MUTANT FACTOR VIII COMPOSITIONS AND METHODS

Abstract

In one aspect, present invention provides a recombinant mutant human factor VIII having increased expression and/or secretion as compared to wild-type factor VIII. In certain embodiments, the recombinant factor VIII includes one or more amino acid substitution(s) selected from the group consisting of 186, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152. In other aspects, the present invention provides FVIII encoding nucleic acids, FVIII-expression vectors, as well as methods of using the modified FVIII genes in the treatment of FVIII deficiencies, such as hemophilia A.

Description

[0001] This application is a continuation application of U.S. application Ser. No. 14/893,878, filed Nov. 24, 2015, which is a National Stage Application of International Application PCT/US2014/043777, filed Jun. 24, 2014, which claims priority to U.S. Provisional Patent Application No. 61/838,867, filed on Jun. 24, 2013. The entirety of the aforementioned applications are incorporated herein by reference.

SEQUENCE LISTING

[0003] This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference herein in its entirety. The ASCII text file was created on Sep. 13, 2018, is named Sequence.txt and is 81,419 bytes in size.

FIELD

[0004] The present invention relates to recombinant human factor VIII mutants exhibiting higher expression levels than the corresponding wild-type human factor VIII. The present invention also relates to methods of making and using the recombinant human factor VIII mutants.

BACKGROUND

[0005] Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein factor VIII. In patients with Hemophilia A, the blood does not clot properly resulting in excessive bleeding when the hemophiliac is injured. Treatment consists of replacement therapy using preparations of (purified) plasma or the recombinant protein. Blood clotting begins when platelets adhere to the cut wall of an injured blood vessel at a lesion site. Subsequently, in a cascade of enzymatically regulated reactions, soluble fibrinogen molecules are converted by the enzyme thrombin to insoluble strands of fibrin that hold the platelets together in a thrombus. At each step in the cascade, a protein precursor is converted to a protease that cleaves the next protein precursor in the series. Cofactors are required at most of the steps.

[0006] Factor VIII circulates as an inactive, non-covalent, metal ion-dependent heterodimer precursor in blood, bound tightly and non-covalently to von Willebrand factor. This procofactor form of the protein contains a heavy chain (HC) comprised of A1(a1)A2(a2)B domains and a light chain (LC) comprised of (a3)A3C1C2 domains, with the lower case a representing short (.about.30-40 residue) segments rich in acidic residues. Factor VIII is proteolytically activated by proteolytic cleavages at the A1 A2, A2B and A3A3 junctions catalyzed by thrombin or factor Xa, which serves to dissociate it from von Willebrand factor and activate its procoagulant function in the cascade. In its active form, the protein factor VIIIa is a cofactor that increases the catalytic efficiency of the serine protease factor IXa in the membrane-dependent conversion of zymogen factor X to the serine protease, factor Xa factor by several orders of magnitude.

[0007] Gene therapy has been proposed as treatment modality for supplementing deficiencies in clotting factors in hemophiliacs and there have been attempts to engineer FVIII constructs that are suitable for treatment of humans. For example, Connelly, et al. reported that treatment of FVIII-deficient mice with human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII (Connelly, et al., Blood, Vol. 91, No. 9 (1998), pp. 3273-3281). Sarkar, et al. reported that use of AAV8 serotype in combination with FVIII corrected plasma FVIII activity in mouse models (Sarkar, et al., Blood, Vol. 103, No. 4 (2004), pp. 1253-1260).

[0008] However, as in many areas of gene therapy, theory is much more straightforward than successful, effective implementation. Difficulties in implementation of gene therapy techniques include problems encountered in the use of viruses as gene vectors and insufficient expression levels of FVIII. For example, human FVIII secretes very inefficiently and the yield is logs lower comparing to similar proteins such as factor V. Further, while viruses are effective as gene vectors because they can be used to transduce cells leading to protein expression in vivo, the proteins coating the virus particle may activate the body's immune system.

[0009] Thus, in view of the foregoing, there is a need for approaches that can efficiently express the target FVIII protein in sufficient quantity to reduce the required dose of viral vector to tolerable levels.

SUMMARY

[0010] The present invention provides modified factor VIII (FVIII) proteins, FVIII encoding nucleic acids, and FVIII-expression vectors, as well as methods of using the modified FVIII genes in the treatment of FVIII deficiencies, such as hemophilia A.

[0011] In one aspect, present invention provides a mutant human factor VIII having increased expression or secretion as compared to wild-type factor VIII.

[0012] In one embodiment, the recombinant mutant human factor VIII includes one or more amino acid substitution(s) selected from the group consisting of I86, Y105, A108, D115, Q117, F129, G132, H134, M147, L152 and combinations thereof.

[0013] In another embodiment, the recombinant mutant human factor VIII includes one or more amino acid substitution(s) selected from the group consisting of I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T, L152P and combinations thereof.

[0014] In another embodiment, the recombinant mutant human factor VIII includes amino acid substitutions in each of the amino acids I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152.

[0015] In another embodiment, the recombinant mutant human factor VIII includes the amino acid substitutions I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P.

[0016] In another embodiment, the recombinant mutant human factor VIII includes one or more amino acid substitution(s) selected from the group consisting of I86, A108, G132, M147, L152 and combinations thereof.

[0017] In another embodiment, the recombinant mutant human factor VIII includes one or more amino acid substitution(s) selected from the group consisting of I86V, A108S, G132K, M147T, L152P and combinations thereof.

[0018] In another embodiment, the recombinant mutant human factor VIII includes amino acid substitutions in each of I86V, A108S, G132K, M147T and L152P.

[0019] In another embodiment, the recombinant mutant human factor VIII includes the amino acid substitutions I86V, A108S, G132K, M147T and L152P.

[0020] In other embodiments, the human factor VIII mutants further include a deletion in the B domain of human factor VIII.

[0021] In other embodiments, the human factor VIII mutants further include the a2 and/or a3 domain(s) of human factor VIII.

[0022] In another aspect, the present invention provides isolated polynucleotide sequences encoding the human factor VIII mutants described herein.

[0023] In yet another aspect, the present invention provides an expression vector operatively linked to the polynucleotides encoding the human factor VIII mutants described herein.

[0024] In a further aspect, the present invention provides a pharmaceutical composition comprising an expression vector operatively linked to the polynucleotides encoding the human factor VIII mutants described herein.

[0025] In another aspect, the present invention provides a method for treating a patient with a factor VIII deficiency comprising administering to a patient in need thereof a pharmaceutical composition comprising an expression vector operatively linked to the polynucleotides encoding a human factor VIII mutant described herein in an amount effective for treating the factor VIII deficiency.

[0026] In yet another aspect, the present invention provides a method for expressing a human factor VIII polypeptide mutant comprising: (a) transforming a host cell with an expression vector operatively linked to a polynucleotides encoding a human factor VIII mutant according to the present invention; (b) growing the host cell under conditions suitable for expressing the human factor VIII polypeptide mutant; and (c) purifying the human factor VIII polypeptide mutant from host cells expressing said mutant.

BRIEF DESCRIPTION OF DRAWINGS

[0027] FIG. 1 illustrates the structural domains of the human factor VIII heavy and light chains, including several heavy chain constructions utilized in the present invention.

[0028] FIG. 2 is a graph showing an alignment of the first 200 amino acids of the secreted human factor VIII heavy chain alongside a modified factor VIII with 10 exemplary substitutions for enhanced secretion.

[0029] FIG. 3 is a graph showing the alignment of the first 200 amino acids of the secreted human factor VIII heavy chain alongside a modified human factor VIII mutant with 5 exemplary substitutions for enhanced secretion.

[0030] FIG. 4 summarizes exemplary amino acids determined to affect human factor VIII secretion.

[0031] FIGS. 5 and 6 depict exemplary AAV vectors for expressing B-domain deleted human factor VIII mutants or human factor VIII heavy chain.

[0032] FIG. 7 shows a comparison of the secretion of different human factor VIII mutants in BHK cells (panel A) or 293 cells (panel B).

[0033] FIG. 8 shows a comparison of the secretion of different human factor VIII mutants in secretion in vivo. Plasmids pAAV-CB-hBDDF8 (carrying human factor VIII with B-domain deleted), pAAV-CB-hBDDF8-X10 (carrying hF8-BDD with 10 substitutions), pAAV-CB-hBDD-F8-X5 (carrying hBDDF8 with 5 substitutions) or pAAV-CB-hBDD-F8-G3 1 2K (hF8 with G132K substitutions) were injected in factor VIII double knock-out Balb/c mice.

[0034] FIG. 9 show a comparison of the secretion of different human factor VIII mutants in 293 cells. Amino acids in hBDD-F8-X10 were reverted back to their corresponding wild type amino acids. In hBDD-F8-X10-6p, 6 of 10 amino acids in hBDD-F8-X10 differing from wild type F8 were reverted back to their wild type amino acids.

[0035] FIG. 10 shows a comparison of the expression and secretion of a mutant factor VIII (F8) heavy chain (HC1690-X10) in an AAV vector construct with a wild type hF8-HC1690 AAV vector construct (AAV/hF8HC1690) in vivo.

[0036] FIG. 11 shows a comparison of the secretion of different G132 factor VIII heavy chain mutants in 293 cells.

[0037] FIG. 12 shows a comparison of the secretion of different factor VIII heavy chain mutants in expression/secretion in factor VIII double knock-out Balb/c mice.

[0038] FIG. 13 shows a comparison of the secretion of different L152 factor VIII heavy chain mutants in 293 cells.

[0039] FIG. 14 shows a comparison of the secretion of different A108 factor VIII heavy chain mutants in 293 cells.

[0040] FIG. 15 shows a comparison of the secretion of different M147 factor VIII heavy chain mutants in 293 cells.

[0041] FIG. 16 shows that there was no formation of neutralizing antibodies against 5 mutated amino acids in the F8-/- rat model.

DETAILED DESCRIPTION

[0042] Various terms relating to the biological molecules of the present invention are used hereinabove and also throughout the specification and claims.

[0043] The phrase "secretion enhanced factor VIII (seFVIII, seF8)" refers to a modified FVIII (F8) which has been genetically altered such that the encoded protein exhibits at least 10% or 20% or 50% or 100% increase in secretion when compared to unmodified FVIII. The nucleotide sequences described herein are readily obtainable from GenBank. For human FVIII, see Accession No. NG-011403.1.

[0044] The phrase "BDD" refers to a "B-domainless" factor VIII (F8 or FVIII) mutant lacking the B domain.

[0045] The phrase "one or more" followed by a list of elements or species is intended to encompass any permutation of elements or species in the list. Thus, for example, the phrase "one or more substitution mutations selected from the group consisting of A, B, C, D, E and F" may include any combination of substitution mutations containing A, B, C, D, E and/or F.

[0046] As used herein, ranges may be expressed from one particular integer value to another particular integer value. When such a range is expressed, it should understand that any and all integer values within that range define separate embodiments according to the present invention and that the full scope of embodiments includes within the range further includes any and all sub-ranges between any pair of integer values in the initial range.

[0047] With reference to nucleic acids of the invention, the term "isolated nucleic acid", when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates. For example, the "isolated nucleic acid" may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote. The nucleic acid codons can be optimized for enhanced expression in the mammalian cells.

[0048] With respect to RNA molecules of the invention, the term "isolated nucleic acid" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure" form (the term "substantially pure" is defined below).

[0049] With respect to protein, the term "isolated protein" or "isolated and purified protein" is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure" form.

[0050] The term "promoter region" refers to the transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns.

[0051] The term "vector" refers to a small carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell where it will be replicated. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell.

[0052] The term "operably linked" means that the regulatory sequences necessary for expression of a coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.

[0053] The term "substantially pure" refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, of the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0054] The phrase "consisting essentially of when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

[0055] The term "oligonucleotide," as used herein refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application for which the oligonucleotide is used. The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

[0056] The term "percent identical" is used herein with reference to comparisons among nucleic acid or amino acid sequences. Nucleic acid and amino acid sequences are often compared using computer programs that align sequences of nucleic or amino acids thus defining the differences between the two. Comparisons of nucleic acid sequences may be performed using the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wis. For convenience, the default parameters (gap creation penalty=12, gap extension penalty=4) specified by that program are intended for use herein to compare sequence identity. Alternately, the Blastn 2.0 program provided by the National Center for Biotechnology Information (found on the world wide web at ncbi.nlm.nih.gov/blast/; Altschul, et al., 1990, J Mol Biol 215:403-410) using a gapped alignment with default parameters, may be used to determine the level of identity and similarity between nucleic acid sequences and amino acid sequences.

[0057] A "corresponding" nucleic acid or amino acid or sequence of either, as used herein, is one present at a site in a factor VIII or hybrid factor VIII molecule or fragment thereof that has the same structure and/or function as a site in the factor VIII molecule of another species, although the nucleic acid or amino acid number may not be identical. A sequence "corresponding to" another factor VIII sequence substantially corresponds to such sequence, and hybridizes to the human factor VIII DNA sequence designated SEQ ID NO:1 under stringent conditions. A sequence "corresponding to" another factor VIII sequence also includes a sequence that results in the expression of a factor VIII or claimed procoagulant hybrid factor VIII or fragment thereof and would hybridize to a nucleic molecule comprising SEQ ID NO:1 but for the redundancy of the genetic code.

[0058] A "unique" amino acid residue or sequence, as used herein, refers to an amino acid sequence or residue in the factor VIII molecule of one species that is different from the homologous residue or sequence in the factor VIII molecule of another species.

[0059] "Specific activity," as used herein, refers to the activity that will correct the coagulation defect of human factor VIII-deficient plasma. Specific activity is measured in units of clotting activity per milligram total factor VIII protein in a standard assay in which the clotting time of human factor VIII deficient plasma is compared to that of normal human plasma. One unit of factor VIII activity is the activity present in one milliliter of normal human plasma. In the assay, the shorter the time for clot formation, the greater the activity of the factor VIII being assayed. Hybrid human/porcine factor VIII has coagulation activity in a human factor VIII assay. This activity, as well as that of other hybrid or hybrid equivalent factor VIII molecules or fragments thereof, may be less than, equal to, or greater than that of either plasma-derived or recombinant human factor VIII.

[0060] "Subunits" of human or animal factor VIII, as used herein, are the heavy and light chains of the protein. The heavy chain of factor VIII contains three domains, A1, A2, and B. The light chain of factor VIII also contains three domains, A3, C1, and C2.

[0061] The terms "epitope", "antigenic site", and "antigenic determinant", as used herein, are used synonymously and are defined as a portion of the human, animal, hybrid, or hybrid equivalent factor VIII or fragment thereof that is specifically recognized by an antibody. It can consist of any number of amino acid residues, and it can be dependent upon the primary, secondary, or tertiary structure of the protein. In accordance with this disclosure, a hybrid factor VIII, hybrid factor VIII equivalent, or fragment of either that includes at least one epitope may be used as a reagent in the diagnostic assays described below. In some embodiments, the hybrid or hybrid equivalent factor VIII or fragment thereof is not cross-reactive or is less cross-reactive with all naturally occurring inhibitory factor VIII antibodies than human or porcine factor VIII.

[0062] The term "immunogenic site", as used herein, is defined as a region of the human or animal factor VIII, hybrid or hybrid equivalent factor VIII, or fragment thereof that specifically elicits the production of antibody to the factor VIII, hybrid, hybrid equivalent, or fragment in a human or animal, as measured by routine protocols, such as immunoassay, e.g., ELISA, or the Bethesda assay, described herein. It can consist of any number of amino acid residues, and it can be dependent upon the primary, secondary, or tertiary structure of the protein. In some embodiments, the hybrid or hybrid equivalent factor VIII or fragment thereof is nonimmunogenic or less immunogenic in an animal or human than human or porcine factor VIII.

[0063] "Factor VIII deficiency," as used herein, includes deficiency in clotting activity caused by production of defective factor VIII, by inadequate or no production of factor VIII, or by partial or total inhibition of factor VIII by inhibitors. Hemophilia A is a type of factor VIII deficiency resulting from a defect in an X-linked gene and the absence or deficiency of the factor VIII protein it encodes.

[0064] In one aspect, the present invention relates to a recombinant factor VIII mutant molecule (e.g., protein or nucleic acid) characterized by increased expression and/or secretion as compared to wild-type factor VIII.

[0065] Exemplary human factor VIII cDNA (nucleotide) and predicted amino acid sequences are shown in SEQ ID NOs: 1 and 2, respectively. Human factor VIII is synthesized and secreted as an approximately 300 kDa single chain protein of 2332 amino acids with internal sequence homologies defining a series of structural "domains" as follows: NH2-A1-a1-A2-a2-B-a3-A3-C1-C2-COOH (FIG. 4). As used herein, a factor VIII "domain" is defined by a continuous sequence of amino acids characterized by e.g., internal amino acid sequence identity to structurally related domains and by sites of proteolytic cleavage by thrombin. Further, the terms "domainless" or "lacking a domain" should be understood to mean that at least 95% or 100% of the domain has been deleted. Unless otherwise specified, factor VIII domains are defined by the following amino acid residues in the human factor VIII amino acid sequence set forth in SEQ ID NO:2:

[0066] A1, residues Ala1-Arg372

[0067] A2, residues Ser373-Arg740

[0068] B, residues Ser741-Arg1648;

[0069] a3, residues P1640-Arg1649;

[0070] A3, residues Ser1690-Ile2032;

[0071] C1, residues Arg2033-Asn2172; and

[0072] C2, residues Ser2173-Tyr2332

[0073] The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is usually referred to as the factor VIII light chain activation peptide (FIG. 1). Factor VIII is proteolytically activated by thrombin or factor Xa, which dissociates it from von Willebrand factor, forming factor VIIIa, which has procoagulant function. The biological function of factor VIIIa is to increase the catalytic efficiency of factor IXa toward factor X activation by several orders of magnitude. Thrombin-activated factor Villa is a 160 kDa A1/A2/A3-C1-C2 heterotrimer that forms a complex with factor IXa and factor X on the surface of platelets or monocytes.

[0074] A cDNA sequence encoding the wild-type human factor VIII has the nucleotide sequence set forth in SEQ ID NO:1. In SEQ ID NO:1, the first 57 nucleotides of the factor VIII open reading frame encodes a signal peptide sequence which is typically cleaved off from the mature factor VIII protein represented by SEQ ID NO:2.

[0075] Preferred recombinant factor VIII mutants include or encode one or more amino acid substitutions in the region from as 86 to as 152 of the wild-type human factor VIII amino acid sequence set forth in SEQ ID NO:2. Substitutions within any of these positions may employ any of the other 19 amino acids.

[0076] With reference to mutants described herein, the notion represented by "(amino acid a)-(SEQ ID NO:2 amino acid #b)-(amino acid c)" should be understood to mean that wild type amino acid a (one-letter code) at amino acid number b of SEQ ID NO:2 has been mutated to amino acid c.

[0077] In certain preferred embodiments, the human factor VIII polypeptide mutant comprises amino acid substitution(s) in one or more amino acids in SEQ ID NO:2 selected from the group consisting of I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152. Further, the human factor VIII mutants may include any permutation of mutations encompassing these ten amino acid sites. Exemplary human factor VIII mutants are described in FIG. 4.

[0078] An exemplary recombinant factor VIII of this invention includes a point mutation involving a substitution at I86 of SEQ ID NO:2. A preferred substitution includes valine (i.e., I86V). Further preferred substitutions include leucine (I86L) and methionine (I86M).

[0079] Another exemplary recombinant factor VIII includes a point mutation involving a substitution at Y105 of SEQ ID NO:2. The substitution may include any of the other 19 amino acids. Preferred substitutions include Y105F and Y105W.

[0080] Another exemplary recombinant factor VIII includes a point mutation involving a substitution of at positions A108 of SEQ ID NO:2. A preferred substitution includes: A108S. Further preferred substitutions include A108S, A108G, A108T and A108P.

[0081] Another exemplary recombinant factor VIII includes a point mutation involving a substitution of at positions D115 of SEQ ID NO:2. A preferred substitution includes: D115E. Further preferred substitutions include D115N, D115H, D115Q, D115R and D115K.

[0082] Another exemplary recombinant factor VIII includes a point mutation involving a substitution of at positions Q117 of SEQ ID NO:2. A preferred substitution includes: Q117H. Further preferred substitutions include Q117N, Q117E, Q117D, Q117R and Q117K.

[0083] Another exemplary recombinant factor VIII includes a point mutation involving a substitution of at positions F129 of SEQ ID NO:2. A preferred substitution include: F129L. Further preferred substitutions include F129V, F129I, F129M, F129P, F129T and F129K.

[0084] Another exemplary recombinant factor VIII includes a point mutation involving a substitution of at positions G132 of SEQ ID NO:2. A preferred substitution include: G132K. Further preferred substitutions include G132E, G132D, G132R, G132T, G132M, G132N, G132S and G132W.

[0085] Another exemplary recombinant factor VIII includes a point mutation involving a substitution of at positions H134 of SEQ ID NO:2. A preferred substitution includes: H134Q. Further preferred substitutions include H134G, H134Y, H134N, H134E, H134D, H134R and H134K.

[0086] Another exemplary recombinant factor VIII includes a point mutation involving a substitution of at positions M147 of SEQ ID NO:2. A preferred substitution includes: M147T. Further preferred substitutions include M147A, M147G, M147S and M147P.

[0087] Another exemplary recombinant factor VIII includes a point mutation involving a substitution of at positions L152 of SEQ ID NO:2. A preferred substitution includes: L152P. Further preferred substitutions include L152S, L152G and L152T.

[0088] Another exemplary recombinant factor VIII includes multiple substitutions of one or more amino acid residues at positions I86, A108, G132, M147, L152 of SEQ ID NO:2. The substitution(s) can include any permutation of mutations encompassing these five amino acid sites. Specific embodiments may include mutations at one or more substitution mutations selected from the group consisting of I86V, A108S, G132K, M147T, L152P. Further preferred embodiments include 2, 3, 4 or 5 substitutions, including any combination (or permutation) of substitutions selected from I86V, A108S, G132K, M147T and L152P.

[0089] Exemplary recombinant factor VIII mutants include point mutation(s) involving substitution(s) at one or more amino acid residues in SEQ ID NO:2 selected from the group consisting of I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152. The substitution(s) can include any permutation of mutations encompassing these ten amino acid sites. Specific embodiments may include mutations at one or more substitution mutations selected from the group consisting of I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P. Further preferred embodiments include 2, 3, 4 or up to 9 substitutions, including any combination (or permutation) of substitutions selected from I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P.

[0090] The nucleic acids encoding the above mentioned factor VIII substitutions are included in this invention and include all possible nucleic acids encoding the breadth of substitution mutants described herein.

[0091] Compared to wild type factor VIII in production (in cell lines or in vivo), the above described factor VIII mutants may exhibit increases in factor VIII secretion of between 5% to 10,000 fold, 10% to 2,000 fold, 50% up to 500 fold, 2 to 200 fold, 5 to 100 fold, 10 to 50 fold, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 500 fold, at least 2,000 fold or at least 10,000 fold.

[0092] In certain embodiments, suitable mutant factor VIII sequences may be further modified to include, delete, or modify other factor VIII sequences to confer properties with regard to other attributes, including, without limitation, antigenicity, circulating half-life, protein secretion, affinity for factor IXa and/or factor X, altered factor VIII-inactivation cleavage sites, stability of the activated factor VIII form, immunogenicity, and shelf-life.

[0093] In certain specific embodiments, the mutant factor VIII may be modified to produce a B-domain deleted (BDD) or "B-domainless" factor VIII product. FIG. 1 shows an exemplary BDD factor VIII embodiment containing amino acid residues 1-740 and 1690-2332 of SEQ ID NO:2. Preferably, the recombinant B-domainless factor VIII contains one or multiple substitutions at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147, L152 as described herein.

[0094] In one embodiment, a B-domainless recombinant factor VIII is produced, whereby the B-domain is replaced with a DNA linker segment and at least one codon is replaced with a codon encoding an amino acid residue having the same charge as a corresponding residue of porcine factor VIII (see, e.g., U.S. Patent Application Publication No. 2004/0197875 to Hauser, et al.).

[0095] In another embodiment, a B-domainless recombinant factor VIII is produced having a truncated factor IX intron 1 inserted in one or more locations (see, e.g., U.S. Pat. No. 6,800,461 to Negrier and U.S. Pat. No. 6,780,614 to Negrier). This recombinant factor VIII can be used for yielding higher production of the recombinant factor VIII in vitro as well as in a transfer vector for gene therapy (see, e.g., U.S. Pat. No. 6,800,461 to Negrier). In a particular embodiment, the recombinant factor VIII can be encoded by a nucleotide sequence having a truncated factor IX intron 1 inserted in two locations and having a promoter that is suitable for driving expression in hematopoietic cell lines, and specifically in platelets (see, e.g., U.S. Pat. No. 6,780,614 to Negrier).

[0096] A second example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a chimeric human/animal factor VIII that contains one or more animal amino acid residues as substitution(s) for human amino acid residues that are responsible for the antigenicity of human factor VIII. In particular, animal (e.g., porcine) residue substitutions can include, without limitation, one or more of the following: R484A, R488G, P485A, L486S, Y487L, Y487A, S488A, S488L, R489A, R489S, R490G, L491S, P492L, P492A, K493A, G494S, V495A, K496M, H497L, L498S, K499M, D500A, F501A, P502L, 1503M, L504M, P505A, G506A, E507G, 1508M, 1508A, M2199I, F2200L, L2252F, V2223A, K2227E and/or L225I (U.S. Pat. No. 5,859,204 to Lollar, U.S. Pat. No. 6,770,744 to Lollar, and U.S. Patent Application Publication No. 2003/0166536 to Lollar). Preferably, the recombinant chimeric factor VIII contains one or multiple substitutions at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152 as described herein.

[0097] In a further embodiment, the mutant factor VIII is modified to confer greater stability of activated factor VIII by virtue of fused A2 and A3 domains. In particular, a factor VIII can be modified by substituting cysteine residues at positions 664 and 1826, (i.e., Y664C, T1826C) resulting in a mutant factor VIII forming a Cys664-Cys1826 disulfide bond covalently linking the A2 and A3 domains (Gale, et al., "An Engineered Interdomain Disulfide Bond Stabilizes Human Blood Coagulation Factor Villa," J. Thrombosis and Haemostasis 1(9):1966-1971 (2003)). Preferably, the recombinant fused domain (A2-A3) factor VIII contains one or multiple substitutions at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and L152 as described herein.

[0098] In a further embodiment, a mutant factor VIII in accordance with the present invention (e.g., containing one or multiple substitutions at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152) is further modified to confer altered inactivation cleavage sites. For example, Arg336 or Arg562 may be substituted used to decrease the mutant factor VIII's susceptibility to cleavage enzymes that normally inactivate the wild type factor VIII (see, e.g., Amano, et al., "Mutation at Either Arg336 or Arg562 in Factor VIII is Insufficient for Complete Resistance to Activated Protein C (APC)-Mediated Inactivation: implications for the APC Resistance Test," Thrombosis & Haemostasis 79(3):557-63 (1998)).

[0099] In a further embodiment, a mutant factor VIII in accordance with the present invention (e.g., containing one or multiple substitutions at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152) is further modified to confer enhanced affinity for factor IXa (see, e.g., Fay, et al., "Factor VIIIa A2 Subunit Residues 558-565 Represent a Factor IXa Interactive Site," J. Biol. Chem. 269(32):20522-7 (1994); Bajaj, et al., "Factor IXa: Factor VIIIa Interaction. Helix 330-338 of Factor IXa Interacts with Residues 558-565 and Spatially Adjacent Regions of the A2 Subunit of Factor VIIIa," J. Biol. Chem. 276(19):16302-9 (2001); and Lenting, et al., "The Sequence Glu1811-Lys1818 of Human Blood Coagulation Factor VIII Comprises a Binding Site for Activated Factor IX," J. Biol. Chem. 271(4):1935-40 (1996)) and/or factor X (see, e.g., Lapan, et al., "Localization of a Factor X Interactive Site in the A1 Subunit of Factor VIIIa," J. Biol. Chem. 272:2082-88 (1997)).

[0100] In yet another further embodiment, a mutant factor VIII in accordance with the present invention (e.g., containing one or multiple substitutions at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152) is further modified to further enhance secretion of factor VIII (see, e.g., Swaroop, et al., "Mutagenesis of a Potential Immunoglobulin-Binding Protein-Binding Site Enhances Secretion of Coagulation Factor VIII," J. Biol. Chem. 272(39):24121-4 (1997)).

[0101] In a further embodiment, a mutant factor VIII in accordance with the present invention (e.g., containing one or multiple substitutions at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152) is further modified to confer increased circulating half-life. This may be achieved through various approaches, including, without limitation, by reducing interactions with heparan sulfate (Sarafanov, et al., "Cell Surface Heparan Sulfate Proteoglycans Participate in Factor VIII Catabolism Mediated by Low Density Lipoprotein Receptor-Related Protein," J. Biol. Chem. 276(15):11970-9 (2001)) and/or low-density lipoprotein receptor-related protein ("LRP") (Saenko, et al., "Role of the Low Density Lipoprotein-Related Protein Receptor in Mediation of Factor VIII Catabolism," J. Biol. Chem. 274(53):37685-92 (1999); and "The Light Chain of Factor VIII Comprises a Binding Site for Low Density Lipoprotein Receptor-Related Protein," J. Biol. Chem. 274(34):23734-9 (1999)).

[0102] An eighth example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a modified factor VIII encoded by a nucleotide sequence modified to code for amino acids within known, existing epitopes to produce a recognition sequence for glycosylation at asparagine residues (see, e.g., U.S. Pat. No. 6,759,216 to Lollar). Such modification can be useful escaping detection by existing inhibitory antibodies (low antigenicity factor VIII) and decreasing the likelihood of developing inhibitory antibodies (low immunogenicity factor VIII). In one representative embodiment, the modified factor VIII is mutated to incorporate a consensus amino acid sequence for N-linked glycosylation, such as N-X-S/T (see U.S. Pat. No. 6,759,216 to Lollar).

[0103] A ninth example of a suitable mutant factor VIII that can be modified in accordance with the present invention is a modified factor VIII that is a procoagulant-active factor VIII having various mutations (see, e.g., U.S. Patent Application Publication No. 2004/0092442 to Kaufman, et al.). One example of this embodiment relates to a modified factor VIII that has been modified to (i) delete the von Willebrand factor binding site, (ii) add a mutation at Arg 740, and (iii) add an amino acid sequence spacer between the A2- and A3-domains, where the amino acid spacer is of a sufficient length so that upon activation, the procoagulant-active factor VIII protein becomes a heterodimer (see U.S. Patent Application Publication No. 2004/0092442 to Kaufman, et al).

[0104] Further, the mutant factor VIII can be modified to take advantage of various advancements regarding recombinant coagulation factors generally (see, e.g., Saenko, et al., "The Future of Recombinant Coagulation Factors," J. Thrombosis and Haemostasis 1:922-930 (2003)).

[0105] The recombinant factor VIII of the present invention can be modified at position I86, Y105, A108, D115, Q117, F129, G132, H134, M147, L152, as well as be modified to be B-domainless, to be chimeric, to have fused A2-A3 domains, to have altered inactivation cleavage sites, to have enhanced factor IXa and/or factor X affinity, to have enhanced specific activity, to have an increased circulating half-life, to have mutant glycosylation sites, or to possess any two or more of such modifications in addition to the modifications at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

[0106] Another aspect of the present invention relates to a method of making a recombinant factor VIII having increased specific activity compared to that of a wild-type factor VIII. This method involves altering the amino acid sequence of a wild-type factor VIII to yield a recombinant factor VIII. Alteration of the amino acid sequence of the wild-type factor VIII can include, for example, introducing at least one point mutation in or near at least one calcium binding site of the wild-type factor VIII. Thereafter, using protein analysis techniques well-known in the art, a determination can be made as to whether the recombinant factor VIII has increased specific activity compared to that of the wild-type factor VIII.

[0107] The recombinant factor VIII is preferably produced in a substantially pure form. In a particular embodiment, the substantially pure recombinant factor VIII is at least about 80% pure, more preferably at least 90% pure, most preferably at least 95% pure, 98% pure, 99% pure or 99.9% pure. A substantially pure recombinant factor VIII can be obtained by conventional techniques well known in the art. Typically, the substantially pure recombinant factor VIII is secreted into the growth medium of recombinant host cells. Alternatively, the substantially pure recombinant factor VIII is produced but not secreted into growth medium. In such cases, to isolate the substantially pure recombinant factor VIII, the host cell carrying the recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove cell debris. The supernatant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the substantially pure recombinant factor VIII is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the recombinant factor VIII. If necessary, a protein fraction (containing the substantially pure recombinant factor VIII) may be further purified by high performance liquid chromatography ("HPLC").

[0108] Another aspect of the present invention relates to an isolated nucleic acid molecule that encodes a recombinant mutant factor VIII as described herein. The isolated nucleic acid molecule encoding the recombinant mutant factor VIII can be an RNA or DNA. The nucleic acid codons can be further optimized for enhanced expression.

[0109] In one embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2 as modified with one of the substitutions at positions identified in this invention (i.e., possessing one to three nucleotide substitutions within codon 86 (nt256-258), codon 105 (nt:313-315), codon 108 (nt:322-324), codon 115 (nt:343-345), codon 117 (nt:349-351), codon 129 (nt:385-387), codon 132 (nt:394-396), codon 134 (nt:400-402), codon 147 (nt:439-441) and/or codon 152 (nt:454-456) of SEQ ID NO:1 (the first 57 nucleotides are not counted since they encode the signal peptides). The isolated nucleic acid molecule may have one or multiple changes in these positions in any combination.

[0110] In another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a B-domainless factor VIII of the type described above, as modified with one or multiple substitutions at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

[0111] In another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a chimeric human/porcine of the type described above, as modified with one or multiple substitutions at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

[0112] In a further embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a fused A2-A3 domain factor VIII of the type described above, as modified with one or multiple substitutions at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

[0113] In another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII whose inactivation sites have been modified as described above, as further modified with one or multiple substitutions at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

[0114] In yet another embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII whose affinity for factor IXa and/or factor X has been enhanced, along with one or multiple substitutions at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

[0115] In a still further embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII whose affinity for various serum-binding proteins has been altered to increase its circulating half-life and further modified with one or multiple substitutions at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

[0116] In a further embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII that has increased secretion in culture, as further modified with one or multiple substitutions at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

[0117] In a further embodiment, the isolated nucleic acid molecule can have a nucleotide sequence encoding a factor VIII that possesses one or more non-naturally occurring glycosylation site(s) along with one or multiple substitutions at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

[0118] In yet another embodiment, the isolated nucleic acid molecule encodes a recombinant factor VIII that is modified with one or more substitutions at position(s) I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152 and is further modified to possess any combination of the following modification: modified to be B-domainless, modified to be chimeric, modified to have fused A2-A3 domains, modified to have one or more altered inactivation cleavage site(s), modified to have enhanced factor IXa and/or factor X affinity, modified to have enhanced secretion, modified to have an increased circulating half-life, and modified to possess one or more non-naturally occurring glycosylation site(s).

[0119] Another aspect of the present invention relates to an expression vector for expressing the mutant factor VIII polynucleotides described herein. As used herein, the term "expression vector" refers to a viral or non-viral vector that comprise a polynucleotide encoding the novel peptide of the present invention in a form suitable for expression of the polynucleotide in a host cell. One type of non-viral vector is a "plasmid," which includes a circular double-stranded DNA loop into which additional DNA segments can be ligated. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

[0120] When preparing an expression vector, the transgene sequences may be inserted into a plasmid containing suitable bacterial sequences for replication in bacterial, as well as eukaryotic cells. Any convenient plasmid may be employed, which can include markers allowing for selection in a bacterium, and generally one or more unique, conveniently located restriction sites. The selection of a vector will depend on the preferred transformation technique and target host for transformation.

[0121] Expression vectors for expressing mutant factor VIII polypeptides include one or more regulatory sequences operably linked to the polynucleotide sequence to be expressed. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce the mutant factor VIII proteins described herein.

[0122] As used herein, the term "control sequences" or "regulatory sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The term "control/regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Control/regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0123] A nucleic acid sequence is "operably linked" to another nucleic acid sequence when the former is placed into a functional relationship with the latter. For example, a DNA for a presequence or secretory leader peptide is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0124] Suitable expression vectors for directing expression in mammalian cells generally include a promoter, as well as other transcription and translation control sequences known in the art. In certain embodiments, the mammalian expression vector is capable of directing expression of the polynucleotide preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the polynucleotide). Tissue-specific regulatory elements are known in the art and may include, for example, liver cell-specific promoters and/or enhancers (e.g., albumin promoter, a-I antitrypsin promoter, apoE enhancer). Alternatively, a constitutive promoter (e.g., HCMV) active in virtually any cell type may be used.

[0125] In certain preferred embodiments, the expression vectors are viral vectors. Viral vectors typically have one or more viral genes removed and include a gene/promotor cassette inserted into a viral genome insertion site for insertion of exogenous transgenes, including the mutant factor VIII genes described herein. The necessary functions of the removed gene(s) may be supplied by cell lines which have been engineered to express the gene products of the early genes in trans. Exemplary viral vectors include, but are not limited to, adeno-associated viral (AAV) vectors, retroviral vectors, including lentiviral vectors, adenoviral vectors, herpes viral vectors, and alphavirus vectors. Other viral vectors include astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, togavirus viral vectors and the like. The viral vector may comprise any suitable nucleic acid construct, such as a DNA or RNA construct and may be single stranded, double stranded, or duplexed.

[0126] Once the DNA construct of the present invention has been prepared, it is ready to be incorporated into a host cell. Accordingly, another aspect of the present invention relates to a method of making a recombinant cell comprising a factor VIII nucleic acid. Basically, this entails introducing the DNA construct into cells via transformation, transduction, electroporation, calcium phosphate precipitation, liposomes and the like and selecting for cells that have incorporated the DNA episomally or integrated into the host genome.

[0127] Thus, a further aspect of the present invention relates to a host cell including an isolated nucleic acid molecule encoding the recombinant factor VIII of the present invention. The host cell can contain the isolated nucleic acid molecule as a DNA molecule in the form of an episomal plasmid or stably integrated into the host cell genome. Further, the host cell can constitute an expression system for producing the recombinant mutant factor VIII protein. Suitable host cells can be, without limitation, animal cells (e.g., baby hamster kidney ("BHK") cells), Chinese hamster ovary cells ("CHO"), bacterial cells (e.g., E. coli), insect cells (e.g., Sf9 cells), fungal cells, yeast cells (e.g., Saccharomyces or Schizosaccharomyces), plant cells (e.g., Arabidopsis or tobacco cells), algal cells and the like.

[0128] Another aspect of the present invention relates to a method of making a recombinant factor VIII of the present invention. This method involves growing a host cell of the present invention under conditions whereby the host cell expresses the recombinant factor VIII. The recombinant factor VIII is then isolated. In one embodiment, the host cell is grown in vitro in a growth medium. In a particular embodiment, suitable growth media can include, without limitation, a growth medium containing a von Willebrand Factor (referred to herein as "VWF"). In this embodiment, the host cell can contain a transgene encoding a VWF or the VWF can be introduced to the growth medium as a supplement. VWF in the growth medium will allow for greater expression levels of the recombinant factor VIII. Once the recombinant factor VIII is secreted into the growth medium, it can then be isolated from the growth medium using techniques well-known by those of ordinary skill in the relevant recombinant DNA and protein arts (including those described herein). In another embodiment, the method of making the recombinant factor VIII of the present invention further involves disrupting the host cell prior to isolation of the recombinant factor VIII. In this embodiment, the recombinant factor VIII is isolated from cellular debris.

[0129] When recombinantly produced, the factor VIII protein or polypeptide (or fragment or mutant thereof) is expressed in a recombinant host cell, typically, although not exclusively, a eukaryote. In certain preferred embodiments, eukaryotic host cells, such as mammalian cells, are used to produce mutant factor VIII polypeptides as described herein. Mammalian cells suitable for carrying out the present invention include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), CHOP, and NS-1 cells.

[0130] Another aspect of the present invention relates to a method for treating patient with a factor VIII deficiency. In one embodiment, this involves administering to a patient in need thereof a recombinant mutant factor VIII (as described herein) in an amount effective for treating the factor VIII deficiency.

[0131] In a particular embodiment, the recombinant factor VIII, alone, or in the form of a pharmaceutical composition (i.e., in combination with stabilizers, delivery vehicles, and/or carriers) is infused into patients intravenously according to the same procedure that is used for infusion of human or animal factor VIII. A suitable effective amount of the recombinant factor VIII can include, without limitation, between about 10 to about 500 units/kg body weight of the patient.

[0132] In another embodiment, a method for treating patient with a factor VIII deficiency comprises administering to a patient in need thereof a pharmaceutical composition comprising an expression vector encoding a mutant factor VIII, or a functional fragments thereof, in an amount effective for treating the factor VIII deficiency. In certain embodiments, the recombinant factor VIII can be administered by transplanting cells genetically engineered to produce the recombinant factor VIII, typically via implantation of a device containing such cells. Such transplantation typically involves using recombinant dermal fibroblasts, a non-viral approach (Roth, et al., New Engl. J. Med. 344:1735-1742 (2001)).

[0133] Administration of FVIII-encoding expression vectors to factor VIII deficient patients can result in sufficient expression of FVIII polypeptide to functionally reconstitute the coagulation cascade. The expression vector(s) may be administered alone or in combination with other therapeutic agents in a pharmaceutically acceptable or biologically compatible composition.

[0134] The FVIII-encoding polynucleotide can be employed as a single chain molecule containing both heavy and light chain portions (FIG. 10) or split into two or multiple molecules (e.g., heavy and light chain; FIG. 11) in viral or non-viral vectors for delivery into host cells of the patient.

[0135] In a preferred embodiment of the invention, the expression vector comprising nucleic acid sequences encoding the mutant FVIII mutants is a viral vector. Viral vectors which may be used in the present invention include, but are not limited to, adenoviral vectors (with or without tissue specific promoters/enhancers), adeno-associated virus (AAV) vectors of multiple serotypes (e.g., AAV-1 to AAV-12, and others) and hybrid AAV vectors, lentivirus vectors and pseudo-typed lentivirus vectors [e.g., Ebola virus, vesicular stomatitis virus (VSV), and feline immunodeficiency virus (FIV)], herpes simplex virus vectors, vaccinia virus vectors, retroviral vectors, lentiviral vectors, non-viral vectors and others.

[0136] In certain preferred embodiments, methods are provided for the administration of a viral vector comprising nucleic acid sequences encoding a mutant FVIII, or a functional fragment thereof involve the use of AAV vectors or lentiviral vectors. Most preferably, only the essential parts of vector e.g., the ITR and LTR elements, respectively are included. Direct delivery of vectors or ex-vivo transduction of human cells and followed by infusion into the body will result in expression of mutant FVIIIs thereby exerting a beneficial therapeutic effect on hemostasis by enhancing pro-coagulation activity.

[0137] Recombinant AAV and lentiviral vectors have found broad utility for a variety of gene therapy applications. Their utility for such applications is due largely to the high efficiency of in vivo gene transfer achieved in a variety of organ contexts. AAV and lentiviral particles may be used to advantage as vehicles for effective gene delivery. Such virions possess a number of desirable features for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also demonstrated no sustained toxicity and immune responses were minimal or undetectable. AAV are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints and hematopoietic stem cells. It is likely that non-viral vectors based on plasmid DNA or minicircles will be also suitable gene transfer vectors for a large gene as that encoding FVIII.

[0138] It is desirable to introduce a vector that can provide, for example, sufficient expression of a desired gene and minimal immunogenicity. Improved AAV and lentiviral vectors and methods for producing such vectors have been described in detail in a number of references, patents, and patent applications, including Wright J. F. (Hum Gene Ther 20:698-706, 2009) which describes the production of clinical grade vectors. Lentiviral vector can be produced at CHOP and the other vectors are available through the Lentivirus vector production core laboratory by NHLBI Gene Therapy Resource Program (GTRP)-Lentivirus Vector Production Core Laboratory.

[0139] For some applications, an expression construct may further comprise regulatory elements which serve to drive expression in a particular cell or tissue type. Such regulatory elements are known to those of skill in the art and discussed in depth in Sambrook, et al. (1989) and Ausubel, et al. (1992). The incorporation of tissue specific regulatory elements in the expression constructs of the present invention provides for at least partial tissue tropism for the expression of the mutant FVIIIs or functional fragments thereof. For example, nucleic acid sequences encoding mutant FVIII under the control of a cytomegalovirus (CMV) promoter can be employed for skeletal muscle expression or the hAAT-ApoE and others for liver specific expression. Hematopoietic specific promoters in lentiviral vectors may also be used to advantage in the methods of the present invention.

[0140] In a preferred embodiment, the mutant FVIII sequence is provided as a component of a viral vector packaged in a capsid. In a particularly preferred embodiment, an AAV vector is used for in vivo delivery of the mutant FVIIIs (Gnatenko, et al., Br. J. Haematol. 104:27-36 (1999)). In this case, the AAV vector includes at least one mutant FVIII and associated expression control sequences for controlling expression of the mutant FVIII sequence. As shown in FIGS. 10 and 11, exemplary AAV vectors for expressing mutant FVIII sequences may include promoter-enhancer regulatory regions for FVIII expression and cis-acting ITRs functioning to enable promote replication and packaging of the mutant FVIII nucleic acids into AAV capsids and integration of the mutant FVIII nucleic acid into the genome of a target cell. Preferably, the AAV vector has its rep and cap genes deleted and replaced by the mutant hFVIII sequence and its associated expression control sequences. As shown in FIGS. 10 and 11, the mutant FVIII sequence is typically inserted adjacent to one or two (i.e., flanked by) AAV TRs or TR elements adequate for viral replication. Other regulatory sequences suitable for facilitating tissue-specific expression of the mutant hFVIII sequence in the target cell may also be included.

[0141] The viral capsid component of the packaged viral vectors may be a parvovirus capsid. AAV Cap and chimeric capsids are preferred. Examples of suitable parvovirus viral capsid components are capsid components from the Parvoviridae family, such as an autonomous parvovirus or a dependovirus. For example, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV 9, AAV10, AAV 11 or AAV12 capsid; one skilled in the art would know there are likely other variants not yet identified that perform the same or similar function), or may include components from two or more AAV capsids. A full complement of AAV Cap proteins includes VP1, VP2, and VP3. The ORF comprising nucleotide sequences encoding AAV VP capsid proteins may comprise less than a full complement AAV Cap proteins or the full complement of AAV Cap proteins may be provided.

[0142] One or more of the AAV Cap proteins may be a chimeric protein, including amino acid sequences AAV Caps from two or more viruses, preferably two or more AAVs, as described in Rabinowitz et al., U.S. Pat. No. 6,491,907. For example, the chimeric virus capsid can include an AAV I Cap protein or subunit and at least one AAV2 Cap or subunit. The chimeric capsid can, for example, include an AAV capsid with one or more B19 Cap subunits, e.g., an AAV Cap protein or subunit can be replaced by a B19 Cap protein or subunit. For example, the Vp3 subunit of the AAV capsid can be replaced by the Vp2 subunit of B19.

[0143] Packaging cells may be cultured to produce packaged viral vectors of the invention. The packaging cells may include heterologous (1) viral vector function(s), (2) packaging function(s), and (3) helper function(s). The viral vector functions typically include a portion of a parvovirus genome, such as an AAV genome, with rep and cap deleted and replaced by the mutant FVIII sequence and its associated expression control sequences as described above.

[0144] In certain embodiments, the viral vector functions may suitably be provided as duplexed vector templates, as described in U.S. Patent Publication No. 2004/0029106 to Samulski et al. Duplexed vectors are dimeric self-complementary (sc) polynucleotides (typically, DNA). For example, the DNA of the duplexed vectors can be selected so as to form a double-stranded hairpin structure due to intrastrand base pairing. Both strands of the duplexed DNA vectors may be packaged within a viral capsid. The duplexed vector provides a function comparable to double-stranded DNA virus vectors and can alleviate the need of the target cell to synthesize complementary DNA to the single-stranded genome normally encapsidated by the virus.

[0145] The TR(s) (resolvable and non-resolvable) selected for use in the viral vectors are preferably AAV sequences (from any AAV serotype). Resolvable AAV ITRs need not have a wild-type TR sequence (e.g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as the TR mediates the desired functions, e.g., virus packaging, integration, and/or provirus rescue, and the like. The TRs may be synthetic sequences that function as AAV inverted terminal repeats, such as the "double-D sequence" as described in U.S. Pat. No. 5,478,745 to Samulski et al. Typically, but not necessarily, the TRs are from the same parvovirus, e.g., both TR sequences are from AAV2.

[0146] The packaging functions include capsid components. The capsid components are preferably from a parvoviral capsid, such as an AAV capsid or a chimeric AAV capsid function. Examples of suitable parvovirus viral capsid components are capsid components from the family Parvoviridae, such as an autonomous parvovirus or a Dependovirus. For example, the capsid components may be selected from AAV capsids, e.g., AAV1-AAV12 and other novel capsids as yet unidentified or from non-human primate sources. Capsid components may include components from two or more AAV capsids.

[0147] In certain embodiments, one or more of the VP capsid proteins may comprise chimeric proteins, comprising amino acid sequences from two or more viruses, preferably two or more AAVs, as described in Rabinowitz et al., U.S. Pat. No. 6,491,907. For example, the chimeric virus capsid can include a capsid region from an adeno-associated virus (AAV) and at least one capsid region from a B19 virus. The chimeric capsid can, for example, include an AAV capsid with one or more B19 capsid subunits, e.g., an AAV capsid subunit can be replaced by a B19 capsid subunit. For example, the VP1, VP2 or VP3 subunit of the AAV capsid can be replaced by the VP1, VP2 or VP3 subunit of B19. As another example, the chimeric capsid may include an AAV type 2 capsid in which the type 2 VP1 subunit has been replaced by the VP1 subunit from an AAV type 1, 3, 4, 5, or 6 capsid, preferably a type 3, 4, or 5 capsid. Alternatively, the chimeric parvovirus has an AAV type 2 capsid in which the type 2 VP2 subunit has been replaced by the VP2 subunit from an AAV type 1, 3, 4, 5, or 6 capsid, preferably a type 3, 4, or 5 capsid. Likewise, chimeric parvoviruses in which the VP3 subunit from an AAV type 1, 3, 4, 5 or 6 (more preferably, type 3, 4 or 5) is substituted for the VP3 subunit of an AAV type 2 capsid are preferred. As a further alternative, chimeric parvoviruses in which two of the AAV type 2 subunits are replaced by the subunits from an AAV of a different serotype (e.g., AAV type 1, 3, 4, 5 or 6) are preferred. In exemplary chimeric parvoviruses according to this embodiment, the VP1 and VP2, or VP1 and VP3, or VP2 and VP3 subunits of an AAV type 2 capsid are replaced by the corresponding subunits of an AAV of a different serotype (e.g., AAV type 1, 3, 4, 5 or 6). Likewise, in other preferred embodiments, the chimeric parvovirus has an AAV type 1, 3, 4, 5 or 6 capsid (preferably the type 2, 3 or 5 capsid) in which one or two subunits have been replaced with those from an AAV of a different serotype, as described above for AAV type 2.

[0148] The packaged viral vector generally includes the mutant FVIII sequence and expression control sequences flanked by TR elements sufficient to result in packaging of the vector DNA and subsequent expression of the mutant FVIII sequence in the transduced cell. The viral vector functions may, for example, be supplied to the cell as a component of a plasmid or an amplicon. The viral vector functions may exist extrachromosomally within the cell line and/or may be integrated into the cells' chromosomal DNA.

[0149] In a preferred embodiment, the mutant FVIII described herein are used for gene therapy of FVIII associated disorders, such as hemophilia A. In this case, expression of the mutant FVIII transgene can enhance clotting in a subject otherwise vulnerable to uncontrolled bleeding due to factor VIII deficiency (e.g., intraarticular, intracranial, or gastrointestinal hemorrhage), including hemophiliacs who have developed antibodies to human factor VIII. The target cells of the vectors are cells capable of expressing polypeptides with FVIII activity, such as those of the hepatic system of a mammal, endothelial cells and other cells with the proper cellular machinery to process the precursor to yield protein with FVIII activity.

[0150] In particular embodiments, the present invention provides a pharmaceutical composition comprising a vector of the present invention including a modified gene of FVIII in a pharmaceutically-acceptable carrier and/or other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.

[0151] The treatment dosages of recombinant factor VIII that should be administered to a patient in need of such treatment will vary depending on the severity of the factor VIII deficiency. Generally, dosage level is adjusted in frequency, duration, and units in keeping with the severity and duration of each patient's bleeding episode. Accordingly, the recombinant factor VIII is included in a pharmaceutically acceptable carrier, delivery vehicle, or stabilizer in an amount sufficient to deliver to a patient a therapeutically effective amount of the protein to stop bleeding, as measured by standard clotting assays.

[0152] Factor VIII is classically defined as that substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A. The coagulant activity in vitro of purified and partially-purified forms of factor VIII is used to calculate the dose of recombinant factor VIII for infusions in human patients and is a reliable indicator of activity recovered from patient plasma and of correction of the in vivo bleeding defect. There are no reported discrepancies between standard assay of novel factor VIII molecules in vitro and their behavior in the dog infusion model or in human patients, according to Lusher, et al., New Engl. J. Med. 328:453-459 (1993); Pittman, et al., Blood 79:389-397 (1992); and Brinkhous, et al., Proc. Natl. Acad. Sci. 82:8752-8755 (1985).

[0153] Usually, the desired plasma factor VIII activity level to be achieved in the patient through administration of the recombinant factor VIII is in the range of 30-200% of normal. In one embodiment, administration of the therapeutic recombinant factor VIII is given intravenously at a preferred dosage in the range from about 5 to 500 units/kg body weight, and particularly in a range of 10-100 units/kg body weight, and further particularly at a dosage of 20-40 units/kg body weight; the interval frequency is in the range from about 8 to 24 hours (in severely affected hemophiliacs); and the duration of treatment in days is in the range from 1 to 10 days or until the bleeding episode is resolved. See, e.g., Roberts, H. R., and M. R. Jones, "Hemophilia and Related Conditions--Congenital Deficiencies of Prothrombin (Factor II, Factor V, and Factors VII to XII)," Ch. 153, 1453-1474, 1460, in Hematology, Williams, W. J., et al., ed. (1990). Patients with inhibitors may require a different amount of recombinant factor VIII than their previous form of factor VIII. For example, patients may require less recombinant factor VIII because of its higher specific activity than the wild-type VIII and its decreased antibody reactivity. As in treatment with human or plasma-derived factor VIII, the amount of therapeutic recombinant factor VIII infused is defined by the one-stage factor VIII coagulation assay and, in selected instances, in vivo recovery is determined by measuring the factor VIII in the patient's plasma after infusion. It is to be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed recombinant factor VIII.

[0154] Treatment can take the form of a single intravenous administration of the recombinant factor VIII or periodic or continuous administration over an extended period of time, as required. Alternatively, therapeutic recombinant factor VIII can be administered subcutaneously or orally with liposomes in one or several doses at varying intervals of time.

[0155] For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and will preferably be in solid or liquid particulate form. As an injection medium, it is preferred to use water that contains the additives usual for injection solutions, such as stabilizing agents, salts or saline, and/or buffers.

[0156] Exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. Physiologically-acceptable carriers include pharmaceutically-acceptable carriers. Pharmaceutically acceptable carriers are those which are that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing undesirable biological effects which outweigh the advantageous biological effects of the material. A pharmaceutical composition may be used, for example, in transfection of a cell ex vivo or in administering a viral vector or cell directly to a subject.

[0157] Recombinant virus vectors comprising the modified gene of FVIII are preferably administered to the cell in a biologically-effective amount. If the virus vector is administered to a cell in vivo (e.g., the virus is administered to a subject as described below), a biologically-effective amount of the virus vector is an amount that is sufficient to result in transduction and expression of the transgene in a target cell.

[0158] The cells transduced with a viral vector are preferably administered to the subject in a "therapeutically-effective amount" in combination with a pharmaceutical carrier. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0159] Dosages of the cells to administer to a subject will vary upon the age, condition and species of the subject, the type of cell, the nucleic acid being expressed by the cell, the mode of administration, and the like. Typically, at least about 10.sup.2 to about 10.sup.8, preferably about 10.sup.3 to about 10.sup.8 cells, will be administered per dose. Preferably, the cells will be administered in a therapeutically-effective amount.

[0160] Administration of the vector to a human subject or an animal in need thereof can be by any means known in the art for administering virus vectors. Exemplary modes of administration include rectal, transmucosal, topical, transdermal, inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, and the like, as well as direct tissue or organ injection, alternatively, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one may administer the virus in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0161] In other preferred embodiments, the inventive vector comprising the mutant FVIII gene is administered intramuscularly, more preferably by intramuscular injection or by local administration. The vectors disclosed herein may be administered to the lungs of a subject by any suitable means, but are preferably administered by administering an aerosol suspension of respirable particles comprised of the inventive parvovirus vectors, which the subject inhales. The respirable particles may be liquid or solid. Aerosols of liquid particles comprising the inventive parvovirus vectors may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729.

[0162] Dosages of the virus vector with the mutant FVIII gene will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular viral vector, and the gene to be delivered, and can be determined in a routine manner. Exemplary doses for achieving therapeutic effects are virus titers of at least about 10.sup.5, 10.sup.6, 10.sup.7 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, 10.sup.15 transducing units or more, preferably about 10.sup.8-10.sup.13 transducing units, yet more preferably 10.sup.12 transducing units. The mutant FVIII genes may be administered as components of a DNA molecule having regulatory elements appropriate for expression in the target cells. The mutant FVIII genes may be administered as components of viral plasmids, such as rAAV vectors. Viral particles may be administered as viral particles alone, whether as an in vivo direct delivery to the portal vasculature or as an ex vivo treatment comprising administering the vector viral particles in vitro to cells from the animal receiving treatment followed by introduction of the transduced cells back into the donor.

[0163] The present invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference.

Example 1: Construction, Expression and Purification of B-Domainless Factor VIII Mutants

[0164] Plasmid pAAV-CB-F8 carries a B-domainless human factor VIII (hF8) cDNA under the control of a CB promoter (beta-actin promoter with a CMV enhancer). This plasmid, consistent with the construction shown in FIG. 10, was used as template for making various hF8 mutants. A hF8 cDNA fragment encoding the substitution mutations I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P was synthesized chemically and used to replace the corresponding region of pAAV-CB-F8. The resulting plasmid (pAAV-CB-F8-X10) expresses a mutant factor VIII protein with the above 10 mutations (F8-X10; SEQ ID NO: 3).

[0165] A factor VIII cDNA fragment encoding the substitution mutations I86V, A108S, G132K, M147T, L152P was synthesized chemically and used to replace the corresponding region of pAAV-CB-F8. The resulting plasmid (pAAV-CB-F8-X5) expresses a mutant factor VIII protein with the above 5 mutations (F8-X5; SEQ ID NO:4).

[0166] Site-directed mutagenesis was used to introduce individual mutations corresponding to I86, Y105, A108, D115, Q117, F129, G132, H134, M147, L152 of human factor VIII in the pAAV-CB-F8 plasmid. The resulting plasmids include pAAV-CB-F8-I86V, pAAV-CB-F8-Y105F, pAAV-CB-F8-A108S, pAAV-CB-F8-D115E, pAAV-CB-F8-Q117H, pAAV-CB-F8-F129L, pAAV-CB-F8-G132K, pAAV-CB-F8-H134Q and pAAV-CB-F8-M147T, pAAV-CB-F8-L152P.

[0167] Site-directed mutagenesis was also used to revert back individual mutations in amino acids 86, 105, 108, 115, 117, 129, 132, 134, 147, 152 of hF8-X10 in the pAAV-CB-F8-X10 plasmid to wild-type human amino acids. The resulting plasmids include pAAV-CB-F8-X10-V861, pAAV-CB-F8-X10-F105Y, pAAV-CB-F8-X10-S108A, pAAV-CB-F8-X10-E115D, pAAV-CB-F8-X10-HI17Q, pAAV-CB-F8-X10-L129F, pAAV-CB-F8-X10-K132G, pAAV-CB-F8-X10-Q134H, pAAV-CB-F8-X10-T147M, pAAV-CB-F8-X10-P152L. These mutants were tested as described Example 4 (FIG. 7) below.

[0168] A set of degenerated oligonucleotides with NNN corresponding to G132 position was used to create all 19 mutations corresponding to G132. The resulting factor VIII mutant plasmids include pAAV-CB-F8-G132A, pAAV-CB-F8-G1321, pAAV-CB-F8-G132V etc. The last letter indicates the amino acid substitution at that particular position. A similar strategy can be used to generate other substitutions in accordance with the present invention.

[0169] The above described plasmids contain an AAV-ITR and can be used to generate AAV vectors. A liver specific promoter can be used to replace the CB promoter to reduce the size of the expression cassette and to improve vector packaging and expression in the liver. Other tissue specific promoter can also be used as well.

[0170] Sequences between the FVIII heavy chain and the FVIII stop codon were removed from the FVIII expression vectors. The resulting heavy chain (HC) mutants contain the first 745 amino acid of FVIII and lack B domain (BDD) and light chain (LC) sequences. The plasmid pAAV-CB-HC-X10 contains mutations corresponding to I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P in the heavy chain. The plasmid pAAV-CB-HC-X5 contains mutations corresponding to I86V, A108S, G132K, M147T, L152P in the heavy chain. The plasmid pAAV-CB-HC-G132K contains the G132K mutation in the heavy chain. Alternatively, acidic region 3 (a3) can be added to these constructs to obtain HC mutants in an HC1690 background.

Example 2: Comparison of Different Factor VIII Mutants for Secretion in Tissue Culture Cells

[0171] Plasmids pAAV-CB-hBDD-F8 (wt), pAAV-CB-hBDD-F8-X10, pAAV-CB-hBDD-F8-X5 and pAAV-CB-hBDD-F8-G312K were separately transfected in BHK cells (panel A) or 293 cells (panel B). Secreted F8 in the media was harvested and assayed by aPTT assay at 48 hours post transfection. The expression/secretion by wild type human BDD-F8(hBDD) was set as 100%. As shown in FIG. 7, the hF8 mutants were secreted at about 2-8.5 fold higher expression levels than the wild type hF8.

Example 3: Comparison of Human Factor VIII Mutant Secretion In Vivo

[0172] Plasmids pAAV-CB-hBDDF8 (B-domain deleted (BDD) wt hF8), pAAV-CB-hBDDF8-X10 (hF8-BDD with 10 substitutions; SEQ ID NO:3), pAAV-CB-hBDD-F8-X5(hF8 with 5 substitutions; SEQ ID NO:4) and pAAV-CB-hBDD-F8-G312K (hF8 with G132K substitution) were separately injected to Balb/c and F8 double knock-out mice. Secreted F8 in the blood was collected and assayed by aPTT assay at 48 hours post injection. The expression/secretion by wild type human F8 (hBDD-F8) was set as 100%. All mutants described here outperform the wild type factor VIII. As shown in FIG. 8, the hF8 mutants were secreted at about 1.2-5.2 fold higher expression levels than the wild type hF8.

Example 4: Comparison of Different Factor VIII Mutants in Secretion

[0173] Plasmids encoding amino acids in hBDD-F8-X10 were modified to revert back the mutant substitutions to their corresponding wild type amino acids as indicated in the table. For example, hBDD-F8-X10-V861 means that "V86" in hBDD-F8-X10 was changed back to "I". In hBDD-F8-X10-6p, 6 of 10 mutant amino acids in hBDD-F8-X10 were reverted back to their corresponding wild type amino acids. The above plasmids were separately transfected in 293 cells. Secreted F8 in the media was harvested and assayed by aPTT assay at 48 hours post transfection. The expression/secretion by hBDD-F8-X10 was set as 100%. As shown in FIG. 9, all of the revertants exhibited reduced expression and secretion as compared to the hBDD-F8-X10 mutant.

Example 5: Comparison of Different Factor VIII Mutants in Secretion

[0174] AAV8 vector AAV-apoEhAAT-hF8HC1690 (carrying human factor VIII heavy chain aa#1-745 and a3 sequence, apoEhAAT: human alpha one antitrypsin promoter with apo E enhancer) and AAV-apoEhAAT-hF8-HC-X10 (hF8HC with 10 substitutions) were each separately injected in F8 knock-out mice with a light chain expression vector (dose: each vector 1E11 particles/mouse). Secreted F8 in the blood was collected and assayed by aPTT assay the indicated times. The expression/secretion by wild type human F8 heavy chain (hF8-HC1690) was set as 100%. The hF8-HC1690-X10 mutant described here outperform the wild type heavy chain expression. As shown in FIG. 10, the hF8-HC1690-X10 mutant was secreted at about 6-20 fold higher expression levels compared to wild type hF8HC.

Example 6: Comparison of G132 Factor VIII Heavy Chain Mutants in Secretion in 293 Cells

[0175] Plasmid pAAV-CB-hF8-HC1690 (carrying human factor VIII heavy chain aa#1-745 and a3 sequence; see FIG. 1) or plasmids with indicated substitutions at G132 were transfected in 293 cells with an F8 light chain (LC) expression plasmid. Secreted F8 in the media was collected and assayed by aPTT assay at 48 hours post transfection. The expression/secretion by wild type human F8 heavy chain (hF8-HC1690) was set as 100%. As shown in FIG. 11, the F8 G132HC1690 mutants were secreted at about 1.4-3.4 fold higher expression levels compared to wild type hF811C.

Example 7: Comparison of Different Factor VIII Heavy Chain Mutants in Expression/Secretion In Vivo

[0176] Plasmids pAAV-CB-hF8-IIC1690 (carrying human factor VIII heavy chain aa#1-745 and acidic region 3 (a3) sequence; see FIGS. 1, 6), pAAV-CB-hF8-HC1690-X10 (hF8 heavy chain with 10 substitutions; SEQ ID NO:5), pAAV-CB-hF8-HC1690-X5 (hF8 heavy chain with 5 substitutions along with a3 sequence; SEQ ID NO:6) and pAAV-CB-hF8-HC1690-G312K (hF8 heavy chain with G132K substitution and a3 sequence) were separately injected in Balb/c and F8 double knock-out mice along with a light chain expression plasmid. Secreted F8 in the blood was collected and assayed by aPTT assay at 48 hours post injection. The expression/secretion by wild type human F8 heavy chain (hF8-HC1690) was set as 100%. As shown in FIG. 12, the hF8 mutants were secreted at about 2.5-4.5 fold higher expression levels than the wild type hF8HC.

Example 8: Comparison of L152 Factor VIII Heavy Chain Mutants in Secretion in 293 Cells

[0177] Plasmid pAAV-CB-hF8-HC1690 (carrying human factor VIII heavy chain aa#1-745 and a3 sequence; see FIGS. 1, 6) or plasmids with indicated substitutions at L152 were separately transfected in 293 cells with a hF8 light chain expression plasmid. Secreted hF8 in the media was collected and assayed by aPTT assay at 48 hours post transfection. The expression/secretion by wild type human F8 heavy chain (hF8-HC1690) was set as 100%. The hF8-L152HC mutants are identified by their specific amino acid substitutions in the Figure. As shown in FIG. 13, the hF8-L152HC mutants were secreted at about 1.4-3.5 fold higher expression levels compared to wild type hF8HC

Example 9: Comparison of A108 Factor VIII Heavy Chain Mutants in Secretion in 293 Cells

[0178] Plasmid pAAV-CB-hF8-HC1690 (carrying human factor VIII heavy chain aa#1-745 and a3 sequence; see FIG. 1) or plasmids with indicated substitutions at A108 were separately transfected in 293 cells with a hF8 light chain expression plasmid. Secreted hF8 in the media was collected and assayed by aPTT assay at 48 hours post transfection. The hF8-A108HC mutants are identified by their specific amino acid substitutions in the Figure. The expression/secretion by wild type human F8 heavy chain (hF8-HC1690) was set as 100%. As shown in FIG. 14, the majority of hF8-A108HC mutants were secreted at higher expression levels compared to wild type hF8HC.

Example 10: Comparison of M147 Factor VIII Heavy Chain Mutants in Secretion in 293 Cells

[0179] Plasmid pAAV-CB-hF8-HC1690 (carrying human factor VIII heavy chain aa#1-745 and a3 sequence; see FIG. 1) or plasmids with indicated substitutions at M147 were separately transfected in 293 cells with a hF8 light chain expression plasmid. Secreted hF8 in the media was collected and assayed by hF8 ELISA at 48 hours post transfection. The hF8-M147HC mutants are identified by their specific amino acid substitutions in the Figure. The expression/secretion by wild type human F8 heavy chain (hF8-HC1690) was set as 100%. As shown in FIG. 15, the majority of hF8-M1471-IC mutants were secreted at higher expression levels compared to wild type hF8HC.

Example 11: Neutralizing Antibodies Against 5 Mutated Amino Acids were not Detected in the F8-/- Rat Model

[0180] F8-/- rats in a WAG/RijYcb background with single mutation in the A1 domain (Leu176Pro) were administered AAV-TTR-hF8-X10 at 1.times.10.sup.12 viral particles/per rat. Of 3 injected rats, two were confirmed to develop rat anti-human factor VIII inhibitory antibodies against factor VIII as determined by a Bethesda assay. Panel A is representative of inhibitor levels in rat plasma at week 8. Panel B shows the activity of F8 remains in the supernatant after antibody absorption. To determine whether the inhibitory antibodies specifically target the 5 mutated amino acids, we used an excess of biotinylated recombinant human F8 (1.2 1.tg of Bio-F8) to saturate the inhibitory antibodies against regular FVIII in rat plasma. Then 30 1.1.1 of streptavidin agarose was used to pull down antigen-antibody complexes by rotation at room temperature for 1 hour and then centrifuged at 10,000 rpm for 2 min. An equivalent amount of biotinylated BSA (Bio-BSA) was used as a control. 200 ng of BDD-F8-X5 concentrate from a stably expressing cell line was then added to the rat plasma pretreated with Bio-F8 or Bio-BSA. Rat plasma without pretreatment was used as control. The F8 activity in the supernatant was determined by one-stage aPTT assay. As shown in FIG. 15, neutralizing antibodies against the five mutated amino acids were not detected in this F8-/- rat model.

[0181] The above Examples show that the mutant factor VIII products of the present invention express and/or secrete better than wild type factor VIII, therefore they can decrease the production cost and improve transgene expression levels when using a gene transfer vector. They can also allow lower vector doses to be administered and higher factor VIII expression levels.

[0182] The above description is for the purpose of teaching a person of ordinary skill in the art how to practice the present invention. It is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence effective to meet the objectives there intended, unless the context specifically indicates the contrary.

Sequence CWU 1

1

917056DNAHomo sapiens 1atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct 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 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 accaagaagc 2280ttctcccaga attcaagaca ccgtagcact aggcaaaagc aatttaatgc caccacaatt 2340ccagaaaatg acatagagaa gactgaccct tggtttgcac acagaacacc tatgcctaaa 2400atacaaaatg tctcctctag tgatttgttg atgctcttgc gacagagtcc tactccacat 2460gggctatcct tatctgatct ccaagaagcc aaatatgaga ctttttctga tgatccatca 2520cctggagcaa tagacagtaa taacagcctg tctgaaatga cacacttcag gccacagctc 2580catcacagtg gggacatggt atttacccct gagtcaggcc tccaattaag attaaatgag 2640aaactgggga caactgcagc aacagagttg aagaaacttg atttcaaagt ttctagtaca 2700tcaaataatc tgatttcaac aattccatca gacaatttgg cagcaggtac tgataataca 2760agttccttag gacccccaag tatgccagtt cattatgata gtcaattaga taccactcta 2820tttggcaaaa agtcatctcc ccttactgag tctggtggac ctctgagctt gagtgaagaa 2880aataatgatt caaagttgtt agaatcaggt ttaatgaata gccaagaaag ttcatgggga 2940aaaaatgtat cgtcaacaga gagtggtagg ttatttaaag ggaaaagagc tcatggacct 3000gctttgttga ctaaagataa tgccttattc aaagttagca tctctttgtt aaagacaaac 3060aaaacttcca ataattcagc aactaataga aagactcaca ttgatggccc atcattatta 3120attgagaata gtccatcagt ctggcaaaat atattagaaa gtgacactga gtttaaaaaa 3180gtgacacctt tgattcatga cagaatgctt atggacaaaa atgctacagc tttgaggcta 3240aatcatatgt caaataaaac tacttcatca aaaaacatgg aaatggtcca acagaaaaaa 3300gagggcccca ttccaccaga tgcacaaaat ccagatatgt cgttctttaa gatgctattc 3360ttgccagaat cagcaaggtg gatacaaagg actcatggaa agaactctct gaactctggg 3420caaggcccca gtccaaagca attagtatcc ttaggaccag aaaaatctgt ggaaggtcag 3480aatttcttgt ctgagaaaaa caaagtggta gtaggaaagg gtgaatttac aaaggacgta 3540ggactcaaag agatggtttt tccaagcagc agaaacctat ttcttactaa cttggataat 3600ttacatgaaa ataatacaca caatcaagaa aaaaaaattc aggaagaaat agaaaagaag 3660gaaacattaa tccaagagaa tgtagttttg cctcagatac atacagtgac tggcactaag 3720aatttcatga agaacctttt cttactgagc actaggcaaa atgtagaagg ttcatatgac 3780ggggcatatg ctccagtact tcaagatttt aggtcattaa atgattcaac aaatagaaca 3840aagaaacaca cagctcattt ctcaaaaaaa ggggaggaag aaaacttgga aggcttggga 3900aatcaaacca agcaaattgt agagaaatat gcatgcacca caaggatatc tcctaataca 3960agccagcaga attttgtcac gcaacgtagt aagagagctt tgaaacaatt cagactccca 4020ctagaagaaa cagaacttga aaaaaggata attgtggatg acacctcaac ccagtggtcc 4080aaaaacatga aacatttgac cccgagcacc ctcacacaga tagactacaa tgagaaggag 4140aaaggggcca ttactcagtc tcccttatca gattgcctta cgaggagtca tagcatccct 4200caagcaaata gatctccatt acccattgca aaggtatcat catttccatc tattagacct 4260atatatctga ccagggtcct attccaagac aactcttctc atcttccagc agcatcttat 4320agaaagaaag attctggggt ccaagaaagc agtcatttct tacaaggagc caaaaaaaat 4380aacctttctt tagccattct aaccttggag atgactggtg atcaaagaga ggttggctcc 4440ctggggacaa gtgccacaaa ttcagtcaca tacaagaaag ttgagaacac tgttctcccg 4500aaaccagact tgcccaaaac atctggcaaa gttgaattgc ttccaaaagt tcacatttat 4560cagaaggacc tattccctac ggaaactagc aatgggtctc ctggccatct ggatctcgtg 4620gaagggagcc ttcttcaggg aacagaggga gcgattaagt ggaatgaagc aaacagacct 4680ggaaaagttc cctttctgag agtagcaaca gaaagctctg caaagactcc ctccaagcta 4740ttggatcctc ttgcttggga taaccactat ggtactcaga taccaaaaga agagtggaaa 4800tcccaagaga agtcaccaga aaaaacagct tttaagaaaa aggataccat tttgtccctg 4860aacgcttgtg aaagcaatca tgcaatagca gcaataaatg agggacaaaa taagcccgaa 4920atagaagtca cctgggcaaa gcaaggtagg actgaaaggc tgtgctctca aaacccacca 4980gtcttgaaac gccatcaacg ggaaataact cgtactactc ttcagtcaga tcaagaggaa 5040attgactatg atgataccat atcagttgaa atgaagaagg aagattttga catttatgat 5100gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta ttttattgct 5160gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct aagaaacagg 5220gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt tactgatggc 5280tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact cctggggcca 5340tatataagag cagaagttga agataatatc atggtaactt tcagaaatca ggcctctcgt 5400ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca aggagcagaa 5460cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa agtgcaacat 5520catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt ctctgatgtt 5580gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg ccacactaac 5640acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct gtttttcacc 5700atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa ctgcagggct 5760ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt ccatgcaatc 5820aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca aaggattcga 5880tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt cagtggacat 5940gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt 6000gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt ggaatgcctt 6060attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag caataagtgt 6120cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac agcttcagga 6180caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc aatcaatgcc 6240tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt 6300cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat ctctcagttt 6360atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa ttccactgga 6420accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa tatttttaac 6480cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat tcgcagcact 6540cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag 6600agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa tatgtttgcc 6660acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc ctggagacct 6720caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat gaaagtcaca 6780ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa ggagttcctc 6840atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag 6900gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga cccaccgtta 6960ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc cctgaggatg 7020gaggttctgg gctgcgaggc acaggacctc tactga 705622332PRTHomo sapiens 2Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp 195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser 210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His 245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu 260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile 275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly 290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg 325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe 355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His 370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro 405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr 420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile 435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile 450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys 485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys 500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys 515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala 530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe 565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln 580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe 595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser 610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg 740 745 750Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn 770 775 780Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro785 790 795 800His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe 805 810 815Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser 820 825 830Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val 835 840 845Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser865 870 875 880Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala 885 890 895Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His 900 905 910Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp945 950 955 960Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys 965 970 975Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu 1010 1015 1020Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu 1025 1030 1035Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp 1040 1045 1050Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly 1070 1075 1080Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys 1085 1090 1095Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His 1100 1105 1110Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln 1115 1120 1125Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe 1130 1135 1140Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr 1145 1150 1155Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn 1160 1165 1170Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr 1190 1195 1200Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr Val Thr 1205 1210 1215Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg 1220 1225 1230Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr Ala Pro Val Leu 1235 1240 1245Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys 1250 1255 1260His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu 1265 1270 1275Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys 1280 1285 1290Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln

Gln Asn Phe Val Thr 1295 1300 1305Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu 1310 1315 1320Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser Thr 1325 1330 1335Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser 1355 1360 1365Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala 1370 1375 1380Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser 1385 1390 1395Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser 1400 1405 1410Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val 1415 1420 1425Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu 1430 1435 1440Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln Arg Glu 1445 1450 1455Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys 1460 1465 1470Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys 1490 1495 1500Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu 1505 1510 1515Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile 1520 1525 1530Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540 1545Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555 1560Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys Glu 1565 1570 1575Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His 1595 1600 1605Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu 1610 1615 1620Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln 1625 1630 1635Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr 1640 1645 1650Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile 1655 1660 1665Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675 1680Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr 1685 1690 1695Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1700 1705 1710Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro 1715 1720 1725Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe 1730 1735 1740Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1745 1750 1755Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760 1765 1770Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1775 1780 1785Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg 1790 1795 1800Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1805 1810 1815Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1820 1825 1830Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His 1835 1840 1845Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu 1850 1855 1860Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu 1865 1870 1875Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1880 1885 1890Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu 1895 1900 1905Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly 1910 1915 1920Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln 1925 1930 1935Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1940 1945 1950His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys 1955 1960 1965Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe 1970 1975 1980Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val 1985 1990 1995Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu 2000 2005 2010Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala 2015 2020 2025Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr 2030 2035 2040Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 2045 2050 2055Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val 2060 2065 2070Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly 2075 2080 2085Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile 2090 2095 2100Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn 2105 2110 2115Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser 2120 2125 2130Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2135 2140 2145Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155 2160Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu 2165 2170 2175Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser 2180 2185 2190Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val 2210 2215 2220Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met 2225 2230 2235Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr 2240 2245 2250Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly 2255 2260 2265His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe 2270 2275 2280Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 2285 2290 2295Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp 2300 2305 2310Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala 2315 2320 2325Gln Asp Leu Tyr 233039720DNAArtificial SequencepAAV-CB-hBDD-F8-X10 3aattcccatc atcaataata taccttattt tggattgaag ccaatatgat aatgaggggg 60tggagtttgt gacgtggcgc ggggcgtggg aacggggcgg gtgacgtagt agtctctaga 120ggtccccagc gaccttgacg ggcatctgcc cggcatttct gacagctttg tgaactgggt 180ggccgagaag gaatgggagt tgccgccaga ttctgacatg gatctgaatc tgattgagca 240ggcacccctg accgtggccg agaagctgca tcgctggcgt aatagcgaag aggcccgcac 300cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaattcca gacgattgag 360cgtcaaaatg taggtatttc catgagcgtt tttcctgttg caatggctgg cggtaatatt 420gttctggata ttaccagcaa ggccgatagt ttgagttctt ctactcaggc aagtgatgtt 480attactaatc aaagaagtat tgcgacaacg gttaatttgc gtgatggaca gactctttta 540ctcggtggcc tcactgatta taaaaacact tctcaggatt ctggcgtacc gttcctgtct 600aaaatccctt taatcggcct cctgtttagc tcccgctctg attctaacga ggaaagcacg 660ttatacgtgc tcgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc 720gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 780tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 840tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 900tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 960gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 1020ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 1080aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 1140aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 1200ggtacatatg attgacatgc tagttttacg attaccgttc atcgcctgca gggggggggg 1260ggggggggtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 1320aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagag 1380agggagtggc caactccatc actaggggtt cctagatctg aattcggtac gtacctctgg 1440tcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac cccgcccatt 1500gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 1560atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 1620aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 1680catgacctta tgggactttc ctacttggca gtacatctac tcgaggccac gttctgcttc 1740actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 1800ttttgtgcag cgatgggggc gggggggggg gggggggggg cgcgcgccag gcggggcggg 1860gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg 1920cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg 1980aagcgcgcgg cgggcgggag cgggatcagc caccgcggtg gcggcctaga gtcgacgagg 2040aactgaaaaa ccagaaagtt aactggtaag tttagtcttt ttgtctttta tttcaggtcc 2100cggatccggt ggtggtgcaa atcaaagaac tgctcctcag tggatgttgc ctttacttct 2160aggcctgtac ggaagtgtta cttctgctct aaaagctgcg gaattgtacc cgcggccgct 2220tttcaaaatg caaatagagc tctccacctg cttctttctg tgccttttgc gattctgctt 2280tagtgccacc agaagatact acctgggtgc agtggaactg tcatgggact atatgcaaag 2340tgatctcggt gagctgcctg tggacgcaag atttcctcct agagtgccaa aatcttttcc 2400attcaacacc tcagtcgtgt acaaaaagac tctgtttgta gaattcacgg atcacctttt 2460caacatcgct aagccaaggc caccctggat gggtctgcta ggtcctacca tccaggctga 2520ggtttacgac acggtggtcg ttaccctgaa gaacatggct tctcatcccg ttagtcttca 2580cgctgtcggc gtctccttct ggaaatcttc cgaaggcgct gaatatgagg atcacaccag 2640ccaaagggag aaggaagacg ataaagtcct tcccggtaaa agccaaacct acgtctggca 2700ggtcctgaaa gaaaatggtc caacagcctc tgacccacca tgtcttacct actcatacct 2760gtctcacgtg gacctggtga aagacctgaa ttcgggcctc attggagccc tactagtatg 2820tagagaaggg agtctggcca aggaaaagac acagaccttg cacaaattta tactactttt 2880tgctgtattt gatgaaggga aaagttggca ctcagaaaca aagaactcct tgatgcagga 2940tagggatgct gcatctgctc gggcctggcc taaaatgcac acagtcaatg gttatgtaaa 3000caggtctctg ccaggtctga ttggatgcca caggaaatca gtctattggc atgtgattgg 3060aatgggcacc actcctgaag tgcactcaat attcctcgaa ggtcacacat ttcttgtgag 3120gaaccatcgc caggcgtcct tggaaatctc gccaataact ttccttactg ctcaaacact 3180cttgatggac cttggacagt ttctactgtt ttgtcatatc tcttcccacc aacatgatgg 3240catggaagct tatgtcaaag tagacagctg tccagaggaa ccccaactac gaatgaaaaa 3300taatgaagaa gcggaagact atgatgatga tcttactgat tctgaaatgg atgtggtcag 3360gtttgatgat gacaactctc cttcctttat ccaaattcgc tcagttgcca agaagcatcc 3420taaaacttgg gtacattaca ttgctgctga agaggaggac tgggactatg ctcccttagt 3480cctcgccccc gatgacagaa gttataaaag tcaatatttg aacaatggcc ctcagcggat 3540tggtaggaag tacaaaaaag tccgatttat ggcatacaca gatgaaacct ttaagactcg 3600tgaagctatt cagcatgaat caggaatctt gggaccttta ctttatgggg aagttggaga 3660cacactgttg attatattta agaatcaagc aagcagacca tataacatct accctcacgg 3720aatcactgat gtccgtcctt tgtattcaag gagattacca aaaggtgtaa aacatttgaa 3780ggattttcca attctgccag gagaaatatt caaatataaa tggacagtga ctgtagaaga 3840tgggccaact aaatcagatc ctcggtgcct gacccgctat tactctagtt tcgttaatat 3900ggagagagat ctagcttcag gactcattgg ccctctcctc atctgctaca aagaatctgt 3960agatcaaaga ggaaaccaga taatgtcaga caagaggaat gtcatcctgt tttctgtatt 4020tgatgagaac cgaagctggt acctcacaga gaatatacaa cgctttctcc ccaatccagc 4080tggagtgcag cttgaggatc cagagttcca agcctccaac atcatgcaca gcatcaatgg 4140ctatgttttt gatagtttgc agttgtcagt ttgtttgcat gaggtggcat actggtacat 4200tctaagcatt ggagcacaga ctgacttcct ttctgtcttc ttctctggat ataccttcaa 4260acacaaaatg gtctatgaag acacactcac cctattccca ttctcaggag aaactgtctt 4320catgtcgatg gaaaacccag gtctatggat tctggggtgc cacaactcag actttcggaa 4380cagaggcatg accgccttac tgaaggtttc tagttgtgac aagaacactg gtgattatta 4440cgaggacagt tatgaagata tttcagcata cttgctgagt aaaaacaatg ccattgaacc 4500aagaagcttc tcccagaatt caagacaccc tagcactagg caaaagcaat ttaatgccac 4560cacaccacca gtcttgaaac gccatcaacg cgaaataact cgtactactc ttcagtcaga 4620tcaagaggaa attgactatg atgataccat atcagttgaa atgaagaagg aagattttga 4680catttatgat gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta 4740ttttattgct gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct 4800aagaaacagg gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt 4860tactgatggc tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact 4920cctggggcca tatataagag cagaagttga agataatatc atggtaactt tcagaaatca 4980ggcctctcgt ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca 5040aggagcagaa cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa 5100agtgcaacat catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt 5160ctctgatgtt gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg 5220ccacactaac acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct 5280gtttttcacc atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa 5340ctgcagggct ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt 5400ccatgcaatc aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca 5460aaggattcga tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt 5520cagtggacat gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct 5580ctatccaggt gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt 5640ggaatgcctt attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag 5700caataagtgt cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac 5760agcttcagga caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc 5820aatcaatgcc tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc 5880aatgattatt cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat 5940ctctcagttt atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa 6000ttccactgga accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa 6060tatttttaac cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat 6120tcgcagcact cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt 6180gggaatggag agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa 6240tatgtttgcc acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc 6300ctggagacct caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat 6360gaaagtcaca ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa 6420ggagttcctc atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg 6480caaagtaaag gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga 6540cccaccgtta ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc 6600cctgaggatg gaggttctgg gctgcgaggc acaggacctc tactgacaat tgacgctgat 6660cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 6720ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 6780cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 6840gggaggattg ggaagacaat agcaggcatg ctggggagag atctaggaac ccctagtgat 6900ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc 6960cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg 7020agtggccaac cccccccccc ccccccctgc aggcgattct cttgtttgct ccagactctc 7080aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg 7140aatttatcag ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt 7200tctcacccgt ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag 7260ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag 7320ggtcataatg tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat 7380tttgctaatt ctttgccttg cctgtatgat ttattggatg ttggaattcc tgatgcggta 7440ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat 7500ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 7560ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 7620ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 7680gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 7740cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 7800tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 7860gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 7920tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 7980tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 8040ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 8100atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 8160cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 8220attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 8280gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 8340ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 8400gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 8460agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 8520gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 8580gtctcgcggt atcattgcag cactggggcc agatggtaag

ccctcccgta tcgtagttat 8640ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 8700tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 8760tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 8820catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 8880gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 8940aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 9000gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 9060gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 9120gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 9180atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 9240cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 9300cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 9360agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 9420tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 9480gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 9540catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 9600agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 9660ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 972049720DNAArtificial SequencepAAV-CB-hBB-F8-X5 4aattcccatc atcaataata taccttattt tggattgaag ccaatatgat aatgaggggg 60tggagtttgt gacgtggcgc ggggcgtggg aacggggcgg gtgacgtagt agtctctaga 120ggtccccagc gaccttgacg ggcatctgcc cggcatttct gacagctttg tgaactgggt 180ggccgagaag gaatgggagt tgccgccaga ttctgacatg gatctgaatc tgattgagca 240ggcacccctg accgtggccg agaagctgca tcgctggcgt aatagcgaag aggcccgcac 300cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaattcca gacgattgag 360cgtcaaaatg taggtatttc catgagcgtt tttcctgttg caatggctgg cggtaatatt 420gttctggata ttaccagcaa ggccgatagt ttgagttctt ctactcaggc aagtgatgtt 480attactaatc aaagaagtat tgcgacaacg gttaatttgc gtgatggaca gactctttta 540ctcggtggcc tcactgatta taaaaacact tctcaggatt ctggcgtacc gttcctgtct 600aaaatccctt taatcggcct cctgtttagc tcccgctctg attctaacga ggaaagcacg 660ttatacgtgc tcgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc 720gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 780tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 840tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 900tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 960gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 1020ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 1080aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 1140aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 1200ggtacatatg attgacatgc tagttttacg attaccgttc atcgcctgca gggggggggg 1260ggggggggtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 1320aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagag 1380agggagtggc caactccatc actaggggtt cctagatctg aattcggtac gtacctctgg 1440tcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac cccgcccatt 1500gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 1560atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 1620aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 1680catgacctta tgggactttc ctacttggca gtacatctac tcgaggccac gttctgcttc 1740actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 1800ttttgtgcag cgatgggggc gggggggggg gggggggggg cgcgcgccag gcggggcggg 1860gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg 1920cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg 1980aagcgcgcgg cgggcgggag cgggatcagc caccgcggtg gcggcctaga gtcgacgagg 2040aactgaaaaa ccagaaagtt aactggtaag tttagtcttt ttgtctttta tttcaggtcc 2100cggatccggt ggtggtgcaa atcaaagaac tgctcctcag tggatgttgc ctttacttct 2160aggcctgtac ggaagtgtta cttctgctct aaaagctgcg gaattgtacc cgcggccgct 2220tttcaaaatg caaatagagc tctccacctg cttctttctg tgccttttgc gattctgctt 2280tagtgccacc agaagatact acctgggtgc agtggaactg tcatgggact atatgcaaag 2340tgatctcggt gagctgcctg tggacgcaag atttcctcct agagtgccaa aatcttttcc 2400attcaacacc tcagtcgtgt acaaaaagac tctgtttgta gaattcacgg atcacctttt 2460caacatcgct aagccaaggc caccctggat gggtctgcta ggtcctacca tccaggctga 2520ggtttatgat acagtggtcg ttacacttaa gaacatggct tcccatcctg tcagtcttca 2580tgctgttggt gtatcctact ggaaatcttc tgagggagct gaatatgatg atcagaccag 2640tcaaagggag aaagaagatg ataaagtctt ccctggtaaa agccatacat atgtctggca 2700ggtcctgaaa gagaatggtc caacagcctc tgacccacca tgccttacct actcatatct 2760ttctcatgtg gacctggtaa aagacttgaa ttcaggcctc attggagccc tactagtatg 2820tagagaaggg agtctggcca aggaaaagac acagaccttg cacaaattta tactactttt 2880tgctgtattt gatgaaggga aaagttggca ctcagaaaca aagaactcct tgatgcagga 2940tagggatgct gcatctgctc gggcctggcc taaaatgcac acagtcaatg gttatgtaaa 3000caggtctctg ccaggtctga ttggatgcca caggaaatca gtctattggc atgtgattgg 3060aatgggcacc actcctgaag tgcactcaat attcctcgaa ggtcacacat ttcttgtgag 3120gaaccatcgc caggcgtcct tggaaatctc gccaataact ttccttactg ctcaaacact 3180cttgatggac cttggacagt ttctactgtt ttgtcatatc tcttcccacc aacatgatgg 3240catggaagct tatgtcaaag tagacagctg tccagaggaa ccccaactac gaatgaaaaa 3300taatgaagaa gcggaagact atgatgatga tcttactgat tctgaaatgg atgtggtcag 3360gtttgatgat gacaactctc cttcctttat ccaaattcgc tcagttgcca agaagcatcc 3420taaaacttgg gtacattaca ttgctgctga agaggaggac tgggactatg ctcccttagt 3480cctcgccccc gatgacagaa gttataaaag tcaatatttg aacaatggcc ctcagcggat 3540tggtaggaag tacaaaaaag tccgatttat ggcatacaca gatgaaacct ttaagactcg 3600tgaagctatt cagcatgaat caggaatctt gggaccttta ctttatgggg aagttggaga 3660cacactgttg attatattta agaatcaagc aagcagacca tataacatct accctcacgg 3720aatcactgat gtccgtcctt tgtattcaag gagattacca aaaggtgtaa aacatttgaa 3780ggattttcca attctgccag gagaaatatt caaatataaa tggacagtga ctgtagaaga 3840tgggccaact aaatcagatc ctcggtgcct gacccgctat tactctagtt tcgttaatat 3900ggagagagat ctagcttcag gactcattgg ccctctcctc atctgctaca aagaatctgt 3960agatcaaaga ggaaaccaga taatgtcaga caagaggaat gtcatcctgt tttctgtatt 4020tgatgagaac cgaagctggt acctcacaga gaatatacaa cgctttctcc ccaatccagc 4080tggagtgcag cttgaggatc cagagttcca agcctccaac atcatgcaca gcatcaatgg 4140ctatgttttt gatagtttgc agttgtcagt ttgtttgcat gaggtggcat actggtacat 4200tctaagcatt ggagcacaga ctgacttcct ttctgtcttc ttctctggat ataccttcaa 4260acacaaaatg gtctatgaag acacactcac cctattccca ttctcaggag aaactgtctt 4320catgtcgatg gaaaacccag gtctatggat tctggggtgc cacaactcag actttcggaa 4380cagaggcatg accgccttac tgaaggtttc tagttgtgac aagaacactg gtgattatta 4440cgaggacagt tatgaagata tttcagcata cttgctgagt aaaaacaatg ccattgaacc 4500aagaagcttc tcccagaatt caagacaccc tagcactagg caaaagcaat ttaatgccac 4560cacaccacca gtcttgaaac gccatcaacg cgaaataact cgtactactc ttcagtcaga 4620tcaagaggaa attgactatg atgataccat atcagttgaa atgaagaagg aagattttga 4680catttatgat gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta 4740ttttattgct gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct 4800aagaaacagg gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt 4860tactgatggc tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact 4920cctggggcca tatataagag cagaagttga agataatatc atggtaactt tcagaaatca 4980ggcctctcgt ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca 5040aggagcagaa cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa 5100agtgcaacat catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt 5160ctctgatgtt gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg 5220ccacactaac acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct 5280gtttttcacc atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa 5340ctgcagggct ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt 5400ccatgcaatc aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca 5460aaggattcga tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt 5520cagtggacat gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct 5580ctatccaggt gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt 5640ggaatgcctt attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag 5700caataagtgt cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac 5760agcttcagga caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc 5820aatcaatgcc tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc 5880aatgattatt cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat 5940ctctcagttt atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa 6000ttccactgga accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa 6060tatttttaac cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat 6120tcgcagcact cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt 6180gggaatggag agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa 6240tatgtttgcc acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc 6300ctggagacct caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat 6360gaaagtcaca ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa 6420ggagttcctc atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg 6480caaagtaaag gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga 6540cccaccgtta ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc 6600cctgaggatg gaggttctgg gctgcgaggc acaggacctc tactgacaat tgacgctgat 6660cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 6720ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 6780cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 6840gggaggattg ggaagacaat agcaggcatg ctggggagag atctaggaac ccctagtgat 6900ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc 6960cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg 7020agtggccaac cccccccccc ccccccctgc aggcgattct cttgtttgct ccagactctc 7080aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg 7140aatttatcag ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt 7200tctcacccgt ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag 7260ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag 7320ggtcataatg tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat 7380tttgctaatt ctttgccttg cctgtatgat ttattggatg ttggaattcc tgatgcggta 7440ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat 7500ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 7560ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 7620ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 7680gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 7740cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 7800tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 7860gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 7920tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 7980tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 8040ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 8100atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 8160cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 8220attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 8280gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 8340ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 8400gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 8460agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 8520gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 8580gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 8640ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 8700tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 8760tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 8820catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 8880gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 8940aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 9000gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 9060gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 9120gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 9180atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 9240cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 9300cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 9360agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 9420tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 9480gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 9540catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 9600agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 9660ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 972057757DNAArtificial SequencepAAV-CB-hHC1690-X10 5aattcccatc atcaataata taccttattt tggattgaag ccaatatgat aatgaggggg 60tggagtttgt gacgtggcgc ggggcgtggg aacggggcgg gtgacgtagt agtctctaga 120ggtccccagc gaccttgacg ggcatctgcc cggcatttct gacagctttg tgaactgggt 180ggccgagaag gaatgggagt tgccgccaga ttctgacatg gatctgaatc tgattgagca 240ggcacccctg accgtggccg agaagctgca tcgctggcgt aatagcgaag aggcccgcac 300cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaattcca gacgattgag 360cgtcaaaatg taggtatttc catgagcgtt tttcctgttg caatggctgg cggtaatatt 420gttctggata ttaccagcaa ggccgatagt ttgagttctt ctactcaggc aagtgatgtt 480attactaatc aaagaagtat tgcgacaacg gttaatttgc gtgatggaca gactctttta 540ctcggtggcc tcactgatta taaaaacact tctcaggatt ctggcgtacc gttcctgtct 600aaaatccctt taatcggcct cctgtttagc tcccgctctg attctaacga ggaaagcacg 660ttatacgtgc tcgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc 720gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 780tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 840tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 900tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 960gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 1020ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 1080aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 1140aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 1200ggtacatatg attgacatgc tagttttacg attaccgttc atcgcctgca gggggggggg 1260ggggggggtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 1320aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagag 1380agggagtggc caactccatc actaggggtt cctagatctg aattcggtac gtacctctgg 1440tcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac cccgcccatt 1500gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 1560atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 1620aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 1680catgacctta tgggactttc ctacttggca gtacatctac tcgaggccac gttctgcttc 1740actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 1800ttttgtgcag cgatgggggc gggggggggg gggggggggg cgcgcgccag gcggggcggg 1860gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg 1920cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg 1980aagcgcgcgg cgggcgggag cgggatcagc caccgcggtg gcggcctaga gtcgacgagg 2040aactgaaaaa ccagaaagtt aactggtaag tttagtcttt ttgtctttta tttcaggtcc 2100cggatccggt ggtggtgcaa atcaaagaac tgctcctcag tggatgttgc ctttacttct 2160aggcctgtac ggaagtgtta cttctgctct aaaagctgcg gaattgtacc cgcggccgct 2220tttcaaaatg caaatagagc tctccacctg cttctttctg tgccttttgc gattctgctt 2280tagtgccacc agaagatact acctgggtgc agtggaactg tcatgggact atatgcaaag 2340tgatctcggt gagctgcctg tggacgcaag atttcctcct agagtgccaa aatcttttcc 2400attcaacacc tcagtcgtgt acaaaaagac tctgtttgta gaattcacgg atcacctttt 2460caacatcgct aagccaaggc caccctggat gggtctgcta ggtcctacca tccaggctga 2520ggtttacgac acggtggtcg ttaccctgaa gaacatggct tctcatcccg ttagtcttca 2580cgctgtcggc gtctccttct ggaaatcttc cgaaggcgct gaatatgagg atcacaccag 2640ccaaagggag aaggaagacg ataaagtcct tcccggtaaa agccaaacct acgtctggca 2700ggtcctgaaa gaaaatggtc caacagcctc tgacccacca tgtcttacct actcatacct 2760gtctcacgtg gacctggtga aagacctgaa ttcgggcctc attggagccc tactagtatg 2820tagagaaggg agtctggcca aggaaaagac acagaccttg cacaaattta tactactttt 2880tgctgtattt gatgaaggga aaagttggca ctcagaaaca aagaactcct tgatgcagga 2940tagggatgct gcatctgctc gggcctggcc taaaatgcac acagtcaatg gttatgtaaa 3000caggtctctg ccaggtctga ttggatgcca caggaaatca gtctattggc atgtgattgg 3060aatgggcacc actcctgaag tgcactcaat attcctcgaa ggtcacacat ttcttgtgag 3120gaaccatcgc caggcgtcct tggaaatctc gccaataact ttccttactg ctcaaacact 3180cttgatggac cttggacagt ttctactgtt ttgtcatatc tcttcccacc aacatgatgg 3240catggaagct tatgtcaaag tagacagctg tccagaggaa ccccaactac gaatgaaaaa 3300taatgaagaa gcggaagact atgatgatga tcttactgat tctgaaatgg atgtggtcag 3360gtttgatgat gacaactctc cttcctttat ccaaattcgc tcagttgcca agaagcatcc 3420taaaacttgg gtacattaca ttgctgctga agaggaggac tgggactatg ctcccttagt 3480cctcgccccc gatgacagaa gttataaaag tcaatatttg aacaatggcc ctcagcggat 3540tggtaggaag tacaaaaaag tccgatttat ggcatacaca gatgaaacct ttaagactcg 3600tgaagctatt cagcatgaat caggaatctt gggaccttta ctttatgggg aagttggaga 3660cacactgttg attatattta agaatcaagc aagcagacca tataacatct accctcacgg 3720aatcactgat gtccgtcctt tgtattcaag gagattacca aaaggtgtaa aacatttgaa 3780ggattttcca attctgccag gagaaatatt caaatataaa tggacagtga ctgtagaaga 3840tgggccaact aaatcagatc ctcggtgcct gacccgctat tactctagtt tcgttaatat 3900ggagagagat ctagcttcag gactcattgg ccctctcctc atctgctaca aagaatctgt 3960agatcaaaga ggaaaccaga taatgtcaga caagaggaat gtcatcctgt tttctgtatt 4020tgatgagaac cgaagctggt acctcacaga gaatatacaa cgctttctcc ccaatccagc 4080tggagtgcag cttgaggatc cagagttcca agcctccaac atcatgcaca gcatcaatgg 4140ctatgttttt gatagtttgc

agttgtcagt ttgtttgcat gaggtggcat actggtacat 4200tctaagcatt ggagcacaga ctgacttcct ttctgtcttc ttctctggat ataccttcaa 4260acacaaaatg gtctatgaag acacactcac cctattccca ttctcaggag aaactgtctt 4320catgtcgatg gaaaacccag gtctatggat tctggggtgc cacaactcag actttcggaa 4380cagaggcatg accgccttac tgaaggtttc tagttgtgac aagaacactg gtgattatta 4440cgaggacagt tatgaagata tttcagcata cttgctgagt aaaaacaatg ccattgaacc 4500aagaagcttc tcccagaatc caccagtctt gaaacgccat caacgcgaaa taactcgtac 4560tactcttcag tcagatcaag aggaaattga ctatgatgat accatatcag ttgaaatgaa 4620gaaggaagat tttgacattt atgatgagga tgaaaatcag agcccccgca gctagcaatt 4680gtagtctgaa cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 4740cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 4800aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 4860ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggagagatc 4920taggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 4980gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag 5040cgagcgcgca gagagggagt ggccaacccc cccccccccc cccctgcagg cgattctctt 5100gtttgctcca gactctcagg caatgacctg atagcctttg tagagacctc tcaaaaatag 5160ctaccctctc cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt 5220tgactgtctc cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg 5280catttaaaat atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc 5340ccgcaaaagt attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg 5400aggctttatt gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg 5460gaattcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg 5520tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca 5580acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 5640gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 5700agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 5760tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 5820ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 5880taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 5940tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 6000gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 6060atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 6120ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 6180cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 6240ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 6300aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 6360ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 6420gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 6480ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 6540gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 6600ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 6660tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 6720cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 6780tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 6840atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 6900tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 6960tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 7020ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 7080cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 7140ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 7200gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 7260tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 7320gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 7380ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 7440tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 7500ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 7560tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 7620attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 7680tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 7740ccgattcatt aatgcag 775767757DNAArtificial SequencepAAV-CB-hHC1690-X5 6aattcccatc atcaataata taccttattt tggattgaag ccaatatgat aatgaggggg 60tggagtttgt gacgtggcgc ggggcgtggg aacggggcgg gtgacgtagt agtctctaga 120ggtccccagc gaccttgacg ggcatctgcc cggcatttct gacagctttg tgaactgggt 180ggccgagaag gaatgggagt tgccgccaga ttctgacatg gatctgaatc tgattgagca 240ggcacccctg accgtggccg agaagctgca tcgctggcgt aatagcgaag aggcccgcac 300cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaattcca gacgattgag 360cgtcaaaatg taggtatttc catgagcgtt tttcctgttg caatggctgg cggtaatatt 420gttctggata ttaccagcaa ggccgatagt ttgagttctt ctactcaggc aagtgatgtt 480attactaatc aaagaagtat tgcgacaacg gttaatttgc gtgatggaca gactctttta 540ctcggtggcc tcactgatta taaaaacact tctcaggatt ctggcgtacc gttcctgtct 600aaaatccctt taatcggcct cctgtttagc tcccgctctg attctaacga ggaaagcacg 660ttatacgtgc tcgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc 720gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 780tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 840tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 900tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 960gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 1020ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 1080aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 1140aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 1200ggtacatatg attgacatgc tagttttacg attaccgttc atcgcctgca gggggggggg 1260ggggggggtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 1320aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagag 1380agggagtggc caactccatc actaggggtt cctagatctg aattcggtac gtacctctgg 1440tcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac cccgcccatt 1500gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 1560atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 1620aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 1680catgacctta tgggactttc ctacttggca gtacatctac tcgaggccac gttctgcttc 1740actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 1800ttttgtgcag cgatgggggc gggggggggg gggggggggg cgcgcgccag gcggggcggg 1860gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg 1920cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg 1980aagcgcgcgg cgggcgggag cgggatcagc caccgcggtg gcggcctaga gtcgacgagg 2040aactgaaaaa ccagaaagtt aactggtaag tttagtcttt ttgtctttta tttcaggtcc 2100cggatccggt ggtggtgcaa atcaaagaac tgctcctcag tggatgttgc ctttacttct 2160aggcctgtac ggaagtgtta cttctgctct aaaagctgcg gaattgtacc cgcggccgct 2220tttcaaaatg caaatagagc tctccacctg cttctttctg tgccttttgc gattctgctt 2280tagtgccacc agaagatact acctgggtgc agtggaactg tcatgggact atatgcaaag 2340tgatctcggt gagctgcctg tggacgcaag atttcctcct agagtgccaa aatcttttcc 2400attcaacacc tcagtcgtgt acaaaaagac tctgtttgta gaattcacgg atcacctttt 2460caacatcgct aagccaaggc caccctggat gggtctgcta ggtcctacca tccaggctga 2520ggtttatgat acagtggtcg ttacacttaa gaacatggct tcccatcctg tcagtcttca 2580tgctgttggt gtatcctact ggaaatcttc tgagggagct gaatatgatg atcagaccag 2640tcaaagggag aaagaagatg ataaagtctt ccctggtaaa agccatacat atgtctggca 2700ggtcctgaaa gagaatggtc caacagcctc tgacccacca tgccttacct actcatatct 2760ttctcatgtg gacctggtaa aagacttgaa ttcaggcctc attggagccc tactagtatg 2820tagagaaggg agtctggcca aggaaaagac acagaccttg cacaaattta tactactttt 2880tgctgtattt gatgaaggga aaagttggca ctcagaaaca aagaactcct tgatgcagga 2940tagggatgct gcatctgctc gggcctggcc taaaatgcac acagtcaatg gttatgtaaa 3000caggtctctg ccaggtctga ttggatgcca caggaaatca gtctattggc atgtgattgg 3060aatgggcacc actcctgaag tgcactcaat attcctcgaa ggtcacacat ttcttgtgag 3120gaaccatcgc caggcgtcct tggaaatctc gccaataact ttccttactg ctcaaacact 3180cttgatggac cttggacagt ttctactgtt ttgtcatatc tcttcccacc aacatgatgg 3240catggaagct tatgtcaaag tagacagctg tccagaggaa ccccaactac gaatgaaaaa 3300taatgaagaa gcggaagact atgatgatga tcttactgat tctgaaatgg atgtggtcag 3360gtttgatgat gacaactctc cttcctttat ccaaattcgc tcagttgcca agaagcatcc 3420taaaacttgg gtacattaca ttgctgctga agaggaggac tgggactatg ctcccttagt 3480cctcgccccc gatgacagaa gttataaaag tcaatatttg aacaatggcc ctcagcggat 3540tggtaggaag tacaaaaaag tccgatttat ggcatacaca gatgaaacct ttaagactcg 3600tgaagctatt cagcatgaat caggaatctt gggaccttta ctttatgggg aagttggaga 3660cacactgttg attatattta agaatcaagc aagcagacca tataacatct accctcacgg 3720aatcactgat gtccgtcctt tgtattcaag gagattacca aaaggtgtaa aacatttgaa 3780ggattttcca attctgccag gagaaatatt caaatataaa tggacagtga ctgtagaaga 3840tgggccaact aaatcagatc ctcggtgcct gacccgctat tactctagtt tcgttaatat 3900ggagagagat ctagcttcag gactcattgg ccctctcctc atctgctaca aagaatctgt 3960agatcaaaga ggaaaccaga taatgtcaga caagaggaat gtcatcctgt tttctgtatt 4020tgatgagaac cgaagctggt acctcacaga gaatatacaa cgctttctcc ccaatccagc 4080tggagtgcag cttgaggatc cagagttcca agcctccaac atcatgcaca gcatcaatgg 4140ctatgttttt gatagtttgc agttgtcagt ttgtttgcat gaggtggcat actggtacat 4200tctaagcatt ggagcacaga ctgacttcct ttctgtcttc ttctctggat ataccttcaa 4260acacaaaatg gtctatgaag acacactcac cctattccca ttctcaggag aaactgtctt 4320catgtcgatg gaaaacccag gtctatggat tctggggtgc cacaactcag actttcggaa 4380cagaggcatg accgccttac tgaaggtttc tagttgtgac aagaacactg gtgattatta 4440cgaggacagt tatgaagata tttcagcata cttgctgagt aaaaacaatg ccattgaacc 4500aagaagcttc tcccagaatc caccagtctt gaaacgccat caacgcgaaa taactcgtac 4560tactcttcag tcagatcaag aggaaattga ctatgatgat accatatcag ttgaaatgaa 4620gaaggaagat tttgacattt atgatgagga tgaaaatcag agcccccgca gctagcaatt 4680gtagtctgaa cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 4740cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 4800aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 4860ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggagagatc 4920taggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 4980gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag 5040cgagcgcgca gagagggagt ggccaacccc cccccccccc cccctgcagg cgattctctt 5100gtttgctcca gactctcagg caatgacctg atagcctttg tagagacctc tcaaaaatag 5160ctaccctctc cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt 5220tgactgtctc cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg 5280catttaaaat atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc 5340ccgcaaaagt attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg 5400aggctttatt gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg 5460gaattcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg 5520tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca 5580acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 5640gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 5700agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 5760tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 5820ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 5880taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 5940tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 6000gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 6060atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 6120ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 6180cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 6240ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 6300aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 6360ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 6420gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 6480ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 6540gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 6600ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 6660tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 6720cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 6780tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 6840atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 6900tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 6960tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 7020ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 7080cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 7140ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 7200gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 7260tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 7320gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 7380ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 7440tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 7500ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 7560tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 7620attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 7680tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 7740ccgattcatt aatgcag 77577200PRTHomo sapiens 7Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala 195 2008200PRTHomo sapiens 8Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser Ser Glu Gly Ala 100 105 110Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala 195 2009200PRTHomo sapiens 9Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro 20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala Ser His Pro Val 85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ser Ser Glu Gly Ala 100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val 115 120 125Phe Pro Gly Lys Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn 130 135 140Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu 165 170 175Leu Val Cys Arg Glu

Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu 180 185 190His Lys Phe Ile Leu Leu Phe Ala 195 200

Claims

1-63. (canceled)

64. An isolated polynucleotide encoding a polypeptide, wherein the polypeptide comprises wildtype human factor VIII with one or more amino acid substitution(s) at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

65. The isolated polynucleotide of claim 64, wherein the amino acid substitutions are selected from the group consisting of I86V, I86L, I86M, Y105F, Y105W, A108S, A108G, A108T, A108P, D115E, D115N, D115H, D115Q, D115R, D115K, Q117H, Q117N, Q117E, Q117D, Q117R, Q117K, F129L, F129V, F1291, F129M, F129P, F129T, F129K, G132K, G132E, G132D, G132R, G132T, G132M, G132N, G132S, G132W, H134Q, H134G, H134Y, H134N, H134E, H134D, H134R, H134K, M147T, M147A, M147G, M147S, M147P, L152P, L152S, L152G and L152T.

66. The isolated polynucleotide of claim 64, wherein the polypeptide comprises amino acid substitutions A108S, M147T, and L152P.

67. The isolated polynucleotide of claim 65, wherein the amino acid substitutions are selected from the group consisting of I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P.

68. The isolated polynucleotide of claim 66, wherein the polypeptide comprises the amino acid substitution I86V.

69. The isolated polynucleotide of claim 65, wherein the amino acid substitutions are selected from the group consisting of I86V, A108S, G132K, M147T and L152P.

70. The isolated polynucleotide of claim 65, wherein the amino acid substitutions are selected from the group consisting of A108S, M147T, and L152P.

71. The isolated polynucleotide of claim 70, wherein the polypeptide further comprises amino acid substitutions I86V and G132K.

72. The isolated polynucleotide of claim 64, wherein the polypeptide comprises a deletion in the B domain.

73. The isolated polynucleotide of claim 64, wherein the polypeptide comprises the a2 and/or a3 domain(s) of human factor VIII.

74. An isolated polynucleotide comprising SEQ ID NO: 1, wherein the polynucleotide encodes a human factor VIII polypeptide comprising one or more amino acid substitution(s) at positions I86, Y105, A108, D115, Q117, F129, G132, H134, M147 and/or L152.

75. The isolated polynucleotide of claim 74, wherein the amino acid substitutions are selected from the group consisting of I86V, I86L, I86M, Y105F, Y105W, A108S, A108G, A108T, A108P, D115E, D115N, D115H, D115Q, D115R, D115K, Q117H, Q117N, Q117E, Q117D, Q117R, Q117K, F129L, F129V, F1291, F129M, F129P, F129T, F129K, G132K, G132E, G132D, G132R, G132T, G132M, G132N, G132S, G132W, H134Q, H134G, H134Y, H134N, H134E, H134D, H134R, H134K, M147T, M147A, M147G, M147S, M147P, L152P, L152S, L152G and L152T.

76. The isolated polynucleotide of claim 75, wherein the amino acid substitutions are selected from the group consisting of I86V, Y105F, A108S, D115E, Q117H, F129L, G132K, H134Q, M147T and L152P.

77. The isolated polynucleotide of claim 74, wherein the polypeptide comprises a deletion in the B domain.

78. An expression vector comprising the polynucleotide of claim 64.

79. A host cell comprising the polynucleotide of claim 64.

80. A host cell comprising the expression vector of claim 79.

81. A pharmaceutical composition comprising the expression vector of claim 80.

82. A method for treating a patient with a factor VIII deficiency comprising: administering to the patient in need thereof the pharmaceutical composition of claim 81 in an amount effective for treating the factor VIII deficiency.

83. A method for expressing a human factor VIII polypeptide mutant comprising: (a) transforming a host cell with an expression vector comprising the polynucleotide of claim 64; (b) growing the host cell under conditions suitable for expressing the human factor VIII polypeptide mutant; and (c) purifying the human factor VIII polypeptide mutant from host cells expressing said mutant.