ADENO-ASSOCIATED VIRUS VECTOR DELIVERY OF MICRODYSTROPHIN TO TREAT MUSCULAR DYSTROPHY

RELATED APPLICATIONS

This application is related to Indian Provisional Application 202021033689 filed on 6 ^th Aug, 2020 and is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to gene therapy vectors which are useful in the treatment of dystrophic diseases, especially Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

BACKGROUND OF THE INVENTION

Muscular dystrophy is a collection of heterogeneous genetic disorders characterized by weak skeletal muscles and their progressive degeneration. Duchenne Muscular Dystrophy (DMD) is one of the most common forms of muscular dystrophy, affecting 1 in every 3500-5000 live male births arising due to mutations in the DMD gene which encodes for Dystrophin protein.

A milder form of the disease called Becker muscular dystrophy (BMD) is distinguished from DMD by delayed onset, later dependence on wheelchair support, and longer life span. BMD is caused by mutations maintaining the reading frame and the most critical parts of the gene, leading to a truncated but still functional dystrophin protein (Muntoni F et al., Lancet Neurol, 2003. 2(12): 731-40). Currently available therapies for DMD only aim to delay the disease progression. Most of the patients are treated with corticosteroids like prednisone to reduce the inflammation, extending the duration of gait and delaying the appearance of scoliosis when the patients become wheelchair bound. Additionally, some other novel therapeutic approaches aiming to restore dystrophin expression like translational read- through therapy (gentamicin and ataluren), RNA-splicing based exon skipping therapy using anti-sense oligonucleotides and cell therapy are being explored or developed. However, these suffer from drawbacks like non-significant effect to improve mobility or quality of life, targeting of certain group of patient population due to specificity and not significant increase in dystrophin expression though posing higher risk. These limitations have prompted the development of gene-augmentation and/or gene replacement therapies for DMD. In order to prevent muscle degeneration, around 30% of normal levels of dystrophin protein is likely to be required and therefore, different gene transfer approaches for DMD aim to compensate for dystrophin loss-of-function and offer the potential to treat all patients using a single medication.

Nevertheless, enormous gene size and abundance of various muscle tissues throughout body makes it herculean task as the former creates a stumbling block for viral vector packaging and the latter beseeches for whole-body homogenous gene-expression.

To tackle these hitches, researchers have constructed extremely truncated microdystrophin genes (MD1, A3990 and mDys5R) as well as explored various Adeno- associated virus (AAV) vectors for body-wide systemic gene transfer.

The current invention relates to development of an AAV based viral vector system containing an expression cassette of micro-dystrophin (abbreviated as p-dys like MD1 and A3990) that is packaged in the optimized viral capsids for muscle tissue tropism (i.e. different serotypes of AAV; AAV9 and AAVpol). Such vector can be used for the treatment of DMD and Becker’s Muscular Dystrophy (BMD).

OBJECTS OF THE INVENTION

The principal object of the present invention is to provide recombinant AAV vector comprising polynucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.

Another object of the present invention is to provide recombinant AAV vector comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein under regulation of muscle-specific control element.

Another object of the present invention relates to viral genome of recombinant AAV particle which comprises: i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5- 12-hCK promoter which drives transcription of micro-dystrophin (MD1 and A3990); and iv) SV 40 Poly A transcription terminator.

In one object functional micro-dystrophin is MD1. In another object functional microdystrophin is A3990.

Another object of the present invention relates to viral genome of recombinant AAV particle comprising nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding: i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5- 12-hCK promoter which drives transcription of micro-dystrophin (MD1 and A3990); and iv) SV 40 Poly A transcription terminator.

Another object of the present invention is to provide pharmaceutical formulation comprising:

(a) AAV vector genome comprising nucleotide sequence encoding microdystrophin gene (MD1 and A3990); and

(b) A pharmaceutically acceptable carrier, diluents, excipients, or buffer.

Another object of the present invention aims at alleviating or curing devastating Duchenne muscular dystrophy (DMD) as well as Becker muscular dystrophy (BMD) by expressing a shorter but functional dystrophin polypeptide called microdystrophin gene (MD1 and A3990).

Another object of the present invention is to provide a method of treatment for muscular dystrophy or dystrophinopathy comprises administering a therapeutically effective amount of the recombinant AAV vector containing nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.

SUMMARY OF THE INVENTION

The principal aspect of the present invention is to provide recombinant AAV vector comprising polynucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.

Another aspect of the present invention is to provide recombinant AAV vector comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein under regulation of muscle-specific control element. Another aspect of the present invention relates to viral genome of recombinant AAV particle which comprises; i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990) gene; iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5- 12-hCK promoter which drives transcription of micro-dystrophin (MD1 and A3990); and iv) SV 40 Poly A transcription terminator.

In one aspect functional micro-dystrophin is MD1. In another aspect functional microdystrophin is A3990.

Another aspect of the present invention relates to viral genome of recombinant AAV particle comprising nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding: i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5- 12-hCK promoter which drives transcription of micro-dystrophin (MD1 and A3990); and iv) SV 40 Poly A transcription terminator. Another aspect of the present invention is to provide pharmaceutical formulation comprising:

(a) AAV vector genome comprising nucleotide sequence encoding microdystrophin gene (MD1 and A3990); and

(b) A pharmaceutically acceptable carrier, diluents, excipients, or buffer.

Another aspect of the present invention aims at alleviating or curing devastating Duchenne muscular dystrophy (DMD) as well as Becker muscular dystrophy (BMD) by expressing a shorter but functional dystrophin polypeptide called micro dystrophin (MD1 and A3990).

Another aspect of the present invention is to provide a method of treatment for muscular dystrophy or dystrophinopathy comprises administering a therapeutically effective amount of the recombinant AAV vector containing nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.

BRIEF DESCRIPTION OF DRAWINGS

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein: Figure 1: Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-MD1 with its transgene and other regulatory elements.

Figure 2: Schematic representation of (A) vector map & (B) cassette of plntas- hMHCK7-MDl with its transgene and other regulatory elements.

Figure 3: Schematic representation of (A) vector map & (B) cassette of plntas-Sk- CRM4-mDes-MDl with its transgene and other regulatory elements.

Figure 4: Schematic representation of (A) vector map & (B) cassette of plntas-Sk- CRM4-hDes-MDl with its transgene and other regulatory elements.

Figure 5: Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-hDes-MDl with its transgene and other regulatory elements.

Figure 6: Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-hCK-MDl with its transgene and other regulatory elements.

Figure 7: Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-A3990 with its transgene and other regulatory elements.

Figure 8: Schematic representation of (A) vector map & (B) cassette of plntas- hMHCK7-A3990 with its transgene and other regulatory elements.

Figure 9: Schematic representation of (A) vector map & (B) cassette of plntas-Sk- CRM4-mDes-A3990 with its transgene and other regulatory elements.

Figure 10: Schematic representation of (A) vector map & (B) cassette of plntas-Sk- CRM4-hDes-A3990 with its transgene and other regulatory elements.

Figure 11: Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-hDes-A3990 with its transgene and other regulatory elements.

Figure 12: Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-hCK-A3990 with its transgene and other regulatory elements.

Figure 13: Schematic representation of (A) vector map & (B) cassette of plntas-ACG- R2C9wt.

Figure 14: Schematic representation of (A) vector map & (B) cassette of plntas-ACG- R2Cpolwt. Figure 15: Determination of Promoter Activity: Comparison of a Specific Promoter Activity amongst Different Cell Lines, viz. C2C12, RCDMD, HEK293FT and Huh- 7. Black dotted line represents 100% Flue activity cutoff derived under the regulation of CMV promoter.

Figure 16: Determination of Promoter Activity: Comparison of Activity of All Promoters within a Specific Cell Line to Understand the Cell Line Specific Activity. Black dotted line represents 100% Flue activity cutoff derived under the regulation of CMV promoter.

DETAILED DESCRIPTION OF THE INVENTION

The rAAV vectors are designed to express microdystrophin (MD1 and A3990) protein in mammalian, and more particularly, human cells. These micro-dystrophin (MD1 and A3990) are particularly well suited for treatment of DMD and BMD. As described herein, rAAV micro-dystrophin (MD1 and A3990) constructs have been developed which have demonstrated high yield, expression levels, and/or activity.

Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

It is to be noted that the term "a" or "an" refers to one or more. As such, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of or “consisting essentially of language.

There are several naturally occurring (“wild-type”) serotypes and over 100 known variants of AAV, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid proteins, and thus in their gene delivery properties. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.

For the purposes of the disclosure herein, the terminology “AAV” is an abbreviation for Adeno-associated virus, including, without limitation, the virus itself and derivatives thereof. Except where otherwise indicated, the terminology refers to all subtypes or serotypes and both replication-competent and recombinant forms. The term “AAV” includes, without limitation, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3 A (AAV-3 A or AAV3A), AAV type 3B (AAV-3B or AAV3B), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), AAV type 10 (AAV- 10 or AAV10 or AAVrhlO or AAV rhlO or AAV-rhlO), avian AAV, bovine AAV, porcine AAV like AAVpol, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infect primates, "non-primate AAV" refers to AAV that infect non-primate mammals, "bovine AAV" refers to AAV that infect bovine mammals, etc.

An "AAV vector" as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

The term "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation/capsid proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

The terminology "helper virus" for AAV refers to a virus that allows efficient AAV (e.g. wild-type AAV) replication and packaging in a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the 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.

The term "gene" refers to a polynucleotide that performs a function of some kind in the cell. For example, a gene can contain an open reading frame that is capable of encoding a gene product. One example of a gene product is a protein, which is transcribed and translated from the gene. Another example of a gene product is an RNA, e.g. a functional RNA product, e.g., an aptamer, an interfering RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a non-coding RNA (ncRNA), a guide RNA for nucleases, etc., which is transcribed but not translated. The terminology "operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter initiates transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

The term "pharmaceutically acceptable" refers to compounds and compositions which are suitable for administration to humans and/or animals without undue adverse side effects such as toxicity and/or irritation and/or allergic response and commensurate with a reasonable benefit/risk ratio.

The principal embodiment of the present invention is to provide recombinant AAV vector comprising polynucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.

Another embodiment of the present invention is to provide recombinant AAV vector comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein under the regulation of muscle-specific control element.

Another embodiment of the present invention relates to viral genome of recombinant AAV particle which comprises; i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5- 12-hCK promoter which drives transcription of micro-dystrophin gene (MD1 and A3990); and iv) SV 40 Poly A transcription terminator.

In one embodiment functional micro-dystrophin is MD1. In another embodiment functional micro-dystrophin is A3990.

Another embodiment of the present invention relates to viral genome of recombinant AAV particle comprising nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding: i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5- 12-hCK promoter which drives transcription of micro-dystrophin (MD1 and A3990); and iv) SV 40 Poly A transcription terminator. Another embodiment of the present invention is to provide pharmaceutical formulation comprising:

(a) AAV vector genome comprising nucleotide sequence encoding microdystrophin gene (MD1 and A3990); and

(b) A pharmaceutically acceptable carrier, diluents, excipients, or buffer.

Another embodiment of the present invention aims at alleviating or curing the devastating Duchenne muscular dystrophy (DMD) as well as Becker muscular dystrophy (BMD) by expressing a shorter but functional dystrophin polypeptide called micro dystrophin (MD1 and A3990).

Another embodiment of the present invention is to provide a method of treatment for muscular dystrophy or dystrophinopathy comprises administering a therapeutically effective amount of the recombinant AAV vector containing nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.

According to one embodiment, said sequence can be an isolated nucleic acid encoding a microdystrophin having substantial homology or identity (60%, 70%, 80%, 90% 95% or even 99%) to the peptides disclosed herein, especially of sequence SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12.

Preferably, the nucleotide sequence of an isolated nucleic acid encoding a peptide of the invention is “substantially homologous/identical”, that is, about 60% homologous/identical , more preferably about 70% homologous/identical, even more preferably about 80% homologous/identical, more preferably about 90% homologous/identical, even more preferably, about 95% homologous/identical, and even more preferably about 97%, 98% or even 99% homologous/identical to a nucleotide sequence of an isolated nucleic acid encoding the functional microdystrophin, especially of sequence SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12.

Vectors having ITRs from a different source than its capsid are termed "pseudotyped". In certain embodiment inverted terminal repeats (ITR) from AAV2 may be selected for generation of pseudotyped AAV and in certain embodiments, ITRs from a source other than AAV2 may be selected for this construct to generate another pseudotyped AAV. Alternatively, ITRs from the same source as the capsid may be selected. In brief, any homologous or heterologous combination of AAV ITRs - AAV capsid could be used to generate recombinant AAV or pseudotyped recombinant AAV. In certain embodiments, ITRs may be selected to generate a self-complementary AAV, such as defined infra.

In certain embodiment the DNA sequence packaged inside the virus contains ITR sequences at 5’ and 3’ end along with different muscle-specific promoters described as follows:

• SPc5-12 promoter: is a synthetic muscle-specific promoter containing enhancer element with binding sites for the various skeletal muscle-specific transcription factors like MEF-1, MEF-2, TEF-1 and SRE followed by chicken alpha-actin promoter (reported in Patent US6410228B1). • hMHCK7 promoter: is a hybrid promoter consisting of enhance and promoter elements of human muscle creatine kinase (hCK) and mouse alpha-myosin heavychain gene (MyHC), respectively (reported in Trask et al., JBC, 1988 and Patent application W02018170408A1).

• Sk-CRM4-mDes promoter: contains muscle-specific transcription factor binding sites for EF2, CEBP, LRF, MyoD and SREBP followed by mouse desmin enhancer and promoter (reported in Patent application WO2015110449).

• Sk-CRM4-hDes promoter: contains muscle-specific transcription factor binding sites for EF2, CEBP, LRF, MyoD and SREBP followed by human desmin enhancer and promoter (reported in Li and Paulin, JBC, 1991 and Patent application WO2015110449).

• SPc5-12-hDes promoter: contains enhancer element with binding sites for the various skeletal muscle-specific transcription factors like MEF-1, MEF-2, TEF-1 and SRE followed by human desmin (hDes) promoter (reported in Li and Paulin, JBC, 1991 and Patent US6410228B1).

• SPc5-12-hCK promoter: contains enhancer element with binding sites for the various skeletal muscle-specific transcription factors like MEF-1, MEF-2, TEF-1 and SRE followed by hCK promoter (reported in Trask et al., JBC, 1988 and Patent US6410228B1).

All promoters are downstream linked with intron and Kozak sequence followed by the nucleotide sequence encoding microdystrophin (MD1 or A3990) along with SV40 Poly (A) transcription termination sequence. The construct is intended to produce a fully functional microdystrophin polypeptide sequence; MD1 encoding N-terminal Actin-Binding Domain, Hinge regions - Hl , H2 and H4 and Rod domains - R1 -3 and R24, Cys-rich region and A3990 encoding N-terminal Actin-Binding Domain, Hinge regions - Hl, H3 and H4 and Rod domains - Rl-2 and R22-24, and Cys-rich region; that will provide continuous source of functional microdystrophin in the muscle tissues of DMD patients, augmenting with non- functional dystrophin protein.

For use in producing an AAV viral vector (e.g., a recombinant (r) AAV), the expression cassettes can be carried on any suitable vector, e.g. a plasmid, which is delivered either to a packaging host cell (encoding rep and cap of AAV) along with helper plasmid or helper virus carrying adenovirus helper element or a mammalian cell line along with helper and packaging plasmids (transient triple transfection). The plasmids useful may be engineered such that they are suitable for replication and packaging in prokaryotic cells, mammalian cells, or both. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by one of skill in the art.

EXPERIMENTAL DETAILS

Vector Designing

Twelve transducing plasmid vector constructs (1) pIntas-SPc5-12-MDl, (2) plntas- SPc5-12-A3990, (3) pIntas-hMHCK7-MDl, (4) pIntas-hMHCK7-A3990, (5) plntas- Sk-CRM4-mDes-MDl, (6) pIntas-Sk-CRM4-mDes-A3990, (7) pintas- Sk-CRM4- hDes-MDl, (8) pintas- Sk-CRM4-hDes-A3990, (9) pIntas-SPc5-12-hDes-MDl, (10) pIntas-SPc5-12-hDes-A3990, (11) _P Intas-SPc5-12-hCK-MDl and (12) pIntas-SPc5- 12-hCK-A3990 were synthesized by combining six different muscle-specific promoter (SPc5-12, hMHCK7, Sk-CRM4-mDes, Sk-CRM4-hDes, SPc5-12-hDes or SPc5-12- hCK) with two different p-dys gene (MD1 or A3990) to identify potential vector candidates for high level of expression of p-dys (either MD1 or A3990) in muscle and good genome packaging in different serotypes of AAV i.e. AAV9 and/or AAVpol.

The vectors were chemically synthesized and propagated in E. coli for bulk DNA preparation. The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also form the part of this specification and are intended to be included within the scope of it.

EXAMPLE 1; PLASMID VECTOR DESIGNING

A gene cassette was synthesized and cloned in the vector backbone (reporter plasmid and micro-dystrophin (pDys) variants plasmid MD1 or A3990) carrying a high-copy- number functional origin of replication for E. coli and kanamycin resistance gene as selection marker by GenScript in trans-conformation that comprises:

(1) 5’ and 3’ ITR from AAV2 (as disclosed in US6521225B1)

(2) Genetic promoter/enhancer: Cassette has one of the genetic promoters selected from A to F as mentioned below:

A. SPc5-12 Promoter: A chimeric synthetic muscle-specific promoter (as disclosed in US6410228B1) that contains enhancer element with binding sites for the various skeletal muscle-specific transcription factors like MEF-1, MEF-2, TEF-1 and SRE followed by chicken alpha-actin promoter.

B. SPc5-12-hDes Promoter: A chimeric synthetic muscle-specific promoter (as described in Li and Paulin, JBC, 1991 and US6410228B1) that contains enhancer element of SPc5-12 followed by human desmin (hDes) promoter. C. SPc5-12-hCK Promoter: A chimeric synthetic promoter (as described in Trask et al., JBC, 1988 and US6410228B1) that contains that contains enhancer element of SPc5-12 followed by human muscle creatine kinase (hCK) promoter.

D. hMHCK7 Promoter: A chimeric promoter (as described Trask et al., JBC, 1988 and W02018170408A1) that contains enhancer element of hCK followed by alphamyosin heavy-chain gene (MyHC) promoter.

E. Sk-CRM4-mDes Promoter: A chimeric synthetic muscle-specific promoter (as described in WO2015110449) that contains muscle-specific transcription factor binding sites for EF2, CEBP, LRF, MyoD and SREBP followed by mouse desmin enhancer and promoter.

F. Sk-CRM4-hDes Promoter: A chimeric synthetic muscle-specific promoter (as described in Li and Paulin, JBC, 1991 and WO2015110449) that contains musclespecific transcription factor binding sites for EF2, CEBP, LRF, MyoD and SREBP followed by human desmin enhancer and promoter.

(3) Chimeric Intron: A chimera between introns from human P-globin and immunoglobulin heavy chain genes (As described in US6461606).

(4) Kozak Sequence: A vertebrate nucleic acid consensus sequence that works for strong initiation of translation in eukaryotic mRNA transcripts.

(5) Coding sequence (CDS): Coding sequences for micro-dystrophin (pDys) variants MD1 or A3990 or reporter genes i.e. synthetic luc2 version of the firefly luciferase gene FLuc (Genbank Accession number MK484108.1) and enhanced Green fluorescent protein (eGFP), connected with 2A peptide from porcine teschovirus-1 polyprotein i.e. P2A. All the above sequences were codon optimized for human expression before synthesis.

MD1 encodes N-terminal actin-binding domain, hinge regions - Hl, H2 and H4 and rod domains - Rl-3, and R24, and cys-rich region and A3990 encodes N-terminal actin-binding domain, hinge regions - Hl, H3 and H4 and rod domains - Rl-2, and R22-24, and cys-rich region.

Reporter constructs encoding FLuc-P2A-eGFP were generated for the promoter screening in vitro and in vivo.

(6) SV40 Polyadenylation Signal: It is mammalian terminator that promotes both polyadenylation and transcription termination (As described in US5635177).

Sequence Labels for the elementary transducing cassette:

5’ITR - Promoter - Chimeric intron - CDS - SV40 Poly (A) - 3’ITR

Using various combinations of promoters as described above and CDS (MD1 or A3990 or reporter genes i.e. FLuc-P2A-eGFP) and keeping all other genetic elements constant, we generated constructs as mentioned below:

1. Plasmid vector: pintas- SPc5-12-MDl

Combining the gene sequences of promoter SPc5-12 and pDys MD1 in the elementary transducing cassette, we generated transfer plasmid pIntas-SPc5-12-MDl (SEQ ID NO: 01, Figure 1) that will encode -132 kDa fully functional microdystrophin protein, MD1.

2. Plasmid vector: pintas- SPc5- 12- A3990

Combining the gene sequences of promoter SPc5-12 and pDys A3990 in the elementary transducing cassette, we generated transfer plasmid pIntas-SPc5-12- A3990 (SEQ ID NO: 07, Figure 7) that encodes -146 kDa fully functional microdystrophin protein, A3990.

3. Reporter Plasmid vector: pIntas-SPc5-12-FLuc-eGFP Combining the gene sequences of promoter SPc5-12 and reporter sequence FLuc- P2A-eGFP in the elementary transducing cassette, we generated reporter transfer plasmid p!ntas-SPc5-12-FLuc-eGFP. The construct was proposed to be used for screening of skeletal muscle-specific promoter/enhancer combinations while exploiting either FLuc activity or eGFP or both as a measure of promoter/enhancer activity in muscle, kidney and liver cell lines of human and mouse origin viz. HEK- 293 FT (Human Embryonic Kidney cells), HepG2 (Human Liver cells)/Huh-7 (Human hepatoma-derived cell line), RCDMD (Human DMD muscle cells) and C2C12 (mouse myoblast cells) in vitro and also in wt-mice.

4. Plasmid vector: pintas- SPc5-12-hDes-MDl

Combining the gene sequences of promoter SPc5-12-hDes and pDys MD1 in the elementary transducing cassette, we generated transfer plasmid pIntas-SPc5-12-hDes- MD1 (SEQ ID NO: 05, Figure 5) that encoded -132 kDa fully functional microdystrophin protein, MD1.

5. Plasmid vector: pintas- SPc5-12-hDes-A3990

Combining the gene sequences of promoter SPc5-12-hDes and pDys A3990 in the elementary transducing cassette, we generated transfer plasmid pIntas-SPc5-12-hDes- A3990 (SEQ ID NO: 11, Figure 11) that encodes -146 kDa fully functional microdystrophin protein, A3990.

6. Reporter Plasmid vector: pIntas-SPc5-12-hDes-FLuc-eGFP

Combining the gene sequences of promoter SPc5-12-hDes and reporter sequence FLuc-P2A-eGFP in the elementary transducing cassette, we generated reporter transfer plasmid pIntas-SPc5-12-hDes-FLuc-eGFP. The construct was proposed to be used for screening of skeletal muscle-specific promoter/enhancer combinations while exploiting either FLuc activity or eGFP or both as a measure of promoter/enhancer activity in muscle, kidney and liver cell lines of human and mouse origin viz. HEK- 293 FT (Human Embryonic Kidney cells), HepG2 (Human Liver cells)/Huh-7 (Human hepatoma-derived cell line), RCDMD (Human DMD muscle cells) and C2C12 (mouse myoblast cells) in vitro and also in wt-mice.

7. Plasmid vector: pintas- SPc5-12-hCK-MDl

Combining the gene sequences of promoter SPc5-12-hCK and pDys MD1 in the elementary transducing cassette, we generated transfer plasmid p!ntas-SPc5-12-hCK- MD1 (SEQ ID NO: 06, Figure 6) that will encode —132 kDa fully functional microdystrophin protein, MD1.

8. Plasmid vector: pintas- SPc5-12-hCK-A3990

Combining the gene sequences of promoter SPc5-12-CK and pDys A3990 in the elementary transducing cassette, we generated transfer plasmid pIntas-SPc5-12-hCK- A3990 (SEQ ID NO: 12, Figure 12) that encodes ~146 kDa fully functional microdystrophin protein, A3990.

9. Reporter Plasmid vector: pIntas-SPc5-12-hCK-FLuc-eGFP

Combining the gene sequences of promoter SPc5-12-hCK and reporter sequence FLuc-P2A-eGFP in the elementary transducing cassette, we generated reporter transfer plasmid pIntas-SPc5-12-hCK-FLuc-eGFP. The construct was proposed to be used for screening of skeletal muscle-specific promoter/enhancer combinations while exploiting either FLuc activity or eGFP or both as a measure of promoter/enhancer activity in muscle, kidney and liver cell lines of human and mouse origin viz. HEK- 293 FT (Human Embryonic Kidney cells), HepG2 (Human Liver cells)/Huh-7 (Human hepatoma-derived cell line), RCDMD (Human DMD muscle cells) and C2C12 (mouse myoblast cells) in vitro and also in wt-mice. 10. Plasmid vector: pIntas-hMHCK7-MDl

Combining the gene sequences of promoter hMHCK7 and pDys MD1 in the elementary transducing cassette, we generated transfer plasmid pintas- hMHCK7- MD1 (SEQ ID NO: 02, Figure 2) that will encode —132 kDa fully functional microdystrophin protein, MD1.

11. Plasmid vector: pIntas-hMHCK7-A3990

Combining the gene sequences of promoter hMHCK7 and pDys A3990 in the elementary transducing cassette, we generated transfer plasmid pIntas-hMHCK7- hCK-A3990 (SEQ ID NO: 08, Figure 8) that encodes ~146 kDa fully functional microdystrophin protein, A3990.

12. Reporter Plasmid vector: pIntas-hMHCK7-FLuc-eGFP

Combining the gene sequences of promoter hMHCK7 and reporter sequence FLuc- P2A-eGFP in the elementary transducing cassette, we generated reporter transfer plasmid pIntas-hMHCK7-FLuc-eGFP. The construct was proposed to be used for screening of skeletal muscle-specific promoter/enhancer combinations while exploiting either FLuc activity or eGFP or both as a measure of promoter/enhancer activity in muscle, kidney and liver cell lines of human and mouse origin viz. HEK- 293 FT (Human Embryonic Kidney cells), HepG2 (Human Liver cells)/Huh-7 (Human hepatoma-derived cell line), RCDMD (Human DMD muscle cells) and C2C12 (mouse myoblast cells) in vitro and also in wt-mice.

13. Plasmid vector: pIntas-Sk-CRM4-mDes-MDl

Combining the gene sequences of promoter Sk-CRM4-mDes and pDys MD1 in the elementary transducing cassette, we generated transfer plasmid pIntas-Sk-CRM4- mDes-MDl (SEQ ID NO: 03, Figure 3) that will encode —132 kDa fully functional microdystrophin protein, MD1. 14. Plasmid vector: pIntas-Sk-CRM4-mDes-A3990

Combining the gene sequences of promoter Sk-CRM4-mDes and pDys A3990 in the elementary transducing cassette, we generated transfer plasmid p!ntas-Sk-CRM4- mDes-hCK-A3990 (SEQ ID NO: 09, Figure 9) that encodes ~146 kDa fully functional microdystrophin protein, A3990.

15. Reporter Plasmid vector: pIntas-Sk-CRM4-mDes-FLuc-eGFP

Combining the gene sequences of promoter Sk-CRM4-mDes and reporter sequence FLuc-P2A-eGFP in the elementary transducing cassette, we generated reporter transfer plasmid pIntas-Sk-CRM4-mDes-FLuc-eGFP. The construct was proposed to be used for screening of skeletal muscle-specific promoter/enhancer combinations while exploiting either FLuc activity or eGFP or both as a measure of promoter/enhancer activity in muscle, kidney and liver cell lines of human and mouse origin viz. HEK-293 FT (Human Embryonic Kidney cells), HepG2 (Human Liver cells)/Huh-7 (Human hepatoma-derived cell line), RCDMD (Human DMD muscle cells) and C2C12 (mouse myoblast cells) in vitro and also in wt-mice.

16. Plasmid vector: pintas- Sk-CRM4-hDes-MDl

Combining the gene sequences of promoter Sk-CRM4-hDes and pDys MD1 in the elementary transducing cassette, we generated transfer plasmid pIntas-Sk-CRM4- hDes-MDl (SEQ ID NO: 04, Figure 4) that will encode —132 kDa fully functional microdystrophin protein, MD1.

17. Plasmid vector: pintas- Sk-CRM4-hDes-A3990

Combining the gene sequences of promoter Sk-CRM4-hDes and pDys A3990 in the elementary transducing cassette, we generated transfer plasmid pIntas-Sk-CRM4- hDes-hCK-A3990 (SEQ ID NO: 10, Figure 10) that encodes ~146 kDa fully functional microdystrophin protein, A3990. 18. Reporter Plasmid vector: pIntas-Sk-CRM4-hDes-FLuc-eGFP

Combining the gene sequences of promoter Sk-CRM4-hDes and reporter sequence FLuc-P2A-eGFP in the elementary transducing cassette, we generated reporter transfer plasmid pintas- Sk-CRM4-hDes-Fluc-eGFP. The construct was proposed to be used for screening of skeletal muscle-specific promoter/enhancer combinations while exploiting either FLuc activity or eGFP or both as a measure of promoter/enhancer activity in muscle, kidney and liver cell lines of human and mouse origin viz. HEK-293 FT (Human Embryonic Kidney cells), HepG2 (Human Liver cells)/Huh-7 (Human hepatoma-derived cell line), RCDMD (Human DMD muscle cells) and C2C12 (mouse myoblast cells) in vitro and also in wt-mice.

19. Packaging Plasmid vector: pIntas-ACG-R2C9

The packaging plasmid vector pIntas-ACG-R2C9 (SEQ ID NO: 13, Figure 13) consists of an AAV2 genomic expression cassette lacking 5’- and 3’-ITRs as well as AAV2 capsid i.e. consists of 5’UTR (untranslated Region), replicase (Rep2) coding sequence, spacer sequence and 3’UTR of AAV2 (AAV2 genome - Genbank Accession: NC_001401.2) along with AAV9 capsid coding sequence, AAV9 genome - Genbank Accession: AY530579.1) placed between Rep2 and 3’ UTR of AAV2 in a vector backbone with a functional origin of replication for E. coli and ampicillin as selection marker. Additionally, ACG was used as start codon instead of ATG.

20. Packaging Plasmid vector: pIntas-ACG-R2Cpol

The packaging plasmid vector pintas- AC G-R2Cpol (SEQ ID NO: 14, Figure 14) consists of an AAV2 genomic expression cassette lacking 5’- and 3’-ITRs as well as AAV2 capsid i.e. consists of 5’UTR (untranslated Region), Replicase (Rep2) coding sequence, spacer sequence and 3’UTR of AAV2 (AAV2 genome - Genbank Accession: NC_001401.2) along with AAVpol capsid coding sequence (AAVpol genome - Genbank Accession: FJ688147.1) placed between Rep2 and 3’ UTR of AAV2 in a vector backbone with a functional origin of replication for E. coli and ampicillin as selection marker. Additionally, ACG was used as start codon instead of ATG.

21. Helper Plasmid : pintas Helper

The helper plasmid vector plntas-Helper contains the sequences of E2A, E4 and VA helper genes from Adenovirus Type 5 (Genbank Accession: AF369965.1). The expression of the said genes is driven by inherent viral promoters.

EXAMPLE 2; TRANSFORMATION, BANKING AND BULK DNA PREPARATION

Transformation, Clone Selection and Banking:

The plasmid DNAs were transformed in an A. coli strain DH5 -alpha, ToplO and/or in Stbl3 or Stbl4. Transformants were scored over LB-kanamycin or LB-ampicillin plates. Plasmid DNAs were isolated from the transformants using miniprep (QIAprep Spin Miniprep Kit; Cat No. 27104) and restriction digestion analysis was performed to characterize the plasmid for the presence of gene of interest and vector integrity. Among several correct transformants, a high performing (high purity, yield as well as high content of covalently closed circular plasmid) clone was selected and final glycerol banks were prepared.

Large scale DNA Preparation:

A vial from the glycerol stock was grown over night at 37 °C for 16 to 18 hours in LB medium containing desired antibiotic (kanamycin or ampicillin) and temperature shift was done at 42°C for 4 to 6 hours (optional). Bacterial pellet was subsequently harvested by pelleting the biomass via centrifugation at 4 °C and large scale plasmid preparation was performed using maxiprep (Qiagen plasmid Maxi Kits; Cat No.12162, 12163 and 12165) or gigaprep (Qiagen Hi speed plasmid Giga EF Kit; Cat No.1054575) as per manufacturer’s recommendation. Briefly, bacterial pellets were re-suspended in re-suspension buffer using a pipette followed by alkaline lysis, neutralization and column purification.

In Vitro Evaluation of Promoter Activity Using Reporter Plasmid Vectors:

In vitro promoter activity was assessed in cell lines from different origins including C2C12, RCDMD, HEK293FT, and Huh-7 or HepG2 by a promoter reporter assay (as described in Piekarowicz et. al., Mol Ther Methods Clin Dev. 2019) for screening promoter for muscle-specific expression.

EXAMPLE 3: CELL LINE MAINTENANCE

Different cell lines, viz. C2C12 (mouse myoblast cells), RCDMD (Immortalized Human Duchenne Muscular Dystrophy Skeletal Muscle Cells), HEK293FT (Human Embryonic Kidney cells) Huh-7 (human hepatocyte cell) or HepG2 (Human Hepatic cells), were propagated in their preferred media in 75 cm ² cell culture treated flasks (T75, Corning, 430641U) at 37°C, 5% CO2.

EXAMPLE 4: SINGLE DNA TRANSIENT TRANSFECTION IN DIFFERENT CELL LINES

Single DNA transient transfection in different cell lines was done and promoter activity and transfection efficacy was evaluated by measuring luciferase expression as well as eGFP-expression. Briefly, different cells (C2C12, RCDMD, HEK293FT, Huh- 7 or HepG2) was plated in 24- well plates after trypsinization of T75 flask and 24 hours later transfection was performed at 70-80% cell confluency with the six reporter plasmid vector (plasmids pIntas-SPc5-12-Fluc-eGFP, pintas- SPc5-12-hDes-Fluc- eGFP, pIntas-SPc5-12-hCK-Fluc-eGFP, _P Intas-hMHCK7-FLuc-eGFP, plntas-Sk- CRM4-mDes-Fluc-eGFP and pIntas-Sk-CRM4-hDes-Fluc-eGFP), separately in MEM using lipofectamine 3000 as the transfection agent (Invitrogen, Cat No. L3000- 001) as per manufacturer’s instruction. Four hours post transfection; the media was changed with cell line specific complete media. Two separate plasmid vectors expressing FLuc and GFP respectively under regulation of CMV promoter were used as positive transfection controls, while keeping two wells with non-transfected cells as negative control. The assay was done in two technical replicates for each parameter to be measured. After 48 hours of transfection, the cells were harvested.

EXAMPLE 5: DETERMINATION OF PROMOTER ACTIVITY AND TRANSFECTION EFFICACY

Post 48 hours of transfection, culture supernatant was aspirated, cell lysis was performed with glo-lysis buffer and promoter activity was measured in a firefly luciferase assay (Promega, Cat No. E2610) using cell lysate as per manufacturer’s instruction. Comparison of promoter activity amongst different cell lines, viz. C2C12, RCDMD, HEK293FT and Huh-7 was established in cell lysates using luciferase reporter assay (Figure 15 and Figure 16). Total protein content in cell lysate was determined and FLuc activity was recorded as activity per pg of protein. Luminescence signals from Flue positive control for each cell line was adjusted to 100% and correspondingly percentage promoter activity for all the promoters was determined. Data presented in Figure 15 and Figure 16 indicates that all the six promoters were able to drive high muscle-specific expression of Flue in C2C12 and RCDMD (> 100% compared to CMV promoter) with minimal activity in liver (Huh- 7) and kidney (HEK293FT) cell lines. While highest expression was driven by plntas- Sk-CRM4-mDes-FLuc-eGFP and pIntas-Sk-CRM4-hDes-FLuc-eGFP in mouse myoblast cell line (C2C12), the same was not true for human muscle cell line (RCDMD). pintas- SPc5-12-FLuc-eGFP, pIntas-SPc5-12-hCK-Fluc-eGFP and plntas- hMHCK7-FLuc-eGFP reporter constructs showed FLuc activity higher than positive control in both C2C12 and RCDMD. On tx'he basis of the data obtained from RCDMD, the promoters were ranked as follows:

SPc5-12 > SPc5-12-hCK > hMHCK7 > SPc5-12-hDes > Sk-CRM4-mDes > Sk- CRM4-hDes.

The best candidate promoter would be the one which gave good microdystrophin expression both in mouse and human muscle tissue. Based on this, the promoters selected for further studies with micro-dystrophin constructs were pIntas-SPc5-12- MD1, pIntas-SPc5-12-A3990, pIntas-SPc5-12-hCK-MDl, pIntas-SPc5-12-hCK- A3990, pIntas-hMHCK7-MDl, and pIntas-hMHCK7-A3990.

In vivo Evaluation of Muscle-Specific Promoter Activity Using Recombinant Reporter AAV

Recombinant reporter AAV was generated, purified, titrated for viral genome and capsid particles and promoter driven systemic expression was evaluated in vivo using Balb/c / C57BL/6 / SCID-mdx mouse. (As described in Sarcar et a/ Nat. Comm. 2019) with some modifications as mentioned below.

EXAMPLE 6: GENERATION OF RECOMBINANT AAV9 AND/OR AAVpol

HEK 293 cells were propagated and maintained in 1000 mL shake flask in a suitable mammalian cell culture media. An exemplary option of such kind of media can be DMEM medium (Gibco, Cat No. Al 5169-01) or BalanCD medium (Irvine Scientific, Cat No. 94137). The cells were seeded at 0.8 million/mL in 1000 mL shake flask with 300 mL media and maintained at 37 °C, 125 rpm and 8% CO2 for 48 hours or until the cell density reaches between 2.0-3.5 million/mL.

A triple co-transfection of HEK 293 cells was done with the either of the AAV reporter transgene plasmid (as identified in EXAMPLE 5) along with packaging plasmid vector (p!ntas-ACG-R2C9 or pintas- AC G-R2Cpol : plasmids encoding capsid proteins for AAV serotype 9 or AAV serotype pol) and helper plasmid (plntas- Helper) in the ratio of 1: 1:2. The transfection of DNA was done using PEI-MAX (Polysciences Inc., Cat no. 24765-1). Transfected HEK 293 cells were maintained at 37 °C, 125 rpm and 8% CO2 for 72 to 96 hours in CO2 shaker incubator. At the termination time culture was harvested and stored in -80 °C freezer till the purification.

EXAMPLE 7; PURIFICATION OF rAAV9 OR rAAVpol REPORTER VIRUS

HEK293 cell culture harvests post transfection (from Example 6) were lysed and clarified by centrifugation followed by 0.2 pm filtration. The AAV preparations were purified by methods using affinity chromatography, anion exchange chromatography, CsCl or lodixanol gradient purification (separation of filled capsid with empty capsid). The purified product was characterized for vector genome titer.

EXAMPLE 8: VECTOR GENOME ANALYSIS

Quantification of AAV genomes (vector genomes or vg/ml) was done using gene specific primers (FLuc and/or eGFP) and probes on Droplet Digital PCR (ddPCR). EXAMPLE 9: IN-VIVO BIOLUMINESCENCE ANALYSIS

In-vivo screening of the AAV vectors containing different promoters driving FLuc expression was performed in Balb/c / C57BL/6 / SCID-mdx mice. Six to eight weeks old animals were dehaired one day before intramuscular or intravenous (tail vein) administration of recombinant reporter AAV9 or AAVpol (50-100 pl of purified reporter AAV vectors [1 x 10 ¹⁰ - 6 x 10 ¹¹ vg/mouse]). The experimental animals were live-imaged using non-invasive in vivo bioluminescence optical imaging system (IVIS, Perkin Elmer) at 2, 4 and 6 weeks post AAV-administration within one minute after intravenous administration of a D-luciferin substrate at a dose of 150 mg/kg of body weight. Six weeks post AAV-administration, animals were euthanized by overdosing with anesthesia within 1 -minute post D-luciferin administration and quantitative image analysis of individual organs was performed. In-vivo bioluminescence was expressed in photons (ph) s-1 cm-2 steradian (sr)-l and displayed as a pseudo-color overlay onto a grayscale animal image using a rainbow color scale.

EXAMPLE 10: TRANSDUCTION EFFICIENCY AND BIODISTRIBUTION

After final imaging post euthanasia, various organs and tissues were collected from mice and genomic DNA was extracted from 30 mg of each organ/tissue using DNeasy Blood & Tissue Kit protocol (Qiagen, Chatsworth, CA, USA). 100 ng of genomic DNA from each sample was subjected to ddPCR and transduction efficiency and bio distribution in the organs/tissues was determined by quantifying FLuc transgene copy numbers. The data was expressed as mean AAV copy number/100 ng of genomic DNA. EXAMPLE 11: IN-VIVO ANALYSIS OF THERAPEUTIC EFFICACY

Four- weeks-old SCID/mdx mice were injected intravenously with 1-5 X 10 ¹¹ VG therapeutic AAV vector (AAV9/AAVpol) encoding pDes-MDl and / or pDes- A3990. Control mice were injected with vehicle either carrying empty AAV vector or no AAV vector. Additionally, the age matched C57BL/6 mice were used as positive control. Four/eight/sixteen weeks post-injection, the treated, control mice and C57BL/6 mice were subjected to a treadmill test to determine correction of the dystrophic phenotype.

EXAMPLE 12: IN-VITRO ANALYSIS OF MUSCLE CONTRACTILE PROPERTIES

Animals (AAV vector treated and control SCID-mdx mice and/or C57BL/6 mice) injected with AAV vectors (AAV9/AAVpol) encoding MD1 or p Des- 3990 were euthanized 16 weeks post vector administration and the extensor digitorum longus muscle was isolated from each mouse. Force test (an in vitro muscle test using an Aurora 1200 A) was performed on freshly isolated muscles after electrical stimulation at 30 °C chamber filled with Krebs-Ringer bicarbonate buffer solution containing 10 mM glucose and continuously gassed with 99% O2. The electrical stimulation was induced with a series of 150 Hz pulses for 0.5 s (evoking titanic force of 0.2 ms pulses).