EA046419B1 - MINIATURIZED DYSTROPHINS AND THEIR APPLICATIONS - Google Patents

Description

Link to related applications

This invention claims benefit to U.S. Provisional Application No. 62/749982, filed October 24, 2018, which is incorporated herein by reference in its entirety.

1. Technical field of the invention

The subject matter disclosed herein generally relates to polynucleotides, polypeptides, cells, vectors, applications and kits related to miniaturized dystrophin.

2. State of the art

Duchenne muscular dystrophy (DMD) is a recessively inherited muscle-wasting disorder that affects approximately 1 in 3,500 men. DMD is caused by mutations in the dystrophin gene, which is located on the X chromosome. Mutations in this gene result in aberrant or absent expression of the dystrophin protein.

Dystrophin is a key component of a protein complex that is responsible for regulating the integrity and function of muscle cells. Patients with DMD typically lose the ability to physically support themselves during childhood and become increasingly weaker over time. This progressive skeletal muscle wasting and cardiac dysfunction usually lead to loss of ambulation and premature death, primarily due to cardiac or respiratory failure. Treatments for DMD have been attempted in the past. However, available treatment options have been significantly limited due to the large size of the complementary DNA (cDNA) (approximately 13.9 kilobase pairs (kb)) of wild-type dystrophin, which cannot be administered to patients with DMD and cannot be expressed in them with using standard viral vectors, including adeno-associated virus (AAV), which cannot transport more than 4.9 kb. heterologous DNA. Therefore, there is a need to develop a recombinant dystrophin gene that can be efficiently packaged into a gene therapy vector. Adeno-associated virus (AAV) vectors have been shown to be useful in gene therapy approaches aimed at correcting genetic deficiencies resulting in reduced or absent protein expression levels (Nathwani et al. - A.M. Keeler et al.), and are potentially useful for gene knockdown , genome editing or modification, and modulation of non-coding RNA (Valdmanis et al., 2017). Packaging of the entire muscle-specific dystrophin isoform cDNA into a single rAAV capsid cannot be easily achieved due to the large size of the dystrophin cDNA. Previous studies have focused on developing small genetic constructs that express only specific domains of dystrophin. See US Patent Nos. 6,869,777 and 8,501,920, each of which is incorporated by reference. However, these approaches have had only limited success. There remains a need for more precise and effective gene therapy tools to treat patients with mutations in the dystrophin gene, and in particular the need to develop a recombinant dystrophin gene that can be efficiently packaged into a gene therapy vector.

3. Essence of the invention

The present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes a miniaturized dystrophin polypeptide comprising, from N-terminus to C-terminus, a hinge 1 (H1) domain, a spectrin repeat 1 (R1) domain ), spectrin repeat domain 3 (R3), hinge 2 (H2) domain, spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain, where The miniaturized dystrophin polypeptide does not contain dystrophin spectrin repeat 2.

In some embodiments, the miniaturized dystrophin polypeptide does not comprise a spectrin repeat 2 (R2) domain, a spectrin repeat 4 (R4) domain, a spectrin repeat 5 (R5) domain, a spectrin repeat 6 (R6) domain, a spectrin repeat 7 (R7) domain spectrin repeat 8 (R8), spectrin repeat domain 9 (R9), spectrin repeat domain 10 (R10), spectrin repeat domain 11 (R11), spectrin repeat domain 12 (R12), spectrin repeat domain 13 (R13), spectrin repeat domain 14 (R14), spectrin repeat domain 15 (R15), spectrin repeat domain 18 (R18), spectrin repeat domain 19 (R19), spectrin repeat domain 20 (R20), spectrin repeat domain 21 (R21), spectrin repeat domain 22 ( R22), spectrin repeat domain 23 (R23), or any combination thereof. In some embodiments, the R1 domain is directly fused to the R3 domain via a peptide bond. In some embodiments, the R1 domain and the R3 domain are fused through the amino acids arginine-valine (ARG-VAL, RV). In some embodiments, the H2 domain and the R16 domain are fused via a linker.

In some embodiments, the linker comprises an amino acid sequence that is at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, is at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 75 (IHTVREE TMMVMTEDMP LEI), and the amino acid sequence is capable of improving neuronal Nitric Oxide Synthase signal transduction. nNOS). In some embodiments, disclosed herein is a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence comprising the sequence of SEQ ID NO: 75 (IHTVREE TMMVMTEDMP LEI), wherein the nucleic acid molecule is at least about 80% at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 100 (ATCCACACCGTGCGGGAAGAGACAATGATGGTCATGACAGAGGAC ATGCCCCTGGAAATC), and the amino acid sequence is capable of improving nNOS signal transmission. In some embodiments, the amino acid sequence is a linker connecting the first dystrophin domain to the second dystrophin domain. In some embodiments, the first dystrophin domain is an H2 domain and the second dystrophin domain is an R16 domain. In some embodiments, the amino acid sequence further comprises the sequence set forth in SEQ ID NO: 74 at the N-terminus.

In some embodiments, the nucleic acid molecule encodes a miniaturized dystrophin polypeptide. In some embodiments, the miniaturized dystrophin polypeptide comprises, from N-terminus to C-terminus, a hinge 1 (H1) domain, a spectrin repeat 1 (R1) domain, a spectrin repeat 3 (R3) domain, a hinge 2 (H2) domain, a spectrin domain repeat 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain. In some embodiments, the R1 domain and the R3 domain are fused through the amino acids ARG-VAL (RV), and wherein the H2 domain and the R16 domain are fused through the amino acid sequence set forth in SEQ ID NOs: 74-75, in combination. In some embodiments, (i) the H1 domain and the R1 domain are directly fused, (ii) the R3 domain and the H2 domain are directly fused, (iii) the R16 and R17 domains are directly fused, (iv) the R17 and R24 domains are directly fused, or (v) the R24 and H4 domains are fused directly, or (vi) any combination thereof. In some embodiments, the miniaturized dystrophin polypeptide further comprises an ABD1 domain and/or a CR domain. In some embodiments, the miniaturized dystrophin polypeptide consists essentially of or consists of, from N-terminus to C-terminus, an ABD1 domain, an H1 domain, an R1 domain, RV amino acids, an R3 domain, an H2 domain, the amino acid sequence set forth in SEQ ID NO : 74-75, R16 domain, R17 domain, R24 domain, H4 domain and CR domain of dystrophin.

In some embodiments, the H1 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96 % is at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 69. In some embodiments, the R1 domain is an amino acid sequence that at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least is at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 70. In some embodiments, the R3 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 % or about 100% identical to the sequence of SEQ ID NO: 72. In some embodiments, the H2 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 73. In some embodiments, the R16 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 76. In some embodiments, the R17 domain is an amino acid sequence , which is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, is at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 77. In some embodiments, the R24 domain is an amino acid sequence that is at least about 80% identical to at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about about 99% or about 100% identical to the sequence of SEQ ID NO: 78. In some embodiments, the H4 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least

- 2046419 is at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 79. In some embodiments, the miniaturized dystrophin polypeptide further comprises at the N-terminus an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97 % is at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 68. In some embodiments, the miniaturized dystrophin polypeptide further comprises, at its C terminus, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 80. In some embodiments, the miniaturized dystrophin polypeptide contains an amino acid sequence that is at least about 80%, at least about 85% identical , at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or is about 100% identical to the sequence of SEQ ID NO: 118. In some embodiments, the nucleotide sequence comprises a sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% , is at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequence of SEQ ID NO: 133.

In some embodiments, the miniaturized dystrophin polypeptide exhibits one or more properties selected from the group consisting of (i) lower CD4 proliferation compared to BXA-027741, (ii) lower CD8 proliferation compared to BXA-027741, (iii) higher expression of miniaturized dystrophin polypeptide than BXA-027741, and (iv) any combination thereof. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a miniaturized dystrophin polypeptide comprising a spectrin repeat 1 (R1) domain and a spectrin repeat 16 (R16) domain, wherein the R1 domain and the R16 domain are fused through a linker comprising the amino acid sequence set forth in SEQ ID NO: 84 (IHTVREETMMVMTEDMPLEI). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a miniaturized dystrophin polypeptide comprising, from N-terminus to C-terminus, a hinge 1 (H1) domain, a spectrin repeat 1 (R1) domain, a spectrin repeat 16 (R16) domain , spectrin repeat domain 17 (R17), hinge 3 (H3) domain, spectrin repeat domain 23 (R23), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain, where the R1 domain and the R16 domain are fused by amino acid sequence given in SEQ ID NO: 84 (IHTVREETMMVMTEDMPLEI). In some embodiments, (i) the H1 domain and the R1 domain are directly fused, (ii) the R16 and R17 domains are directly fused, (iii) the R17 and H3 domains are directly fused, (iv) the R23 and R24 domains are directly fused, or (v) the R24 and H4 fused directly, or (vi) any combination thereof.

In some embodiments, the miniaturized dystrophin polypeptide does not comprise a spectrin repeat 2 (R2) domain, a spectrin repeat 3 (R3) domain, a spectrin repeat 4 (R4) domain, a spectrin repeat 5 (R5) domain, a spectrin repeat 6 (R6) domain spectrin repeat 7 (R7), spectrin repeat domain 8 (R8), spectrin repeat domain 9 (R9), spectrin repeat domain 10 (R10), spectrin repeat domain 11 (R11), spectrin repeat domain 12 (R12), spectrin repeat domain 13 (R13), spectrin repeat domain 14 (R14), spectrin repeat domain 15 (R15), spectrin repeat domain 18 (R18), spectrin repeat domain 19 (R19), spectrin repeat domain 20 (R20), spectrin repeat domain 21 ( R21) and/or spectrin repeat domain 22 (R22).

In some embodiments, the miniaturized dystrophin polypeptide further comprises an ABD1 domain and/or a CR domain. In some embodiments, the miniaturized dystrophin polypeptide consists essentially of or consists of, from the N-terminus to the C-terminus, an ABD1 domain, an H1 domain, an R1 domain, the amino acid sequence set forth in SEQ ID NO: 84, an R16 domain, an R17 domain, H3 domain, R23 domain, R24 domain, H4 domain and CR domain of dystrophin. In some embodiments, the H1 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96 % is at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 82. In some embodiments, the R1 domain is an amino acid sequence that at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least at least about 98%, at least about 99% or about

- 3 046419 is 100% identical to the sequence of SEQ ID NO: 83. In some embodiments, the R16 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 85. In some embodiments, the R17 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 86. In some embodiments, the H3 domain is an amino acid sequence , which is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, is at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 87. In some embodiments, the R23 domain is an amino acid sequence that is at least about 80% identical to at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about about 99% or about 100% identical to the sequence of SEQ ID NO: 88. In some embodiments, the R24 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 89. In some embodiments, the H4 domain is an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 90.

In some embodiments, the miniaturized dystrophin polypeptide further comprises, at its N-terminus, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, is at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 81. In some embodiments, the miniaturized dystrophin polypeptide further comprises at the C-terminus an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, is at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 91. In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 132 or SEQ ID NO: 132, where the C-terminal domain is deleted. In some embodiments, the nucleotide sequence comprises a sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least is at least about 99% or about 100% identical to the nucleotide sequence of SEQ ID NO: 147 or SEQ ID NO: 148 or SEQ ID NO: 149.

In some embodiments, the miniaturized dystrophin polypeptide exhibits higher expression of the miniaturized dystrophin polypeptide than BXA-196481.

In some embodiments, expression of the miniaturized dystrophin polypeptide is at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least at least about 1.9 times, at least about 2 times, at least about 2.1 times, at least about 2.2 times, at least about 2.3 times, at least about 2.4 times, at least about 2.5 times, at least about 2.6 times, at least about 2.7 times, at least about 2.8 times, at least about about 2.9-fold or at least about 3-fold higher than the expression of the BXA-196481 polypeptide.

In some embodiments, the nucleic acid molecule further comprises a promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter drives expression of the therapeutic protein in hepatocytes, muscle cells, endothelial cells, neuronal cells, sinusoidal cells, or any combination thereof. In some embodiments, the promoter is selected from

- 4 046419 group consisting of mouse thyretin promoter (mTTR), human endogenous factor VIII promoter (F8), human alpha-1 antitrypsin promoter (hAAT), human minimal albumin promoter, mouse albumin promoter, tristetraproline (TTP) promoter, CASI, synapsin 1 gene promoter, CAG promoter, cytomegalovirus (CMV) promoter, α1-antitrypsin (AAT), muscle creatine kinase (MCK), alpha-myosin heavy chain (aMHC), myoglobin (MB), desmin (DES), SPc512, 2R5Sc5-12, dMCK, tMCK and phosphoglycerate kinase (PGK) promoter.

In some embodiments, the nucleic acid molecule further comprises an intronic sequence. In some embodiments, the intronic sequence is located 5' from the nucleotide sequence encoding the miniaturized dystrophin polypeptide. In some embodiments, the intronic sequence is located 3' from the promoter. In some embodiments, the intronic sequence includes a synthetic intronic sequence. In some embodiments, the nucleic acid molecule further comprises a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element is located 3' of the nucleotide sequence encoding the miniaturized dystrophin polypeptide. In some embodiments, the post-transcriptional regulatory element comprises a mutant hepatitis virus post-transcriptional regulatory element (WPRE), a miRNA binding site, or a target nuclear DNA sequence, or any combination thereof.

In some embodiments, the nucleic acid molecule further comprises a 3 ^r UTR poly(A) tail sequence. In some embodiments, the 3 ^r UTR poly(A) tail sequence is selected from the group consisting of bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof. In some embodiments, the 3UTR poly(A) tail sequence includes a bGH poly(A).

In some embodiments, the nucleic acid molecule further comprises an enhancer sequence.

In some embodiments, the nucleic acid molecule further comprises a first ITR and/or a second ITR. In some embodiments, the first ITR and the second ITR are identical. In some embodiments, the first ITR and/or the second ITR are derived from an adeno-associated virus.

In some embodiments, the nucleic acid molecule comprises a heterologous fragment. In some embodiments, the heterologous fragment is selected from the group consisting of albumin or a fragment thereof, an immunoglobulin Fc region, a human chorionic gonadotropin β-subunit C-terminal peptide (CTP), a PAS sequence, a HAP sequence, transferrin or a fragment thereof, an albumin-binding fragment, or its derivative and any combination thereof.

In some embodiments, a vector is provided containing a nucleic acid molecule disclosed herein. In some embodiments, the vector is selected from the group consisting of an adenoviral vector, a retroviral vector, a poxvirus vector, a baculovirus vector, and a herpes virus-based vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the AAV vector is AAV9.

In some embodiments, a nucleic acid molecule or vector disclosed herein is formulated with a delivery vehicle. In some embodiments, the delivery vehicle includes a lipid nanoparticle. In some embodiments, the delivery vehicle is selected from the group consisting of liposomes, non-lipid polymer molecules, endosomes, and any combination thereof. In some embodiments, the nucleic acid molecule or vector disclosed herein is formulated for intravenous, transdermal, intradermal, subcutaneous, pulmonary or oral delivery, or any combination thereof. In some embodiments, the nucleic acid molecule or vector disclosed herein is formulated for intravenous delivery. In some embodiments, a polypeptide encoded by a nucleic acid molecule or vector disclosed herein is provided. In some embodiments, a host cell is provided containing a nucleic acid molecule described herein. In some embodiments, the cell is a CHO cell, HEK293 cell, HBK cell, COS cell, NSO cell, or HT1080 cell.

In some embodiments, a pharmaceutical composition is provided comprising (a) a nucleic acid disclosed herein, a vector disclosed herein, a polypeptide disclosed herein, or a host cell disclosed herein; and (b) a pharmaceutically acceptable excipient.

In some embodiments, a kit is provided comprising a nucleic acid disclosed herein, a vector disclosed herein, a polypeptide disclosed herein

- 5 046419 document, a host cell disclosed herein or a pharmaceutical composition disclosed herein, and instructions for administering the nucleic acid molecule to a subject in need thereof.

In some embodiments, a method of producing a miniaturized dystrophin polypeptide is provided, comprising: culturing a host cell as disclosed herein under suitable conditions and isolating the miniaturized dystrophin polypeptide.

In some embodiments, a method of expressing a miniaturized dystrophin polypeptide in a subject in need thereof is provided, comprising administering to the subject a nucleic acid disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein. .

In some embodiments, a method of treating a subject suffering from a disease or condition is provided, comprising administering to the subject a nucleic acid disclosed herein, a vector disclosed herein, a polypeptide disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, the disease or condition is a disease caused by dystrophin deficiency. In some embodiments, the disease is sarcopenia, heart disease, cachexia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy (XLDC), facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, limb-lumbar dystrophy, oculopharyngeal muscular dystrophy, muscular dystrophy

Emery-Dreyfus, distal muscular dystrophy and/or congenital muscular dystrophy. In some embodiments, the method of any one of claims 86 to 89, wherein the nucleic acid molecule, vector, polypeptide, host cell or pharmaceutical composition is administered intravenously, transdermally, intradermally, subcutaneously, orally or pulmonaryly, or any combination thereof. In some embodiments, the nucleic acid molecule, vector, polypeptide, host cell, or pharmaceutical composition is administered intravenously, transdermally, intradermally, subcutaneously, orally, or pulmonaryly, or any combination thereof.

In some embodiments, the method further includes administering a second agent to the subject.

In some embodiments, the subject is a human. In some embodiments, administration of a nucleic acid molecule, vector, polypeptide, host cell, or pharmaceutical composition to a subject results in increased dystrophin protein expression compared to dystrophin protein expression in the subject prior to administration, wherein dystrophin protein expression is increased by at least about 2-fold, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times, at least about about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times.

In some embodiments, disclosed herein is a nucleic acid molecule comprising a nucleotide sequence that encodes a miniaturized dystrophin polypeptide comprising a hinge 2 (H2) domain and a spectrin repeat 16 (R16) domain, wherein the H2 domain and the R16 domain are fused through a linker containing an amino acid the sequence given in SEQ ID NO: 84 (IHTVREETMMVMTEDMPLEI).

4. Brief description of drawings

Fig. 1 shows a schematic representation of the full-length human dystrophin protein. ABD1: actin binding domain-1; H# (eg H1): hinge area; R# (eg R1): spectrin-like repeat domains; ABD2: actin binding domain-2; CR: cysteine-rich domain; Conc: C-terminal domain of a protein.

Fig. 2 shows schematic representations of the miniaturized dystrophin polypeptides BXA-027741, BXA-027742, BXA-027743, BXA-027744, BXA-196480, and BXA-196481, and, by way of reference, the full-length human dystrophin protein of FIG. 1.

Fig. 3 shows the ratio of miniaturized dystrophin polypeptide expression to miniaturized dystrophin mRNA expression in isogenic induced pluripotent stem cell (iPSC)-derived human cardiac myocytes (iCM) (carrying a premature stop codon E2035X in the dystrophin gene, which prevents endogenous dystrophin expression) after plasmid transfection. expressing said miniaturized dystrophin polypeptides.

Fig. 4 shows a schematic representation of a miniaturized dystrophin polypeptide

- 6 046419

BXA-027743 (SEQ ID NO: 132), as well as the amino acid sequence of its J1 and J7 junction regions, which are said to have relatively low immunogenicity.

Fig. 5 shows a schematic representation of the miniaturized dystrophin polypeptide BXA-027741 (SEQ ID NO: 129) with arrows pointing to its junction regions J10, J11 and J9.

Fig. 6A is a histogram showing the proportion of samples, among the 40 sample cell panel tested, that were treated with the various junction peptides as indicated and had proliferating CD4+ cells (each colored square represents a sample from one patient). Fig. 6B is a histogram showing the proportion of samples, among the 40 sample cell panel tested, that were treated with the various junction peptides as indicated and had proliferating CD8+ cells (each colored square represents a sample from one patient).

Fig. 7 shows a schematic representation of the miniaturized dystrophin polypeptide BXA-196473 (SEQ ID NO: 119).

Fig. 8A shows a schematic representation of the miniaturized dystrophin polypeptide BXA-196477 (SEQ ID NO: 118). RV: arginine-valine dipeptide between R1 and R3; SEAQ: serine glutamic acid-alanine-glutamine peptide. Fig. 8B shows the three-dimensional fold around the original junction region 10 between R1 and R3 of the miniaturized dystrophin polypeptide BXA027741 (SEQ ID NO: 129) (left) and the three-dimensional fold around the version 3 junction region 10 of the miniaturized dystrophin polypeptide BXA-196477 (SEQ ID NO: 118) ( on right).

Fig. 9 shows the ratio of expression of miniaturized dystrophin polypeptide and miniaturized dystrophin mRNA in human isogenic induced pluripotent stem cell (iPSC)-derived cardiac myocytes (iCM), (carrying the premature stop codon E2035X in the dystrophin gene, which prevents endogenous expression of dystrophin) after transfection of plasmids expressing the indicated miniaturized dystrophin polypeptides.

Fig. 10 shows a schematic representation of the miniaturized dystrophin polypeptide BXA-196474 (SEQ ID NO: 120).

Fig. 11 shows a schematic representation of the miniaturized dystrophin polypeptide BXA-196475 (SEQ ID NO: 121).

Fig. 12 shows a schematic representation of the miniaturized dystrophin polypeptide BXA-196476 (SEQ ID NO: 122).

Fig. 13 shows a schematic representation of the miniaturized dystrophin polypeptide BXA-196478 (SEQ ID NO: 124).

Fig. 14 shows a schematic representation of the miniaturized dystrophin polypeptide BXA-196479 (SEQ ID NO: 125).

Fig. 15. Conduction rates in human DMD hiPSC-CMs infected with AAV9 constructs expressing the miniaturized dystrophin polypeptides BXA-196477 and BXA213788. Fig. 15A shows a photograph of the multielectrode array (MEA) used in the experiments (left) and a photograph of individual MEA electrodes (right). Fig. 15B shows the signal from 28 MEA electrodes under experimental conditions. Fig. 15C shows a graph illustrating the conduction of a pulse through the matrix. Fig. 15D shows schematic drawings of the miniaturized dystrophin polypeptides BXA-196477 (SEQ ID NO: 118) and BXA-213788 (SEQ ID NO: 152). Fig. 15E shows a graph plotting the conduction rate of human DMD hiPSC-CMs expressing the miniaturized dystrophin polypeptides BXA-196477 (SEQ ID NO: 118) and BXA-213788 as a function of time after transfection. Fig. 15F shows a histogram showing the expression of miniaturized dystrophin polypeptides in cells in which conduction velocity was measured.

Fig. 16 shows a diagram summarizing the experimental setup when performing in vivo studies.

Fig. 17. Target interaction of AAV9-BXA-196477 or AAV9-BXA-213788. Fig. 17B shows immunofluorescence imaging of the expression of miniaturized dystrophin and wheat germ agglutinin (WGA) polypeptides in muscle tissue of mice treated with AAV9-BXA-196477 or AAV9-BXA-213788. Fig. 17A presents histograms showing the relative number of cells in various muscles that are positive for miniaturized dystrophin. Diagonal bars = % BXA-196477 cells positive for miniaturized dystrophin as a proportion of WGA-positive cells; Longitudinal bars = % miniaturized dystrophin BXA-213788 as the proportion of WGA-positive cells.

Fig. 18. Target interaction of AAV9-BXA-196477 or AAV9-BXA-213788 determined in 4-week-old mdx ^4cv mice. Fig. 18A shows schematic drawings of miniaturized dystrophin polypeptides BXA-196477 (SEQ ID NO: 118) and BXA-213788 (SEQ ID NO: 152). Fig. 18B is a histogram showing the relative abundance of viral genomes in muscle tissue of mdx ^4cv mice treated with AAV9-BXA-196477 or AAV9-BXA-213788. Fig. 18C

- 7 046419 presents a histogram showing the relative amount of miniaturized dystrophin mRNA in the heart of mdx ^4cv mice treated with AAV9-BXA-196477 or AAV9-BXA-213788. Fig. 18D is a bar graph showing the relative amount of miniaturized dystrophin protein in muscle tissue of mice treated with AAV9-BXA-196477 or AAV9-BXA-213788. Dia = aperture; Gas = gastrocnemius muscle; TA = tibialis anterior muscle. Treated mice were compared with wild type and untreated mdx ^4cv mice.

Fig. 19A is a bar graph showing the amount of creatine kinase in the serum of mdx ^4cv mice (4 weeks old) treated with AAV9-BXA-196477 or AAV9-BXA-213788. Fig. 19B is a bar graph showing the amount of creatine kinase in the serum of mdx ^4cv mice (12 weeks old) treated with AAV9-BXA-196477 or AAV9-BXA-213788. Treated mice were compared with wild type and untreated mdx ^4cv mice.

Fig. 20. Peak tetanic contraction at 2 months of age. Fig. 20A is a graph showing contractile properties (force in Newton meters; Nm) of mdx ^4cv mice treated with AAV9-BXA-196477 or AAV9-BXA-213788. Fig. 20B is a histogram showing the peak of tetanic contraction in treated mice. Fig. 20C is a bar graph showing TA mass in treated mice. Fig. 20D is a bar graph showing the TA strength/mass ratio in treated mice. Treated mice were compared with wild type and untreated mdx ^4cv mice.

Fig. 21. Protection against damage caused by contractions. Fig. 21A shows a graphical representation of the experimental setup. Fig. 21B is a graph showing contractile properties (maximum isometric torque) of muscles in mice treated with AAV9BXA-196477 or AAV9-BXA-213788. Treated mice were compared with wild type and untreated mdx ^4cv mice.

Fig. 22A shows a schematic representation of miniaturized dystrophin polypeptides BXA-027741 (SEQ ID NO: SEQ ID NO: 129). Fig. 22B is a bar graph showing the immunogenicity of connecting peptides 1 to 12.

Fig. 23A is a bar graph showing the MHC I/CD8+ immune response to junctional peptides. Peptide J11v3 here is the same peptide as peptide #3 in FIG. 22V.

Fig. 23B is a bar graph showing the MHC II/CD4+ immune response to junctional peptides. Peptide J11v3 here is the same peptide as peptide #3 in FIG. 22V.

5. Detailed description of the invention.

5.1 Overview

The present description relates to new miniaturized dystrophins or genes encoding them. Miniaturized dystrophins can be functionally linked to the regulatory cassette. The present invention also provides methods for treating a subject suffering from muscular dystrophy, sarcopenia, heart failure or cachexia. In addition, the present invention provides methods for the prophylactic treatment of a subject at risk of developing muscular dystrophy, sarcopenia, heart failure, or cachexia. Methods of treating a subject who has or is at risk for developing muscular dystrophin, sarcopenia, heart failure, or cachexia may involve administering to the subject a pharmaceutical composition comprising the miniaturized dystrophin gene and a delivery vehicle.

5.2. Definitions

For easier understanding of the present invention, definitions of certain terms are first given. Each of the following terms used in this invention, unless otherwise expressly provided herein, has the meaning set forth below. Additional definitions are provided in the appendix.

The term and/or, as used herein, is to be understood as specifically disclosing each of the two specified features or components, with or without the other. Thus, the term and/or, as used herein in a phrase such as A and/or B, is intended to include A and B, A or B, A (only) and B (only). Likewise, the term and/or, when used in a phrase such as A, B and/or C, is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (only); B (only); and C (only).

It is understood that wherever aspects are described herein by the expression comprising, similar aspects described by the terms consisting of and/or consisting essentially of are also provided.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide those skilled in the art with a common dictionary of many of the terms used in this invention.

- 8 046419 ni.

Units, prefixes and symbols are designated in their accepted Systeme International de Unites (SI) form. Numeric ranges include numbers that define a range. The headings presented herein are not intended to limit the various aspects of the invention, which may be derived by reference to the description as a whole.

Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Dystrophin (DMD) is a large, X-linked human gene encoding dystrophin. The dystrophin protein is a cytoskeletal protein with a molecular mass of 427 kDa, which is localized on the cytoplasmic side of the sarcolemma and is enriched in costameres in muscle fibers. The dystrophin protein has four main functional domains: the amino-terminal actin-binding domain (ABD1); a central rod-shaped domain containing a number of rod-shaped regions called spectrin repeat domains and hinge regions; cysteine-rich domain; and a carboxyl-terminal domain.

As used herein, the term miniaturized dystrophin polypeptide or miniaturized dystrophin peptide refers to a polypeptide that is smaller in size than the full-length wild-type dystrophin polypeptide. In some embodiments, the miniaturized dystrophin polypeptide is capable of altering (increasing or decreasing, as appropriate) a measurable measure of muscle physiology and anatomy in a DMD animal model by at least about 10 or 20% of that of the wild type, such that the score approaches that of the wild type (e.g., the mdx mouse has a measurable measure of muscle physiology or anatomy that is 50% of that of the wild type, and this measure increases by at least 60% of that of the wild type; or the mdx mouse has measurable a measure of muscle physiology or anatomy that is 150% of that of the wild type, and this score is reduced to a maximum of 140% of that of the wild type). In some embodiments, the miniaturized dystrophin polypeptide is capable of altering a measurable measure of muscle physiology and anatomy in a DMD animal model by at least about 30% of that of the wild type. In some embodiments, the miniaturized dystrophin polypeptide is capable of altering a measurable measure of muscle physiology or anatomy in a DMD animal model to a level similar to that of the wild type (eg, YYY 4%). As used herein, the term spectrin repeats or spectrin-like repeats refers to peptides of approximately 100 amino acids that are responsible for the rod-like shape of many structural proteins, including, but not limited to, dystrophin, with spectrin repeats typically present in multiple copies . Spectrin repeats can include mutations of natural peptide sequences, such as conservative and/or non-conservative changes in amino acid sequence, as well as additions or deletions of up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids to/from the end of a spectrin repeat or within a spectrin repeat. In some embodiments, each spectrin repeat (each of R1 to R24) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 %, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a natural spectrin repeat (each of natural R1 to R24).

As used herein, the term spectrin repeat encoding sequences refers to nucleic acid sequences encoding spectrin repeat peptides. The term includes natural and synthetic nucleic acid sequences encoding spectrin repeats (eg, both naturally occurring and mutated spectrin repeat peptides).

As used herein, the term spectrin repeat domain refers to a region in a miniaturized dystrophin polypeptide that contains spectrin repeats of a miniaturized dystrophin polypeptide. The term fusion refers to a first amino acid sequence that is linked in frame to a second amino acid sequence to which it is not normally associated in nature, forming a fusion protein/polypeptide. These fusion amino acid sequences, which typically exist as separate proteins, can be combined in a fusion polypeptide, or amino acid sequences that typically exist in the same protein can be arranged in a new arrangement in a fusion polypeptide. The fusion protein is created, for example, by chemical synthesis of peptides or by recombinant DNA technology, by which a polynucleotide is created and then translated, with sections of the peptide being encoded in the desired relationship. The fusion protein may also comprise a second amino acid sequence linked to the first amino acid sequence by a covalent non-peptide bond or non-covalent bond. In some embodiments, the fusion of two polypeptides is achieved through a linker. Linkers can be amino acids or other chemical structures. In some embodiments, the linkers may be synthetic.

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In some embodiments, the fusion between two polypeptides is a direct fusion, that is, without an intermediate linker. The term directly fused or direct fused refers to the connection between two polypeptide chains via a peptide bond. For example, a first amino acid is directly fused to a second amino acid when the first amino acid is fused to a second amino acid via a peptide bond. Heterologous and heterologous fragment, when referring to a polypeptide fragment or polynucleotide fragment that is part of a larger polypeptide or polynucleotide, respectively, describes a polypeptide or polynucleotide that is derived from a polypeptide or polynucleotide different from the rest of the polypeptide or polynucleotide molecule. The additional heterologous component of the polypeptide or polynucleotide may be from the same organism as the remainder of the polypeptide or polynucleotide, respectively, described herein, or the additional components may be from a different organism. For example, a heterologous polypeptide may be synthetic or derived from a different species, a different cell type of an individual, or the same or a different cell type of different individuals. In one aspect, a heterologous fragment is a polypeptide fused to another polypeptide to form a polypeptide. In another aspect, the heterologous fragment is a non-polypeptide, such as a PEG, conjugated to a polypeptide or protein.

As used herein, the term muscle cell refers to a cell derived from muscle tissue, including, but not limited to, cells derived from skeletal muscle, smooth muscle (eg, from the digestive tract, bladder and blood vessels), and cardiac muscle. The term includes muscle cells in vitro, ex vivo and in vivo. Thus, for example, an isolated cardiomyocyte will represent the same muscle cell as the cell as it exists in muscle tissue present in a subject in vivo. The term also includes both terminally differentiated and undifferentiated muscle cells such as myocytes, myotubes, myoblasts, cardiomyocytes and cardiomyoblasts.

As used herein, the term muscle-specific with respect to a gene regulatory element (eg, enhancer sequence, promoter sequence) means that the regulatory element controls transcriptional activity primarily in muscle cells or tissues (eg, 20:1) versus with transcriptional activity controlled by a regulatory element in other tissues. Assays to determine muscle specificity of a regulatory element are known in the art (eg, in vitro assays using murine muscle and liver cells transfected with an expression vector containing the regulatory element of interest driving beta-galactoside reporter expression).

As used herein, the term adeno-associated virus or AAV includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7 , AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, AAV primate, non-primate AAV and ovine AAV, AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78: 6381 (2004)) and Moris et al. (Virol. 33: 375 (2004)) and any other AAV currently known or discovered later. See, for example, Fields et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). AAV belongs to the Dependoparvovirus in the Parvoviridae family of viruses. For example, the AAV may be AAV derived from a naturally occurring wild-type virus, AAV derived from a recombinant AAV (rAAV) genome packaged in a capsid derived from capsid proteins encoded by a naturally occurring cap gene, and/or an rAAV genome packaged in a capsid , derived from capsid proteins encoded by the non-naturally occurring cap gene. As used herein, the term AAV may be used to refer to the virus itself or its derivatives. The term covers all subtypes and both naturally occurring and recombinant forms, unless explicitly stated otherwise. Primate AAV refers to AAV that infects primates, non-primate AAV refers to AAV that infects non-primate mammals, bovine AAV refers to AAVs that infect bovine mammals, etc. See, for example, BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (3rd ed., Lippincott-Raven Publishers).

The term rAAV refers to recombinant AAV. In some embodiments, the recombinant AAV has an AAV genome in which some or all of the rep and cap genes are replaced with heterologous polynucleotide sequences. As used herein, the term AAV vector or adeno-associated virus vector refers to an rAAV containing a polynucleotide sequence not derived from AAV (ie, a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell. In general, a heterologous polynucleotide is flanked by at least one and typically two AAV inverted terminal repeat (ITR) sequences.

Capsidless or capsidless (or variants thereof) viral (e.g., AAV) genome or nucleic acid molecule refers to a genome or nucleic acid molecule free of capsid

- 10 046419 yes. In some embodiments, the capsidless genome or nucleic acid molecule does not contain sequences encoding, for example, the AAV Rep protein.

AAV, or AAV viral particle, or AAV vector, or rAAV vector particle refers to a viral particle consisting of at least one AAV capsid protein (typically all wild-type AAV capsid proteins) and an encapsidated polynucleotide. If the particle contains a heterologous polynucleotide (that is, a polynucleotide different from the wild-type AAV genome, such as a transgene that is to be delivered into a mammalian cell), it is usually called an rAAV vector particle or simply an AAV vector. The term helper virus for AAV refers to the virus that which allows AAV (eg, wild-type AAV) to replicate and be packaged by a mammalian cell. There are many such AAV helper viruses known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia virus. Adenoviruses span a number of different subgroups, but the most widely used adenovirus is type 5 subgroup C. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and are available from repositories such as ATCC. Viruses of the herpesvirus family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

As used herein, the term inverted terminal repeat (ITR) refers to a single-stranded sequence of nucleotides followed by a reverse complementary sequence in the direction of transcription. The intermediate nucleotide sequence between the initial sequence and the reverse complementary sequence can be of any length, including zero. The AAV genome typically contains inverted terminal repeats (ITRs) at both ends, with each end typically being palindromic and capable of forming a hairpin. The terms polynucleotide and nucleic acid are used interchangeably herein and refer to a biopolymer consisting of multiple nucleotide monomers covalently linked into a chain.

As used herein, the term tropism refers to the ability of a virus (e.g., AAV) to infect only one or a few specific cell types and its ability to interact only with specific cell surface fragments to achieve cell entry, optionally and preferably followed by expression (e.g., transcription and , if necessary, translation) of sequences carried by a virus (eg, AAV) into the cell (eg, for a recombinant virus, expression of heterologous nucleotide sequence(s)).

As used herein, the term transduction refers to the entry of a virus (eg, AAV) into a cell and the transfer of genetic material contained within the virus into the cell to obtain expression from the viral genome. Typically, a virus (eg AAV) enters cells according to its tropism.

Administration refers to the physical administration of a therapeutic agent to a subject using any of the various delivery methods and systems known to those skilled in the art. Examples of routes of administration, for example, for AAV therapy include intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal , oral, rectal, topical, epidermal, transmucosal, intranasal, vaginal, rectal and sublingual administration. Administration may also be performed, for example, once, multiple times and/or over one or more extended periods.

The term treatment or therapy of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, ameliorating, attenuating, inhibiting, delaying or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical parameters associated with the disease. A therapeutically effective amount, therapeutic dose, effective dose or effective dosage as used herein means an amount or dose that achieves a therapeutic goal as described herein. One skilled in the art will also appreciate that a therapeutically effective amount, etc. may be administered as a single dose or may be achieved by administering multiple doses (ie, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses). The ability of a therapeutic agent to promote disease regression or inhibit the development or relapse of disease can be assessed using various methods known to the skilled practitioner, for example, in humans during clinical trials, in animal model systems that predict the effectiveness of treatment in patients, or by analysis activity of the drug in in vitro assays.

Subject includes any person or non-human animal. The term does not refer

- 11 046419 human-related animals include, but are not limited to, vertebrates such as non-human primates, sheep, dogs and rodents such as mice, rats and guinea pigs. In some embodiments, the subject is a human. The terms subject and patient are used interchangeably throughout this document.

As used herein, the terms μg and μM are used interchangeably with μί and μM, respectively.

The use of an alternative (for example, or) should be understood to mean one, both, or any combination of the alternatives. When used herein, the indefinite articles a or an are to be understood as referring to one or more of any listed or renumbered component or entity.

About or About: As used herein, the term about or about when applied to one or more values of interest refers to a value that is similar to the stated reference value and is within the range of values that fall within the limits of 25, 20, 19, 18, 17, 16. 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less in any direction (more than or less than) of the stated reference value, unless otherwise stated or otherwise not obvious from the context (unless such amount exceeds 100% of the possible value). When the term about or about is applied herein to a specific meaning, a meaning without the term about or about is also disclosed herein.

As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the specified range and, as appropriate, fractions thereof (e.g., one tenth and one hundredth of an integer), unless otherwise stated. Various aspects of the invention are described in more detail in the following subsections.

5.3. Polynucleotides and polypeptides.

5.3.1. Miniaturized dystrophin

The present invention relates to a nucleic acid molecule containing a nucleotide sequence that encodes a miniaturized dystrophin polypeptide. In some embodiments, the miniaturized dystrophin polypeptide comprises at least three dystrophin hinge domains and at least five spectrin repeat domains. Dystrophin is a rod-shaped cytoplasmic protein that connects the muscle fiber cytoskeleton to the surrounding extracellular matrix through the cell membrane. This protein is found mainly in the muscles used for movement (skeletal muscle) and in the heart (cardio) muscle. Nerve cells in the brain contain small amounts of dystrophin. In skeletal and cardiac muscle, dystrophin is part of a group of proteins (protein complex) that work together to strengthen muscle fibers and protect them from injury during muscle contraction and relaxation. The dystrophin complex acts as an anchor, connecting the structural framework of each muscle cell (cytoskeleton) to the network of proteins and other molecules outside the cell (extracellular matrix). The dystrophin complex may also play a role in cell signaling by interacting with proteins that send and receive chemical signals.

The DMD gene, encoding the full-length dystrophin protein, is one of the longest known human genes, spanning 2.3 million base pairs (0.08% of the human genome) at the Xp21 locus. The primary transcript in muscle is approximately 2100 kb in size and takes 16 h to transcribe; mature mRNA is 14.0 kb in size. The muscle transcript of 79 exons encodes a protein of 3685 amino acid residues.

The present description discloses the amino acid and nucleotide sequences for dystrophin. The amino acid sequence constituting wild-type human dystrophin, isoform Dp427m, is known as UniProt identifier No. NP_003997.1 and is shown in Table. 1.

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Table 1

Amino acid sequence of full-length dystrophin protein (NP 003997.1) SEQ ID NO: 1

MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDL LEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLT LGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTS WSDGLALNALIHSHRPDLFDWNS VVCQQSATQRLEHAFNIARYQLGIEKLLDPED VDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMH YSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSF

IGS SLMESEVNLDRYQTALEEVLSWLLS AEDTLQ AQGEISNDVEVVKDQFHTHEGY MMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASM EKQSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQ HKVLQEDLEQEQVRVNSLTH MVVVVDESSGDHATAALEEQLKVLGDRWANICR WTEDRWVLLQDILLKWQRLTEEQCLF S AWLSEKED A VNKIHTTGFKDQNEMLS S LQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKTEAWLDNFARCWD NLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVTTVTTREQILVKHAQ EELPPPPP QKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQSPEFAIFRKEGNFSDLKEKVNA IEREKAEKFRKLQDASRSAQALVEQMVNEGVNADSIKQASEQLNSRWIEFCQLLS ERLNWLEYQNNIIAFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKICKDE VNRLSGLQPQIERL KIQSIALKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREKEL QTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSL QEQQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEE QMNKLRKIQNHIQTLKKWMAEVDVFLKEEWPAL GDSEILKKQLKQCRLLVSDIQ TIQPSLNSVNEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQQVYARKEALKG GLEKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKRAKEEA QQKEAKVKLLTESVNSVIAQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLE EVW ACWHELLSYLEKANKWLNEVEFKLKTTENIPGGAEEISEVLDSLENLMRHSE DNPNQIRILAQTLTDGGVMDELINEELETFNSRWRELHEEAVRRQKLLEQSIQSAQ ETEI<SLHLIQESLTFIDI<QLAAYIADI<VDAAQMPQEAQI<IQSDLTSHEISLEEMI<I< HNQGKEAAQRVLSQIDVAQKKL QDVSMKFRLFQKPANFEQRLQESKMILDEVKM HLPALETKSVEQEVVQSQLNHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTENP KELDERVTALKLHYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATD MELTKRSAVEGMPSNLDSEVAWGKATQKEIEKQKVHLKSITEVGEALKTVLGKK ET LVEDKLSLLNSNWIAVTSRAEEWLNLLLEYQKHMETFDQNVDHITKWIIQADT

- 13 046419

LLDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRDQAANLMANRGDHCRKLVE PQISELNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEIQQGVNLKEEDF NKDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNALKDLRSQ RRKKALEISHQWYQYK RQADDLLKCLDDIEKKLASLPEPRDERKIKEIDRELQKK KEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIESKFAQFRRLNFAQIHTV REETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLF KQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQW EKV NKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYK WYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCK QLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQV KLLVEELPLRQ GILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRAL PEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEGPFDV QETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQEL RAKQPDLAPGLTTIGASPTQTVTLVTQPV VTKETAISKLEMPSSLMLEVPALADFN RAWTELTDWLSLLDQVIKSQRVMVGDLEDINEMIIKQKATMQDLEQRRPQLEELI TAAQNLKNKTSNQEARTIITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWL EAKEEAEQVLGQARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQT NVDV ANDLALKLLRDYSADDTRKVHMITENINASWRSIHKRVSEREAALEETHRLLQQF PLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGEIEAH TDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEAS SDQWKRLHLSLQELLVWLQLKDDELSRQAPIGG DFPAVQKQNDVHRAFKRELKT KEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTE WEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLID SLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRW KLLQV AVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQ TTCWDHPKMTELYQ SLADLNN VRF S AYRTAMKLRRLQKALCLDLLSLS AACD AL DQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYD TGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCD QRRLGLLLHDSIQI PRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLH RVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHY PMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMET PVTLINFWPVDSAPASSPQLS HDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESI DDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAE YDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQI LEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLR VVG SQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM

Various other dystrophin isoforms resulting from alternative splicing are known in the art. In some embodiments, the constructs contain the nucleotide sequences presented in table. 2.

In addition, this document discloses the nucleotide sequence encoding the full-length dystrophin protein.

- 14 046419

table 2

Nucleotide sequence of full-length dystrophin protein (isoform Dp427m) SEQ ID NO: 2 GGGATTCCCTCACTTTCCCCCTACAGGACTCAGATCTGGGAGGCAATTACCTTC GGAGAAAAACGAATAGGAAAAACTGAAGTGTTACTTTTTTTAAAGCTGCTGAA GTTTGTTGGTTTCTCATTGTTTTTAAGCCTACTGGAGCAATAAAGTTTGAAGAA CTTTTACCAGGTTTTTTTTAT CGCTGCCTTGATATACACTTTTCAAAATGCTTTG GTGGGAAGAAGTAGAGGACTGTTATGAAAGAGAAGATGTTCAAAAGAAAACA TTCACAAAATGGGTAAATGCACAATTTTCTAAGTTTGGGAAGCAGCATATTGA GAACCTCTTCAGTGACCTACAGGATGGGAGGCGCCTCCTAGACCTCCTCGAAG GCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCACAAGGTTCATGC CCTGAAC AATGTCAACAAGGCACTGCGGGTTTTGCAGAACAATAATGTTGATT TAGTGAATATTGGAAGTACTGACATCGTAGATGGAAATCATAAACTGACTCTT GGTTTGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTAATGAAAAA TATCATGGCTGGATTGCAACAAACCAACAGTGAAAAGATTCTCCTGAGCTGGG TCCGACAATCAACTCGTAATTATCCACAGGTTAATGTAATCAACT TCACCACC AGCTGGTCTGATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCCAGA CCTATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAGCCACACAACGACTGG AACATGCATTCAACATCGCCAGATATCAATTAGGCATAGAGAAACTACTCGAT CCTGAAGATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTACAT CACATCACTCTTCCAAGTTTTGCCTCAACAAG TG AGCATTGAAGCCATCCAGGAAGT GGAAATGTTGCCAAGGCCACCTAAAGTGACTAAAGAAGAACATTTTCAGTTAC ATCATCA AATGCACTATTCTCAACAGATCACGGTCAGTCTAGCACAGGGATATGAGAGAA CTTCTTC CCCTAAGCCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGTCACCA CCTCTGA CCCTACACGGAGCCCATTTCCTTCACAGCATTTGGAAGCTCCTG AAGACAAGT CATTTGG CAGTTCATTGATGGAGAGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAG AAGAAGT ATTATCGTGGCTTCTTTCTGCTGAGGACACATTGCAAGCACAAGGAGAGATTT CTAATGA TGTGGAAGTGGTGAAAGACCAGTTTCATACTCATGAGGGGTACATGATGGATT TGACAGC CCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTAAGCT GATTGGAA CAGGAAA ATTATCAGAAGATGAAGAAACTGAAGTACAAGAGCAGATGAATCTCCTAAAT TCAAGATG GGAATGCCTCAGGGTAGCTAGCATGGAAAAACAAAGCAATTTACATAGAGTTT TAATGGA TCTCCAGAATCAGAAACTGAAAGAGTTGAATGACTGGCTAACAAAAACAGAA GAAAGAAC AAGGAAAATGGAGGAAGAGCCTCTTGGACCTGATCTTGAAGACCTAAAACGC CAAGTACA ACAACATAAGGTGCTTCAAGAAGATCTAGAACAAGAACAAGTCAGGGTCAAT TC TCTCAC TCACATGGTGGTGGTAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTT TGGAAGA ACAACTTAAGGTATTGGGAGATCGATGGGCAAACATCTGTAGATGGACAGAA GACCGCTG

- 15 046419

GGTTCTTTTACAAGACATCCTTCTCAAATGGCAACGTCTTACTGAAGAACAGT GCCTTTT

TAGTGCATGGCTTTCAGAAAAGAAGATGCAGTGAACAAGATTCACACAACT GGCTTTAA

AGATCAAAATGAAATGTTATCAAGTCTTCAAAAACTGGCCGTTTTAAAAGCGG ATCTAGA

AAAGAAAAAGCAATCCATGGGCAAACTGTATTCACTCAAACAAGATCTTTCTTT CAACACT

GAAGAATAAGTCAGTGACCCAGAAGACGGAAGCATGGCTGGATAACTTTGCC CGGTGTTG

GGATAATTTAGTCCAAAAACTTGAAAAGAGTACAGCACAGATTTCACAGGCTG TCACCAC

CACTCAGCCATCACTAACACAGACAACTGTAATGGAAACAGTAACTACGGTG ACCACAAG

GGAACAGATCCTGGTAAAGCATGCTCAAGAGGAACTTCCACCACCACCTCCCCC AAAAGAA

GAGGCAGATTACTGTGGATTCTGAAATTAGGAAAAGGTTGGATGTTGATATAA CTGAACT

TCACAGCTGGATTACTCGCTCAGAAGCTGTGTTGCAGAGTCCTGAATTTGCAA

TCTTTCG

GAAGGAAGGCAACTTCTCAGACTTAAAAGAAAAAGTCAATGCCATAGAGCGA GAAAAAGC

TGAGAAGTTCAGAAAACTGCAAGATGCCCAGCAGATCAGCTCAGGCCCTGGTG

GAACAGAT

GGTGAATGAGGGTGTTAATGCAGATAGCATCAAACAAGCCTCAGAACAACTG AACAGCCG

GTGGATCGAATTCTGCCAGTTGCTAAGTGAGAGACTTAACTGGCTGGAGTATC AGAACAA

CATCATCGCTTTCTATAATCAGCTACAACAATTGGAGCAGATGACAACTACTG

CTGAAAA

CTGGTTGAAAATCCAACCCACCACCCCATCAGAGCCAACAGCAATTAAAAGTC AGTTAAA

AATTTGTAAGGATGAAGTCAACCGGCTATCAGGTCTTCAACCTCAAATTGAAC GATT AAA

AATTCAAAGCATAGCCCTGAAAGAGAAAGGACAAGGACCCATGTTCCTGGAT GCAGACTT

TGTGGCCTTTACAAATCATTTTAAGCAAGTCTTTTCTGATGTGCAGGCCAGAGA GAGAAAGA

GCTACAGACAATTTTTGACACTTTGCCACCAATGCGCTATCAGGAGACCATGA

GTGCCAT

CAGGACATGGGTCCAGCAGTCAGAAACCAAACTCTCCATACCTCAACTTAGTG TCACCGA

CTATGAAATCATGGAGCAGAGACTCGGGGAATTGCAGGCTTTACAAAGTTCTC TGCAAGA

GCAACAAAGTGGCCTATACTATCTCAGCACCACTGTGAAAGAGATGTCGAAGA AAGCGCC

CTCTGAAATTAGCCGGAAATATCAATCAGAATTTGAAGAAATTGAGGGACGCT GGAAGAA

GCTCTCCTCCCAGCTGGTTGAGCATTGTCAAAAGCTAGAGGAGCAAATGAATA AACTCCG

- 16 046419

AAAAATTCAGAATCACATACAAACCCTGAAGAAATGGATGGCTGAAGTTGAT GTTTTTCT

GAAGGAGGAATGGCCTGCCCTTGGGGATTCAGAAATTCTAAAAAAGCAGCTG AAACAGTG

CAGACTTTTAGTCAGTGATATTCAGACAATTCAGCCCAGTCTAAACAGTGTCA ATGAAGG

TGGGCAGAAGATAAAGAATGAAGCAGAGCCAGAGTTTGCTTCGAGACTTGAG ACAGAACT

CAAAGAACTTAACACTCAGTGGGATCACATGTGCCAACAGGTCTATGCCAGAA AGGAGGC

CTTGAAGGGAGGTTTGGAGAAAACTGTAAGCCTCCAGAAAGATCTATCAGAG ATGCACGA

ATGGATGACACAAGCTGAAGAAGAGTATCTTGAGAGAGATTTTGAATATAAA ACTCCAGA

TGAATTACAGAAAGCAGTTGAAGAGATGAAGAGAGCTAAAGAAGAGGCCCAA CAAAAAGA

AGCGAAAGTGAAACTCCTTACTGAGTCTGTAAATAGTGTCATAGCTCAAGCTC CACCTGT

AGCACAAGAGCCTTAAAAAAAGGAACTTGAAACTCTAACCACCAACTACCA GTGGCTCTG

CACTAGGCTGAATGGGAAATGCAAGACTTTGGAAGAAGTTTGGGCATGTTGGC ATGAGTT

ATTGTCATACTTGGAGAAAGCAAACAAGTGGCTAAATGAAGTAGAATTTAAAC TTAAAAC

CACTGAAAACATTCCTGGCGGAGCTGAGGAAATCTCTGAGGTGCTAGATTCAC TTGAAAA

TTTGATGCGACATTCAGAGGATAACCCAAATCAGATTCGCATATTGGCACAGA CCCTAAC

AGATGGCGGAGTCATGGATGAGCTAATCAATGAGGAACTTGAGACATTTAATT CTCGTTG

GAGGGAACTACATGAAGAGGCTGTAAGGAGGCAAAAGTTGCTTGAACAGAGC ATCCAGTC

TGCCCAGGAGACTGAAAAATCCTTACACTTAATCCAGGAGTCCCCTCACATTCA TTGACAA

GCAGTTGGCAGCTTATATTGCAGACAAGGTGGACGCAGCTCAAATGCCTCAGG AAGCCCA

GAAAATCCAATCTGATTTGACAAGTCATGAGATCAGTTTAGAAGAAATGAAGA AACATAA

TCAGGGGAAGGAGGCTGCCCAAAGAGTCCTGTCTCAGATTGATGTTGCACAGA AAAAAATT

ACAAGATGTCTCCATGAAGTTTCGATTATTCCAGAAACCAGCCAATTTTGAGC TGCGTCT

ACAAGAAAGTAAGATGATTTTAGATGAAGTGAAGATGCACTTGCCTGCATTGG AAACAAA

GAGTGTGGAACAGGAAGTAGTACAGTCACAGCTAAATCATTGTGTGAACTTGT ATAAAAG

TCTGAGTGAAGTGAAGTCTGAAGTGGAAATGGTGATAAAGACTGGACGTCAG ATTGTACA

GAAAAAGCAGACGGAAAATCCCAAAGAACTTGATGAAAGAGTAACAGCTTTG AAATTGCA

- 17 046419

TTATAATGAGCTGGGAGCAAAGGTAACAGAAAGAAAGCAACAGTTGGAGAAA TGCTTGAA

ATTGTCCCGTAAGATGCGAAAGGAAATGAATGTCTTGACAGAATGGCTGGCAG CTACAGA

TATGGAATTGACAAAGAGATCAGCAGTTGAAGGAATGCCTAGTAATTTGGATT CTGAAGT

TGCCTGGGGAAAGGCTACTCAAAAAGAGATTGAGAAACAGAAGGTGCACCTG AAGAGTAT

CACAGAGGTAGGAGAGGCCTTGAAAACAGTTTTGGGCAAGAAGGAGACGTTG GTGGAAGA

TAAACTCAGTCTTCTGAATAGTAACTGGATAGCTGTCACCTCCCGAGCAGAAG AGTGGTT

AAATCTTTTGTTGGAATACCAGAAACACATGGAAACTTTTGACCAGAATGTGG ACCACAT

CACAAAGTGGATCATTCAGGCTGACACACTTTTGGATGAATCAGAGAAAAAG AAACCCCA

GCAAAAAGAAGACGTGCTTAAGCGTTTAAAAGGCAGAACTGAATGACATACGC CCAAAGGT

GGACTCTACACGTGACCAAGCAGCAAACTTGATGGCAAACCGCGGTGACCAC TGCAGGAA

ATTAGTAGAGCCCCAAATCTCAGAGCTCAACCATCGATTTGCAGCCATTTCAC ACAGAAT

TAAGACTGGAAAGGCCTCCATTCCTTTGAAGGAATTGGAGCAGTTTAACTCAG ATATACA

AAAATTGCTTGAACCACTGGAGGCTGAAATTCAGCAGGGGGTGAATCTGAAA GAGGAAGA

CTTCAATAAAGATATGAATGAAGACAATGAGGGTACTGTAAAAGAATTGTTGC AAAGAGG

AGACAACTTACAACAAAGAATCACAGATGAGAGAAAGAGAGAGGAAATAAA GATAAAACA

GCAGCTGTTACAGACAAAACATAATGCTCTCAAGGATTTGAGGTCTCAAAGAA GAAAAAA

GGCTCTAGAAATTTCTCATCAGTGGTATCAGTACAAGAGGCAGGCTGATGATC TCCTGAA

ATGCTTGGATGACATTGAAAAAAAATTAGCCAGCCTACCTGAGCCCAGAGATG AAAGGAA

AATAAAGGAAATTGATCGGGAATTGCAGAAGAAGAAAGAGGAGCTGAATGCA GTGCGTAG

GCAAGCTGAGGGCTTGTCTGAGGATGGGGCCGCAATGGCAGTGGAGCCAACT CAGATCCA

GCTCAGCAAGCGCTGGCGGGAAATTGAGAGCAAATTTGCTCAGTTTCGAAGAC TCAACTT

TGCACAAATTCACACTGTCCGTGAAGAAACGATGATGGTGATGACTGAAGACA TGCCTTT

GGAAATTTCTTATGTGCCTTCTACTTATTTGACTGAAATCACTCATGTCTCACA AGCCCT

ATTAGAAGTGGAACAACTTCTCAATGCTCCTGACCTCTGTGCTAAGGACTTTG AAGATCT

CTTTAAGCAAGAGGAGTCTCTGAAGAATATAAAAGATAGTCTACAACAAAGCT CAGGTCG

- 18 046419

GATTGACATTATTCATAGCAAGAAGACAGCAGCATTGCAAAGTGCAACGCCTG TGGAAAG

GGTGAAGCTACAGGAAGCTCTCTCCCAGCTTGATTTCCAATGGGAAAAAGTTA ACAAAAT

GTACAAGGACCGACAAGGGCGATTTGACAGATCTGTTGAGAAATGGCGGCGT TTTCATTA

TGATATAAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAA AGACACA

AATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAGGAACTCC AGGATGG

CATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAA ATAATTCA

GCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTG AATCTGCG

GTGGCAGGAGGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAGGCTAGAAGAA CAAAAGAA

TATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGA AGCAGA

TAACATTGCTAGTATCCCACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAA AAGCTTGA

GCAAGTCAAGTTACTGGTGGAAGAGTTGCCCCTGCGCCAGGGAATTCTCAAAC AATTAAA

TGAAACTGGAGGACCCGTGCTTGTAAGTGCTCCCATAAGCCCAGAAGAGCAA GATAAACT

TGAAAATAAGCTCAAGCAGACAAATCTCCAGTGGATAAAGGTTTCCAGAGCTT TACCTGA

GAAACAAGGAGAAATTGAAGCTCAAATAAAAGACCTTGGGCAGCTTGAAAAA AAGCTTGA

AGACCTTGAAGAGCAGTTAAATCATCTGCTGCTGTGGTTATCTCCTATTAGGA ATCAGTT

GGAAATTTATAACCAACCAAACCAAGAAGGACCATTTGACGTTCAGGAAACT GAAATAGC

AGTTCAAGCTAAACAACCGGATGTGGAAGAGATTTTGTCTAAAGGGCAGCATT TGTACAA

GGAAAAACCAGCCACTCAGCCAGTGAAGAGGAAGTTAGAAGATCTGAGCTCT GAGTGGAA

GGCGGTAAACCGTTTACTTCAAGAGCTGAGGGCAAAGCAGCCTGACCTAGCTC CTGGACT

GACCACTATTGGAGCCTCTCCTACTCAGACTGTTACTCTGGTGACACAACCTGT GGTTAC

TAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGG TACCTGC

TTCTGGCAGATTTCAACCGGGCTTGGACAGAACTTACCGACTGGCTTTCTCTGCT TGATCA

AGTTATAAAATCACAGAGGGTGATGGTGGGTGACCTTGAGGATATCAACGAG ATGATCAT

CAAGCAGAAGGCAACAATGCAGGATTTGGAACAGAGGCGTCCCCAGTTGGAA GAACTCAT

TACCGCTGCCCAAAATTTGAAAAACAAGACCAGCAATCAAGAGGCTAGAACA ATCATTAC

- 19 046419

GGATCGAATTGAAAGAATTCAGAATCAGTGGGATGAAGTACAAGAACACCTT CAGAACCG

GAGGCAACAGTTGAATGAAATGTTAAAGGATTCAACACAATGGCTGGAAGCT AAGGAAGA

AGCTGAGCAGGTCTTAGGACAGGCCAGAGCCAAGCTTGAGTCATGGAAGGAG GGTCCCTA

TACAGTAGATGCAATCCAAAAGAAAATCACAGAAACCAAGCAGTTGGCCAAA GACCTCCG

CCAGTGGCAGACAAATGTAGATGTGGCAAATGACTTGGCCCTGAAACTTCTCC GGGATTA

TTCTGCAGATGATACCAGAAAAGTCCACATGATAACAGAGAATATCAATGCCT CTTGGAG

AAGCATTCATAAAAGGGTGAGTGAGCGAGAGGCTGCTTTGGAAGAAACTCAT AGATTACT

GCAACAGTTCCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTG AAACAAC

TGCCAATGTCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCA AGGGAGT

AAAAGAGCTGATGAAACAATGGCAAGACCTCCAAGGTGAAATTGAAGCTCAC ACAGATGT

TTATCACAACCTGGATGAAAACAGCCAAAAAATCCTGAGATCCCTGGAAGGTT CCGATGA

TGCAGTCCTGTTACAAAGACGTTTGGATAACATGAACTTCAAGTGGAGTGAAC TTCGGAA

AAAGTCTCTCAACATTAGGTCCCATTTGGAAGCCAGTTCTGACCAGTGGAAGC GTCTGCA

CCTTTCTCTGCAGGAACTTCTGGTGTGGCTACAGCTGAAAGATGATGAATTAA GCCGGCA

GGCACCTATGGAGGCGACTTTCCAGCAGTTCAGAAGCAGAACGATGTACATA GGGCCTT

CAAGAGGGAATTGAAAACTAAAGAACCTGTAATCATGAGTACTCTTGAGACTG TACGAAT

ATTTCTGACAGAGCAGCCTTTGGAAGGACTAGAGAAACTCTACCAGGAGCCCA GAGAGCT

GCCTCCTGAGGAGAGAGCCCAGAATGTCACTCGGCTTCTACGAAAGCAGGCTG AGGAGGT

CAATACTGAGTGGGAAAAATTGAACCTGCACTCCGCTGACTGGCAGAGAAAA ATAGATGA

GACCCTTGAAAGACTCCAGGAACTTCAAGAGGCCACGGATGAGCTGGACCTC AAGCTGCG

CCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCGTGGGCGATCTCCTCATTG ACTCTCT

CCAAGATCACCTCGAGAAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTG AAAGAGAA

CGTGAGCCACGTCAATGACCTTGCTCGCCAGCTTACCACTTTGGGCATTCAGCT CTCACC

GTATAACCTCAGCACTCTGGAAGACCTGAACACCAGATGGAAGCTTCTGCAGG TGGCCGT

CGAGGACCGAGTCAGGCAGCTGCATGAAGCCCACAGGGACTTTGGTCCAGCA TCTCAGCA

- 20 046419

CTTTCTTTCCACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAAACA AAGTGCC

CTACTATATCAACCACGAGACTCAAACAACTTGCTGGGACCATCCCAAAAATGA CAGAGCT

CTACCAGTCTTTAGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTGC CATGAA

ACTCCGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTTGAGCCTGTCAGCTG CATGTGA

TGCCTTGGACCAGCACAACCTCAAGCAAAATGACCAGCCCATGGATATCCTGC AGATTAT

TAATTGTTTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAATTTGG TCAACGT

CCCTCTCTGCGTGGATATGTGTCTGAACTGGCTGCTGAATGTTTATGATACGGG ACGAAC

AGGGAGGATCCGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAAAGC ACATTT

GGAAAGACAAGTACAGATACCTTTTCAAGCAAGTGGCAAGTTCAACAGGATTTT GTGACCA

GCGCAGGCTGGGCCTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGG GTGAAGT

TGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTGTCCGGAGCTGCTTCCAAT

TTGCTAA

TAATAAGCCAGAGATCGAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAAC CCCAGTC

CATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTGCAGAAACTGCCAAGC ATCAGGC

CAAATGTAACATCTGCAAAGAGTGTCCAATCATTGGATTCAGGTACAGGAGTC TAAAGCA

CTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTCGAGTTGCAAAAGG CCATAA

AATGCACTATCCCATGGTGGAATATTGCACTCCGACTACATCAGGAGAAGATG TTCGAGA

CTTTGCCAAGGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGAAGC ATCCCCG

AATGGGCTACCTGCCAGTGCAGACTGTCTTAGAGGGGGACAACATGGAAACTC CCGTTAC

TCTGATCAACTTCTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTTC ACACGA

TGATACTCATTCACGCATTGAACATTATGCTAGCAGGCTAGCAGAAATGGAAA ACAGCAA

TGGATCTTATCTAAATGATAGCATCTCTCCTAATGAGAGCATAGATGATGAAC ATTTGTT

AATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCCCCCCTGAGCCAGCCTC GTAGTCC

TGCCCAGATCTTGATTTCCTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGA ATCCTAGC

AGATCTTGAGGAAGAAAACAGGAATCTGCAAGCAGAATATGACCGTCTAAAG CAGCAGCA

CGAACATAAAGGCCTGTCCCCACTGCCGTCCCCTCCTGAAATGATGCCCACCT CTCCCCA

- 21 046419

GAGTCCCCGGGATGCTGAGCTCATTGCTGAGGCCAAGCTACTGCGTCAACACA AAGGCCG

CCTGGAAGCCAGGATGCAAATCCTGGAAGACCACAATAAACAGCTGGAGTCA CAGTTACA

CAGGCTAAGGCAGCTGCTGGAGCAACCCCAGGCAGAGGCCAAAGTGAATGGC ACAACGGT

GTCCTCTCCTTCTACCTCTCTACAGAGGTCCGACAGCAGTCAGCCTATGCTGCT CCGAGT

GGTTGGCAGTCAAACTTCGGACTCCATGGGTGAGGAAGATCTTCTCAGTCCTC CCCAGGA

CACAAGCACAGGGTTAGAGGAGGTGATGGAGCAACTCAACAACTCCTTCCCT AGTTCAAG

AGGAAGAAATACCCCTGGAAAGCCAATGAGAGAGGACACAATGTAGGAAGTC TTTTCCAC

ATGGCAGATGATTTGGGCAGAGCGATGGAGTCCTTAGTATCAGTCATGACAGA TGAAGAA

GGAGCAGAATAAATGTTTTACAACTCCTGATTCCCGCATGGTTTTTATAATATT CATACA

ACAAAGAGGATTAGACAGTAAGAGTTTACAAGAAATAAATCTATTTTTGTG AAGGGTA

GTGGTATTATACTGTAGATTTCAGTAGTTTCTAAGTCTGTTATTGTTTTGTTAAC AATGG

CAGGTTTTACACGTCTATGCAATTGTACAAAAAAAGTTATAAGAAAACTACAT GTAAAATC

TTGATAGCTAAATAACTTGCCATTTCTTTATATGGAACGCATTTTGGGTTGTTT AAAAAT

TTATAACAGTTATAAAGAAAGATTGTAAACTAAAGTGTGCTTTATAAAAAAAA GTTGTTT

ATAAAAACCCCTAAAAACAAAACAAACACACACACACACATACACACACA CACACAAA

ACTTTGAGGCAGCGCATTGTTTTGCATCCTTTTGGCGTGATATCCATATGAAAT TCATGG

CTTTTTCTTTTTTTGCATATTAAAGATAAGACTTCCTCTACCACCACACCAAAT GACTAC

TACACACTGCTCATTTGAGAACTGTCAGCTGAGTGGGGCAGGCTTGAGTTTTC ATTTCAT

ATATCTATATGTCTATAAGTATATAAATACTATAGTTATATAGATAAAGAGAT ACGAATT

TCTATAGACTGACTTTTTCCATTTTTTTAAATGTTCATGTCACATCCTAATAGAA AGAAAT

TACTTCTAGTCAGTCATCCAGGCTTACCTGCTTGGTCTAGAATGGATTTTTCCC GGAGCC

GGAAGCCAGGAGGAAACTACACCACACTAAAACATTGTCTACAGCTCCAGAT GTTTCTCA

TTTTAAACAACTTTCCACTGACAACGAAAGTAAAGTAAAGTATTGGATTTTTTT AAAGGG

AACATGTGAATGAATACACAGGACTTATTATATCAGAGTGAGTAATCGGTTGG TTGGTTG

ATTGATTGATTGATTGATACATTCAGCTTCCTGCTGCTAGCAATGCCACGATTT AGATTT

- 22 046419

AATGATGCTTCAGTGGAAATCAATCAGAAGGTATTCTGACCTTGTGAACATCA GAAGGTA

TTTTTTAACTCCCAAGCAGTAGCAGGACGATGATAGGGCTGGAGGGCTATGGA TTCCCAG

CCCATCCCTGTGAAGGAGTAGGCCACTCTTTAAGTGAAGGATTGGATGATTGT TCATAAT

ACATAAAGTTCTCTGTAATTACAACTAAATTATTATGCCCTCTTCTCACAGTCA AAAGGA

ACTGGGTGGTTTGGTTTTGTTGCTTTTTTAGATTTATTGTCCCATGTGGGATGA GTTTT

TAAATGCCACAAGACATAATTTAAAATAAATAAACTTTGGGAAAAGGTGTAA GACAGTAG

CCCACATTTGTGATACTGACAGGTATCAACCCAGAAGCCCATGAACTGTGTTT CCA

TCCTTTGCATTTCTCTGCGAGTAGTTCCACACAGGTTTGTAAGTAAGTAAGAAA GAAGGC

AAATTGATTCAAATGTTACAAAAAAACCCTTCTTTGGTGGATTAGACAGGTTA AATATATA

AACAAACAAACAAAATTGCTCAAAAAAGAGGAGAAAAGCTCAAGAGGAAA AGCTAAGGA

CTGGTAGGAAAAGCTTTACTCTTTCATGCCATTTTATTTCTTTTTGATTTTTAA ATCAT

TCATTCAATAGATACCACCGTGTGACCTATAATTTTGCAAATCTGTTACCTCTG ACATCA

AGTGTAATTAGCTTTTGGAGAGTGGGCTGACATCAAGTGTAATTAGCTTTTGG AGAGTGG

GTTTTGTCCATTATTAATAATTAATTAATTAACATCAAACACGGCTTCTCATGC TATTTC

TACCTCACTTTGGTTTTGGGGTGTTCCTGATAATTGTGCACACCTGAGTTCACA

GCTTCA

CCACTTGTCCATTGCGTTATTTTCTTTTTCCTTTATAATTCTTTCTTTTTCCTTCA

TAAT

TTTCAAAAGAAAACCCAAAGCTCTAAGGTAACAAATTACCAAATTTACATGAAG

ATTTGGT

TTTTGTCTTGCATTTTTTTCCTTTATGTGACGCTGGACCTTTTCTTTACCCAAGG

ATTTT

TAAAACTCAGATTTAAAACAAGGGTTACTTTACATCCTACTAAGAAGTTTAA GTAAGTA

AGTTTCATTCTAAAATCAGAGGTAAATAGAGTGCATAAATAATTTTGTTTTAAT

CTTTTT

GTTTTTCTTTTAGACACATTAGCTCTGGAGTGAGTCTGTCATAATATTTGAACA

AAAATT

GAGAGCTTTATTGCTGCATTTTAAGCATAATTAATTTGGACATTATTTCGTGTT GTGTTC

TTTATAACCACCGAGTATTAAACTGTAAATCATAATGTAACTGAAGCATAAAC ATCACAT

GGCATGTTTTGTCATTGTTTTCAGGTACTGAGTTCTTACTTGAGTATCATAATA TATTGT

GTTTT AACACCAACACTGTAACATTTACGAATTATTTTTTTTAAACTTCAGTTTT

ACTGCA

TTTTCACAACATATCAGACTTCACCAAATATATGCCTTACTATTGTATTATAGT ACTGCT

TTACTGTGTATCTCAATAAAGCACGCAGTTATGTTAC_________________

The full-length wild-type dystrophin protein (isoform Dp427m) contains 24 spectrin-like repeats, at least four hinge regions, an actin-binding domain (ABD1), a cysteine-rich domain (CR), and a C-terminal domain (C-conc). The polypeptide sequence of each domain is shown in Table. 3 and the nucleotide sequence of each domain is shown in Table. 4.

- 23 046419

Table 3

Amino acid sequences of dystrophin domains

Description and sequence ID Subsequence ABD I (SEQ ID NO: 3) MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTR VHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHW QVKNVMKNIMAGLQQTNSEKIL LSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVC QQSATQRLEHAFNIARYQLGIE KLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVE Hinge 1 (SEQ ID NO: 4) MLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAY TQAAYVTTSDPTRSPFPSQHL EAPEDKSFGSSLMES Spectrin repeat 1 (SEQ ID NO: 5) EVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGY MMDLTAHQGRVGNILQLGSKLI GTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLH Spectrin repeat 2 (SEQ ID NO: 6) RVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQH KVLQEDLEQEQVRVNSLTHMVVV VDESSGDHATAALEEQLKVLGDRWANICRWTEDRWVLLQDI Spectrin repeat 3 (SEQ ID NO: 7) LLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQNEMLSSLQKLAV LKADLEKKKQSMGKLYSLKQDL LSTLKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQA Hinge 2 (SEQ ID NO: 8) VTTTQPSLTQTTVMETVTTVTTREQILVKHAQEELPPPPPQKKRQITVD Spectrin repeat 4 (SEQ ID NO: 9) SEIRKRLDVDITELHSWITRSEAVLQSPEFAIFRKEGNFSDLKEKVNAIER EKAEKFRKLQDASRSAQAL VEQMVNEGVNADSIKQASEQLNSRWIEFCQLLSERLNWLEY Spectrin repeat 5 (SEQ ID NO: 10) QNNIIAFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKICKDEVN RLSGLQPQIERLKIQSIALK EKGQGPMFLDADFVAFTNHFKQVFSDVQAREKELQTIFD Spectrin repeat 6 (SEQ ID NO: 11) TLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQ SSLQEQQSGLYYLSTTVKEMS KKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQ Spectrin repeat 7 (SEQ ID NO: 12) MNKLRKIQNHIQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQCRLL VSDIQTIQPSLNSVNEGGQKIK NEAEPEFASRLETELKELNTQWDHMCQQVYARKEALKGG

- 24 046419

Spectrin repeat 8 (SEQ IDNO: 13) LEKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKR AKEEAQQKEAKVKLLTESVNSVI AQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEV Spectrin repeat 9 (SEQ IDNO: 14) WACWHELLSYLEKANKWLNEVEFKLKTTENIPGGAEEISEVLDSLENL MRHSEDNPNQIRILAQTLTDGG VMDELINEELETFNSRWRELHEEAVRRQKLLEQS Spectrin repeat 10 (SEQ IDNO: 15) IQSAQETEI<SLHLIQESLTFIDI<QLAAYIADI<VDAAQMPQEAQI<IQSDLT SHEISLEEMKKHNQGKEAAQ RVLSQIDVAQI<I<LQDVSMI<FRL Spectrin repeat AND (SEQ IDNO: 16) FQKPANFE Q RLQESKMILDEVKMHLPALETKSVEQEVVQSQLNHCVNLYKSLSEVKS EVEMVIKTGRQIV QKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEKC Spectrin repeat 12 (SEQ IDNO: 17) LKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEVAWGK ATQKEIEKQKVHLKSITEVGEALK TVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLLLEY Spectrin repeat 13 (SEQ IDNO: 18) QKHMETFDQNVDHITKWIIQADTLLDESEKKKPQQKEDVLKRLKAELN DIRPKVDSTRDQAANLMANRGD HCRKLVEPQISELNHRFAAISHRIKTGKASIPLK Spectrin repeat 14 (SEQ IDNO: 19) ELEQFNSDIQKLLEPLEAEIQQGVNLKEEDFNKDMNEDNEGTVKELLQR GDNLQQRITDERKREEIKIKQ QLLQTKHNALKDLRSQRRKKALEI Spectrin repeat 15 (SEQ Ш NO: 20) SHQWYQYI<RQADDLLI<CLDDIEI<I<LASLPEPRDERI< H<EIDRELQI<I<I<E ELNAVRRQAEGLSEDGAAMAV EPTQIQLSKRWREIESKFAQFRRLNFAQ L3 (20-mer linker) (SEQ IDNO: 21) IHTVREETMMVMTEDMPLEI Spectrin repeat 16 (SEQ Ш NO: 22) SYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKD SLQQSSGRIDIIHSKKTAAL QSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRS Spectrin repeat 17 (SEQ IDNO: 23) VEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQ DGIGQRQTVVRTLNATGEEIIQQ SSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQ Spectrin repeat 18 (SEQ Ш NO: 24) KNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEE LPLRQGILKQLNETGGPVLV SAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQL Spectrin repeat 19 (SEQ IDNO: 25) EKKLEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEGPFDVQETEIAVQAK QPDVEEILSKGQHLYKEKPAT QPVKRKLEDLS SEWKAVNRLLQELRAKQPDL Hinge 3 (SEQ Ш NO: 26) APGLTTIGASPTQT VTL VTQP VVTKET AISKLEMP S SLMLE Spectrin repeat 20 (SEQ Ш NO: 27) VPALADFNRAWTELTDWLSLLDQVIKSQRVMVGDLEDINEMIIKQKAT MQDLEQRRPQLEELITAAQNLK NKTSNQEARTIITDRIERIQNQWDEVQEHLQNRRQQLNEM Spectrin repeat 21 (SEQ Ш NO: 28) LKDSTQWLEAKEEAEQVLGQARAKLESWKEGPYTVDAIQKKITETKQL AKDLRQWQTNVDVANDLALKLL RDYSADDTRKVHMITENINASWRSIHKRVSEREAALEET

- 25 046419

Spectrin repeat 22 (SEQ Ш NO: 29) HRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELM KQWQDLQGEIEAHTDVYHNLDE NSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEAS Spectrin repeat 23 (SEQ IDNO: 30) SDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRA FKRELKTKEPVIMSTLETVRIFL TEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHS ADWQRKIDET Spectrin repeat 24 (SEQ IDNO: 31) LERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKA LRGEIAPLKENVSHVNDLARQL TTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHE Hinge 4 (SEQ Ш NO: 32) AHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKM TELYQSLADLNN VRF S AYRTAM KL CR (SEQ Ш NO: 33) RRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRL EQEHNNLVNVPLCVDMCLNWL LNVYDTGRTGRIRVL SFKTGIISLCKAHLEDKYRYLFKQ VAS STGFCDQ RRLGLLLHDSIQIPRQLGEVA SFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRV AAAETAKHQAKCNICKECPII GFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVR DFAKVLKNKFRTKRYFAKHPRM GYLPVQTVLEGDNMET C-conc. (SEQ Ш NO: 34) PVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLND SISPNESIDDEHLLIQHYCQS LNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQ QHEHKGLSPLPSPPEMMPT SPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLL EQPQAEAKVNGTTVSSPSTSL QRSDSSQPMLLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSF PSSRGRNTPGKPMREDTM

Table 4

Nucleotide sequences of dystrophin domains

Description and sequence ID Subsequence 5' untranslated region (SEQIDNO:35) gggattccct cactttcccc ctacaggact cagatctggg aggcaattac cttcggagaa 60 aaacgaatag gaaaaactga agtgttactt tttttaaagc tgctgaagtt tgttggtttcl20 tcattgtttt taagcctact ggagcaataa agtttgaaga acttttacca ggtttttt ttl 80 atcgctgcct tgatatacac ttttcaaa 208 ABDI (SEQ ID NO:36) atgctttggt gggaagaagt agaggactgt tatgaaagag aagatgttca aaagaaaaca 60 ttcacaaaat gggtaaatgc acaattttct aagtttggga agcagcatat tgagaacctcl20 ttcagtgacc tacaggatgg gaggcgcctc ctagacctcc tcgaaggcct gacagggcaal80 aaactgccaa aagaaaaagg atccacaaga gttcatgccc tgaacaatgt caacaaggca240 ctgcgggttt tgcagaacaa taatgttgat ttagtgaata ttggaagtac tgacatcgta300 gatggaaatc ataaactgac tcttggtttg atttggaata taatcctcca ctggcaggtc 360 aaaaatgtaa tgaaaaatat catggctgga ttgcaacaaa ccaacagtga aaagattctc420 ctgagctggg tccgacaatc aactcgtaat tatccacagg ttaatgtaat caacttcacc480 accagctggt ctgatggcct ggctttgaat gctctcatcc atagtcatag gccagaccta540

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tttgactgga atagtgtggt ttgccagcag tcagccacac aacgactgga acatgcattc600 aacatcgcca gatatcaatt aggcatagag aaactactcg atcctgaaga tgttgatacc660 acctatccag ataagaagtc catcttaatg tacatcacat cactcttcca agttttgcct720 caacaagtga gcattgaagc catccaggaa gtggaa756 Hinge 1 (SEQ IDNO:37) atgttgccaa ggccacctaa agtgactaaa gaagaacatt ttcagttaca tcatcaaatg 60 cactattctc aacagatcac ggtcagtcta gcacagggat atgagagaac ttcttcccctl20 aagcctcgat tcaagagcta tgcctacaca caggctgctt atgtcaccac ctctgaccctl80 acac ggagcc catttccttc acagcatttg gaagctcctg aagacaagtc atttggcagt240 tcattgatgg agagt 255 Spectrin repeat 1 (SEQ IDNO:38) gaagtaaacc tggaccgtta tcaaacagct ttagaagaag tattatcgtg gcttctttct 60 gctgaggaca cattgcaagc acaaggag atttctaatg atgtggaagt ggtgaaagacl20 cagtttcata ctcatgaggg gtacatgatg gatttgacag cccatcaggg ccgggttggtl80 a atattctac aattgggaag taagctgatt ggaacaggaa aattatcaga agatgaagaa240 actgaagtac aagagcagat gaatctccta aattcaagat gggaatgcct cagggtagct300 agcatggaaa aacaaagcaa tttacat327 Spectrin repeat 2 (SEQ ID 140:3 9) agagttttaa tggatctcca gaatcagaaa ctgaaagagt tgaatgactg gctaacaaaa 60 acagaagaaa gaacaaggaa aatggaggaa gagcctcttg gacctgatct tgaagacctal20 aaacgccaag tacaacaaca taaggtgctt caagaagatc tagaacaaga acaagtcaggl80 gt caattctc tcactcacat ggtggtggta gttgatgaat ctagtggaga tcacgcaact240 gctgctttgg aagaacaact taaggtattg ggagatcgat gggcaaacat ctgtagatgg300 acagaagacc gctgggttct tttacaagac ate 333 Spectrin repeat 3 (SEQ Ш NO:40) cttctcaaat ggcaacgtctactgaagaa cagtgccttt ttagtgcatg gctttcagaa 60 aaagaagatg cagtgaacaa gattcacaca actggcttta aagatcaaaa tgaaatgttal20 teaagtette aaaaactggc cgttttaaaa geggatetag aaaagaaaaa gcaatccatgl80 gg caaactgt attcactcaa acaagatctt ctttcaacac tgaagaataa gtcagtgacc240 cagaagacgg aagcatggct ggataacttt gcccggtgtt gggataattt agtccaaaaa300 ettgaaaaga gtacagcaca gatttcacag get 333 Hinge 2 (SEQ IDNO:41) gtcaccacca ctcagccatc actaacacag acaactgtaa tggaaacagt aactacggtg 60 accacaaggg aacagatcct ggtaaagcat gctcaagagg aacttccacc accacctcccl20 caaaagaaga ggcagattac tgtggatl47 Spectrin repeat 4 (SEQ Ш NO:42) tetgaaatta ggaaaaggtt ggatgttgat ataactgaac ttcacagctg gattactcgc 60 teagaagetg tgttgcagag teetgaattt gcaatctttc ggaaggaagg caacttctcal20 gaettaaaag aaaaagtcaa tgccatagag egagaaaaag etgagaagtt cagaaaactgl80 ca agatgcca gcagatcagc tcaggccctg gtggaacaga tggtgaatga gggtgttaat240 gcagatagca tcaaacaagc ctcagaacaa ctgaacagcc ggtggatcga attctgccag300 ttgctaagtg agagaettaa ctggctggag tat 333 Spectrin repeat 5 (SEQ IDNO:43) cagaacaaca teategettt ctataatcag ctacaacaat tggagcagat gacaactact 60 gctgaaaact ggttgaaaat ccaacccacc accccatcag agccaacagc aattaaaagtl20 cagttaaaaa tttgtaagga tgaagtcaac cggctatcag gtcttcaacc tcaaattgaal80 egattaaa aa ttcaaagcat agccctgaaa gagaaaggac aaggacccat gttcctggat240 gcagactttg tggcctttac aaatcatttt aagcaagtct tttctgatgt gcaggccaga300 gagaaagagc tacagacaat ttttgac327

- 27 046419

Spectrin repeat 6 (SEQ Ш NO:44) actttgccac caatgcgcta tcaggagacc atgagtgcca tcaggacatg ggtccagcag 60 tcagaaacca aactctccat acctcaactt agtgtcaccg actatgaaat catggagcagl20 agactcgggg aattgcaggc tttacaaagt tctctgcaag agcaacaaag tggcctatacl80 tatctcagca ccactgtgaa agagatgtcg aagaaagcgc cctctgaaat tagccggaaa240 tatcaatcag aatttgaaga aattgaggga cgctggaaga agctctcctc ccagctggtt300 gagcattgtc aaaagctaga ggagcaa327 Spectrin repeat 7 (SEQ ZDNO:45) atgaataaac tccgaaaaat tcagaatcac atacaaaccc tgaagaaatg gatggctgaa 60 gttgatgttt ttctgaagga ggaatggcct gcccttgggg attcagaaat tctaaaaaagl20 cagctgaaac agtgcagact tttagtcagt gatattcaga caattcagcc cagtctaaacl80 a gtgtcaatg aaggtgggca gaagataaag aatgaagcag agccagagtt tgcttcgaga240 cttgagacag aactcaaaga acttaacact cagtgggatc acatgtgcca acaggtctat300 gccagaaagg aggccttgaa gggaggt327 Spectrin repeat 8 (SEQ Ш NO:46) ttggagaaaa ctgtaagcct ccagaaagat ctatcagaga tgcacgaatg gatgacacaa 60 gctgaagaag agtatcttga gagagatttt gaatataaaa ctccagatga attacagaaal20 gcagttgaag agatgaagag agctaaagaa gaggcccaac aaaaagaagc gaaagtgaaal80 ctccttactg agtctgtaaa tagtgtcata gctcaagctc cacctgtagc acaagaggcc240 ttaaaaaagg aacttgaaac tctaaccacc aactaccagt ggctctgcac taggctgaat300 gggaaatgca agactttgga agaagtt327 Spectrin repeat 9 (SEQ Ш NO:47) tgggcatgtt ggcatgagtt attgtcatac ttggagaaag caaacaagtg gctaaatgaa 60 gtagaattta aacttaaaac cactgaaaac attcctggcg gagctgagga aatctctgagl20 gtgctagatt cacttgaaaa tttgatgcga cattcagagg ataacccaaa tcagattcgcl80 at attggcac agaccctaac agatggcgga gtcatggatg agctaatcaa tgaggaactt240 gagacattta attctcgttg gagggaacta catgaagagg ctgtaaggag gcaaaagttg300 cttgaacaga gc312 Spectrin repeat 10 (SEQ IDNO:48) atccagtctg cccaggagac tgaaaaatcc ttacacttaa tccaggagtc cctcacattc 60 attgacaagc agttggcagc ttatattgca gacaaggtgg acgcagctca aatgcctcagl20 gaagcccaga aaatccaatc tgatttgaca agtcatgaga tcagtttaga agaaatgaagl80 aa acataatc aggggaagga ggctgcccaa agagtcctgt ctcagattga tgttgcacag240 aaaaaattac aagatgtctc catgaagttt cgatta276 Spectrin repeat I (SEQ Ш NO:49) ttccagaaac cagccaattt tgagctgcgt ctacaagaaa gtaagatgat tttagatgaa 60 gtgaagatgc acttgcctgc attggaaaca aagagtgtgg aacaggaagt agtacagtcal20 cagctaaatc attgtgtgaa cttgtataaa agtctgagtg aagtgaagtc tgaag tggaal80 atggtgataa agactggacg tcagattgta cagaaaaagc agacggaaaa tcccaaagaa240 cttgatgaaa gagtaacagc tttgaaattg cattataatg agctgggagc aaaggtaaca300 gaaagaaagc aacagttgga gaaatgc327 Spectrin repeat 12 (SEQ ZDNO:50) ttgaaattgt cccgtaagat gcgaaaggaa atgaatgtct tgacagaatg gctggcagct 60 acagatatgg aattgacaaa gagatcagca gttgaaggaa tgcctagtaa tttggattctl20 gaagttgcct ggggaaaggcactcaaaaa gagattgaga aacagaaggt gcacctgaagl80 agt atcacag agtaggaga ggccttgaaa acagttttgg gcaagaagga gacgttggtg240 gaagataaac tcagtcttct gaatagtaac tggatagctg tcacctcccg agcagaagag300 tggttaaatc ttttgttgga atac 324

- 28 046419

Spectrin repeat 13 (SEQ IDNO:51) cagaaacaca tggaaacttt tgaccagaat gtggaccaca tcacaaagtg gatcattcag 60 gctgacacac ttttggatga atcagagaaa aagaaacccc agcaaaaaga agacgtgcttl20 aagcgtttaa aggcagaact gaatgacata cgcccaaagg tggactctac acgtgaccaal80 gcag caaact tgatggcaaa ccgcggtgac cactgcagga aattagtaga gccccaaatc240 tcagagctca accatcgatt tgcagccatt tcacacagaa ttaagactgg aaaggcctcc300 attcctttga ag312 Spectrin repeat 14 (SEQ IDNO:52) gaattggagc agtttaactc agatatacaa aaattgcttg aaccactgga ggctgaaatt 60 cagcaggggg tgaatctgaa agaggaagac ttcaataaag atatgaatga agacaatgagl20 ggtactgtaa aagaattgtt gcaaagagga gacaacttac aacaaagaat cacagatgagl80 agaaagagag aggaaataaa gataaaacag cagctgttac agacaaaaca taatgctctc240 aaggatttga ggtctcaaag aagaaaaaag gctagaaa tt 282 Spectrin repeat 15 (SEQ IDNO:53) tctcatcagt ggtatcagta caagaggcag gctgatgatc tcctgaaatg cttggatgac 60 attgaaaaaa aattagccag cctacctgag cccagagatg aaaggaaaat aaaggaaattl20 gatcgggaat tgcagaagaa gaaagaggag ctgaatgcag tgcgtaggca agctgaggcl80 ttgtctgagg atggggccgc aatggcagtg gagccaactc agatccagct cagcaagcgc240 tggcgggaaa ttgagagcaa atttgctcag tttcgaagac tcaactttgc acaa294 L3 (20-mer linker) (SEQ IDNO:54) attcacactg tccgtgaaga aacgatgatg gtgatgactg aagacatgcc tttggaaatt 60 Spectrin repeat 16 (SEQ IDNO:55) tcttatgtgc cttctactta tttgactgaa atcactcatg tctcacaagc cctattagaa 60 gtggaacaac ttctcaatgc tcctgacctc tgtgctaagg actttgaaga tctctttaagl20 caagaggagt ctctgaagaa tataaaagat agtctacaac aaagctcagg tcggattgacl80 attatt cata gcaagaagac agcagcattg caaagtgcaa cgcctgtgga aagggtgaag240 ctacaggaag ctctctccca gcttgatttc caatgggaaa aagttaacaa aatgtacaag300 gaccgacaag ggcgatttga cagatct327 Spectrin repeat 17 (SEQ IDNO:56) gttgagaaat ggcggcgttt tcattatgat ataaagatat ttaatcagtg gctaacagaa 60 gctgaacagt ttctcagaaa gacacaaatt cctgagaatt gggaacatgc taaatacaaal20 tggtatctta aggaactcca ggatggcatt gggcagcggc aaactgttgt cagaacattgl80 aatgcaactg gggaagaaat aattcagcaa tcctcaaaaa cagatgccag tattctacag240 gaaaaattgg gaagcctgaa tctgcggtgg caggaggtct gcaaacagct gtcagacaga300 aaaaagaggc tagaagaaca a321 Spectrin repeat 18 (SEQ IDNO:57) AAGAATAATTCT TGTCAGAATT TCAAAAGAGAGAT TTGTTTATG GTTGGAGAA 60 Gcagataaca ttgctagta CCCCACTGAAAG AGCAAGCAAGAAAGAAAGEAGEAG20 AGTTACT GGTGGAAGAGAG TTGCCCCCTGC GCCAGGGAAT TCTCAACAACAAL80 TTAAATGAAA CGGCCCCCA TAAGCCAAGCAAGAT240 AAACTTGAAA ATAAGCTCAA GCAGACAAAT CTCCAGTGGA TAAAGGGTTTC CAGAGCTTA300 CCTGAGAAAC AAGGAAAT TGAAGCTCAA ATAAAAGGGGGGCAGCT 1351 Spectrin repeat 19 (SEQ IDNO:58) gaaaaaaagc ttgaagacct tgaagagcag ttaaatcatc tgctgctgtg gttatctcct 60 attaggaatc agttggaaat ttataaccaa ccaaaccaag aaggaccatt tgacgttcagl20 gaaactgaaa tagcagttca agctaaacaa ccggatgtgg aagagatttt gtcta aagggl80 cagcatttgt acaaggaaaa accagccact cagccagtga agaggaagtt agaagatctg240 agctctgagt ggaaggcggt aaaccgttta cttcaagagc tgaggcaaa gcagcctgac300 cta303

- 29 046419

Hinge 3 (SEQ IDNO:59) gctcctggac tgaccactat tggagcctct cctactcaga ctgttactct ggtgacacaa 60 cctgtggtta ctaaggaaac tgccatctcc aaactagaaa tgccatcttc cttgatgttgl20 gag123 Spectrin repeat 20 (SEQ Ш NO :60) gtacctgctc tggcagattt caaccgggct tggacagaac ttaccgactg gctttctctg 60 cttgatcaag ttataaaatc acagaggtg atggtgggtg accttgagga tatcaacgagl20 atgatcatca agcagaaggc aacaatgcag gatttggaac agaggcgtcc ccagttggaal80 gaactcatta ccgctgccca aaatttgaaa aacaagacca gcaatcaaga ggctagaaca240 atcattacgg atcgaattga aagaattcag aatcagtggg atgaagtaca agaacacctt300 cagaaccgga ggcaacagtt gaatgaaatg 330 Spectrin repeat 21 (SEQ IDNO:61) ttaaaggatt caacacaatg gctggaagct aaggaagaag ctgagcaggt cttaggacag 60 gccagagcca agcttgagtc atggaaggag ggtccctata cagtagatgc aatccaaaagl20 aaaatcacag aaaccaagca gttggccaaa gacctccgcc agtggcagac aaatgtagatl80 gtgg caaatg acttggccct gaaacttctc cgggattatt ctgcagatga taccagaaaa240 gtccacatga taacagagaa tatcaatgcc tcttggagaa gcattcataa aagggtgagt300 gagcgagagg ctgctttgga agaaact327 Spectrin repeat 22 (SEQ Ш NO:62) catagattac tgcaacagtt ccccctggac ctggaaaagt ttcttgcctg gcttacagaa 60 gctgaaacaa ctgccaatgt cctacaggat gctacccgta aggaaaggct cctagaagacl20 tccaagggag taaaagagct gatgaaacaa tggcaagacc tccaaggtga aattgaagctl80 cacacaga tg tttatcacaa cctggatgaa aacagccaaa aaatcctgag atccctggaa240 ggttccgatg atgcagtcct gttacaaaga cgtttggata acatgaactt caagtggagt300 gaacttcgga aaaagtctct caacattagg tcccatttgg aagccagt 348 Spectrin repeat 23 (SEQ IDNO:63) tctgaccagt ggaagcgtct gcacctttct ctgcaggaac ttctggtgtg gctacagctg 60 aaagatgatg aattaagccg gcaggcacct attggaggcg actttccagc agttcagaagl20 cagaacgatg tacatagggc cttcaagagg gaattgaaaa ctaaagaacc tgtaatcatgl8 0 agtactcttg agactgtacg aatatttctg acagagcagc ctttggaagg actagagaaa240 ctctaccagg agcccagaga gctgcctcct gaggagagag cccagaatgt cactcggctt300 ctacgaaagc aggctgagga ggtcaatact gagtgggaaa aattgaacct gcactccgct360 gact ggcaga gaaaaataga tgagacc387 Spectrin repeat 24 (SEQ Ш NO: 64) cttgaaagac tccaggaact tcaagaggcc acggatgagc tggacctcaa gctgcgccaa 60 gctgaggtga tcaagggatc ctggcagccc gtgggcgatc tcctcattga ctctctccaal20 gatcacctcg agaaagtcaa ggcacttcga ggagaaattg cgcctctgaa agagaacgtgl80 agccacgtca atgaccttgc tcgccagctt accactttgg gcattcagct ctcaccgtat240 aacctcagca ctctggaaga cctgaacacc agatggaagc ttctgcaggt ggccgtcgag300 gaccgagtca ggcagctgca tgaa 324 Hinge 4 (SEQ Π)ΝΟ:6 5) gcccacaggg actttggtcc agcatctcag cactttcttt ccacgtctgt ccaggtccc 60 tgggagagag ccatctcgcc aaacaaagtg ccctactata tcaaccacga gactcaaacal20 acttgctggg accatcccaa aatgacagag ctctaccagt ctttagctga cctgaataatl80 gtcagattct cagcttatag gactgccatg aaactc216 CR (SEQ Ш NO:66) cgaagactgc agaaggccct ttgcttggat ctcttgagcc tgtcagctgc atgtgatgcc 60 ttggaccagc acaacctcaa gcaaaatgac cagcccatgg atatcctgca gattattaatl20 tgtttgacca ctatttatga ccgcctggag caagagcaca acaatttggt caacgtccctl80 ct ctgcgtgg atatgtgtct gaactggctg ctgaatgttt atgatacggg acgaacaggg240 agatccgtg tcctgtcttt taaaactggc atcatttccc tgtgtaaagc acatttggaa300

- 30 046419

gacaagtaca gatacctttt caagcaagtg gcaagttcaa caggattttg tgaccagcgc360 agctgggcc tccttctgca tgattctatc caaattccaa gacagttggg tgaagttgca420 tcctttgggg gcagtaacat tgagccaagt gtccggagct gcttccaatt tgctaataat480 aag ccagaga tcgaagcggc cctcttccta gactggatga gactggaacc ccagtccatg540 gtgtggctgc ccgtcctgca cagagtggct gctgcagaaa ctgccaagca tcaggccaaa600 tgtaacatct gcaaagagtg tccaatcatt ggattcaggt acaggagtct aaagcacttt660 aattatgaca tctgccaaag ctgctttttt tctggtcgag ttgcaaaagg ccataaaatg720 cactatccca tggtggaata ttgcactccg actacatcag gagaagatgt tcgagacttt780 gccaaggtac taaaaaacaa atttcgaacc aaaaggtatt ttgcgaagca tccccgaatg840 ggctacctgc cagtgcagac tgtcttagag ggggacaaca tggaaact C-conc. (SEQ Ш NO:67) cccgttactc tgatcaactt ctggccagta gattctgcgc ctgcctcgtc ccctcagctt 60 tcacacgatg atactcattc acgcattgaa cattatgcta gcaggctagc agaaatggaal20 aacagcaatg gatcttatct aaatgatagc atctctccta atgagagcat agatgatgaal80 cattt gttaa tccagcatta ctgccaaagt ttgaaccagg actcccccct gagccagcct240 cgtagtcctg cccagatctt gatttcctta gagagtgagg aaagagggga gctagagaga300 atcctagcag atcttgagga agaaaacagg aatctgcaag cagaatatga ccgtctaaag360 cagcagcacg aacata aagg cctgtcccca ctgccgtccc ctcctgaaat gatgcccacc420 tctccccaga gtccccggga tgctgagctc attgctgagg ccaagctact gcgtcaacac480 aaaggccgcc tggaagccag gatgcaaatc ctggaagacc acaataaaca gctggagtca540 cagttacaca ggctaaggca gctgctggag caaccccagg cagaggccaa agtgaatggc600 acaacggtgt cctctccttc tacctctcta cagaggtccg acagcagtca gcctatgctg660 ctccgagtgg ttgg cagtca aacttcggac tccatgggtg aggaagatct tctcagtcct720 ccccaggaca caagcacagg gttagaggag gtgatggagc aactcaacaa ctccttccct780 agttcaagag gaagaaatac ccctggaaag ccaatgagag aggacacaat gtag

The present invention relates to a miniaturized dystrophin polypeptide that is smaller than the full-length dystrophin protein, i.e. isoform Dp427m, and which is not identical to naturally occurring dystrophin protein isoforms, or to a nucleic acid molecule containing a nucleotide sequence encoding a miniaturized dystrophin polypeptide. Where the present disclosure discloses miniaturized dystrophin polypeptides, the present disclosure also discloses a nucleic acid molecule comprising a nucleotide sequence encoding the corresponding disclosed miniaturized dystrophin polypeptide, and vice versa. In some embodiments, a nucleic acid molecule encoding a miniaturized dystrophin polypeptide is suitable for gene therapy. Accordingly, a nucleic acid molecule encoding a miniaturized dystrophin polypeptide is designed not only to conform to a gene therapy vector, e.g., an AAV vector, or to be suitable for recombinant expression, but also to reduce any unwanted immune response (e.g., humoral immune response and /or cellular immune response, for example, CD4 and/or CD8) directed against the miniaturized dystrophin polypeptide when administered or expressed in vivo. In some embodiments, the miniaturized dystrophin polypeptide of the present invention comprises, from N-terminus to C-terminus, a hinge 1 (H1) domain, a spectrin repeat 1 (R1) domain, a spectrin repeat 3 (R3) domain, a hinge 2 (H2) domain , spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain. In some embodiments, the miniaturized dystrophin polypeptide does not contain a dystrophin spectrin repeat 2 (R2) domain. In other embodiments, the miniaturized dystrophin polypeptide does not contain a spectrin repeat domain 2 (R2) and (1) a spectrin repeat domain 4 (R4), (2) a spectrin repeat domain 5 (R5), (3) a spectrin repeat domain 6 (R6), (4) spectrin repeat domain 7 (R7), (5) spectrin repeat domain 8 (R8), (6) spectrin repeat domain 9 (R9), (7) spectrin repeat domain 10 (R10), (8) spectrin repeat domain 11 (R11), (9) spectrin repeat domain 12 (R12), (10) spectrin repeat domain 13 (R13), (11) spectrin repeat domain 14 (R14), (12) spectrin repeat domain 15 (R15), ( 13) spectrin repeat domain 18 (R18), (14) spectrin repeat domain 19 (R19), (15) spectrin repeat domain 20 (R20), (16) spectrin repeat domain 21 (R21), (17) spectrin repeat domain 22 (R22) or (18) spectrin repeat domain 23 (R23), or (19) any combination thereof. In some embodiments, the miniaturized dystrophin polypeptide does not comprise a spectrin repeat 2 (R2) domain, a spectrin repeat 4 (R4) domain, a spectrin repeat 5 (R5) domain, a spectrin repeat 6 (R6) domain, a spectrin repeat 7 (R7) domain spectrin repeat 8 (R8), spectrin repeat domain 9 (R9), spectrin repeat domain 10 (R10), spectrin repeat domain 11 (R11), spectrin repeat domain 12 (R12), spectrin repeat domain 13 (R13), spectrin repeat domain 14 (R14), spectrin repeat domain 15 (R15), spectrin repeat domain 18 (R18), spectrin repeat domain 19 (R19), spectrin repeat domain 20 (R20), spectrin repeat domain 21 (R21), spectrin repeat domain 22 ( R22) and spectrin repeat domain 23 (R23).

In some embodiments, the miniaturized dystrophin polypeptide of the present invention comprises, from N-terminus to C-terminus, a hinge 1 (H1) domain, a spectrin domain

- 31 046419 repeat 1 (R1), spectrin repeat domain 3 (R3), hinge 2 (H2), spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24) and hinge 4 (H4) dystrophin domain, where the R1 domain is directly fused to the R3 domain via a peptide bond, for example, see miniaturized dystrophin polypeptide BXA-027741. In some embodiments, the miniaturized dystrophin polypeptide of the present invention comprises, from N-terminus to C-terminus, hinge 1 (H1) domain, spectrin repeat 1 (R1) domain, spectrin repeat 3 (R3) domain, hinge 2 (H2), spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain, where the R1 domain and the R3 domain are fused through the amino acids ARG-VAL (RV).

In some embodiments, the miniaturized dystrophin polypeptide of the present invention comprises, from N-terminus to C-terminus, hinge 1 (H1) domain, spectrin repeat 1 (R1) domain, spectrin repeat 3 (R3) domain, hinge 2 (H2), spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain, where the H2 domain and the R16 domain are fused by a linker. The linker can be any linker known in the art. In other embodiments, the linker may be selected from any of the linkers disclosed herein. In other embodiments, the linker may be the linker from section 5.3.2. In some embodiments, the linker may comprise an amino acid sequence that is at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97% , is at least about 98%, at least about 99%, or about 100% identical to the sequence shown in SEQ ID NO: 75 (IHTVREE TMMVMTEDMP LEI). In some embodiments, the miniaturized dystrophin polypeptide of the present invention comprises, from N-terminus to C-terminus, hinge 1 (H1) domain, spectrin repeat 1 (R1) domain, spectrin repeat 3 (R3) domain, hinge 2 (H2), spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain, where the H2 domain and the R16 domain are fused by a linker. In some embodiments, the linker may comprise an amino acid sequence that is at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97% , is at least about 98%, at least about 99%, or about 100% identical to the sequences shown in SEQ ID NO: 74-75, in combination (SEAQ IHTVREE TMMVMTEDMP LEI).

In some embodiments, the miniaturized dystrophin polypeptide of the present invention comprises, from N-terminus to C-terminus, hinge 1 (H1) domain, spectrin repeat 1 (R1) domain, spectrin repeat 3 (R3) domain, hinge 2 (H2), spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain, where the H2 domain and the R16 domain are fused by a linker. In some embodiments, the linker may comprise an amino acid sequence that is at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97% , is at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 74 (SEAQ).

In other embodiments, the miniaturized dystrophin polypeptide of the present invention comprises, from N-terminus to C-terminus, hinge 1 (H1) domain, spectrin repeat 1 (R1) domain, spectrin repeat 3 (R3) domain, hinge 2 (H2), spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24), and hinge 4 (H4) domain of dystrophin, where the R1 domain and the R3 domain are fused through the ARG-VAL amino acids and where the H2 domain and the domain R16 are fused through a linker, for example, SEQ ID NO: 74-75 in combination. In some embodiments, the miniaturized dystrophin polypeptide of the present invention comprises, from N-terminus to C-terminus, hinge 1 (H1) domain, spectrin repeat 1 (R1) domain, spectrin repeat 3 (R3) domain, hinge 2 (H2), spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24) and hinge 4 (H4) domain of dystrophin, where the R1 domain and the R3 domain are fused through the amino acids ARG-VAL, where the H2 domain and the R16 are fused through a linker, for example, SEQ ID NO: 74-75 in combination, and wherein (i) the H1 domain and the R1 domain are directly fused, (ii) the R3 domain and the H2 domain are directly fused, (iii) the R16 and R17 domains are directly fused , (iv) the R17 and R24 domains are directly fused, or (v) the R24 and H4 domains are directly fused, or (vi) any combination thereof. In some embodiments, a miniaturized dystrophin polypeptide useful for the present invention comprises, from N-terminus to C-terminus, hinge 1 (H1) domain, spectrin repeat 1 (R1) domain, spectrin repeat 3 (R3) domain, hinge 2 (H2), spectrin repeat domain 16 (R16), spectrin repeat domain 17 (R17), spectrin repeat domain 24 (R24) and hinge 4 (H4) domain of dystrophin, where the R1 domain and the R3 domain are fused through the amino acids ARG-VAL, where the H2 domain and the R16 are fused through a linker, for example, SEQ ID NO: 74-75 in combination, and wherein (i) the H1 domain and the R1 domain are directly fused, (ii) the R3 domain and the H2 domain are directly fused, (iii) the R16 and R17 domains are directly fused , (iv) domains

- 32 046419

R17 and R24 are directly fused, and (v) the R24 and H4 domains are directly fused. In some embodiments, a miniaturized dystrophin polypeptide useful for the present invention further comprises an ABD1 domain (optionally at the N-terminus) and/or a CR domain (optionally at the N-terminus). In some embodiments, the miniaturized dystrophin polypeptide consists essentially of or consists of, from N-terminus to C-terminus, an ABD1 domain, an H1 domain, an R1 domain, RV amino acids, an R3 domain, an H2 domain, an amino acid sequence as set forth in SEQ ID NO: 74-75 in combination, R16 domain, R17 domain, R24 domain, H4 domain, CR domain of dystrophin. Each domain in miniaturized dystrophin polypeptides may have one or more changes compared to the corresponding wild-type domain.

For example, miniaturized dystrophin BXA-196477 consists of the following protein domains in order.

Table 5

Amino acid sequence and domain structure of the miniaturized dystrophin polypeptide BXA-196477

SEQ ID NO: Description Subsequence 68 ABDI MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIEN LFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNK ALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQV KNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFT TSWSDGLALNALIHSHRPDLFDWNSV VCQQSATQRLEHAF NIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQ VSIEAIQEVE 69 Hinge 1 MLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSP KPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSL MES 70 Spectrin-1 EVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKD QFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEE TEVQEQMNLLNSRWECLRVASMEKQSNLH 71 LI (RV linker) RV 72 Spectrin-3 LLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQNEML SSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVT QKTEAWLDNFARCWDNLVQKLEKSTAQISQA 73 Hinge 2 VTTTQPSLTQTTVMETVTTVTTREQILVKHAQEELPPPPPQK KRQITVD 74 L2 (SEAQ linker) SEAQ 75 L3 (20-dimensional linker) IHTVREETMMVMTEDMPLEI 76 Spectrin-16 SYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQ EESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQE ALSQLDFQWEKVNKMYKDRQGRFDRS 77 Spectrin-17 VEI<WRRFHYDII<IFNQWLTEAEQFLRI<TQIPENWEHAI<YI< WYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQE KLGSLNLRWQEVCKQLSDRKKRLEEQ 78 Spectrin-24 LERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQD HLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNL STLEDLNTRWKLLQVAVEDRVRQLHE 79 Hinge 4 AHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTT CWDHPKMTELYQSLADLNNVRFSAYRTAMKL 80 CR RRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINC LTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTG RIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRL GLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIE AALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQ AKCNIC KECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMV EYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPV QTVLEGDNMETDTM

In some embodiments, the H1 domain of a miniaturized dystrophin polypeptide is an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 96%, at least

- 33046419 is at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 69. In some embodiments, the R1 domain is an amino acid sequence that at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least is about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 70. In some embodiments, the R3 domain is an amino acid a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% , is at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 72. In some embodiments, the domain H2 is an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 73. B In some embodiments, the R16 domain is an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% , at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO : 76. In some embodiments, the R17 domain is an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical sequence SEQ ID NO: 77. In some embodiments, the R24 domain is an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85% , at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or is about 100% identical to the sequence of SEQ ID NO: 78. In some embodiments, the H4 domain is an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least by about 85%, by at least about 90%, by at least about 95%, by at least about 96%, by at least about 97%, by at least about 98%, by at least about 99% or about 100% identical to the sequence of SEQ ID NO: 79. In some embodiments, the miniaturized dystrophin polypeptide further comprises at the N-terminus a polypeptide sequence that is at least about 70%, at least about 75%, at least at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 68. In some embodiments, the miniaturized dystrophin polypeptide further comprises, at its C-terminus, a polypeptide sequence that is at least about 70% identical to at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 80.

- 34 046419

Table 5B

Amino acid sequence and domain structure of the miniaturized dystrophin polypeptide BXA-027743

SEQ Ш NO: Description Subsequence 81 ABDI MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIEN LFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNK ALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQ VKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINF TTSWSDGLALNALIHSHRPDLFDWNS VVCQQSATQRLEHA FNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVE 82 Hinge 1 MLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSP KPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSL MES 83 Spectrin-1 EVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKD QFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEE TEVQEQMNLLNSRWECLRVASMEKQSNLH 84 L1 IHTVREETMMVMTEDMPLEI 85 Spectrin16 SYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQ EESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQE ALSQLDFQWEKVNKMYKDRQGRFDRS 86 Spectrin17 VEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYK WYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQE KLGSLNLRWQEVCKQLSDRKKRLEEQ 87 Hinge 3 APGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSLMLE 88 Spectrin23 SDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQK QNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKL YQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSAD WQRKIDET 89 Spectrin24 LERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQD HLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNL STLEDLNTRWKLLQVAVEDRVRQLHE 90 Hinge 4 AHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTT CWDHPKMTELYQSLADLNNVRFSAYRTAMKL 91 CR/C-conc RRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINC LTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTG RIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRL GLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIE AALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQ AKCNIC KECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMV EYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPV QTVLEGDNMETPAQILISLESEERGELERILADLEEENRNLQ AEYDRLKQQHEHKGLSPLPSPPDTM

In some embodiments, the present invention provides a miniaturized dystrophin polypeptide comprising a spectrin repeat 1 (R1) domain and a spectrin repeat 16 (R16) domain, wherein the R1 domain and the R16 domain are fused through a linker comprising the amino acid sequence set forth in SEQ ID NO: 84 ( IHTVREETMMVMTEDMPLEI). In some embodiments, the present invention provides a miniaturized dystrophin polypeptide comprising, from N-terminus to C-terminus, hinge 1 (H1) domain, spectrin repeat 1 (R1) domain, spectrin repeat 16 (R16) domain, spectrin repeat 17 domain ( R17), hinge 3 (H3) domain, spectrin repeat domain 23 (R23), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain, wherein the R1 domain and the R16 domain are fused by the amino acid sequence set forth in SEQ ID NO : 84 (IHTVREETMMVMTEDMPLEI). In some embodiments, the present invention includes a nucleic acid molecule comprising a nucleotide sequence that encodes a miniaturized dystrophin polypeptide comprising a spectrin repeat 1 (R1) domain and a spectrin repeat 16 (R16) domain, wherein the R1 domain and the R16 domain are fused through a linker containing an amino acid the sequence given in SEQ ID NO: 84 (IHTVREETMMVMTEDMPLEI). In some embodiments, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes a miniaturized dystrophin polypeptide comprising an N-terminal to C-terminal hinge 1 (H1) domain, a spectrin repeat 1 (R1) domain, a spectrin repeat 16 domain ( R16), spectrin repeat domain 17 (R17), hinge 3 (H3) domain, spectrin repeat domain 23 (R23), spectrin repeat domain 24 (R24), and dystrophin hinge 4 (H4) domain, where the R1 domain and the R16 domain are merged

- 35 046419 you through the amino acid sequence given in SEQ ID NO: 84 (IHTVREETMMVMTEDMPLEI).

In some embodiments, the miniaturized dystrophin polypeptide of the present invention has the following characteristics: (i) the H1 domain and the R1 domain are directly fused, (ii) the R16 and R17 domains are directly fused, (iii) the R17 and H3 domains are directly fused, (iv) the R23 domains and R24 are directly fused, or (v) the R24 and H4 domains are directly fused, or (vi) any combination thereof. In some embodiments, the miniaturized dystrophin polypeptide of the present invention does not contain spectrin repeat domain 2 (R2), spectrin repeat domain 3 (R3), spectrin repeat domain 4 (R4), spectrin repeat domain 5 (R5), spectrin repeat domain 6 (R6 ), spectrin repeat domain 7 (R7), spectrin repeat domain 8 (R8), spectrin repeat domain 9 (R9), spectrin repeat domain 10 (R10), spectrin repeat domain 11 (R11), spectrin repeat domain 12 (R12), spectrin repeat domain 13 (R13), spectrin repeat domain 14 (R14), spectrin repeat domain 15 (R15), spectrin repeat domain 18 (R18), spectrin repeat domain 19 (R19), spectrin repeat domain 20 (R20), spectrin domain repeat 21 (R21) and/or spectrin repeat 22 (R22) domain. In some embodiments, the miniaturized dystrophin polypeptide further comprises an ABD1 domain and/or a CR domain.

In some embodiments, the miniaturized dystrophin polypeptide comprises, consists essentially of, or consists of, from N-terminus to C-terminus, an ABD1 domain, an H1 domain, an R1 domain, the amino acid sequence set forth in SEQ ID NO: 84 (IHTVREETMMVMTEDMPLEI), a domain R16 domain, R17 domain, H3 domain, R23 domain, R24 domain, H4 domain and CR domain of dystrophin.

In some embodiments, the H1 domain useful for a miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 82. In other embodiments, the R1 domain useful for a miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80 %, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, is at least about 99% or about 100% identical to the sequence of SEQ ID NO: 83.

In some embodiments, the R16 domain useful for a miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 85.

In some embodiments, the R17 domain useful for a miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 86.

In other embodiments, the H3 domain useful for a miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 87.

In some embodiments, the R23 domain useful for a miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 88.

In other embodiments, the R24 domain useful for a miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 89.

- 36 046419

In some embodiments, the H4 domain useful for a miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 90.

In some embodiments, the miniaturized dystrophin polypeptide further comprises, at its N-terminus, an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100 % identical to the sequence of SEQ ID NO: 81. In some embodiments, the miniaturized dystrophin polypeptide further comprises, at its C-terminus, an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least at least about 99% or about 100% identical to the sequence of SEQ ID NO: 91.

In some embodiments, the nucleic acid sequence encoding each domain may be as follows.

Table 6A

Nucleotide sequence (and domain structure) encoding the miniaturized dystrophin polypeptide BXA-196477

SEQ ID NO: Description Nucleotide sequence 92 5'UTR CCGCCTTCGGCACCATTCCTCACGACACCCAAATATGGCACG GGTGAGGAATGGTGGGGAGTTATTTTAGAGCGGTGAGGAAGG TGGGCAGGCAGCAGGTGTTGGCGCTCTAAAAATAACTCCCGG GAGTTATTTTAGAGCGGAGGAATGGTGGACACCCAAATAGGC GACGGTTCCTCACCCGTCGCCATATTTGGGTGTCCGCCCTCGG CCGGGGCCGCATTCCTGGGGCCGGGCGG TGCTCCCGCCCGCC TCGATAAAAGGCTCCGGGGCCGGCGGCGGCCCACGAGCTACC CGGAGGAGCGGGAGGCTCTAGACCACC 93 ABDI ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAG GACGTGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAG TTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGC GACCTGCAGGACGGCAGACGGCTGCTGGACCTGCTGGAAGGC CTGACCGGCCAGAAGCTGCCCAAAGAAGGGCAGCACCAG AGTGCACGCCCTGAACAACGTGAACAAGGC CCTGCGGGTGCT GCAGAACAACAACGTGGACCTGGTGAACATCGGCAGCACCGA CATCGTGGACGGCAACCACAAGCTGACCCTGGGCCTGATCTG GAACATCATCCTGCACTGGCAGGTCAAAACGTGATGAAGAA CATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGATCCT GCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGT CAACGTGATCAACTTCACCACCTC TTGGAGCGACGGCCTGGC CCTGAACGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGA CTGGAACAGCGTGGTCTGCCAGCAGAGCGCCACCCAGCGGCT GGAACACGCCTTCAATATCGCCAGATACCAGCTGGGCATCGA GAAGCTGCTGGATCCCGAGGACGTGGACACCACCTACCCCGA CAAGAAATCCATCCTGATGTATATCACCAGCCTGTTCCAGGTG GGTGTCCATCGAGGCCATCCAGGAAGTGGAA 94 Hinge 1 ATGCTGCCCAGACCCCCCAAAGTGACCAAAGAGGAACACTTC CAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTG TCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGCCC CGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGACC ACCAGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTG GAAGCCCCCGAGGACAAGAGCTTCGGCAGCAG CCTGATGGAA AGC 95 Spectrin-1 GAAGTGAACCTGGACAGATACCAGACCGCCCTGGAAGAGGTG CTGAGCTGGCTGCTGAGCGCCGAGGATACACTGCAGGCTCAG GGCGAGATCAGCAACGACGTGGAAGTCGTGAAGGACCAGTTC CACACCCACGAGGGCTACATGATGGACCTGACAGCCCACCAG GGCAGAGTGGGCAACATCCTGCAGCTGGGCTCCAAGCTGATC GGCACCGGCAAGCTGAGCGAGGACGAAGA GACAGAGGTGCA

- 37 046419

GGAACAGATGAACCTGCTGAACAGCAGATGGGAGTGCCTGCG GGTGGCCAGCATGGAAAAGCAGAGCAACCTGCAT 96 LI (RV linker) AGGGTC 97 Spectrin-3 CTGCTGAAGTGGCAGCGGCTGACCGAGGAACAGTGCCTGTTT AGCGCCTGGCTGTCCGAGAAAGAGGACGCCGTGAACAAGATC CACACCACCGGCTTCAAGGACCAGAACGAGATGCTGAGCAGC CTGCAGAAACTGGCCGTGCTGAAGGCCGACCTGGAAAAGAAA AAGCAGTCCATGGGCAAGCTGTACTCCCTGAAGCAGGACCTG CTGTCCACCCTGAAGAACAAGAGCGTGACCC AGAAAACCGAG GCCTGGCTGGACAACTTCGCCCGGTGCTGGGACAACCTGGTG CAGAAGCTGGAAAAGTCCACCGCCCAGATCTCCCAGGCC 98 Hinge 2 GTGACCACAACACAGCCCAGCCTGACCCAGACCACCGTGATG GAAACCGTGACAACAGTGACCACCCGGGAACAGATCCTCGTG AAGCACGCCCAGGAAGAACTGCCCCCTCCACCCCCCCAGAAG AAACGGCAGATCACAGTGGAC 99 L2 (SEAQ linker) AGTGAAGCTCAG 100 L3 (20-dimensional linker) ATCCACACCGTGCGGGAAGAGACAATGATGGTCATGACAGAG GACATGCCCCTGGAAATC 101 Spectrin16 AGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCATGTG TCCCAGGCCCTGCTGGAAGTGGAACAGCTGCTGAACGCCCCC GACCTGTGCGCCAAGGATTTCGAGGACCTGTTCAAGCAGGAA GAGAGCCTGAAGAATATCAAGGATAGCCTGCAGCAGAGCAG CGGCCGGATCGACATCATCCACAGCAAGAAAACAGCCGCCCT GCAGAGCGCCACCCCCGTGGAAAGAGTGAAACT GCAGGAAG CCCTGTCCCAGCTGGACTTCCAGTGGGAGAAAGTGAACAAAA TGTACAAGGACCGGCAGGGCAGATTCGACCGCAGC 102 Spectrin17 GTGGAAAAGTGGCGGCGGTTCCACTACGACATCAAGATCTTC AACCAGTGGCTGACAGAGGCCGAGCAGTTCCTGAGAAAGACC CAGATCCCCGAGAACTGGGAGCACGCCAAGTACAAGTGGTAT CTGAAAGAGCTGCAGGACGGCATCGGCCAGAGACAGACAGT CGTGCGGACCCTGAATGCCACCGGCGAGGAAATCATCCAGCA GTCCAGCAAGACCGACGCCAGCATTCTGCAGGAAA AGCTGGG CAGCCTGAACCTGCGGTGGCAGGAAGTGTGCAAGCAGCTGTC CGACCGGAAGAAGCGGCTGGAAGAACAG 103 Spectrin24 CTGGAACGGCTGCAGGAACTGCAGGAGGCCACCGACGAGCTG GACCTGAAACTGAGACAGGCCGAAGTGATCAAGGGCAGCTG GCAGCCAGTGGGCGACCTGCTGATCGACTCCCTGCAGGACCA TCTGGAAAAAGTGAAGGCCCTGAGAGGCGAGATCGCCCCCCT GAAAGAAAACGTGTCCCACGTGAACGACCTGGCCCGGCAGCT GACAACACTGGGCATTCAGCTGAGCCCCTACA ACCTGAGCAC ACTGGAAGATCTGAACACCCGGTGGAAGCTGCTGCAGGTGGC CGTGGAAGATAGAGTGCGGCAGCTGCACGAG 104 Hinge 4 GCCCACAGAGATTTTGGCCCTGCCTCCCAGCACTTCCTGAGCA CCAGTGTGCAGGGCCCTTGGGAGAGAGCCATCTCCCCTAACA AGGTGCCCTACTACATCAACCACGAGACACAGACCACCTGAC TGAACTGGGACCACCCCAAGCTGACCGACGACCTGACCACCT

- 38 046419

CAGCTGACCGACGACGACGACCTGACGACGACGACGACGAC GACGACGACGACGAC 105 CR/C-conc CGGAGACTGCAGAAAGCTCTGTGCCTGGATCTGCTGTCCCTGT CCGCCGCCTGTGATGCCCTGGACCAGCACAATCTGAAGCAGA ACGACCAGCCCATGGATATCCTGCAGATCATCAACTGCCTGA CCACCATCTACGACCGGCTGGAACAGGAACACAACAATCTCG TGAACGTGCCCCTGTGCGTGGACATGTGCCTGAATTGGCTGCT GAATGTGTACGACACCGGCCGGACAGGCCGGATC AGAGTGCT GTCCTTCAAGACCGGCATCATCAGCCTGTGCAAAGCCCACCT GGAAGATAAGTACCGGTATCTGTTCAAACAGGTGGCCTCTAG CACCGGCTTTTGCGACCAGAAGGCTGGGCCTGCTGCTGCA CGACAGCATCCAGATCCCTAGACAGCTGGGCGAAGTGGCCAG CTTTGGCGGCAGCAACATCGAGCCTAGCGTGCGGAGCTGCTT CCAGTTCGCCAACAACAAGCCCGAGATC GAGGCCGCCCTGTT CCTGGACTGGATGAGACTGGAACCCCAGAGCATGGTGTGGCT GCCCGTGCTGCATAGAGTGGCCGCTGCCGAGACAGCCAAGCA CCAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCATCGG CTTCCGGTACAGAAGCCTGAAGCACTTCAACTACGATATCTGC CAGAGCTGTTTCTTCAGCGGCAGGGTGGCCAAGGGCCACAAA ATGCACTACCCCATGGTGGAATACT GCACCCCCACCACAAGC GGCGAGGATGTGCGGGATTTCGCCAAGGTGCTGAAAAACAAG TTCCGGACCAAGCGGTACTTCGCCAAACACCCCCGGATGGGC TACCTGCCTGTGCAGACAGTGCTGGAAGGCGACAACATGGAA ACCGACACCATGTGATGATGATTTGGGCAGAGCGATGGAGTC CTTAGTATCAGTCATGACAGATGAAGAAGGAGCAGAATAAAT GTTTTACAACTCCTG ATTCCCGCATGCGGCCAGCTTATCGATA CCGTCGAAATAAAAGATCCTTATTTTCATTGGATCTGTGTGTT GGTTTTTTGTGTG

In some embodiments, the nucleic acid sequence encoding the H1 domain of the miniaturized dystrophin polypeptide is a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 94. In some embodiments, the nucleic acid sequence encoding the R1 domain is a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98 % is at least about 99% or about 100% identical to the sequence of SEQ ID NO: 95. In some embodiments, the nucleic acid sequence encoding the R3 domain is a sequence that is at least about 60% identical to by about 70%, by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95%, by at least about 96%, by at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 97. In some embodiments, the nucleic acid sequence encoding the H2 domain is a sequence that is at least at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 98. In some embodiments, the nucleic acid sequence encoding the domain R16 is a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 101. B In some embodiments, the nucleic acid sequence encoding the R17 domain is a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 102. In some embodiments, the nucleic acid sequence encoding the R24 domain is a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, according to

- 39046419 is at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 103. In some embodiments, the nucleic acid sequence encoding the H4 domain is a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 104. In some embodiments, the nucleic acid sequence encoding the ABD1 domain of the miniaturized dystrophin polypeptide is a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least is at least about 99% or about 100% identical to the sequence of SEQ ID NO: 93. In some embodiments, the nucleic acid sequence encoding the CR/C-terminal polypeptide is a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% , is at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 105.

Table 6B

Nucleotide sequence (and domain structure) encoding the miniaturized dystrophin polypeptide BXA-027743

SEQ ID NO: Description Nucleotide sequence 106 5'UTR CCTGCGGCCGCCTTCGGCACCATTCCTCACGACACCCAAATAT GGCGACGGGTGAGGAATGGTGGGGAGTTATTTTTTAGAGCGGT GAGGAAGGTGGGCAGGCAGCAGGTGTTGGCGCTCTAAAAATA ACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGGTGGACACC CAAATATGGCGACGGTTCCTCACCCGTCGCCATATTTGGGTGT CCGCCCTCGGCCGGGGCCGCATTCCT GGGGGCCGGGCGGTGC TCCCGCCCGCCTCGATAAAAGGCTCCGGGGCCGGCGGCGGCC CACGAGCTACCCGGAGGAGCGGGAGGCTCTAGACCACC 107 ABDI ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAG GACGTGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAG TTCAGCAAGTTCGGCAAGCAGCACATCGAGAACCTGTTCAGC GACCTGCAGGACGGCAGACGGCTGCTGGACCTGCTGGAAGGC CTGACCGGCCAGAAGCTGCCCAAAGAAGGGCAGCACCAG AGTGCACGCCCTGAACAACGTGAACAAGGC CCTGCGGGTGCT GCAGAACAACACGTGGACCTGGTGAACATCGGCAGCACCG ACATCGTGGACGGCAACCACAAGCTGACCCTGGGCCTGATCT GGAACATCATCCTGCACTGGCAGGTCAAAAACGTGATGAAGA

- 40 046419

ACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGATCC TGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGG TCAACGTGATCAACTTCACCACCTCTTGGAGCGACGGCCTGG CCCTGAACGCCCTGATCCACAGCCACCGGCCCGACCTGTTCG ACTGGAACAGCGTGGTCTGCCAGCAGAGCGCCACCCAGCGGC TGGAACACGCCTTCAATATCGCCAGATACCAG CTGGGCATCG AGAAGCTGCTGGATCCCGAGGACGTGGACACCACCTACCCCG ACAAGAAATCCATCCTGATGTATATCACCAGCCTGTTCCAGGT GCTGCCCCAGGTGTCCATCGAGGCCATCCAGGAAGTGGA A 108 Hinge 1 ATGCTGCCCAGACCCCCCAAAGTGACCAAAGAGGAACACTTC CAGCTGCACCACCAGATGCACTACAGCCAGCAGATCACCGTG TCCCTGGCTCAGGGCTACGAGCGGACCAGCAGCCCCAAGCCC CGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACC ACCAGCGACCCCACCAGAAGCCCATTCCCCAGCCAGCATCTG GAAGCCCCCGAGGACAAGAGCTTCGGCAGCAGC CTGATGGA AAGC 108 Spectrin -1 GAAGTGAACCTGGACAGATACCAGACCGCCCTGGAAGAGGT GCTGTCCTGGCTGCTGAGCGCCGAGGATACACTGCAGGCCCA GGGCGAGATCAGCAACGACGTGGAAGTGGTGAAAGACCAGT TCCACACCCACGAGGGCTACATGATGGACCTGACCGCCCACC AGGGCAGAGTGGGCAACATCCTGCAGCTGGGCAGCAAGCTG ATCGGCACCGGCAAGCTGAGCGAGGACGAA GAGACAGAGGT GCAGGAACAGATGAACCTGCTGAACAGCAGATGGGAGTGCCT GCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCAC 110 LI ATCCACACCGTGCGGGAAGAGACAATGATGGTGATGACCGAG GACATGCCCCTGGAAATC 111 Spectrin -16 AGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTG TCCCAGGCTCTGCTGGAAGTGGAACAGCTGCTGAACGCCCCC GACCTGTGCGCCAAGGACTTCGAGGATCTGTTCAAGCAGGAA GAGAGCCTGAAGAATATCAAGGACTCCCTGCAGCAGTCCAGC GGCCGGATCGACATCATCCACAGCAAGAAAACAGCCGCCCTG CAGTCCGCCACCCCCGTGGAAAGAGTGAAG CTGCAGGAGCC CTGAGCCAGCTGGACTTCCAGTGGGAGAAAGTGAACAAGATG TACAAGGACCGGCAGGGCAGATTCGACCGCAGC 112 Spectrin -17 GTGGAAAAGTGGCGGCGGTTCCACTACGACATCAAGATCTTC AACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGAGAAAGACC CAGATCCCCGAGAACTGGGAGCACGCCAAGTACAAGTGGTAT CTGAAAGAACTGCAGGATGGCATCGGCCAGCGGCAGACCGTG GTGCGCACACTGAATGCCACCGGCGAGGAAATCATCCAGCAG AGCAGCAAGACCGACGCCAGCATCCTGCAGGAAAAG CTGGG CTCCCTGAACCTGCGGTGGCAGGAAGTGTGCAAGCAGCTGAG CGACCGGAAGAAGAGGCTGGAAGAACAG

- 41 046419

FROM Hinge 3 GCCCCTGGCCTGACCACCATCGGCGCCAGCCCTACCCAGACC GTGACCCTGGTGACACAGCCCGTGGTGACAAAAGAGACAGCC ATCTCCAAGCTGGAAATGCCCAGCTCCCTGATGCTGGAA 114 Spectrin -23 TCTGACCAGTGGAAGCGTCTGCACCTTTCTCTGCAGGAACTTC TGGTGTGGCTACAGCTGAAAGATGATGAATTAAGCCGGCAGG CACCTATTGGAGGCGACTTTCCAGCAGTTCAGAAGCAGAACG ATGTACATAGGGCCTTCAAGAGGGAATTGAAAACTAAAGAAC CTGTAATCATGAGTACTCTTGAGACTGTACGAATATTTCTGAC AGAGCAGCCTTTGGAAGGACTAGAGAAACTCTAC CAGGAGCC CAGAGAGCTGCCTCCTGAGGAGAGAGCCCAGAATGTCACTCG GCTTCTACGAAAGCAGGCTGAGGAGGTCAATACTGAGTGGGA AAAATTGAACCTGCACTCCGCTGACTGGCAGAGAAAAATAGA TGAGACC 115 Spectrin -24 CTGGAAAGGCTGCAGGAACTGCAGGAGGCCACCGACGAGCT GGACCTGAAGCTGCGGCAGGCCGAAGTGATCAAGGGCAGCT GGCAGCCCGTGGGCGACCTGCTGATCGACAGCCTGCAGGACC ACCTGGAAAAAGTGAAGGCCCTGAGAGGCGAGATCGCCCCCC TGAAAGAAAACGTGTCCCACGTGAACGACCTGGCCCGGCAGC TGACCACCCTGGGCATCCAGCTGAGCCCCTACAA CCTGAGCA CCCTGGAAGATCTGAACACCCGGTGGAAGCTGCTGCAGGTGG CCGTGGAAGATAGAGTGCGGCAGCTGCACGAG 116 Hinge 4 GCCCACAGAGACTTTGGCCCTGCCAGCCAGCACTTCCTGAGC ACCTCTGTGCAGGGACCCTGGGAGAGAGCCATCAGCCCCAAC AAGGTGCCCTACTACATCAACCACGAGACACAGACCACCTGT GATGGTGACCACCCCAGCTACGACCGACGACCACCCCAGCTG ACGACCGACGACCACCCCAGCTGACGACCGAC 117 CR/Cconc AGGCGGCTGCAGAAAGCTCTGTGCCTGGATCTGCTGAGCCTG AGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAG AACGACCAGCCCATGGATATCCTGCAGATCATCAACTGCCTG ACCACAATCTACGACAGGCTGGAACAGGAACACAACAATCTG GTCAACGTGCCCCTGTGCGTGGACATGTGCCTGAATTGGCTGC TGAATGTGTACGACACCGGCCGGACCGGCAGA ATCCGGGTGC TGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGCCCACC TGGAAGATAAGTACCGCTACCTGTTCAAACAGGTGGCCAGCT CCACCGGCTTTTGCGACCAGCGGAGACTGGGCCTGCTGCTGC ACGACAGCATCCAGATCCCCAGACAGCTGGGCGAGGTGGCCT CCTTCGGCGGCAGCAACATTGAGCCCAGCGTGCGGAGCTGCT TCCAGTTCGCCAACAACAAGCCC GAGATCGAGGCCGCCCTGT TCCTGGACTGGATGAGACTGGAACCCCAGAGCATGGTGTGGC TGCCCGTGCTGCATCGGGTGGCCGCTGCCGAGACAGCCAAGC ACCAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCATCG GCTTCCGGTACAGAAGCCTGAAGCACTTCAACTACGATATCT GCCAGAGCTGCTTCTTCAGCGGCAGAGTGGCCAAGGGCCACA AAATGCACTACCCCAT GGTGGAATACTGCACCCCCACCACCA GCGGCGAGGATGTGCGGGACTTCGCCAAGGTGCTGAAAAACA AGTTCCGGACCAAGCGGTACTTTGCCAAGCACCCCCGGATGG GCTACCTGCCCGTGCAGACAGTGCTGGAAGGCGACAACATGG AAACCCCTGCCCAGATCCTGATCAGCCTGGAAAGCGAGGAAC GGGGCGAGCTGGAACGGATCCTGGCCGATCTGGAAGAGGAA AACCGGAACCTGCAGGCCGAGTACGACCGGCTGAAGCAGCA GCACGAGCACAAGGGCCTGAGCCCCCTGCCTAGCCCCCCTGA CACCATGTGA

Various miniaturized dystrophin polypeptides of the present invention are shown in table. 7.

Table 7

Amino acid sequences of miniaturized dystrophin constructs

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SEQ ID NO and description Subsequence SEQ ID NO:118 - BXA- 196477 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHRVLLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKD QNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQK TEAWLDNFARCWDNLVQKLEKSTAQIS QAVTTTQPSLTQTTVMETVTT VTTREQILVKHAQEELPPPPPQKKRQITVDSEAQIHTVREETMMVMTED MPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESL I<NII<DSLQQSSGRIDIIHSI<I<TAALQSATPVERVI<LQEALSQLDFQWEI< VNI<MYI<DRQGRFDRSVEI <WRRFHYDII<IFNQWLTEAEQFLRI<TQIPEN WEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQE KLGSLNLRWQEVCKQLSDRKKRLEEQLERLQELQEATDELDLKLRQAE VII<GSWQPVGDLLIDSLQDHLEI<VI<ALRGEIAPLI<ENVSHVNDLARQLT TLGI QLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQH FLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLN NVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMD ILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIR VLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIP RQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVW LPVLHRVAAAET AKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSG RVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPR MGYLPVQTVLEGDNMETDTM SEQ ID NO:119 - MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ

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BXA- 196473 QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLL NSRWECLR VASMEKQSNLHLLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQ NEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKT EAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVTTV TTREQILVKHAQEELPPPPPQKKRQITVDIHTVREETMM VMTEDMPLEIS YVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDS LQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMY KDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAK YKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSS KTDASILQEKLGSLN LRWQEVCKQLSDRKKRLEEQLERLQELQEATDELDLKLRQAEVIKGSW QPVGDLLIDSLQDHLEI<VI<ALRGEIAPLI<ENVSHVNDLARQLTTLGIQL SPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSV QGPWERAISPNKVPYYINHETQTTCWDHPK MTELYQSLADLNNVRFSA YRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINC LTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKT GIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEV ASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQ SMVWLPVLHR VAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKG HI<MHYPMVEYCTPTTSGEDVRDFAI<VLI<NI<FRTI<RYFAI<HPRMGYLP VQTVLEGDNMETDTM SEQ ID NO: 120 - BXA- 196474 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHLLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQ NEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKT EAWLDNFARCWDNLVQKLEKSTAQISQ AVTTTQPSLTQTTVMETVTTV TTREQILVKHAQEELPPPPPQKKRQITVDSEAQIHTVREETMMVMTEDM PLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLK NIKDSLQQSSGRroilHSKKTAALQSATPVERVKLQEALSQLDFQWEKV NKMYKDRQGRFDRSVEKWRRFHYDI KIFNQWLTEAEQFLRKTQIPENW EHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL GSLNLRWQEVCKQLSDRKKRLEEQKNILQELQEATDELDLKLRQAEVI I<GSWQPVGDLLIDSLQDHLEI<VI<ALRGEIAPLI<ENVSHVNDLARQLTT LGIQLSPY NLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHF LSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNN VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDI LQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRV LSFKTGIISLCKAHLEDKYRYLF KQVASSTGFCDQRRLGLLLHDSIQIPR QLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWL PVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGR VAI<GHI<MHYPMVEYCTPTTSGEDVRDFAI<VLI<NI<FRTI<RYFAI<HPR MGYLPVQTVLEGDNME TTPCDDDLGRAMESLVSVMTDEEGAE

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SEQ ID NO:121 - BXA- 196475 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHLLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQ NEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKT EAWLDNFARCWDNLVQKLEKSTAQISQ AVTTTQPSLTQTTVMETVTTV TTREQILVKHAQEELPPPPPQKKRQITVDSEAQIHTVREETMMVMTEDM PLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLK NII<DSLQQSSGRIDIIHSI<I<TAALQSATPVERVI<LQEALSQLDFQWEI<V NI<MYI<DRQGRFDRSVEI <WRRFHYDII<IFNQWLTEAEQFLRI<TQIPENW EHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL GSLNLRWQEVCKQLSDRKKRLEEQKLERLQELQEATDELDLKLRQAEV II<GSWQPVGDLLIDSLQDHLEI<VI<ALRGEIAPLI<ENVSHVNDLARQLTT LGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHF LSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNN VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDI V AI<GHI<MHYPMVEYCTPTTSGEDVRDFAI<VLI<NI<FRTI<RYFAI<HPR MGYLPVQTVLEGDNMETDTM SEQ ID NO: 122 - BXA- 196476 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHLLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQ NEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKT EAWLDNFARCWDNLVQKLEKSTAQISQ AVTTTQPSLTQTTVMETVTTV TTREQILVKHAQEELPPPPPQKKRQITVDSEAQIHTVREETMMVMTEDM PLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLK NII<DSLQQSSGRIDIIHSI<I<TAALQSATPVERVI<LQEALSQLDFQWEI<V NI<MYI<DRQGRFDRSVEI <WRRFHYDII<IFNQWLTEAEQFLRI<TQIPENW EHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL GSLNLRWQEVCKQLSDRKKRLEEQKNILERLQELQEATDELDLKLRQA EVII<GSWQPVGDLLIDSLQDHLEI<VI<ALRGEIAPLI<ENVSHVNDLARQL TTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQ HFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADL NNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPM DILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRI

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RVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQI PRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMV WLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFS GRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHP R MGYLPVQTVLEGDNMETDTM SEQ ID NO: 123 - BXA196477-Q MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHRVLLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKD QNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQK TEAWLDNFARCWDNLVQKLEKSTAQIS QAVTTTQPSLTQTTVMETVTT VTTREQILVKHAQEELPPPPPQKKRQITVDSEAQIHTVREETMMVMTED MPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESL I<NII<DSLQQSSGRIDIIHSI<I<TAALQSATPVERVI<LQEALSQLDFQWEI< VNI<MYI<DRQGRFDRSVEI <WRRFHYDII<IFNQWLTEAEQFLRI<TQIPEN WEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQE KLGSLNLRWQEVCKQLSDRKKRLEEQLERLQELQEATDELDLKLRQAE VII<GSWQPVGDLLIDSLQDHLEI<VI<ALRGEIAPLI<ENVSHVNDLARQLT TLGI QLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQH FLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLN NVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMD ILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIR VLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIP RQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVW LPVLHRVAAAET AKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSG RVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPR MGYLPVQTVLEGDNMETDTM SEQ ID NO: 124 - BXA- 196478 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHRVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHT TGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNK SVTQKTEAWLDNFARCWDNLVQKLEKSTA QISQAVTTTQPSLTQTTVM ETVTTVTTREQILVKHAQEELPPPPPQKKRQITVDSEAQIHTVREETMM VMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLF I<QEESLI<NII<DSLOQSSGRIDIIHSI<I<TAALOSATPVERVI<LQEALSQLD FQWEI<VNI<MYI<DRQGR FDRSVEI<WRRFHYDII<IFNQWLTEAEQFLRI< TQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTD ASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQLERLQELQEATDELDL

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I<LRQAEVII<GSWQPVGDLLIDSLQDHLEI<VI<ALRGEIAPLI<ENVSHVN DLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRD FGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQ SLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQ NDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDT GRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLL HDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLE PQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDIC QSCFF SGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKR YFAKHPRMGYLPVQTVLEGDNMETDTM SEQ ID NO: 125 - BXA- 196479 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHLLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQ NEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKT EAWLDNFARCWDNLVQKLEKSTAQISQ AVTTTQPSLTQTTVMETVTTV TTREQILVKHAQEELPPPPPQKKRQITVDSEAQIHTVREETMMVMTEDM PLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLK NII<DSLQQSSGRIDIIHSI<I<TAALQSATPVERVI<LQEALSQLDFQWEI<V NI<MYI<DRQGRFDRSVEI <WRRFHYDII<IFNQWLTEAEQFLRI<TQIPENW EHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL GSLNLRWQEVCKQLSDRKKRLEEQLERLQELQEATDELDLKLRQAEVI I<GSWQPVGDLLIDSLQDHLEI<VI<ALRGEIAPLI<ENVSHVNDLARQLTT LGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHF LSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNN VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDI V AI<GHI<MHYPMVEYCTPTTSGEDVRDFAI<VL1<NI<FRTI<RYFAI<HPR MGYLPVQTVLEGDNMETDTM SEQ ID NO: 126 - BXA- 196480 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQAL LEVEQLLNAPDLCAI<DFEDLFI<QEESLI<NII<DSLQQSSGRIDIIHSI<I<TA ALQSATPVERVKLQEALSQLDFQW EKVNKMYKDRQGRFDRSVEKWR RFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQR

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QTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKK RLEEQAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSLMLELERL QELQEATDELDL1<LRQAEVII<GSWQPVGDLLIDSLQDHLEI<VI<ALRGEI APLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQ VAVED RVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTC WDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAA CDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDM CLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASST GF CDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIE AALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFR YRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFA KVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPAQILISLESEERG ELERIL ADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPDTM SEQ ID NO: 127 BXA- 196480- option MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSLEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHRVLIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVS QALLEVEQLLNAPDLCAI<DFEDLFI<QEESLI<NII<DSLQQSSGRIDIIHSI< KTAALQSATPVERVKLQEALSQLDFQW EKVNKMYKDRQGRFDRSVEK WRRFHYDII<IFNQWLTEAEQFLRI<TQIPENWEHAI<YI<WYLI<ELQDGIG QRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDR KKRLEEQKNDLAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSL MLEVERLQELQEATDEL DL1<LRQAEVII<GSWQPVGDLLIDSLQDHLEI< VKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKL LQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYIN HETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLD LLSLSAACDALDQHNLKQND QPMDILQIINCLTTIYDRLEQEHNNLVNV PLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFK QVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFA NNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICK ECPIIGFRYRSLKHFNYDICQSCFFS GRVAKGHKMHYPMVEYCTPTTSG EDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPAQILI SLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPDT M SEQ ID NO: 128 - BXA- 196481 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSLEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHSYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDL FKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQL

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DFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLR KTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKT DASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQLEGLEKLYQEPRELPP EERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQ RKIDETLERLQELQE ATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKE NVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQL HEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPK MTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSL SAACDALD QHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWL LNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQ RRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFL DWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRY RSLK HFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKN KFRTKRYFAKHPRMGYLPVQTVLEGDNMETPAQILISLESEERGELERIL ADLEEENRNLQ AE YDRLKQQHEHKGLSPLP SPPDTM SEQ GO NO: 129 - BXA- 027741 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHLLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQ NEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKT EAWLDNFARCWDNLVQKLEKSTAQISQ AVTTTQPSLTQTTVMETVTTV TTREQILVKHAQEELPPPPPQKKRQITVDIHTVREETMMVMTEDMPLEIS YVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDS LQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMY KDRQGRFDRSVEKWRRFHYDIKIFNQWLT EAEQFLRKTQIPENWEHAK YKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLN LRWQEVCKQLSDRKKRLEEQLERLQELQEATDELDLKLRQAEVIKGSW QPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQL SPYNLSTLEDLNTRWKLLQV AVEDRVRQLHEAHRDFGPASQHFLSTSV QGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSA YRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINC LTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKT GIISLCKAHLEDKYRYLFKQVASSTGFCDQRR LGLLLHDSIQIPRQLGEV ASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHR VAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKG HI<MHYPMVEYCTPTTSGEDVRDFAI<VL1<NI<FRTI<RYFAI<HPRMGYLP VQTVLEGDNMETPAQILISLESEERGEL ERIL ADLEEENRNLQ AEYDRLK QQHEHKGLSPLPSPPDTM SEQ ID NO:130 - BXA- 027744 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN

- 49 046419

LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQAL LEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKK TA ALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWR RFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQR QTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKK RLEEQAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSS LMLEVPAL ADFNRAWTELTDWLSLLDQVIKSQRVMVGDLEDINEMIIKQKATMQDL EQRRPQLEELITAAQNLKNKTSNQEARTIITDRIERIQNQWDEVQEHLQN RRQQLNEMLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQ DHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPY NLSTLEDLN TRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNK VPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQ KALCLDLLLSSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEH NNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLED KY RYLFKQ VAS STGFCDQRRLGLLLHD SIQIPRQLGEVASFGGSNIEPS V RSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQ AKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEY CTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNM ET PAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSP LPSPPDTM SEQ ID NO:131 - BXA- 027742 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPD LEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAAL EEQLKVLGDRWANICRWTEDRWVLLQ DILLKWQRLTEEQCLFSAWLS EKEDAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSL KQDLLSTLKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQAIHT VREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCA KDFEDLFKQEESLKNIKDSLQQSSGRIDIIHS KKTAALQSATPVERVKLQ EALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTE AEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEII QQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQLERLQELQE ATDELDLKLRQAEVIKGSWQ PVGDLLIDSLQDHLEKVKALRGEIAPLKE NVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQL HEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPK MTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALD QHNLKQNDQPMDILQIINCLTTIYDRLEQE HNNLVNVPLCVDMCLNWL LNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQ RRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFL DWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLK HFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPT TSGEDVRDFAKVLKN KFRTKRYFAKHPRMGYLPVQTVLEGDNMETDTM

- 50 046419

SEQ ID

NO:132 BXA027743

MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIG STDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSW VRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQS ATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQ QVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTS SPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMM DLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLR VASMEKQSNLHIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQAL LEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTA ALQSATPVERVKLQEALSQLDFQWEKVNKMY KDRQGRFDRSVEKWR RFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQR QTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKK RLEEQAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSLMLESDQ WKRLHLSLQELLVWLQLKDDELSRQ APIGGDFPAVQKQNDVHRAFKR ELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLL RI<QAEEVNTEWEI<LNLHSADWQRI<IDETLERLQELQEATDELDLI<LRQ AEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLAR QLTTLGIQLSPYNLSTLEDLNTR WKLLQVAVEDRVRQLHEAHRDFGPA SQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLA DLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQ PMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRT GRIRVLSFKTGIISLCKAHLEDKYRYLFKQVAS STGFCDQRRLGLLLHDS IQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQS MVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSC FFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFA KHPRMGYLPVQTVLEGDNMETPAQILISLESEERGE LERILADLEEENRN LQAEYDRLKQQHEHKGLSPLPSPPDTM

In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 118-132, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to one of the sequences , given in SEQ ID NO: 118 (ВХА-196477), SEQ ID NO: 119 (ВХА-196473), SEQ ID NO: 120 (ВХА-196474), SEQ ID NO: 121 (BXA196475), SEQ ID NO: 122 (BXA-196476), SEQ ID NO: 124 (BXA-196478) or SEQ ID NO: 125 (BXA196479). In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 118, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 119, where the amino acid sequence, when expressed, has at least one dystrophin activity. In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 120, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%

- 51 046419 by at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, according to is at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 121, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 122, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 123, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 124, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 125, wherein the amino acid sequence, when expressed, has at least one dystrophin activity.

In certain embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 126, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In certain embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 127, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In certain embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 128, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In certain embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 129, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In certain embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 130, where

- 52 046419 amino acid sequence, when expressed, has at least one dystrophin activity. In certain embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 131, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In certain embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 132, wherein the amino acid sequence, when expressed, has at least one dystrophin activity. In some embodiments, the miniaturized dystrophin polypeptide encoded by the nucleic acid molecule has formula (I):

H1-R1-L1-R3 -H2-L2-L3 -RI 6-R17-R24-H4 (I) where H1 is dystrophin hinge 1 domain; R1 is dystrophin spectrin repeat domain 1; L1 is Arg-Val (RV) amino acids; R3 is spectrin repeat domain 3; H2 is dystrophin hinge 2 domain; L2 is the amino acid sequence shown in SEQ ID NO: 74 (SEAQ); L3 is the amino acid sequence shown in SEQ ID NO: 75 (IHTVREE TMMVMTEDMP LEI); R16 is dystrophin spectrin repeat 16; R17 is dystrophin spectrin repeat 17; R24 is dystrophin spectrin repeat 24; H4 is dystrophin hinge 4 domain; and (-) represents a peptide bond.

In some embodiments, the miniaturized dystrophin polypeptide encoded by the nucleic acid molecule exhibits one or more properties selected from the group consisting of (i) lower CD4 proliferation compared to BXA-027741, (ii) lower CD8 proliferation compared to BXA -027741, and (iv) any combination thereof.

In some embodiments, the miniaturized dystrophin polypeptide encoded by the nucleic acid molecule has formula (II):

H1-R1-L-R16-R17-H3-R23-R24-H4 (II) where: H1 is dystrophin hinge 1 domain; R1 is dystrophin spectrin repeat domain 1; L represents amino acids SEQ ID NO: 84 (IHTVREETMMVMTEDMPLEI); R16 is dystrophin spectrin repeat 16; R17 is dystrophin spectrin repeat 17; H3 is dystrophin hinge 3 domain; R23 is dystrophin spectrin repeat 23; R24 is dystrophin spectrin repeat 24; H4 is dystrophin hinge 4 domain; and (-) represents a peptide bond.

In some embodiments, the miniaturized dystrophin polypeptide encoded by the nucleic acid molecule comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 132. In some embodiments, the nucleotide sequence includes a sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75 %, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, is at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequence of SEQ ID NO: 147.

In some embodiments, the miniaturized dystrophin polypeptide exhibits a higher level of expression of the miniaturized dystrophin polypeptide than BXA196481. In some other embodiments, the expression level of the miniaturized dystrophin polypeptide is at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9 times, at least about 2 times, at least about 2.1 times, at least about 2.2 times, at least about 2.3 times, at least at least about 2.4 times, at least about 2.5 times, at least about 2.6 times, at least about 2.7 times, at least about 2.8 times, at least

- 53 046419 at least about 2.9 times or at least about 3 times higher than the expression level of the BXA-196481 polypeptide.

In some embodiments, miniaturized dystrophin polypeptides may be encoded by nucleotide sequences. Some examples of nucleotide sequences are presented in table. 8.

Table 8

Nucleotide sequences of dystrophin constructs

SEQ Ш NO and description Subsequence SEQ Ш NO: 133 - VHA- 196477 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGACCACC AGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGAGCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAACG

- 54 046419

ACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCTCCAAGCTGATCGGCACCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCATAG GGTCCTGCT GAAGTGGCAGCGGCTGACCGAGGAACAGTGCCTGTTT AGCGCCTGGCTGTCCGAGAAAGAGGACGCCGTGAACAAGATCCACA CCACCGGCTTCAAGGACCAGAACGATGCTGAGCAGCCTGCAGAA ACTGGCCGTGCTGAAGGCCGACCTGGAAAAGAAAAAGCAGTCCATG GGCAAGCTGTACTCCCTGAAGCAGGACCTGCTGTCCACCCTGAAGA ACAAGAGCGTGACCCAGAAAACCGA GGCCTGGCTGGACAACTTCGC CCGGTGCTGGGACAACCTGGTGCAGAAGCTGGAAAAGTCCACCGCC CAGATCTCCCAGGCCGTGACCACAACACAGCCCAGCCTGACCCAGA CCACCGTGATGGAAACCGTGACAACAGTGACCACCCGGGAACAGAT CCTCGTGAAGCACGCCCAGGAAGAACTGCCCCCTCCACCCCCCCAG AAGAAACGGCAGATCACAGTGGACAGTGAAGCTCAGATCC ACACCG TGCGGGAAGAGACAATGATGGTCATGACAGAGGACATGCCCCTGGA AATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCATGTGT CCCAGGCCCTGCTGGAAGTGGAACAGCTGCTGAACGCCCCCGACCT GTGCGCCAAGGATTTCGAGGACCTGTTCAAGCAGGAAGAGAGCCTG AAGAATATCAAGGATAGCCTGCAGCAGAGCAGCGGCCGGATCGACA TCATCC ACAGCAAGAAAACAGCCGCCCTGCAGAGCGCCACCCCCGT GGAAAGAGTGAAACTGCAGGAAGCCCTGTCCCAGCTGGACTTCCAG TGGGAGAAAGTGAACAAAATGTACAAGGACCGGCAGGGCAGATTCG ACCGCAGCGTGGAAAAGTGGCGGCGGTTCCACTACGACATCAAGAT CTTCAACCAGTGGCTGACAGAGGCCGAGCAGTTCCTGAGAAAGACC CAGATCCCCGAGAACTGGGAGC ACGCCAAGTACAAGTGGTATCTGA AAGAGCTGCAGGACGGCATCGGCCAGAGACAGACAGTCGTGCGGAC CCTGAATGCCACCGGCGAGGAAATCATCCAGCAGTCCAGCAAGACC GACGCCAGCATTCTGCAGGAAAAGCTGGGCAGCCTGAACCTGCGGT GGCAGGAAGTGTGCAAGCAGCTGTCCGACCGGAAGAAGCGGCTGGA AGAACAGCTGGAACGGCTGCAGGAACTGCAG GAGGCCACCGACGA GCTGGACCTGAAACTGAGACAGGCCGAAGTGATCAAGGGCAGCTGG CAGCCAGTGGGCGACCTGCTGATCGACTCCCTGCAGGACCATCTGGA AAAAGTGAAGGCCCTGAGAGGCGAGATCGCCCCTGAAAGAAAC GTGTCCCACGTGAACGACCTGGCCCGGCAGCTGACAACACTGGGCA TTCAGCTGAGCCCCTACAACCTGAGCACACTGGAAGATCTGAACA CC CGGTGGAAGCTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGC TGCACGAGGCCCACAGAGATTTTGGCCCTGCCTCCCAGCACTTCCTG AGCACCAGTGTGCAGGGCCCTTGGGAGAGAGCCATCTCCCCTAACA AGGTGCCCTACTACATCAACCACGAGACACAGACCACCTGTTGGGA CCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCTGACCTGAAC AACGTGCGG TTCAGCGCCTACCGGACCGCCATGAAGCTGCGGAGAC TGCAGAAAGCTCTGTGCCTGGATCTGCTGTCCCTGTCCGCCGCCTGT GATGCCCTGGACCAGCACAATCTGAAGCAGAACGACCAGCCCATGG ATATCCTGCAGATCATCAACTGCCTGACCACCATCTACGACCGGCTG GAACAGGAACACAACAATCTCGTGAACGTGCCCCTGTGCGTGGACA TGTGCCTGAATTGGCTGCTGAA TGTGTACGACACCGGCCGGACAGGC

- 55 046419

CGGATCAGAGTGCTGTCCTTCAAGACCGGCATCATCAGCCTGTGCAA AGCCCACCTGGAAGATAAGTACCGGTATCTGTTCAAACAGGTGGCCT CTAGCACCGGCTTTTGCGACCAGAGAAGGCTGGGCCTGCTGCTGCAC GACAGCATCCAGATCCCTAGACAGCTGGGCGAAGTGGCCAGCTTTG GCGGCAGCAACATCGAGCCTAGCGTGCGGAGCTGCTTCCAGTTCGCC AACA ACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGA GACTGGAACCCCAGAGCATGGTGTGGCTGCCCGTGCTGCATAGAGT GGCCGCTGCCGAGACAGCCAAGCACCAGGCCAAGTGCAACATCTGC AAAGAGTGCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGCACTT CAACTACGATATCTGCCAGAGCTGTTTCTTCAGCGGCAGGGTGGCCA AGGGCCACAAAATGCACT ACCCCATGGTGGAATACTGCACCCCCAC CACAAGCGGCGAGGATGTGCGGGATTTCGCCAAGGTGCTGAAAAAC AAGTTCCGGACCAAGCGGTACTTCGCCAAACACCCCCGGATGGGCT ACCTGCCTGTGCAGACAGTGCTGGAAGGCGACAACATGGAAACCGA CACCATGTGATGATGATTTGGGCAGAGCGATGGAGTCCTTAGTATCA GTCATGACAGATGAAGAAGGAGCAGAATAAATGTTT TACAACTCCT GATTCCCGCATGCGGCCAGCTTATCGATACCGTCGAAATAAAAGATC CTTATTTTCATTGGATCTGTGTGTTGGTTTTTTGTGTG SEQ ID NO: 134 - BXA- 196473 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGACCACC AGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGAGCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAACG ACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCTCCAAGCTGATCGGCA CCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCATCT GCTGAAGTGGCAGCGGCTGACCGAGGAACAGTGCCTGTTTAGCGCC

- 56 046419

TGGCTGTCCGAGAAAGAGGACGCCGTGAACAAGATCCACACCACG GCTTCAAGGACCAGAACGAGATGCTGAGCAGCCTGCAGAAACTGGC CGTGCTGAAGGCCGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAG CTGTACTCCCTGAAGCAGGACCTGCTGTCCACCCTGAAGAACAAGA GCGTGACCCAGAAAACCGAGGCCTGGCTGGACAACTTCGCCCGGTG CTGGGACAAC CTGGTGCAGAAGCTGGAAAAGTCCACCGCCCAGATC TCCCAGGCCGTGACCACAACACAGCCCAGCCTGACCCAGACCACG TGATGGAAACCGTGACAACAGTGACCACCCGGGAACAGATCCTCGT GAAGCACGCCCAGGAAACTGCCCCCTCCACCCCCCCAGAAGAAA CGGCAGATCACAGTGGACATCCACACCGTGCGGGAAGAGACAATGA TGGTCATGACAGAGGACATGCCC CTGGAAATCAGCTACGTGCCCAG CACCTACCTGACCGAGATCACCCATGTGTCCCAGGCCCTGCTGGAAG TGGAACAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGATTTCGA GGACCTGTTCAAGCAGGAAGAGCCTGAAGAATATCAAGGATAGC CTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAAA CAGCCGCCCTGCAGAGCGCCACCCCCGTGGAAAGAG C GGCCAGAGACAGACAGTCGTGCGGACCCTGAATGCCACCGGCGAG GAAATCATCCAGCAGTCCAGCAACCGACGCCAGCATTCTGCAGG AAAAGCTGGGCAGCCTGAACCTGCGGTGGCAGGAAGTGTGCAAGCA GCTGTCCGACCGGAAGAAGCGGCTGGAAGAACAGCTGGAACGGCTG CAGGAACTGCAGGAGGCCACCGACGAGCTGGACCTGAAACTGAGAC AGGCCGAAGTGATCAAG GGCAGCTGGCAGCCAGTGGGCGACCTGCT GATCGACTCCCTGCAGGACCATCTGGAAAAAGTGAAGGCCCTGAGA GGCGAGATCGCCCCCCTGAAAGAAAACGTGTCCCACGTGAACGACC TGGCCCGGCAGCTGACAACACTGGGCATTCAGCTGAGCCCCTACAA CCTGAGCACACTGGAAGATCTGAACACCCGGTGGAAGCTGCTGCAG GTGGCCGTGGAAGATAGAGTGCGGCAGCTGC ACGAGGCCCACAGAG ATTTTGGCCCTGCCTCCCAGCACTTCCTGAGCACCAGTGTGCAGGGC CCTTGGGAGAGAGCCATCTCCCCTAACAAGGTGCCCTACTACATCAA CCACGAGACACAGACCACCTGTTGGGACCACCCCAAGATGACCGAG CTGTACCAGAGCCTGGCTGACCTGAACAACGTGCGGTTCAGCGCCTA CCGGACCGCCATGAAGCTGCGGAGACTGCAGAAAGCTCTGTGCC TG GATCTGCTGTCCCTGTCCGCCGCCTGTGATGCCCTGGACCAGCACAA TCTGAAGCAGAACGACCAGCCCATGGATATCCTGCAGATCATCAACT GCCTGACCACCATCTACGACCGGCTGGAACAGGAACACAACAATCT CGTGAACGTGCCCCTGTGCGTGGACATGTGCCTGAATTGGCTGCTGA ATGTGTACGACACCGGCCGGACAGGCCGGATCAGAGTGCTGTCCTTC AAGA CCGGCATCATCAGCCTGTGCAAAGCCCACCTGGAAGATAAGT ACCGGTATCTGTTCAAACAGGTGGCCTCTAGCACCGGCTTTTGCGAC CAGAGAAGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCTA GACAGCTGGGCGAAGTGGCCAGCTTTGGCGGCAGCAACATCGAGCC TAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATC GAGGCCGCCCTGTTCCTG ACTGGATGAGACTGGAACCCCAGAGCA

- 57 046419

TGGTGTGGCTGCCCGTGCTGCATAGAGTGGCCGCTGCCGAGACAGCC AAGCACCAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCATCG GCTTCCGGTACAGAAGCCTGAAGCACTTCAACTACGATATCTGCCAG AGCTGTTTCTTCAGCGGCAGGGTGGCCAAGGGCCACAAAATGCACT ACCCCATGGTGGAATACTGCACCCCCACCACAAGCGGCGAGGATGT GCGGGATTTCGCC AAGGTGCTGAAAAACAAGTTCCGGACCAAGCGG TACTTCGCCAAACACCCCCGGATGGGCTACCTGCCTGTGCAGACAGT GCTGGAAGGCGACAACATGGAAACCGACACCATGTGATGATGATTT GGGCAGAGCGATGGAGTCCTTAGTATCAGTCATGACAGATGAAGAA GGAGCAGAATAAATGTTTTACAACTCCTGATTCCCGCATGCGGCCAG CTTATCGATACCGTCGAAATA AAAGATCCTTATTTTCATTGGATCTGT GTGTTGGTTTTTTGTGTG SEQ ID NO: 135 - BXA- 196474 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGACCACC AGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGAGCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAACG ACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCTCCAAGCTGATCGGCA CCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCATCT GCTGAAGTGGCAGCGGCTGACCGAGGAACAGTGCCTGTTTAGCGCC TGGCTGTCCGAGAAAGAGGACGCCGTGAACAAGATCCACACCACCG GCTTCAAGGACCAGAACGAGATGCTGAGCAGCCTGCA GAAACTGGC CGTGCTGAAGGCCGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAG CTGTACTCCCTGAAGCAGGACCTGCTGTCCACCCTGAAGAACAAGA GCGTGACCCAGAAAACCGAGGCCTGGCTGGACAACTTCGCCCGGTG CTGGGACAACCTGGTGCAGAAGCTGGAAAAGTCCACCGCCCAGATC

- 58 046419

TCCCAGGCCGTGACCACAACACAGCCCAGCCTGACCCAGACCACG TGATGGAAACCGTGACAACAGTGACCACCCGGGAACAGATCCTCGT GAAGCACGCCCAGGAAGAACTGCCCCCTCCACCCCCCCAGAAGAAA CGGCAGATCACAGTGGACAGTGAAGCTCAGATCCACACCGTGCGGG AAGACAAATGATGGTCATGACAGAGGACATGCCCCTGGAAATCAG CTACGTGCC CAGCACCTACCTGACCGAGATCACCCATGTGTCCCAGG CCCTGCTGGAAGTGGAACAGCTGCTGAACGCCCCCGACCTGTGCGCC AAGGATTTCGAGGACCTGTTCAAGCAGGAAGAGAGCCTGAAGAATA TCAAGGATAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCA CAGCAAGAAAACAGCCGCCCTGCAGAGCGCCACCCCCGTGGAAAGA GTGAAACTGCAGGAAGCCCTG TCCCAGCTGGACTTCCAGTGGGAGA AAGTGAACAAAATGTACAAGGACCGGCAGGGCAGATTCGACCGCAG CGTGGAAAAGTGGCGGCGGTTCCACTACGACATCAAGATCTTCAAC CAGTGGCTGACAGAGGCCGAGCAGTTCCTGAGAAAGACCCAGATCC CCGAGAACTGGGAGCACGCCAAGTACAAGTGGTATCTGAAAGAGCT GCAGGACGGCATCGGCCAGACAGACAGTCGTG CGGACCCTGAAT GCCACCGGCGAGGAAATCATCCAGCAGTCCAGCAAGACCGACGCCA GCATTCTGCAGGAAAAGCTGGGCAGCCTGAACCTGCGGTGGCAGGA AGTGTGCAAGCAGCTGTCCGACCGGAAGAAGCGGCTGGAAGAACAG AAGAACATCCTGCAGGAACTGCAGGAGGCCACCGACGAGCTGGACC TGAAACTGAGACAGGCCGAAGTGATCAAGGGCAGCTGGCAGCCAGT G GGCGACCTGCTGATCGACTCCCTGCAGGACCATCTGGAAAAAGTG AAGGCCCTGAGAGGCGAGATCGCCCCCCTGAAAGAAAACGTGTCCC ACGTGAACGACCTGGCCCGGCAGCTGACAACACTGGGCATTCAGCT GAGCCCCTACAACCTGAGCACACTGGAAGATCTGAACACCCGGTGG AAGCTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGCTGCACG AGGCCCACAGAGATTTT GGCCCTGCCTCCCAGCACTTCCTGAGCACC AGTGTGCAGGGCCCTTGGGAGAGAGCCATCTCCCCTAACAAGGTGC CCTACTACATCAACCACGAGACACAGACCACCTGTTGGGAACCACCCC AAGATGACCGAGCTGTACCAGAGCCTGGCTGACCTGAACAACGTGC GGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGAGACTGCAGAA AGCTCTGTGCCTGGATCTGCTGTCCCTG TCCGCCGCCTGTGATGCCCT GGACCAGCACAATCTGAAGCAGAACGACCAGCCCATGGATATCCTG CAGATCATCAACTGCCTGACCACCATCTACGACCGGCTGGAACAGG AACACAACAATCTCGTGAACGTGCCCCTGTGCGTGGACATGTGCCTG AATTGGCTGCTGAATGTGTACGACACCGGCCGGACAGGCCGGATCA GAGTGCTGTCCTTCAAGACCGGCATCATCAGCCTGTGCAAA AAC CCCAGAGCATGGTGTGGCTGCCCGTGCTGCATAGAGTGGCCGCT GCCGAGACAGCCAAGCACCAGGCCAAGTGCAACATCTGCAAAGAGT GCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGCACTTCAACTAC GATATCTGCCAGAGCTGTTTCTTCAGCGGCAGGGTGGCCAAGGGCCA CAAAATGCACTACCCCATGGTGGAATACTGCACCCCCACCACAAGC GGCGAGGATGTGCG ATTTCGCCAAGGTGCTGAAAACAAGTTCC

- 59 046419

GGACCAAGCGGTACTTCGCCAAACACCCCCGGATGGGCTACCTGCCT GTGCAGACAGTGCTGGAAGGCGACAACATGGAAACCACACCATGTG ATGATGATTTGGGCAGAGCGATGGAGTCCTTAGTATCAGTCATGACA GATGAAGAAGGAGCAGAATAAATGTTTTACAACTCCTGATTCCCGC ATGCGGCCAGCTTATCGATACCGTCGAAATAAAAGATCCTTATTTTTC ATTGGATCTGTGTG TTGGTTTTTTTGTGTG SEQ ID NO: 136 - BXA- 196475 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGACCACC AGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGAGCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAACG ACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCTCCAAGCTGATCGGCA CCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCATCT GCTGAAGTGGCAGCGGCTGACCGAGGAACAGTGCCTGTTTAGCGCC TGGCTGTCCGAGAAAGAGGACGCCGTGAACAAGATCCACACCACCG GCTTCAAGGACCAGAACGAGATGCTGAGCAGCCTGCA GAAACTGGC CGTGCTGAAGGCCGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAG CTGTACTCCCTGAAGCAGGACCTGCTGTCCACCCTGAAGAACAAGA GCGTGACCCAGAAAACCGAGGCCTGGCTGGACAACTTCGCCCGGTG CTGGGACAACCTGGTGCAGAAGCTGGAAAAGTCCACCGCCCAGATC TCCCAGGCCGTGACCACAACACAGCCCAGCCTGACCCAGACCACG TG ATGGAAACCGTGACAACAGTGACCACCCGGGAACAGATCCTCGT GAAGCACGCCCAGGAAGAACTGCCCCCTCCACCCCCCCAGAAGAAA CGGCAGATCACAGTGGACAGTGAAGCTCAGATCCACACCGTGCGGG AAGACAAATGATGGTCATGACAGAGGACATGCCCCTGGAAATCAG CTACGTGCCCAGCACCTACCTGACCGAGATCACCCCATGTGTCCCAGG

- 60 046419

CCCTGCTGGAAGTGGAACAGCTGCTGAACGCCCCCGACCTGTGCGCC AAGGATTTCGAGGACCTGTTCAAGCAGGAAGAGAGCCTGAAGAATA TCAAGGATAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCA CAGCAAGAAAACAGCCGCCCTGCAGAGCGCCACCCCCGTGGAAAGA GTGAAACTGCAGGAAGCCCTGTCCCAGCTGGACTTCCAGTGGGAGA AAGTGAACAAAA TGTACAAGGACCGGCAGGGCAGATTCGACCGCAG CGTGGAAAAGTGGCGGCGGTTCCACTACGACATCAAGATCTTCAAC CAGTGGCTGACAGAGGCCGAGCAGTTCCTGAGAAAGACCCAGATCC CCGAGAACTGGGAGCACGCCAAGTACAAGTGGTATCTGAAAGAGCT GCAGGACGGCATCGGCCAGACAGACAGTCGTGCGGACCCTGAAT GCCACCGGCGAGGAAATCATCCAGCA GTCCAGCAAGACCGACGCCA GCATTCTGCAGGAAAAGCTGGGCAGCCTGAACCTGCGGTGGCAGGA AGTGTGCAAGCAGCTGTCCGACCGGAAGAAGCGGCTGGAAGAACAG AAGCTGGAACGGCTGCAGGAACTGCAGGAGGCCACCGACGAGCTGG ACCTGAAACTGAGACAGGCCGAAGTGATCAAGGGCAGCTGGCAGCC AGTGGGCGACCTGCTGATCGACTCCCTGCAGGACCATCTGG AAAAA GTGAAGGCCCTGAGAGGCGAGATCGCCCCCCTGAAAGAAAACGTGT CCCACGTGAACGACCTGGCCCGGCAGCTGACAACACTGGGCATTCA GCTGAGCCCCTACAACCTGAGCACACTGGAAGATCTGAACACCCGG TGGAAGCTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGCTGC ACGAGGCCCACAGAGATTTTGGCCCTGCCTCCCAGCACTTCCTGAGC ACCAGTGTGCAGG GCCCTTGGGAGAGAGCCATCTCCCCTAACAAGG TGCCCTACTACATCAACCACGAGACACAGACCACCTGTTGGGACCAC CCCAAGATGACCGAGCTGTACCAGAGCCTGGCTGACCTGAACAACG TGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGAGACTGCA GAAAGCTCTGTGCCTGGATCTGCTGTCCCTGTCCGCCGCCTGTGATG CCCTGGACCAGCACAATCTGAAGCAGA ACGACCAGCCCATGGATAT CCTGCAGATCATCAACTGCCTGACCACCATCTACGACCGGCTGGAAC AGGAACACAACAATCTCGTGAACGTGCCCCTGTGCGTGGACATGTG CCTGAATTGGCTGCTGAATGTGTACGACACCGGCCGGACAGGCCGG ATCAGAGTGCTGTCCTTCAAGACCGGCATCATCAGCCTGTGCAAAGC CCACCTGGAAGATAAGTACCGGTATCTGTTCAAACAGGTGGCC TCTA GCACCGGCTTTTGCGACCAGAGAAGGCTGGGCCTGCTGCTGCACGA CAGCATCCAGATCCCTAGACAGCTGGGCGAAGTGGCCAGCTTTGGC GGCAGCAACATCGAGCCTAGCGTGCGGAGCTGCTTCCAGTTCGCCA ACAACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGAG ACTGGAACCCCAGAGCATGGTGTGGCTGCCCGTGCTGCATAGAGTG GCCGCTG CCGAGACAGCCAAGCACCAGGCCAAGTGCAACATCTGCA AAGAGTGCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGCACTTC AACTACGATATCTGCCAGAGCTGTTTCTTCAGCGGCAGGGTGGCCAA GGGCCACAAAATGCACTACCCCATGGTGGAATACTGCACCCCCACC ACAAGCGGCGAGGATGTGCGGGATTTCGCCAAGGTGCTGAAAAACA AGTTCCGGACCAAGCGGTACT TCGCCAAACACCCCCGATGGGCTA CCTGCCTGTGCAGACAGTGCTGGAAGGCGACAACATGGAAACCGAC ACCATGTGATGATGATTTGGGCAGAGCGATGGAGTCCTTAGTATCAG TCATGACAGATGAAGAAGGAGCAGAATAAATGTTTTTACAACTCCTG ATTCCCGCATGCGGCCAGCTTATCGATACCGTCGAAATAAAAGATCC TTATTTTCATTGGATCTGTGTGTTGGTTTTTTGTG TG

- 61 046419

SEQ ID

NO: 137 BXA196476

ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGACCACC AGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGAGCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAACG ACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCTCCAAGCTGATCGGCA CCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCATCT GCTGAAGTGGCAGCGGCTGACCGAGGAACAGTGCCTGTTTAGCGCC TGGCTGTCCGAGAAAGAGGACGCCGTGAACAAGATCCACACCACCG GCTTCAAGGACCAGAACGAGATGCTGAGCAGCCTGCA GAAACTGGC CGTGCTGAAGGCCGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAG CTGTACTCCCTGAAGCAGGACCTGCTGTCCACCCTGAAGAACAAGA GCGTGACCCAGAAAACCGAGGCCTGGCTGGACAACTTCGCCCGGTG CTGGGACAACCTGGTGCAGAAGCTGGAAAAGTCCACCGCCCAGATC TCCCAGGCCGTGACCACAACACAGCCCAGCCTGACCCAGACCACG TG ATGGAAACCGTGACAACAGTGACCACCCGGGAACAGATCCTCGT GAAGCACGCCCAGGAAGAACTGCCCCCTCCACCCCCCCAGAAGAAA CGGCAGATCACAGTGGACAGTGAAGCTCAGATCCACACCGTGCGGG AAGACAAATGATGGTCATGACAGAGGACATGCCCCTGGAAATCAG CTACGTGCCCAGCACCTACCTGACCGAGATCACCATGTGTCCCAGG CCCTGCTGGAAGTG GAACAGCTGCTGAACGCCCCCGACCTGTGCGCC AAGGATTTCGAGGACCTGTTCAAGCAGGAAGAGAGCCTGAAGAATA TCAAGGATAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCA CAGCAAGAAAACAGCCGCCCTGCAGAGCGCCACCCCCGTGGAAAGA GTGAAACTGCAGGAAGCCCTGTCCCAGCTGGACTTCCAGTGGGAGA AAGTGAACAAAATGTACAAGGACCGGCA GGGCAGATTCGACCGCAG

- 62 046419

CGTGGAAAAGTGGCGGCGGTTCCACTACGACATCAAGATCTTCAAC CAGTGGCTGACAGAGGCCGAGCAGTTCCTGAGAAAGACCCAGATCC CCGAGAACTGGGAGCACGCCAAGTACAAGTGGTATCTGAAAGAGCT GCAGGACGGCATCGGCCAGAGACAGACAGTCGTGCGGACCCTGAAT GCCACCGGCGAGGAAATCATCCAGCAGTCCAGCAAGACCGACGCCA GCATTCTGCA GGAAAAGCTGGGCAGCCTGAACCTGCGGTGGCAGGA AGTGTGCAAGCAGCTGTCCGACCGGAAGAAGCGGCTGGAAGAACAG AAGAACATCCTGGAACGGCTGCAGGAACTGCAGGAGGCCACCGACG AGCTGGACCTGAAACTGAGACAGGCCGAAGTGATCAAGGGCAGCTG GCAGCCAGTGGGCGACCTGCTGATCGACTCCCTGCAGGACCATCTGG AAAAAGTGAAGGCCCTGAGAGGCGAG ATCGCCCCCCTGAAAGAAAA CGTGTCCCACGTGAACGACCTGGCCCGGCAGCTGACAACACTGGGC ATTCAGCTGAGCCCCTACAACCTGAGCACACTGGAAGATCTGAACA CCCGGTGGAAGCTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCA GCTGCACGAGGCCCACAGATTTTGGCCCTGCCTCCCAGCACTTCC TGAGCACCAGTGTGCAGGGCCCTTGGGAGAGAGCCATCTCC CCTAA CAAGGTGCCCTACTACATCAACCACGAGACACAGACCACCTGTTGG GACCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCTGACCTGA ACAACGTGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGAG ACTGCAGAAAGCTCTGTGCCTGGATCTGCTGTCCCTGTCCGCCGCCT GTGATGCCCTGGACCAGCACAATCTGAAGCAGAACGACCAGCCCAT GGATATCCT GCAGATCATCAACTGCCTGACCACCATCTACGACCGGC TGGAACAGGAACACAACAATCTCGTGAACGTGCCCCTGTGCGTGGA CATGTGCCTGAATTGGCTGCTGAATGTGTACGACACCGGCCGGACAG GCCGGATCAGAGTGCTGTCCTTCAAGACCGGCATCATCAGCCTGTGC AAAGCCCACCTGGAAGATAAGTACCGGTATCTGTTCAAACAGGTGG CCTCTAGCACCGGCTTTG CGACCAGAGAAGGCTGGGCCTGCTGCTG CACGACAGCATCCAGATCCCTAGACAGCTGGGCGAAGTGGCCAGCT TTGGCGGCAGCAACATCGAGCCTAGCGTGCGGAGCTGCTTCCAGTTC GCCAACAACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGA TGAGACTGGAACCCCAGAGCATGGTGTGGCTGCCCGTGCTGCATAG AGTGGCCGCTGCCGAGACAGCCAAGCACC AGGCCAAGTGCAACATC TGCAAAGAGTGCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGC ACTTCAACTACGATATCTGCCAGAGCTGTTTCTTCAGCGGCAGGGTG GCCAAGGGCCACAAAATGCACTACCCCATGGTGGAATACTGCACCC CCACCACAAGCGGCGAGGATGTGCGGGATTTCGCCAAGGTGCTGAA AAACAAGTTCCGGACCAAGCGGTACTTCGCCAAACACCCCCGGA TG GGCTACCTGCCTGTGCAGACAGTGCTGGAAGGCGACAACATGGAAA CCGACACCATGTGATGATGATTTGGGCAGAGCGATGGAGTCCTTAGT ATCAGTCATGACAGATGAAGAAGGAGCAGAATAAATGTTTTACAAC TCCTGATTCCCGCATGCGGCCAGCTTATCGATACCGTCGAAATAAAA __GATCCTTATTTTCATTGGATCTGTGTGTTGGTTTTTTGTGTG_______ ATGCTGTG GTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG SEQ Ш TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA NO 138 - GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC

GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC BHA- TGCCCAAAGAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT

GAACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG

- 63 046419

196477Q

AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTGGAGCGACGGCCTGGCCCTGAA CGCCCTGATCCACAG CCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGTCCATCGAGGCCA TCCAGGAAGTGGAAATGCTGCCCA GACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGACCACC AGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGACAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAG TGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGAGCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAACG ACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCTCCAAGCTGATCGGCACCGGCAAGCTGAGCGAGGACGAAG AGACAGAGG TGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCATAG GGTCCTGCTGAAGTGGCAGCGGCTGACCGAGGAACAGTGCCTGTTT AGCGCCTGGCTGTCCGAGAAAGAGGACGCCGTGAACAAGATCCACA CCACCGGCTTCAAGGACCAGAACGAGATGCTGAGCAGCCTGCAGAA ACTGGCCGTGCTGAAGGCCGACCT GGAAAAGAAAAAGCAGTCCATG GGCAAGCTGTACTCCCTGAAGCAGGACCTGCTGTCCACCCTGAAGA ACAAGAGCGTGACCCAGAAAACCGAGGCCTGGCTGGACAACTTCGC CCGGTGCTGGGACAACCTGGTGCAGAAGCTGGAAAAGTCCACCGCC CAGATCTCCCAGGCCGTGACCACAACACAGCCCAGCCTGACCCAGA CCACCGTGATGGAAACCGTGACAACAGTGACCACCCGGGAAC AGAT CCTCGTGAAGCACGCCCAGGAAACTGCCCCCTCCACCCCCCCAG AAGAAACGGCAGATCACAGTGGACAGTGAAGCTCAGATCCACACCG TGCGGGAAGACAATGATGGTCATGACAGAGGACATGCCCCTGGA AATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCACCCATGTGT CCCAGGCCCTGCTGGAAGTGGAACAGCTGCTGAACGCCCCCGACCT GTGCGC CAAGGATTTCGAGGACCTGTTCAAGCAGGAAGAGAGCCTG AAGAATATCAAGGATAGCCTGCAGCAGAGCAGCGGCCGGATCGACA TCATCCACAGCAAGAAAACAGCCGCCCTGCAGAGCGCCACCCCCGT GGAAAGAGTGAAACTGCAGGAAGCCCTGTCCCAGCTGGACTTCCAG TGGGAGAAAGTGAACAAAATGTACAAGGACCGGCAGGGCAGATTCG ACCGCAGCGTGGAAAAGTGGC GGCGGTTCCACTACGACATCAAGAT CTTCAACCAGTGGCTGACAGAGGCCGAGCAGTTCCTGAGAAAGACC CAGATCCCCGAGAACTGGGAGCACGCCAAGTACAAGTGGTATCTGA AAGAGCTGCAGGACGCATCGGCCAGAGACAGACAGTCGTGCGGAC CCTGAATGCCACCGGCGAGGAAATCATCCAGCAGTCCAGCAAGACC GACGCCAGCCATTCTGCAGGAAAAGCTGGGCAGC CTGAACCTGCGGT

- 64 046419

GGCAGGAAGTGTGCAAGCAGCTGTCCGACCGGAAGAAGCGGCTGGA AGAACAGCTGGAACGGCTGCAGGAACTGCAGGAGGCCACCGACGA GCTGGACCTGAAACTGAGACAGGCCGAAGTGATCAAGGGCAGCTGG CAGCCAGTGGGCGACCTGCTGATCGACTCCCTGCAGGACCATCTGGA AAAAGTGAAGGCCCTGAGAGGCGAGATCGCCCCCCTGAAAGAAAAC GTGTCCCACG TGAACGACCTGGCCCGGCAGCTGACAACACTGGGCA TTCAGCTGAGCCCCTACAACCTGAGCACACTGGAAGATCTGAACACC CGGTGGAAGCTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGC TGCACGAGGCCCACAGAGATTTTGGCCCTGCCTCCCAGCACTTCCTG AGCACCAGTGTGCAGGGCCCTTGGGAGAGAGCCATCTCCCCTAACA AGGTGCCCTACTACATCAACCA CGAGACACAGACCACCTGTTGGGA CCACCCCAAGATGACCGAGCTGTACCAGAGCCTGGCTGACCTGAAC AACGTGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGAGAC TGCAGAAAGCTCTGTGCCTGGATCTGCTGTCCCTGTCCGCCGCCTGT GATGCCCTGGACCAGCACAATCTGAAGCAGAACGACCAGCCCATGG ATATCCTGCAGATCATCAACTGCCTGACCACCATCTAC GACCGGCTG GAACAGGAACACAACAATCTCGTGAACGTGCCCCTGTGCGTGGACA TGTGCCTGAATTGGCTGCTGAATGTGTACGACACCGGCCGGACAGGC CGGATCAGAGTGCTGTCCTTCAAGACCGGCATCATCAGCCTGTGCAA AGCCCACCTGGAAGATAAGTACCGGTATCTGTTCAAACAGGTGGCCT CTAGCACCGGCTTTTGCGACCAGAAGGCTGGGCCTGCTGCT GCAC GACAGCATCCAGATCCCTAGACAGCTGGGCGAAGTGGCCAGCTTTG GCGGCAGCAACATCGAGCCTAGCGTGCGGAGCTGCTTCCAGTTCGCC AACAACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGA GACTGGAACCCCAGAGCATGGTGTGGCTGCCCGTGCTGCATAGAGT GGCCGCTGCCGAGACAGCCAAGCACCAGGCCAAGTGCAACATCTGC AAAGAG TGCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGCACTT CAACTACGATATCTGCCAGAGCTGTTTCTTCAGCGGCAGGGTGGCCA AGGGCCACAAAATGCACTACCCCATGGTGGAATACTGCACCCCCAC CACAAGCGGCGAGGATGTGCGGGATTTCGCCAAGGTGCTGAAAAAC AAGTTCCGGACCAAGCGGTACTTCGCCAAACACCCCCGGATGGGCT ACCTGCCTGGGCAGACAGTGCT AAGGCGACAACATGGAAACCGA CACCATGTGATGATGATTTGGGCAGAGCGATGGAGTCCTTAGTATCA GTCATGACAGATGAAGAAGGAGCAGAATAAATGTTTTTACAACTCCT GATTCCCGCATGCGGCCAGCTTATCGATACCGTCGAAATAAAAGATC CTTATTTTCATTGGATCTGTGTGTTGGTTTTTTGTGTG SEQ ID NO: 139 - BXA- 196478 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA

- 65 046419

CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCATCGAGGCCA TCCAGGA AGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGACCACC AGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGACAAGAGCTTCGGCAGC AGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGAGCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAACG ACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCTCCAAGCTGATCGGCACCGGCAAGCTGAGCGA GGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCATAG AGTACTGCTCCAAGACATCCTGCTGAAGTGGCAGCGGCTGACCGAG GAACAGTGCCTGTTTAGCGCCTGGCTGTCCGAGAAAGAGGACGCCG TGAACAAGATCCACACCACCGGCTTCAAGGACCAGAACGAGATGCT GAGCA GCCTGCAGAAACTGGCCGTGCTGAAGGCCGACCTGGAAAAG AAAAAGCAGTCCATGGGCAAGCTGTACTCCCTGAAGCAGGACCTGC TGTCCACCCTGAAGAACAAGAGCGTGACCCAGAAAACCGAGGCCTG GCTGGACAACTTCGCCCGGTGCTGGGACAACCTGGTGCAGAAGCTG GAAAAGTCCACCGCCCAGATCTCCCAGGCCGTGACCACAACACAGC CCAGCCTGACCCAGACCAC CGTGATGGAAACCGTGACAACAGTGAC CACCCGGGAACAGATCCTCGTGAAGCACGCCCAGGAAGAACTGCCC CCTCCACCCCCCCAGAAGAAACGGCAGATCACAGTGGACAGTGAAG CTCAGATCCACACCGTGCGGGAAGAGACAATGATGGTCATGACAGA GGACATGCCCCTGGAAATCAGCTACGTGCCCAGCACCTACCTGACCG AGATCACCCATGTGTCCCAGCCCTGCTG GAAGTGGAACAGCTGCTG AACGCCCCCGACCTGTGCGCCAAGGATTTCGAGGACCTGTTCAAGCA GGAAGAGAGCCTGAAGAATATCAAGGATAGCCTGCAGCAGAGCAGC GGCCGGATCGACATCATCCACAGCAAGAAAACAGCCGCCCTGCAGA GCGCCACCCCCGTGGAAAGAGTGAAACTGCAGGAAGCCCTGTCCCA GCTGGACTTCCAGTGGGAGAAAGTGAACAAAATGTACAAGGACC GG CAGGGCAGATTCGACCGCAGCGTGGAAAAGTGGCGGCGGTTCCACT ACGACATCAAGATCTTCAACCAGTGGCTGACAGAGGCCGAGCAGTT CCTGAGAAAGACCCAGATCCCCGAGAACTGGGAGCACGCCAAGTAC AAGTGGTATCTGAAAGAGCTGCAGGACGGCATCGGCCAGAGACAGA CAGTCGTGCGGACCCTGAATGCCACCGGCGAGGAAATCATCCAGCA GTCCAGCAAGACCGA CGCCAGCATTCTGCAGGAAAAGCTGGGCAGC CTGAACCTGCGGTGGCAGGAAGTGTGCAAGCAGCTGTCCGACCGGA AGAAGCGGCTGGAAGAACAGCTGGAACGGCTGCAGGAACTGCAGG AGGCCACCGACGAGCTGGACCTGAAACTGAGACAGGCCGAAGTGAT CAAGGGCAGCTGGCAGCCAGTGGGCGACCTGCTGATCGACTCCCTG CAGGACCATCTGGAAAAAGTGAAGGCCCTGAGAG GCGAGATCGCCC

- 66 046419

CCCTGAAAGAAAACGTGTCCCACGTGAACGACCTGGCCCGGCAGCT GACAACACTGGGCATTCAGCTGAGCCCCTACAACCTGAGCACACTG GAAGATCTGAACACCCGGTGGAAGCTGCTGCAGGTGGCCGTGGAAG ATAGAGTGCGGCAGCTGCACGAGGCCCACAGAGATTTTGGCCCTGC CTCCCAGCACTTCCTGAGCACCAGTGTGCAGGGCCCTTGGGAGAGA GCCATCTCCCC TAACAAGGTGCCCTACTACATCAACCACGAGACACA GACCACCTGTTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGC CTGGCTGACCTGAACAACGTGCGGTTCAGCGCCTACCGGACCGCCAT GAAGCTGCGGAGACTGCAGAAAGCTCTGTGCCTGGATCTGCTGTCCC TGTCCGCCGCCTGTGATGCCCTGGACCAGCACAATCTGAAGCAGAAC GACCAGCCCATGGATATCCT GCAGATCATCAACTGCCTGACCACCAT CTACGACCGGCTGGAACAGGAACACAACAATCTCGTGAACGTGCCC CTGTGCGTGGACATGTGCCTGAATTGGCTGCTGAATGTGTACGACAC CGGCCGGACAGGCCGGATCAGAGTGCTGTCCTTCAAGACCGGCATC ATCAGCCTGTGCAAAGCCCACCTGGAAGATAAGTACCGGTATCTGTT CAAACAGGTGGCCTCTAGCACCGGCTTTTGCGAC CAGAGAAGGCTG GGCCTGCTGCTGCACGACAGCATCCAGATCCCTAGACAGCTGGGCG AAGTGGCCAGCTTTGGCGGCAGCAACATCGAGCCTAGCGTGCGGAG CTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGT TCCTGGACTGGATGAGACTGGAACCCCAGAGCATGGTGTGGCTGCC CGTGCTGCATAGAGTGGCCGCTGCCGAGACAGCCAAGCACCAGG CC AAGTGCAACATCTGCAAAGAGTGCCCCATCATCGGCTTCCGGTACAG AAGCCTGAAGCACTTCAACTACGATATCTGCCAGAGCTGTTTCTTCA GCGGCAGGGTGGCCAAGGGCCACAAAATGCACTACCCCATGGTGGA ATACTGCACCCCCACCAAGCGGCGAGGATGTGCGGGATTTCGCC AAGGTGCTGAAAAACAAGTTCCGGACCAAGCGGTACTTCGCCAAAC ACCCCCGGATG CTACCTGCCTGTGCAGACAGTGCTGGAAGGCGA CAACATGGAAACCGACACCATGTGATGATGATTTGGGCAGAGCGAT GGAGTCCTTAGTATCAGTCATGACAGATGAAGAAGGAGCAGAATAA ATGTTTTTACAACTCCTGATTCCCGCATGCGGCCAGCTTATCGATACC GTCGAAATAAAAGATCCTTATTTTCATTGGATCTGTGTGTTGGTTTTTT TGTGTG SEQ ID NO: 140 - BXA- 196479 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA

- 67 046419

TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGACCACC AGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGACAAGAG CTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGAGCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAACG ACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCTCCAAGCTGATCGGCACCCA AGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCATCT GCTGAAGTGGCAGCGGCTGACCGAGGAACAGTGCCTGTTTAGCGCC TGGCTGTCCGAGAAAGAGGACGCCGTGAACAAGATCCACACCACCG GCTTCAAGGACCAGAACGAGATGCTGAGCAGCCTGCAGAAACT GGC CGTGCTGAAGGCCGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAG CTGTACTCCCTGAAGCAGGACCTGCTGTCCACCCTGAAGAACAAGA GCGTGACCCAGAAAACCGAGGCCTGGCTGGACAACTTCGCCCGGTG CTGGGACAACCTGGTGCAGAAGCTGGAAAAGTCCACCGCCCAGATC TCCCAGGCCGTGACCACAACACAGCCCAGCCTGACCCAGACCACG TGATGGAAA CCGTGACAACAGTGACCACCCGGGAACAGATCCTCGT GAAGCACGCCCAGGAAGAACTGCCCCCTCCACCCCCCCAGAAGAAA CGGCAGATCACAGTGGACAGTGAAGCTCAGATCCACACCGTGCGGG AAGACAAATGATGGTCATGACAGAGGACATGCCCCTGGAAATCAG CTACGTGCCCAGCACCTACCTGACCGAGATCACCCATGTGTCCCAGG CCCTGCTGGAAGTGGAACAG CTGCTGAACGCCCCCGACCTGTGCGCC AAGGATTTCGAGGACCTGTTCAAGCAGGAAGAGAGCCTGAAGAATA TCAAGGATAGCCTGCAGCAGAGCAGCGGCCGGATCGACATCATCCA CAGCAAGAAAACAGCCGCCCTGCAGAGCGCCACCCCCGTGGAAAGA GTGAAACTGCAGGAAGCCCTGTCCCAGCTGGACTTCCAGTGGGAGA AAGTGAACAAAATGTACAAGGACCGGCAGGGCAG ATTCGACCGCAG CGTGGAAAAGTGGCGGCGGTTCCACTACGACATCAAGATCTTCAAC CAGTGGCTGACAGAGGCCGAGCAGTTCCTGAGAAAGACCCAGATCC CCGAGAACTGGGAGCACGCCAAGTACAAGTGGTATCTGAAAGAGCT GCAGGACGGCATCGGCCAGAGACAGACAGTCGTGCGGACCCTGAAT GCCACCGGCGAGGAAATCCATCCAGCAGTCCAGCAAGACCGACGCCA GCATTCTGCAGGAAAAGCTGGCAGCCTGAACCTGCGGTGGCAGGA AGTGTGCAAGCAGCTGTCCGACCGGAAGAAGCGGCTGGAAGAACAG CTGGAACGGCTGCAGGAACTGCAGGAGGCCACCGACGAGCTGGACC TGAAACTGAGACAGGCCGAAGTGATCAAGGGCAGCTGGCAGCCAGT GGGCGACCTGCTGATCGACTCCCTGCAGGACCATCTGGAAAAAGTG AAGGCCCTGAGAGGC GAGATCGCCCCCCTGAAAGAAAACGTGTCCC ACGTGAACGACCTGGCCCGGCAGCTGACAACACTGGGCATTCAGCT GAGCCCCTACAACCTGAGCACACTGGAAGATCTGAACACCCGGTGG AAGCTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGCTGCACG AGGCCCACAGATTTTGGCCCTGCCTCCCAGCACTTCCTGAGCACC AGTGTGCAGGGCCCTTGGGAGAGAGCCAT CTCCCCTAACAAGGTGC

- 68 046419

CCTACTACATCAACCACGAGACACAGACCACCTGTTGGGACCACCCCC AAGATGACCGAGCTGTACCAGAGCCTGGCTGACCTGAACAACGTGC GGTTCAGCGCCTACCGGACCGCCATGAAGCTGCGGAGACTGCAGAA AGCTCTGTGCCTGGATCTGCTGTCCCTGTCCGCCGCCTGTGATGCCCT GGACCAGCACAATCTGAAGCAGAACGACCAGCCCATGGATATCCTG CAGATCAT CAACTGCCTGACCACCATCTACGACCGGCTGGAACAGG AACACAACAATCTCGTGAACGTGCCCCTGTGCGTGGACATGTGCCTG AATTGGCTGCTGAATGTGTACGACACCGGCCGGACAGGCCGGATCA GAGTGCTGTCCTTCAAGACCGGCATCATCAGCCTGTGCAAAGCCCAC CTGGAAGATAAGTACCGGTATCTGTTCAAACAGGTGGCCTCTAGCAC CGGCTTTTGCGACCAGAGAA GGCTGGGCCTGCTGCTGCACGACAGC ATCCAGATCCCTAGACAGCTGGGCGAAGTGGCCAGCTTTGGCGGCA GCAACATCGAGCCTAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAAC AAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGAGACTGG AACCCCAGAGCATGGTGTGGCTGCCCGTGCTGCATAGAGTGGCCGCT GCCGAGACAGCCAAGCACCAGGCCAAGTGCA ACATCTGCAAAGAGT GCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGCACTTCAACTAC GATATCTGCCAGAGCTGTTTCTTCAGCGGCAGGGTGGCCAAGGGCCA CAAAATGCACTACCCCATGGTGGAATACTGCACCCCCACCACAAGC GGCGAGGATGTGCGGGATTTCGCCAAGGTGCTGAAAAACAAGTTCC GGACCAAGCGGTACTTCGCCAAACACCCCCGGATGGGCTACCTGC CT GTGCAGACAGTGCTGGAAGGCGACAACATGGAAACCGACACCATGT GATGATGATTTGGGCAGAGCGATGGAGTCCTTAGTATCAGTCATGAC AGATGAAGAAGGAGCAGAATAAATGTTTTACAACTCCTGATTCCCG CATGCGGCCAGCTTATCGATACCGTCGAAATAAAAGATCCTTATTTT CATTGGATCTGTGTGTTGGTTTTTTGTGTG SEQ ID NO: 141 - BXA- 196480 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCTCAGGGCTACGAGCGGACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACCACC AGCGACCCCACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA

- 69 046419

CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGTCCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCCCAGGGCGAGATCAGCAACG ACGTGGAAGTGGTGAAAGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACCGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCAGCAAGCTGATCGGCACCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTG CAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAT CCACACCGTGCGGGAAGAGACAATGATGGTGATGACCGAGGACATG CCCCTGGAAATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCAC CCACGTGTCCCAGGCTCTGCTGGAAGTGGAACAGCTGCTGAACGCCC CCGACCTGTGCGCCAAGGACTTCGA GGATCTGTTCAAGCAGGAAGA GAGCCTGAAGAATATCAAGGACTCCCTGCAGCAGTCCAGCGGCCGG ATCGACATCATCCACAGCAAGAAAACAGCCGCCCTGCAGTCCGCCA CCCCCGTGGAAAGAGTGAAGCTGCAGGAAGCCCTGAGCCAGCTGGA CTTCCAGTGGGAGAAAGTGAACAAGATGTACAAGGACCGGCAGGGC AGATTCGACCGCAGCGTGGAAAAGTGGCGGCGGTTCC ACTACGACA TCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGAG AAAGACCCAGATCCCCGAGAACTGGGAGCACGCCAAGTACAAGTGG TATCTGAAAGAACTGCAGGATGGCATCGGCCAGCGGCAGACCGTGG TGCGCACACTGAATGCCACCGGCGAGGAAATCATCCAGCAGAGCAG CAAGACCGACGCCAGCATCCTGCAGGAAAAGCTGGGCTCCCTGAAC CTGC GGTGGCAGGAAGTGTGCAAGCAGCTGAGCGACCGGAAGAAGA GGCTGGAAGAACAGGCCCCTGGCCTGACCACCATCGGCGCCAGCCC TACCCAGACCGTGACCCTGGTGACACAGCCCGTGGTGACAAAAGAG ACAGCCATCTCAAGCTGGAAATGCCCAGCTCCCTGATGCTGGAACT GGAAAGGCTGCAGGAACTGCAGGAGGCCACCGACGAGCTGGACCTG AAGCTGCGGCAGGCCGAAGTG ATCAAGGGCAGCTGGCAGCCCGTGG GCGACCTGCTGATCGACAGCCTGCAGGACCACCTGGAAAAAGTGAA GGCCCTGAGAGGCGAGATCGCCCCCCTGAAAGAAAACGTGTCCCAC GTGAACGACCTGGCCCGGCAGCTGACCACCCTGGGCATCCAGCTGA GCCCCTACAACCTGAGCACCCTGGAAGATCTGAACACCCGGTGGAA GCTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAG CTGCACGAG GCCCACAGAGACTTTGGCCCTGCCAGCCAGCACTTCCTGAGCACCTC TGTGCAGGGACCCTGGGAGAGAGCCATCAGCCCCAACAAGGTGCCC TACTACATCAACCACGAGACACAGACCACCTGTTGGGACCACCCCA AGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAATGTGCG GTTCAGCGCCTACCGGACCGCCATGAAGCTGAGGCGGCTGCAGAAA GC TCTGTGCCTGGATCTGCTGAGCCTGAGCGCCGCCTGCGACGCCCT GGACCAGCACAACCTGAAGCAGAACGACCAGCCCATGGATATCCTG CAGATCATCAACTGCCTGACCACAATCTACGACAGGCTGGAACAGG AACAAACAATCTGGTCAACGTGCCCCTGTGCGTGGACATGTGCCTG AATTGGCTGCTGAATGTGTACGACACCGGCCGGACCGGCAGAATCC GGGTGCTGAGCTTC AAGACCGGCATCATCAGCCTGTGCAAGGCCCA CCTGGAAGATAAGTACCGCTACCTGTTCAAACAGGTGGCCAGCTCCA CCGGCTTTTGCGACCAGCGGAGACTGGGCCTGCTGCTGCACGACAGC ATCCAGATCCCCAGACAGCTGGGCGAGGTGGCCTCCTTCGGCGGCA GCAACATTGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAAC AAGCCCGAGATCGAGGCCGCCCTGTTC CTGGACTGGATGAGACTGG

- 70 046419

AACCCCAGAGCATGGTGTGGCTGCCCGTGCTGCATCGGGTGGCCGCT GCCGACAGCCAAGCACCAGGCCAAGTGCAACATCTGCAAAGAGT GCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGCACTTCAACTAC GATATCTGCCAGAGCTGCTTCTTCAGCGGCAGAGTGGCCAAGGGCC ACAAAATGCACTACCCCATGGTGGAATACTGCACCCCCACCACCAG CGGCGAGGATGTGCGGG ACTTCGCCAAGGTGCTGAAAAACAAGTTC CGGACCAAGCGGTACTTTGCCAAGCACCCCCGGATGGGCTACCTGCC CGTGCAGACAGTGCTGGAAGGCGACAACATGGAAACCCCTGCCCAG ATCCTGATCAGCCTGGAAAGCGAGGAACGGGGCGAGCTGGAACGGA TCCTGGCCGATCTGGAAGAGGAAAACCGGAACCTGCAGGCCGAGTA CGACCGGCTGAAGCAGCAGCACGAGCACA AGGGCCTGAGCCCCCTG CCTAGCCCCCCTGACACCATGTGATGATGATTTGGGCAGAGCGATGG AGTCCTTAGTATCAGTCATGACAGATGAAGAAGGAGCAGAATAAAT GTTTTACAACTCCTGATTCCCGCATGCGGCCAGCTTATCGATACCGT CGAAATAAAAGATCCTTATTTTCATTGGATCTGTGTGTTGGTTTTTTG TGTG SEQ ID NO: 142 BXA- 196480- option ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCTCAGGGCTACGAGCGGACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACCACC AGCGACCCCACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGTCCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCCCAGGGCGAGATCAGCAACG ACGTGGAAGTGGTGAAAGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACCGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCAGCAAGCTGATCGGCA CCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACCG GGTGCTGATCCACACCGTGCGGGAAGAGACAATGATGGTGATGACC GAGGACATGCCCCTGGAAATCAGCTACGTGCCCAGCACCTACCTGA CCGAGATCACCCACGTGTCCCAGGCTCTGCTGGA AGTGGAACAGCT

- 71 046419

GCTGAACGCCCCCGACCTGTGCGCCAAGGACTTCGAGGATCTGTTCA AGCAGGAAGAGAGCCTGAAGAATATCAAGGACTCCCTGCAGCAGTC CAGCGGCCGGATCGACATCATCCACAGCAAGAAAACAGCCGCCCTG CAGTCCGCCACCCCCGTGGAAAGAGTGAAGCTGCAGGAAGCCCTGA GCCAGCTGGACTTCCAGTGGGAGAAAGTGAACAAGATGTACAAGGA CCGGCAGGGCAG ATTCGACCGCAGCGTGGAAAAGTGGCGGCGGTTC CACTACGACATCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGC AGTTCCTGAGAAAGACCCAGATCCCCGAGAACTGGGAGCACGCCCAA GTACAAGTGGTATCTGAAAGAACTGCAGGATGGCATCGGCCAGCGG CAGACCGTGGTGCGCACACTGAATGCCACCGGCGAGGAAATCATCC AGCAGAGCAGCAAGACCGACGCCAG CATCCTGCAGGAAAAGCTGGG CTCCCTGAACCTGCGGTGGCAGGAAGTGTGCAAGCAGCTGAGCGAC CGGAAGAAGAGGCTGGAAGAACAGAAGAACGACCTGGCCCCTGGC CTGACCACCATCGGCGCCAGCCCTACCCAGACCGTGACCCTGGTGAC ACAGCCCGTGGTGACAAAAGAGACAGCCATCTCCAAAGCTGGAAATG CCCAGCTCCCTGATGCTGGAAGTGGAAAGGCTGCAGGAACT GCAGG AGGCCACCGACGAGCTGGACCTGAAGCTGCGGCAGGCCGAAGTGAT CAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACAGCCTG CAGGACCACCTGGAAAAAGTGAAGGCCCTGAGAGGCGAGATCGCCC CCCTGAAAGAAAACGTGTCCCACGTGAACGACCTGGCCCGGCAGCT GACCACCCTGGGCATCCAGCTGAGCCCCTACAACCTGAGCACCCTGG AAGATCTGA ACACCCGGTGGAAGCTGCTGCAGGTGGCCGTGGAAGA TAGAGTGCGGCAGCTGCACGAGGCCCACAGAGACTTTGGCCCTGCC AGCCAGCACTTCCTGAGCACCCTGTGCAGGGACCCTGGGAGAGAG CCATCAGCCCCAACAAGGTGCCCTACTACATCAACCACGAGACACA GACCACCTGTTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGC CTGGCCGACCTGAACAATGTG CGGTTCAGCGCCTACCGGACCGCCAT GAAGCTGAGGCGGCTGCAGAAAGCTCTGTGCCTGGATCTGCTGAGC CTGAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGA ACGACCAGCCCATGGATATCCTGCAGATCATCAACTGCCTGACCACA ATCTACGACAGGCTGGAACAGGAACACAACAATCTGGTCAACGTGC CCCTGTGCGTGGACATGTGCCTGAATTGGCTGCTGAA TGTGTACGAC ACCGGCCGGACCGGCAGAATCCGGGTGCTGAGCTTCAAGACCGGCA TCATCAGCCTGTGCAAGGCCCACCTGGAAGATAAGTACCGCTACCTG TTCAAACAGGTGGCCAGCTCCACCGGCTTTTGCGACCAGCGGAGACT GGGCCTGCTGCTGCACGACAGCATCCAGATCCCCAGACAGCTGGGC GAGGTGGCCTCCTTCGGCGGCAGCAACATTGAGCCCAGCTGCGGA GCTGC TTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTG TTCCTGGACTGGATGAGACTGGAACCCCAGAGCATGGTGTGGCTGCC CGTGCTGCATCGGGTGGCCGCTGCCGAGACAGCCAAGCACCAGGCC AAGTGCAACATCTGCAAAGAGTGCCCCATCATCGGCTTCCGGTACAG AAGCCTGAAGCACTTCAACTACGATATCTGCCAGCTGCTTCTTCA GCGGCAGAGTGGCCAAG GGCCACAAAATGCACTACCCCATGGTGGA ATACTGCACCCCCACCACCAGCGGCGAGGATGTGCGGGACTTCGCC AAGGTGCTGAAAAACAAGTTCCGGACCAAGCGGTACTTTGCCAAGC ACCCCCGGATGGGCTACCTGCCCGTGCAGACAGTGCTGGAAGGCGA CAACATGGAAACCCCTGCCCAGATCCTGATCAGCCTGGAAAGCGAG GAACGGGGCGAGCTGGAACGGATCCTGGCCGAT CTGGAAGAGGAAA

- 72 046419

ACCGGAACCTGCAGGCCGAGTACGACCGGCTGAAGCAGCAGCACGA GCACAAGGGCCTGAGCCCCCTGCCTAGCCCCCCTGACACCATGTGAT GATGATTTGGGCAGAGCGATGGAGTCCTTAGTATCAGTCATGACAG ATGAAGAAGGAGCAGAATAAATGTTTTACAACTCCTGATTCCCGCAT GCGGCCAGCTTATCGATACCGTCGAAATAAAAGATCCTTATTTTCAT TGGATCTGTG TGTTGGTTTTTTTGTGTG SEQ ID NO: 143 - BXA- 196481 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCTCAGGGCTACGAGCGGACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACCACC AGCGACCCCACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGTCCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCCCAGGGCGAGATCAGCAACG ACGTGGAAGTGGTGAAAGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACCGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCAGCAAGCTGATCGGCA CCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAG CTACGTGCCCAGCACCTACCTGACCGAGATCACCCACGTGTCCCAGG CTCTGCTGGAAGTGGAACAGCTGCTGAACGCCCCCGACCTGTGCGCC AAGGACTTCGAGGATCTGTTCAAGCAGGAA GAGAGCCTGAAGAATA TCAAGGACTCCCTGCAGCAGTCCAGCGGCCGGATCGACATCATCCAC AGCAAGAAAACAGCCGCCCTGCAGTCCGCCACCCCCGTGGAAAGAG TGAAGCTGCAGGAAGCCCTGAGCCAGCTGGACTTCCAGTGGGAGAA AGTGAACAAGATGTACAAGGACCGGCAGGGCAGATTCGACCGCAGC GTGGAAAAGTGGCGGCGGTTCCACTACGACATCAAGATCTTC AACC AGTGGCTGACCGAGGCCGAGCAGTTCCTGAGAAAGACCCAGATCCC CGAGAACTGGGAGCACGCCAAGTACAAGTGGTATCTGAAAGAACTG CAGGATGGCATCGGCCAGCGGCAGACCGTGGTGCGCACACTGAATG CCACCGGCGAGGAAATCATCCAGCAGAGCAGCAAGACCGACGCCAG CATCCTGCAGGAAAAGCTGGGCTCCCTGAACCTGCGGTGGCAGGAA

- 73 046419

GTGTGCAAGCAGCTGAGCGACCGGAAGAAGAGGCTGGAAGAACAG CTGGAAGGACTGGAAAAGCTGTACCAGGAACCCAGAGAGCTGCCCC CCGAGGAACGGGCCCAGAACGTGACCCGGCTGCTGCGGAAACAGGC CGAAGAGGTGAACACCGAGTGGGAGAAGCTGAACCTGCACTCCGCC GACTGGCAGCGGAAGATCGACGAGACACTGGAAAGGCTGCAGGAA CTGCAGGAGGC CACCGACGAGCTGGACCTGAAGCTGCGGCAGGCCG AAGTGATCAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGA CAGCCTGCAGGACCACCTGGAAAAAGTGAAGGCCCTGAGAGGCGAG ATCGCCCCCCTGAAAGAAAACGTGTCCCACGTGAACGACCTGGCCC GGCAGCTGACCACCCTGGGCATCCAGCTGAGCCCCTACAACCTGAG CACCCTGGAAGATCTGAACACCCGGTG GAAGCTGCTGCAGGTGGCC GTGGAAGATAGAGTGCGGCAGCTGCACGAGGCCCACAGAGACTTTG GCCCTGCCAGCCAGCACTTCCTGAGCACCTCTGTGCAGGGACCCTGG GAGAGAGCCATCAGCCCCAACAAGGTGCCCTACTACATCAACCACG AGACACAGACCACCTGTTGGGACCACCCCAAGATGACCGAGCTGTA CCAGAGCCTGGCCGACCTGAACAATGTGCGGTTCAGCG CCTACCGG ACCGCCATGAAGCTGAGGCGGCTGCAGAAAGCTCTGTGCCTGGATC TGCTGAGCCTGAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCT GAAGCAGAACGACCAGCCCATGGATATCCTGCAGATCATCAACTGC CTGACCACAATCTACGACAGGCTGGAACAGGAACACAACAATCTGG TCAACGTGCCCCTGTGCGTGGACATGTGCCTGAATTGGCTGCTGAAT GTGTACG ACACCGGCCGGACCGGCAGAATCCGGGTGCTGAGCTTCA AGACCGGCATCATCAGCCTGTGCAAGGCCCACCTGGAAGATAAGTA CCGCTACCTGTTCAAACAGGTGGCCAGCTCCACCGGCTTTTGCGACC AGCGGAGACTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCAG ACAGCTGGGCGAGGTGGCCTCCTTCGGCGGCAGCAACATTGAGCCC AGCGTGCGGAGCTGCTTCCAG TTCGCCAACAACAAGCCCGAGATCG AGGCCGCCCTGTTCCTGGACTGGATGAGACTGGAACCCCAGAGCAT GGTGTGGCTGCCCGTGCTGCATCGGGTGGCCGCTGCCGAGACAGCC AAGCACCAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCATCG GCTTCCGGTACAGAAGCCTGAAGCACTTCAACTACGATATCTGCCAG AGCTGCTTCTTCAGCGGCAGAGTGGCCAAGGGCC ACAAAATGCACT ACCCCATGGTGGAATACTGCACCCCCACCACCAGCGGCGAGGATGT GCGGGACTTCGCCAAGGTGCTGAAAACAAGTTCCGGACCAAGCGG TACTTTGCCAAGCACCCCCGGATGGGCTACCTGCCCGTGCAGACAGT GCTGGAAGGCGACAACATGGAAACCCCTGCCCAGATCCTGATCAGC CTGGAAAGCGAGGAACGGGGCGAGCTGGAACGGATCCTGGCCGATC T GGAAGAGGAAAACCGGAACCTGCAGGCCGAGTACGACCGGCTGAA GCAGCAGCACGAGCACAAGGGCCTGAGCCCCCTGCCTAGCCCCCCT GACACCATGTGATGATGATTTGGGCAGAGCGATGGAGTCCTTAGTAT CAGTCATGACAGATGAAGAAGGAGCAGAATAAATGTTTTACAACTC CTGATTCCCGCATGCGGCCAGCTTATCGATACCGTCGAAATAAAAGA TCCTTATTTT CATTGGATCTGTGTGTTGGTTTTTTGTGTG SEQ ID NO: 144 - ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT

- 74 046419

VHA027741

GAACAAGGCCCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTC ACCACCTCTTGGAGCGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCC CCAGCAGGTGTCCATCGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCCCAGGGCTACGAGAACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCTACGTGACCACC AGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCT AAGCCC CCGAGGACAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGAGCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGCAACG ACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCTC CAAGCTGATCGGCACCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCATCT GCTGAAGTGGCAGCGGCTGACCGAGGAACAGTGCCTGTTTAGCGCC TGGCTGTCCGAGAAAGAGGACGCCGTGAACAAGATCCACACCACG GCTTCAAGGACCAGAACGAGATG CTGAGCAGCCTGCAGAAACTGGC CGTGCTGAAGGCCGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAG CTGTACTCCCTGAAGCAGGACCTGCTGTCCACCCTGAAGAACAAGA GCGTGACCCAGAAAACCGAGGCCTGGCTGGACAACTTCGCCCGGTG CTGGGACAACCTGGTGCAGAAGCTGGAAAAGTCCACCGCCCAGATC TCCCAGGCCGTGACCACAACACAGCCCAGCCTGACC T GGAACAGCTGCTGAACGCCCCCGACCTGTGCGCCAAGGATTTCGA GGACCTGTTCAAGCAGGAAGAGAGCCTGAAGAATATCAAGGATAGC CTGCAGCAGAGCAGCGGCCGGATCGACATCATCCACAGCAAGAAAA CAGCCGCCCTGCAGAGCGCCACCCCCGTGGAAAGAGTGAAACTGCA GGAAGCCCTGTCCCAGCTGGACTTCCAGTGGGAGAAAGTGAACAAA ATGTACAAGGACCGG CAGGGCAGATTCGACCGCAGCGTGGAAAAGT GGCGGCGGTTCCACTACGACATCAAGATCTTCAACCAGTGGCTGACA GAGGCCGAGCAGTTCCTGAGAAAGACCCAGATCCCCGAGAACTGGG AGCACGCCAAGTACAAGTGGTATCTGAAAGAGCTGCAGGACGGCAT CGGCCAGAGACAGACAGTCGTGCGGACCCTGAATGCCACCGGCGAG GAAATCATCCAGCAGTCCAGCAAGACC GACGCCAGCATTCTGCAGG

- 75 046419

AAAAGCTGGGCAGCCTGAACCTGCGGTGGCAGGAAGTGTGCAAGCA GCTGTCCGACCGGAAGAAGCGGCTGGAAGAACAGCTGGAACGGCTG CAGGAACTGCAGGAGGCCACCGACGAGCTGGACCTGAAACTGAGAC AGGCCGAAGTGATCAAGGGCAGCTGGCAGCCAGTGGGCGACCTGCT GATCGACTCCCTGCAGGACCATCTGGAAAAAGTGAAGGCCCTGAGA GGCGAGATCGCCC CCCTGAAAGAAAACGTGTCCCACGTGAACGACC TGGCCCGGCAGCTGACAACACTGGGCATTCAGCTGAGCCCCTACAA CCTGAGCACACTGGAAGATCTGAACACCCGGTGGAAGCTGCTGCAG GTGGCCGTGGAAGATAGAGTGCGGCAGCTGCACGAGGCCCACAGAG ATTTTGGCCCTGCCTCCCAGCACTTCCTGAGCACCAGTGTGCAGGGC CCTTGGGAGAGCCATCTCCCCTAAC AAGGTGCCCTACTACATCAA CCACGAGACACAGACCACCTGTTGGGACCACCCCAAGATGACCGAG CTGTACCAGAGCCTGGCTGACCTGAACAACGTGCGGTTCAGCGCCTA CCGGACCGCCATGAAGCTGCGGAGACTGCAGAAAGCTCTGTGCCTG GATCTGCTGTCCCTGTCCGCCGCCTGTGATGCCCTGGACCAGCACAA TCTGAAGCAGAACGACCAGCCCATGGATATCCTGCAGAT CA GAGAAGGCTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCTA GACAGCTGGGCGAAGTGGCCAGCTTTGGCGGCAGCAACATCGAGCC TAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAACAAGCCCGAGATC GAGGCCGCCCTGTTCCTGGACTGGATGAGACTGGAACCCCAGAGCA TGGTGTGGCTGCCCGTGCTGCATAGAGTGGCCGCTGCCGAGACAGCC AAGCACCAGGCCAAG TGCAACATCTGCAAAGAGTGCCCCATCATCG GCTTCCGGTACAGAAGCCTGAAGCACTTCAACTACGATATCTGCCAG AGCTGTTTCTTCAGCGGCAGGGTGGCCAAGGGCCACAAAATGCACT ACCCCATGGTGGAATACTGCACCCCCACCACAAGCGGCGAGGATGT GCGGGATTTCGCCAAGGTGCTGAAAAACAAGTTCCGGACCAAGCGG TACTTCGCCAAACACCCCCGGATGGGCTACCT GCCTGTGCAGACAGT GCTGGAAGGCGACAACATGGAAACCCCTGCCCAGATCCTGATCAGC CTGGAATCCGAGGAACGGGGCGAGCTGGAACGGATCCTGGCCGATC TGGAAGAGGAAAACCGGAACCTGCAGGCCGAGTACGACCGGCTGAA GCAGCAGCACGAGCACAAGGGCCTGAGCCCCCTGCCTAGCCCCCCT GACACCATGTGATGATGATTTGGGCAGAGCGATGGAGTCCTTA GTAT CAGTCATGACAGATGAAGAAGGAGCAGAATAAATGTTTTACAACTC CTGATTCCCGCATGCGGCCAGCTTATCGATACCGTCGA SEQ ID NO: 145 - BXA- 027744 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA

- 76 046419

TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTGGAGCGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGA CACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGCAGGTGTCCATCGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCTCAGGGCTACGAGCGGACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCT ACACCCAGGCCGCCTACGTGACCACC AGCGACCCCACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGACAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGTCCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCCCAGGGCGAGATCAGCAACG ACGTGGAAGTGGTGAAAGACCAGTTCCACACCCACGA GGGCTACAT GATGGACCTGACCGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCAGCAAGCTGATCGGCACCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAT CCACACCGTGCGGGAAGACAATGATGGTGATGACCGAGGACATG CCCCT GGAAATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCAC CCACGTGTCCCAGGCTCTGCTGGAAGTGGAACAGCTGCTGAACGCCC CCGACCTGTGCGCCAAGGACTTCGAGGATCTGTTCAAGCAGGAAGA GAGCCTGAAGAATATCAAGGACTCCCTGCAGCAGTCCAGCGGCCGG ATCGACATCATCCACAGCAAGAAAACAGCCGCCCTGCAGTCCGCCA CCCCCGTGGAAAGAGTGA AGCTGCAGGAAGCCCTGAGCCAGCTGGA CTTCCAGTGGGAGAAAGTGAACAAGATGTACAAGGACCGGCAGGGC AGATTCGACCGCAGCGTGGAAAAGTGGCGGCGGTTCCACTACGACA TCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGAG AAAGACCCAGATCCCCGAGAACTGGGAGCACGCCAAGTACAAGTGG TATCTGAAAGAACTGCAGGATGGCATCGGC CAGCGGCAGACCGTGG TGCGCACACTGAATGCCACCGGCGAGGAAATCATCCAGCAGAGCAG CAAGACCGACGCCAGCATCCTGCAGGAAAAGCTGGGCTCCCTGAAC CTGCGGTGGCAGGAAGTGTGCAAGCAGCTGAGCGACCGGAAGAAGA GGCTGGAAGAACAGGCCCCTGGCCTGACCACCATCGGCGCCAGCCC TACCCAGACCGTGACCCTGGTGACACAGCCCGTGGTGACAAAAG AG ACAGCCATCTCCAAGCTGGAAATGCCCAGCTCCCTGATGCTGGAAGT ACCTGCTCTGGCAGATTTCAACCGGGCTTGGACAGAACTTACCGACT GGCTTTCTCTGCTTGATCAAGTTATAAAATCACAGAGGGTGATGGTG GGTGACCTTGAGGATATCAACGAGATGATCATCAAGCAGAAGGCAA CAATGCAGGATTTGGAACAGAGGCGTCCCCAGTTGGAAGAACTCAT TACCGCTGCCCAAA ATTTGAAAAACAAGACCAGCAATCAAGAGGCT AGAACAATCATTACGGATCGAATTGAAAGAATTCAGAATCAGTGGG ATGAAGTACAAGAACACCTTCAGAACCGGAGGCAACAGTTGAATGA AATGCTGGAAAGGCTGCAGGAACTGCAGGAGGCCACCGACGAGCTG GACCTGAAGCTGCGGCAGGCCGAAGTGATCAAGGGCAGCTGGCAGC CCGTGGGCGACCTGCTGATCGACAGCCTGCA GGACCACCTGGAAAA

- 77 046419

AGTGAAGGCCCTGAGAGGCGAGATCGCCCCCCTGAAAGAAAACGTG TCCCACGTGAACGACCTGGCCCGGCAGCTGACCACCCTGGGCATCCA GCTGAGCCCCTACAACCTGAGCACCCTGGAAGATCTGAACACCCGG TGGAAGCTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGCTGC ACGAGGCCCACAGAGACTTTGGCCCTGCCAGCCAGCACTTCCTGAGC ACCTCTGTGCAGG GACCCTGGGAGAGAGCCATCAGCCCCAACAAGG TGCCCTACTACATCAACCACGAGACACAGACCACCTGTTGGGACCAC CCCAAGATGACCGAGCTGTACCAGAGCCTGGCCGACCTGAACAATG TGCGGTTCAGCGCCTACCGGACCGCCATGAAGCTGAGGCGGCTGCA GAAAGCTCTGTGCCTGGATCTGCTGAGCCTGAGCGCCGCCTGCGACG CCCTGGACCAGCACAACCTGAAGCAGAAC GACCAGCCCATGGATAT CCTGCAGATCATCAACTGCCTGACCACAATCTACGACAGGCTGGAAC AGGAACACAACAATCTGGTCAACGTGCCCCTGTGCGTGGACATGTG CCTGAATTGGCTGCTGAATGTGTACGACACCGGCCGGACCGGCAGA ATCCGGGTGCTGAGCTTCAAGACCGGCATCATCAGCCTGTGCAAGGC CCACCTGGAAGATAAGTACCGCTACCTGTTCAAACAGGTGGCCAG CT CCACCGGCTTTTGCGACCAGCGGAGACTGGGCCTGCTGCTGCACGAC AGCATCCAGATCCCCAGACAGCTGGGCGAGGTGGCCTCCTTCGGCG GCAGCAACATTGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAAC AACAAGCCCGAGATCGAGGCCGCCCTGTTCCTGGACTGGATGAGAC TGGAACCCCAGAGCATGGTGTGGCTGCCCGTGCTGCATCGGGTGGCC GCTG CCGAGACAGCCAAGCACCAGGCCAAGTGCAACATCTGCAAAG AGTGCCCCATCATCGGCTTCCGGTACAGAAGCCTGAAGCACTTCAAC TACGATATCTGCCAGAGCTGCTTCTTCAGCGGCAGAGTGGCCAAGGG CCACAAAATGCACTACCCCATGGTGGAATACTGCACCCCCACCACCA GCGGCGAGGATGTGCGGGACTTCGCCAAGGTGCTGAAAAACAAGTT CCGGACCAAGCGGTACTTT GCCAAGCACCCCCGGATGGGCTACCTGC CCGTGCAGACAGTGCTGGAAGGCGACAACATGGAAACCCCTGCCCA GATCCTGATCAGCCTGGAAAGCGAGGAACGGGGCGAGCTGGAACGG ATCCTGGCCGATCTGGAAGAGGAAAACCGGAACCTGCAGGCCGAGT ACGACCGGCTGAAGCAGCAGCACGAGCACAAGGGCCTGAGCCCCCT GCCTAGCCCCCCTGACACCATGTGATGATGATTTG GGCAGAGCGATG GAGTCCTTAGTATCAGTCATGACAGATGAAGAAGGAGCAGAATAAA TGTTTTACAACTCCTGATTCCCGCATGCGGCCAGCTTATCGATACCGT CGAAATAAAAGATCCTTATTTTCATTGGATCTGTGTGTTGGTTTTTTG TGTG SEQ ID NO: 146 - BXA- 027742 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTCAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGATCTGCTGGAAGGCCTGACCGGACAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTCGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAAGTGAAGAACGT GATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAG ATCCTGCTGAGCTGGGTGCGCCAGAGCACCAGAAACTACCCCCCAAG TGAACGTGATCAACTTCACCACCTCTT GGAGCGACGGCCTGGCCCTG

- 78 046419

AATGCCCTGATCCACAGCCACAGACCCGACCTGTTCGACTGGAACA GCGTCGTGTGTCAGCAGAGCGCCACCCAGAGGCTGGAACACGCCTT CAATATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGACCCC GAGGATGTGGACACCTACCCCGACAAGAAATCCATCCTGATGT ATATCACCAGCCTGTTCCAGGTGCTGCTCAGCAGGTGTCCATCGAG GCCATCCAGGA AGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCA AAGAGGAACACTTCCAGCTGCACCACCAGATGCACTACTCTCAGCA GATCACCGTGTCCCTGGCCCAGGGCTACGAGAGAACCAGCAGCCCC AAGCCCCGGTTCAAGAGCTACGCCTATACCCAGGCCGCCTACGTGAC CACCAGCGACCCTACCAGAAGCCCATTCCCCAGCCAGCATCTGGAA GCCCCCGAGGACAAGAGCTTCGGCA GCAGCCTGATGGAAAGCGAAG TGAACCTGGATAGATACCAGACCGCCCTGGAAGAGGTGCTGTCCTG GCTGCTGAGCGCCGAGGATACACTGCAGGCTCAGGGCGAGATCAGC AACGACGTGGAAGTCGTGAAGGACCAGTTCCACACCCACGAGGGCT ACATGATGGACCTGACAGCCCACCAGGGCAGAGTGGGCAACATTCT GCAGCTGGGCTCCAAGCTGATCGGCACCGGCAAGCT GAGCGAGGAC GAAGAGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGA TGGGAGTGCCTGAGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGC ACCGGGTGCTGATGGATCTGCAGAACCAGAAGCTGAAAGAGCTGAA CGACTGGCTGACCAAGACCGAGGAACGGACCCGGAAGATGGAAGA GGAACCCCTGGGACCAGACCTGGAAGATCTGAAAAGACAGGTGCAG CAGCAC AAAGTGCTGCAGGAAGATCTGGAACAGGAACAGGTGCGCG TGAACAGCCTGACCCACATGGTCGTGGTGGTGGACGAGAGCAGCGG CGATCATGCCACAGCCGCTCTGGAAGAACAGCTGAAGGTGCTGGGC GACAGATGGGCCAACATCTGCCGGTGGACCGAGGATAGATGGGTGC TGCTGCAGGACATCCTGCTGAAGTGGCAGCGGCTGACAGAGGAACA GTCCTGTTCTCCGC CTGGCTGTCCGAGAAAGAGGACGCCGTGAACA AAATCCACACCACCGGCTTCAAGGACCAGAACGAGATGCTGAGCAG CCTGCAGAAACTGGCCGTGCTGAAGGCCGACCTGGAAAAGAAAAAG CAGTCCATGGGCAAGCTGTACTCCCTGAAGCAGGACCTGCTGTCCAC CCTGAAGAACAAGAGCGTGACCCAGAAAACCGAGGCCTGGCTGGAC AACTTCGCCCGGTGCTGGGACAACCTGG CAGAAGCTGGAAAAGT CCACCGCCCAGATCTCCCAGGCCATTCACACCGTGCGGGAAGAGAC AATGATGGTCATGACTGAGGACATGCCCCTGGAAATCAGCTACGTG CCCTCCACCTACCTGACCGAGATCACCCATGTGTCCCAGGCCCTGCT GGAAGTGGAACAGCTGCTGAACGCCCCCGATCTGTGCGCCAAGGAC TTCGAGGATCTGTTCAAGCAGGAAGAGCCTGAAGAATATC AAGG ACTCTCTGCAGCAGTCCAGCGGCAGAATCGACATCATCCACAGCAA GAAAACAGCCGCCCTGCAGTCCGCCACCCCCGTGGAAAGAGTGAAA CTGCAGGAAGCCCTGTCCCAGCTGGACTTCCAGTGGGAGAAAGTGA ACAAGATGTACAAGGACCGGCAGGGCAGATTCGACCGCAGCGTGGA AAAGTGGCGGCGGTTCCACTACGACATCAAGATCTTCAACCAGTGG CTGACCGA GGCCGAACAGTTCCTGAGAAAGACCCAGATCCCCGAGA ACTGGGAGCACGCCAAGTACAAGTGGTATCTGAAAGAACTGCAGGA TGGCATCGGCCAGAGACAGACCGTCGTGCGGACACTGAATGCCACC GGCGAGGAAATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCC TGCAGGAAAAGCTGGGCAGCCTGAACCTGAGATGGCAGGAAGTGTG CAAGCAGCTGTCCGACCGGAAGAAGC GGCTGGAAGAACAGCTGGAA

- 79 046419

CGGCTGCAGGAACTGCAGGAGGCCACCGACGAGCTGGACCTGAAAC TGAGACAGGCCGAAGTGATCAAGGGCAGCTGGCAGCCAGTGGGCGA CCTGCTGATCGATTCTCTGCAGGACCACCTGGAAAAAGTGAAGGCCC TGAGAGGCGAGATCGCCCCCCTGAAAGAAAACGTGTCCCACGTGAA CGACCTGGCCCGGCAGCTGACAACACTGGGCATCCAGCTGAGCCCC TACAACCTGA GCACACTGGAAGATCTGAACACCCGGTGGAAGCTGC TGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGCTGCACGAGGCCCA CAGAGATTTTGGCCCTGCCTCCCAGCACTTCCTGAGCACCTCAGTGC AGGGCCCTTGGGAGAGAGCCATCTCCCCTAACAAGGTGCCCTACTAC ATCAACCACGAGACACAGACCACCTGTTGGGACCACCCCAAGATGA CCGAGCTGTACCAGAGCCTGGCCGAC CTGAACAATGTGCGGTTCAG CGCCTACCGGACCGCCATGAAGCTGCGGAGACTGCAGAAAGCTCTG TGCCTGGATCTGCTGTCCCTGAGCGCCGCCTGTGATGCCCTGGACCA GCACAATCTGAAGCAGAACGACCAGCCCATGGATATCCTGCAGATC ATTAACTGCCTGACCACCATCTACGACCGGCTGGAACAGGAACACA ACAACCTCGTGAACGTGCCCCTGTGCGTGGACATGTGCCTGAA TTGG CTGCTGAATGTGTACGACACCGGCCGGACAGGCCGGATCAGAGTGC TGAGCTTCAAGACCGGCATCAGCCTGTGCAAAGCCCATCTGGAA GATAAGTACCGCTACCTGTTCAAACAGGTGGCCAGCTCTACCGGCTT CTGCGATCAGAGAAGGCTGGGCCTGCTGCTGCATGACTCCATCCAGA TCCCCAGACAGCTGGGCGAGGTGGCCTCTTTCGGCGGCAGCAATATC GA GCCTAGCGTGCGGAGCTGCTTCCAGTTTCGCCAACAACAAGCCCG AGATCGAGGCCGCCCTGTTTCTGGACTGGATGAGACTGGAACCCCA GAGCATGGTGTGGCTGCCCGTGCTGCATAGAGTGGCCGCTGCCGAG ACAGCCAAGCACCAGGCCAAGTGCAATATCTGCAAAGAGTGCCCCA TCATCGGCTTCCGGTACAGAAGCCTGAAGCACTTCAACTACGATATC TGCCAGAGCTGTTTC TTCAGCGGCAGGGTGGCCAAGGGCCACAAAA TGCACTACCCCATGGTGGAATACTGCACCCCCACCACAAGCGGAGA GGATGTGCGGGACTTCGCCAAGGTGCTGAAAAACAAGTTCCGGACC AAGCGGTATTTCGCCAAACACCCCCGGATGGGCTACCTGCCTGTGCA GACAGTGCTGGAAGGCGACAACATGGAAACCGACACCATGTGATGA TGATTTGGGCAGAGCGATGGAGTCCTTA GTATCAGTCATGACAGATG AAGAAGGAGCAGAATAAATGTTTTACAACTCCTGATTCCCGCATGCG GCCAGCTTATCGATACCGTCGAAATAAAAGATCCTTATTTTCATTGG ATCTGTGTGTTGGTTTTTTGTGTG SEQ ID NO: 147 - BXA- 027743 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGC CCTGCGGGTGCTGCAGAACAACAACGTGGACCTGGTGAACATCGGC AGCACCGACATCGTGGACGGCAAC CACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCA GGTCAAAAACGTGATGAAGAACA TCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGATCCTGCTGAG CTGGGTGCGCCAGAGCACCCGGAA

- 80 046419

CTACCCCCAGGTCAACGTGATCAACTTCACCACCTCTTGGAGCGACG GCCTGGCCCTGAACGCCCTGATC CACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCGTGGTCTGCCA GCAGAGCGCCACCCAGCGGCTGG AACACGCCTTCAATATCGCCAGATACCAGCTGGGCATCGAGAAGCT GCTGGATCCCGAGGACGTGGACAC CACCTACCCCGACAAGAAATCCATCCTGATGTATATCACCAGCCTGT TCCAGGTGCTGCCCCAGCAGGTG TCCATCGAGGCCATCCAGGAAGTGGAAATGCTGCCCAGACCCCCCA AAGTGACCAAAGAGGAACACTTCC AGCTGCACCACCAGATGCACTACAGCCAGCAGATCACGTGTCCCTG GCTCAGGGCTACGAGCGGACCAG CAGCCCCAAGCCCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCT ACGTGACCACCAGCGACCCCACC AGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCCCCGAGGACAAGA GCTTCGGCAGCAGCCTGATGGAAA GCGAAGTGAACCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCT GTCCTGGCTGCTGAGCGCCGAGGA TACACTGCAGGCCCAGGGCGAGATCAGCAACGACGTGGAAGTGGTG AAAGACCAGTTCCACACCCACGAG GGCTACATGATGGACCTGACCGCCCACCAGGGCAGAGTGGGCAACA TCCTGCAGCTGGGCAGCAAGCTGA TCGGCACCGGCAAGCTGAGCGAGGACGAAGAGACAGAGGTGCAGG AACAGATGAACCTGCTGAACAGCAG ATGGGAGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTG CACATCCACACCGTGCGGGAAGAG ACAATGATGGTGATGACCGAGGACATGCCCCTGGAAATCAGCTACG TGCCCAGCACCTACCTGACCGAGA TCACCCACGTGTCCCAGGCTCTGCTGGAAGTGGAACAGCTGCTGAAC GCCCCCGACCTGTGCGCCAAGGA CTTCGAGGATCTGTTCAAGCAGGAAGAGAGCCTGAAGAATATCAAG GACTCCCTGCAGCAGTCCAGCGGC CGGATCGACATCATCCACAGCAAGAAAACAGCCGCCCTGCAGTCCG CCACCCCCGTGGAAAGAGTGAAGC TGCAGGAAGCCCTGAGCCAGCTGGACTTCCAGTGGGAGAAAGTGAA CAAGATGTACAAGGACCGGCAGGG CAGATTCGACCGCAGCGTGGAAAAGTGGCGGCGGTTCCACTACGAC ATCAAGATCTTCAACCAGTGGCTG ACCGAGGCCGAGCAGTTCCTGAGAAAGACCCAGATCCCCGAGAACT GGGAGCACGCCAAGTACAAGTGGT ATCTGAAAGAACTGCAGGATGGCATCGGCCAGCGGCAGACCGTGGT GCGCACACTGAATGCCACCGGCGA GGAAATCATCCAGCAGAGCAGCAAGACCGACGCCAGCATCCTGCAG GAAAAGCTGGGCTCCCTGAACCTG CGGTGGCAGGAAGTGTGCAAGCAGCTGAGCGACCGGAAGAAGAGG CTGGAAGAACAGGCCCCTGGCCTGA CCACCATCGGCGCCAGCCCTACCCAGACCGTGACCCTGGTGACACA GCCCGTGGTGACAAAAGAGACAGC

- 81 046419

CATCTCCAAGCTGGAAATGCCCAGCTCCCTGATGCTGGAATCTGACC AGTGGAAGCGTCTGCACCTTTCT CTGCAGGAACTTCTGGTGTGGCTACAGCTGAAAGATGATGAATTAA GCCGGCAGGCACCTATTGGAGGCG ACTTTCCAGCAGTTCAGAAGCAGAACGATGTACATAGGGCCTTCAA GAGGGAATTGAAAACTAAAGAACC TGTAATCATGAGTACTCTTGAGACTGTACGAATATTTCTGACAGAGC AGCCTTTGGAAGGACTAGAGAAA CTCTACCAGGAGCCCAGAGAGCTGCCTCCTGAGGAGAGAGCCCAGA ATGTCACTCGGCTTCTACGAAAGC AGGCTGAGGAGGTCAATACTGAGTGGGAAAAATTGAACCTGCACTC CGCTGACTGGCAGAGAAAAATAGA TGAGACCCTGGAAAGGCTGCAGGAACTGCAGGAGGCCACCGACGAG CTGGACCTGAAGCTGCGGCAGGCC GAAGTGATCAAGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCG ACAGCCTGCAGGACCACCTGGAAA AAGTGAAGGCCCTGAGAGGCGAGATCGCCCCCCTGAAAGAAAACGT GTCCCACGTGAACGACCTGGCCCG GCAGCTGACCACCCTGGGCATCCAGCTGAGCCCCTACAACCTGAGC ACCCTGGAAGATCTGAACACCCGG TGGAAGCTGCTGCAGGTGGCCGTGGAAGATAGAGTGCGGCAGCTGC ACGAGGCCCACAGAGACTTTGGCC CTGCCAGCCAGCACTTCCTGAGCACCTCTGTGCAGGGACCCTGGGAG AGAGCCATCAGCCCCAACAAGGT GCCCTACTACATCAACCACGAGACACAGACCACCTGTTGGGACCAC CCCAAGATGACCGAGCTGTACCAG AGCCTGGCCGACCTGAACAATGTGCGGTTCAGCGCCTACCGGACCG CCATGAAGCTGAGGCGGCTGCAGA AAGCTCTGTGCCTGGATCTGCTGAGCCTGAGCGCCGCCTGCGACGCC CTGGACCAGCACAACCTGAAGCA GAACGACCAGCCCATGGATATCCTGCAGATCATCAACTGCCTGACCA CAATCTACGACAGGCTGGAACAG GAACACACAATCTGGTCAACGTGCCCCTGTGCGTGGACATGTGCCT GAATTGGCTGCTGAATGTGTACG ACACCGGCCGGACCGGCAGAATCCGGGTGCTGAGCTTCAAGACCGG CATCATCAGCCTGTGCAAGGCCCA CCTGGAAGATAAGTACCGCTACCTGTTCAAACAGGTGGCCAGCTCCA CCGGCTTTTGCGACCAGCGGAGA CTGGGCCTGCTGCTGCACGACAGCATCCAGATCCCCAGACAGCTGG GCGAGGTGGCCTCCTTCGGCGGCA GCAACATTGAGCCCAGCGTGCGGAGCTGCTTCCAGTTCGCCAACAAC AAGCCCGAGATCGAGGCCGCCCT GTTCCTGGACTGGATGAGACTGGAACCCCAGAGCATGGTGTGGCTG CCCGTGCTGCATCGGGTGGCCGCT GCCGAGACAGCCAAGCACCAGGCCAAGTGCAACATCTGCAAAGAGT GCCCCATCATCGGCTTCCGGTACA GAAGCCTGAAGCACTTCAACTACGATATCTGCCAGAGCTGCTTCTTC AGCGGCAGAGTGGCCAAGGGCCA

- 82 046419

CAAAATGCACTACCCCATGGTGGAATACTGCACCCCCACCACCAGC GGCGAGGATGTGCGGGACTTCGCC AAGGTGCTGAAAAACAAGTTCCGGACCAAGCGGTACTTTGCCAAGC ACCCCCGGATGGGCTACCTGCCCG TGCAGACAGTGCTGGAAGGCGACAACATGGAAACCCCTGCCCAGAT CCTGATCAGCCTGGAAAGCGAGGA ACGGGGCGAGCTGGAACGGATCCTGGCCGATCTGGAAGAGGAAAAC CGGAACCTGCAGGCCGAGTACGAC CGGCTGAAGCAGCAGCACGAGCACAAGGGCCTGAGCCCCCTGCCTA GCCCCCCTGACACCATGTGATGAT GATTTGGGCAGAGCGATGGAGTCCTTAGTATCAGTCATGACAGATG AAGAAGGAGCAGAATAAATGTTTT ACAACTCCTGATTCCCGCATGCGGCCAGCTTATCGATACCGTCGAAA TAAAAGATCCTTATTTTCATTGG ATCTGTGTGTTGGTTTTTTTGTGTG SEQ ID NO: 148 - BXA- 212371 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCTCAGGGCTACGAGCGGACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACCACC AGCGACCCCACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGTCCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCCCAGGGCGAGATCAGCAACG ACGTGGAAGTGGTGAAAGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACCGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCAGCAAGCTGATCGGCA CCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAT CCACACCGTGCGGGAAGAGACAATGATGGTGATGACCGAGGACATG CCCCTGGAAATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCAC CCACGTGTCCCAGGCTCTGCTGGAAGTGGAACAGCT GCTGAACGCCC

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CCGACCTGTGCGCCAAGGACTTCGAGGATCTGTTCAAGCAGGAAGA GAGCCTGAAGAATATCAAGGACTCCCTGCAGCAGTCCAGCGGCCGG ATCGACATCATCCACAGCAAGAAAACAGCCGCCCTGCAGTCCGCCA CCCCCGTGGAAAGAGTGAAGCTGCAGGAAGCCCTGAGCCAGCTGGA CTTCCAGTGGGAGAAAGTGAACAAGATGTACAAGGACCGGCAGGGC AGATTCGACCGC AGCGTGGAAAAGTGGCGGCGGTTCCACTACGACA TCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGAG AAAGACCCAGATCCCCGAGAACTGGGAGCACGCCAAGTACAAGTGG TATCTGAAAGAGCTGCAGGACGGCATCGGCCAGCGGCAGACAGTGG TCCGCACCCTGAATGCCACCGGCGAGGAAATCATCCAGCAGAGCAG CAAGACCGACGCCAGCATCCTGCAGG AAAAGCTGGGCAGCCTGAAC CTGCGGTGGCAGGAAGTGTGCAAGCAGCTGAGCGACCGGAAGAAGC GGCTGGAAGAACAGGCCCCTGGCCTGACCACAATCGGCGCCAGCCC TACCCAGACCGTGACCCTGGTGACACAGCCCGTGGTGACAAAAGAG ACAGCCATCAGCAAGCTGGAAATGCCCAGCAGCCTGATGCTGGAAA GCGACCAGTGGAAGCGGCTGCACCTGAGCCTGCAGGA ACTGCTGGT CTGGCTGCAGCTGAAGGACGACGAGCTGAGCAGACAGGCCCCCATC GGCGGCGATTTCCCCGCCGTGCAGAAACAGAACGACGTGCACCGGG CCTTCAAGCGCGAGCTGAAAACAAAAGAACCCGTGATCATGAGCAC CCTGGAAACCGTGCGGATCTTCCTGACCGAGCAGCCCCTGGAAGGC CTGGAAAAGCTGTACCAGGAACCCAGAGAGCTGCCCCCCGAGGAAC GGGCC CAGAACGTGACCAGACTGCTGCGGAAGCAGGCCGAAGAGGT CAACACCGAGTGGGAAGCTGAACCTGCACAGCGCCGACTGGCAG CGGAAGATCGACGACACTGGAACGGCTGCAGGAACTGCAGGAG GCCACCGACGAGCTGGACCTGAAGCTGAGACAGGCCGAAGTGATCA AGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACTCCCTGCA GGACCACCTGGAAAAAG TGAAGGCCCTGCGGGGCGAGATCGCCCCC CTGAAAGAAAACGTGTCCCACGTGAACGACCTGGCCCGGCAGCTGA CCACCCTGGGCATCCAGCTGAGCCCCTACAACCTGTCCACCCTGGAA GATCTGAACACCCGGTGGAAGCTGCTGCAGGTGGCCGTGGAAGATA GAGTGCGGCAGCTGCACGAGGCCCACAGAGACTTTGGCCCTGCCAG CCAGCACTTCCTGAGCACCCTGTGCAGGGACC CTGGGAGAGAGCC ATCAGCCCCAACAAGGTGCCCTACTACATCAACCACGACACAGA CCACCTGTTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGCCT GGCCGACCTGAACAATGTGCGGTTCAGCGCCTACCGGACCGCCATG AAGCTGAGGCGGCTGCAGAAAGCTCTGTGCCTGGATCTGCTGAGCCT GAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGA AC GACCAGCCCATGGATATCCTGCAGATCATCAACTGCCTGACCACAAT CTACGACAGGCTGGAACAGGAACACAACAATCTGGTCAACGTGCCC CTGTGCGTGGACATGTGCCTGAATTGGCTGCTGAATGTGTACGACAC CGGCCGGACCGGCAGAATCCGGGTGCTGAGCTTCAAGACCGGCATC ATCAGCCTGTGCAAGGCCCACCTGGAAGATAAGTACCGCTACCTGTT CAAACAGGTG GCCAGCTCCACCGGCTTTTGCGACCAGCGGAGACTG GGCCTGCTGCTGCACGACAGCATCCAGATCCCCAGACAGCTGGGCG AGGTGGCCTCCTTCGGCGGCAGCAACATTGAGCCCAGCGTGCGGAG CTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGT TCCTGGACTGGATGAGACTGGAACCCCAGAGCATGGTGTGGCTGCC CGTGCTGCATCGGGTGGCCG CTGCCGAGACAGCCAAGCACCAGCC

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AAGTGCAACATCTGCAAAGAGTGCCCCATCATCGGCTTCCGGTACAG AAGCCTGAAGCACTTCAACTACGATATCTGCCAGAGCTGCTTCTTCA GCGGCAGAGTGGCCAAGGGCCACAAAATGCACTACCCCATGGTGGA ATACTGCACCCCCACCACCAGGCGAGGATGTGCGGGACTTCGCC AAGGTGCTGAAAAACAAGTTCCGGACCAAGCGGTACTTTGCCAAGC ACCCCCGGATGGG CTACCTGCCCGTGCAGACAGTGCTGGAAGGCGA CAACATGGAAACCGACACCATGTGATGATGATTTGGGCAGAGCGAT GGAGTCCTTAGTATCAGTCATGACAGATGAAGAAGGAGCAGAATAA ATGTTTTTACAACTCCTGATTCCCGCATGCGGCCAGCTTATCGATACC GTCGAAATAAAAGATCCTTATTTTCATTGGATCTGTGTGTTGGTTTTTT TGTGTG SEQ ID NO: 149 - BXA- 212372 ATGCTGTGGTGGGAGGAAGTGGAAGATTGCTACGAGCGCGAGGACG TGCAGAAGAAAACCTTCACCAAATGGGTGAACGCCCAGTTCAGCAA GTTCGGCAAGCAGCACATCGAGAACCTGTTCAGCGACCTGCAGGAC GGCAGACGGCTGCTGGACCTGCTGGAAGGCCTGACCGGCCAGAAGC TGCCCAAAGAAGGGCAGCACCAGAGTGCACGCCCTGAACAACGT GAACAAGGCCC TGCGGGTGCTGCAGAACAACAACGTGGACCTGGTG AACATCGGCAGCACCGACATCGTGGACGGCAACCACAAGCTGACCC TGGGCCTGATCTGGAACATCATCCTGCACTGGCAGGTCAAAAACGTG ATGAAGAACATCATGGCCGGCCTGCAGCAGACCAACAGCGAGAAGA TCCTGCTGAGCTGGGTGCGCCAGAGCACCCGGAACTACCCCCAGGTC AACGTGATCAACTTCACCACCTCTTAG CGACGGCCTGGCCCTGAA CGCCCTGATCCACAGCCACCGGCCCGACCTGTTCGACTGGAACAGCG TGGTCTGCCAGCAGAGCGCCACCCAGCGGCTGGAACACGCCTTCAA TATCGCCAGATACCAGCTGGGCATCGAGAAGCTGCTGGATCCCGAG GACGTGGACACCACCTACCCCGACAAGAAATCCATCCTGATGTATAT CACCAGCCTGTTCCAGGTGCTGCCCCAGGTGTCCAT CGAGGCCA TCCAGGAAGTGGAAATGCTGCCCAGACCCCCCAAAGTGACCAAAGA GGAACACTTCCAGCTGCACCACCAGATGCACTACAGCCAGCAGATC ACCGTGTCCCTGGCTCAGGGCTACGAGCGGACCAGCAGCCCCAAGC CCCGGTTCAAGAGCTACGCCTACACCCAGGCCGCCTACGTGACCACC AGCGACCCCACCAGAAGCCCATTCCCCAGCCAGCATCTGGAAGCCC CCGAGGA CAAGAGCTTCGGCAGCAGCCTGATGGAAAGCGAAGTGAA CCTGGACAGATACCAGACCGCCCTGGAAGAGGTGCTGTCCTGGCTG CTGAGCGCCGAGGATACACTGCAGGCCCAGGGCGAGATCAGCAACG ACGTGGAAGTGGTGAAAGACCAGTTCCACACCCACGAGGGCTACAT GATGGACCTGACCGCCCACCAGGGCAGAGTGGGCAACATCCTGCAG CTGGGCAGCAAGCTGATCGGCA CCGGCAAGCTGAGCGAGGACGAAG AGACAGAGGTGCAGGAACAGATGAACCTGCTGAACAGCAGATGGG AGTGCCTGCGGGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAT CCACACCGTGCGGGAAGAGACAATGATGGTGATGACCGAGGACATG CCCCTGGAAATCAGCTACGTGCCCAGCACCTACCTGACCGAGATCAC CCACGTGTCCCAGGCTCTGCTGGAAGTGGAACAGCT GCTGAACGCCC CCGACCTGTGCGCCAAGGACTTCGAGGATCTGTTCAAGCAGGAAGA GAGCCTGAAGAATATCAAGGACTCCCTGCAGCAGTCCAGCGGCCGG ATCGACATCATCCACAGCAAGAAAACAGCCGCCCTGCAGTCCGCCA CCCCCGTGGAAAGAGTGAAGCTGCAGGAAGCCCTGAGCCAGCTGGA

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CTTCCAGTGGGAGAAAGTGAACAAGATGTACAAGGACCGGCAGGGC AGATTCGACCGCAGCGTGGAAAAGTGGCGGCGGTTCCACTACGACA TCAAGATCTTCAACCAGTGGCTGACCGAGGCCGAGCAGTTCCTGAG AAAGACCCAGATCCCCGAGAACTGGGAGCACGCCAAGTACAAGTGG TATCTGAAAGAGCTGCAGGACGGCATCGGCCAGCGGCAGACAGTGG TCCGCAC CCTGAATGCCACCGGCGAGGAAATCATCCAGCAGAGCAG CAAGACCGACGCCAGCATCCTGCAGGAAAAGCTGGGCAGCCTGAAC CTGCGGTGGCAGGAAGTGTGCAAGCAGCTGAGCGACCGGAAGAAGC GGCTGGAAGAACAGGCCCCTGGCCTGACCACAATCGGCGCCAGCCC TACCCAGACCGTGACCCTGGTGACACAGCCCGTGGTGACAAAAGAG ACAGCCATCAGCAAGCTGGAAA TGCCCAGCAGCCTGATGCTGGAAA GCGACCAGTGGAAGCGGCTGCACCTGAGCCTGCAGGAACTGCTGGT CTGGCTGCAGCTGAAGGACGACGAGCTGAGCAGACAGGCCCCCATC GGCGGCGATTTCCCCGCCGTGCAGAAACAGAACGACGTGCACCGGG CCTTCAAGCGCGAGCTGAAAACAAAAGAACCCGTGATCATGAGCAC CCTGGAAACCGTGCGGATCTCCTGACCGAGCAG CCCCTGGAAGGC CTGGAAAAGCTGTACCAGGAACCCAGAGAGCTGCCCCCCGAGGAAC GGGCCCAGAACGTGACCAGACTGCTGCGGAAGCAGGCCGAAGAGGT CAACACCGAGTGGGAGAAGCTGAACCTGCACAGCGCCGACTGGCAG CGGAAGATCGACGAGACACTGGAACGGCTGCAGGAACTGCAGGAG GCCACCGACGAGCTGGACCTGAAGCTGAGACAGGCCGAAGTGATCA AGGGCAGCTGGCAGCCCGTGGGCGACCTGCTGATCGACTCCCTGCA GGACCACCTGGAAAAAGTGAAGGCCCTGCGGGGCGAGATCGCCCCC CTGAAAGAAAACGTGTCCCACGTGAACGACCTGGCCCGGCAGCTGA CCACCCTGGGCATCCAGCTGAGCCCCTACAACCTGTCCACCCTGGAA GATCTGAACACCCGGTGGAAGCTGCTGCAGGTGGCCGTGGAAGATA GAGTGCGGCAGCTGCAC GAGGCCCACAGAGACTTTGGCCCTGCCAG CCAGCACTTCCTGAGCACCTCTGTGCAGGGACCCTGGGAGAGAGCC ATCAGCCCCAACAAGGTGCCCTACTACATCAACCAGGAGACACAGA CCACCTGTTGGGACCACCCCAAGATGACCGAGCTGTACCAGAGGCCT GGCCGACCTGAACAATGTGCGGTTCAGCGCCTACCGGACCGCCATG AAGCTGAGGCGGCTGCAGAAAGCTCTGTG CCTGGATCTGCTGAGCCT GAGCGCCGCCTGCGACGCCCTGGACCAGCACAACCTGAAGCAGAAC GACCAGCCCATGGATATCCTGCAGATCATCAACTGCCTGACCACAAT CTACGACAGGCTGGAACAGGAACACAACAATCTGGTCAACGTGCCC CTGTGCGTGGACATGTGCCTGAATTGGCTGCTGAATGTGTACGACAC CGGCCGGACCGGCAGAATCCGGGTGCTGAGCTTCAAGACC GGCATC ATCAGCCTGTGCAAGGCCCACCTGGAAGATAAGTACCGCTACCTGTT CAAACAGGTGGCCAGCTCCACCGGCTTTTGCGACCAGCGGAGACTG GGCCTGCTGCTGCACGACAGCATCCAGATCCCCAGACAGCTGGGCG AGGTGGCCTCCTTCGGCGGCAGCAACATTGAGCCCAGCGTGCGGAG CTGCTTCCAGTTCGCCAACAACAAGCCCGAGATCGAGGCCGCCCTGT TC CTGGACTGGATGAGACTGGAACCCCAGAGCATGGTGTGGCTGCC CGTGCTGCATCGGGTGGCCGCTGCCGAGACAGCCAAGCACCAGGCC AAGTGCAACATCTGCAAAGAGTGCCCCATCATCGGCTTCCGGTACAG AAGCCTGAAGCACTTCAACTACGATATCTGCCAGAGCTGCTTCTTCA GCGGCAGAGTGGCCAAGGGCCACAAAATGCACTACCCCATGGTGGA ATACTGCACCCCCACCACCAG CGGCGAGGATGTGCGGGACTTCGCC AAGGTGCTGAAAAACAAGTTCCGGACCAAGCGGTACTTTGCCAAGC ACCCCCGGATGGGCTACCTGCCCGTGCAGACAGTGCTGGAAGGCGA CAACATGGAAACCGACACCATGTGATGATGAGAAGTCTTTTTCCACAT GGCAGATGAAATAAAAGATCCTTATTTTCATTGGATCTGTGTGTTGG _TTTTTTGTGTG

SEQ ID NO: 148 encodes construct BXA-212371, which is similar to construct BXA027743 except that the C-terminus is deleted.

SEQ ID NO: 149 encodes the construct BXA-212372, which is similar to BXA-212371 but with a shorter 3'UTR.

In some embodiments, the miniaturized dystrophin polypeptide comprises or is a construct shown in Table. 9.

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Table 9

Amino acid sequence of the miniaturized dystrophin polypeptide BXA-213788

SEQ Ш NO: Subsequence 152 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNI GSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLS WVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQ SATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLP QQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERT SSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEV NLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYM MDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECL RVASMEKQSNLHIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQA LLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKT AALQSATPVERVKLQEALSQLDFQWEKVNK MYKDRQGRFDRSVEKW RRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQ RQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRK KRLEEQKNDLAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSLM LESDQWKRLHLSLQELLVWLQLK DDELSRQAPIGGDFPAVQKQNDVH RAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQ NVTRLLRI<QAEEVNTEWEI<LNLHSADWQRI<IDETLERLQELQEATDEL DLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSH VNDLARQLTTLGIQLSPYNL STLEDLNTRWKLLQVAVEDRVRQLHEAH RDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTEL YQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNL KQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVY DTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQ VASSTGFCDQRRLGL LLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMR LEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNY DICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRT KRYFAKHPRMGYLPVQTVLEGDNMET

In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 152, wherein the amino acid sequence, when expressed, has at least one dystrophin activity.

In some embodiments, the miniaturized dystrophin polypeptide comprises or is a construct shown in Table. 10.

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Table 10

Amino acid sequence of the miniaturized dystrophin polypeptide BXA-213780 J11V3

SEQ Ш NO: Subsequence 153 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDG RRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNI GSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLS WVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFD WNSVVCQQ SATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLP QQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERT SSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEV NLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYM MDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECL RVASMEKQSNLHRVLLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFK DQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQ KTEAWLDNFARCWDNLVQKLEKSTAQISQ AVTTTQPSLTQTTVMETVT TVTTREQILVKHAQEELPPPPPQKKRQITVDSEIHTVREETMMVMTEDM PLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLK NIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKV NKMYKDRQGRFDRSVEKWRRFHYDIKIF NQWLTEAEQFLRKTQIPENW EHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKL GSLNLRWQEVCKQLSDRKKRLEEQLERLQELQEATDELDLKLRQAEVI I<GSWQPVGDLLIDSLQDHLEI<VI<ALRGEIAPLI<ENVSHVNDLARQLTT LGIQLSPYNLSTLED LNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHF LSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNN VRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDI LQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRV LSFKTGIISLCKAHLEDKYRYLFKQVA SSTGFCDQRRLGLLLHDSIQIPR QLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWL PVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGR VAI<GHI<MHYPMVEYCTPTTSGEDVRDFAI<VLI<NI<FRTI<RYFAI<HPR MGYLPVQTVLEGDNMET

SEQ ID NO: 153 encodes construct BXA-213780J11V3, which is similar to construct BXA196477 except that amino acids A and Q of the internal SEAQ linker (SEQ ID NO: 74), as well as the last three amino acids at the C-terminus, are deleted in BXA-21378OJ11V3 .

In some embodiments, the miniaturized dystrophin polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 153, wherein the amino acid sequence, when expressed, has at least one dystrophin activity.

In some embodiments, the miniaturized dystrophin BXA-213780 J11V3 may be encoded by the nucleotide sequence shown in table. eleven.

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Table 11

Nucleotide sequence of the miniaturized _________________dystrophin polypeptide BXA-21378OJ11V3___________________

SEQ ID NO: Subsequence 154 atgctgtggtgggaggaagtggaagattgctacgagcgcgaggacgtgcagaagaaaaccttcaccaaatggg tgaacgcccagttcagcaagttcggcaagcagcacatcgagaacctgttcagcgacctgcaggacggcagacg gctgctggacctgctggaaggcctgaccggccaagagctgcccaaagagaagggcagc accagagtgcacg ccctgaacaacgtgaacaaggccctgcgggtgctgcagaacaacaacgtggacctggtgaacatcggcagcac cgacatcgtggacggcaaccacaagctgaccctgggcctgatctggaacatcatcctgcactggcaggtcaaaa acgtgatgaagaacatcatggccggcctgcagcagaccaacagcgagaaga tcctgctgagctgggtgcgcca gagcacccggaactacccccaggtcaacgtgatcaacttcaccacctcttggagcgacggcctggccctgaacg ccctgatccacagccaccggcccgacctgttcgactggaacagcgtggtctgccagcagagcgccacccagcg gctggaacacgccttcaatatcgccagataccagctg ggcatcgagaagctgctggatcccgaggacgtggaca ccacctaccccgacaagaaatccatcctgatgtatatcaccagcctgttccaggtgctgccccagcaggtgtccat cgaggccatccaggaagtggaaatgctgcccagaccccccaaagtgaccaaagaggaacacttccagctgcac caccagatgcactacagccagcagatcaccgt gtccctggcccagggctacgagagaaccagcagccccaag ccccggttcaagagctacgcctatacccaggccgcctacgtgaccaccagcgaccctaccagaagcccattccc cagccagcatctggaagcccccgaggacaagagcttcggcagcagcctgatggaaagcgaagtgaacctgga cagataccagaccgccctggaagaggtgctgagctgg ect gctgaacagcagatgggagtgcctgcgggtggccag catggaaaagcagagcaacctgcatagggtcctgctgaagtggcagcggctgaccgaggaacagtgcctgttta gcgcctggctgtccgagaaagaggacgccgtgaacaagatccacaccaccggcttcaaggaccagaacgaga tgctgagcagcctgcagaaactggccgtgctgaaggccgacctggaaaagaaaaagcagtccatgggcaagct gtactccctgaagcaggacctgctgtccaccctgaagaacaagagcgtgacccagaaaaccgaggcctggctg gacaacttcgcccggtgctgggacaacctggtgcagaagctggaaaagtccaccgcccaga tctcccaggccg tgaccacaacacagcccagcctgacccagaccaccgtgatggaaaccgtgacaacagtgaccacccgggaac agatcctcgtgaagcacgcccaggaagaactgccccctccacccccccagaagaaacggcagatcacagtgg acagtgaaatccacaccgtgcgggaagagacaatgatggtcatgacagaggacatgcccctggaa atcagctac gtgcccagcacctacctgaccgagatcacccatgtgtcccaggccctgctggaagtggaacagctgctgaacgc ccccgacctgtgcgccaaggatttcgaggacctgttcaagcaggaagagagcctgaagaatatcaaggatagcc tgcagcagagcagcggccggatcgacatcatccacagcaagaaaacagccgccctg cagagcgccaccccc gtggaaagagtgaaactgcaggaagccctgtcccagctggacttccagtgggagaaagtgaacaaaatgtacaa ggaccggcagggcagattcgaccgcagcgtggaaaagtggcggcggttccactacgacatcaagatcttcaac cagtggctgacagaggccgagcagttcctgagaaagacccagat ccccgagaactgggagcacgccaagtac aagtggtatctgaaagagctgcaggacggcatcggccagagacagacagtcgtgcggaccctgaatgccaccg gcgaggaaatcatccagcagtccagcaagaccgacgccagcattctgcaggaaaagctgggcagcctgaacct gcggtggcaggaagtgtgcaagcagctgtccgaccgga agaagcggctggaagaacagctggaacggctgc aggaactgcaggaggccaccgacgagctggacctgaaactgagacaggccgaagtgatcaagggcagctgg cagccagtgggcgacctgctgatcgactccctgcaggaccatctggaaaaagtgaaggccctgagaggcgaga tcgcccccctgaaagaaaacgtgtcccacgtgaac gacctggcccggcagctgacaacactgggcattcagctg agcccctacaacctgagcacactggaagatctgaacacccggtggaagctgctgcaggtggccgtggaagata gagtgcggcagctgcacgaggcccacagagattttggccctgcctcccagcacttcctgagcaccagtgtgcag ggcccttggagagagccatctcc cctaacaaggtgccctactacatcaaccacgagacacagaccacctgttg ggaccaccccaagatgaccgagctgtaccagagcctggctgacctgaacaacgtgcggttcagcgcctaccgg accgccatgaagctgcggagactgcagaaagctctgtgcctggatctgctgtccctgtccgccgcctgtgatgcc ctggaccagca caatctgaagcagaacgaccagcccatggatatcctgcagatcatcaactgcctgaccaccatc tacgaccggctggaacaggaacacaacaatctcgtgaacgtgcccctgtgcgtggacatgtgcctgaattggctg ctgaatgtgtacgacaccggccggacaggccggatcagagtgctgtccttcaagaccggcatcatcagcctgt g caaagcccacctggaagataagtaccggtatctgttcaaacaggtggcctctagcaccggcttttgcgaccagag aaggctgggcctgctgctgcacgacagcatccagatccctagacagctgggcgaagtggccagctttggcggc agcaacatcgagcctagcgtgcggagctgcttccagttcgccaacaacaagccc gagatcgaggccgccctgtt cctggactggatgagactggaaccccagagcatggtgtggctgcccgtgctgcatagagtggccgctgccgag acagccaagcaccaggccaagtgcaacatctgcaaagagtgccccatcatcggcttccggtacagaagcctgaa gcacttcaactacgatatctgccagagctgtttcttcagcggcagggt ggccaagggccacaaaatgcactaccc catggtggaatactgcacccccaccacaagcggcgaggatgtgcgggatttcgccaaggtgctgaaaaacaagt tccggaccaagcggtacttcgccaaacacccccggatgggctacctgcctgtgcagacagtgctggaaggcga caacatggaaacctgaGAAGTCTTTTCACATGGCAGATG AAATAAAAGATC CTTATTTTCATTGGATCTGTGTGTTGGTTTTTGTGTG

In some embodiments, the miniaturized dystrophin polypeptide has a connecting region between a hinge 2 (H2) domain and a spectrin repeat 16 R16 (R16) domain that contains the amino acid sequence set forth in Table 1. 12.

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Table 12

Amino acid sequences of connecting regions

SEQ Ш NO Subsequence SEQ Ш NO: 155 LPPPPPQKKRQITVDIHTVREETMMVMTEDMPLEI SEQ Ш NO: 156 LPPPPPQKKRQITVDSIHTVREETMMVMTEDMPLEI SEQ Ш NO: 157 LPPPPPQKKRQITVDSEIHTVREETMMVMTEDMPLEI SEQ Ш NO: 158 LPPPPPQKKRQITVDQIHTVREETMMVMTEDMPLEI SEQ Ш NO: 159 LPPPPPQKKRQITVDAQIHTVREETMMVMTEDMPLEI SEQ Ш NO: 160 LPPPPPQKKRQITVIHTVREETMMVMTEDMPLEI SEQ Ш NO: 161 LPPPPPQKKRQITVDHTVREETMMVMTEDMPLEI SEQ ID NO: 162 LPPPPPQKKRQITVHTVREETMMVMTEDMPLEI SEQ ID NO: 163 LPPPPPQKKRQITVDSQIHTVREETMMVMTEDMPLEI SEQ ID NO: 164 LPPPPPQKKRQITVDSHTVREETMMVMTEDMPLEI SEQ ID NO: 165 LPPPPPQKKRQITVQIHTVREETMMVMTEDMPLEI SEQ ID NO: 166 QKKRQITVDSEAQIHTVREETMMV

In other embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequences set forth in SEQ ID NO: 133-149, where the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In other embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequences set forth in SEQ ID NO: 133 (BXA-196477 ), SEQ ID NO: 134 (VHA-196473), SEQ ID NO: 135 (VHA -196474), SEQ ID NO: 136 (VHA-196475), SEQ ID NO: 137 (VHA-196476), SEQ ID NO: 139 (ВХА-196478), SEQ ID NO: 140 (ВХА-196479), SEQ ID NO: 148 (ВХА-212371) or SEQ ID NO: 149 (ВХА-212372). In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 133, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence

- 90 046419 you, which is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 134, where the polypeptide miniaturized dystrophin, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 135. In some embodiments, the nucleotide sequence encoding a miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80% identical to SEQ ID NO: 135. %, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, is at least about 99% or about 100% identical to the sequence of SEQ ID NO: 136. In some embodiments, the nucleotide sequence encoding a miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60% identical to about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97 % is at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 137. In some embodiments, the nucleotide sequence encoding a miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 138, wherein the miniaturized dystrophin polypeptide, when expressed in nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 139, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 140, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 141, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 142, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about

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70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% is at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 143, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 144, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 145, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 146, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 147, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 148, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity. In some embodiments, the nucleotide sequence encoding the miniaturized dystrophin polypeptide includes a nucleic acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 149, wherein the miniaturized dystrophin polypeptide, when expressed as a nucleotide sequence, has at least one dystrophin activity.

5.3.2. Linker

Also provided herein is an amino acid sequence or a nucleotide sequence encoding an amino acid sequence. In some embodiments, the present invention provides an amino acid sequence encoded by a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to the sequence of SEQ ID NO: 100, where the amino acid sequence includes SEQ ID NO: 75 (IHTVREE TMMVMTEDMP LEI). In some embodiments, the present invention provides an amino acid sequence encoded by a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about

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96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 99-100 in combination, wherein the amino acid sequence includes SEQ ID NO : 74-75 in combination (SEAQIHTVREE TMMVMTEDMP LEI).

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes an amino acid sequence comprising the sequence of SEQ ID NO: 75 (IHTVREE TMMVMTEDMP LEI), wherein the nucleic acid molecule is at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 100 (ATCCACCGTGCGGGAAGAGACAATGATGGTCATGACAGAGGACATGCCCCTGGA AATC).

In some embodiments, the amino acid sequence may be used as a linker connecting one or more dystrophin domains. In some embodiments, the linker connects the first dystrophin domain and the second dystrophin domain. In some embodiments, the first and second dystrophin domains that can be joined are an R1 domain and an R16 domain. In other embodiments, the first and second dystrophin domains that may be joined are an R3 domain and an R16 domain. In other embodiments, the first and second dystrophin domains that may be joined are an H2 domain and an R16 domain.

In some embodiments, the invention includes a nucleic acid molecule comprising a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical sequence SEQ ID NO: 99 (AGTGAAGCTCAG).

In some embodiments, the invention includes a nucleic acid molecule comprising a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical sequence SEQ ID NO: 100 (ATCCACACCGTGCGGGAAGAGACAATGATGGTCATGACAGAGGACATGCCCCTG GAAATC).

In some embodiments, the invention includes a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid nucleotide sequence that is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO: 99 and 100 in combination, (AGTGAAGCTCAGATCCACACCGTGCGGGAAGAGACAATGATGGTCATGACAGA ACATGCCCCTGGAAATC).

In some embodiments, a linker is placed into the miniaturized dystrophin polypeptide described herein by connecting two domains of the miniaturized dystrophin polypeptide (eg, the H2 and R16 domains). In some embodiments, the linker is located between the H2 and R16 domains in a miniaturized dystrophin polypeptide comprising ABD1-H1-R1-R3-H2-L-R16-R17-R24-H4-CR-C conc, where ABD1 is an actin binding domain 1, H1 is a hinge 1 domain, R1 is a spectrin repeat domain 1, R3 is a spectrin repeat domain 3, H2 is a hinge 2 domain, L is a linker, e.g., SEQ ID NO: 74 or SEQ ID NO: 75 (or both in combination) (or a sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90% , at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to these sequences), R16 is the spectrin repeat domain 16, R17 is the spectrin repeat domain 17, R24 is the spectrin domain 24, H4 is the hinge 4 domain, CR is the cysteine-rich domain, and C-terminal is the optional C-terminal domain (or its Part). The present invention also provides a linker containing IHTVREETMMVMTEDMPLEI (SEQ ID NO: 84). In some embodiments, a linker comprising the sequence set forth in SEQ ID NO: 75 (IHTVREETMMVMTEDMPLEI) (or a sequence that is at least about 60%, at least about 70%, at least about 80%, at least at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about about 99% or about 100% identical to the sequence shown in SEQ ID NO: 84 (IHTVREETMMVMTEDMPLEI)), placed in the described herein

- 93 046419 miniaturized dystrophin polypeptide by joining two miniaturized dystrophin polypeptide domains (eg, R1 and R16 domains). In some embodiments, the linker is located between the R1 and R16 domains in a miniaturized dystrophin polypeptide comprising ABD1-H1-R1-L-R16-R17-H3-R23-R24-H4-CR-C conc, where ABD1 is actin binding domain 1, H1 is hinge 1 domain, R1 is spectrin repeat domain 1, L is linker, e.g. SEQ ID NO: 84, R16 is spectrin repeat domain 16, R17 is spectrin repeat domain 17, H3 is hinge 3 domain , R23 is the spectrin domain 23, R24 is the spectrin domain 24, H4 is the hinge domain 4, CR is the cysteine-rich domain, and C-terminal is the C-terminal domain (or part thereof).

5.3.3. Non-coding polynucleotides

In some aspects, provided herein are nucleic acid molecules, such as DNA or RNA, comprising a nucleotide sequence encoding a miniaturized dystrophin polypeptide.

In some embodiments, the nucleic acid molecules described herein contain non-coding components. In some embodiments, the nucleic acid molecules described herein comprise promoters. Some exemplary regulatory sequences for expression in mammalian host cells include viral elements that mediate high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), adenovirus (eg, adenoviral major late promoter (AdMLP)) and polyoma virus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the beta globin promoter may be used. Additionally, regulatory elements consisting of sequences from other sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and T-cell leukemia virus type 1 long terminal repeat (Takebe, Y. et al. (1988) Mol. Cell Biol 8: 466-472). In some embodiments, the regulatory sequence comprises a tissue-specific promoter. In some embodiments, a tissue-specific promoter drives expression of a gene of interest in a tissue selected from the group consisting of heart, liver, lung, eye, nervous system, lymphatic system, central nervous system, nerve cells, muscle and stem cells. In some embodiments, the promoters disclosed herein are tissue-specific promoters. In some embodiments, the promoter drives expression of the therapeutic protein in hepatocytes, muscle cells, endothelial cells, sinusoidal cells or neuronal cells, or any combination thereof. In some embodiments, the promoter is selected from the group consisting of a synapsin 1 gene promoter, a mouse thyretin promoter (mTTR), a human endogenous factor VIII (F8) promoter, a human alpha-1 antitrypsin (hAAT) promoter, a minimal human albumin promoter, a murine albumin promoter, tristetraproline (TTP) promoter, CASI promoter, CAG promoter, cytomegalovirus (CMV) promoter, α1-antitrypsin (AAT) promoter, muscle creatine kinase (MCK) promoter, alpha myosin heavy chain (aMHC) promoter, myoglobin (MB) promoter, desmin promoter (DES), SPc5-12 promoter, 2R5Sc5-12 promoter, dMCK promoter, tMCK promoter, α-synuclein promoter and phosphoglycerate kinase (PGK) promoter.

In some embodiments, the nucleic acid molecules described herein contain an intronic sequence. In some embodiments, the intronic sequence is located 5' from the nucleotide sequence encoding the miniaturized dystrophin polypeptide. In some embodiments, the intronic sequence is located 3' from the promoter. In some embodiments, the intronic sequence includes a synthetic intronic sequence.

In some embodiments, the nucleic acid molecules described herein comprise a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element is located 3' of the nucleotide sequence encoding the miniaturized dystrophin polypeptide. In some embodiments, the post-transcriptional regulatory element comprises a mutant hepatitis virus post-transcriptional regulatory element (WPRE), a miRNA binding site, or a target nuclear DNA sequence, or any combination thereof.

In some embodiments, the nucleic acid molecules disclosed herein contain a 3 ^r UTR poly(A) tail sequence. In some embodiments, the 3UTR poly(A) tail sequence is selected from the group consisting of bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof. In some embodiments, the 3'UTR poly(A) tail sequence includes a bGH poly(A).

In some embodiments, the nucleic acid molecules described herein

- 94 046419 slots contain an enhancer sequence. In some embodiments, the nucleic acid molecules described herein comprise a first inverted terminal repeat (ITR) and/or a second ITR. In some embodiments, the first ITR and the second ITR are identical. In some embodiments, the first ITR and/or the second ITR are derived from an adeno-associated virus. In some embodiments, the first ITR is derived from an adeno-associated virus and the second ITR is derived from an adeno-associated virus.

It is further recognized that the nucleic acid molecule may contain additional elements that facilitate translation of the polypeptide. Such sequences include, for example, Kozak sequences attached to the 5' end of a polypeptide encoding a polynucleotide. The Kozak consensus sequence is a sequence that occurs on eukaryotic mRNA that plays a role in initiating the translation process and has the consensus (gee)gccRccAUGG (SEQ ID NO: 150); where (1) a lowercase letter denotes the most common base in a position where the base may, however, vary; (2) capital letters indicate highly conserved bases, that is, the AUGG sequence is constant or rarely, if at all, changes, except for the IUPAC ambiguity code 'R', which indicates that a purine (adenine or guanine) is commonly observed at that position; and (3) the sequence in parentheses ((gee)) has an undefined meaning.

In one non-limiting embodiment, the nucleic acid molecule includes a functional variant of the Kozak sequence or fragment thereof. A functional variant of the Kozak sequence or fragment thereof will retain the ability to increase protein translation relative to the level of translation from the sequence lacking the leader sequence. Such a functional fragment may contain at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 contiguous Kozak sequence nucleotides or a sequence given in SEQ ID NO: 150 or SEQ ID NO: 151 (gccaccATGG). Alternatively, the functional variant may include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the Kozak sequence or sequence given in SEQ ID NO: 150 or SEQ ID NO: 151.

5.3.4. Heterologous fragments

In some embodiments, the polypeptides of the present invention may further comprise an additional element, such as a heterologous fragment. Such elements may promote expression of the polypeptide, promote secretion of the polypeptide, improve stability of the polypeptide, provide more efficient purification of the polypeptide, and/or modulate the activity of the polypeptide. In some embodiments, the heterologous fragment is a polypeptide fragment. In other embodiments, the heterologous fragment is a non-polypeptide fragment. In some embodiments, the polypeptide contains a heterologous fragment fused to the polypeptide.

In some embodiments of the present invention, the polypeptide described herein includes one or more additional heterologous fragments. In some embodiments, the heterologous fragments are half-life extending fragments. In some embodiments, the heterologous fragment includes albumin or a fragment thereof, an immunoglobulin Fc region, a human chorionic gonadotropin β-subunit C-terminal peptide (CTP), a PAS sequence, a HAP sequence, transferrin or a fragment thereof, or an albumin-binding fragment, or derivative, or any combination thereof.

In some embodiments, the polypeptides disclosed herein contain one or more additional heterologous fragments. In some embodiments, the heterologous fragments are half-life extending fragments. In some embodiments, the heterologous fragment includes albumin, an immunoglobulin constant region or a portion thereof, an immunoglobulin binding polypeptide, immunoglobulin G (IgG), an albumin binding polypeptide (ABP), a PASylation fragment, a HESylation fragment, XTEN, a PEGylation fragment or an Fc region, or any combination of them.

5.4. Cells

In certain aspects, provided herein are cells (eg, host cells) expressing (eg, recombinantly) the proteins described herein, and expression vectors containing nucleotides encoding the proteins described herein.

In some embodiments, the host cell contains nucleic acid molecules described herein. In some embodiments, the host cell contains the vectors described herein.

In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from the group consisting of a mammalian cell, an insect cell, a yeast cell, a transgenic mammalian cell, and a plant cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial

- 95 046419 cell.

In some embodiments, the host cell is a mammalian cell. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 cells (a mouse myeloma cell line that does not produce endogenous immunoglobulin chains), CRL7O3O, COS (eg COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB/20, BMT10, HBK, NSO, HT1080 and HsS78Bst.

5.5. Vectors.

5.5.1. Adeno-associated virus (AAV).

5.5.1.1. Review

Provided herein are vectors (eg, expression vectors) containing nucleic acid molecules containing nucleotide sequences encoding miniaturized dystrophin protein for recombinant expression in host cells and cells that are the target of therapeutic intervention. As used herein, the term vector is intended to mean a nucleic acid molecule capable of carrying another nucleic acid to which it is linked; or an object containing such a nucleic acid molecule capable of carrying another nucleic acid. One type of vector is a plasmid, which designates a circular loop of double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Particular vectors or polynucleotides that are part of vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell and thus replicate along with the host genome. In addition, specific vectors are capable of controlling the expression of genes to which they are functionally linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). Typically, expression vectors for use in recombinant DNA techniques are often present in the form of plasmids. As used herein, the terms plasmid and vector may sometimes be used interchangeably, depending on the context, since plasmid is the most common form of vector. However, other forms of expression vectors are also disclosed herein, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions. In some embodiments, polynucleotides disclosed herein are expressed by an adeno-associated virus (AAV). AAV is a non-enveloped, single-stranded DNA virus belonging to the family Parvoviridae. Unlike most other members of the Parvoviridae family, AAV is replication defective and is only able to replicate efficiently in the presence of a helper virus such as an adenovirus or herpes virus. AAV was first discovered in the mid-1960s as a contaminant of adenovirus viral preparations. See Atchison R W, Casto V S, HAMMON W M. Science. 149(3685), 754-756 (1965). Since then, safer and more efficient ways to use AAV as a recombinant DNA vector have been developed. See, for example, Hermonat P.L. and Muzyczka N. Proc Natl Acad Sci USA. 81(20), 6466-6470 (1984). 3. Laughlin C.A., et al. Gene, 23(1), 65-73 (1983). Matsushita T., et al. Gene Ther. 5(7), 938-945 (1998). Xiao X., et al. Journal of Virology. 72(3), 2224-2232 (1998). A small number of AAV genomes have been shown to integrate into the host chromosome. See Cheung AK, Hoggan MD, Hauswirth WW, et al. Integration of the adeno-associated vims genome into cellular DNA in latently infected human Detroit 6 cells. J Virol 1980; 33:739-748. AAV is immunologically distinct from any known adenoviral antigen. The AAV capsid contains a single-stranded DNA (ssDNA) genome. See Rose JA, Berns KI, Hoggan MD, et al. Proc Natl Acad Sci USA 1969;64:863-869.

AAV has a 4.7-kb single-stranded DNA genome encoding the replication gene (rep) and encapsidation protein (cap) genes flanked by two inverted terminal repeats (ITRs). It predominantly does not integrate and forms stable episomes in nondividing tissue. Despite its high seroprevalence in the adult population, AAV has not been associated with any disease in humans. See Gonçalves, M. Virol. J. 2, 43 (2005). AAV's stable tissue expression, lack of pathogenicity, and ease of obtaining high titers have made it a very attractive vector and popular platform for gene transfer.

Recombinant AAV (rAAV) is a genetically modified AAV in which typically part or all of the rep and cap genes are replaced with heterologous transgenic sequences. Recombinant AAVs can also drive long-term transgene expression in postmitotic cells, most likely because the recombinant AAV genome is retained largely as circular episomes in the nucleus. The only cis-acting DNA element in rAAV required for rAAV production is the inverted terminal repeats (ITRs).

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AAV, while the rep, cap and adenoviral helper genes can be provided in transposition. Thus, in some embodiments disclosed herein, rAAVs contain only heterologous transgenic DNA flanked by ITR sequences and the genome is encapsidated within a serotype-specific AAV capsid.

AAV has unique features that make it attractive as a vector system for delivering foreign DNA into cells. Infection of cells in culture by AAV is generally not cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Moreover, AAV infects many different mammalian cell types, allowing it to target many different tissues in vivo. AAV also has additional advantages that make it a particularly attractive viral gene delivery system, including stimulation of an immune response that is relatively mild compared to other forms of gene delivery, and persistent expression in both dividing and quiescent cells on a nonintegrating, nonintegrating basis. episomal vector DNA. In addition, AAV withstands the conditions used to inactivate adenovirus (56 to 65°C for several hours), making cold preservation of rAAV vaccines less critical.

Viral DNA replication is not required for integration into the host cell genome and therefore does not require a helper virus for this process. The AAV proviral genome is infectious like cloned DNA in plasmids, making the creation of recombinant genomes possible. Moreover, since the signals that control AAV genome replication, encapsidation, and integration are contained in the ITR of the AAV genome, the internal approximately 4.7 kb. genome (encoding replication proteins and structural capsid proteins, rep-cap) can be replaced by foreign DNA, such as a gene expression cassette containing a promoter, DNA of interest and a polyadenylation signal.

AAV vectors may include additional elements that function in cis or trans. In specific embodiments, the AAV vector that includes the vector genome also has one or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' end of the donor sequence; an expression control element that controls transcription (eg, a promoter or enhancer) of the donor sequence, such as a constitutive or regulated control element, or a tissue-specific expression control element; intron sequence, filler polynucleotide sequence; and/or a polyadenine sequence located at the 3' end of the donor sequence. In some embodiments, the AAV is replicated by a helper virus. A number of such AAV helper viruses are known in the art, including adenoviruses, herpeviruses, and poxviruses such as vaccinia virus. Adenoviruses span many different subgroups, although adenovirus type 5, subgroup C is commonly used. Numerous human, non-human mammalian and avian adenoviruses are known and available from repositories such as ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

Typical AAV vectors include capsid protein sequences from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RH10, Rh74, or AAV-2i8, or a variant capsid protein of AAV1, AAV2, AAV3, AAV4 , AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8. Recombinant AAV vectors of the invention also include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV2i8 and variants thereof. Specific capsid protein variants include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV2i8 capsid protein variants, such as a capsid protein sequence with an amino acid substitution, deletion, or insertion/addition . In one embodiment, the AAV vector is AAV9.

In some aspects, the invention relates to AAVs having different tissue-infecting abilities (eg, tissue tropisms). In some embodiments, the AAV variant capsid polypeptides further exhibit increased transduction or tropism in one or more human stem cell types compared to the non-variant parental capsid polypeptides. In some embodiments, human stem cell types include, but are not limited to, embryonic stem cells, adult tissue stem cells (i.e., somatic stem cells), bone marrow stem cells, progenitor cells, induced pluripotent stem cells, and reprogrammed stem cells. In some embodiments, adult stem cells may include organoid stem cells (ie, stem cells derived from any organ or organ system of interest in the body). In some embodiments, the target AAV tissue is the gonad, diaphragm, heart, stomach, liver, spleen, pancreas, muscle, or kidney. In some embodiments, the AAV targets organs of the body, which include, but are not limited to, skin, hair, nails, sensory receptors, sweat gland, sebaceous glands, bone, muscle, brain, spinal cord, nerve, pituitary gland, pineal gland, hypothalamus , thyroid gland,

- 97 046419 parathyroid gland, thymus, adrenal glands, pancreas (islet tissue), heart, blood vessels, lymph nodes, lymphatic vessels, thymus, spleen, tonsils, nose, pharynx, larynx, trachea, bronchi, lungs, mouth, pharynx, esophagus, stomach, small intestine, large intestine, rectum, anal canal, teeth, salivary glands, tongue, liver, gall bladder, pancreas, appendix, kidneys, ureters, bladder, urethra, testicles, vas deferens, urethra, prostate, penis, scrotum, ovaries, uterus, fallopian tubes, vagina, vulva and mammary glands (breasts). Organ systems of the body include, but are not limited to, the integumentary system, skeletal system, muscular system, nervous system, endocrine system, cardiovascular system, lymphatic system, respiratory system, digestive system, urinary system and reproductive system. In some embodiments, transduction and/or tropism of AAV with variant capsid polypeptides is increased by about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, 65, about 70, about 75, about 80, about 85, about 90, about 95, about 99, or about 100% compared to AAV having non-variant capsid polypeptides. In some embodiments, transduction and/or tropism is increased from about 5 to about 80%, from about 10 to about 70%, from about 20 to about 60%, or from about 30 to about 60%.

5.5.1.2. AAV replication, capsid and assembly genes

The single-stranded AAV genome includes three genes: rep (replication), cap (capsid), and aap (assembly). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing.

The rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) that are required for replication and packaging of the viral genome.

Expression of the cap gene results in the formation of viral capsid proteins (VP1; VP2; VP3), which form the outer capsid shell that protects the viral genome and is also actively involved in cell binding and internalization. It is estimated that the viral envelope consists of 60 proteins that form an icosahedral structure. The aap gene encodes assembly-activating protein (AAP) in an alternative reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly and to play a role in the nucleolar localization of VP proteins in some AAV serotypes.

In some embodiments, one or more of the rep, cap, or aap genes are naturally occurring, e.g., the rep, cap, or aap genes include all or a portion of the parvovirus rep, cap, or aap genes. In some embodiments, one or more rep, cap, or aap genes comprise a synthetic sequence. In one embodiment, the rep gene contains a synthetic sequence. In one embodiment, the cap gene contains a synthetic sequence. In one embodiment, the aap gene comprises a synthetic sequence. In one embodiment, the rep and cap genes contain a synthetic sequence. In one embodiment, the rep and aap genes comprise a synthetic sequence. In one embodiment, the cap and aap genes comprise a synthetic sequence. In one embodiment, the rep, cap, and aap genes comprise a synthetic sequence.

In some embodiments, rep is derived from an AAV genome selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and any combination thereof. In a specific embodiment, rep is derived from the AAV1 genome. In a specific embodiment, rep is derived from the AAV2 genome. In a specific embodiment, rep is derived from the AAV3 genome. In a specific embodiment, rep is derived from the AAV4 genome. In a specific embodiment, rep is derived from the AAV5 genome. In a specific embodiment, rep is derived from the AAV6 genome. In a specific embodiment, rep is derived from the AAV7 genome. In a specific embodiment, rep is derived from the AAV8 genome. In a specific embodiment, rep is derived from the AAV9 genome. In a specific embodiment, rep is derived from the AAV10 genome. In a specific embodiment, rep is derived from the AAV11 genome.

In some embodiments, cap is derived from an AAV genome selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and any combination thereof. In a specific embodiment, cap is derived from the AAV1 genome. In a specific embodiment, cap is derived from the AAV2 genome. In a specific embodiment, cap is derived from the AAV3 genome. In a specific embodiment, cap is derived from the AAV4 genome. In a specific embodiment, cap is derived from the AAV5 genome. In a specific embodiment, cap is derived from the AAV6 genome. In a specific embodiment, cap is derived from the AAV7 genome. In a specific embodiment, cap is derived from the AAV8 genome. In a specific embodiment, cap is derived from the AAV9 genome. In a specific embodiment, cap is derived from the AAV10 genome. In a specific embodiment, cap is derived from the AAV11 genome.

In some embodiments, the aap is derived from an AAV genome selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and any combination thereof. IN

- 98 046419 in a specific embodiment, aap is derived from the AAV1 genome. In a specific embodiment, aap is derived from the AAV2 genome. In a specific embodiment, aap is derived from the AAV3 genome. In a specific embodiment, aap is derived from the AAV4 genome. In a specific embodiment, aap is derived from the AAV5 genome. In a specific embodiment, aap is derived from the AAV6 genome. In a specific embodiment, aap is derived from the AAV7 genome. In a specific embodiment, aap is derived from the AAV8 genome. In a specific embodiment, aap is derived from the AAV9 genome. In a specific embodiment, aap is derived from the AAV10 genome. In a specific embodiment, aap is derived from the AAV11 genome.

It should be understood that a particular AAV genome described herein may have genes derived from different AAV genomes (eg, genomes from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11). Thus, AAVs are described herein that contain any possible permutation/combination of rep, cap, or aap.

In some embodiments disclosed herein, the AAV is recombinant AAV (rAAV). In some embodiments, rAAV lacks one or more of the rep gene, the cap gene, and the aap gene. In some embodiments, rAAV lacks the rep gene. In some embodiments, rAAV lacks the cap gene. In some embodiments, rAAV lacks the aap gene. In some embodiments, rAAV lacks the rep gene and lacks the cap gene. In some embodiments, rAAV lacks the rep gene and lacks the aap gene. In some embodiments, rAAV lacks the cap gene and lacks the aap gene. In some embodiments, rAAV lacks the rep gene, the cap gene, and the aap gene.

In some embodiments disclosed herein, the rAAV is modified such that one or more of the rep gene, the cap gene, and the aap gene is mutated such that the expression of one or more AAV genes is altered. In some embodiments, the rep gene is mutated. In some embodiments, the cap gene is mutated. In some embodiments, the aap gene is mutated. In some embodiments, the rep gene and the cap gene are mutated. In some embodiments, the rep gene and the aap gene are mutated. In some embodiments, the cap gene and the aap gene are mutated. In some embodiments, the cap gene, the rep gene, and the aap gene are mutated.

5.5.1.3. Inverted terminal repeats

In some embodiments, the AAV comprises a first ITR, such as a 5' ITR, and a second ITR, such as a 3' ITR. Typically, ITRs are involved in the replication and rescue or excision of parvovirus (e.g., AAV) DNA from prokaryotic plasmids (Samulski et al., 1983, 1987; Senapathy et al., 1984; Gottlieb and Muzyczka, 1988). In addition, ITRs have been reported to be the minimal sequences required for the integration of AAV proviruses and for the packaging of AAV DNA into virions (McLaughlin et al., 1988; Samulski et al., 1989). These elements are necessary for efficient propagation of the parvovirus genome.

In some embodiments, the ITR comprises a naturally occurring ITR, for example, the ITR comprises all or part of a parvovirus ITR. In some embodiments, the ITR comprises a synthetic sequence. In one embodiment, the first ITR or second ITR contains a synthetic sequence. In another embodiment, each of the first ITR and the second ITR contains a synthetic sequence. In some embodiments, the first ITR or the second ITR comprises a naturally occurring sequence. In another embodiment, each of the first ITR and the second ITR contains a naturally occurring sequence. In some embodiments, the ITR includes an ITR from the AAV genome. In some embodiments, the ITR is an AAV genome ITR selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and any combination thereof. In a specific embodiment, the ITR is the ITR of the AAV2 genome. In another embodiment, the ITR is a synthetic genetically engineered sequence containing at its 5' and 3' ends ITRs derived from one or more AAV genomes. In some embodiments, the ITRs are derived from the same genome, such as the genome of the same virus, or from different genomes, such as the genomes of two or more different AAV genomes. In some embodiments, the ITRs are derived from the same AAV genome. In a particular embodiment, the two ITRs present in the nucleic acid molecule of the invention are the same and may, in particular, be the AAV2 ITR. In one particular embodiment, the first ITR and the second ITR are identical.

In some embodiments, the ITRs form hairpin loop structures. In one embodiment, the first ITR forms a hairpin structure. In another embodiment, the second ITR forms a hairpin structure. In yet another embodiment, both the first ITR and the second ITR form hairpin structures. In some embodiments, the ITR in a nucleic acid molecule disclosed herein is a transcriptionally activated ITR. A transcriptionally activated ITR may include all or a portion of a wild-type ITR that has been transcriptionally activated by inclusion of at least one transcriptionally active element. Various types of transcriptionally active elements are suitable in this context. In some countries

- 99 046419 ant implementation of the transcriptionally active element is a constitutive transcriptionally active element. Constitutive transcriptionally active elements provide a constant level of gene transcription and can be used when it is desired that the transgene be expressed on a continuous basis. In other embodiments, the transcriptionally active element is an inducible transcriptionally active element. Inducible transcriptionally active elements typically exhibit low activity in the absence of an inducer (or inducing state) and are activated in the presence of an inducer (or switch to an inducing state). Inducible transcriptionally active elements can be used when expression is required only at certain times or in certain locations, or when titration of the level of expression using an inducing agent is desired. Transcriptionally active elements can also be tissue specific; that is, they are active only in certain tissues or cell types.

Transcriptionally active elements can be incorporated into the ITR in a variety of ways. In some embodiments, the transcriptionally active element is included at the 5' end of any portion of the ITR or at the 3' end of any portion of the ITR. In other embodiments, the transcriptionally active element of the transcriptionally activated ITR is located between two ITR sequences. If a transcriptionally active element includes two or more elements that must be separated, these elements may alternate with parts of the ITR. In some embodiments, the ITR hairpin structure is removed and replaced with inverted repeats of the transcription element. This last arrangement will create a stud that simulates the removed part in the structure. Multiple tandem transcriptionally active elements may also be present in a transcriptionally activated ITR, and they may be contiguous or spaced apart. In addition, protein binding sites (eg, Rep binding sites) can be introduced into transcriptionally active elements of transcriptionally activated ITRs. The transcriptionally active element may include any sequence that allows controlled transcription of DNA by RNA polymerase to produce RNA, and may comprise, for example, a transcriptionally active element as defined below.

Transcriptionally activated ITRs provide both transcriptional activation and ITR function to a nucleic acid molecule in a nucleotide sequence of relatively limited length, which effectively maximizes the length of the transgene that can be transferred to and expressed from the nucleic acid molecule. Incorporation of a transcriptionally active element into an ITR can be accomplished in a variety of ways. Comparison of the ITR sequence and the sequence requirements of the transcriptionally active element can provide insight into the way the element is encoded in the ITR. For example, transcriptional activity can be added to the ITR by making certain changes to the ITR sequence that replicate functional elements of the transcriptionally active element. There are a number of techniques in the art to effectively add, remove and/or change specific nucleotide sequences at specific sites (see, for example, Deng and Nickoloff (1992) Anal. Biochem. 200: 81-88). Another method for creating transcriptionally activated ITRs involves introducing a restriction site at the desired location in the ITR. In addition, several transcriptionally active elements can be included in a transcriptionally activated ITR using methods known in the art.

By way of illustration, transcriptionally activated ITRs can be generated by the inclusion of one or more transcriptionally active elements, such as: TATA box, GC box, CCAAT box, Sp1 site, Inr region, CRE (cAMP regulatory element) site, ATF- site 1/CRE, APBbox, APBa-box, CArG-box, CCAC-box, or any other element involved in transcription, as known in the art.

5.5.1.4. Gene of interest and other sequences

Some aspects of the present invention relate to methods of administering AAV therapy to a subject. In some embodiments, the AAV contains a gene of interest (GOI). In some embodiments, the GOI is a nucleic acid molecule comprising a nucleotide sequence described herein that encodes a miniaturized dystrophin polypeptide described herein.

The expressed GOI can be either a protein-coding DNA segment with any necessary control elements (e.g., promoters, operators) desired by the user, or a non-coding DNA segment whose transcription produces all or part of some RNA-containing molecule, such as a ribozyme or antisense molecule.

In some embodiments, the AAV contains more than one GOI. In AAVs with more than one GOI, some embodiments include elements such as IRES or 2A to co-express them from a single promoter. In some embodiments, AAV contains two genes of interest separated by an IRES element. In some embodiments, AAV contains two genes of interest separated by a 2A element. In some embodiments it is possible to

- 100 046419 The AAV stem contains three genes of interest separated by an IRES element between the genes of interest (eg, GOI-IRES-GOI-IRES-GOI). In some embodiments, the AAV contains three genes of interest separated by 2A elements between the genes of interest. In some embodiments, AAV comprises a regulatory sequence. In some embodiments, AAV comprises non-coding regulatory DNA. In some embodiments, the AAV genome contains regulatory sequences that control the expression of antibody chain genes in the host cell. The term regulatory sequence includes promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Those skilled in the art will appreciate that the design of AAV, including the choice of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, etc. In some embodiments, the AAV genome contains mRNA splice donor/acceptor sites. Some regulatory sequences for expression in mammalian host cells include viral elements that mediate high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (eg, adenovirus core late promoter (AdMLP) and polyoma virus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or β-globin promoter can be used. In addition, regulatory elements consisting of sequences from various sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and human T cell leukemia virus type 1 long terminal repeat (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472). In some embodiments, the regulatory sequence comprises a tissue-specific promoter In some embodiments, a tissue-specific promoter drives expression of a gene of interest in a tissue selected from the group consisting of heart, liver, lung, eye, nervous system, lymphatic system, muscle and stem cells.

5.5.1.5. AAV drugs

In some embodiments, the AAV vector is formulated with a delivery vehicle. In some embodiments, the delivery vehicle includes a lipid nanoparticle. In some embodiments, the delivery vehicle is selected from the group consisting of liposomes, non-lipid polymer molecules, endosomes, and any combination thereof.

5.5.2. Non-AAV vectors

Also provided herein is a vector that contains the polynucleotides described above operably linked to a promoter. A nucleotide sequence is operably linked to an expression control sequence (eg, a promoter) where the expression control sequence controls and regulates transcription and translation of the sequence. The term operably linked, as applied to a nucleotide sequence, includes the presence of an appropriate start signal (eg, ATG) before the nucleotide sequence to be expressed, and maintenance of the correct reading frame to ensure expression of the sequence under the control of the expression control sequence and production of the desired product encoded by the sequence. If the gene that is desired to be inserted into the recombinant nucleic acid molecule does not contain a corresponding start signal, such a start signal may be inserted before the gene. A vector is a replicon, such as a plasmid, phage or cosmid, to which another nucleic acid segment can be attached to cause replication of the attached segment. The promoter may be or is identical to that of bacteria, yeast, insects or mammals. In some embodiments, the vector may be a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage, or eukaryotic viral DNA. Numerous other vector backbones known in the art to be useful for protein expression may be used. Such vectors include, but are not limited to: adenoviral vector, retroviral vector, poxviral vector, baculovirus vector, herpes viral vector, simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), and Moloney murine leukemia virus. In addition, one class of vectors contains DNA elements derived from viruses, such as bovine papillomavirus, polyoma virus, baculovirus, retroviruses, or Semliki Forest virus. Such vectors can be obtained commercially or assembled from sequences described by methods well known in the art.

In some embodiments, a vector described herein is formulated with a delivery vehicle. In some embodiments, the delivery vehicle includes a lipid nanoparticle. In some embodiments, the delivery vehicle is selected from the group consisting of liposomes, non-lipid polymer molecules, endosomes, and any combination thereof.

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5.6. Pharmaceutical compositions

The various polypeptides and polynucleotides disclosed herein (also referred to herein as active compounds) may be included in pharmaceutical compositions suitable for administration. Such compositions typically contain a polypeptide or polynucleotides and a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active compounds is well known in the art. Unless any conventional medium or agent is incompatible with the active compound, it is intended for use in the compositions. Additional active compounds may also be included in the compositions.

In some embodiments, a pharmaceutical composition is disclosed comprising (a) a polypeptide as described herein and (b) a pharmaceutically acceptable excipient.

In some embodiments, a pharmaceutical composition is disclosed, comprising (a) a composition comprising a polypeptide as described herein, and (b) a pharmaceutically acceptable excipient.

In some embodiments, a pharmaceutical composition is disclosed comprising (a) a polynucleotide as described herein and (b) a pharmaceutically acceptable excipient.

In some embodiments, a pharmaceutical composition is disclosed comprising (a) a vector (eg, rAAV) as described herein, and (b) a pharmaceutically acceptable excipient.

In some embodiments, a pharmaceutical composition is disclosed comprising (a) a host cell, as described herein, and (b) a pharmaceutically acceptable excipient.

The pharmaceutical composition herein is formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral, transdermal (topical), and transmucosal, and any combination thereof. Another route of administration involves pulmonary administration. In addition, it may be desirable to administer a therapeutically effective amount of the pharmaceutical composition locally to the area in need of treatment. This can be achieved, for example, by local or regional infusion or perfusion during surgery, topical application, injection, catheter, suppository or implant (for example, implants formed from porous, non-porous or gel-like materials, including membranes such as siallastic membranes or fibers) and the like. In another embodiment, a therapeutically effective amount of the pharmaceutical composition is delivered in vesicles, such as liposomes (see, for example, Langer, Science 249: 1527-33, 1990 and Treat et al., in Liposomes in Therapy of Infectious Disease, and Cancer, Lopez Berestein and Fidler (eds.), Liss, NY, pp. 353-65, 1989).

In yet another embodiment, a therapeutically effective amount of the pharmaceutical composition may be delivered in a controlled release system. In one example, a pump may be used (see, for example, Langer, Science 249: 1527-33, 1990; Sefton, Crit. Rev. Biomed. Eng. 14: 201-40, 1987; Buchwald et al., Surgery 88: 507 -16, 1980; Saudek et al., N Engl J Med 321: 574-79, 1989). In another example, polymeric materials can be used (see, for example, Levy et al., Science 228: 190-92, 1985; During et al., Ann. Neural. 25: 351-56, 1989; Howard et al., J Neurosurg 71: 105-12, 1989). Other controlled release systems such as those discussed by Langer (Science 249: 1527-33, 1990) may also be used.

Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Pharmaceutically acceptable carriers used in parenteral formulations include

- 102 046419 aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, binding or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose injection, and lactated Ringer's injection. Non-aqueous parenteral vehicles include vegetable fatty oils, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobials at bacteriostatic or fungistatic concentrations may be added to parenteral products packaged in multi-dose containers that include phenols or cresols, mercuries, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride . Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). Metal ion binders, or chelating agents, include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible carriers; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid to adjust the pH.

Solutions or suspensions used for parenteral, intradermal or subcutaneous use may include the following components: a sterile diluent such as water for injection, saline, fatty oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity agents such as sodium chloride or dextrose. The pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. The drug for parenteral administration may be contained in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include saline, bacteriostatic water, Cremophor ELS (BASF; Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and liquid to the extent that it can be easily drawn up using a syringe. It must be stable under the conditions of production and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases it will be preferable to include in the composition isotonic agents, for example, sugars, polyhydric alcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including an absorption delaying agent in the composition, for example aluminum monostearate and gelatin.

Sterile injection solutions can be prepared by incorporating the active compound in the required amount into an appropriate diluent with one or a combination of the ingredients listed above, if necessary, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains the basic dispersion medium and other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injection solutions, the preparation methods may be vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredient from its solution, previously sterilized by filtration. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be through mucous membranes or through the skin. For transmucosal or percutaneous administration, the formulation uses penetrants suitable for the permeable barrier. Such penetrants are generally known in the art and include, for example, transmucosal agents, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can

- 103 046419 carried out using nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, ointments, gels or creams as is generally known in the art. The compounds may also be formulated as suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are formulated with carriers that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such compositions will be apparent to those skilled in the art. Materials are also available commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. They can be prepared by methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811. It is particularly preferable to formulate compositions for oral or parenteral administration in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for a subject to be treated, with each dosage unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specification of unit dosage forms herein is dictated by and is directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of creating such a functional compound for the treatment of individuals. The pharmaceutical compositions may be included in a container, package or dispenser along with instructions for use.

5.7. Application and methods.

5.7.1. Methods for producing miniaturized dystrophin

The present invention also discloses methods for producing a miniaturized dystrophin polypeptide, comprising: culturing a host cell as disclosed herein under suitable conditions and isolating the miniaturized dystrophin polypeptide.

As used herein, the term isolated polynucleotide or nucleic acid molecule means a molecule that is separated from other nucleic acid molecules that are present in a natural source (eg, mouse or human) of the nucleic acid molecule. Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant methods, or substantially free of chemical precursors or other chemical compounds when chemically synthesized. For example, the expression essentially free includes preparations of polynucleotide or nucleic acid molecules containing less than about 15, 10, 5, 2, 1, 0.5 or 0.1% (especially less than about 10%) of other material , for example, cellular material, culture medium, other nucleic acid molecules, chemical precursors and/or other chemicals. In a specific embodiment, the nucleic acid molecule(s) encoding a polypeptide described herein is isolated or purified.

Polynucleotides can be prepared and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. Nucleotide sequences encoding the polypeptides described herein, for example, the polypeptides described in table. 3 and 4, and modified versions of these polypeptides can be determined using methods well known in the art, that is, nucleotide codons that are known to encode specific amino acids are assembled so as to generate nucleic acid encoding the polypeptides. Such a polynucleotide encoding a polypeptide can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6), which, in brief, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding a polypeptide, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides using PCR.

Alternatively, a polynucleotide encoding a polypeptide described herein can be obtained from nucleic acid from a suitable source (eg, a hybridoma) using methods well known in the art (eg, PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizing to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the polypeptide of interest. Such PCR amplification methods can be used to produce nucleic acids containing a sequence encoding, for example, IL2, a linker sequence, or IL2Ra. Amplified nucleic acids can be cloned into vectors for expression in cells

- 104 046419 hosts and for further cloning, for example, to obtain polypeptides.

If a clone containing the nucleic acid encoding a particular polypeptide is not available, but the sequence of the polypeptide molecule is known, the nucleic acid encoding the polypeptide can be chemically synthesized or obtained from a suitable source (for example, a cDNA library or a cDNA library derived from a nucleic acid, preferably a polypeptide A+RNA isolated from any tissue or cells expressing proteins of interest, for example, from hybridoma cells selected to express a polypeptide described herein) by PCR amplification using synthetic primers hybridized to the 3' and 5' ends sequence, or by cloning using an oligonucleotide probe specific for a particular gene sequence to identify, for example, a cDNA clone from a cDNA library encoding polypeptides. The amplified nucleic acids obtained by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

DNA encoding the polypeptides disclosed herein can be readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the polypeptides disclosed herein). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors which are then transfected into host cells such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells (e.g. CHO cells from the CHO GS SYSTEM™ (Lonza) ) or myeloma cells that do not otherwise produce immunoglobulin protein, to produce polypeptide synthesis in recombinant host cells.

5.7.1. Therapeutic uses and methods

Miniaturized dystrophin polypeptides, polynucleotides encoding miniaturized dystrophin polypeptides, vectors (eg, rAAV) carrying polynucleotides encoding miniaturized dystrophin polypeptides, and the methods described herein have a variety of in vitro and in vivo applications. Thus, for example, a nucleotide sequence encoding a miniaturized dystrophin polypeptide, e.g., a vector, e.g., an AAV vector, or polypeptides described herein, can be introduced into cells in culture, in vitro or ex vivo, or into human subjects, e.g., in vivo, for the treatment of diseases. Accordingly, disclosed herein are therapeutic methods using any of the miniaturized dystrophin nucleic acid molecules disclosed herein, polypeptides disclosed herein, host cells disclosed herein, vectors disclosed herein, or pharmaceutical compositions, disclosed herein, or any combination thereof.

In some embodiments, disclosed herein is a method for expressing a miniaturized dystrophin polypeptide in a subject in need thereof, comprising administering to the subject a nucleic acid disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition. disclosed in this document. In some embodiments, disclosed herein is a method of treating a subject suffering from a disease or condition, comprising administering to the subject a nucleic acid disclosed herein, a vector disclosed herein, a polypeptide disclosed herein, a host cell disclosed herein , or a pharmaceutical composition disclosed herein. In some embodiments, the disease is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy (XLDC), facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy dystrophy, Emery-Dreyfus muscular dystrophy, distal muscular dystrophy and/or congenital muscular dystrophy. In other embodiments, the disease to be treated is sarcopenia, heart disease, cachexia.

In some embodiments, a nucleic acid molecule disclosed herein, a polypeptide disclosed herein, a vector (e.g., rAAV) disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein, administered intravenously, transdermally, intradermally, subcutaneously, orally or pulmonaryly, or any combination thereof. In some embodiments, a nucleic acid molecule disclosed herein, a polypeptide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein is administered topically, transmucosally. membrane of the epidermis, intranasal, oral, vaginal, rectal, sublingual, local, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal , epidural or intrathoracic route. In some embodiments, the nucleic acid molecule

- 105 046419 acid, vector (eg, rAAV), host cell as described herein, or polypeptide is administered intravenously.

In some embodiments, the method of treatment further includes administering a second agent to the subject.

As used herein, the term subject includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject with cancer. The term non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. In some embodiments, the subject is a human.

In some embodiments, administration of a nucleic acid molecule, vector (e.g., rAAV), polypeptide, host cell, or pharmaceutical composition to a subject results in increased dystrophin protein expression compared to dystrophin protein expression in the subject prior to administration, wherein dystrophin protein expression is increased by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 100% 8 times, at least about 9 times, at least about 10 times, at least about 11 times, at least about 12 times, at least about 13 times, at least about 14 times , at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, according to at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times. In some aspects of the present invention, the method comprises or further comprises administering AAV therapy to a subject. In some embodiments, AAV therapy includes administration of recombinant AAV. Any recombinant AAV known in the art and/or described herein can be used in the methods of the present invention. In some embodiments, AAV therapy comprises administering an AAV selected from the group consisting of AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV and any combination of them. In some embodiments, the AAV therapy includes administration of AAV type 1. In some embodiments, the AAV therapy includes administration of AAV type 2. In some embodiments, the AAV therapy includes administration of AAV type 3. In some embodiments, the AAV therapy includes administration of AAV type 4. In some embodiments, the AAV therapy includes administration of AAV type 3. In some embodiments, the AAV therapy includes administration of AAV type 4. in embodiments, the AAV therapy includes administration of AAV type 5. In some embodiments, the AAV therapy includes administration of AAV type 6. In some embodiments, the AAV therapy includes administration of AAV type 7. In some embodiments, the AAV therapy includes administration of AAV type 8. In some embodiments, the AAV therapy includes administration of AAV type 9. In some embodiments, the AAV therapy includes administration of AAV type 10. In some embodiments, the AAV therapy includes administration of AAV type 11. In some embodiments, the AAV therapy includes administration of AAV type 12. In some embodiments, the AAV therapy includes administration of AAV type 11. In some embodiments, the AAV therapy includes administration of AAV type 12. in embodiments, therapy for AAV includes administration of AAV type 13. In some embodiments, treating the subject with miniaturized dystrophin nucleic acid molecules disclosed herein, polypeptides disclosed herein, host cells disclosed herein, vectors disclosed herein, or the pharmaceutical compositions disclosed herein, or any combination thereof, does not cause significant inflammatory reactions, for example, immune-mediated pneumonitis, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypophysitis, immune-mediated hypothyroidism and hyperthyroidism, or other immune-mediated adverse reactions. In some embodiments, treating a subject with miniaturized dystrophin nucleic acid molecules disclosed herein, polypeptides disclosed herein, host cells disclosed herein, vectors disclosed herein, pharmaceutical compositions disclosed herein, or any their combination does not cause significant cardiac disorders, for example, ventricular arrhythmia; eye diseases, for example, iridocyclitis; infusion-related reactions; increasing amylase levels, increasing lipase levels; nervous system disorders, such as dizziness, peripheral and sensory neuropathy; diseases of the skin and subcutaneous tissue, for example, rash, itching, exfoliative dermatitis, erythema multiforme, vitiligo or psoriasis; respiratory, thoracic and mediastinal diseases, such as cough; fatigue; nausea; decreased appetite; constipation; arthralgia; or diarrhea.

5.8. Sets

The present invention also discloses kits containing one or more nucleic acid molecules, as disclosed herein, one or more vectors (eg, rAAV), such as

- 106 046419 disclosed herein, one or more polypeptides as disclosed herein, or one or more host cells as disclosed herein, or any combination thereof. In some embodiments, the kit also contains instructions for administering any of the above, or a combination thereof, to a subject in need thereof. The terms set and system as used herein are intended to refer to at least one or more nucleic acid molecules as disclosed herein, one or more vectors (e.g., rAAV) as disclosed herein, one or more polypeptides, as disclosed herein, or one or more host cells as disclosed herein, or any combination thereof, which in particular embodiments are in combination with one or more other types of elements or components (e.g., other types of biochemical reagents , containers, packaging such as packaging intended for commercial sale, instructions for use and the like).

In some embodiments, a kit is disclosed comprising (a) one or more miniaturized dystrophin polypeptides as described herein, a composition comprising a miniaturized dystrophin polypeptide as described herein, a nucleic acid encoding a miniaturized dystrophin polypeptide as described herein , vector (eg, rAAV) and/or host cell; and (b) and instructions for administering any of the foregoing to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) a miniaturized dystrophin polypeptide as described herein, and (b) and instructions for administering the miniaturized dystrophin polypeptide to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) a composition comprising a miniaturized dystrophin polypeptide as described herein, and (b) and instructions for administering the composition to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) a nucleic acid encoding a miniaturized dystrophin polypeptide as described herein, and (b) and instructions for administering the nucleic acid to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) a vector as described herein, and (b) and instructions for administering the vector to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) an AAV vector as described herein, and (b) and instructions for administering the vector to a subject in need thereof. In some embodiments, a kit is disclosed comprising (a) a host cell, as described herein, and (b) and instructions for administering the host cell to a subject in need thereof.

In a specific embodiment, provided herein is a pharmaceutical package or kit including one or more containers filled with one or more ingredients of the pharmaceutical compositions described herein, such as one or more miniaturized dystrophin peptides provided herein. In some embodiments, the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein. In some embodiments, the kits may contain a T cell mitogen, such as, for example, phytohemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and an anti-CD28 antibody. Such container(s) may not necessarily be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting the agency's approval of the manufacture, use, or sale for administration to humans. Also provided herein are kits that can be used in the above methods. In one embodiment, the kit includes a miniaturized dystrophin polypeptide described herein, preferably a purified miniaturized dystrophin polypeptide, in one or more containers. In a specific embodiment, the kits described herein contain a substantially isolated miniaturized dystrophin polypeptide as a control. In another specific embodiment, the kits described herein further contain a control protein that does not react with the miniaturized dystrophin polypeptide antigen. In another specific embodiment, the kits described herein contain one or more elements for determining the binding of a miniaturized dystrophin polypeptide to a dystrophin antigen (e.g., the miniaturized dystrophin polypeptide may be conjugated to a detectable substrate, such as a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound or second antibody that recognizes the first antibody may be conjugated to the detectable substrate). In specific embodiments, the kit provided herein may include a recombinantly produced or chemically synthesized miniaturized dystrophin polypeptide. The antigen to the miniaturized dystrophin polypeptide disclosed herein, as provided in the kit, can also be attached to a solid support. In a more specific embodiment, the means detects

- 107 046419 A variation of the kit described above includes a solid support to which a miniaturized dystrophin polypeptide antigen is attached. Such a kit may also include an unbound reporter-labeled anti-human antibody or an anti-mouse/rat antibody. In this embodiment, binding of the miniaturized dystrophin polypeptide to an antigen can be detected by binding of said reporter-labeled antibody.

The practice of the present invention will utilize, unless otherwise indicated, conventional methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the competence of those skilled in the art. Such methods are fully explained in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. US Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., NY); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); Crooks, Antisense drug technology: Principles, strategies and applications, 2 ^nd Ed. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

All references cited above, as well as all references cited herein, and amino acid or nucleotide sequences (eg, GenBank numbers and/or Uniprot numbers) are incorporated herein by reference in their entirety. The following examples are offered as illustrations and not as limitations.

6. Examples.

6.1. New miniaturized dystrophins with reduced immunogenicity and increased stability

Mutations in the dystrophin gene often lead to disruption of the stability of the corresponding dystrophin protein, which in turn leads to proteasomal degradation of the unstable dystrophin protein and dystrophic pathophysiology. Likewise, miniaturization of dystrophin-encoding DNA to accommodate the limited packaging capacity of AAV may impair the stability of the corresponding miniaturized dystrophin protein.

The stability of various miniaturized dystrophin proteins was examined by comparing dystrophin protein:mRNA ratios in cells transfected with the corresponding dystrophin expression vectors. Male cardiac myocytes (iCM) were generated that were derived from human isogenic induced-pluripotent stem cells (iPSCs) that carry a premature stop codon E2035X in the dystrophin gene, which prevents the expression of endogenous dystrophin. These cells were transfected with various cassettes expressing miniaturized dystrophin proteins, and dystrophin protein:mRNA ratios were examined after the transfected cells were cultured in vitro for 24 days. Protein levels were examined using Meso Scale Discovery (MSD) ELISA assay and mRNA levels were examined using qrtPCR.

The miniaturized dystrophins tested and the test results are presented in FIG. 2 and 3, respectively. Data showed that miniaturized dystrophin peptides BXA027741 (SEQ ID NO: 129) and BXA-027743 (SEQ ID NO: 132) provided the best protein stability.

Subsequently, the immunogenicity of new compounds generated within these two most stable designs was tested using an in silico immunogenicity prediction tool. The new junction regions of the BXA-027743 construct (M/linker R16 (J1 junction), R17/H3 and H3/R23 (J7 junction)) (see FIG. 4 and not shown) were found to have minimal immunogenic risk, based on the above in silico approach. The new connecting regions in the BXA-027741 construct (R1/R3 (J10 connecting region), H2/linker R16 (J11 connecting region) and R17/R24 (J9 connecting region)) (see Fig. 5) were analyzed in a similar way. The J9 junction was found to have minimal immunogenic risk, while the J10 and J11 junctions could be improved.

The immunogenic potential of the above junction regions of the BXA-027741 construct and variant junction regions therein was tested using an in vitro T cell proliferation assay as described below. Briefly, samples of peripheral blood mononuclear cells (PBMC) were isolated from healthy human volunteers by Ficoll gradient centrifugation (GE Healthcare) and characterized for their expression.

- 108 046419 human lymphocyte antigen (HLA) class I and II using a combination of polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes (Prolmmune, Sarasota, FL). A panel of PBMC samples from 40 donors having an HLA expression profile closely matching those of the global population was used for further analysis. PBMC samples were labeled with CFSE (Invitrogen, Carlsbad, CA) to monitor proliferation and plated in six replicates of 200,000 cells per well in 96-well plates in RPMI (Lonza, Basel, Switzerland) containing 10% human AB (Bioreclamation, Westbury, NY. ), nonessential amino acids, and penicillin-streptomycin (both Gibco/Fisher Scientific).

BXA-027741 junction peptides, variants, and control peptides were cultured with a panel of 40 PBMC samples at a concentration of 1 μM for 7 days, after which the medium was washed off and cells were labeled with anti-CD4 and anti-CD8 APC monoclonal antibody (BD Biosciences, Franklin Lake , NJ). After removing unbound antibodies by washing, cells were fixed with 3.7% formaldehyde (Sigma, St. Louis, MO) in PBS and analyzed by flow cytometry to determine the percentage of proliferating CD4+ cells or CD8+ cells.

Percentage of samples (among 40 donor samples) that showed a positive response after seven days of culture with various BXA-027741 junction peptides and their variants, defined as a significant increase in the number of proliferating CD4+ or CD8+ T cells compared to PBMC incubated in medium without peptides with connecting regions or control peptides is shown in FIG. 6A (CD4+) and FIG. 6B (CD8+). The following peptides were used as control: (1) Avastin scaffold peptide; (2) VL6-VL CDR3 peptide; and (3) peptide PADRE-61309.

J10 junction version 3 (J10v3) was found to be superior to J10 junction and other versions tested, and that J11 junction version 12 (J11v12) was found to be generally superior to J11 junction in terms of immunogenic risk.

The C-terminal domain of miniaturized dystrophin BXA-027741 was then removed to generate miniaturized dystrophin BXA-196473 (SEQ ID NO: 119) (Fig. 7) to better accommodate the limited packaging capacity of AAV. J10v3 was then used to replace J10 and J11v12 was used to replace J11 in miniaturized dystrophin BXA-196473, resulting in miniaturized dystrophin BXA-196477 (SEQ ID NO: 118) (FIGS. 8A and 8B). To create J10v3 and J11v12, RV amino acids were inserted between amino acids 446 and 447, and SEAQ amino acids were inserted between amino acids 606 and 607, respectively, in BXA-196473 (SEQ ID NO: 1 19) (Fig. 8A and 8B).

BHA-196473 (SEQ ID NO: 119) was further modified with other versions of the connecting sections tested as follows:

BHA-196474 (SEQ ID NO: 120): Joint 9 Version 2 (J9v2) and Joint 11 Version 12 (J11v12).

BHA-196475 (SEQ ID NO: 121): Joint 9 Version 5 (J9v5) and Joint 11 Version 12 (J11v12).

BHA-196476 (SEQ ID NO: 122): Joint 9 Version 6 (J9v6) and Joint 11 Version 12 (J11v12).

BHA-196478 (SEQ ID NO: 124): Joint 10 Version 6 (J10v6) and Joint 11 Rev 12 (J11v12).

BHA-196479 (SEQ ID NO: 125): Joint Section 11, Version 12 (J11v12).

J9v2 is a LER to KNI amino acid change between the R17 domain and the R24 domain, that is, amino acids 843 to 845 in BXA-196473.

J9v5 is a K insertion between the R17 domain and the R24 domain, that is, between amino acids 842 and 843 in BXA-196473.

J9v6 is a KNI insertion between the R17 domain and the R24 domain, that is, between amino acids 842 and 843 in BXA-196473.

J10v3 is an RV insertion between the R1 and R3 domains, that is, between amino acids 446 and 447 in BXA-196473.

J10v6 is an RVLLQDI insertion between the R1 and R3 domains, that is, between amino acids 446 and 447 in BXA-196473.

J11v12 is a SEAQ insertion between the H2 domain and the linker upstream of the R16 domain, that is, between amino acids 606 and 607 in BXA-196473.

The resulting miniaturized dystrophins were then tested for stability as described previously (Fig. 9). The results showed that the miniaturized dystrophin construct BXA-196477 (which contained J10V3 and J11V12) not only had the lowest immunogenic risk, but was also the most stable. In addition, the BXA-212371 miniaturized dystrophin polypeptide construct was modified by removing the last three C-terminal amino acids and inserting a KNDL variant junction amino acid sequence (J2V10; between spectrin repeat domain 17 (R17) and hinge 3 (H3) domain) after the amino acid position 682 in BXA-212371 (resulting in miniaturized dystrophin BXA-213788, SEQ ID NO: 152),

- 109 046419 which has been found to have a lower immunogenic risk.

6.2. Physiology in vitro

DMD iCMs have lower amplitude of #1+ channels. prolonged cFPD (Q-T interval) and greater heart rate variability when compared with isogenic wild-type iCM when using multielectrode arrays. DMD iCMs also have higher Ca2+ transduction and lower impedance than wild-type iCMs, with each method confirming heart rate variability in unstimulated cells.

We investigated whether expression of miniaturized dystrophin BXA-196477 in DMD iCMs harboring the E2035X mutation could mitigate the DMD phenotype and improve the physiological characteristics of the cells. Multielectrode recording, contraction impedance analysis, and Ca2+ transients are used to measure the effect of miniaturized dystrophin expression. We also removed the C-terminal domain of BXA-027743 to generate BXA-212371 to better fit the limited packaging capacity of AAV. The functional ability of BXA-212371 to attenuate the DMD phenotype and improve the physiological characteristics of the same cells is examined using the same analytical procedures as proposed for BXA196477.

The hiPSC-CMs used in this study were iCell DMDs (E2035X) purchased from Cellular Dynamics International, Madison, Wisconsin. Human ventricular fibroblasts were purchased from Lonza, Walkersville, Maryland. iCell hiPSC-CMs have been reported to have electrophysiological properties similar to primary adult cardiomyocytes and respond similarly to a range of cardiac ion channel inhibitors as well as adrenergic and muscarinic receptor agonists and antagonists. The inventors' own work has shown that co-culturing hiPSC-CM with fibroblasts provides a more consistent preparation for electrophysiological studies using multielectrode arrays (MEA). hiPSC-CMs were cultured with 7% CO ₂ in 6-well culture plates treated with 0.1% gelatin for 7 days, then trypsinized and diluted with adult cardiac fibroblasts at a ratio of approximately 5:1. Suspensions of hiPSC-CMs and fibroblasts were then cocultured on laminin-coated 9-well multielectrode array (MEA) plates (256-9wellMEA300/30iR-ITO-mq; Multichannel Systems). hiPSC CMs were infected with AAV9-mDys expression constructs (expressing miniaturized dystrophins BXA-196477 and BXA-213788, Figure 15D) at an MOI of 1 x 10 ⁶ for 48 h, and hiPSC-CMs were then assessed for effects on extracellular field potentials ( FP)) 7 days and 9 days after infection. Both BXA-196477 and BXA-213788 significantly improved conduction velocity by ~80% compared to DMD SM (two-way ANOVA ***P < 0.001 followed by Tukey's test n = 6) (Figure 15E). Dystrophin ELISA confirmed dystrophin expression in the cells (Figure 15F). MEA technology allows spatiotemporal analysis of excitable cells or tissues using an array of extracellular electrodes embedded in a substrate on which cells can be cultured or tissues placed. Extracellular field potentials (FPs) are recorded by each electrode and correspond to cellular action potentials. Assessing FP morphology, duration, and conduction velocity provides a picture of ion channel activity following treatment, as well as the effects on repolarization and conductance. After 7 days of culturing hiPSC-CMs on MEA plates, cells formed a spontaneously contracting monolayer over recording electrodes inserted into each well. Spontaneous FPs were recorded from 28 electrodes per well (diameter 30 μm, center-to-center distance 300 μm) at a sampling rate of 10 kHz using USB-MEA256-System software and MS Rack for data acquisition (multi-channel systems). After a 20-minute equilibration period in humidified medium at 37°C with a constant supply of 5% CO2 and 95% _O2, each well was transfected with AAV9-mDys expression constructs in 300 μl of maintenance medium.

The effects of miniaturized dystrophin expression on FP interpulse intervals (IPI) were monitored and recorded. Data were analyzed using custom software written in MatLab (Mathworks). Beat rate (bpm), serving as a surrogate for heart rate, was calculated using BR = 60,000/IPI, where IPI is the averaged IPI (ms) of the 100-second steady-state recording in each condition. The electrophysiological parameters measured are field potential duration, repolarization surrogate, and field potential conduction velocity. The field potential duration was adjusted to account for beat rate changes (FPD). All treatments were performed in at least 3 replicates.

Conduction velocity was quantified by measuring the activation time of the field potential for each electrode inserted into the MEA well during a synchronized single propagated beat. Digitized field potential recordings from each electrode were smoothed by least squares using a 21-point smoothing polynomial ( Savitsky & Golay, 1964 ), with a time window of 2.1 ms. The activation time is the peak value of the negative derivative of each field potential waveform. Time between the time of the earliest asset

- 110 046419 tion and the time of the latest activation represents the conduction time for propagation of the field potential through the monolayer of cardiomyocytes, and the distance between the two corresponding electrodes represents the conduction distance. The conduction time of the potential divided by the conduction distance for each potential propagation event represents the conduction velocity for each contraction of monolayer cardiomyocytes in the MEA well (Fig. 15).

The data show that both miniaturized dystrophin designs significantly improved conduction velocity (Figure 15).

6.3. In vivo restoration of skeletal muscle physiology

Dystrophin-deficient skeletal muscles produce less specific force (force per unit area) and are very susceptible to contraction-induced damage. Restoring dystrophin expression may alleviate these disorders. Dystrophic mdx mice at 2 weeks of age are systemically treated with retroorbital injection of either 2e14 vg/kg AAV9-C5-12-BXA-196477 or AAV9-C5-12-BXA-212371. The physiology of the limb muscles is examined at 8 weeks of age. Briefly, the mouse's knee is clamped, the foot is placed in the stirrup, and the stirrup is moved while the muscles are maximally contracted using a needle electrode. This test measures the peak twitch and the magnitude of the muscle's tetanic force, as well as the damage caused by the contraction.

6.4. In vivo prevention of dystrophic pathology

Skeletal muscle in the mdx mouse model of DMD typically undergoes necrosis and regeneration beginning at approximately 3–4 weeks of age. Regenerated muscles tend to be more variable in size and contain nuclei that are centrally located in frozen cross-sections. Additionally, fibrosis becomes more prevalent in regenerated muscles. Myofiber size, proportion of centrally located nuclei, and fibrosis were measured in untreated mdx muscle and mdx muscle treated with either AAV9-C5-12-BXA-196477 or AAV9-C5-12-BXA-212371. The proportion of muscle fibers expressing miniaturized dystrophins is also quantified. The diaphragm, gastrocnemius, tibialis anterior and cardiac muscles are examined using the HALO and Columbus computer imaging programs.

6.5. In vivo study

Research plan.

2-week-old mdx ^4cv mice were treated with 2e14vg/kg AAV9-BXA-196477 or AAV9-BXA213788. Two weeks after administration, n=3 mice were sacrificed to study the target effects of miniaturized dystrophin (biodistribution and expression levels). At 2 months of age, the right tibialis anterior muscle was examined for strength and resistance to contraction-induced injury (n=10), similar to that previously described (Khairallah et. al., 2012). Mice were sacrificed at 3 months of age and examined for dystrophin targeting and prevention (FIG. 16).

Target impact.

Immunofluorescence. Muscles were frozen in OCT in 2-methylbutane in liquid _N2 . 10 μm frozen sections were immunostained with antibodies to dystrophin (Mandys106; DSHB; 1:200) and Alexa-647-conjugated wheat germ agglutinin. Alexa-488 IgG2a antibody was used to label the primary anti-dystrophin antibody. Sections were acquired using a Phoenix Opera High Content confocal screening microscope (Perkin-Elmer) and quantified using Columbus software. Briefly, Alexa-647 images were inverted and M-mode was used to search for cells and calculate morphology in a selected population of cells in <2000 μm ² cross sections. Next, the intensity of Alexa-488 was calculated and a population with an intensity greater than that of control mdx ^4cv muscles was selected. More than 90% of the muscles from 4-week and 12-week mice were positive for miniaturized dystrophin (Figure 17). mRNA - To isolate total RNA, tissue was homogenized using a Qiagen Tissuelyzer and RNA was isolated using the Qiagen RNeasy 96 Universal tissue kit (Qiagen catalog number 74881). Tissue (~15) was placed into microcollection tubes from the RNeasy kit containing 750 μl of Qiazol reagent and one 5 mm steel bead, homogenized using a Tissuelyzer at 30 Hz for 2 min and repeated until homogenization was complete, followed by centrifugation at 6000xg for 1 min at 4°C. 150 ml of chloroform was added to each tube and the samples were shaken vigorously for 15 s. After a 3-min incubation at room temperature, samples were centrifuged at 6000xg for 15 min at 4°C. The aqueous phase was withdrawn (~360 μl) and transferred to a new tube containing 1 volume of 70% RNase-free EtOH. All samples were transferred to an RNeasy 96 96-well plate, the plates were sealed with AirPore tape and centrifuged at 5600xg for 4 min at room temperature. 400 μl of buffer RW1 was added to each well, the plate was resealed and centrifuged for 4 min at 5600xg. During centrifugation, a DNase stock solution was prepared by adding 550 μL of RNase-free water per DNase vial. 670 μl of DNase I stock solution was diluted with 7.3 ml of RDD buffer, mixed and stored at 4°C. When centrifugation was completed, the flow was discarded and 80 μl of DNase I mixture was added directly.

- 111 046419 in the center of each well, and the plate was incubated at room temperature for 15 minutes. After incubation, 400 μl of RW1 was added to each well and the plate was sealed and centrifuged for 4 min at 5600xg. The flow was discarded and 800 μl of RPE buffer was added to each well. The plate was sealed again and centrifuged for 4 min at 5600xg. This process was repeated and the plate was centrifuged for 10 min at 5600xg. Each sample was then eluted into an unused tube by adding 60 μl of RNase-free water to the center of each well and centrifuging for 4 min at 5600xg. To improve recovery, 60 μl was reapplied to the plate and centrifuged for an additional 4 min at 5600xg. RNA yield was quantified using a nanodrop 8000 instrument. RT-PCR - For cDNA synthesis and subsequent PCR, 1 μg of RNA was added to 1 well of a 96-well plate in 10 μl of H2O (Plate-Axygen, PCR-96-CS). 10 μl of master mix (High Capacity cDNA Reverse Transcription kit, Applied Biosystem) was added to each well and the plate was centrifuged at 1000 rpm. cDNA synthesis was carried out at 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, followed by holding at 4°C. For qPCR, each sample was analyzed in duplicate with primer/probe sets (forward primer 5'TGGAAGATTGCTACGAGCGC-3'; reverse primer 5'-CAGGTCGCTGAACAGGTTCT-3'; probe^AMGCAAGTTCGGCAAGCAGCACA-MGBNFG) in 384-well transparent reaction plates (applied biosystem; catalog number 4483285). To each reaction mixture, 2 µl of DNA and 8 µl of master mix (5 µl of rapid improved master mix, 0.5 µl of 20X FAM probe/primer mix and 2.5 µl of water) were added and the plates were centrifuged for 1 min at 1000 rpm Samples were incubated at 95°C for 20 s followed by 40 cycles of 95°C for 1 s and 60°C for 20 s using the ViiA7 system and Quant Studio real-time PCR software for data analysis (Applied Biosystem ).

Vector genome/genomic DNA isolation and qPCR - For genomic DNA isolation, tissue was homogenized using a Qiagen Tissuelyzer and genomic DNA was isolated using the Qiagen DNeasy 96 Blood and Tissue Kit (Qiagen catalog number 69581). Tissue (~10 mg) was placed in a 96-well plate (costar assay block 1 ml, catalog number 3958) containing 200 µl ATL buffer with proteinase K and one 5 mm steel bead, homogenized using a Tissuelyzer 30 Hz, 2 min, repeat until completion of homogenization. Genomic DNA extraction was carried out in accordance with the manufacturer's instructions. For qPCR, each sample was analyzed in duplicate with sets of primers/probes (forward primer 5'-TGGAAGATTGCTACGAGCGC-3'; reverse primer 5'-CAGGTCGCTGAACAGGTTCT-3'; probe-6FAM-GCAAGTTCGGCAAGCAGCACA-MGBNFG) in 384-well transparent reaction plates (applied biosystem ; catalog number 4483285). To each reaction mixture, 2 μl of genomic DNA and 8 μl of master mix (5 μl of rapid improved master mix, 0.5 μl of 20X FAM probe/primer mix and 2.5 μl of water) were added and the plates were centrifuged for 1 min at 1000 rpm Samples were incubated at 95°C for 20 s followed by 40 cycles of 95°C for 1 s and 60°C for 20 s using the ViiA7 system and Quant Studio real-time PCR software for data analysis (Applied Biosystem ).

MSD-ELISA - Protein expression was determined using an ELISA assay. 384-well multi-assay plates (Meso Scale Discovery, catalog number L21XB-4) were precoated with Manex 1011b monoclonal antibody at 2 μg/mL in bicarbonate buffer overnight. The plates were then blocked with blocking buffer (5% BSA in PBS) for 4 hours with shaking at room temperature. Tissue (~20 mg) was homogenized in ripa buffer at a concentration of 1 mg tissue/10 μl lysis buffer (Sigma, catalog number R0278) with protease inhibitor cocktail tablet (Roche, catalog number 04 693 159001) using a Qiagen Tissuelyzer at 30 Hz for 5 min and repeat this step until homogenization is complete. Tissue lysate in ripa was diluted 1 to 3 in binding buffer (1% BSA, 0.05% Tween 20, 20 mM Tris pH 7.5 in PBS). Tissue lysates and sulfo-conjugated Mandys 106 (0.2 μg/ml) were added to precoated 384-well plates and incubated at 40°C with shaking overnight. The plates were washed with PBS with 0.05% Tween 20 and 40 μl of MSD Reading Buffer T with surfactant (cat. no. R92TC-1) was added. The plates were then read on an MSD Sector 6000 machine.

The data showed that enough virus was administered to achieve expression (mRNA and protein) in >90% of striated muscle cells at 4 weeks of age (Fig. 18). Creatine kinase. - Creatine kinase (CK) levels as an indicator of muscle damage were measured in serum using commercially available kits. SC was measured at 4 weeks of age (2 weeks after delivery). In addition, SC was measured at 3 months of age, i.e., 1 month after the contraction injury protocol (see below). ****P<0.001 one-way ANOVA followed by Tukey's test, n=10 versus mdx ^4cv . The data showed that in mice treated with BXA-196477 or BXA-213788, creatine kinase levels and therefore muscle damage were significantly reduced (Fig. 19). Functional studies.

The contractile properties of the tibialis anterior (TA) muscle were tested using an in vivo setup (foot plate) according to the manufacturer's instructions.

- 112 -

Claims (4)

diator (Aurora Scientific). Briefly, the peak of tetanic contraction was reached in the force curve at 150 Hz (force measured in torque in newton meters). Peak tetanic contraction was similar in wild-type mice, mdx ^4cv mice, and mdx ^4cv mice treated with BXA-196477 or BXA213788. However, TA muscle mass is greater in mdx ^4cv mice (****P < 0.001 one-way ANOVA followed by Tukey's test, n = 10), such that the normalization of peak tetanic force to TA mass decreased in mdx ^4cv mice and remained at the level of wild-type mice type in treated mice (***P<0.001 vs. mdx ^4cv ; one-way ANOVA followed by Tukey's test, n=10) (Fig. 20). Tibialis anterior (TA) muscle damage was measured using an in vivo instrument (stand) according to the manufacturer's instructions (Aurora Scientific). During peak tetanic contraction at 150 Hz (maximum isometric torque), the stand was rotated from 90 to 135° to exert muscle tension. This contraction was repeated every minute for 20 contractions as previously described (Khairallah et. al., 2012). Maximum isometric torque immediately before muscle contraction decreased significantly with each contraction in mdx ^4cv mice (P<0.0001 vs. wild-type; two-way ANOVA; n=9-10). In contrast, both BXA-196477 and BXA-213788 prevented contraction-induced injury similar to wild-type levels (****P<0.0001 BXA-213788 and **P<0.01 BXA-196477 versus mdx ^4cv followed by Tukey's test n=9-10). Data show that both miniaturized dystrophin constructs protect TA muscles from contraction-induced damage (Figure 21). In vitro and in vivo expression of the miniaturized dystrophin constructs described in the examples herein was under the control of the C5-12 promoter (see US 2004/0175727), from which the first seven and last thirteen nucleotides were deleted. The AAV used was AAV9, where the ITRs were from AAV2. 6.6. Additional immunogenicity studies Immunogenicity of new junction regions (see Table 12: SEQ ID NO: 156 to 166; SEQ ID NO: 155 was the original sequence) created between the hinge 2 (H2) domain and R16 spectrin repeat 16 (R16) in the BXA- polypeptide 027741 miniaturized dystrophin was tested using an in silico immunogenicity prediction tool. The novel junction region of SEQ ID NO: 157 (see FIG. 22) was determined to have minimal immunogenic risk based on the above in silico approach. The junction sequences tested are numbered as follows: SEQ ID NO: 155 (1), SEQ ID NO: 156 (2), SEQ ID NO: 157 (3), SEQ ID NO: 158 (4), SEQ ID NO: 159 ( 5), SEQ ID NO: 160 (6), SEQ ID NO: 161 (7), SEQ ID NO: 162 (8), SEQ ID NO: 163 (9), SEQ ID NO: 164 (10), SEQ ID NO: 165 (11), SEQ ID NO: 166 (12) (See FIG. 22B). The immunogenic potential of the above junctions of construct BXA027741 and variant junctions therein were tested using an in vitro T cell proliferation assay as described above. The data show that the junction region J11v3 (SEQ ID NO: 157, No. 3 in Fig. 22B) caused significantly less proliferation of CD8+ (Fig. 23A) and CD4+ T cells (Fig. 23B) compared to other compounds tested and is therefore the best (Fig. 23). The miniaturized dystrophin polypeptide BXA-196477 was modified to carry the J11v3 junction, the last three amino acids at the C-terminus were removed and the 3'UTR was truncated, resulting in the miniaturized dystrophin polypeptide BXA-213780 (SEQ ID NO: 153) and the nucleotide sequence SEQ ID NO: 154. CLAIM 1. A nucleic acid molecule containing a nucleotide sequence that encodes a miniaturized dystrophin polypeptide containing, from N-terminus to C-terminus, hinge 1 (H1) domain, spectrin repeat domain 1 (R1), spectrin repeat domain 3 (R3), hinge 2 (H2) domain, spectrin repeat 16 (R16) domain, spectrin repeat 17 (R17) domain, spectrin repeat 24 (R24) domain, and dystrophin hinge 4 (H4) domain, wherein the miniaturized dystrophin polypeptide does not contain dystrophin spectrin repeat 2, wherein the R1 domain and the R3 domain are fused through ARG-VAL (RV) amino acids, and where the H2 domain and the R16 domain are fused through the amino acid sequence set forth in SEQ ID NO: 74-75, and where (i) the H1 domain and the R1 domain are directly fused , (ii) the R3 domain and the H2 domain are directly fused, (iii) the R16 and R17 domains are directly fused, (iv) the R17 and R24 domains are directly fused, or (v) the R24 and H4 domains are directly fused. 2. The nucleic acid molecule according to claim 1, wherein the miniaturized dystrophin polypeptide further comprises an ABD1 domain and/or a CR domain. 3. The nucleic acid molecule according to claim 1 or 2, wherein the miniaturized dystrophin polypeptide consists of, from the N-terminus to the C-terminus, an ABD1 domain, an H1 domain, an R1 domain, RV amino acids, an R3 domain, an H2 domain, the amino acid sequence given in SEQ ID NO: 74-75, R16 domain, R17 domain, R24 domain, H4 domain and CR domain of dystrophin. 4. Nucleic acid molecule according to any one of claims 1-3, where the polypeptide is miniaturized - 113 -