid transgene comprising portions of human Factor VIII gene sequence and portions of porcine Factor VIII gene sequence. In some instances, the gene sequence comprises all or a portion of the human Factor cDNA as set forth in GenBank Accession No. 192448441 as updated Jul. 17, 2017, wherein said portion would encode a function portion of the human Factor VIII protein. In some instances, the gene sequence comprises a sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to all or a portion of the human Factor cDNA as set forth in GenBank Accession No. 192448441 as updated Jul. 17, 2017, wherein said portion would encode a functional portion of the human Factor VIII protein. In some instances, the gene sequence may encode all or a functional portion of the human Factor VIII protein as set forth in GenBank Accession No. 192448441 as updated Jul. 17, 2017, reflecting the protein transcribed from transcript variant 1 of the Factor VIII gene. This protein is approximately 300 kDa and contains a series of homology-defined domains designated A1-A2-B-ap-A3-C.sub.1-C.sub.2. In some instances, the exogenous Factor VIII gene sequence is modified relative to wild-type protein sequence to result in increased protein expression, increased protein stability, reduced immunogenicity, or a combination of one or more thereof.

[0041] In some instances, the sequence of one or more of the Factor VIII protein domains may be deleted. In one example, the B domain of Factor VIII is deleted. The B domain of Factor VIII has no known function and can be deleted without loss of coagulant activity. Deletion of the B-domain has been shown to increase factor VIII protein production in heterologous systems (Toole et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:5939-5942). In addition, wildtype porcine Factor VIII protein having the B-domain deleted may have 10-100-fold higher expression and secretion than the human Factor FVIII gene sequence, both in vitro and in vivo. (See, for example, Dooriss, K. L., et al., Comparison of factor VIII transgenes bioengineered for improved expression in gene therapy of hemophilia A. Hum Gene Ther. 20:465-478 (2009). A B-domain deleted form of human Factor VIII protein (Lind et al. (1995) Eur. J. Biochem. 232:19-27) has been approved for clinical use.

[0042] In some instances, the exogenous Factor VIII gene sequence may include protein modifications to reduce immunogenicity of the protein thereby reducing the risk of an immune response due to therapy. For example, alanine substitutions may be included as described in Healey, J. F., et al., The comparative immunogenicity of human and porcine factor VIII in haemophilia A mice. Thromb Haemost. 102:35-41 (2009) and Lubin, I. M., et al., Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis. J Biol Chem. 272:30191-30195 (1997), which are incorporated by reference herein in their entirety.

[0043] In some instances, one or more of the human Factor VIII protein domain sequences may be substituted with the sequence of the corresponding porcine Factor VIII protein domain sequences. For example, one or more porcine Factor VIII domains may be substituted for one or more human Factor VIII domains. For example, inclusion of the porcine Factor VIII domains A1 and ap-A3 may increase expression of the expressed Factor VIII protein. See, for example, Doering, C. B., et al., Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion. J Biol Chem. 279:6546-6552 (2004). In some embodiments, the exogenous Factor VIII gene sequence may comprise the human Factor VIII A2 and C2 domains and the porcine Factor VIII A1, A3, and C1 domains.

[0044] In some instances, the exogenous Factor VIII gene sequence may comprise a modified Factor VIII sequence comprising a B domain-deleted (BDD) Factor VIII transgene having the sequence of the human A2 and C2 domains and the porcine A1, A3, and C1 domains, and also include three alanine substitutions in the A2 domain to reduce immunogenicity, as described in Lubin, I. M., et al., Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis. J Biol Chem. 1997; 272(48):30191-5. This modified Factor VIII protein is referred to as the ET3 transgene in this disclosure, including in the Examples below. In some instances, the ET3 transgene is expressed at a comparable level to that of wild-type porcine Factor VIII protein while having 91% identity to the amino acid sequence of wild-type human Factor VIII protein. In one example, the exogenous Factor VIII gene sequence may comprise a human/porcine Factor VIII transgene as described in Doering, C. B., et al., Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A, Mol. Ther. 17(7):1145-1154 (2009), which is incorporated herein by reference in its entirety.

[0045] In some instances, the Factor VIII transgene sequence may comprise one of the modified Factor VIII sequences described in Brown et al. (2018) Target-Cell-Directed Bioengineering Approaches for Gene Therapy of Hemophilia A. Mol. Ther. Methods Clin. Dev., 2018. 9:57-69, which is incorporated herein by reference in its entirety for all purposes. Factor VIII polypeptides, including tissue-specific codon optimized variants, are described therein. Modified Factor VIII transgene sequences used in the methods described herein can be any one of SEQ ID NOs: 7-16 (as described in Brown et al.). For example, Factor VIII transgene sequences that can be used in the methods described herein include a B-domain deleted (BDD) human Factor VIII polypeptide (HSQ) as set forth in SEQ ID NO: 15, a BDD chimeric human/porcine Factor VIII polypeptide (ET3) as set forth in SEQ ID NO: 11, or an ancestral Factor VIII polypeptide (An53) as set forth in SEQ ID NO: 7.

[0046] In some instances, the exogenous Factor VIII gene sequence may be modified for expression in a particular organ or tissue type. For example, the gene sequence may be optimized for expression in myeloid tissue. In some embodiments, the Factor VIII transgene may comprise myeloid codon optimized ET3 (mcoET3) as set forth in SEQ ID NO: 12 or myeloid codon optimized HSQ (mcoHSQ) as set forth in SEQ ID NO: 16. Alternatively, the Factor VIII transgene may be optimized for expression in liver tissue. In some embodiments, the Factor VIII transgene may comprise liver codon optimized ET3 (lcoET3) as set forth in SEQ ID NO: 10; liver codon optimized An53 as set forth in SEQ ID NO: 8; or liver codon optimized (lcoHSQ) as set forth in SEQ ID NO: 14.

[0047] In some instances, the exogenous Factor VIII gene sequence may comprise one of the modified Factor VIII sequences described in U.S. Pat. No. 7,635,763, which is incorporated herein by reference in its entirety for all purposes. Regions of the porcine Factor VIII polypeptide that comprises the A1 and ap-A3 regions, and variants and fragments thereof, are described therein that impart high-level expression to both the porcine and human Factor VIII polypeptide. The exogenous Factor VIII gene sequence encoded by the viral vector of the provided methods may be the polynucleotides set forth in any one of SEQ ID NOs: 19, 21, 23, 25, or 27 (SEQ ID NOs: 15, 17, 19, 13, or 21 as described in U.S. Pat. No. 7,635,763). The modified Factor VIII protein expressed at high levels in the modified MSC may comprise the amino acid sequences set forth in any one of SEQ ID NOs: 18, 20, 22, 24, or 26 (SEQ ID NOs: 14, 16, 18, 12, or 20 as described in U.S. Pat. No. 7,635,763). Such sequences are summarized in Table 1 below. In some instances, these sequences may be used to construct an exogenous Factor VIII gene sequence encoding a modified factor VIII polypeptide that results in a high level of expression of the encoded modified Factor VIII protein.

TABLE-US-00001 TABLE 1 Exemplary Modified Factor VIII Proteins Modified Factor VIII Protein SEQ ID NO. Description HP44/OL aa: SEQ ID NO: 18 A1.sub.P-A2.sub.P-ap.sub.P-A3.sub.P-C1.sub.H-C2.sub.H nt: SEQ ID NO: 19 porcine A1, A2, ap-A3 domains, porcine- derived linker sequence S F A Q N S R P P S A S A P K P P V L R R H Q R (SEQ ID NO: 30), and human C1 and C2 domains HP46/SQ aa: SEQ ID NO: 20 A1.sub.P-A2.sub.H-ap.sub.H-A3.sub.H-C1.sub.H-C2.sub.H nt: SEQ ID NO: 21 porcine A1 domain, human A2, ap-A3, C1 and C2 domains, and human S F S Q N P P V L K R H Q R linker sequence HP47/OL aa: SEQ ID NO: 22 A1.sub.P-A2.sub.H-ap.sub.p-A3.sub.P-C1.sub.H-C2.sub.H nt: SEQ ID NO: 23 porcine A1, ap-A3 domains, porcine- derived linker sequence S F A Q N S R P P S A S A P K P P V L R R H Q R (SEQ ID NO: 31), and human A2, C1 and C2 domains B-domain deleted aa: SEQ ID NO: 24 Human Factor VIII protein sequence nt: SEQ ID NO: 25 minus B-domain HP63/OL aa: SEQ ID NO: 26 porcine A1 domain and a partially nt: SEQ ID NO: 27 humanized ap-A3 domain that comprises porcine amino acids from about 1690 to about 1804 and from about 1819 to about 2019

[0048] As discussed above, in some instances, the MSC are also modified to express high levels of vWF protein via introduction into the MSC of a vector. In some embodiments coding sequences for vWF can be any one of SEQ ID NOs: 28 or 29. In some instances, the vWF gene sequence in the vector may encode all or a functional portion of the human vWF protein set forth in GenBank Accession No. 1023301060 as updated Aug. 21, 2017. However, in some instances, the vWF gene sequence may include one or more modifications to the wild-type vWF gene sequence to increase protein expression, increase protein stability, reduce immunogenicity, or a combination of one or more thereof, of the vWF protein. For example, the full cDNA sequence of the vWF gene may be too large to be packaged efficiently in certain vectors, such as, for example, a lentiviral vector. Thus, in some instances, one or more exons of the vWF gene may be deleted while still retaining biological function of the expressed protein. In some instances, exons 24-46 of the vWF gene may be deleted as described in U.S. Patent Application Publication No. 2010/0183556. In some instances, the vWF gene sequence may be codon-optimized for efficient expression in the MSC. In some instances, the exogenous vWF gene sequence may modified for expression in a particular organ or tissue type. For example, the gene sequence may be optimized for expression in the liver. Thus, in some instances, the vWF gene sequence may comprise at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identity to the corresponding wild-type vWF gene sequence and comprise modifications to improve expression. In some instances, the vWF gene sequence comprises the truncated human vWF sequence set forth below in this disclosure or a sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical thereto while retaining biological activity of the expressed protein. In some instances, the vWF gene sequence comprises the truncated sheep vWF sequence set forth below in this disclosure or a sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical thereto while retaining biological activity of the expressed protein.

[0049] In some instances, gene-editing may be performed on the MSC to insert, delete, or replace the genomic sequence of one or both of the endogenous genes using engineered nucleases (molecular scissors). Gene-editing nucleases belong to one of three known categories: zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) tools. All operate on the same principle; they are all capable of inducing a double-strand break at a defined genomic sequence that is subsequently corrected by endogenous DNA repair mechanisms. Double-strand breaks can be repaired through homology-driven repair (HDR), in the presence of donor homologous DNA sequences, resulting in gene-editing events.

[0050] In some instances, the MSC may be modified to express high levels of the Factor VIII protein via gene-editing of an endogenous Factor VIII gene sequence of the MSC, wherein the gene-editing introduces one or more modifications to an endogenous Factor VIII gene sequence that increase protein expression, increase protein stability, reduce immunogenicity, or a combination of one or more thereof, of the Factor VIII protein. In some instances, the MSC are modified to express high levels of an exogenous FVIII protein via genome-editing, wherein the gene-editing introduces an exogenous FVIII gene, under the control of a constitutive promoter, into a "safe harbor" region within the genome, such as the AAVS1 site. In some instances, the MSC are modified to express high levels of the vWF protein via gene-editing of an endogenous vWF gene sequence of the MSC, wherein the gene editing introduces one or more modifications to the endogenous vWF gene sequence that increase protein expression, increase protein stability, reduce immunogenicity, or a combination of one or more thereof, of the vWF protein. In some instances, the MSC are modified to express high levels of an exogenous vWF protein via genome-editing, wherein the gene-editing introduces an exogenous vWF gene, under the control of a constitutive promoter, into a "safe harbor" within the genome, such as the AAVS1 site. Exemplary "safe harbor" regions are described in Cerbini, T., et al., Transfection, selection, and colony-picking of human induced pluripotent stem cells TALEN-targeted with a GFP gene into the AAVS1 safe harbor. J Vis Exp. 2015 Feb. 1; (96):52504 and Hong, S. G., et al., Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers. Mol Ther. 2017; 25(1):44-53.

[0051] In some instances, the endogenous Factor VIII gene sequence may be modified by gene-editing to have the type of modifications described above for embodiments where an exogenous Factor VIII gene sequence is introduced via transduction. The discussion of the various modifications described above is thus also applicable to embodiments where the endogenous Factor VIII gene sequence is modified. For example, in some instances, the sequence of one or more protein domains of the endogenous Factor VIII gene sequence may be deleted. In some instances, the B domain of Factor VIII is deleted. In some instances, the endogenous Factor VIII gene sequence may be modified to reduce immunogenicity of the protein thereby reducing the risk of an immune response due to therapy. For example, alanine substitutions may be introduced as described in Healey, J. F., et al., The comparative immunogenicity of human and porcine factor VIII in haemophilia A mice. Thromb Haemost. 102:35-41 (2009) and Lubin, I. M., et al., Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis. J Biol Chem. 272:30191-30195 (1997), which are incorporated by reference herein in their entirety.

[0052] In some instances, the endogenous Factor VIII gene sequence may be modified to substitute one or more of the Factor VIII protein domain sequences with the sequence of the corresponding Factor VIII protein domain sequences from another species. For example, for human subjects, the endogenous Factor VIII gene sequence may be modified to substitute one or more of the human Factor VIII protein domain sequences with the sequence of the corresponding porcine Factor VIII protein domain sequences. For example, substitution with the porcine Factor VIII domains A1 and ap-A3 may increase expression of the expressed Factor VIII protein. See, for example, Doering, C. B., et al., Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion. J Biol Chem. 279:6546-6552 (2004). In some embodiments, the endogenous Factor VIII gene sequence may be modified to comprise the porcine Factor VIII A1, A3, and C1 domains, while retaining the human Factor VIII A2 and C2 domains.

[0053] In some instances, the endogenous Factor VIII gene sequence may be modified to include a B domain deletion, the porcine A1, A3, and C1 domains, and also include three alanine substitutions in the A2 domain to reduce immunogenicity, as described above for the exogenous Factor VIII gene sequence embodiments. In one example, the endogenous Factor VIII gene sequence may be modified to have the sequence of a human/porcine Factor VIII transgene as described in Doering, C. B., et al., Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A, Mol. Ther. 17(7):1145-1154 (2009), which is incorporated herein by reference in its entirety. In some instances, the modified endogenous Factor VIII gene sequence results in expression of a modified Factor VIII protein at a level comparable to that of wild-type porcine Factor VIII protein while having 91% identity to the amino acid sequence of wild-type human Factor VIII protein.

[0054] In some instances, the endogenous Factor VIII gene sequence may be modified to comprise one of the modified Factor VIII sequences described in U.S. Pat. No. 7,635,763, which is incorporated herein by reference in its entirety for all purposes. In some instances, the endogenous Factor VIII gene sequence may comprise the polynucleotides set forth in any one of SEQ ID NOs: 19, 21, 23, 25, or 27 (SEQ ID NOs: 15, 17, 19, 13, or 21 as described in U.S. Pat. No. 7,635,763). The modified Factor VIII protein expressed at high levels in the modified MSC may comprise the amino acid sequences set forth in any one of SEQ ID NOs: 18, 20, 22, 24, or 26 (SEQ ID NOs: 14, 16, 18, 12, or 20 as described in U.S. Pat. No. 7,635,763). Such sequences are summarized in Table 1 above.

[0055] As discussed above, in some instances, the MSC are also modified to express high levels of vWF protein via gene-editing. In some instances, the vWF gene sequence may include one or more modifications to the wild-type vWF gene sequence to increase protein expression, increase protein stability, reduce immunogenicity, or a combination of one or more thereof, of the vWF protein. For example, in some instances, one or more exons of the vWF gene may be deleted while still retaining biological function of the expressed protein. In some instances, exons 24-46 of the vWF gene may be deleted as described in U.S. Patent Application Publication No. 2010/0183556. In some instances, the vWF gene sequence may be codon-optimized for efficient expression in the MSC. In some instances, the exogenous vWF gene sequence may modified for expression in a particular organ or tissue type. For example, the gene sequence may be optimized for expression in the liver. Thus, in some instances, the vWF gene sequence may be modified to comprise at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identity to the corresponding wild-type vWF gene sequence and comprise modifications to improve expression. In some instances, the vWF gene sequence may be modified to comprise the truncated human vWF sequence set forth below in this disclosure or a sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical thereto while retaining biological activity of the expressed protein. In some instances, the vWF gene sequence may be modified to comprise the truncated sheep vWF sequence set forth below in this disclosure or a sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 89% 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical thereto while retaining biological activity of the expressed protein.

[0056] Where the MSC are modified to express high levels of both Factor VIII and vWF, the same method of modification may be used to achieve high expression of both proteins or different methods could be used for each protein. For example, in some instances, the MSC may be modified to express high levels of both Factor VIII and vWF protein via introduction of exogenous gene sequences for both proteins. In another example, the MSC may be modified to express high levels of both Factor VIII and vWF protein via gene-editing of the endogenous gene sequences of both proteins. In some instances, the MSC may be modified to express high levels of Factor VIII via transduction of an exogenous Factor VIII gene sequence and modified to express high levels of vWF via gene-editing of the endogenous vWF gene sequences. In other instances, the MSC may be modified to express high levels of vWF via transduction of an exogenous vWF gene sequence and modified to express high levels of Factor VIII via gene-editing of the endogenous Factor VIII gene sequences.

[0057] A "high level of expression" means that the production/expression of the modified Factor VIII protein or vWF protein is at an increased level as compared to the expression level of the corresponding native Factor VIII protein or vWF protein expressed under the same conditions. An increase in protein expression levels (considered a high level of expression) comprises at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20-fold or greater expression of the modified Factor VIII protein or vWF protein compared to the expression levels of the corresponding Factor VIII protein or vWF protein. Alternatively, "high-level expression" can comprise an increase in protein expression levels of at least 1-25 fold, 1-5 fold, 5-10 fold, 10-15 fold, 15-20 fold, 20-25 fold or greater expression levels of the modified Factor VIII protein or vWF protein when compared to the corresponding Factor VIII protein or vWF protein. Methods for assaying protein expression levels are routine in the art. By "corresponding" Factor VIII protein or vWF protein is intended a Factor VIII protein or vWF protein that comprises an equivalent amino acid sequence. In one example, expression of a modified human Factor VIII protein comprising the A1-A2-ap-A3-C1-C2 domains is compared to a human Factor VIII protein containing corresponding domains A1-A2-ap-A3-C1-C2. In another example, for a fragment of a modified human Factor VIII protein containing domains A1-A2-ap-A3, expression is compared to a fragment of human Factor VIII protein having the corresponding domains A1-A2-ap-A3. Alternatively, in certain instances, expression of a modified Factor VIII protein or vWF protein may be compared to the full-length corresponding proteins. In one example, for a fragment of a modified human Factor VIII protein containing domains A1-A2-ap-A3, expression is compared to human Factor VIII protein having the A1-A2-ap-A3-C1-C2 domains.

[0058] The modified MSC are cultured in vitro to generate an expanded modified MSC population. The expanded modified MSC population provides sufficient numbers of modified MSC for therapeutic use. Culture conditions may be selected based on the type of MSC used in the method. For example, MSC isolated from placental tissue may be grown in culture medium optimized for placental cells. In another example, MSC isolated from amnion tissue may be grown in culture medium optimized for amniotic cells. In another example, MSC isolated from umbilical cord or bone marrow may be grown in culture medium optimized for MSC cells. The modified cells may be grown on plastic culture dishes for at least 2, 3, 4, 5, or 6 passages to generate the expanded modified MSC population. In some instances, all or a portion of the expanded modified MSC population may be cryopreserved.

[0059] Following culturing of the modified MSC to generate expanded modified MSC population, modified MSC from expanded modified MSC population are injected into the subject. The injection may be made at least one of intraperitoneal injection, intravenous injection, or intra-articular injection. Each injection comprises about 10.sup.5 to about 10.sup.9 MSC from the expanded modified MSC population per kilogram weight of the subject. For example, the injection may comprise 10.sup.5 MSC, 10.sup.6 MSC, 10.sup.7 MSC, 10.sup.8 MSC, or 10.sup.9 MSC. The number of cells injected into the subject is based on the amount of protein expressed per cell. This metric is determined empirically for the expanded modified MSC population. In some instances, this metric may be generally predictable based on the nature of the modified MSC (for example, method of modification, Factor VIII gene sequence, vWF gene sequence, vector and vector components).

[0060] In some instances, modified MSC are injected into the subject once, twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times. In some instances, modified MSC are injected into the subject at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. For example, the modified MSC may injected as multiple injections on the same day. In some instances, the modified MSC may be injected into the subject on multiple days. In some instances, the subject is injected with modified MSC on a first day and then the subject may be monitored over a period of time (days or weeks) to determine if there is sufficient protein expression to provide the desired therapeutic benefit. In some instances, the amount of protein expression in the subject's blood of Factor VIII protein, vWF protein, or both, may be monitored. In some instances, the efficiency of the subject's blood to clot may be assessed using routine blood clotting tests known in the art. In some instances, the subject's symptoms relating to joint pain and/or inflammation may be assessed. Where monitoring indicates that the amount of expression of Factor VIII protein alone, or the amount of expression of Factor VIII protein and vWF protein, is insufficient, the subject's disease symptoms are not alleviated, or both, the subject may be injected with modified MSC on a second day. Again, the subject may be monitored over a period of time to determine if there is sufficient protein expression to provide the desired therapeutic benefit. These steps may be repeated for a fourth, fifth, sixth, seventh, eighth, ninth, or tenth injection as needed to achieve the desired therapeutic benefit of alleviating the subject's disease symptoms.

[0061] In some instances, the use of MSC as cellular vehicles to deliver a Factor VIII gene sequence, a vWF gene sequence, or both, to a subject (as opposed to administration of vector directly) may overcome limitations/risks observed to-date in AAV-based clinical trials for hemophilia: 1) the possibility of off-target transduction of troubling cell types, such as germline cells; 2) the inability to treat patients with pre-existing antibodies to the serotype of AAV being employed as a vector; and 3) the transient hepatotoxicity induced by the AAV capsid, that triggers subsequent immune/inflammatory destruction of many of the transduced cells. Although early studies in vitro and in normal and hemophilia A mice, have used unselected stromal cells (isolated based solely upon plastic adherence) as cellular vehicles for delivering exogenous Factor VIII, no attempts have yet been made to use phenotypically-defined MSC/pericytes to deliver FVIII in vivo in any preclinical model of hemophilia A.

[0062] In some instances, the use of MSC as cellular vehicles to deliver a therapeutic gene is also an improvement over the use of hematopoietic stem cells (HSC), as have been used in most cell-based gene therapy trials. The use of MSC eliminates the possibility of insertional leukemogenesis, which is the most serious adverse event seen to-date in clinical gene therapy trials. A successful outcome of the proposed studies targeting hemophilia A thus promises to open the door to safe correction of a variety of congenital disorders using MSC to deliver the therapeutic gene.

EXAMPLES

Example 1. Animal Model

[0063] Applicant re-established a line of sheep that emulates the genetics, inhibitor formation, and clinical symptoms of the severe form of human hemophilia A (HA), including the development of frequent, spontaneous hematomas and crippling hemarthroses, making them unique among the HA models. See Porada, C. D., et al., Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A. J Thromb Haemost, 2010. 8(2): 276-285. Using unique antibodies developed to various regions of the ovine FVIII protein, it was determined that these sheep do not produce any FVIII antigen (FIG. 1), as demonstrated by the complete lack of any staining within the liver of two of the HA sheep, which is in marked contrast to the widespread bright staining that is seen in the liver from a normal/healthy sheep. As such, they should be an excellent model of severe, cross-reacting material (CRM)-negative hemophilia A patients. Additionally, sheep are close in size to humans, their immune system is quite similar to that of humans, and their long lifespan allows long-term efficacy and safety to be addressed.

Example 2. Preliminary Pilot Study--Allogeneic Cells

[0064] A pilot study on 2 pediatric subjects from the HA sheep model described in Example 1. See Porada, et al., Phenotypic correction of hemophilia A in sheep by postnatal intraperitoneal transplantation of FVIII-expressing MSC. Exp Hematol, 2011. 39(12):1124-1135. During the first 3-5 months of life, both animals had received frequent, on-demand infusions of human FVIII (hFVIII) for multiple hematomas and chronic, progressive, debilitating hemarthroses of the leg joints which had resulted in severe defects in posture and gait, rendering them nearly immobile. Thus, for these subjects, FVIII was presented in the context of "danger signals", which is known to trigger a robust host immune response to FVIII and other proteins.

[0065] Haploidentical MSC from the ram that had sired the two HA lambs were used for the therapy. The MSC were modified to introduce via transduction a B domain-deleted, wild-type porcine FVIII cDNA as described in Porada et al., Phenotypic correction of hemophilia A in sheep by postnatal intraperitoneal transplantation of FVIII-expressing MSC. Exp Hematol. 2011; 39(12):1124-1135. MSC were simultaneously transduced with 2 lentivectors; one encoded eGFP for in vivo tracking of donor cells, and the second encoded an expression/secretion optimized porcine FVIII (pFVIII) transgene previously shown to be expressed/secreted from human cells at 10-100 times higher levels than hFVIII or sheep (ovine) FVIII (oFVIII). See Gangadharan et al., High-level expression of porcine factor VIII from genetically modified bone marrow-derived stem cells. Blood, 2006. 107(10):3859-64; Doering et al., Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A. Mol Ther, 2009. 17(7):1145-54; Doering et al., Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion. J Biol Chem, 2004. 279(8):6546-52; Dooriss et al., Comparison of Factor VIII Transgenes Bioengineered for Improved Expression in Gene Therapy of Hemophilia A. Hum Gene Ther, 2009. 20(5):465-78; Ide, L. M., et al., Hematopoietic stem-cell gene therapy of hemophilia A incorporating a porcine factor VIII transgene and nonmyeloablative conditioning regimens. Blood, 2007. 110(8):2855-63; and Johnston et al., Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A. Gene Ther, 2013. 20(6):607-15.

[0066] FVIII/GFP-expressing MSC were then expanded and transplanted by IP injection (30.times.10.sup.6), in the absence of any preconditioning, into the first lamb. Following transplant, this animal's clinical picture improved dramatically, and he enjoyed an event-free clinical course, devoid of spontaneous bleeds, obviating the need for hFVIII infusions. Even more remarkably, the animal's existing hemarthroses resolved, his joints recovered fully, and he regained normal posture and gait, resuming a normal activity level. To the inventors' knowledge, this represents the first report of phenotypic correction of severe HA in a large animal model following transplantation of cells modified to express FVIII, and is the first time that reversal of chronic debilitating hemarthroses has been achieved in any setting.

[0067] Based on this remarkable clinical improvement, the modified MSC were transplanted into the second animal using an identical procedure, but a higher cell dose (125.times.10.sup.6). In similarity to the first animal, hemarthroses present in this second animal at the time of transplant resolved, he resumed normal activity shortly after transplantation, and became factor-independent.

[0068] Interestingly, despite the marked phenotypic improvement in both these animals, no circulating FVIII activity was detectable following the transplant, most likely due to the presence of high-titer inhibitors in these animals. These findings are remarkable, since despite the high titers of antibodies/inhibitors present in these animals, the transplanted allogeneic (haploidentical) MSC persisted and were not eliminated by the recipient's immune system, and the therapeutic effect of the treatment was maintained, i.e., the animals' symptoms of spontaneous joint bleeds, hematomas, and bleeding upon needle stick all improved.

Example 3. Mechanistic Study--Autologous Cells

[0069] Twenty female HA carriers will be artificially inseminated (AI) via laparoscopy, as done in Example 2, with the support of the North Carolina State Theriogenology/Ruminant Medicine team. At 50-70 days of gestation (term: 150 days), amniotic fluid will be collected, and fetal cells from the amniotic fluid will be isolated, cultured, and expanded, using standard methods in our lab. Given the severe phenotype of these animals, we will perform a PCR-based RFLP (see Porada, C. D., et al., J Thromb Haemost, 2010. 8(2):276-85) to identify affected fetuses, allowing us to plan for their subsequent delivery. Following amniocentesis, the animals will be allowed to complete term. When the sheep have nearly completed gestation, the pregnant ewes carrying affected fetuses will be placed under close observation, and ewes will either be induced into labor using intramuscular dexamethasone for natural delivery, or the lambs will be delivered by Caesarian section, with clinical veterinarians assisting in either case. Both approaches have been used previously with success.

[0070] Affected lambs will be treated immediately with recombinant full-length or B-domain deleted ovine FVIII (oFVIII) produced as described in Zakas, P. M., et al., Development and characterization of recombinant ovine coagulation factor VIII. PLoS One, 2012. 7(11):e49481. Although we have found that oFVIII is not a very high-expressing FVIII variant when compared to FVIII from other species, oFVIII protein for transfusion and an oFVIII transgene in the vectors are being used because the consensus in the hemophilia field is that the use of "same-species" FVIII is essential in preclinical gene therapy-based studies to accurately model the potential immune response in the clinical arena.

[0071] While human cells may be needed to perform definitive clinical studies, human cells are not appropriate for these mechanistic studies because using human cells would not allow us to address the critical issue of whether the use of autologous cells results in higher levels of long-term engraftment than we achieved in our pilot study with allogeneic cells, and whether the use of autologous cells may reduce the incidence of inhibitor formation. For this reason, sheep MSC will be used throughout this study.

[0072] It is our goal to treat HA with this MSC-based delivery system during the first 18 months of postnatal human life, since this is the time by which most HA patients would be diagnosed. Sheep develop much faster than humans, and are weaned at 60-90 days of age. We thus know this corresponds to the first 12-18 months for a human, so we will test the MSC-based treatment during the first 2-3 months of life in the sheep.

[0073] Starting at birth, HA lambs will be treated prophylactically 2-3 times per week with recombinant oFVIII. At 4-5 weeks of age, we will collect bone marrow (under oFVIII coverage), and isolate MSC from each affected lamb, as we have done previously. These methods enable us to successfully establish primary sheep MSC that are phenotypically and functionally similar to their human counterparts; these sheep MSC are devoid of hematopoietic cells (they lack CD11b, CD34, and CD45), but they express the MSC markers CD146 and CD90. See FIG. 2. Anti-ovine antibodies to other antigens routinely used to identify MSC, such as CD44, CD105, and CD73, are not commercially available. Immunofluorescence microscopy demonstrated expression of vimentin and .alpha.-smooth muscle actin, known MSC cytoskeleton proteins, and we verified the ability of these sheep MSC were able to differentiate into adipocytes (by Oil-red-o staining) and osteocytes (by calcium deposition and alkaline phosphatase). See FIG. 2.

[0074] We will then subject the isolated MSC to 2-3 rounds of transduction with either the EF1.alpha.-[oFVIII] lentivirus vector (FIG. 3) alone, or simultaneously with the EF1.alpha.-[oFVIII] lentivirus vector and the CAG-[vWF] lentivirus vector (FIG. 4). See De Meyer, S. F., et al., Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor. Blood, 2006. 107(12): 4728-36. The EF1.alpha.-[oFVIII] lentivirus vector contains a B-domain deleted oFVIII gene having the polynucleotide sequence set forth in SEQ ID NO: 33. The CAG-[vWF] lentiviral vector contains a truncated vWF having the polynucleotide sequence set forth in SEQ ID NO: 29. From the pilot study described in Example 2, we know that 2-3 rounds of simultaneous exposure to two different lentivirus vectors results in transduction of 90-95% of sheep MSC with both vectors. The lentivirus vectors for these studies both contain the 3'-modified LTR to produce SIN lentivirus vectors, and the constitutive EF1.alpha. promoter will drive FVIII expression. Due to packaging constraints, it is not possible to utilize the EF1.alpha. promoter in the vWF lentivirus vector. Prior studies have established the ability of vWF to be packaged within a lentivirus vector and expressed at high levels. See De Meyer, S. F., et al., 2006 (above). However, if any difficulties arise packaging vWF-encoding lentivirus vectors, we will utilize truncated vWF cassettes as described in this disclosure, or switch to foamy virus vectors, as they possess a much larger packaging capacity.

[0075] We are including a group in which autologous MSC are transduced with vectors encoding both oFVIII and vWF (ovine vWF; GI:426227037) for two reasons: 1) binding to vWF stabilizes FVIII and prolongs its half-life and, thus, delivery of MSC secreting FVIII complexed with vWF may produce a more pronounced therapeutic effect; and 2) vWF may reduce the immunogenicity of exogenously administered FVIII by preventing both its uptake and presentation by dendritic cells, and its recognition by immune effector cells. We predict that delivering the two proteins in the same vector will result in the release of FVIII:vWF as a complex from the transduced MSC. We will confirm co-localization/complex formation of vector-derived oFVIII and vWF in transduced MSC populations by confocal microscopy prior to performing the proposed transplants. Although MSC do not endogenously produce any vWF, we will add a 6-His tag (SEQ ID NO:32) to the vWF transgene, making it readily distinguishable from any trace endogenous vWF for these in vitro studies.

[0076] Following transduction, the FVIII-expressing MSC will be expanded until the animals have reached 2-3 months of age, at which point the MSC be transplanted autologously into their respective donor HA lamb, via IP injection under ultrasound guidance, with no preconditioning (as in our pilot study [23]), using a dose of 5-10.times.10.sup.6 cells/kg. An aliquot of each cell type will be reserved to determine vector copy number by qPCR and to perform integration site analysis by LM-PCR. Methods described in Porada, C. D., et al., Phenotypic correction of hemophilia A in sheep by postnatal intraperitoneal transplantation of FVIII-expressing MSC. Exp Hematol, 2011. 39(12): 1124-1135 (qPCR) and Russo-Carbolante, E. M., et al., Integration pattern of HIV-1 based lentiviral vector carrying recombinant coagulation factor VIII in Sk-Hep and 293T cells. Biotechnol Lett, 2011. 33(1):23-31 and Tellez, J., et al., High Incidence of Vector Integration Near Cancer Related Genes within Primitive Hematopoietic Stem Cells (HSC) After Fetal Gene Transfer with .gamma.-Retroviral Vectors. Molecular Therapy, 2010. 18(Suppl. 1): p. S331 (LM-PCR). Two experimental groups will be included: 1) autologous MSC transduced with the EF1.alpha.-[oFVIII] lentivector (n=2-3 HA lambs); and 2) autologous MSC transduced with both the EF1.alpha.-[oFVIII] and CAG-vWF lentivectors (n=2-3 HA lambs).

[0077] Following transplantation, prophylactic oFVIII infusions will be discontinued, and any benefit as a result of this MSC-based approach should be readily apparent, given the severe, life-threatening phenotype of these animals. The sheep will be continually monitored for bleeds, and platelet-deficient plasma will be collected monthly until at least 1.5 years of age for coagulation assays, to quantify the plasma levels of oFVIII by chromogenic assay and/or ELISA. The formation/presence of inhibitors will be assessed at each time point by performing the Nijmegen modification of the Bethesda assay (as described in Verbruggen, B., et al., The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability. Thromb Haemost, 1995. 73(2):247-51) and a commercially available kit (Technoclone/DiaPharma Group, Inc.) on an aliquot of plasma collected from the animals. Once we have obtained these values, we will compare the HA lambs that received MSC transduced with the lentivector encoding oFVIII alone to those that received MSC transduced with both the oFVIII and vWF lentivectors, and compare each of these to the historic values from untransplanted HA sheep, and to a reference panel of normal unaffected males. These studies will allow us to: 1) establish what levels of vector-encoded oFVIII are expressed as a result of this postnatal approach; 2) determine the duration of the therapeutic effect of this approach; 3) assess whether using autologous MSC and a lentivector that lacks eGFP avoids the inhibitors seen in the pilot study of Example 2; and 4) establish whether including vWF improves the therapeutic effect of this MSC-based treatment and/or reduces the incidence/titer of inhibitor formation.

[0078] At 1.5 years of age (or sooner, if we see that FVIII levels are dropping), the HA lambs will be euthanized, and all major organs will be harvested. All tissues will be fixed in 4% paraformaldehyde, processed through a sucrose gradient, embedded and frozen in OCT, and sectioned at 5 .mu.m. 8-10 slides/tissue will be stained with an antibody specific to oFVIII and analyzed/quantitated by confocal microscopy for the presence of engrafted MSC to precisely determine the levels and localization (parenchymal vs. perivascular) of engrafted cells that are expressing FVIII, and are therefore providing therapeutic benefit. We will also collect plasma from each of these recipients at the time of euthanasia, and quantitate the circulating levels of vector-derived oFVIII in these sheep using an ELISA specific for oFVIII, correlating levels and patterns of engraftment with circulating FVIII levels. Based on ease of access to the circulation, we hypothesize that maximal plasma FVIII levels will be obtained when MSC lodge in perivascular regions of the engrafted tissues.

[0079] While confocal analysis should provide us with a fairly accurate estimate of the levels of oFVIII+MSC within each tissue, the slides selected for quantitation may or may not be representative of the engraftment levels within the organ as a whole. Therefore, we will also use an ELISA to precisely quantitate the amount of oFVIII within tissue homogenates. A standard curve will be created with known numbers of oFVIII+MSC, thereby establishing how much oFVIII is present on a per-cell basis. Protein extracts will then be prepared from the tissues from each animal and analyzed using this ELISA, comparing the tissue values to that of the standard curve, to precisely quantitate the number of MSC that have engrafted within each tissue and are expressing oFVIII. We will then compare the levels of donor MSC in each tissue with the resultant plasma FVIII levels to determine in which tissues engraftment produces the highest circulating levels of FVIII.

Example 4. Study Assessing Treatment in Subjects with Pre-Existing Inhibitors

[0080] The findings of the study described in Example 3 will be used to determine the ability of this autologous cell-based approach to mediate clinical/phenotypic improvement in recipients with pre-existing inhibitors as a result of on-demand FVIII treatment.

[0081] Three to four HA lambs will be treated on-demand with oFVIII beginning at birth. We expect, based on the pilot study of Example 2, that treating on-demand with FVIII products will result in the formation of inhibitors in almost all HA sheep by 4-5 months of age. We will collect bone marrow and isolate MSC at 4-5 weeks of age, and transduce these cells with either the EF1.alpha.-[oFVIII] lentivector alone, or with both the EF1.alpha.-[oFVIII] and CAG-vWF lentivectors (depending which approach yields the best outcome in the preceding studies), and expand the transduced cells to obtain adequate numbers for transplant.

[0082] Beginning at birth, we will draw blood from these animals every other week to obtain plasma and perform the Nijmegen modification of the Bethesda assay (as above) (Technoclone/DiaPharma Group, Inc.) to assess the development of inhibitors. Once inhibitors have developed, we will transplant the transduced autologous MSC into each animal by ultrasound-guided IP injection, as in Exp. Set #1.1, at a dose of 5-10.times.10.sup.6 cells/kg.

[0083] Following transplantation, we will analyze the animals as detailed in Example 3, continually monitoring them for bleeds, and collecting platelet-deficient plasma monthly until at least 1 year of age for coagulation assays, to quantify the plasma levels of oFVIII by chromogenic assay and/or ELISA, and to quantitate the levels of inhibitors present, to ascertain whether the MSC-based treatment impacts upon the levels of the pre-existing inhibitors. We will then compare the results obtained with these HA lambs with pre-existing inhibitors to those in Example 3 in which the HA lambs lacked inhibitors at the time of MSC infusion.

Example 5. First Line Therapy Study

[0084] We hypothesize that previously untreated patients (PUPS) represent the ideal group to initially target with this MSC-based treatment, because their immune systems are completely naive to exogenous FVIII, and will be exposed to it for the first time when it is released by the transplanted MSC; we anticipate this will reduce/eliminate the risk of inhibitor formation in this population.

[0085] In families with no prior history, HA is normally diagnosed during the first 18 months of life, after the child exhibits abnormal bruising/bleeding after a minor trauma. In families with a history of HA, diagnosis can be made at birth, or even prior to birth [148-158]. Regardless of when diagnosis is made, however, it would not be possible to collect bone marrow MSC from these patients without treating with factor to prevent hemorrhage during the procedure. This same issue exists with the HA sheep, since they present with a severe phenotype and spontaneous bleeding from birth. However, in similarity to patients with a family history of HA, the affected sheep can be diagnosed in utero by amniocentesis (as can be done in human patients), making it possible to collect autologous cells from the amniotic fluid part way through gestation, transduce these cells, expand them, and have them ready to transplant as the first-line therapy at birth, or shortly thereafter. We recently found that MSC-like cells present within the amniotic fluid, "AF-MSC", are readily transduced with lentivirus vectors, they endogenously produce low levels of FVIII (FIG. 5) and vWF (FIG. 6), and they express very high levels of FVIII following lentivector transduction (FIG. 7). These findings suggest that amniotic fluid can replace marrow as a source of autologous MSC for delivering a FVIII transgene, enabling us to test the MSC-based treatment's efficacy as first-line therapy in PUPs.

[0086] To test the efficacy of the MSC-based treatment in PUPs, HA carrier ewes will be bred or artificially inseminated as detailed in Example 3. At 50-60 days of gestation (term: 150 days), amniotic fluid will be collected, AF-MSC isolated, and a PCR-based RFLP performed to detect the HA mutation, as detailed in Example 3. AF-MSC from the affected fetuses will then be subjected to 2-3 rounds of transduction with the EF1.alpha.-[oFVIII] comprising the polynucleotide sequence set forth in SEQ ID NO: 33 (FIG. 3) and expanded. At near term, labor will be induced in the ewes with affected lambs, or a C-section performed to deliver the lambs. Immediately after birth, the HA lamb "PUPs" (n=3-4) will be injected IP with the transduced autologous AF-MSC at a dose of 5-10.times.10.sup.6 cells/kg, as described for the BM-MSC in Example 3.

[0087] Following transplantation, we will analyze the animals as detailed in Example 3 and 4, continually monitoring them for bleeds, and collecting platelet-deficient plasma monthly until at least 1 year of age for: 1) coagulation assays; 2) to quantify the plasma levels of oFVIII by chromogenic assay and/or ELISA; and 3) to perform the Nijmegen modified Bethesda assay to assess the development of inhibitors. We will then compare the results obtained by using this MSC-based treatment as a first-line therapy in these HA lamb "PUPS" to the results obtained in the HA lambs treated prophylactically (Example 3) and to those treated on-demand (Example 4) prior to MSC infusion.

Example 6. Study to Assess Induction of Immune Tolerance to Factor VIII

[0088] We hypothesize that the continued delivery of FVIII to the circulation by the lentivector-modified MSC can serve as a much-needed novel method of inducing immune tolerance to FVIII.

[0089] The aim of this study is to test the ability of this MSC-based approach as a novel method of inducing immune tolerance through the continued delivery of FVIII to the circulation by the genetically-modified MSC. To accomplish this objective, 2-3 HA lambs (more will be added if initial data are not clear-cut) will be treated on-demand with oFVIII, beginning at birth, as we know that treating on-demand with FVIII products results in the formation of inhibitors in almost all HA sheep by 4-5 months of age. As in Example 4, we will isolate BM-MSC at 4-5 weeks of age, and transduce these cells with both the EF1.alpha.-[oFVIII] and CAG-vWF lentivectors, as clinical data indicate that the inclusion/presence of vWF may facilitate ITI [123, 159, 160]. The transduced cells will then be expanded to obtain adequate numbers for subsequent transplant.

[0090] Beginning at birth, we will draw blood from these animals every other week to obtain plasma and perform the Nijmegen modified Bethesda assay (as above) to assess inhibitor induction. Once inhibitors have developed, we will transplant transduced autologous MSC, at a dose of 10' cells/kg, into the peritoneal cavity of each animal, as in Example 3. This procedure will be repeated each 4-5 days until we observe a drop in inhibitor titer (as detailed below); a maximum of 10 infusions will be given initially.

[0091] Following transplantation, we will analyze the animals as in Example 3, continually monitoring for bleeds (as the repeated MSC-based treatment should produce clinical/phenotypic improvement), and collecting platelet deficient plasma bi-weekly for .gtoreq.3 months, to perform coagulation assays, to quantify the oFVIII plasma levels by chromogenic assay and/or ELISA, and to quantitate the levels of inhibitors present, to ascertain whether the repeated infusion of MSC expressing high levels of FVIII can break the existing inhibitors and induce tolerance to FVIII. To further confirm that this cell-based ITI has overcome the existing inhibitors, we will assess the restoration of normal FVIII pharmacokinetics, using well-established methodology (plasma FVIII recovery.gtoreq.66% of expected and a .gtoreq.6 h half-life, determined following a 72-hour FVIII-exposure-free period).

Example 7. Comparison of Recombinant Factor VIII Infusion Therapy with MSC-Based Tolerance Induction

[0092] One of the only clinical options for HA patients who develop inhibitors is immune tolerance induction (ITI), which involves the long-term administration of high doses of FVIII protein. ITI is extremely expensive, is only effective in a percentage of patients with inhibitors, and the mechanism for its success is unknown. To-date, no preclinical HA model has been used to study and/or optimize ITI. Given the high incidence of inhibitor formation in the HA sheep and their lack of any cross-reactive material, they represent an excellent model in which to investigate ITI. We propose to perform a head-to-head comparison of traditional ITI, using repeated high-dose recombinant oFVIII to the MSC-based ITI protocol developed/tested in Example 6.

[0093] To achieve these goals, 2-3 HA lambs will be treated on-demand with oFVIII, beginning at birth, as described in Example 6, until inhibitors develop. We will then commence a clinically employed, protein-based ITI regimen, infusing the inhibitor animals with a dose of 100 IU/kg/day for 3 months (as described in Oldenburg, J., et al., Primary and rescue immune tolerance induction in children and adults: a multicentre international study with a VWF-containing plasma-derived FVIII concentrate. Haemophilia, 2014. 20(1):83-91). During the course of this ITI protocol, we will analyze the animals as detailed in Example 6, collecting platelet-deficient plasma weekly, to: 1) perform coagulation assays; 2) quantify the plasma levels of oFVIII by chromogenic assay and/or ELISA; and 3) quantitate the levels of inhibitors present, to assess the ability of the protein-based ITI to break existing inhibitors, and define the kinetics with which this happens. To further confirm that ITI has overcome existing inhibitors, we will assess the restoration of normal FVIII pharmacokinetics, as detailed above. The success rate and kinetics of tolerance induction with the protein-based ITI will be compared to those of the cell-based protocol in Example 6, to determine whether the cell-based method is a viable alternative to the time consuming and expensive protein-based method that represents the current state-of-the-art in clinical ITI.

Example 8. Transduction Efficiency with Different Vectors

[0094] The transduction efficiency, FVIII production, and FVIII secretion from human PLC following transduction at an identical multiplicity of infection (MOI) of 7.5 with an identical lentiviral vector (LV) encoding one of the following four different FVIII transgenes: (1) a bioengineered human-porcine hybrid FVIII (ET3) having the polynucleotide sequence set forth in SEQ ID NO: 11; (2) a liver codon-optimized ET3 (lcoET3) having the polynucleotide sequence set forth in SEQ ID NO: 10; (3) a liver codon-optimized human FVIII (lcoHSQ) having the polynucleotide sequence set forth in SEQ ID NO: 14; and (4) a myeloid-codon optimized ET3 (mcoET3) having the polynucleotide sequence set forth in SEQ ID NO: 12 were compared. Brown et al. (2018) Mol. Ther. Methods Clin. Dev. 9:57-69, demonstrated that vectors encoding FVIII, when codon-optimized to the target cells, or tissue, result in a dramatically increase FVIII expression of functional FVIII. Following transduction, PLCs were analyzed by flow cytometry and confocal microscopy to measure transduction efficiency and FVIII production. Conditioned media of PLCs were assayed by aPTT to quantitate FVIII activity. Analysis of the culture supernatants by aPTT demonstrated FVIII activity was readily detectable in supernatants of all transduced cells lines. It also revealed marked differences in the secretion of functional FVIII following transduction with each of these vectors. Specifically, PLCs transduced with mcoET3 (SEQ ID NO: 12), ET3 (SEQ ID NO: 11), lcoET3 (SEQ ID NO: 10), and lcoHSQ (SEQ ID NO: 14) LV secreted 25.+-.9, 19.+-.8, 11.+-.2, and 1.+-.0.1 IU of FVIII/24 h/10.sup.6 cells, respectively (FIG. 10). PLC population doubling time was not affected by transduction with any of the vectors; nor were phenotype or expression of signaling molecules involved in innate immunity. Importantly, at passage 3 following transduction with any of the 4 lentiviral vectors, PLCs continued to produce and secrete FVIII at similar levels to those observed shortly after transduction, demonstrating stable vector integration and durability/longevity of FVIII expression. The relative levels of FVIII expression by PLCs following transduction with each lentiviral vector were also assessed by immunofluorescence microscopy with an antibody specific to a region of FVIII that is conserved in all 4 FVIII transgenes. These analyses confirmed the results of the aPTT analyses on the supernatants from these cells, with mcoET3-PLC exhibiting the brightest/highest intensity staining for FVIII, followed by PLC transduced with ET3 (SEQ ID NO: 11), then those transduced with lcoET3 (SEQ ID NO: 10) and with lcoHSQ (SEQ ID NO: 14) (data not shown).

[0095] The gene transfer efficiency of these gene-modified cells was assessed by determining the final proviral/vector copy number (VCN) using a commercially available qPCR-based kit (Lenti-X Provirus Quantitation Kit, Takara Bio USA, Inc., Mountain View, Calif.). To ensure that only integrated copies were detected by the assay, qPCR for VCN was performed in PLCs that had been passaged at least three times after transduction. After transducing the cells at the same MOI (7.5) with each lentiviral vector, the VCNs for mcoET3-PLC, lcoHSQ-PLC, lcoET3-PLC, and ET3-PLC were all around 1.

Example 9. Optimizing Factor VIII Expression in Placental Cells

[0096] The aim of this study was to investigate the suitability of placental cells (PLC) as cellular delivery vehicles for FVIII. The expression of phenotypical markers was determined in three different master cells banks (101, 103, and 104) of placental cells (PLCs), each of which was derived from a different human donor by the Regenerative Medicine Clinical Core (RMCC) at WFIRM following GMP-compliant standard operating procedures (SOPs) established by the RMCC for PLC. Expression of CD29, CD44, CD73, CD90, CD105, HLA-ABC, HLA-E, CD31, CD34, CD35, CD144, HLA-G, HLA-DR/DP/DQ, and ABO blood group were determined using flow cytometric analysis. These markers were selected to confirm that the PLC isolated possessed a phenotype characteristic of MSC from other tissues (CD29, CD44, CD73, CD90, CD105), to assess their potential for stimulating an immune response upon transplantation (HLA-ABC, HLA-E, CD35, CD144, HLA-G, HLA-DR/DP/DQ, and ABO blood group), and to discern whether they expressed markers indicative of endothelial properties (CD31, CD34). No statistically significant differences (p<0.05) were found in expression of phenotypic markers between PLCs derived from three different master cell banks (101, 103, and 104). PLCs from each of the master cell banks expressed CD29, CD44, CD73, CD90, CD 105, HLA-ABC, and HLA-E (FIG. 8A); had negligible amounts (<1%) of CD31, CD34, CD35, CD144, HLA-G, and HLA-DR/DP/DQ (data not shown); and were devoid of ABO blood group (data not shown). Collectively, these findings support the conclusion that PLC are an MSC-like population and that they should exhibit minimal immunogenicity upon transplantation.

[0097] PLCs derived from each of the master cell banks [MCBs] (101, 103, and 104) were assessed for their ability to express FVIII protein constitutively. Immunofluorescence microscopy with a primary antibody specific to hFVIIIc and a fluorochrome-conjugated secondary antibody and flow cytometric analysis with fluorochrome-conjugated antibodies to were used to determine the levels of constitutively expressed FVIII protein and define the phenptype of the PLCs, respectively. As shown in FIG. 8A, these cells expressed markers characteristic of MSC from bone marrow and other tissues. All three MCBs endogenously expressed detectable amounts of FVIII by immunofluorescence microscopy, and MCB 103 expressed the highest levels, as indicated by the brightest fluorescence intensity (data not shown). The fold increase of FVIII expression over isotype control for PLCs derived from each of the MCBs is presented as relative mean fluorescence intensity (MFI) as shown in FIG. 8B.

[0098] The activated partial thromboplastin time (aPTT or PTT) assay is a functional measure of the intrinsic and common pathways of the coagulation cascade (i.e. it characterizes blood coagulation). The aPTT assay was used to quantitate levels of functional FVIII secreted by PLCs. PLCs were plated at the same density and cultured for 24 hours in Phenol Red-free alpha-MEM AmnioMax Complete Medium (ThermoFisher Scientific, Raleigh, N.C.). Supernatants were collected and the number of cells present counted, and then the levels of secreted functional FVIII were measured by the Clinical Hematology Laboratory at Wake Forest Baptist Health using a commercial aPTT assay. Levels of FVIII were normalized by adjusting to account for the number of cells present at the time of supernatant collection, and expressing FVIII activity on a per cell basis. The data from this analysis is shown in FIG. 8C

[0099] FVIII mRNA levels in the PLCs derived from three different master cell banks (101, 103, and 104) were evaluated by qPCR using primers specific to human FVIII. Relative expression of endogenous mRNA for FVIII was calculated by comparing the threshold cycle (CT) value for FVIII with the CT of each master cell bank's respective internal reference gene, GAPDH. The relative expression of endogenous FVIII mRNA was 0.01.+-.0.0005, 0.075.+-.0.007, and 0.011.+-.0.0002, for PLCs 101, 103, and 104, respectively.

Example 10. Suitability of PLCs as a Transgenic FVIII Production Platform

[0100] PLC 101, 103, and 104 were transduced at the same MOI (7.5) using a lentiviral vector-(LV) encoding mcoET3 (mcoET3-PLC) as described in Example 4 above. Vector copy number (VCN) was determined, as described in detail above. The VCN was found to be similar between the three different PLC MCBs (0.71-0.75). After transduction, the relative levels of expression of FVIII by the 3 MCB PLCs were assessed by immunofluorescence microscopy after staining with a primary antibody specific to hFVIIIc and a fluorochrome-conjugated secondary antibody. All 3 MCBs expressed high levels of FVIII after transduction with the mcoET3 lentiviral vector, but MCB 103 exhibited the highest levels of FVIII protein, as evidence by the brightest/highest fluorescence intensity (data not shown). The secretion of FVIII was determined using aPTT performed on 24-hour culture supernatants harvested from PLCs that were plated at the same density and normalized for the number of cells present at the time of the supernatant collection, as described in detail in the preceding paragraphs (FIG. 9A). Levels of FVIII in the culture supernatant increased significantly (p<0.05) in PLCs derived from all 3 MCBs (101, 103, and 104) when compared with respective non-transduced PLC counterparts (FIG. 9B). No significant differences were found between the different transduced cells.

[0101] The effect of transduction of PLCs with LV encoding mcoET3 on phenotype or molecules involved in immunity was assessed. Expression of CD29, CD44, CD73, CD90, CD 105, CD58, CD112, CD155, CD47, HLA-ABC, HLA-E, HLA-G, and HLA-DR/DP/DQ, were determined by flow cytometric analysis, as described above. No statistically significant differences (p<0.05) were found between transduced and non-transduced cells (FIGS. 8A-8C). Additionally, PLC population doubling time was not affected by transduction (data not shown). Both non-transduced and transduced PLCs expressed CD47, a molecule involved in immune evasion (FIG. 8B). Transduced cells did not significantly upregulate the expression of HLA-DR/DP/DQ (FIG. 8C).

[0102] To further examine whether transduction of the PLC with the mcoET3 lentiviral vector had the potential to alter the immunogenicity of these cells, we examined the levels of expression of various Toll-like Receptors (TLRs) on the PLC prior to and following transduction, as these molecules play a key role in innate immunity, and their upregulation could potentially trigger an immune response to the transduced cells upon transplantation. To address this possibility, the effect of PLC transduction with the mcoET3 lentiviral vector on TLR-3, TLR-4, TLR-7, TLR-8, and TLR-9 expression was assessed. TLR expression on transduced (t) and non-transduced (n) PLCs (101, 103, and 104) was determined using flow cytometric analysis. No significant differences in expression of TLR molecules was detected in the PLC populations. As shown in FIG. 11A, there was no difference in the levels of expression of any of these TLRs in transduced vs. non-transduced PLCs, confirming that transduction of these 3 MCB PLCs with the mcoET3 lentiviral vector did not lead to upregulation of any of these immune-stimulating molecules.

[0103] In order to evaluate the demands of PLC transduction with mcoET3 and increased Factor VIII expression on the secretory and endoplasmic reticulum pathways, expression of stress molecules MICA/B, ULBP-1, ULBP-2, and ULBP-3 was determined in transduced (t) and non-transduced (n) PLCs (101, 103, and 104). Flow cytometric analysis demonstrated that no significant expression or alteration/upregulation of MICA/B or ULBP-1 were found before or after transduction with mcoET3 lentiviral vector (FIG. 11B).

[0104] The production of interferon-gamma (IFN-.gamma.) by mcoET3 transduced and non-transduced PLCs was measured using a high-sensitivity ELISA (assay range: 0.16-10.0 pg/mL). PLCs were cultured for 24 hours in AmnioMax Complete Medium (ThermoFisher). Supernatants were collected and IFN-.gamma. production was determined. No IFN-.gamma. was detected in any of the culture supernatants of mcoET3-transduced or non-transduced PLCs (data not shown).

[0105] All features of the described systems are applicable to the described methods mutatis mutandis, and vice versa.

[0106] All patents, patent publications, patent applications, journal articles, books, technical references, and the like discussed in the instant disclosure are incorporated herein by reference in their entirety for all purposes.

[0107] It is to be understood that the figures and descriptions of the disclosure have been simplified to illustrate elements that are relevant for a clear understanding of the disclosure. It should be appreciated that the figures are presented for illustrative purposes and not as construction drawings. Omitted details and modifications or alternative embodiments are within the purview of persons of ordinary skill in the art.

[0108] It can be appreciated that, in certain aspects of the disclosure, a single component may be replaced by multiple components, and multiple components may be replaced by a single component, to provide an element or structure or to perform a given function or functions. Except where such substitution would not be operative to practice certain embodiments, such substitution is considered within the scope of the disclosure.

[0109] The examples presented herein are intended to illustrate potential and specific implementations of the invention. It can be appreciated that the examples are intended primarily for purposes of illustration for those skilled in the art. There may be variations to these diagrams or the operations described herein without departing from the spirit of the invention. For instance, in certain cases, method steps or operations may be performed or executed in differing order, or operations may be added, deleted or modified.

[0110] Different arrangements of the components depicted in the drawings or described above, as well as components and steps not shown or described are possible. Similarly, some features and sub-combinations are useful and may be employed without reference to other features and sub-combinations. Aspects and embodiments of the invention have been described for illustrative and not restrictive purposes, and alternative embodiments will become apparent to readers of this patent. Accordingly, the present invention is not limited to the embodiments described above or depicted in the drawings, and various embodiments and modifications can be made without departing from the scope of the claims below.

[0111] While exemplary embodiments have been described in some detail, by way of example and for clarity of understanding, those of skill in the art will recognize that a variety of modification, adaptations, and changes may be employed. Hence, the scope of the present invention should be limited solely by the claims.

Sequence CWU 1

1

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aaaccttcaa gactcgcgaa 1380gcaatccagt acgagtccgg aattctgggc ccactgctgt atggggaagt gggagacacc 1440ctgctgatca ttttcaagaa ccaggcctct aggccctaca atatctatcc tcatggcatt 1500acagatgtgt ctcccctgca cagtggacgc ctgcctaagg gcgtgaaaca cctgaaggac 1560ctgcctatcc tgccagggga aatttttaag tacaaatgga ctgtgaccgt cgaggatgga 1620ccaactaaga gcgaccccag gtgcctgacc cgctactatt ctagtttcat caatctggaa 1680agagatctgg caagcggact gatcggacca ctgctgattt gttacaaaga gtccgtggat 1740cagcgaggca accagatgat gtctgacaag cggaatgtga tcctgttctc agtctttgac 1800gaaaaccgca gctggtatct gaccgagaac atgcagcgat tcctgccaaa tgcagcagga 1860gtgcagccac aggatcctga gtttcaggct agtaacatca tgcattcaat taatggctac 1920gtgttcgact cactgcagct gagcgtgtgt ctgcacgagg tcgcttactg gtatatcctg 1980agcgtcggag cacagacaga tttcctgtcc gtgttctttt ctggctacac ttttaagcat 2040aaaatggtgt atgaggacac actgactctg ttcccttttt ccggcgaaac cgtctttatg 2100tctatggaga atccagggct gtgggtgctg ggatgccaca actccgattt ccggaataga 2160ggaatgactg ccctgctgaa agtgtcaagc tgtgaccgga acaccggcga ctactatgaa 2220gatacatacg aggacatccc aacttatctg ctgtctgaaa acaatgtgat tgagcccaga 2280agcttcagcc agaatccacc cgtgctgaag cgacaccagc gggaaatcac cctgactacc 2340ctgcagtcag agcaggaaga gattgattac gacgatacca tcagcattga aacaaaaagg 2400gaggacttcg atatctatgg ggaagacgag aaccagggac ctcgctcctt ccagaagagg 2460acacgccatt actttattgc tgcagtggag aggctgtggg attatgggat gtcccgctct 2520ccccacgtcc tgcgaaatcg ggcccagagt ggatcagtgc ctcagttcaa gaaagtggtc 2580ttccaggagt ttactgacgg gagctttacc cagcctctgt accggggaga actgaacgag 2640cacctgggac tgctgggccc atatatcaga gcagaagtgg aggataacat tatggtcacc 2700ttcaagaatc aggccagtcg gccctactca ttttattcct ctctgatcag ctacgaagag 2760gaccagcgcc agggcgcaga accacgaaaa aacttcgtga agcccaatga gaccaaaaca 2820tacttttgga aggtgcagca ccatatggct cctacaaaag acgaattcga ttgcaaggcc 2880tgggcttatt ttagtgacgt ggatctggag aaggacatgc actcagggct gatcggacct 2940ctgctgattt gtcatactaa caccctgaat ccagcacacg gacgacaggt gacagtccag 3000gaattcgctc tgttctttac aatcttcgat gagactaaga gctggtactt cactgaaaac 3060atggagagaa attgcagggc cccttgtaat atccagatgg aagacccaac attcaaggag 3120aactacagat ttcatgctat taatggctat gtgatggata ctctgccagg gctggtcatg 3180gcacaggacc agagaatcag gtggtacctg ctgtctatgg ggagtaacga gaatatccac 3240agcattcatt tctccggaca cgtgtttact gtcaggaaga aagaagagta taaaatggcc 3300gtgtacaacc tgtatccagg cgtgttcgaa accgtcgaga tgctgccaag caaggcagga 3360atctggcgag tggaatgcct gattggcgag cacctgcatg ctgggatgag taccctgttt 3420ctggtgtact caaaacagtg tcagacacct ctgggaatgg catctggcca tatccgggat 3480ttccagatta ccgcaagtgg acagtacgga cagtgggctc caaagctggc aagactgcac 3540tatagcggct ccatcaacgc ctggtctaca aaagagccct ttagttggat taaggtggac 3600ctgctggccc ctatgatcat tcatggcatc aaaactcagg gggctaggca gaagttcagt 3660tcactgtaca tcagccagtt tatcatcatg tactccctgg atgggaagaa atggcagacc 3720taccgcggga atagcacagg aactctgatg gtgttctttg gaaacgtcga cagctccggc 3780atcaagcaca acattttcaa tcctccaatc attgcccgct acatccgact gcaccccacc 3840cattattcaa ttcgaagcac actgcggatg gaactgatgg gctgcgatct gaactcttgt 3900agtatgcctc tggggatgga gtctaaggcc atcagtgacg ctcagattac cgcatctagt 3960tacttcacca atatgtttgc cacatggtca ccaagccagg ctaggctgca cctgcaggga 4020agaacaaacg cctggaggcc tcaggtgaac aatccaaagg agtggctgca ggtggatttc 4080cagaaaacta tgaaggtcac cggaatcaca actcagggcg tgaaatcact gctgaccagc 4140atgtatgtga aggagtttct gatttcaagc tcccaggacg gccaccattg gacactgttc 4200ctgcagaacg ggaaggtgaa agtcttccag ggaaatcagg attcttttac accagtggtc 4260aacagtctgg acccccctct gctgactcgg tacctgagaa tccaccccca gagctgggtc 4320catcagattg cactgcgact ggaagtgctg ggatgcgagg cacagcagct gtattga 437784359DNAArtificial Sequencesynthetic lcoAn53 8atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgcagttctc attctctgcc 60accaggagat actacctggg cgccgtggag ctgagctggg actacatgca gtctgacctg 120ctgtctgagc tgcatgtgga caccaggttc ccccccagag tgccccgaag cttccccttc 180aacaccagcg tgatgtacaa gaagaccgtg ttcgtggagt tcactgacca cctgttcaac 240atcgccaagc ccaggccccc ctggatgggc ctgctgggcc ccaccatcag agccgaggtg 300tacgacaccg tggtcatcac cctgaagaac atggccagcc accccgtctc cctgcacgcc 360gtgggggtga gctactggaa ggcctctgag ggcgccgagt acgacgacca gaccagccag 420agggagaagg aggacgacaa ggtgttccct ggggaaagcc acacctacgt gtggcaggtc 480ctgaaggaga acggccccat ggcctctgac cccccatgcc tgacctacag ctacctgagc 540cacgtggacc tggtgaagga cctgaactct ggcctgattg gggccctgct ggtgtgcagg 600gagggcagcc tggccaagga gagaacccag accctgcacc agttcgtgct gctgttcgcc 660gtgttcgacg agggcaagag ctggcactct gaaaccaagg atagcctgac tcaggccatg 720gactctgcct ctgccagggc ctggcccaag atgcacaccg tcaacggcta cgtcaacagg 780agcctgcctg gcctgattgg ctgccacagg aagagcgtgt actggcatgt gatcggcatg 840ggcaccaccc ctgaggtgca cagcatcttc ctggagggcc acaccttcct ggtcaggaac 900cacaggcagg ccagcctgga gatcagcccc atcaccttcc tgaccgccca gaccctgctg 960atggacctgg gccagttcct gctgttctgc cacatctcca gccaccagca cgacggcatg 1020gaggcctacg tgaaagtgga cagctgccct gaggagcccc agctgaggat gaagaacaac 1080gaggaggagg aggactatga tgacgacctg tatgacagcg agatggacgt ggtcaggttc 1140gacgacgaca acagcccccc tttcatccag atcaggagcg tggccaagaa gcaccccaag 1200acctgggtgc actacatcgc tgctgaggag gaggactggg actatgcccc ctccgtgctg 1260acccctgatg acaggagcta caagagccag tacctgaaca atggccccca gaggattggc 1320aggaagtaca agaaagtcag gttcatggcc tacactgatg aaaccttcaa gaccagggag 1380gccatccagt acgagtctgg catcctgggc cccctgctgt acggggaggt gggggacacc 1440ctgctgatca tcttcaagaa ccaggccagc aggccctaca acatctaccc ccatggcatc 1500accgacgtga gccccctgca cagcggaagg ctgcctaagg gggtgaagca cctgaaagac 1560ctgcccatcc tgcctgggga gatcttcaag tacaagtgga ctgtgactgt ggaggacggc 1620cccaccaaga gcgaccccag gtgcctgacc agatactaca gcagcttcat caacctggag 1680agggacctgg cctctggcct gattggcccc ctgctgatct gctacaagga gtctgtggac 1740cagaggggca accagatgat gagcgacaag aggaacgtga tcctgttctc tgtcttcgac 1800gagaacagga gctggtacct gaccgagaac atgcagaggt tcctgcccaa cgcagctggg 1860gtgcagccac aggaccccga gttccaggcc agcaacatca tgcacagcat caatggctac 1920gtgttcgaca gcctgcagct gagcgtgtgc ctgcacgagg tggcctactg gtacatcctg 1980agcgtcggcg cccagaccga cttcctgagc gtgttcttct ctggctacac cttcaagcac 2040aagatggtgt atgaggacac cctgaccctg ttccccttca tgagcatgga gaaccctggc 2100ctgtgggtgc tgggctgcca caacagcgac ttcaggaaca ggggcatgac tgccctgctg 2160aaagtctcca gctgtgaccg gaacaccggg gactactacg aggacacata cgaggacatc 2220ccaacttacc tgctgagcga aaacaatgtg atcgagccca ggagcttctc tcagaacccc 2280ccagtgctga agaggcacca gagggagatc accctgacca ccctgcagtc tgagcaggag 2340gagatcgact atgatgacac catcagcatt gagacaaaga gggaggactt cgacatctac 2400ggggaggacg agaaccaggg acccaggagc ttccagaaga ggaccaggca ctacttcatt 2460gctgctgtgg agaggctgtg ggactatggc atgtcccgca gcccccatgt gctgaggaac 2520agggcccagt ctggcagcgt gccccagttc aagaaagtcg tgttccagga gttcaccgac 2580ggcagcttca cccagcccct gtacagaggg gagctgaacg agcacctggg cctgctgggc 2640ccctacatca gggccgaggt ggaggacaac atcatggtga ccttcaagaa ccaggccagc 2700aggccctaca gcttctacag cagcctgatc agctacgagg aggaccagag gcagggggct 2760gagcccagga agaactttgt gaagcccaat gaaaccaaga cctacttctg gaaggtgcag 2820caccacatgg cccccaccaa ggacgagttc gactgcaagg cctgggccta cttctctgac 2880gtggacctgg agaaggacat gcactctggc ctgattggcc ccctgctgat ttgccacacc 2940aacaccctga accctgccca tggcaggcag gtgactgtgc aggagttcgc cctgttcttc 3000accatcttcg atgaaaccaa gagctggtac ttcactgaga acatggagag gaactgcagg 3060gccccctgca acatccagat ggaggacccc accttcaagg agaactacag gttccatgcc 3120atcaatggct acgtgatgga caccctgcct ggcctggtca tggcccagga ccagaggatc 3180aggtggtatc tgctgagcat gggcagcaac gagaacatcc acagcatcca cttctctggc 3240cacgtgttca ctgtgaggaa gaaggaggag tacaagatgg ccgtgtacaa cctgtaccct 3300ggggtgttcg aaaccgtgga gatgctgccc agcaaggccg gcatctggag ggtggagtgc 3360ctgattgggg agcacctgca cgccggcatg agcaccctgt tcctggtgta cagcaaacag 3420tgccagaccc ccctgggcat ggcctctggc cacatcaggg acttccagat cactgcctct 3480ggccagtacg gccagtgggc ccccaagctg gccaggctgc actactccgg aagcatcaat 3540gcctggagca ccaaggagcc cttcagctgg atcaaagtgg acctgctggc ccccatgatc 3600atccacggca tcaagaccca gggggccagg cagaagttct ccagcctgta catcagccag 3660ttcatcatca tgtacagcct ggacggcaag aagtggcaga cctacagggg caacagcacc 3720ggcaccctga tggtgttctt cggcaacgtg gacagcagcg gcatcaagca caacatcttc 3780aaccccccca tcatcgccag atacatcagg ctgcacccca cccactacag catcaggagc 3840accctgagga tggagctgat gggctgtgac ctgaacagct gcagcatgcc cctgggcatg 3900gagagcaagg ccatctctga cgcccagatc actgcctcca gctacttcac caacatgttt 3960gccacctgga gccccagcca ggccaggctg cacctgcagg gcaggacaaa tgcctggagg 4020ccccaggtca acaaccccaa ggagtggctg caggtggact tccagaagac catgaaggtg 4080actgggatca ccacccaggg ggtgaagagc ctgctgacca gcatgtacgt gaaggagttc 4140ctgatctcca gcagccagga cggccaccat tggaccctgt tcctgcagaa tggcaaggtg 4200aaggtgttcc agggcaacca ggacagcttc acccctgtgg tcaacagcct ggaccccccc 4260ctgctgacca gatacctgag gatccacccc cagagctggg tgcaccagat cgccctgagg 4320ctggaggtgc tgggctgtga ggcccagcag ctgtactga 435994404DNAArtificial Sequencesynthetic lcoET3 with CpGs 9atgcagctgg aactgtctac ctgtgtgttt ctgtgtctgc tgcctctggg gttttctgct 60atccgccgct actatctggg agccgtggag ctgtcctggg actacaggca gagcgagctg 120ctgagagaac tgcacgtgga taccagattc ccagctaccg ctccaggagc tctgcctctg 180ggcccatccg tgctgtacaa gaaaaccgtc ttcgtggagt ttaccgacca gctgttcagc 240gtggccaggc caagaccacc ttggatggga ctgctgggac caaccatcca ggctgaggtg 300tacgataccg tggtcgtgac cctgaaaaac atggcctccc atcccgtgag cctgcacgct 360gtcggggtgt ccttctggaa gtccagcgag ggagccgagt acgaagacca tacctcccag 420cgcgagaaag aagacgataa ggtgctgcct ggcaaaagcc agacctatgt ctggcaggtg 480ctgaaggaga acggaccaac cgctagcgac ccaccatgcc tgacctactc ttatctgtcc 540cacgtcgatc tggtgaagga cctgaattcc ggactgatcg gagctctgct ggtgtgtaga 600gagggaagcc tgaccagaga aagaacccag aacctgcatg agttcgtcct gctgttcgcc 660gtgtttgacg aagggaagag ctggcactct gcccgcaatg actcctggac cagagctatg 720gatccagctc ctgctagagc tcagcctgct atgcacaccg tcaacggcta cgtgaatcgg 780tctctgccag gactgatcgg ctgccataag aaaagcgtct attggcacgt gatcggaatg 840ggcaccagcc ccgaggtgca ttctatcttc ctggaaggcc acacctttct ggtcaggcac 900catagacagg cctctctgga gatctcccct ctgaccttcc tgaccgctca gacctttctg 960atggacctgg ggcagttcct gctgttttgc catatctctt cccaccatca cggaggaatg 1020gaggctcacg tcagggtgga atcctgtgct gaggaaccac agctgagaag aaaggctgat 1080gaggaagagg actacgacga taacctgtat gacagcgata tggacgtcgt gcgcctggac 1140ggcgacgatg tcagcccttt catccagatc cggtctgtgg ccaagaaaca tccaaagacc 1200tgggtccact acatcgccgc tgaagaggaa gattgggact atgcccccct ggtgctggct 1260cctgacgata gatcctacaa aagccagtat ctgaacaatg ggccccagcg catcggacgg 1320aagtacaaga aagtgaggtt catggcctat accgacgaga cctttaagac cagagaggct 1380atccagcacg aatccgggat cctgggacct ctgctgtacg gcgaagtggg ggataccctg 1440ctgatcatct tcaagaacca ggcctccagg ccatacaata tctatcccca tggcatcacc 1500gacgtgagac cactgtacag caggagactg cccaaggggg tcaaacacct gaaggatttc 1560cccatcctgc ctggagagat ctttaagtat aaatggaccg tcaccgtgga agacgggcct 1620accaagtccg atccacgctg cctgacccgg tactatagct ctttcgtgaa catggagaga 1680gacctggcta gcggactgat cggacccctg ctgatctgtt acaaagagag cgtggaccag 1740aggggcaacc agatcatgtc tgataagaga aatgtcatcc tgttctccgt gtttgacgag 1800aaccgcagct ggtacctgac cgagaacatc cagcggttcc tgccaaatcc agctggagtg 1860cagctggagg acccagaatt tcaggcttcc aacatcatgc atagcatcaa tggctacgtg 1920ttcgatagcc tgcagctgtc tgtctgcctg cacgaggtgg cctactggta tatcctgtcc 1980atcggcgctc agaccgactt cctgtccgtg ttctttagcg ggtacacctt taagcataaa 2040atggtgtatg aggataccct gaccctgttc cccttttctg gcgagaccgt gttcatgtcc 2100atggaaaacc ctggcctgtg gatcctgggg tgccacaaca gcgacttcag gaatagagga 2160atgaccgccc tgctgaaagt gtccagctgt gataagaata ccggcgatta ctatgaggac 2220tcttacgaag atatctccgc ttatctgctg agcaagaaca atgccatcga gcccaggtct 2280ttcgctcaga actccagacc tccaagcgct tctgctccta agccacctgt gctgagaaga 2340catcagaggg acatctccct gcctaccttc cagccagagg aagataaaat ggactacgac 2400gatatcttca gcaccgagac caagggggaa gattttgaca tctatggaga ggacgaaaac 2460caggatccaa gatccttcca gaagagaacc agacactact ttatcgccgc tgtggagcag 2520ctgtgggact atgggatgtc cgaaagccca cgggccctga ggaacagagc tcagaatgga 2580gaggtgcccc gcttcaagaa agtcgtgttc cgggagtttg ccgacggcag ctttacccag 2640ccatcttaca ggggggagct gaacaagcat ctggggctgc tgggacccta tatcagagcc 2700gaggtcgaag ataacatcat ggtgaccttc aagaatcagg cttctcgccc ctactccttt 2760tattcttccc tgatctccta ccctgacgat caggagcagg gcgccgaacc taggcacaac 2820ttcgtgcagc caaatgagac cagaacctac ttttggaagg tgcagcatca catggctccc 2880accgaggatg aattcgactg caaagcttgg gcctattttt ccgatgtcga cctggagaag 2940gacgtgcata gcggcctgat cgggcctctg ctgatctgtc gcgccaacac cctgaatgct 3000gctcacggaa gacaggtcac cgtgcaggag ttcgctctgt tctttaccat ctttgacgaa 3060accaagagct ggtacttcac cgagaacgtg gaaaggaatt gcagagcccc ctgtcatctg 3120cagatggagg accctaccct gaaggaaaac tacaggttcc acgccatcaa tggatatgtc 3180atggataccc tgcccggcct ggtcatggct cagaaccagc gcatccggtg gtacctgctg 3240tctatgggat ccaacgagaa tatccatagc atccacttct ctggccatgt cttttccgtg 3300aggaagaaag aggaatacaa aatggccgtg tacaatctgt atcctggggt cttcgagacc 3360gtggaaatgc tgccaagcaa agtgggaatc tggagaatcg agtgcctgat cggcgaacac 3420ctgcaggccg ggatgagcac caccttcctg gtgtactcta agaaatgtca gaccccactg 3480gggatggcct ccggacatat ccgcgacttc cagatcaccg ctagcggaca gtacggacag 3540tgggctccaa agctggctag actgcactat tctggctcca tcaacgcctg gtctaccaaa 3600gagccattct cctggatcaa ggtggacctg ctggccccca tgatcatcca cggaatcaaa 3660acccagggcg ctaggcagaa gttcagctct ctgtacatct cccagtttat catcatgtat 3720agcctggacg ggaagaaatg gcagacctac agaggcaatt ccaccgggac cctgatggtc 3780ttctttggaa acgtggattc cagcggcatc aagcacaaca tcttcaatcc acccatcatc 3840gcccgctaca tccggctgca tcctacccac tatagcatca ggtctaccct gagaatggag 3900ctgatgggat gcgacctgaa cagctgttct atgccactgg gcatggagtc caaggctatc 3960agcgatgccc agatcaccgc ttcttcctac ttcaccaata tgtttgctac ctggtcccca 4020agcaaggcta gactgcacct gcagggaaga tccaacgctt ggagacccca ggtgaacaat 4080cctaaggagt ggctgcaggt cgacttccag aaaaccatga aggtcaccgg ggtgaccacc 4140cagggagtga aatctctgct gacctccatg tacgtcaagg agttcctgat cagctcttcc 4200caggacggcc accagtggac cctgttcttt cagaacggca aggtcaaagt gttccagggg 4260aatcaggact cttttacccc cgtcgtgaac tccctggatc ctccactgct gaccaggtac 4320ctgagaatcc atcctcagag ctgggtgcac cagatcgctc tgagaatgga ggtcctggga 4380tgcgaagctc aggacctgta ttga 4404104403DNAArtificial Sequencesynthetic lcoET3 CpGs removed 10atgcagctgg aactgtctac ctgtgtgttt ctgtgtctgc tgcctctggg gttttctgct 60atcaggagat actatctggg agctgtggag ctgtcctggg actacaggca gtctgagctg 120ctgagagaac tgcatgtgga taccagattc ccagctacag ctccaggagc tctgcctctg 180ggcccatctg tgctgtacaa gaaaacagtc tttgtggagt ttacagacca gctgttctct 240gtggccaggc caagaccacc ttggatggga ctgctgggac caaccatcca ggctgaggtg

300tatgatacag tggtggtgac cctgaaaaac atggcctccc atcctgtgag cctgcatgct 360gtgggggtgt ccttctggaa gtcctctgag ggagctgagt atgaagacca tacctcccag 420agggagaaag aagatgataa ggtgctgcct ggcaaaagcc agacctatgt ctggcaggtg 480ctgaaggaga atggaccaac tgcttctgac ccaccatgcc tgacctactc ttatctgtcc 540catgtggatc tggtgaagga cctgaattct ggactgattg gagctctgct ggtgtgtaga 600gagggaagcc tgaccagaga aagaacccag aacctgcatg agtttgtcct gctgtttgct 660gtgtttgatg aagggaagag ctggcactct gccaggaatg actcctggac cagagctatg 720gatccagctc ctgctagagc tcagcctgct atgcacacag tcaatggcta tgtgaatagg 780tctctgccag gactgattgg ctgccataag aaatctgtct attggcatgt gattggaatg 840ggcaccagcc ctgaggtgca ttctatcttc ctggaaggcc acacctttct ggtcaggcac 900catagacagg cctctctgga gatctcccct ctgaccttcc tgacagctca gacctttctg 960atggacctgg ggcagttcct gctgttttgc catatctctt cccaccatca tggaggaatg 1020gaggctcatg tcagggtgga atcctgtgct gaggaaccac agctgagaag aaaggctgat 1080gaggaagagg actatgatga taacctgtat gactctgata tggatgtggt gaggctggat 1140ggggatgatg tcagcccttt catccagatc aggtctgtgg ccaagaaaca tccaaagacc 1200tgggtccact acattgctgc tgaagaggaa gattgggact atgcccccct ggtgctggct 1260cctgatgata gatcctacaa aagccagtat ctgaacaatg ggccccagag gattggaagg 1320aagtacaaga aagtgaggtt catggcctat acagatgaga cctttaagac cagagaggct 1380atccagcatg aatctgggat cctgggacct ctgctgtatg gagaagtggg ggatacctgc 1440tgatcatctt caagaaccag gcctccaggc catacaatat ctatccccat ggcatcacag 1500atgtgagacc actgtacagc aggagactgc ccaagggggt caaacacctg aaggatttcc 1560ccatcctgcc tggagagatc tttaagtata aatggacagt cacagtggaa gatgggccta 1620ccaagtctga tccaaggtgc ctgaccagat actatagctc ttttgtgaac atggagagag 1680acctggcttc tggactgatt ggacccctgc tgatctgtta caaagagtct gtggaccaga 1740ggggcaacca gatcatgtct gataagagaa atgtcatcct gttctctgtg tttgatgaga 1800acaggagctg gtacctgaca gagaacatcc agaggttcct gccaaatcca gctggagtgc 1860agctggagga cccagaattt caggcttcca acatcatgca tagcatcaat ggctatgtgt 1920ttgatagcct gcagctgtct gtctgcctgc atgaggtggc ctactggtat atcctgtcca 1980ttggagctca gacagacttc ctgtctgtgt tctttagtgg gtacaccttt aagcataaaa 2040tggtgtatga ggataccctg accctgttcc ccttttctgg ggagacagtg ttcatgtcca 2100tggaaaaccc tggcctgtgg atcctggggt gccacaactc tgacttcagg aatagaggaa 2160tgacagccct gctgaaagtg tccagctgtg ataagaatac aggggattac tatgaggact 2220cttatgaaga tatctctgct tatctgctga gcaagaacaa tgccattgag cccaggtctt 2280ttgctcagaa ctccagacct ccatctgctt ctgctcctaa gccacctgtg ctgagaagac 2340atcagaggga catctccctg cctaccttcc agccagagga agataaaatg gactatgatg 2400atatcttcag cacagagacc aagggggaag attttgacat ctatggagag gatgaaaacc 2460aggatccaag atccttccag aagagaacca gacactactt tattgctgct gtggagcagc 2520tgtgggacta tgggatgtct gaaagcccaa gggccctgag gaacagagct cagaatggag 2580aggtgcccag attcaagaaa gtggtgttca gagagtttgc tgatggcagc tttacccagc 2640catcttacag gggggagctg aacaagcatc tggggctgct gggaccctat atcagagctg 2700aggtggaaga taacatcatg gtgaccttca agaatcaggc ttctaggccc tactcctttt 2760attcttccct gatctcctac cctgatgatc aggagcaggg agctgaacct aggcacaact 2820ttgtgcagcc aaatgagacc agaacctact tttggaaggt gcagcatcac atggctccca 2880cagaggatga atttgactgc aaagcttggg cctatttttc tgatgtggac ctggagaagg 2940atgtgcattc tggcctgatt gggcctctgc tgatctgtag ggccaacacc ctgaatgctg 3000ctcatggaag acaggtcaca gtgcaggagt ttgctctgtt ctttaccatc tttgatgaaa 3060ccaagagctg gtacttcaca gagaatgtgg aaaggaattg cagagccccc tgtcatctgc 3120agatggagga ccctaccctg aaggaaaact acaggttcca tgccatcaat ggatatgtca 3180tggataccct gcctggcctg gtcatggctc agaaccagag gatcagatgg tacctgctgt 3240ctatgggatc caatgagaat atccatagca tccacttctc tggccatgtc ttttctgtga 3300ggaagaaaga ggaatacaaa atggctgtgt acaatctgta tcctggggtc tttgagacag 3360tggaaatgct gccaagcaaa gtgggaatct ggagaattga gtgcctgatt ggggaacacc 3420tgcaggctgg gatgagcacc accttcctgg tgtactctaa gaaatgtcag accccactgg 3480ggatggcctc tggacatatc agggacttcc agatcacagc ttctggacag tatggacagt 3540gggctccaaa gctggctaga ctgcactatt ctggctccat caatgcctgg tctaccaaag 3600agccattctc ctggatcaag gtggacctgc tggcccccat gatcatccat ggaatcaaaa 3660cccagggagc taggcagaag ttcagctctc tgtacatctc ccagtttatc atcatgtata 3720gcctggatgg gaagaaatgg cagacctaca gaggcaattc cactgggacc ctgatggtct 3780tctttggaaa tgtggattcc tctggcatca agcacaacat cttcaatcca cccatcattg 3840ccaggtacat caggctgcat cctacccact atagcatcag gtctaccctg agaatggagc 3900tgatgggatg tgacctgaac agctgttcta tgccactggg catggagtcc aaggctatct 3960ctgatgccca gatcacagct tcttcctact tcaccaatat gtttgctacc tggtccccaa 4020gcaaggctag actgcacctg cagggaagat ccaatgcttg gagaccccag gtgaacaatc 4080ctaaggagtg gctgcaggtg gacttccaga aaaccatgaa ggtcacaggg gtgaccaccc 4140agggagtgaa atctctgctg acctccatgt atgtcaagga gttcctgatc agctcttccc 4200aggatggcca ccagtggacc ctgttctttc agaatggcaa ggtcaaagtg ttccagggga 4260atcaggactc ttttacccca gtggtgaact ccctggatcc tccactgctg accaggtacc 4320tgagaatcca tcctcagagc tgggtgcacc agattgctct gagaatggag gtcctgggat 4380gtgaagctca ggacctgtat tga 4403114404DNAArtificial Sequencesynthetic NoCoET3 11atgcagctag agctctccac ctgtgtcttt ctgtgtctct tgccactcgg ctttagtgcc 60atcaggagat actacctggg cgcagtggaa ctgtcctggg actaccggca aagtgaactc 120ctccgtgagc tgcacgtgga caccagattt cctgctacag cgccaggagc tcttccgttg 180ggcccgtcag tcctgtacaa aaagactgtg ttcgtagagt tcacggatca acttttcagc 240gttgccaggc ccaggccacc atggatgggt ctgctgggtc ctaccatcca ggctgaggtt 300tacgacacgg tggtcgttac cctgaagaac atggcttctc atcccgttag tcttcacgct 360gtcggcgtct ccttctggaa atcttccgaa ggcgctgaat atgaggatca caccagccaa 420agggagaagg aagacgataa agtccttccc ggtaaaagcc aaacctacgt ctggcaggtc 480ctgaaagaaa atggtccaac agcctctgac ccaccatgtc ttacctactc atacctgtct 540cacgtggacc tggtgaaaga cctgaattcg ggcctcattg gagccctgct ggtttgtaga 600gaagggagtc tgaccagaga aaggacccag aacctgcacg aatttgtact actttttgct 660gtctttgatg aagggaaaag ttggcactca gcaagaaatg actcctggac acgggccatg 720gatcccgcac ctgccagggc ccagcctgca atgcacacag tcaatggcta tgtcaacagg 780tctctgccag gtctgatcgg atgtcataag aaatcagtct actggcacgt gattggaatg 840ggcaccagcc cggaagtgca ctccattttt cttgaaggcc acacgtttct cgtgaggcac 900catcgccagg cttccttgga gatctcgcca ctaactttcc tcactgctca gacattcctg 960atggaccttg gccagttcct actgttttgt catatctctt cccaccacca tggtggcatg 1020gaggctcacg tcagagtaga aagctgcgcc gaggagcccc agctgcggag gaaagctgat 1080gaagaggaag attatgatga caatttgtac gactcggaca tggacgtggt ccggctcgat 1140ggtgacgacg tgtctccctt tatccaaatc cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac gcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atccgcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga acctaggagc 2280tttgcccaga attcaagacc ccctagtgcg agcgctccaa agcctccggt cctgcgacgg 2340catcagaggg acataagcct tcctactttt cagccggagg aagacaaaat ggactatgat 2400gatatcttct caactgaaac gaagggagaa gattttgaca tttacggtga ggatgaaaat 2460caggaccctc gcagctttca gaagagaacc cgacactatt tcattgctgc ggtggagcag 2520ctctgggatt acgggatgag cgaatccccc cgggcgctaa gaaacagggc tcagaacgga 2580gaggtgcctc ggttcaagaa ggtggtcttc cgggaatttg ctgacggctc cttcacgcag 2640ccgtcgtacc gcggggaact caacaaacac ttggggctct tgggacccta catcagagcg 2700gaagttgaag acaacatcat ggtaactttc aaaaaccagg cgtctcgtcc ctattccttc 2760tactcgagcc ttatttctta tccggatgat caggagcaag gggcagaacc tcgacacaac 2820ttcgtccagc caaatgaaac cagaacttac ttttggaaag tgcagcatca catggcaccc 2880acagaagacg agtttgactg caaagcctgg gcctactttt ctgatgttga cctggaaaaa 2940gatgtgcact caggcttgat cggccccctt ctgatctgcc gcgccaacac cctgaacgct 3000gctcacggta gacaagtgac cgtgcaagaa tttgctctgt ttttcactat ttttgatgag 3060acaaagagct ggtacttcac tgaaaatgtg gaaaggaact gccgggcccc ctgccatctg 3120cagatggagg accccactct gaaagaaaac tatcgcttcc atgcaatcaa tggctatgtg 3180atggatacac tccctggctt agtaatggct cagaatcaaa ggatccgatg gtatctgctc 3240agcatgggca gcaatgaaaa tatccattcg attcatttta gcggacacgt gttcagtgta 3300cggaaaaagg aggagtataa aatggccgtg tacaatctct atccgggtgt ctttgagaca 3360gtggaaatgc taccgtccaa agttggaatt tggcgaatag aatgcctgat tggcgagcac 3420ctgcaagctg ggatgagcac gactttcctg gtgtacagca agaagtgtca gactcccctg 3480ggaatggctt ctggacacat tagagatttt cagattacag cttcaggaca atatggacag 3540tgggccccaa agctggccag acttcattat tccggatcaa tcaatgcctg gagcaccaag 3600gagccctttt cttggatcaa ggtggatctg ttggcaccaa tgattattca cggcatcaag 3660acccagggtg cccgtcagaa gttctccagc ctctacatct ctcagtttat catcatgtat 3720agtcttgatg ggaagaagtg gcagacttat cgaggaaatt ccactggaac cttaatggtc 3780ttctttggca atgtggattc atctgggata aaacacaata tttttaaccc tccaattatt 3840gctcgataca tccgtttgca cccaactcat tatagcattc gcagcactct tcgcatggag 3900ttgatgggct gtgatttaaa tagttgcagc atgccattgg gaatggagag taaagcaata 3960tcagatgcac agattactgc ttcatcctac tttaccaata tgtttgccac ctggtctcct 4020tcaaaagctc gacttcacct ccaagggagg agtaatgcct ggagacctca ggtgaataat 4080ccaaaagagt ggctgcaagt ggacttccag aagacaatga aagtcacagg agtaactact 4140cagggagtaa aatctctgct taccagcatg tatgtgaagg agttcctcat ctccagcagt 4200caagatggcc atcagtggac tctctttttt cagaatggca aagtaaaggt ttttcaggga 4260aatcaagact ccttcacacc tgtggtgaac tctctagacc caccgttact gactcgctac 4320cttcgaattc acccccagag ttgggtgcac cagattgccc tgaggatgga ggttctgggc 4380tgcgaggcac aggacctcta ctga 4404124404DNAArtificial Sequencesynthetic mcoET3 12atgcagctgg agctctcaac ctgtgtgttc ctctgcctgc tccccctggg attttcagct 60atcaggagat actatctggg agcagtggaa ctgtcctggg actacaggca gtcagagctg 120ctcagagaac tgcatgtgga tactaggttc cctgcaacag ctcctggagc actgccactg 180ggaccttcag tgctgtacaa gaaaactgtc tttgtggagt ttacagacca gctgttcagt 240gtggccaggc ccaggccccc ctggatgggg ctgctgggac ccaccatcca ggctgaagtg 300tatgatactg tggtggtgac cctgaaaaac atggcctctc atccagtcag cctgcatgct 360gtgggagtga gcttctggaa gagcagtgag ggagctgagt atgaagacca tacctcacag 420agggagaaag aagatgataa ggtgctgcca ggaaaaagcc agacctatgt gtggcaggtg 480ctgaaggaga atggccctac agcttcagat cctccctgcc tcacatactc ttatctgagc 540catgtggatc tggtgaagga cctcaatagt ggcctgattg gggcactgct ggtgtgcaga 600gaggggtccc tcacaaggga aagaactcag aacctgcatg agtttgtcct gctctttgct 660gtgtttgatg agggaaagtc ctggcactca gcaaggaatg acagctggac cagggctatg 720gacccagcac cagccagagc tcagccagct atgcacactg tcaatggcta tgtgaatagg 780tccctgcctg gactcattgg ctgccataag aaatcagtct attggcatgt gattggaatg 840ggcaccagcc cagaggtgca ttccatcttc ctggaaggcc acacatttct ggtcaggcac 900catagacagg ccagcctgga gatcagccca ctgactttcc tcacagcaca gacatttctg 960atggacctgg ggcagttcct gctcttttgc catatctcaa gtcaccatca tggagggatg 1020gaggctcatg tcagggtgga aagctgtgca gaggaacctc agctgaggag gaaggcagat 1080gaggaagagg actatgatga taacctgtat gactcagata tggatgtggt gaggctggat 1140ggagatgatg tcagcccatt catccagatc aggtcagtgg ctaagaaaca ccctaagacc 1200tgggtccact acattgcagc tgaagaggaa gattgggact atgcacccct ggtgctggcc 1260ccagatgata gaagttacaa atctcagtat ctgaacaatg ggccccagag gattggaagg 1320aagtacaaga aagtgaggtt catggcttat actgatgaga cctttaagac aagagaggca 1380atccagcatg aaagtggcat cctgggacca ctgctctatg gagaagtggg ggataccctg 1440ctcatcatct tcaagaacca ggcctcaagg ccttacaata tctatcccca tggcatcaca 1500gatgtgaggc ctctctacag caggagactg cccaagggag tcaaacacct caaggatttc 1560cccatcctgc caggggaaat cttcaagtat aaatggacag tcactgtgga agatgggcca 1620actaagtcag atcctaggtg cctgaccagg tactattcta gctttgtgaa catggagagg 1680gacctggctt caggactgat tggacctctg ctcatctgct acaaagaatc agtggaccag 1740aggggcaacc agatcatgag tgataagaga aatgtcatcc tgttctcagt gtttgatgag 1800aataggagtt ggtatctgac agaaaacatc cagaggttcc tgcctaatcc tgcaggagtg 1860cagctggagg acccagaatt tcaggcttca aacatcatgc atagtatcaa tggctatgtg 1920tttgatagtc tgcagctctc tgtctgcctg catgaggtgg cctactggta tatcctcagc 1980attggagctc agactgactt cctgagtgtg ttcttttcag gctacacatt caagcataag 2040atggtctatg aagataccct gacactcttc cccttttctg gggagactgt gtttatgagc 2100atggaaaacc caggcctgtg gattctgggg tgccacaaca gtgacttcag gaatagaggg 2160atgactgctc tgctcaaagt gtcctcatgt gataagaata ctggagatta ctatgaggac 2220tcttatgaag atatcagtgc atatctgctc tccaaaaaca atgccattga gcccaggtca 2280tttgctcaga acagtagacc accttctgca agtgcaccaa agcctccagt gctgaggaga 2340caccagaggg acatcagcct gccaaccttc cagcctgagg aagataaaat ggactatgat 2400gatatcttct ccactgagac caagggggaa gattttgaca tctatggaga ggatgaaaac 2460caggacccca ggtccttcca gaagaggacc agacactact ttattgcagc tgtggagcag 2520ctgtgggact atggcatgtc tgaatcacct agagctctga ggaacagagc acagaatggg 2580gaggtgccca ggttcaagaa agtggtgttc agagaatttg cagatggctc ttttacccag 2640cctagctaca ggggggagct caacaagcat ctggggctgc tgggacccta tatcagagca 2700gaggtggaag ataacatcat ggtgacattc aagaatcagg cctcaagacc ctacagtttt 2760tatagttctc tgatcagcta cccagatgat caggagcagg gggctgaacc aaggcacaac 2820tttgtgcagc ctaatgagac aagaacttac ttttggaagg tccagcatca catggctccc 2880acagaggatg agtttgactg caaggcctgg gcatattttt ctgatgtgga cctggagaag 2940gatgtgcata gtggcctcat tgggccactg ctcatctgca gggcaaacac actgaatgct 3000gcacatggca ggcaggtcac tgtgcaggag tttgccctgt tctttacaat ctttgatgaa 3060actaagtcct ggtacttcac agagaatgtg gaaaggaatt gcagagcccc ctgccatctc 3120cagatggagg acccaactct gaaggaaaac tacaggttcc atgctatcaa tggatatgtc 3180atggatacac tgccaggcct ggtgatggca cagaaccaga ggatcaggtg gtatctgctc 3240agcatggggt ccaatgagaa tatccattct atccacttct caggacatgt cttttcagtg 3300aggaagaaag aggaatataa aatggctgtg tacaatctgt atccaggggt ctttgagaca 3360gtggaaatgc tgcctagcaa agtggggatc tggagaattg agtgcctcat tggagaacac 3420ctgcaggcag ggatgtccac cacatttctg gtgtactcaa agaaatgcca gactcccctg 3480gggatggcaa gtggacatat cagggacttc cagatcactg catcaggaca gtatggacag 3540tgggcaccaa agctggctag gctccactat agtggctcta tcaatgcttg gagtaccaaa 3600gagcctttct cttggatcaa ggtggatctg ctggccccca tgatcatcca tggaatcaaa 3660acacagggag ctagacagaa gttcagctcc ctgtacatca gtcagtttat catcatgtat 3720tctctggatg ggaagaaatg gcagacctac aggggcaata gcactgggac actgatggtc 3780ttctttggaa atgtggattc aagtggcatc aagcacaaca tcttcaatcc tcccatcatt 3840gccaggtaca tcagactgca tcccacacac tattcaatca ggagtactct cagaatggag 3900ctgatggggt gtgacctcaa cagctgctcc atgccactgg gaatggaatc caaggcaatc 3960tcagatgccc agatcactgc ttctagctac ttcaccaata tgtttgcaac atggtcaccc 4020agtaaagcaa ggctgcacct ccagggaagg tccaatgctt ggagacccca ggtgaacaat 4080ccaaaggagt ggctgcaggt ggactttcag aaaaccatga aggtcacagg ggtgactacc 4140cagggagtga aaagtctgct cacctctatg tatgtcaagg agttcctgat ctcctcaagt 4200caggatggcc accagtggac actgttcttt cagaatggca aggtcaaagt gttccagggg 4260aatcaggaca gctttacacc agtggtgaac agcctggacc cccctctgct cactagatat 4320ctgagaatcc atccacagag ctgggtgcac cagattgcac tcagaatgga ggtcctgggc 4380tgtgaagccc aggacctgta ttga 4404134374DNAArtificial Sequencesynthetic lcoHSQ with CpGs 13atgcagatcg aactgtctac ctgtttcttt ctgtgcctgc tgcggttttg tttttccgct 60accagaagat actacctggg agccgtcgaa ctgagctggg attacatgca gtctgacctg 120ggagagctgc ccgtggacgc tagattccca cctagagtcc ctaagtcctt ccccttcaac 180accagcgtgg tctacaagaa aaccctgttc gtggagttta ccgaccacct gttcaacatc 240gctaagccta gaccaccatg gatgggactg ctgggaccaa ccatccaggc cgaggtgtac 300gacaccgtgg tcatcaccct gaaaaacatg gcttctcacc ccgtgtccct gcatgctgtg 360ggcgtctcct actggaaggc cagcgaaggg gctgagtatg acgatcagac cagccagcgg 420gaaaaagagg acgataaggt gttccctggc gggtcccata cctacgtgtg gcaggtcctg 480aaggagaatg gaccaatggc ttccgaccct ctgtgcctga cctactctta tctgtcccac 540gtggacctgg tcaaggatct gaacagcggc ctgatcgggg ctctgctggt gtgtcgcgaa 600gggtccctgg ccaaggagaa aacccagacc ctgcataagt tcatcctgct gttcgccgtg 660tttgacgaag gaaaaagctg gcactctgag accaagaact ctctgatgca ggacagggat 720gccgcttccg ccagagcttg gcccaagatg cacaccgtga acggctacgt caataggagc 780ctgcctggac tgatcggctg ccacagaaag tccgtgtatt ggcatgtcat cggaatgggc 840accacccctg aagtgcacag catcttcctg gaggggcata cctttctggt ccgcaaccac 900cggcaggcta gcctggagat ctctccaatc accttcctga ccgcccagac cctgctgatg 960gacctgggac agttcctgct gttttgccac atctccagcc accagcatga tggcatggag 1020gcttacgtga aagtcgactc ctgtcccgag gaacctcagc tgaggatgaa gaacaatgag 1080gaagccgaag actatgacga tgacctgacc gacagcgaga tggatgtggt ccgcttcgat 1140gacgataact ctccctcctt tatccagatc cggtccgtgg ccaagaaaca ccctaagacc 1200tgggtccatt acatcgccgc tgaggaagag gactgggatt atgctccact ggtgctggcc 1260cccgacgata gatcctacaa aagccagtat ctgaacaatg gaccccagag gatcggcaga 1320aagtacaaga aagtgaggtt catggcttat accgatgaga cctttaagac cagagaagcc 1380atccagcacg agtccgggat cctgggacct ctgctgtacg gcgaagtggg ggacaccctg 1440ctgatcatct tcaagaacca ggccagcagg ccttacaata tctatccaca tggcatcacc 1500gatgtgagac ctctgtactc ccgccggctg ccaaagggcg tgaaacacct gaaggacttc 1560ccaatcctgc ccggggaaat ctttaagtat aaatggaccg tcaccgtcga ggatgggccc 1620accaagagcg accctaggtg cctgaccaga tactattctt ccttcgtgaa tatggagaga 1680gacctggctt ccggactgat cggacccctg ctgatctgtt acaaagagag cgtggatcag 1740cgcggcaacc agatcatgtc tgacaagcgg aatgtgatcc tgttcagcgt ctttgacgaa 1800aaccgctctt ggtacctgac cgagaacatc cagcggttcc tgcctaatcc agctggagtg 1860cagctggaag atcccgagtt ccaggcctct aacatcatgc attccatcaa

tggctacgtg 1920ttcgactccc tgcagctgag cgtgtgcctg cacgaggtcg cttactggta tatcctgagc 1980atcggagccc agaccgattt cctgtctgtg ttcttttccg gctacacctt taagcataaa 2040atggtgtatg aggacaccct gaccctgttc ccattttccg gcgaaaccgt gttcatgagc 2100atggagaatc ccgggctgtg gatcctggga tgccacaact ccgatttcag gaatagaggg 2160atgaccgccc tgctgaaagt gagctcttgt gacaagaaca ccggagacta ctatgaagat 2220agctacgagg acatctctgc ttatctgctg tccaaaaaca atgccatcga gcccaggagc 2280ttctctcaga accctccagt gctgaagcgc caccagcggg agatcaccag aaccaccctg 2340cagagcgatc aggaagagat cgactacgac gataccatct ccgtggaaat gaagaaagag 2400gacttcgata tctatgacga agatgagaac cagtctccca ggtccttcca gaagaaaacc 2460agacattact ttatcgccgc tgtggagcgg ctgtgggact atggcatgtc cagctctcct 2520cacgtgctga gaaatagagc tcagtccgga agcgtcccac agttcaagaa agtggtcttc 2580caggagttta ccgacggaag ctttacccag ccactgtacc gcggcgaact gaacgagcac 2640ctggggctgc tgggacccta tatccgggct gaagtggagg ataacatcat ggtcaccttc 2700aggaatcagg ccagcagacc ctactctttt tattccagcc tgatctccta cgaagaggac 2760cagagacagg gagctgaacc aagaaaaaac ttcgtgaagc ctaatgagac caaaacctac 2820ttttggaagg tgcagcacca tatggcccct accaaagacg agttcgattg caaggcctgg 2880gcttatttta gcgacgtgga tctggagaag gacgtccact ccggcctgat cgggccactg 2940ctggtgtgtc ataccaacac cctgaatcca gctcacggaa ggcaggtgac cgtccaggaa 3000ttcgccctgt tctttaccat ctttgatgag accaagagct ggtacttcac cgaaaacatg 3060gagaggaatt gcagagcccc atgtaacatc cagatggaag accccacctt caaggagaac 3120tacagatttc atgctatcaa tgggtatatc atggataccc tgccaggact ggtcatggct 3180caggaccaga ggatcagatg gtacctgctg agcatggggt ctaacgagaa tatccactcc 3240atccatttca gcggacacgt gtttaccgtc cgcaagaaag aagagtacaa gatggccctg 3300tacaacctgt atcccggcgt gttcgaaacc gtcgagatgc tgccttccaa ggctgggatc 3360tggcgggtgg aatgcctgat cggggagcac ctgcatgccg gaatgtctac cctgttcctg 3420gtgtactcca ataagtgtca gacccccctg gggatggcta gcggacatat ccgcgacttc 3480cagatcaccg cttccggaca gtacggacag tgggctccta agctggctag actgcactat 3540tctggctcca tcaacgcttg gtctaccaaa gagcctttct cctggatcaa ggtggacctg 3600ctggctccaa tgatcatcca tggcatcaaa acccaggggg ccaggcagaa gttctcttcc 3660ctgtacatca gccagtttat catcatgtat tctctggatg ggaagaaatg gcagacctac 3720agaggcaatt ccaccgggac cctgatggtg ttctttggca acgtcgacag ctctgggatc 3780aagcacaaca tcttcaatcc ccctatcatc gcccgctaca tccggctgca cccaacccat 3840tattccatcc gcagcaccct gcggatggag ctgatggggt gcgatctgaa cagctgttct 3900atgcccctgg gaatggagtc taaggccatc tccgacgctc agatcaccgc ctccagctac 3960ttcaccaata tgtttgctac ctggtcccca agcaaggcta gactgcatct gcagggaaga 4020agcaacgctt ggagaccaca ggtgaacaat cccaaggagt ggctgcaggt cgacttccag 4080aaaaccatga aggtgaccgg agtcaccacc cagggcgtga aaagcctgct gacctctatg 4140tacgtcaagg agttcctgat ctcttccagc caggacgggc accagtggac cctgttcttt 4200cagaacggaa aggtgaaagt cttccagggc aatcaggatt cctttacccc tgtggtcaac 4260agcctggacc cacccctgct gaccaggtac ctgagaatcc acccacagtc ctgggtgcat 4320cagatcgctc tgaggatgga agtcctgggc tgcgaggccc aggacctgta ttga 4374144374DNAArtificial Sequencesynthetic lcoHSQ CpGs removed 14atgcagattg aactgtctac ctgtttcttt ctgtgcctgc tgaggttttg tttttctgct 60accagaagat actacctggg agctgtggaa ctgagctggg attacatgca gtctgacctg 120ggagagctgc ctgtggatgc tagattccca cctagagtcc ctaagtcctt ccccttcaac 180acctctgtgg tctacaagaa aaccctgttt gtggagttta cagaccacct gttcaacatt 240gctaagccta gaccaccatg gatgggactg ctgggaccaa ccatccaggc agaggtgtat 300gacacagtgg tcatcaccct gaaaaacatg gcttctcacc ctgtgtccct gcatgctgtg 360ggagtctcct actggaaggc ctctgaaggg gctgagtatg atgatcagac cagccagagg 420gaaaaagagg atgataaggt gttccctgga gggtcccata cctatgtgtg gcaggtcctg 480aaggagaatg gaccaatggc ttctgaccct ctgtgcctga cctactctta tctgtcccat 540gtggacctgg tcaaggatct gaactctggc ctgattgggg ctctgctggt gtgtagggaa 600gggtccctgg ccaaggagaa aacccagacc ctgcataagt tcatcctgct gtttgctgtg 660tttgatgaag gaaaaagctg gcactctgag accaagaact ctctgatgca ggacagggat 720gctgcttctg ccagagcttg gcccaagatg cacacagtga atggctatgt caataggagc 780ctgcctggac tgattggctg ccacagaaag tctgtgtatt ggcatgtcat tggaatgggc 840accacccctg aagtgcacag catcttcctg gaggggcata cctttctggt caggaaccac 900aggcaggcta gcctggagat ctctccaatc accttcctga cagcccagac cctgctgatg 960gacctgggac agttcctgct gttttgccac atctccagcc accagcatga tggcatggag 1020gcttatgtga aagtggactc ctgtcctgag gaacctcagc tgaggatgaa gaacaatgag 1080gaagctgaag actatgatga tgacctgaca gactctgaga tggatgtggt caggtttgat 1140gatgataact ctccctcctt tatccagatc aggtctgtgg ccaagaaaca ccctaagacc 1200tgggtccatt acattgctgc tgaggaagag gactgggatt atgctccact ggtgctggcc 1260cctgatgata gatcctacaa aagccagtat ctgaacaatg gaccccagag gattggcaga 1320aagtacaaga aagtgaggtt catggcttat acagatgaga cctttaagac cagagaagcc 1380atccagcatg agtctgggat cctgggacct ctgctgtatg gggaagtggg ggacaccctg 1440ctgatcatct tcaagaacca ggccagcagg ccttacaata tctatccaca tggcatcaca 1500gatgtgagac ctctgtactc caggaggctg ccaaaggggg tgaaacacct gaaggacttc 1560ccaatcctgc ctggggaaat ctttaagtat aaatggacag tcacagtgga ggatgggccc 1620accaagtctg accctaggtg cctgaccaga tactattctt cctttgtgaa tatggagaga 1680gacctggctt ctggactgat tggacccctg ctgatctgtt acaaagagtc tgtggatcag 1740aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt ctttgatgaa 1800aacaggtctt ggtacctgac agagaacatc cagaggttcc tgcctaatcc agctggagtg 1860cagctggaag atcctgagtt ccaggcctct aacatcatgc attccatcaa tggctatgtg 1920tttgactccc tgcagctgtc tgtgtgcctg catgaggtgg cttactggta tatcctgagc 1980attggagccc agacagattt cctgtctgtg ttcttttctg gctacacctt taagcataaa 2040atggtgtatg aggacaccct gaccctgttc ccattttctg gagaaactgt gttcatgagc 2100atggagaatc ctgggctgtg gatcctggga tgccacaact ctgatttcag gaatagaggg 2160atgacagccc tgctgaaagt gagctcttgt gacaagaaca caggagacta ctatgaagat 2220agctatgagg acatctctgc ttatctgctg tccaaaaaca atgccattga gcccaggagc 2280ttctctcaga accctccagt gctgaagagg caccagaggg agatcaccag aaccaccctg 2340cagtctgatc aggaagagat tgactatgat gataccatct ctgtggaaat gaagaaagag 2400gactttgata tctatgatga agatgagaac cagtctccca ggtccttcca gaagaaaacc 2460agacattact ttattgctgc tgtggagagg ctgtgggact atggcatgtc cagctctcct 2520catgtgctga gaaatagagc tcagtctgga tctgtcccac agttcaagaa agtggtcttc 2580caggagttta cagatggaag ctttacccag ccactgtaca ggggagaact gaatgagcac 2640ctggggctgc tgggacccta tatcagggct gaagtggagg ataacatcat ggtcaccttc 2700aggaatcagg ccagcagacc ctactctttt tattccagcc tgatctccta tgaagaggac 2760cagagacagg gagctgaacc aagaaaaaac tttgtgaagc ctaatgagac caaaacctac 2820ttttggaagg tgcagcacca tatggcccct accaaagatg agtttgattg caaggcctgg 2880gcttattttt ctgatgtgga tctggagaag gatgtccact ctggcctgat tgggccactg 2940ctggtgtgtc ataccaacac cctgaatcca gctcatggaa ggcaggtgac agtccaggaa 3000tttgccctgt tctttaccat ctttgatgag accaagagct ggtacttcac agaaaacatg 3060gagaggaatt gcagagcccc atgtaacatc cagatggaag accccacctt caaggagaac 3120tacagatttc atgctatcaa tgggtatatc atggataccc tgccaggact ggtcatggct 3180caggaccaga ggatcagatg gtacctgctg agcatggggt ctaatgagaa tatccactcc 3240atccatttct ctggacatgt gtttacagta aggaagaaag aagagtacaa gatggccctg 3300tacaacctgt atcctggggt gtttgaaaca gtggagatgc tgccttccaa ggctgggatc 3360tggagggtgg aatgcctgat tggggagcac ctgcatgctg gaatgtctac cctgttcctg 3420gtgtactcca ataagtgtca gacccccctg gggatggctt ctggacatat cagggacttc 3480cagatcacag cttctggaca gtatggacag tgggctccta agctggctag actgcactat 3540tctggctcca tcaatgcttg gtctaccaaa gagcctttct cctggatcaa ggtggacctg 3600ctggctccaa tgatcatcca tggcatcaaa acccaggggg ccaggcagaa gttctcttcc 3660ctgtacatca gccagtttat catcatgtat tctctggatg ggaagaaatg gcagacctac 3720agaggcaatt ccacagggac cctgatggtg ttctttggca atgtggacag ctctgggatc 3780aagcacaaca tcttcaatcc ccctatcatt gccaggtaca tcagactgca cccaacccat 3840tattccatca ggagcaccct gagaatggag ctgatggggt gtgatctgaa cagctgttct 3900atgcccctgg gaatggagtc taaggccatc tctgatgctc agatcacagc ctccagctac 3960ttcaccaata tgtttgctac ctggtcccca agcaaggcta gactgcatct gcagggaaga 4020agcaatgctt ggagaccaca ggtgaacaat cccaaggagt ggctgcaggt ggacttccag 4080aaaaccatga aggtgacagg agtcaccacc cagggagtga aaagcctgct gacctctatg 4140tatgtcaagg agttcctgat ctcttccagc caggatgggc accagtggac cctgttcttt 4200cagaatggaa aggtgaaagt cttccagggc aatcaggatt cctttacccc tgtggtcaac 4260agcctggacc cacccctgct gaccaggtac ctgagaatcc acccacagtc ctgggtgcat 4320cagattgctc tgaggatgga agtcctgggc tgtgaggccc aggacctgta ttga 4374154374DNAArtificial Sequencesynthetic NoCoHSQ 15atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggaccacct tttcaacatc 240gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac gcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atccgcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga acctaggagc 2280ttctctcaga atccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta ctga 4374164375DNAArtificial Sequencesynthetic mcoHSQ 16atgcagattg agctcagcac ctgcttcttt ctgtgcctgc tcaggttctg cttttcagcc 60acaaggagat actatctggg agctgtggaa ctgtcatggg attacatgca gagtgacctg 120ggagagctcc ctgtggatgc taggttcccc ccaagggtcc caaagtcttt cccttttaat 180accagtgtgg tctataagaa aacactcttt gtggaattta ctgatcacct gttcaacatt 240gcaaagccaa ggcctccctg gatgggactg ctgggaccta ccatccaggc tgaggtgtat 300gacactgtgg tcatcacact gaaaaacatg gcatctcacc ctgtcagcct gcatgcagtg 360ggagtcagct actggaaggc ttcagaaggg gcagagtatg atgatcagac aagccagaga 420gaaaaagagg atgataaggt gttcccagga gggagccata cttatgtgtg gcaggtcctg 480aaggagaatg gcccaatggc cagtgaccca ctgtgcctca cctactcata tctgagtcat 540gtggacctgg tcaaggatct caactcaggc ctgattgggg cactgctggt gtgcagggaa 600ggctcactgg ccaaggagaa aacccagaca ctgcataagt tcatcctgct ctttgctgtg 660tttgatgaag ggaaatcttg gcacagtgag accaagaaca gtctgatgca ggacagggat 720gctgcttctg ccagagcttg gcccaagatg cacacagtga atggatatgt caataggtcc 780ctgccaggac tcattggctg ccacagaaag tcagtgtatt ggcatgtcat tggaatgggc 840accacaccag aagtgcacag catcttcctg gaggggcata cctttctggt caggaaccac 900aggcaggcca gcctggagat cagcccaatc accttcctga cagcccagac tctgctcatg 960gatctggggc agttcctgct cttttgccac atcagctccc accagcatga tggaatggag 1020gcatatgtga aagtggactc ctgcccagag gaaccacagc tgaggatgaa gaacaatgag 1080gaagctgaag actatgatga tgacctgaca gactcagaga tggatgtggt caggtttgat 1140gatgataaca gcccctcctt tatccagatc agaagtgtgg ccaagaaaca cccaaagaca 1200tgggtccatt acattgcagc tgaggaagag gactgggatt atgcacctct ggtgctggcc 1260ccagatgata gatcctacaa atcacagtat ctgaacaatg gaccccagag gattggcaga 1320aagtacaaga aagtgaggtt catggcctat actgatgaaa catttaagac tagagaagct 1380atccagcatg agtcaggcat cctgggacca ctgctctatg gagaagtggg ggacaccctg 1440ctcatcatct tcaagaacca ggcttccagg ccatacaata tctatcctca tggcatcaca 1500gatgtgagac cactctactc aaggagactg cctaagggag tcaaacacct caaggacttc 1560cctatcctgc caggggaaat ctttaagtat aaatggactg tgacagtgga ggatgggccc 1620actaagagtg acccaaggtg cctgaccaga tactattcaa gttttgtgaa tatggaaagg 1680gatctggcat caggactgat tggacctctg ctcatctgct acaaagagag tgtggatcag 1740aggggcaacc agatcatgtc agacaagagg aatgtgatcc tgttcagtgt ctttgatgaa 1800aacaggtctt ggtatctgac agagaacatc cagagattcc tgccaaatcc tgcaggggtg 1860cagctggaag atccagagtt tcaggcctca aacatcatgc atagtatcaa tggatatgtg 1920tttgacagtc tgcagctctc tgtgtgcctg catgaagtgg cctactggta tatcctgtcc 1980attggagctc agacagattt cctgagtgtg ttcttttcag gctacacttt taagcataaa 2040atggtctatg aggacacact gactctcttc ccttttagtg gggaaacagt gtttatgagc 2100atggagaatc cagggctgtg gattctggga tgccacaaca gtgatttcag gaatagaggc 2160atgactgctc tgctcaaagt gtctagctgt gacaagaaca caggggacta ctatgaagat 2220tcttatgagg acatcagtgc ttatctgctc tccaaaaaca atgcaattga acccagatca 2280ttcagtcaga atccacctgt gctgaagagg caccagagag agatcactag gactaccctg 2340cagtcagatc aggaagagat tgactatgat gataccatct cagtggaaat gaagaaagag 2400gactttgata tctatgatga agatgagaac cagagtccaa ggtctttcca gaagaaaacc 2460agacattact ttattgctgc agtggagagg ctgtgggatt atggaatgtc ctcaagtcca 2520catgtgctga ggaatagggc acagtctggc agtgtccctc agttcaagaa agtggtcttc 2580caggagttta cagatggcag cttcactcag cctctgtaca ggggagaact caatgagcac 2640ctggggctgc tgggacccta tatcagagct gaagtggagg ataacatcat ggtcaccttc 2700aggaatcagg cttcaagacc ctacagtttt tattctagcc tgatcagcta tgaagaggac 2760cagaggcagg gagctgaacc taggaaaaac tttgtgaagc caaatgagac caaaacatac 2820ttttggaagg tccagcacca catggcacca accaaagatg agtttgattg caaggcatgg 2880gcctattttt cagatgtgga tctggagaag gatgtccaca gtggcctcat tgggcctctg 2940ctggtgtgcc atactaacac cctgaatcca gctcatggca ggcaggtgac agtccaggag 3000tttgcactgt tctttaccat ctttgatgag acaaagtcct ggtacttcac tgaaaacatg 3060gagaggaatt gcagagctcc ttgcaacatc cagatggaag accccacctt caaggagaac 3120tacagatttc atgcaatcaa tgggtatatc atggatacac tgccaggact ggtgatggcc 3180caggaccaga ggatcagatg gtatctgctc agcatggggt ccaatgagaa tatccactct 3240atccatttca gtggacatgt gtttacagtc agaaagaaag aagagtataa aatggccctg 3300tacaacctct atccaggagt gtttgaaaca gtggagatgc tgccaagcaa ggctgggatc 3360tggagggtgg aatgcctcat tggggagcac ctgcatgcag gaatgtcaac cctgtttctg 3420gtctacagta ataagtgcca gacacctctg ggaatggcaa gtggacatat cagggatttc 3480cagatcactg ctagtggaca gtatggacag tgggcaccaa agctggctag actccactat 3540tcaggctcaa tcaatgcttg gtccaccaaa gagccattct catggatcaa ggtggacctg 3600ctggctccta tgatcatcca tggcatcaaa acacaggggg caaggcagaa gttctcctca 3660ctgtacatct ctcagtttat

catcatgtat agcctggatg gcaagaaatg gcagacctac 3720aggggcaata gcacagggac tctgatggtg ttctttggca atgtggacag cagtgggatc 3780aagcacaaca tcttcaatcc cccaatcatt gcaaggtaca tcagactgca ccccacccat 3840tattcaatca ggagtacact caggatggaa ctgatggggt gtgatctcaa cagttgctct 3900atgccactgg gaatggagtc caaggcaatc tcagatgccc agatcactgc tagctcctac 3960ttcactaata tgtttgctac ctggagcccc tccaaagcaa ggctgcacct ccagggaagg 4020agcaatgcat ggaggcctca ggtgaacaat cccaaggaat ggctgcaggt ggatttccag 4080aaaactatga aggtgactgg agtcacaact cagggagtga aaagtctgct cacttctatg 4140tatgtcaagg agttcctgat ctcaagttct caggatggcc accagtggac cctgttcttt 4200cagaatggaa aggtgaaagt cttccagggc aatcaggatt cctttacacc agtggtcaac 4260tcactggacc ctcccctgct cactagatat ctgagaatcc accctcagag ctgggtgcat 4320cagattgctc tcagaatgga agtcctgggc tgtgaggcac aggacctgta ttgag 4375172903DNAArtificial Sequencesynthetic 6x-tRNA 17gtcgactacg tagaatccgg agccgtcttt gtcttccagc tccatctttt ccaccttttg 60cttaggcagt cccccgagtc gtgtcaaggc tgaggagtag aaatggaaca gcactaatat 120taatggcaaa accgttgtga aatagggtta ctttctgttt aagcaaggaa aataaagtaa 180agcaatggga aaaaaattaa aagcaaaagg aatggaggtg ccggggattg aacccggggc 240ctcgtgcatg ctaagcacgc gctctaccac tgagctacac ccccgtactg aaacggttct 300ctcgagagta tattcaagat cagaatctga cccttttgct aggtttcaga accattagtt 360gtaatcagcc aaggtctatt ttatttagtt atttctgata tctcaaattt aggttttgcg 420tccctctttg ctgacagctg agcaaaccgc attctacacc gaaggccctc tattgatggc 480cctgatttaa atcccttccg ccgcctgccg caggtggcta gggtctgagc acacttgaac 540ctcacacccg ccccaggggt agctccttgg ttcccttcag ccccgatgtg tccctcgtgc 600tttgaaatgg aattacagtt ttggttaaaa acatgccttt tccgagttag gaagaatcta 660aatcgactga acgccagtct aaaatttcgg cgttcccaca ccgggagtcg aacccgggcc 720gcctgggtga aaaccaggaa tcctaaccgc tagaccatgt gggagacggc aatagcgact 780ccaagcctag acaaattgag tcttctcggt cggcttccgc ccactccatc gcgttcatcc 840gtaggcgtca aacctgctcc tgcgcctgcg cggagtctgc agcggtttaa accgttcagg 900ttcgcattct actgtttctc tccttgcagg ggcccttgaa tctttcttca atctactctc 960gcgtgcccgg gcgactgggc ataaccctac aggttcatgt ggggtgggtg gcgcgcgcta 1020gcggtgaagg tcactcacaa ttgcgcgctg ggcagacgac ggcagccatt acttttacct 1080cgatcgctgt tttcctggat ccgcacgggt ccaacccgac tcatcccaac caacctgagg 1140tatgaaaacc aggaaagaga gctagcaccg gagcgttggt ggtatagtgg taagcatagc 1200tgccttccaa gcagttgacc cgggttcgat tcccggccaa cgcaagtcgt tttgggtgtt 1260ttttcccccc cccgcctttt ccttttcgtg ttttctgggc cccagcatcg ttgagggttt 1320tcgtgaggtt ttcctgagga aacttccgct ccgaaaggac ccactttccg ctacacccgc 1380gaccacggct ggaccaccgc gctcctgacg gatgcgccct gcaagcccct ccaggcgaga 1440gcaggccggc ctgtgctcag ttttgtagca tcaaaactag gatttctctt gttaccccca 1500gtcactccat tcagttttcg tgtctttccc agctgcatcc atcctttcct cattttcgta 1560tgcagccgac tttttgtgac atctttgtat tcattctctg caattcagct gacctggcca 1620aggaaacaag atcctaagcg tctttccggc ggcgccgtgg cttagttggt taaagcgcct 1680gtctagtaaa caggagatcc tgggttcgaa tcccagcggt gcctccgtgt ttcccccacg 1740cttttgccaa cattaaacat tgtgaggaca gttgcagaaa ctcataactt ccatcctaca 1800tggtttactc acgtacccat ctatcctctc ccggtgcatc tgccacacgc tgttgggttt 1860ttgctcttcg tgcacatggt acttgcgcct cgacctgcag ttacaccagt cgcatcatct 1920gtacagcgct aaaacctagc tgggcgtggt ggtcctgcag tcccagctac tcgggaggct 1980gaggcaggag aatggcttga atccaggagg cggaggtggc agtgagccga gattgcgcca 2040ctgcactcca gcctggtgac agagcgagac tccatctcaa aaaaaaaaaa aaaaaaaaag 2100tcagaattag gtactaaaag ccatatgaca tgcctgaaca gggacttgaa ccctggaccc 2160tcagattaaa agtctgatgc tctaccaact gagctatcca ggcttcttcc ctgctagttt 2220attttaatgc agtaataaat aacagcactt tgttaaaaat aataaaggta taatctgtga 2280cacatccaaa gtggacaaga tgaagagata ataggtatac accaggattc ccctgggcaa 2340actgggacct cttggtaccc tatatattaa gagtctcggg ttttgttttc acttaagcaa 2400atggttaacg aattagcagg ttaagaaaaa ctgtttcccc gtaagaagca gggttctttg 2460gtgttcaatg tggagctccg ccactcccag cccgggtgaa ggaaaactgg gaaacagaat 2520gaatgtgatt atctattcga agataaattt ccacaaagca tgccgtttga tagtagctta 2580taatgtggaa gtaaggcatc ctgtcatccg gccggttagc tcagttggtt agagcgtggt 2640gctaataacg ccaaggtcgc gggttcgatc cccgtactgg ccaagtattc tctgtggctt 2700ttatcaccag aatggatagt aacccagaca tcgatctaaa cgtgtacctg tgtgtttctc 2760caggcttaac tttgccccga gaaaacggat ctgtgaattt ggtgcgccct cgcttactcg 2820acagcggtta atttgaacgg ggacgtttct ttccgctgcc tccaaggcat acccacatcc 2880taccacgatg gtggcggccg caa 2903181467PRTArtificial Sequencesynthetic HP44/OL aa 18Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn 420 425 430Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val 435 440 445Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys 500 505 510Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp 725 730 735Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly 740 745 750Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp 770 775 780Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp785 790 795 800Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly 805 810 815Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln865 870 875 880Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro 930 935 940Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro945 950 955 960Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser 1010 1015 1020Trp Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys 1025 1030 1035His Leu Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe 1040 1045 1050His Ala Ile Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val 1055 1060 1065Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly 1070 1075 1080Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe 1085 1090 1095Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn Leu 1100 1105 1110Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Val 1115 1120 1125Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln Ala 1130 1135 1140Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Lys Cys Gln Thr 1145 1150 1155Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr 1160 1165 1170Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu 1175 1180 1185His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe 1190 1195 1200Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly 1205 1210 1215Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile 1220 1225 1230Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln 1235 1240 1245Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly 1250 1255 1260Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro 1265 1270 1275Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile 1280 1285 1290Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser 1295 1300 1305Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala 1310 1315 1320Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp 1325 1330 1335Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala 1340 1345 1350Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp 1355 1360 1365Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val 1370 1375 1380Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser 1385 1390 1395Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly 1400 1405 1410Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val 1415 1420 1425Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile 1430 1435 1440His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val 1445 1450 1455Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465194401DNAArtificial Sequencesynthetic HP44/OL NT 19atgcagctag agctctccac ctgtgtcttt ctgtgtctct tgccactcgg ctttagtgcc 60atcaggagat actacctggg cgcagtggaa ctgtcctggg actaccggca aagtgaactc 120ctccgtgagc tgcacgtgga caccagattt cctgctacag cgccaggagc tcttccgttg 180ggcccgtcag tcctgtacaa aaagactgtg ttcgtagagt tcacggatca acttttcagc 240gttgccaggc ccaggccacc atggatgggt ctgctgggtc ctaccatcca ggctgaggtt 300tacgacacgg tggtcgttac cctgaagaac atggcttctc atcccgttag tcttcacgct 360gtcggcgtct ccttctggaa atcttccgaa ggcgctgaat atgaggatca caccagccaa 420agggagaagg aagacgataa agtccttccc ggtaaaagcc aaacctacgt ctggcaggtc 480ctgaaagaaa atggtccaac agcctctgac ccaccatgtc ttacctactc atacctgtct 540cacgtggacc tggtgaaaga cctgaattcg ggcctcattg gagccctgct ggtttgtaga 600gaagggagtc tgaccagaga aaggacccag aacctgcacg aatttgtact actttttgct 660gtctttgatg aagggaaaag ttggcactca gcaagaaatg actcctggac acgggccatg 720gatcccgcac ctgccagggc ccagcctgca atgcacacag tcaatggcta tgtcaacagg 780tctctgccag gtctgatcgg atgtcataag aaatcagtct actggcacgt gattggaatg 840ggcaccagcc cggaagtgca ctccattttt cttgaaggcc acacgtttct cgtgaggcac 900catcgccagg cttccttgga gatctcgcca ctaactttcc tcactgctca gacattcctg 960atggaccttg gccagttcct actgttttgt catatctctt cccaccacca tggtggcatg 1020gaggctcacg tcagagtaga aagctgcgcc gaggagcccc agctgcggag gaaagctgat 1080gaagaggaag attatgatga caatttgtac gactcggaca tggacgtggt ccggctcgat 1140ggtgacgacg tgtctccctt tatccaaatc cgctcggttg ccaagaagca tcccaaaacc 1200tgggtgcact acatctctgc agaggaggag gactgggact acgcccccgc ggtccccagc 1260cccagtgaca gaagttataa aagtctctac ttgaacagtg gtcctcagcg aattggtagg 1320aaatacaaaa aagctcgatt cgtcgcttac acggatgtaa catttaagac tcgtaaagct 1380attccgtatg aatcaggaat cctgggacct ttactttatg gagaagttgg agacacactt 1440ttgattatat ttaagaataa agcgagccga ccatataaca tctaccctca tggaatcact 1500gatgtcagcg ctttgcaccc agggagactt ctaaaaggtt ggaaacattt gaaagacatg 1560ccaattctgc caggagagac tttcaagtat aaatggacag tgactgtgga agatgggcca 1620accaagtccg atcctcggtg cctgacccgc tactactcga gctccattaa tctagagaaa 1680gatctggctt cgggactcat tggccctctc ctcatctgct acaaagaatc tgtagaccaa 1740agaggaaacc agatgatgtc agacaagaga aacgtcatcc tgttttctgt attcgatgag 1800aatcaaagct ggtacctcgc agagaatatt cagcgcttcc tccccaatcc ggatggatta 1860cagccccagg atccagagtt ccaagcttct aacatcatgc acagcatcaa tggctatgtt 1920tttgatagct tgcagctgtc ggtttgtttg cacgaggtgg catactggta cattctaagt 1980gttggagcac agacggactt cctctccgtc ttcttctctg gctacacctt caaacacaaa 2040atggtctatg aagacacact caccctgttc cccttctcag gagaaacggt cttcatgtca 2100atggaaaacc caggtctctg ggtccttggg tgccacaact cagacttgcg gaacagaggg 2160atgacagcct tactgaaggt gtatagttgt gacagggaca ttggtgatta ttatgacaac 2220acttatgaag atattccagg cttcttgctg agtggaaaga atgtcattga acctaggagc 2280tttgcccaga attcaagacc ccctagtgcg

agcgctccaa agcctccggt cctgcgacgg 2340catcagaggg acataagcct tcctactttt cagccggagg aagacaaaat ggactatgat 2400gatatcttct caactgaaac gaagggagaa gattttgaca tttacggtga ggatgaaaat 2460caggaccctc gcagctttca gaagagaacc cgacactatt tcattgctgc ggtggagcag 2520ctctgggatt acgggatgag cgaatccccc cgggcgctaa gaaacagggc tcagaacgga 2580gaggtgcctc ggttcaagaa ggtggtcttc cgggaatttg ctgacggctc cttcacgcag 2640ccgtcgtacc gcggggaact caacaaacac ttggggctct tgggacccta catcagagcg 2700gaagttgaag acaacatcat ggtaactttc aaaaaccagg cgtctcgtcc ctattccttc 2760tactcgagcc ttatttctta tccggatgat caggagcaag gggcagaacc tcgacacaac 2820ttcgtccagc caaatgaaac cagaacttac ttttggaaag tgcagcatca catggcaccc 2880acagaagacg agtttgactg caaagcctgg gcctactttt ctgatgttga cctggaaaaa 2940gatgtgcact caggcttgat cggccccctt ctgatctgcc gcgccaacac cctgaacgct 3000gctcacggta gacaagtgac cgtgcaagaa tttgctctgt ttttcactat ttttgatgag 3060acaaagagct ggtacttcac tgaaaatgtg gaaaggaact gccgggcccc ctgccatctg 3120cagatggagg accccactct gaaagaaaac tatcgcttcc atgcaatcaa tggctatgtg 3180atggatacac tccctggctt agtaatggct cagaatcaaa ggatccgatg gtatctgctc 3240agcatgggca gcaatgaaaa tatccattcg attcatttta gcggacacgt gttcagtgta 3300cggaaaaagg aggagtataa aatggccgtg tacaatctct atccgggtgt ctttgagaca 3360gtggaaatgc taccgtccaa agttggaatt tggcgaatag aatgcctgat tggcgagcac 3420ctgcaagctg ggatgagcac gactttcctg gtgtacagca agaagtgtca gactcccctg 3480ggaatggctt ctggacacat tagagatttt cagattacag cttcaggaca atatggacag 3540tgggccccaa agctggccag acttcattat tccggatcaa tcaatgcctg gagcaccaag 3600gagccctttt cttggatcaa ggtggatctg ttggcaccaa tgattattca cggcatcaag 3660acccagggtg cccgtcagaa gttctccagc ctctacatct ctcagtttat catcatgtat 3720agtcttgatg ggaagaagtg gcagacttat cgaggaaatt ccactggaac cttaatggtc 3780ttctttggca atgtggattc atctgggata aaacacaata tttttaaccc tccaattatt 3840gctcgataca tccgtttgca cccaactcat tatagcattc gcagcactct tcgcatggag 3900ttgatgggct gtgatttaaa tagttgcagc atgccattgg gaatggagag taaagcaata 3960tcagatgcac agattactgc ttcatcctac tttaccaata tgtttgccac ctggtctcct 4020tcaaaagctc gacttcacct ccaagggagg agtaatgcct ggagacctca ggtgaataat 4080ccaaaagagt ggctgcaagt ggacttccag aagacaatga aagtcacagg agtaactact 4140cagggagtaa aatctctgct taccagcatg tatgtgaagg agttcctcat ctccagcagt 4200caagatggcc atcagtggac tctctttttt cagaatggca aagtaaaggt ttttcaggga 4260aatcaagact ccttcacacc tgtggtgaac tctctagacc caccgttact gactcgctac 4320cttcgaattc acccccagag ttgggtgcac cagattgccc tgaggatgga ggttctgggc 4380tgcgaggcac aggacctcta c 4401201457PRTArtificial Sequencesynthetic HP46/SQ aa 20Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu785 790 795 800Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His865 870 875 880Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp945 950 955 960Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1010 1015 1020Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1025 1030 1035Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1040 1045 1050Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1055 1060 1065Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1070 1075 1080Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1085 1090 1095Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1100 1105 1110Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1115 1120 1125Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1130 1135 1140Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1145 1150 1155Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1160 1165 1170Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1175 1180 1185Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1190 1195 1200Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1205 1210 1215Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1220 1225 1230Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1250 1255 1260Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1265 1270 1275Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1280 1285 1290Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1295 1300 1305Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1310 1315 1320Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1325 1330 1335Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1340 1345 1350Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1355 1360 1365Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1370 1375 1380Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1385 1390 1395Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1400 1405 1410Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1415 1420 1425Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1430 1435 1440Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1445 1450 1455214368DNAArtificial Sequencesynthetic HP46/SQ NT 21atgcagctag agctctccac ctgtgtcttt ctgtgtctct tgccactcgg ctttagtgcc 60atcaggagat actacctggg cgcagtggaa ctgtcctggg actaccggca aagtgaactc 120ctccgtgagc tgcacgtgga caccagattt cctgctacag cgccaggagc tcttccgttg 180ggcccgtcag tcctgtacaa aaagactgtg ttcgtagagt tcacggatca acttttcagc 240gttgccaggc ccaggccacc atggatgggt ctgctgggtc ctaccatcca ggctgaggtt 300tacgacacgg tggtcgttac cctgaagaac atggcttctc atcccgttag tcttcacgct 360gtcggcgtct ccttctggaa atcttccgaa ggcgctgaat atgaggatca caccagccaa 420agggagaagg aagacgataa agtccttccc ggtaaaagcc aaacctacgt ctggcaggtc 480ctgaaagaaa atggtccaac agcctctgac ccaccatgtc ttacctactc atacctgtct 540cacgtggacc tggtgaaaga cctgaattcg ggcctcattg gagccctgct ggtttgtaga 600gaagggagtc tgaccagaga aaggacccag aacctgcacg aatttgtact actttttgct 660gtctttgatg aagggaaaag ttggcactca gcaagaaatg actcctggac acgggccatg 720gatcccgcac ctgccagggc ccagcctgca atgcacacag tcaatggcta tgtcaacagg 780tctctgccag gtctgatcgg atgtcataag aaatcagtct actggcacgt gattggaatg 840ggcaccagcc cggaagtgca ctccattttt cttgaaggcc acacgtttct cgtgaggcac 900catcgccagg cttccttgga gatctcgcca ctaactttcc tcactgctca gacattcctg 960atggaccttg gccagttcct actgttttgt catatctctt cccaccacca tggtggcatg 1020gaggctcacg tcagagtaga aagctgcgcc gaggagcccc agctgcggag gaaagctgat 1080gaagaggaag attatgatga caatttgtac gactcggaca tggacgtggt ccggctcgat 1140ggtgacgacg tgtctccctt tatccaaatc cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac gcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atccgcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga acctaggagc 2280ttctctcaga atccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900atgccattgg

gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctc 4368221467PRTArtificial Sequencesynthetic HP47/OL aa 22Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro 755 760 765Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp 770 775 780Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp785 790 795 800Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly 805 810 815Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln865 870 875 880Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro 930 935 940Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro945 950 955 960Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser 1010 1015 1020Trp Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys 1025 1030 1035His Leu Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe 1040 1045 1050His Ala Ile Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val 1055 1060 1065Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly 1070 1075 1080Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe 1085 1090 1095Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn Leu 1100 1105 1110Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Val 1115 1120 1125Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln Ala 1130 1135 1140Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Lys Cys Gln Thr 1145 1150 1155Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr 1160 1165 1170Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu 1175 1180 1185His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe 1190 1195 1200Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly 1205 1210 1215Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile 1220 1225 1230Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln 1235 1240 1245Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly 1250 1255 1260Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro 1265 1270 1275Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile 1280 1285 1290Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser 1295 1300 1305Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala 1310 1315 1320Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp 1325 1330 1335Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala 1340 1345 1350Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp 1355 1360 1365Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val 1370 1375 1380Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser 1385 1390 1395Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly 1400 1405 1410Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val 1415 1420 1425Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile 1430 1435 1440His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val 1445 1450 1455Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465234401DNAArtificial Sequencesynthetic HP47/OL NT 23atgcagctag agctctccac ctgtgtcttt ctgtgtctct tgccactcgg ctttagtgcc 60atcaggagat actacctggg cgcagtggaa ctgtcctggg actaccggca aagtgaactc 120ctccgtgagc tgcacgtgga caccagattt cctgctacag cgccaggagc tcttccgttg 180ggcccgtcag tcctgtacaa aaagactgtg ttcgtagagt tcacggatca acttttcagc 240gttgccaggc ccaggccacc atggatgggt ctgctgggtc ctaccatcca ggctgaggtt 300tacgacacgg tggtcgttac cctgaagaac atggcttctc atcccgttag tcttcacgct 360gtcggcgtct ccttctggaa atcttccgaa ggcgctgaat atgaggatca caccagccaa 420agggagaagg aagacgataa agtccttccc ggtaaaagcc aaacctacgt ctggcaggtc 480ctgaaagaaa atggtccaac agcctctgac ccaccatgtc ttacctactc atacctgtct 540cacgtggacc tggtgaaaga cctgaattcg ggcctcattg gagccctgct ggtttgtaga 600gaagggagtc tgaccagaga aaggacccag aacctgcacg aatttgtact actttttgct 660gtctttgatg aagggaaaag ttggcactca gcaagaaatg actcctggac acgggccatg 720gatcccgcac ctgccagggc ccagcctgca atgcacacag tcaatggcta tgtcaacagg 780tctctgccag gtctgatcgg atgtcataag aaatcagtct actggcacgt gattggaatg 840ggcaccagcc cggaagtgca ctccattttt cttgaaggcc acacgtttct cgtgaggcac 900catcgccagg cttccttgga gatctcgcca ctaactttcc tcactgctca gacattcctg 960atggaccttg gccagttcct actgttttgt catatctctt cccaccacca tggtggcatg 1020gaggctcacg tcagagtaga aagctgcgcc gaggagcccc agctgcggag gaaagctgat 1080gaagaggaag attatgatga caatttgtac gactcggaca tggacgtggt ccggctcgat 1140ggtgacgacg tgtctccctt tatccaaatc cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac gcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atccgcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga acctaggagc 2280tttgcccaga attcaagacc ccctagtgcg agcgctccaa agcctccggt cctgcgacgg 2340catcagaggg acataagcct tcctactttt cagccggagg aagacaaaat ggactatgat 2400gatatcttct caactgaaac gaagggagaa gattttgaca tttacggtga ggatgaaaat 2460caggaccctc gcagctttca gaagagaacc cgacactatt tcattgctgc ggtggagcag 2520ctctgggatt acgggatgag cgaatccccc cgggcgctaa gaaacagggc tcagaacgga 2580gaggtgcctc ggttcaagaa ggtggtcttc cgggaatttg ctgacggctc cttcacgcag 2640ccgtcgtacc gcggggaact caacaaacac ttggggctct tgggacccta catcagagcg 2700gaagttgaag acaacatcat ggtaactttc aaaaaccagg cgtctcgtcc ctattccttc 2760tactcgagcc ttatttctta tccggatgat caggagcaag gggcagaacc tcgacacaac 2820ttcgtccagc caaatgaaac cagaacttac ttttggaaag tgcagcatca catggcaccc 2880acagaagacg agtttgactg caaagcctgg gcctactttt ctgatgttga cctggaaaaa 2940gatgtgcact caggcttgat cggccccctt ctgatctgcc gcgccaacac cctgaacgct 3000gctcacggta gacaagtgac cgtgcaagaa tttgctctgt ttttcactat ttttgatgag 3060acaaagagct ggtacttcac tgaaaatgtg gaaaggaact gccgggcccc ctgccatctg 3120cagatggagg accccactct gaaagaaaac tatcgcttcc atgcaatcaa tggctatgtg 3180atggatacac tccctggctt agtaatggct cagaatcaaa ggatccgatg gtatctgctc 3240agcatgggca gcaatgaaaa tatccattcg attcatttta gcggacacgt gttcagtgta 3300cggaaaaagg aggagtataa aatggccgtg tacaatctct atccgggtgt ctttgagaca 3360gtggaaatgc taccgtccaa agttggaatt tggcgaatag aatgcctgat tggcgagcac 3420ctgcaagctg ggatgagcac gactttcctg gtgtacagca agaagtgtca gactcccctg 3480ggaatggctt ctggacacat tagagatttt cagattacag cttcaggaca atatggacag 3540tgggccccaa agctggccag acttcattat tccggatcaa tcaatgcctg gagcaccaag 3600gagccctttt cttggatcaa ggtggatctg ttggcaccaa tgattattca cggcatcaag 3660acccagggtg cccgtcagaa gttctccagc ctctacatct ctcagtttat catcatgtat 3720agtcttgatg ggaagaagtg gcagacttat cgaggaaatt ccactggaac cttaatggtc 3780ttctttggca atgtggattc atctgggata aaacacaata tttttaaccc tccaattatt 3840gctcgataca tccgtttgca cccaactcat tatagcattc gcagcactct tcgcatggag 3900ttgatgggct gtgatttaaa tagttgcagc atgccattgg gaatggagag taaagcaata 3960tcagatgcac agattactgc ttcatcctac tttaccaata tgtttgccac ctggtctcct 4020tcaaaagctc gacttcacct ccaagggagg agtaatgcct ggagacctca ggtgaataat 4080ccaaaagagt ggctgcaagt ggacttccag aagacaatga aagtcacagg agtaactact 4140cagggagtaa aatctctgct taccagcatg tatgtgaagg agttcctcat ctccagcagt 4200caagatggcc atcagtggac tctctttttt cagaatggca aagtaaaggt ttttcaggga 4260aatcaagact ccttcacacc tgtggtgaac tctctagacc caccgttact gactcgctac 4320cttcgaattc acccccagag ttgggtgcac cagattgccc tgaggatgga ggttctgggc 4380tgcgaggcac aggacctcta c 4401241457PRTHomo sapiens 24Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg 35 40 45Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val 50 55 60Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile65 70 75 80Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu145 150 155 160Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170

175Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp225 230 235 240Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265 270Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile 275 280 285Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser 290 295 300Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met305 310 315 320Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760 765Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln 770 775 780Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu785 790 795 800Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe 805 810 815Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp 820 825 830Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His865 870 875 880Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885 890 895Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser 900 905 910Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 915 920 925Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp945 950 955 960Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000 1005Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 1010 1015 1020Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys 1025 1030 1035Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr 1040 1045 1050Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1055 1060 1065Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe 1070 1075 1080Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1085 1090 1095Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1100 1105 1110Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1115 1120 1125Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser 1130 1135 1140Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg 1145 1150 1155Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro 1160 1165 1170Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser 1175 1180 1185Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro 1190 1195 1200Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1205 1210 1215Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1220 1225 1230Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240 1245Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1250 1255 1260Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro 1265 1270 1275Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly 1280 1285 1290Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys 1295 1300 1305Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn 1310 1315 1320Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1325 1330 1335Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1340 1345 1350Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1355 1360 1365Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1370 1375 1380Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu 1385 1390 1395Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp 1400 1405 1410Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr 1415 1420 1425Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala 1430 1435 1440Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1445 1450 1455254371DNAHomo sapiens 25atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggttcacct tttcaacatc 240gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac gcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atccgcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga acctaggagc 2280ttctctcaga atccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta c 4371261467PRTArtificial Sequencesynthetic HP63/OL aa 26Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser 20 25 30Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr 35 40 45Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val 50 55 60Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile 85 90 95Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala 100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser 115 120 125Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu 130 135 140Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr 165 170 175Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu 180 185 190Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg 195 200 205Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu 210 215 220Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly 245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser 260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser 275 280 285Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala 290 295 300Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His 325 330 335His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu 340 345 350Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn 355 360 365Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val 370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala

Ile Gln His Glu 450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp 530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro 755 760 765Ser Thr Arg Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu 770 775 780Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp785 790 795 800Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp 805 810 815Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Arg Thr Arg His 820 825 830Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu 835 840 845Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg 850 855 860Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln865 870 875 880Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro 885 890 895Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn 900 905 910Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro 915 920 925Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro 930 935 940Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro945 950 955 960Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val 965 970 975Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile 980 985 990Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val 995 1000 1005Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser 1010 1015 1020Trp Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys 1025 1030 1035His Leu Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe 1040 1045 1050His Ala Ile Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val 1055 1060 1065Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly 1070 1075 1080Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe 1085 1090 1095Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn Leu 1100 1105 1110Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Val 1115 1120 1125Gly Ile Trp Arg Asn Arg Cys Leu Ile Gly Glu His Leu Gln Ala 1130 1135 1140Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Lys Cys Gln Thr 1145 1150 1155Pro Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr 1160 1165 1170Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu 1175 1180 1185His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe 1190 1195 1200Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly 1205 1210 1215Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile 1220 1225 1230Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln 1235 1240 1245Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly 1250 1255 1260Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro 1265 1270 1275Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile 1280 1285 1290Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser 1295 1300 1305Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala 1310 1315 1320Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp 1325 1330 1335Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala 1340 1345 1350Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp 1355 1360 1365Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val 1370 1375 1380Lys Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser 1385 1390 1395Ser Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly 1400 1405 1410Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val 1415 1420 1425Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile 1430 1435 1440His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val 1445 1450 1455Leu Gly Cys Glu Ala Gln Asp Leu Tyr 1460 1465274398DNAArtificial Sequencesynthetic HP63/OL NT 27atgcagctag agctctccac ctgtgtcttt ctgtgtctct tgccactcgg ctttagtgcc 60atcaggagat actacctggg cgcagtggaa ctgtcctggg actaccggca aagtgaactc 120ctccgtgagc tgcacgtgga caccagattt cctgctacag cgccaggagc tcttccgttg 180ggcccgtcag tcctgtacaa aaagactgtg ttcgtagagt tcacggatca acttttcagc 240gttgccaggc ccaggccacc atggatgggt ctgctgggtc ctaccatcca ggctgaggtt 300tacgacacgg tggtcgttac cctgaagaac atggcttctc atcccgttag tcttcacgct 360gtcggcgtct ccttctggaa atcttccgaa ggcgctgaat atgaggatca caccagccaa 420agggagaagg aagacgataa agtccttccc ggtaaaagcc aaacctacgt ctggcaggtc 480ctgaaagaaa atggtccaac agcctctgac ccaccatgtc ttacctactc atacctgtct 540cacgtggacc tggtgaaaga cctgaattcg ggcctcattg gagccctgct ggtttgtaga 600gaagggagtc tgaccagaga aaggacccag aacctgcacg aatttgtact actttttgct 660gtctttgatg aagggaaaag ttggcactca gcaagaaatg actcctggac acgggccatg 720gatcccgcac ctgccagggc ccagcctgca atgcacacag tcaatggcta tgtcaacagg 780tctctgccag gtctgatcgg atgtcataag aaatcagtct actggcacgt gattggaatg 840ggcaccagcc cggaagtgca ctccattttt cttgaaggcc acacgtttct cgtgaggcac 900catcgccagg cttccttgga gatctcgcca ctaactttcc tcactgctca gacattcctg 960atggaccttg gccagttcct actgttttgt catatctctt cccaccacca tggtggcatg 1020gaggctcacg tcagagtaga aagctgcgcc gaggagcccc agctgcggag gaaagctgat 1080gaagaggaag attatgatga caatttgtac gactcggaca tggacgtggt ccggctcgat 1140ggtgacgacg tgtctccctt tatccaaatc cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga acctaggagc 2280ttctcccaga attcaagaca ccctagcact aggtctcaaa acccaccagt cttgaaacgc 2340catcaacggg aaataactcg tactactctt cagtcagatc aagaggaaat tgactatgat 2400gataccatat cagttgaaat gaagaaggaa gattttgaca tttatgatga ggatgaaaat 2460cagagccccc gcagctttca aaagagaacc cgacactatt tcattgctgc ggtggagcag 2520ctctgggatt acgggatgag cgaatccccc cgggcgctaa gaaacagggc tcagaacgga 2580gaggtgcctc ggttcaagaa ggtggtcttc cgggaatttg ctgacggctc cttcacgcag 2640ccgtcgtacc gcggggaact caacaaacac ttggggctct tgggacccta catcagagcg 2700gaagttgaag acaacatcat ggtaactttc aaaaaccagg cgtctcgtcc ctattccttc 2760tactcgagcc ttatttctta tccggatgat caggagcaag gggcagaacc tcgaaaaaac 2820tttgtcaagc ctaatgaaac caaaacttac ttttggaaag tgcagcatca catggcaccc 2880acagaagacg agtttgactg caaagcctgg gcctactttt ctgatgttga cctggaaaaa 2940gatgtgcact caggcttgat cggccccctt ctgatctgcc gcgccaacac cctgaacgct 3000gctcacggta gacaagtgac cgtgcaagaa tttgctctgt ttttcactat ttttgatgag 3060acaaagagct ggtacttcac tgaaaatgtg gaaaggaact gccgggcccc ctgccatctg 3120cagatggagg accccactct gaaagaaaac tatcgcttcc atgcaatcaa tggctatgtg 3180atggatacac tccctggctt agtaatggct cagaatcaaa ggatccgatg gtatctgctc 3240agcatgggca gcaatgaaaa tatccattcg attcatttta gcggacacgt gttcagtgta 3300cggaaaaagg aggagtataa aatggccgtg tacaatctct atccgggtgt ctttgagaca 3360gtggaaatgc taccgtccaa agttggaatt tggcggaata gatgcctgat tggcgagcac 3420ctgcaagctg ggatgagcac gactttcctg gtgtacagca agaagtgtca gactcccctg 3480ggaatggctt ctggacacat tagagatttt cagattacag cttcaggaca atatggacag 3540tgggccccaa agctggccag acttcattat tccggatcaa tcaatgcctg gagcaccaag 3600gagccctttt cttggatcaa ggtggatctg ttggcaccaa tgattattca cggcatcaag 3660acccagggtg cccgtcagaa gttctccagc ctctacatct ctcagtttat catcatgtat 3720agtcttgatg ggaagaagtg gcagacttat cgaggaaatt ccactggaac cttaatggtc 3780ttctttggca atgtggattc atctgggata aaacacaata tttttaaccc tccaattatt 3840gctcgataca tccgtttgca cccaactcat tatagcattc gcagcactct tcgcatggag 3900ttgatgggct gtgatttaaa tagttgcagc atgccattgg gaatggagag taaagcaata 3960tcagatgcac agattactgc ttcatcctac tttaccaata tgtttgccac ctggtctcct 4020tcaaaagctc gacttcacct ccaagggagg agtaatgcct ggagacctca ggtgaataat 4080ccaaaagagt ggctgcaagt ggacttccag aagacaatga aagtcacagg agtaactact 4140cagggagtaa aatctctgct taccagcatg tatgtgaagg agttcctcat ctccagcagt 4200caagatggcc atcagtggac tttttttcag aatggcaaag taaaggtttt tcagggaaat 4260caagactcct tcacacctgt ggtgaactct ctagacccac cgttactgac tcgctacctt 4320cgaattcacc cccagagttg ggtgcaccag attgccctga ggatggaggt tctgggctgc 4380gaggcacagg acctctac 4398285508DNAHomo sapiens 28atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140aatgaagaat gtccagggga gtgccttgtc acaggtcaat cacacttcaa gagctttgac 1200aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380ggggcaggag ttgccatgga tggccaggac gtccagctcc ccctcctgaa aggtgacctc 1440cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa ctgcacagac 2580catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgcctgcttc 3300tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct gtatgtggag 3780gacatctcgg aaccgccgtt gcacgatttc tactgcagca ggctactgga cctggtcttc 3840ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa ggcctttgtg 3900gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc cgtggtggag 3960taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc gtcagagctg 4020cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac cagcgaggtc 4080ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc ctcccgcatc 4140accctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt tgtccgctac 4200gtccagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg gccccatgcc 4260aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc cttcgtgctg 4320agcagtgtgg

atgagctgga gcagcaaagg gacgagatcg ttagctacct ctgtgacctt 4380gcccctgaag cccctcctcc tactctgccc cccgacatgg cacaagtcac tgtgggcccg 4440gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct ggatgtggcg 4500ttcgtcctgg aaggatcgga caaaattggt gaagccgact tcaacaggag caaggagttc 4560atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt cacggtgctg 4620cagtactcct acatggtgac tgtggagtac cccttcagcg aggcacagtc caaaggggac 4680atcctgcagc gggtgcgaga gatccgctac cagggcggca acaggaccaa cactgggctg 4740gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg ggagcaggcg 4800cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa gaggctgcct 4860ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca ggagctggag 4920aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct cccccgagag 4980gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat ccccaccctc 5040tcccctgcac ctcgtgatga gacgctccag gatggctgtg atactcactt ctgcaaggtc 5100aatgagagag gagagtactt ctgggagaag agggtcacag gctgcccacc ctttgatgaa 5160cacaagtgtc tggctgaggg aggtaaaatt atgaaaattc caggcacctg ctgtgacaca 5220tgtgaggagc ctgagtgcaa cgacatcact gccaggctgc agtatgtcaa ggtgggaagc 5280tgtaagtctg aagtagaggt ggatatccac tactgccagg gcaaatgtgc cagcaaagcc 5340atgtactcca ttgacatcaa cgatgtgcag gaccagtgct cctgctgctc tccgacacgg 5400acggagccca tgcaggtggc cctgcactgc accaatggct ctgttgtgta ccatgaggtt 5460ctcaatgcca tggagtgcaa atgctccccc aggaagtgca gcaagtga 5508296648DNAOvis aries 29atgtttccca ccaggctcgc gaggctgctg cttgctgtgg ccctcacttt gccaggggcc 60ctttgtggag aaggtgctcc tggcaagtca tcgatggccc ggtgcagcct cttcggagct 120gacttcatca acacctttga tgagagcatg tacagctttg cgggagactg tagttacctc 180ctggcagggg attgcaagac acactccttt tcaatcgtag gggacttcca agctggtaga 240agagtgggtc tctctgtgta ccttggggaa tttttcgaca tccatgtgtt tgtcaacggt 300actgtgctgc aggggggcca gcatgtctcc atgccctatg ccaccagagg gctgtacctg 360gacaccgagg ctgggtacca caagctgtcc agcgagtctt atggctttgt ggccaggatc 420gacagcagcg ggaacttcca aatcctgctg tcggacagac acttcaacaa gacctgtggg 480ctgtgcggtg actttaacat cttcgccgaa gatgacttca ggactcaaga aggaaccctg 540acctcagacc cctacgactt tgcaaactcc tgggccctga gcagtgagga gcagcggtgt 600ccacgggtgt cccctcccag cagctcctgc aacatctcct ctgagctgca gaagggcctg 660tgggagcagt gccagcttct gaagacggcc tccgtgttcg cccgctgcca cgccctggtg 720gaccccgagc ctttcgtggc cctgtgtgag cggatgctgt gcgcatgcgc ccaggggctg 780cgctgcccct gcccggtgct cctggagtac gcccgcgcct gcgcccagca agggatgctg 840ctgtacggct gggcggacca cagctcctgc cgaccggact gccccgcggg catggagtac 900aaggagtgtg tgtccccatg ccacaggacc tgccggagcc tgagtatcac cgaagtgtgt 960cgggagcagt gtgtggatgg ctgcagctgc cctgagggac agctcctgga tgaaggccgc 1020tgtgtggaaa gtgccgagtg tccctgtgtg catgctggaa agccataccc tcctggcgcc 1080tccctctcgc gagactgcaa cacctgcatc tgccgaaaca gccagtgggt ctgcagcaat 1140gaggactgtc caggagagtg tctcatcaca ggacaatccc acttcaagag ctttgacgac 1200aggcacttca ccttcagcgg ggtctgccag tacctgctgg cccaggactg ccaggaccac 1260tccttctccg tcatcataga gactgttcag tgtgctgacg accctgatgc ggtctgcacc 1320cgctccgtca ccgtccgcct gcccagcccg caccacggcc tcctgaagct gaagcacggg 1380ggtggagtcg ccctggatgg ccaggacgtc cagattcccc tcctgcaagg tgacctccgg 1440atccagcaca ctgtgacggc ctccctgcac ctcatcttcg gggaggacct gcagatagac 1500tgggacggtc gcgggaggct gctgctgaag ctgtccccgg tctacgcggg gaggacctgc 1560gggctgtgcg ggaattacaa cggcaaccag agggatgact tcctgacgcc cgcgggcctg 1620gtcgagcccc tggtggagca ctttggaaac tcctggaagc tacgtgcaga ctgtgaggac 1680ttgcaggagc agcccagtga cccctgcagc ctcaacccgc gcctgaccaa gttcgcagac 1740caggcctgcg ccatcctgac gtcccgcaag ttcgaggcct gccacagcgc cgtgggcccg 1800ctgccctacc tgcgcaactg ccgcttcgac gtgtgcgcct gctccgatgg cagagactgc 1860ctgtgcgacg cggtggccaa ctacgcggcg gcctgcgcca ggaggggcgt gcacatcggg 1920tggcgggagc ccagcttctg tgcactgagc tgcccacacg gccaggtgta ccagcagtgt 1980gggaccccct gcaacctcac ctgccgctca ctctcccacc cggacgagga atgcactgag 2040gtctgtctgg agggctgctt ctgccctgct gggctcttcc tggatgagac ggggtcctgt 2100gtgcccaagg cccagtgccc ctgttactat gacggcgaga tcttccaacc tgaagacatc 2160ttctcggacc atcacaccat gtgctactgc gaagatggct tcatgcactg ctccacgagc 2220ggagccccgg ggagcctgct gcctgaagca gtcctcagca gccctctgtc ccaccgcagc 2280aaaaggagcc tgtcctgccg gccccccatg gtcaagctgg tgtgccctgc tgacaacccg 2340agggccgaag ggctcgaatg caccaagacc tgccagaact atgacctgga atgcgtgagc 2400acgggctgtg tgtccggctg cctctgcccc ccgggcatgg tccggcatga gaacaggtgt 2460gtggccctgg aaaggtgccc ctgcttccac cagggcagag agtacgcccc cggagacagg 2520gtgaaggccg actgcaacac ctgcgtctgt caggaccgga agtggaactg tacggaccgc 2580gtgtgtgatg ctggctgctc tgccgtgggc ctggctcact acttcacctt tgatgggctc 2640aagtacctgt tcccggggga gtgccagtac gtcctggtac aggaccactg cggtagtaac 2700cctgggacct tccgggtcct ggtggggaat gaggggtgca gcgtcccctc cctgaagtgc 2760aggaagcgca tcaccatcct ggtggaggga ggagagatcg agctgtttga cggggaggtg 2820aacgtgaaga agcccatgaa ggatgagacg cacttcgagg tggtggaatc tggccggtac 2880atcactgtgc tgctgggcaa ggccctctct gtggtctggg acgggcacct ggccatctct 2940gtgttcctga agcggatgta ccaggagcgg gtatgcggcc tgtgtgggaa tttcgatggc 3000gtccagaaca atgacctcac cagtagcagc ctccaagtgg aggaagaccc tgtggacttt 3060gggaattcct ggaaagtgag cccgcattgc gctgacaccc agaaagtgcc gctggactcg 3120gcccctgcca cctgccacaa gaacgtcatg aagcagacca tggtggattc ctcctgcagg 3180gtcctcacca gtgatgtttt ccgggagtgc aacaggctgg tgaaccccga gccgtacctg 3240gatgtttgca tctacgacag ctgctcctgc gagtccatcg gggactgcgc ctgcttctgt 3300gacaccatcg ccgcctacgc ccacgagtgt gcccagcacg gcgaagtggt gacctggagg 3360acagccacac tgtgccccca gaattgtgag gagcggaacc tgaaggagag tgggtaccaa 3420tgtgagtggc gctacaacag ctgtgctccc gcctgtccag tcacgtgcca gcacccagag 3480cccctggcct gccccgtgca gtgcgtggag ggctgccacg cacactgccc gcctgggaaa 3540atcctggacg agcttttgca gacctgtgtc aaccccgagg actgccctgt gtgccaggtg 3600gagggccggc gcttagcctc cgggaagaaa gtgaccctga accctgggga ccctgagcat 3660tgccagctct gtcactgtga tggtgtcagc ctcacttgtg aagcctgcag ggagccagga 3720ggcctgcccg tgccccccac cgaaggcccg gtcagcccca caaccccgta cgtggaggac 3780accccggagc cacccctgca cgacttcttc tgcagcaaac tgctggacct ggtcttcctg 3840ctggacggct cctccaagct gtctgaggcc gacttcgaga cgctgaaggc gttcgtggtg 3900ggcatgatgg agcgtctgca catttcccag aaacgcatcc gtgtggccgt ggtggagtac 3960cacgatggct cccatgccta ccttgcactg caagaccgga agcggccatc cgagctgcgg 4020cgcattgccg ggcaggtgaa gtacgcgggc agcgaggtgg cttccaccag cgaggtcttg 4080aagtacacgc tcttccagat cttcggcagg attgaccggc ccgaggcctc tcgcgtggcc 4140ctgctgctta cggccagcca ggagcccccc aggctggccc ggaacttggt ccgctacctc 4200cagggcctga agaagaagaa ggtctccgtg gtcccggtgg gcatcgggcc ccacgtcagc 4260ctcaagcaga tccgcctcat cgagaagcag gcgtctgaga acaaagcctt tgtgctaagc 4320ggtgtgcacg agctggagca gcggatgaac gagattgtcg gctacctctg tgacctcgcc 4380cccgaggtgc ctgccccgac cccgacgcga catcctctca ttgcgcaggt cactgtggcg 4440ccgcagctcc tgggtccttc gccaccagga cccaagagga gctctgtggt cctggatgtg 4500gcattcctcc tggaaggctc ggatgaggta ggcgaggcca acttcaacag gagcgcagag 4560tttgtggagg aggtgatccg acgcatggac gttggccggg acggcatcca tgtcacggtg 4620ctgcagtact cgtacacggt gaccgtggag cactcgttca gggagccaca gtccaaggag 4680gtggtcctgc agcgactcca tgaagtccgc taccggggtg gcaaccagac gaacacgggg 4740ctggccctgc agtacctgtc ggagcacagc ttctccgcca gccaggggga ccgggagcag 4800gcgcccaacc tggtctacat ggtgacgggc agcccggcct cggacaagat ccagcggatg 4860ccaggagaca tccagctcgt gcccatcggc gtgggccccc gtgtggacgt gcaggagctg 4920gagagggtca gctggcccca gacccccatc ttcatccagg acttcgagag gctcccccga 4980gaagctccgg atctggtgct gcagcggtgc tgctccgaag acggcccgca cctccccacc 5040ctcgcccctg ccccagactg cagccagccc ctggatgtag tcctcctcct ggatggctcc 5100tccacccctc cagcctctta ctttgacgaa atgaagagtt tcgccaaggc tttcatctca 5160aaagccaacc taggccctca gcttacccag gtgtcagtcc tgcagtacgg gagcatcacc 5220aacgtcgacg tatcctggaa tgtgcacgtg gacaaagccc acctgctgag ccttgtggac 5280cccatgcagc gcgagggagg ccccagccga gtcggggagg cgttgtcctt cgcggtgcgc 5340tacatcacgt cccaagtcca cggtgccagg cccggggcct ccaaggtggt ggtgatcctg 5400gtcacaggct cctccatgga ctcagtggag gcggccgccg ctgctgccag atccaaccga 5460gtggctgtgt tccccatcgg gatcggggac cagtatgacg cagcccagct gagggtcttg 5520gcgggcccgg gggccagctc caatgtggca gagctccagc ggattgaaga cctccccagc 5580atggttgccc ttggcaactc cttcttccag aggctatgct ctgggttcgt cagtgtttgc 5640gtggatgagg acgggaatga gaggaggcct ggggacgtct ggaccttgct ggatcagtgc 5700cacacagtga cttgcctgcc agatggccag accttgctga agagtcaccg ggtcaactgt 5760gatcaggggc cacagccatc atgccccgac ggccagatcc cgctcaggat ggaggaagcc 5820tgtggctgcc gctgggcctg tccctgtgtg tgcacaggca gctccactcg gcacatcgtg 5880acctttgatg gacggaattt caagctgacc ggcaactgct catacgttct gtttcacaac 5940aaggagcagg acttggaggt gattctccat aacggattct gcagcgctgg ggcgaggcag 6000gcctgcatga aatctgtgga ggtgaagcac agcggcctct cggttgagct ccgcagcaac 6060atggaggtga tggtgaatgg gagactggtc tctgtccctt acctgggtgg ggacatggag 6120gtcagagtct atggtaccat catgttcgag gtcagattca accttctggg ccacatcctc 6180tccttcaccc cacgtgatga gacactccag gatggctgtg acagtcactt ctgcaaagtc 6240aacaagagag gagagttcat ttgggagaag agggtcatgg gctgcccgcc cttcaacgaa 6300cacaaatgtc tggctgaggg ggggaaagtc atgaaaattc ctggcacgtg ctgtgacaca 6360tgtgaggagc ccgagtgcaa ggacatcaca gccagggtgc agtacatcaa ggtgggagac 6420tgcaaatctg aagaggaagt ggacattaac tactgccagg gaagatgcac cagcaaagcc 6480ctgtactcca tcgacacgga ggacgtgcaa gaccagtgtt cctgctgctc gcccacgcgc 6540acggagccca tgtcagtgcc cctgcgctgc accaacggct ccatcattca ccatgtgatc 6600ctcaacgccc tgcagtgcaa gtgctcatcc aggaagtgcc gcccgtga 66483024PRTArtificial Sequencesynthetic porcine-derived linker sequence 30Ser Phe Ala Gln Asn Ser Arg Pro Pro Ser Ala Ser Ala Pro Lys Pro1 5 10 15Pro Val Leu Arg Arg His Gln Arg 203114PRTArtificial Sequencesynthetic human linker sequence 31Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg1 5 10326PRTArtificial Sequencesynthetic 6-His tag 32His His His His His His1 5334350DNAArtificial SequenceSynthetic truncated ovine (sheep) gene 33atgcacatca agctctgtac ctgcctcttt ctgtgcctct ggccatgcag cttcagtgcc 60atcagaagat actacctggg tgcagtggaa ctgtcctggg actatacgcg aagtgaactg 120ctcagtgagc tgcatgtgaa cacgaggttt cctcccagag tgcccaaacc ttttccattc 180aacacatcag tcatgtacag aaagactgtg tttgtagagt tcacggatca actttttaac 240atcgccaagc ccaggccacc atggatgggt ctgctgggtc cagctatcca ggctgaggtt 300tatgacaccg tggtcattac atttaagaat atggcttctc atcctgttag tcttcatgct 360attggcgtat cctactggaa atcttctgaa ggtgctgcat ataaggatga aaccagccaa 420agggagaagg aagatgacaa agtcattcct ggtaaaagtc atacctacgt ctggcatatc 480ctgaaagaaa acggtccaac agcctctgac ccaccatgtc tcacctactc atatctttct 540catgtggacc tggtgaaaga cgtgaactca ggtctcatcg gagccctgct aatttgtagg 600gaagggactc tgatcaaaga aaggacacag accttgcaca aattcgtact actgtttgct 660gtatttgatg aagggaaaag ttggcactcg ggaaaaaatg agtccttgac acatgttatg 720gattctgcct ctgtactgca caccatcaat ggctatataa acaggtctct gccaggtctg 780attggatgtc ataagaaatc agtctattgg catgtgattg gaatgggcac caccccagaa 840gtgcactcaa ttttcctcga aggccacaca tttctcgtga ggaaccatcg ccaggcttcc 900ttggagatct caccaataac tttccttacc gctcagacag tcctgatgga ctgtggccag 960tttctactgt tttgtcgtat ctcttcccac caacatgatg gtatggaagc ttatgtcaaa 1020gtagatagtt gcccagagga accccgacta tggatgaaaa ataaccaaga agaagattac 1080gatgatggtt tggatgactc tgacatggat gtggtcaggt tcgatggtga cagtgtgcct 1140ccctttatcc aaatgcgctc agttgcaaag aagcatccta aaacctgggt ccactacatt 1200gctgctgaag aggatgactg ggactatgcc ccctcggtcc tcacctccaa tgacagaagt 1260tataaaagtc tgtatctgaa ccacggtcct cagcggattg gtaggaagta cagaaaagta 1320cgatttatag cttacacaga tggaacattt aagactcgtg aagctattca gcatgaatca 1380gggatcctgg ggcctttact ttatggagaa gttggagaca cacttttgat tatatttaag 1440aatcaagcaa gccggccata taacatctac cctcatggaa tcactgatgt cagtcctttg 1500cactcaggga gatttccaaa aggtgtgaaa catttgaaag acatgccaat tctgccagga 1560gaagtgttca agtataaatg gacagtgact gtagaagatg ggccaactaa atcagatcct 1620cggtgtctga cccgatatta ctcgagtttc attaacttag agaaagatct agcttcagga 1680ctcattggcc ccctcctcat ctgttacaaa gaatctgtag atcaaagagg aaaccagatg 1740atgtcagaca agagaaatgt catcctgttt tctgtatttg atgaaaacaa aagttggtac 1800ctcacagaga atattcaacg cttcctcccc agtggagtac agccccagga tccagagttc 1860caagtctcca atgtcatgca cagcatcaat ggctatgttt ttgatagctt gcagctgtcg 1920gtttgtttgc atgaggtggc gtactggtac attctaagtg ttggagccca aattgacttc 1980ctctctgtct tcttctctgg atataccttc aaacacaaaa tggtctatga agacacactc 2040accctattcc ccttctcggg agaaactgtc ttcatgtcaa tggaaaatcc aggtctgtgg 2100gttctggggt gccacaactc agactttcga aacagaggca tgacagcctt actgaaggtt 2160gatagttgtg acaggaacgt tggcgattat tatgacacat atgaagctat tccaaccttc 2220ctgctgagtg aaaacaatgt cattgaaccc agaagcttct cccagaatcc accaagcttg 2280aaacgccatc aaagggagat aacccttact acttttcagc cagagccaga caaaactgac 2340tatgatgata ctttgtcgat tgaaacaaag agagaagatt ttgacattta tggtgaagat 2400gaaaatcagg acccccgcag ctttcaaaag agaacacgcc actattttat tgctgcagtg 2460gagcggctct gggattatgg gatgagtaga tccccccacg cactaagaaa caggtctcag 2520aatggaggag tccctcagtt caaaaaggtg gtgttcgagg aatttactga tggctccttt 2580actcaggccg tataccgtgg acaattaaat gaacacctgg gactcttggg accatatata 2640agagcagaag tggaagacaa tatcatggta actttcaaaa accaggcctc tcgtccctac 2700tccttctatt ctagccttat ttcttataac ggagatcaga gacaaggagc agaacctcga 2760aaaaagtttg tcaagcctaa tgaaacccaa agctactttt ggaaagtgca gcaccatatg 2820gcacccacca aagatgagtt tgactgcaaa gcctgggctt acttttctga tgttgatctg 2880gaaaaagatg tgcactcagg cttgattggc cccattctga tctgccgtgt ggacacgctg 2940agtgctgctc atgggagaca agtgacagta caggaattcg ctctgttttt caccattttt 3000gatgagacca agagctggta ctttgccgaa aacatggcaa ggaactgcgt ggcaccctgc 3060catgtccagc cagaggaccc tactttccaa gaaaagtatc gcttccatgc aatcaatggc 3120tacgtgatgg atacactccc tggcttagtc atggctcagc atcaaaggat taggtggtat 3180ctgctcagca tgggcagcaa tgaaaatatc cattccattc atttcagtgg ccatgtgttc 3240actgtgagaa aaacggagga gtataaaatg gcggtctaca atctctaccc aggtgtcttt 3300gagaccgtgg aaatgctacc atccaaggtt gggacttggc ggatagaatg tcttattggc 3360gagcacctac aagctgggat gagcactctc ttcctggtgt acagcaagga gtgtcaaatt 3420ccactgggaa tggcttctgg acgcattaga gattttcaga ttacagcttc aggacaatat 3480ggacagtggg ccccaaagct ggccagactt cattattctg gatcaatcaa tgcctggagc 3540accaaggatc cctctccttg gatcaaggtg gatctgttgg cgccgatgat tattcacagc 3600atcctgactc agggtgcccg gcagaagttc tccagcctgt acatctctca gtttatcatc 3660atgtacagcc tcgatggaca gcggtggcag ggttatcggg ggaactccac tgggacttta 3720atggtgttct ttggcaatgt ggattcatct ggagtaaaac acaatatttt taaccctcca 3780attattgcta gatatatccg tttgcaccca acgcattaca gcatccgcag cactcttcgc 3840atggagttga tgggctgtga cttaaatagt tgcaacatgc cactgggaat ggagaataaa 3900gctatatcag atacacagat tactgcctca tcccacttaa gcaacatgtt tgccacctgg 3960tctccttcac aagcccgact taaccttcaa gggaggacaa atgcctggag accccaggtg 4020aataatccaa aagagtggct acaagtggac ttccagaaga caatgagagt tacaggaata 4080accactcaag gggtgaaatc tctgcttacc agcatgtatg tgaaggagtt ccttatatcc 4140agtagtcaag agggccataa ctggactcca tttcttcaga atggcaaagt gaaggttttt 4200cagggaaatc aagactcctt cacccccgtg gtgaatactc tagacccccc actgtttacc 4260cgcttccttc ggattcaccc gcagagctgg gtgcaccata tcgccctgag gctggagttt 4320tggggttgtg aggcacagca gcagtactag 4350

Claims

1. A method of treating a subject diagnosed with hemophilia A, comprising: (a) modifying mesenchymal stem/stromal cells (MSC) to express high levels of Factor VIII protein or high levels of both Factor VIII protein and von Willebrand factor (vWF) protein thereby generating modified MSC, the MSC comprising bone-marrow MSC isolated from the subject; (b) generating an expanded modified MSC population by in vitro culturing the modified MSC; and (c) injecting MSC from the expanded modified MSC population into the subject.

2. The method of claim 1, wherein isolating the MSC comprises isolating cells that express at least one of Stro-1 or CD146.

3. The method of claim 1, wherein the subject has received prior treatment with exogenous Factor VIII and has developed an inhibitory immune response that diminishes the effectiveness of the exogenous Factor VIII treatment.

4. A method of treating a subject prenatally diagnosed as having hemophilia A, comprising: (a) modifying mesenchymal stem/stromal cells (MSC) to express high levels of Factor VIII protein or high levels of both Factor VIII protein and von Willebrand factor (vWF) protein thereby generating modified MSC, the MSC comprising MSC isolated from at least one of amniotic fluid, placental tissue, or umbilical cord tissue obtained at the time of the subject's birth or prenatally from the subject's mother; (b) generating an expanded modified MSC population by in vitro culturing the modified MSC; and (c) injecting MSC from the expanded modified MSC population into the subject.

5. The method of claim 4, wherein isolating the MSC comprises isolating cells that express c-kit.

6. The method of claim 4, wherein the isolated MSC express c-kit, CD34, CD90, and CD133.

7. (canceled)

8. (canceled)

9. The method of claim 1, wherein the modified MSC comprise one or both of a Factor VIII gene sequence or a vWF gene sequence comprising one or more modifications that increase protein expression, protein stability, or both, of the Factor VIII protein or vWF protein.

10. (canceled)

11. (canceled)

12. The method claim 1, wherein the MSC are modified by introducing into the MSC a viral vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter, a viral vector comprising a vWF gene sequence operatively linked to a constitutively active promoter, or a viral vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter and a vWF gene sequence operatively linked to a constitutively active promoter.

13. The method claim 1, wherein the MSC are modified via gene editing, wherein the gene editing introduces one or more modifications to one or both of an endogenous Factor VIII gene sequence or an endogenous vWF gene sequence that increase protein expression, protein stability, or both.

14. (canceled)

15. (canceled)

16. The method of claim 12, wherein the Factor VIII gene sequence and the vWF gene sequence are operatively linked to the same constitutively active promoter.

17. (canceled)

18. The method of claim 1, wherein the MSC from the expanded modified MSC population are injected into the subject via at least one of intraperitoneal injection, intravenous injection, or intra-articular injection.

19. The method of claim 1, wherein the expanded modified MSC population are injected into the subject at least once, at least twice, or at least three times.

20. The method of claim 1, wherein the expanded modified MSC population are injected into the subject in an amount of about 10.sup.7 to about 10.sup.9 MSC per kilogram weight of the subject.

21. The method of claim 4, wherein the modified MSC comprise one or both of a Factor VIII gene sequence or a vWF gene sequence comprising one or more modifications that increase protein expression, protein stability, or both, of the Factor VIII protein or vWF protein.

22. The method claim 4, wherein the MSC are modified by introducing into the MSC a viral vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter, a viral vector comprising a vWF gene sequence operatively linked to a constitutively active promoter, or a viral vector comprising a Factor VIII gene sequence operatively linked to a constitutively active promoter and a vWF gene sequence operatively linked to a constitutively active promoter.

23. The method of claim 22, wherein the Factor VIII gene sequence and the vWF gene sequence are operatively linked to the same constitutively active promoter.

24. The method claim 4, wherein the MSC are modified via gene editing, wherein the gene editing introduces one or more modifications to one or both of an endogenous Factor VIII gene sequence or an endogenous vWF gene sequence that increase protein expression, protein stability, or both.

25. The method claim 4, wherein the MSC from the expanded modified MSC population are injected into the subject via at least one of intraperitoneal injection, intravenous injection, or intra-articular injection.

26. The method claim 4, wherein the expanded modified MSC population are injected into the subject at least once, at least twice, or at least three times.

27. The method claim 4, wherein the expanded modified MSC population are injected into the subject in an amount of about 10.sup.7 to about 10.sup.9 MSC per kilogram weight of the subject.