CN117642509A

CN117642509A - Production of recombinant AAV vectors for the treatment of muscular dystrophy

Info

Publication number: CN117642509A
Application number: CN202280049147.2A
Authority: CN
Inventors: M·阿拉姆; L·罗迪诺-克拉帕克
Original assignee: Sarepta Therapeutics Inc
Current assignee: Sarepta Therapeutics Inc
Priority date: 2021-05-17
Filing date: 2022-05-13
Publication date: 2024-03-01

Abstract

The present disclosure provides gene therapy vectors, such as recombinant adeno-associated virus (rAAV), produced in mammalian adherent cells cultured under suspension conditions for expression of human microdystrophin genes. The disclosure also provides compositions and methods of using these rAAV to treat muscular dystrophy, such as duchenne muscular dystrophy.

Description

Production of recombinant AAV vectors for the treatment of muscular dystrophy

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application number 63/253,998 filed on 8 th 10 th 2021, U.S. provisional application number 63/243,944 filed on 14 th 9 th 2021, U.S. provisional application number 63/209,733 filed on 11 th 6 th 2021, and U.S. provisional application number 63/189,676 filed on 17 th 5 th 2021, each of which is incorporated herein by reference in their entirety.

Reference to sequence listing provided in electronic form

The contents of the sequence listing of ASCII text files provided in electronic form (designation: 4140_052pc04_seqlising_st25.txt; size: 60,194 bytes; date of creation: 2022, 5, 13 days) submitted with the present application are incorporated herein by reference in their entirety.

Technical Field

The present disclosure is in the field of gene therapy. More specifically, the present disclosure provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, for expressing miniaturized human microdystrophin (microdystrophin) genes, wherein the AAV is produced from adherent cells cultured under suspension conditions. The present disclosure also provides methods of using these vectors to express minute anti-dystrophin proteins in skeletal muscles (including diaphragm and myocardium), as well as to protect muscle fibers from injury, increase muscle strength, reduce and/or prevent fibrosis in subjects with muscular dystrophy.

Background

The importance of muscle mass and strength to daily activities (e.g., exercise and respiration) and systemic metabolism is self-evident. A deficiency in muscle function can lead to Muscular Dystrophy (MD), which is characterized by muscle weakness and atrophy, and severely affects quality of life. The most typical MD is caused by a mutation in a gene encoding a member of the anti-dystrophin related protein complex (DAPC). These MDs are caused by membrane fragility associated with loss of myomembrane-cytoskeleton tethered by DAPC. Du muscular dystrophy (Duchenne Muscular Dystrophy, DMD) is one of the most damaging muscle diseases, with 1 person suffering from every 5,000 neonates.

DMD is caused by mRNA reduction and a deficiency of anti-dystrophin protein, a 427kD myomembrane protein associated with the anti-dystrophin-associated protein complex (DAPC) due to mutation of the DMD gene (Hoffman et al, cell51:919-28, 1987). DAPC consists of a number of proteins on the muscle myomembrane that form structural links between the extracellular matrix (ECM) and the cytoskeleton through anti-dystrophin (an actin binding protein) and alpha-anti-dystrophin glycans (a laminin binding protein). These structural attachments stabilize the muscle cell membrane during contraction and prevent injury caused by contraction. After loss of dystrophin, membrane fragility can lead to myomembrane tearing and calcium influx, triggering calpain and segmental fiber necrosis (Straub et al, curr Opin. Neurol.10:168-75 (1997)). This uncontrolled cycle of muscle degeneration and regeneration eventually depletes the muscle stem Cell population (Sacco et al, cell 143:1059-1071 (2010); wallace et al, annu Rev Physiol 71:37-57 (2009)), leading to progressive muscle weakness, endomyelitis and fibrotic scarring.

Without the membrane stabilization of anti-dystrophin or mini-anti-dystrophin, DMD will appear as an uncontrolled cycle of tissue injury and repair, ultimately replacing the lost muscle fibers with fibrotic scar tissue through connective tissue hyperplasia. Fibrosis is characterized by excessive deposition of ECM matrix proteins (including collagen and elastin). ECM proteins are produced primarily by cytokines (e.g., tgfβ) released by activated fibroblasts in response to stress and inflammation. Although the main pathological features of DMD are myofibrosis and necrosis, fibrosis has the same effect as a pathological outcome. In DMD patients, excessive generation of fibrotic tissue limits muscle regeneration and leads to progressive muscle weakness. In one study, the presence of fibrosis in the initial DMD muscle biopsies was highly correlated with poor exercise prognosis for 10 years of follow-up (Desguerre et al J Neuropathol Exp Neurol 68:762-767 (2009)). These results indicate that fibrosis is the major factor in DMD muscle dysfunction and underscores the need for early intervention before significant fibrosis occurs.

International publication No. WO2019/245973A1, which is incorporated herein by reference in its entirety, describes the delivery of a minute anti-dystrophin gene with an AAV vector for the treatment of muscular dystrophy (e.g., DMD) in a human subject, however, there remains a need in the art for improved methods of producing such AAV vectors, particularly methods suitable for large scale production of such gene therapy vectors.

Disclosure of Invention

The present disclosure relates to gene therapy vectors (e.g., AAV vectors) produced by the suspension seeding process described herein that express human microdystrophin genes in skeletal muscles (including diaphragm and myocardium), thereby protecting muscle fibers from injury, increasing muscle strength, reducing and/or preventing fibrosis.

The present disclosure provides methods of producing recombinant adeno-associated virus (rAAV) raavrh74.mhck7. Mini-dystrophin in adherent mammalian cells by a suspension seeding process comprising: (a) Culturing cells in an N-2 vessel with a first growth medium comprising serum; (b) removing the cells from the first medium; (c) Inoculating the cells from step (b) into a second medium in the N-1 vessel that does not contain serum or has a serum concentration lower than the first medium; (d) culturing the cells in the N-1 vessel under suspension conditions; and (e) inoculating the third medium in the bioreactor with cells from step (d).

In some aspects, the rAAV used in the methods described herein comprises the human microdystrophin nucleotide sequence of SEQ ID NO. 1. In some aspects, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO. 7. In some aspects, the rAAV comprises a human microdystrophin nucleotide sequence of SEQ ID NO. 1 and an MHCK7 promoter sequence of SEQ ID NO. 7.

In some aspects, the suspension seeding process further comprises: (f) Adherent cells were transfected with transgenic plasmids containing raavrh74.mhck7. Mini-dystrophin constructs, plasmids containing AAV rep genes and AAV cap genes, and adenovirus helper plasmids.

In some aspects, the transgenic plasmid comprising raavrh74.mhck7. The mini-dystrophin construct comprises: the nucleic acid sequence of SEQ ID NO. 9; nucleotides 55-5021 of SEQ ID NO. 3; or nucleotides 1 to 4977 of SEQ ID NO. 8. In some aspects, the plasmid comprising an AAV rep gene and an AAV cap gene comprises an AAV2 rep gene and a rAAVrh74 cap gene. In some aspects, the adenovirus helper plasmid comprises adenovirus 5E2A, E4ORF6 and a VA RNA gene.

In some aspects, the suspension seeding process further comprises: (g) lysing the adherent cells. In some aspects, the adherent cells are lysed by freeze-thawing, solid shearing, hypertonic and/or hypotonic lysis, liquid shearing, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

In some aspects, the suspension seeding process further comprises (h) purifying the rAAV by at least one column chromatography step. In some aspects, the at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

In some aspects, the suspension seeding process further comprises culturing cells with the first growth medium in an N-3 vessel. In some aspects, the suspension seeding process further comprises culturing cells with the first growth medium in an N-4 vessel.

In some aspects, the bioreactor is an adherent bioreactor. In some aspects, the rAAV is purified from a culture produced in an adherent bioreactor.

In some aspects, the third medium in the bioreactor comprises at least one factor that promotes cell adhesion. In some aspects, the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some aspects, the third medium in the bioreactor comprises DMEM and 10% fbs.

In some aspects, the adherent cells are cultured under suspension conditions for about 48-72 hours.

In some aspects, the N-1 container is a suspension shake flask.

In some aspects, the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells. In some aspects, the adherent cells are HeLa cells or HEK-293 cells. In some aspects, the adherent cells are HEK-293 cells. In some aspects, the adherent cells are not suspension-adapted cells. In some aspects, culturing a cell under suspension conditions does not alter the adherence dependence of the cell. In some aspects, the culturing does not alter the cells to create a new cell line.

The present disclosure also provides compositions comprising recombinant adeno-associated virus (rAAV) raavrh74.mhck7. Mini-dystrophin protein, wherein the rAAV is made by any of the methods described herein. In some aspects, the composition comprises: a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO. 9; b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO. 3; and/or c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO. 8.

In some aspects, the present disclosure provides a composition for treating muscular dystrophy in a subject in need thereof comprising a recombinant adeno-associated virus (rAAV) raav.rh74mhck7. A mini-dystrophin, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured in an N-1 vessel under suspension conditions. In some aspects, the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO. 1. In some aspects, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO. 7. In some aspects, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO. 7 and the human microdystrophin nucleotide sequence of SEQ ID NO. 1.

In some aspects, the composition comprises: (a) a rAAV comprising the nucleic acid sequence of SEQ ID NO. 9; (b) a rAAV particle comprising the nucleic acid sequence of SEQ ID NO. 9; (c) rAAV comprising nucleotides 55-5021 of SEQ ID NO. 3; (d) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO. 3; (e) rAAV comprising nucleotides 1-4977 of SEQ ID NO. 8; and/or (f) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO. 8.

The disclosure also provides methods of treating muscular dystrophy in a human subject in need thereof comprising administering to the human subject a composition comprising a rAAV as described herein. In some aspects, the systemic route of administration is used and at about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁵ The rAAV is administered at a dose of vg/kg. In some aspects, the systemic route of administration is intravenous and the dose of rAAV administered is about 2x10 ¹⁴ vg/kg。

In some aspects, the dose of rAAV is administered at a concentration of about 10 mL/kg. In some aspects, the rAAV is administered by injection, infusion, or implantation. In some aspects, the rAAV is administered by infusion within about one hour. In some aspects, the rAAV is administered via the intravenous route via the external Zhou Zhiti vein.

In some aspects, the muscular dystrophy is duchenne muscular dystrophy or Becker muscular dystrophy (Becker's muscular dystrophy). In some aspects, the muscular dystrophy is duchenne muscular dystrophy.

In some aspects, the level of microdystrophin gene expression in cells of the subject after administration of the rAAV is increased as compared to the level of microdystrophin gene expression prior to administration of the rAAV. In some aspects, expression of a microdystrophin gene in the cell is detected by measuring the level of microdystrophin in a muscle biopsy by western blotting before and after administration of the rAAV. In some aspects, expression after administration of the rAAV is at least 55.4% compared to before administration.

In some aspects, the average percentage of micro-dystrophin positive fibers in the muscle tissue of the subject after administration of the rAAV is increased compared to the number of micro-dystrophin positive fibers prior to administration of the rAAV. In some aspects, the average percentage of mini-dystrophin positive fibers is at least 70.5% and the average intensity is at least 116.9% as detected by Immunofluorescence (IF) in muscle biopsies before and after administration of rAAV. In some aspects, the minute dystrophin protein counted by the vector genome is transduced to at least 3.87 average vector genome copies per cell nucleus.

In some aspects, the composition is administered to a patient that is genotyped. In some aspects, the patient is genotyped for the human anti-dystrophin (DMD) gene. In some aspects, the genotyped patient is genotyped for at least one mutation in exons 18-79 of the human anti-dystrophin (DMD) gene.

In some aspects, the method of treating muscular dystrophy further comprises genotyping the DMD gene of the human subject prior to administering the composition to the human subject. In some aspects, genotyping detects at least one mutation in exons 18 to 79 of the DMD gene. In some aspects, the at least one mutation is a frameshift deletion, frameshift repeat, premature termination, or other pathogenic variation resulting in a lack of expression of a human anti-dystrophin protein.

The present disclosure also provides for the use of the compositions described herein for treating muscular dystrophy in a human subject in need thereof. In some aspects, the present disclosure also provides the use of a composition described herein in the manufacture of a medicament for treating muscular dystrophy.

In some aspects, the muscular dystrophy is duchenne muscular dystrophy or becker muscular dystrophy. In some aspects, the muscular dystrophy is duchenne muscular dystrophy.

Drawings

Fig. 1 shows raav.mhck7. Mini-dystrophin constructs. In this construct, the cDNA expression cassette is flanked by AAV2 Inverted Terminal Repeats (ITRs). The construct is characterized by the absence of in-frame rods (R4-R23), while hinges 1, 2 and 4 (H ₁ 、H ₂ And H ₄ ) And a cysteine-rich domain, thereby producing a 138kDa protein. Expression of the mini-dystrophin protein (3579 bp) is directed by the MHCK7 promoter (795 bp). Introns and 5' UTRs are from plasmid pCMV beta (Clontech). The mini-dystrophin cassette has a consensus Kozak in front of the ATG initiator and is used for mRNA terminationA small 53bp synthetic poly-A signal. The human mini-dystrophin cassette contains the (R4-R23/. DELTA.71-78) domain as previously described by Harper et al (Nature Medicine 8:253-261 (2002)).

FIG. 2 provides the nucleic acid sequence of AAVrh74.MHCK7. Mini-dystrophin (SEQ ID NO: 3).

FIG. 3 provides a pNLREP2-Caprh74 AAV helper plasmid map.

FIG. 4 provides the Ad helper plasmid pHELP.

Fig. 5 shows raav.mck. Mini-dystrophin plasmid constructs.

FIG. 6 provides the nucleic acid sequence of rAAV rh74.MCK. Mini-dystrophin (SEQ ID NO: 5).

Figure 7 shows the expression of the mini-dystrophin gene in the muscle fibers of gastrocnemius muscle biopsies measured by immunocytochemistry.

Figures 8A-8C provide western blot images showing that the mini-dystrophin protein is expressed at the correct molecular weight. In fig. 8A and 8B, western blot analysis detected minute anti-dystrophin protein expression in subject 1 (5 years), subject 2 (4 years) and subject 3 (6 years). In fig. 8C, samples of subject 4 (×1:4 diluted (to linear range) because ULDQ (> 80%) exceeded in the primary analysis, the mean value was multiplied by the dilution correction factor to give the final value for comparison with the normal samples. Average mini-dystrophin expression was 182.7% in method 1 and 222.0% in method 2 compared to normal samples.

Figures 9A-9C show that administration of raavrh74.mhck7 in subject 1 (figure 9A), subject 2 (figure 9B) and subject 3 (figure 9C) upregulated DAPC protein, α -and β -actin expression of mini-dystrophin.

Figure 10 provides a graph showing that administration of raavrh74.mhck7. Mini-dystrophin resulted in a sustained significant decrease in Creatine Kinase (CK) values.

Fig. 11 provides a graph showing the average Creatine Kinase (CK) change from baseline to day 270. This data shows that CK decreases significantly over time following administration of raavrh74.mhck7.

Figure 12 provides a graph showing the average NSAA change and average CK change from baseline to day 270. This data shows that NSAA increases significantly over time following administration of raavrh74.mhck7.

FIG. 13 provides the 4977 base nucleic acid sequence of AAVrh74.MHCK7. Minor anti-dystrophin construct (SEQ ID NO: 9). The following molecular elements are described: 5' ITR (bases 1-145); the MHCK7 promoter (190-981 (792 bases)); introns (991-1140 (150 bases)); human minor anti-dystrophin sequence (1151-4729 (3579 bases)); poly a tail (4732-4784 (53 bases)); and 3' ITR (4833-4977 (145 bases)).

Figure 14 shows aavrh74.mhck7. Mini-dystrophin plasmid constructs.

FIG. 15 provides the nucleic acid sequence of an AAVrh74.MHCK7. Mini-dystrophin plasmid construct (SEQ ID NO: 8) comprising a kanamycin resistance gene.

FIG. 16 is a visual representation of the mixed seed-train (seed-train) amplification method described herein.

Figure 17 is a visual representation of AAV particles produced using the mixed vaccination training amplification method described herein.

FIGS. 18A-18B provide graphs showing viability of HEK-293 cells cultured according to the mixed seeding training amplification methods disclosed herein (FIG. 18A) and cultured under adherent conditions only (FIG. 18B).

FIGS. 19A-19B provide graphs showing the viable cell density of HEK-293 cells cultured according to the mixed seeding training expansion methods disclosed herein (FIG. 19A) and cultured under adherent conditions only (FIG. 19B).

Figure 20 is a graph showing the average NSAA scores for cohort 1 (first 11 patients treated with raavrh74.mhck7. Mini-dystrophin) described in example 7. The first 11 patients were 3 points above baseline. Patients aged 6 to 7 (n=9) were 2.9 points above baseline. Each time point represents the 11 patients.

FIGS. 21A-21C show DMD 12 weeks (FIG. 21B) and 24 weeks (FIG. 21C) after treatment with delandistrogene moxeparvovec as compared to normal saline (FIG. 21A) as discussed in example 11 ^mdx Expression of tiny anti-muscular dystrophy proteins in skeletal and cardiac muscles of rats (immunofluorescence). Legend: LTA = left tibialis anterior; HRT = heart.

The bar graphs of fig. 22A-22B depict DMD 12 weeks and 24 weeks after treatment with delandistrogene moxeparvovec as discussed in example 11 ^mdx Quantification of mini-dystrophin expression (immunofluorescence) and vector transduction (vector genome copy) (fig. 22B) in rat muscle tissue. Legend: TA = tibial muscle; HRT = heart; MG = medial gastrocnemius; LG = lateral medial gastrocnemius; DIA = diaphragm; TRI = triceps; PSO = PSO.

FIGS. 23A-23B are bar graphs depicting DMD 12 weeks and 24 weeks after treatment with delandistrogene moxeparvovec as compared to normal saline as discussed in example 11 ^mdx The walking (fig. 23A) and vertical movement (fig. 23B) of the rats increased. Movement of the rats in the moving cages (walking and vertical movements) was measured by breakpoints of the laser beam per hour. Each point represents the number of one animal. Data are presented as mean ± SD; * P =p <0.001；**＝p<0.01. Sd=standard deviation.

FIGS. 24A-24B depict the DMD in comparison to normal saline as discussed in example 11 ^mdx Muscle degeneration was significantly reduced by central nucleation analysis in skeletal muscle 12 and 24 weeks after delandistrogene moxeparvovec gene transfer in rats. FIG. 24A depicts hematoxylin and eosin (H)&E) Dyeing. The bar graph of fig. 24B depicts the percentage of fibers with a central core. Bar graph presented as mean ± SD; * P =p<0.0001. Sd=standard deviation.

FIGS. 25A-25B depict analysis of collagen deposition in skeletal muscle and cardiac muscle as discussed in example 11, showing DMD 12 and 24 weeks after treatment with delandistrogene moxeparvovec, as compared to normal saline ^mdx The fibrosis of the rats was reduced. Figure 25A depicts Ma Senmao 12 weeks after treatmentChromosome staining. Fig. 25B provides a bar graph quantifying collagen deposition in skeletal and cardiac muscles 12 and 24 weeks after treatment. Legend: HRT = heart; MG = medial gastrocnemius; DIA = diaphragm. Data are presented as mean ± SD; * P =p<0.0001；*＝p<0.05. Sd=standard deviation.

The bar graph of fig. 26 shows DMD 1 week and 12 weeks after treatment with delandistrogene moxeparvovec as compared to normal saline as discussed in example 11 ^mdx Serum troponin I levels in rat blood did not change significantly. Bar graph represents average + SD. Each point represents the number of one animal.

FIGS. 27A-27C are bar graphs, as discussed in example 11, which analyze DMD as determined by echocardiography 24 weeks after treatment with delandistrogene moxeparvovec as compared to normal saline ^mdx Cardiac function in rats. Fig. 27A depicts left ventricular end-systole diameter (LVESD) data. Fig. 27B depicts data for ejection fraction (%) (EF). Fig. 27C depicts data of a shortening fraction (%) (FS).

Detailed Description

The present disclosure provides gene therapy vectors, e.g., rAAV, that express human microdystrophin, wherein the rAAV is produced in mammalian adherent cells, and wherein the adherent cells are cultured in an N-1 vessel under suspension conditions.

The present disclosure provides a method of producing recombinant adeno-associated virus (rAAV) raavrh74.mhck7. Mini-dystrophin in adherent mammalian cells by a suspension seeding process comprising: (a) Culturing cells in an N-2 vessel with a first growth medium comprising serum; (b) removing the cells from the first medium; (c) Inoculating the cells from step (b) into a second medium in the N-1 vessel that does not contain serum or has a serum concentration lower than the first medium; (d) culturing the cells in the N-1 vessel under suspension conditions; and (e) inoculating the third medium in the bioreactor with cells from step (d).

The disclosure also provides compositions (e.g., pharmaceutical compositions) comprising the rAAV disclosed herein, and methods of treating muscular dystrophy (e.g., DMD) using the compositions disclosed herein.

Muscle biopsies taken at the earliest age of diagnosis of DMD revealed significant connective tissue hyperplasia. Muscle fibrosis has a number of deleterious effects. It reduces normal transport of endomyral nutrients across connective tissue barriers, reduces blood flow, deprives muscles of vascular-derived nutrients, and functionally leads to loss of early walking ability through limb contractures. Over time, significant fibrosis in the muscle can multiply the difficulty of treatment. This can be observed in muscle biopsies by comparing connective tissue proliferation at successive time points. This process continues to deteriorate, causing the patient to lose walking ability and accelerate loss of control, especially in wheelchair-dependent patients.

The benefits of exon skipping, stop codon readthrough or gene replacement therapy cannot be fully realized without early treatment involving simultaneous adoption of methods for reducing fibrosis. Even small molecule or protein replacement strategies are likely to fail if no means of reducing muscle fibrosis is employed. Previous studies demonstrated that treatment of older mdx mice with aav. Mini-dystrophin, in which fibrosis already exists, failed to achieve complete functional recovery (Liu, M et al Mol Ther 11:245-256 (2005)). It is well known that the progression of DMD cardiomyopathy is accompanied by scarring and fibrosis of the ventricular wall.

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

In the present disclosure, the terms "a" or "an" entity refer to one or more/one or more entities; for example, "a polynucleotide" is understood to mean one or more/one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more" and "at least one/at least one" can be used interchangeably herein.

Furthermore, as used herein, "and/or" should be taken as a specific disclosure of each of two specified features or components with or without another feature or component. Thus, the term "and/or" as used in phrases such as "a and/or B" herein is intended to include "a and B", "a or B", "a" (alone) and "B" (alone). Also, the term "and/or" as used in phrases such as "A, B and/or C" is also intended to encompass the following aspects: 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 (alone); b (alone); and C (alone).

The term "about" as used herein refers to approximately, around … …, or within a region thereof. When the term "about" is used in connection with a range of values, it adjusts the range by extending the boundary above and below the value set. Generally, unless otherwise indicated, the term "about" is used herein to adjust a value up or down (higher or lower) by a difference of 10% above or below the stated value.

The term "at least" preceding a number or series of numbers is understood to include the number adjacent to the term "at least" as well as all subsequent numbers or integers that may be logically included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides in a 21 nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" occurs before a series of numbers or a range, it is to be understood that "at least" can modify each of the series of numbers or the range. "at least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% regardless of the number of significant digits).

Unless otherwise specifically indicated, nucleotide sequences herein are indicated from left to right in the 5 'to 3' direction, only in single strands. Nucleotides and amino acids are herein expressed in the manner recommended by the IUPAC-IUB biochemical nomenclature committee, or (for amino acids) in 37CFR ≡1.822 and the intended usage in single letter codes or three letter codes.

As used herein, "polynucleotide" or "nucleic acid" refers to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides herein are expressed in a direction from the 5 'to the 3' direction. The polynucleotides of the present disclosure may be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are herein indicated in single letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U).

The term "polypeptide" as used herein encompasses peptides and proteins unless otherwise indicated.

The term "coding sequence" or sequence "coding" as used herein refers to a region of DNA or RNA (transcribed region) that "encodes" a particular protein (e.g., insulin or glucokinase). The coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide in vitro or in vivo when under the control of appropriate regulatory regions (e.g., promoters). The boundaries of the coding sequence are defined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. The coding sequence may include, but is not limited to, a cDNA of a prokaryote or eukaryote, genomic DNA of a prokaryote or eukaryote, and synthetic DNA sequences. The transcription termination sequence may be located 3' to the coding sequence.

A gene may comprise several operably linked fragments, such as a promoter, 5 'leader, intron, coding sequence, and 3' untranslated sequence, for example, comprising a polyadenylation site or signal sequence. As used herein, "gene expression" refers to the process by which a gene is transcribed into RNA and/or translated into an active protein.

The term "promoter" as used herein refers to a nucleic acid sequence or fragment having the function of controlling transcription of one or more genes (or coding sequences), which is located upstream of the transcription direction of the transcription initiation site of the gene, and which is structurally identified as the presence of a DNA-dependent RNA polymerase binding site, transcription initiation site and any other DNA sequence, including but not limited to transcription factor binding sites, repressor proteins and activator protein binding sites, as well as any other nucleotide sequence known to those of skill in the art that directly or indirectly regulates transcription from a promoter. "constitutive" promoter refers to a promoter that is active under most physiological and developmental conditions. An "inducible" promoter refers to a promoter regulated by physiological or developmental conditions. "tissue-specific" promoters refer to promoters that are preferentially active in a particular type of differentiated cell/tissue.

The term "enhancer" as used herein is a cis-acting element that stimulates or inhibits transcription of an adjacent gene. Enhancers that inhibit transcription are also referred to as "silencers". Enhancers function (e.g., can be associated with a coding sequence) can be up to several kilobase pairs (kb) in either direction from the coding sequence and downstream of the transcribed region.

The term "operably linked" refers to the placement of a regulatory element nucleotide sequence (e.g., a promoter nucleotide sequence) such that the nucleotide sequence is expressed by the regulatory element.

The term "transgene" as used herein refers to a gene (e.g., a small anti-dystrophin protein) or nucleic acid molecule that is introduced into a cell. An example of a transgene is a nucleic acid encoding a therapeutic polypeptide. In some aspects, the gene may be present, but in some cases, the gene is not normally expressed or is under-expressed in the cell. In this context, "deficient" means that the gene is normally expressed in the cell but may develop a condition and/or disease. In certain aspects, the transgene may increase or over-express gene expression. The transgene may comprise a native sequence in the cell, comprise a sequence that does not occur naturally in the cell, or may comprise a combination of both. In certain aspects, a transgene may include sequences operably linked to regulatory sequences suitable for expression of the gene. In certain aspects, the transgene is not integrated into the genome of the host cell.

The term "AAV" as used herein is a standard abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that only grow in cells where some of the functions are provided by co-infectious helper viruses. Thirteen AAV serotypes have been characterized. General information and reviews on AAV can be found, for example, in Carter,1989,Handbook of Parvoviruses, vol.1, pages 169-228, and Berns,1990, virology, pages 1743-1764, raven Press, (New York). However, it is fully anticipated that these same principles will apply to other AAV serotypes, as it is well known that the various serotypes are quite closely related in structure and function, even at the genetic level. (see, e.g., blacklowe,1988,Parvoviruses and Human Disease, pages 165-174, J.R.Pattison, editions; and Rose, comprehensive Virology3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication characteristics mediated by the homologous rep gene; all AAV serotypes carry three related capsid proteins, for example, the capsid protein expressed in AAV 2. Heteroduplex analysis further indicates the extent of association, which reveals extensive cross-hybridization between serotypes over the length of the genome; and the presence of similar self-annealing fragments at the ends corresponding to the "inverted terminal repeats" (ITRs). Similar patterns of infectivity also indicate that replication function of each serotype is similarly regulated.

The term "adeno-associated vector" or "AAV vector" as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes, e.g., microdystrophin), flanked by AAV Inverted Terminal Repeats (ITRs). Such AAV vectors can replicate and package into infectious viral particles when present in host cells transfected with vectors encoding and expressing rep and cap gene products.

The term "AAV virion", "AAV virion" or "AAV vector particle" as used herein refers to a viral particle composed of at least one AAV capsid protein and an AAV vector encapsulating a polynucleotide. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene delivered to a mammalian cell), it is generally referred to as an "AAV vector particle" or simply an "AAV vector". Thus, producing an AAV vector particle necessarily includes producing an AAV vector. Thus, the vector is contained within an AAV vector particle.

The term "muscle-specific control element" refers to a nucleotide sequence that modulates the expression of a coding sequence that is specifically expressed in muscle tissue. These control elements include enhancers and promoters. The present disclosure provides constructs comprising the muscle-specific control element MCKH7 promoter, MCK promoter and MCK enhancer.

By "muscle cells" or "muscle tissue" is meant cells or cell populations from any kind of muscle, such as skeletal and smooth muscle, such as muscle from the digestive tract, bladder, blood vessels or heart tissue. Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiac myoblasts.

The term "transduction" as used herein refers to the administration/delivery of the coding region of a microdystrophin protein to a recipient cell in vivo or in vitro by a replication defective rAAV of the present disclosure such that the recipient cell expresses the microdystrophin protein.

The term "transfection" of a cell as used herein refers to the introduction of genetic material into a cell for the purpose of genetically modifying the cell. Transfection may be accomplished by a variety of methods known in the art (e.g., transduction or electroporation).

As used herein, "vector" refers to a recombinant plasmid or virus comprising a polynucleotide that is delivered into a host cell in vitro or in vivo. "recombinant" means a distinction from what is normally present in nature.

In the case of a vector or viral capsid, "serotype" is defined as a distinct immunological profile based on capsid protein sequence and capsid structure.

"AAV Cap" refers to AAV Cap proteins, VP1, VP2, and VP3, and analogous proteins thereof.

"AAV Rep" refers to AAV Rep proteins and their analogs.

As used herein, "flanking" in reference to a sequence flanked by other elements means that there is one or more flanking elements upstream and/or downstream (i.e., 5 'and/or 3') relative to the sequence. The term "flanking" does not mean that the sequence must be contiguous. For example, intervening sequences may be present between the nucleic acid encoding the transgene and the flanking elements. "flanking" a sequence (e.g., a transgene) by two other elements (e.g., ITRs) means that one element is located at the 5 'end of the sequence and the other element is located at the 3' end of the sequence; however, intervening sequences may exist between the two elements.

The term "gene therapy" as used herein refers to the insertion of a nucleic acid sequence (e.g., a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a transgene (e.g., a microdystrophin) into cells and/or tissues of an individual to treat a disease or condition. Such transgenes may be exogenous. Exogenous molecules or sequences are understood to be molecules or sequences which are not normally present in the cell, tissue and/or individual to be treated.

The term "genotyping" as used herein refers to a process of determining the composition of a particular allele at one or more locations within the genome of a cell and/or subject, for example, by determining the nucleic acid sequence at that location. Genotyping refers to nucleic acid analysis and/or analysis at the nucleic acid level. In some aspects, the human anti-dystrophin gene (DMD) of a subject is genotyped to characterize mutations in the gene that are particularly suitable for treatment with the compositions disclosed herein. Numerous genotyping techniques are known to those skilled in the art.

The term "stringent" refers to conditions that are commonly understood in the art to be stringent. Hybridization stringency is determined primarily by temperature, ionic strength, and concentration of denaturing agents (e.g., formamide). Examples of stringent conditions for hybridization and washing are: 0.015M sodium chloride, 0.0015M sodium citrate, at 65-68℃or 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide at 42 ℃. See Sambrook et al,Molecular Cloning:A Laboratory Manualversion 2, cold Spring Harbor Laboratory, cold Spring Harbor, n.y. (1989). More stringent conditions (e.g., higher temperature, lower ionic strength, higher formamide or other denaturing agents) may also be used, however, the rate of hybridization will be affected. In cases involving deoxyoligonucleotide hybridization, additional exemplary stringent hybridization conditions include hybridization in 6 XSSC 0.05% sodium pyrophosphate at 37℃for 14 bases Is used), at 48℃for 17 base oligonucleotides, at 55℃for 20 base oligonucleotides and at 60℃for 23 base oligonucleotides. To reduce non-specific and/or background hybridization, other reagents may be included in the hybridization and wash buffers. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate NaDodSO4 (SDS), ficoll, denhardt solution, sonicated salmon sperm DNA (or other non-complementary DNA) and dextran sulfate, other suitable reagents may also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are generally carried out at pH6.8-7.4, however, under the usual ionic strength conditions, the hybridization rate is almost pH independent. See Anderson, m.l.m. et al,Nucleic Acid Hybridisation:A Practical Approachchapter 4, IRL Press Limited (Oxford, england) (1998). Hybridization conditions can be adjusted by those skilled in the art to accommodate these variables and to allow DNA of different sequence relatedness to form hybrids.

The terms "culture medium", "cell culture medium", "tissue culture medium" and "growth medium" as used herein refer to a solution containing nutrients that provide nutrients for growing cultured eukaryotic cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements that are required for minimal growth and/or survival of the cells. The solution may also contain growth and/or survival enhancing components, including hormones and growth factors. The pH and salt concentration of the solution are formulated to be most favorable for cell survival and proliferation. The medium may also be a "defined medium" or a "chemically defined medium" -serum-free medium that does not contain proteins, hydrolysates, or components of unknown composition. The defined medium is free of components of animal origin and all components have a known chemical structure. It will be appreciated by those skilled in the art that the defined medium may comprise recombinant glycoproteins or proteins, such as, but not limited to, hormones, cytokines, interleukins and other signaling molecules.

The term "basal medium formulation" or "basal medium" as used herein refers to any cell culture medium that is modified for culturing cells without supplementation or selective removal of certain components.

The terms "culture", "cell culture" and "eukaryotic cell culture" as used herein refer to a population of eukaryotic cells, which may be surface-attached (i.e., adherent) or suspended, maintained in a medium under conditions suitable for survival and/or growth of the cell population. It will be apparent to those of ordinary skill in the art that these terms as used herein may refer to a combination comprising a population of mammalian cells and a medium in which they are suspended.

The term "batch culture" as used herein refers to a method of culturing cells in which all components ultimately used to culture the cells (including the medium and the cells themselves) are provided at the beginning of the culture process. Batch culture is typically stopped at a certain point in time, and then the cells and/or components in the medium are harvested and optionally purified.

The term "fed-batch culture" as used herein refers to a method of culturing cells in which additional components are provided to the culture at some point after the start of the culture process. Fed-batch culture may be started using basal medium. The medium with the additional components provided to the culture at some point after the start of the culture process is a feed medium. The components provided typically comprise cell nutritional supplements that are depleted during the culturing process. Fed-batch culture is usually stopped at a certain point in time, and then the cells and/or components in the medium are harvested and optionally purified.

The term "perfusion culture" as used herein refers to a method of culturing cells in which additional components are provided to the culture continuously or semi-continuously after the start of the culture process. The components provided typically comprise cell nutritional supplements that are depleted during the culturing process. Typically, a portion of the cells and/or components in the medium are harvested on a continuous or semi-continuous basis and optionally purified.

"growth phase" of a cell culture refers to the period in which cells grow exponentially (log phase), where cells typically divide rapidly. At this stage, the cells are cultured under conditions that maximize cell growth for a period of time, typically between 1-4 days. The determination of the growth cycle of a host cell can be determined according to the particular host cell envisioned without undue experimentation. "time period and conditions under which cell growth is maximized" and like terms refer to culture conditions determined to be most favorable for cell growth and division for a particular cell line. In some aspects, during growth, cells are typically cultured at about 25 ℃ to 40 ℃ in a humidified controlled atmosphere in a nutrient medium containing the necessary additives to achieve optimal growth of a particular cell line.

In some aspects, the cells are maintained in the growth phase for a period of about one to seven days, such as two to six days, such as six days. The length of the growth phase of a particular cell can be determined without undue experimentation. For example, the length of the growth phase is the period of time sufficient to allow a particular cell to proliferate to a viable cell density in the range of about 20% -80% of the maximum possible viable cell density if the culture is maintained under growth conditions. In some aspects, "maximum growth rate" refers to the growth rate of a particular cell line/clone measured during its exponential growth phase when the cells are in fresh medium (e.g., measured when the nutrients are abundant during the culture and no significant inhibition of growth by any component of the culture is observed).

The term "cell viability" as used herein refers to the ability of a cultured cell to survive a given set of culture conditions or experimental changes. The term as used herein also refers to the ratio of the fraction of cells that survive at a particular time to the total number of living and dead cells in the then-current culture.

The term "cell density" as used herein refers to the number of cells present in a given volume of medium.

The term "bioreactor" or "culture vessel" as used herein refers to any vessel used for mammalian cell culture. The bioreactor may be of any size as long as it can be used to culture mammalian cells.

The term "bioreactor operation" as used herein may include a lag phase, log phase or plateau growth phase of one or more of the cell culture cycles.

The terms "N-1 culture vessel", "N-1 seeding training culture vessel (N-1 seed-train culture vessel)", "N-1 vessel", "N1-culture" or "N1 vessel" as used herein refer to a culture vessel prior to an N culture vessel (production culture vessel) for growing cell cultures to a high viable cell density for subsequent seeding into the N (production) culture vessel. Cell cultures grown in N-1 culture vessels can be obtained after culturing cells in several vessels (e.g., N-4, N-3, and N-2 vessels) prior to the N-1 culture vessel.

The terms "N-culture vessel", "production culture vessel", "N-bioreactor" or "production bioreactor" as used herein refer to the cell culture in the bioreactor following the N-1 bioreactor. N culture was used to produce AAV.

The term "seeding" or "seeding" as used herein refers to the process of providing a cell culture to a bioreactor or another vessel. In one aspect, the cells have been previously propagated in another bioreactor or vessel. On the other hand, cells have been frozen and thawed immediately prior to being provided to a bioreactor or vessel. The term refers to any number of cells, including single cells.

rAAV and production of rAAV (or compositions comprising rAAV) (e.g., rAAV rh74.MHCK7. Micro-muscular dystrophy) Good protein) method

The present disclosure provides compositions comprising recombinant adeno-associated virus (rAAV) raav.mhck7. Mini-dystrophin, wherein the rAAV is produced in adherent mammalian cells, and wherein the adherent cells are cultured in an N-1 vessel under suspension conditions. In some aspects, the rAAV belongs to serotype aavrh.74 (e.g., raav.mhck7. Minor anti-dystrophin).

The present disclosure also provides a method of producing recombinant adeno-associated virus (rAAV) raavrh74.mhck7. Mini-dystrophin in adherent mammalian cells by a suspension seeding process comprising: (a) Culturing cells in an N-2 vessel with a first growth medium comprising serum; (b) removing the cells from the first medium; (c) Inoculating the cells from step (b) into a second medium in the N-1 vessel that does not contain serum or has a serum concentration lower than the first medium; (d) culturing the cells in the N-1 vessel under suspension conditions; and (e) inoculating the third medium in the bioreactor with cells from step (d).

In some aspects, the rAAV used in the methods described herein comprises the human microdystrophin nucleotide sequence of SEQ ID NO. 1. In some aspects, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO. 7. In some aspects, the rAAV comprises a human microdystrophin nucleotide sequence of SEQ ID NO. 1 and a MHCK7 promoter sequence of SEQ ID NO. 7.

In some aspects, the N-1 container is a suspension shake flask.

In some aspects, a suspension seeding process for producing raavrh74.mhck7. Tiny anti-dystrophin protein in adherent mammalian cells comprises:

(a) Culturing cells in an N-2 vessel with a first growth medium comprising serum;

(b) Removing cells from the first medium;

(c) Inoculating the cells from step (b) into a second medium in the N-1 vessel that does not contain serum or has a serum concentration lower than the first medium;

(d) Culturing cells in an N-1 vessel under suspension conditions;

(e) Inoculating a third medium with cells from step (d) in a bioreactor,

(f) Transfecting cells with a transgenic plasmid comprising a rAAV rh74.MHCK7. Mini-dystrophin construct, a plasmid comprising an AAV rep gene and an AAV cap gene, and an adenovirus helper plasmid,

(g) Lysing cells

(h) The rAAV is purified by at least one column chromatography step.

In some aspects, rAAV is described in international publication No. WO2019/245973A1, which is expressly incorporated herein by reference in its entirety.

Adeno-associated virus (AAV) is a replication-defective parvovirus, whose single-stranded DNA genome is about 4.7kb in length, including an Inverted Terminal Repeat (ITR) of 145 nucleotides. AAV has a variety of serotypes. The nucleotide sequence of the genome of AAV serotypes is known. For example, the nucleotide sequence of AAV serotype 2 (AAV 2) is shown in Srivastava et al, J virol.45:555-564 (1983), corrected by Ruffing et al, J Gen virol.75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided in GenBank accession nc_002077; the complete genome of AAV-3 is provided in GenBank accession NC-1829; the complete genome of AAV-4 is provided in GenBank accession NC_001829; AAV-5 genome is provided in GenBank accession No. AF085716; the complete genome of AAV-6 is provided in GenBank accession nc_00 1862; at least a portion of the AAV-7 and AAV-8 genomes are provided at GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 for AAV-8); AAV-9 genomes are provided in Gao et al, J.Virol.78:6381-6388 (2004); AAV-10 genomes are provided in mol. Ther.,13 (1): 67-76 (2006); AAV-11 genomes are provided in Virology,330 (2): 375-383 (2004). Cloning of AAVrh.74 serotypes is described in Rodino-Klapac et al, journal of Translational Medicine, 5:45 (2007). Cis-acting sequences that direct viral DNA replication (rep), packaging/packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (designated p5, p19 and p40, respectively, by their relative positions on the map) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p 5 and p 19) are coupled to different splices of a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227) such that the rep gene produces four rep proteins (rep 78, rep 68, rep52, and rep 40). Rep proteins have a variety of enzymatic properties and are ultimately responsible for replication of the viral genome. The cap gene is expressed from the p40 promoter, which encodes three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of three related capsid proteins. The single consensus polyadenylation site is located at map position 95 of the AAV genome. AAV lifecycle and genetics are reviewed in Muzyczka, current Topics in Microbiology and Immunology, 158:97-129 (1992).

AAV has unique features that make it promising as a vector for delivering foreign DNA to cells (e.g., in gene therapy). AAV infection of cells in culture is non-cytopathic and silent and asymptomatic to natural infection of humans and other animals. Furthermore, AAV infects many mammalian cells, making it possible to target a variety of different tissues in vivo. Furthermore, AAV slowly transduces dividing and non-dividing cells and can persist throughout the life cycle of these cells as a transcriptionally active nuclear episome (extrachromosomal element). The proviral genome of AAV is infectious when present in a plasmid in the form of cloned DNA, and thus a recombinant genome can be constructed. In addition, since signals directing AAV replication, genome packaging and integration are contained in ITRs of the AAV genome, a portion or all of the approximately 4.3kb inside the genome (encoding replication and structural capsid protein rep-cap) can be replaced with foreign DNA (e.g., a gene cassette containing a promoter, DNA of interest and polyadenylation signals). rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and powerful virus. It can easily withstand the conditions used to inactivate adenoviruses (56 ℃ to 65 ℃ for several hours), so low temperature storage of AAV is not so important. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

Several studies have demonstrated recombinant AAV-mediated long-term (> 1.5 years) protein expression in muscle. See Clark et al, hum Gene Ther 8:659-669 (1997); kessler et al, proc Nat. Acad Sc. USA 93:14082-14087 (1996); and Xiao et al, J Virol 70:8098-8108 (1996). See also Chao et al, mol Ther 2:619-623 (2000) and Chao et al, mol Ther 4:217-222 (2001). Furthermore, since the muscle is highly vascularized, recombinant AAV transduction results in the appearance of transgene products in the systemic circulation following intramuscular injection, as described by Herzog et al Proc Natl Acad Sci USA 94:5804-5809 (1997) and Murphy et al Proc Natl Acad Sci USA 94:13921-13926 (1997). In addition, lewis et al, J Virol.76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess cytokines necessary for proper antibody glycosylation, folding and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics.

The recombinant AAV genome of the present disclosure comprises a nucleic acid molecule of the present disclosure and one or more nucleic acid molecules flanked by AAV ITRs. AAV DNA in the rAAV genome can be from any AAV serotype from which recombinant viruses can be derived, including, but not limited to, AAV serotypes AAVrh.74, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAVs is disclosed, for example, in WO 01/83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, e.g., marsic et al Molecular Therapy (11): 1900-1909 (2014). As described in the background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote skeletal muscle-specific expression, AAV1, AAV6, AAV8, or aavrh.74 may be used.

The DNA plasmids of the present disclosure comprise the rAAV genomes of the present disclosure. The DNA plasmid is transferred into cells capable of infecting an AAV helper virus (e.g., adenovirus, E1-deficient adenovirus, or herpes virus) to assemble the rAAV genome into infectious viral particles. Techniques for producing rAAV particles are standard in the art, in which AAV genome, rep and cap genes to be packaged, and helper virus functions are provided to cells. Production of rAAV requires the presence of the following components in a single cell (denoted herein as packaging cell): rAAV genome, AAV rep and cap genes that are isolated from (i.e., not in) the rAAV genome, and helper virus functions. AAV rep and cap genes can be from any AAV serotype from which recombinant viruses can be derived, and can be from a different AAV serotype other than the rAAV genome ITR, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAVs is disclosed, for example, in WO01/83692, which is incorporated herein by reference in its entirety.

The method of generating packaging cells is to create a cell line that stably expresses all components necessary for AAV particle production. For example, a plasmid (or plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker (e.g., a neomycin resistance gene) is integrated into the genome of the cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al, proc. Natl. Acad. S6.USA 79:2077-2081 (1982)), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al, gene 23:65-73 (1983)) or direct blunt-ended ligation (Senapathy & Carter, J. Biol. Chem.259:4661-4666 (1984)). The packaging cell line is then infected with a helper virus (e.g., adenovirus). The advantage of this approach is that the cells are selective and suitable for large scale production of rAAV. Examples of other suitable methods are the use of adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and/or rep and cap genes into packaging cells.

General principles for producing rAAV are reviewed in, for example, carter, current Opinions in Biotechnology 1533-539 (1992); and Muzyczka, N., curr. Topics Microbiol. Immunol.158:97-129 (1992). Various methods are described in Ratschn et al, mol.cell.biol.4:2072 (1984); hermonat et al, proc.Natl. Acad. Sci.USA,81:6466 (1984); tratschn et al, mo1.cell.biol.5:3251 (1985); mcLaughlin et al, J.Virol.,62:1963 (1988); and Lebkowski et al mol.cell.biol.,7:349 (1988). Samulski et al, J.Virol.,63:3822-3828 (1989); U.S. Pat. nos. 5,173,414; WO95/13365 and corresponding U.S. Pat. No. 5,658.776; WO95/13392; WO96/17947; PCT/US98/18600; WO97/09441 (PCT/US 96/14423); WO 97/08098 (PCT/US 96/13872); WO97/21825 (PCT/US 96/20777); WO97/06243 (PCT/FR 96/01064); WO99/11764; perrin et al, vaccine 13:1244-1250 (1995); paul et al, human Gene Therapy, 4:609-615 (1993); clark et al, gene Therapy3:1124-1132 (1996); U.S. patent No. 5,786,211; U.S. patent No. 5,871,982; and U.S. Pat. No. 6,258,595. The above documents are incorporated herein by reference in their entirety and particularly emphasize the portions of the documents that are relevant to rAAV production.

Thus, the present disclosure provides packaging cells that produce infectious rAAV. In one aspect, the packaging cell may be a stably transformed cancer cell, such as a HeLa cell, 293 cell, and PerC.6 cell (a cognate 293 line). On the other hand, the packaging cells are untransformed cancer cells such as low passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

The recombinant AAV (i.e., infectious packaged rAAV particles) of the present disclosure comprises a rAAV genome. In an exemplary aspect, both rAAV genomes lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between ITRs of the genomes. Examples of rAAV that can be constructed to include the nucleic acid molecules of the present disclosure are described in international patent application No. PCT/US2012/047999 (WO 2013/016352), which is incorporated herein by reference in its entirety.

In one exemplary aspect, the recombinant AAV vectors of the invention are produced by a triple transfection method (Xiao et al, J Virol 72:2224-2232 (1998)) using the AAV vector plasmid rAAV.MHCK7. Microdystrophin, pNLRep2-Caprh74 and pHELP. The rAAV contains a mini-dystrophin gene expression cassette flanked by AAV2 Inverted Terminal Repeats (ITRs). It is this sequence that is encapsulated into AAVrh74 virions. The plasmid contains the core promoter elements of the mini-dystrophin sequence, MHCK7 enhancer and muscle-specific promoter to drive gene expression. The expression cassette also contains the SV40 intron (SD/SA) to facilitate high-level gene expression and bovine growth hormone polyadenylation signals for efficient transcription termination.

pNLREP2-Caprh74 is an AAV helper plasmid that encodes 4 wild-type AAV2rep proteins and 3 wild-type AAV VP capsid proteins from serotype rh 74. A schematic representation of the pNLREP 2-Capra 74 plasmid is shown in FIG. 3.

pHELP adenovirus helper plasmid is 11,635bp, which is obtained from Applied viruses. This plasmid contains the regions of the adenovirus genome important for AAV replication, namely E2A, E ORF6 and VA RNA (adenovirus E1 function is provided by 293 cells). The adenovirus sequence in this plasmid only accounts for about 40% of the adenovirus genome and does not contain cis-elements critical for replication, such as adenovirus terminal repeats. Thus, such production systems are not expected to produce infectious adenoviruses. A schematic representation of the pHELP plasmid is shown in FIG. 4.

The rAAV disclosed herein can be purified by standard methods in the art, for example by column chromatography or cesium chloride gradient methods. Methods for purifying rAAV vectors from helper viruses are known in the art and include those disclosed, for example, in Clark et al, hum. Gene Ther.10 (6): 1031-1039 (1999); schenpp and Clark, methods mol. Med. 69.427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98/09657.

In one aspect, the disclosure provides a rAAV comprising a muscle-specific control element nucleotide sequence and a nucleotide sequence encoding a mini-dystrophin protein. For example, the nucleotide sequence encodes a functional minor anti-dystrophin protein, wherein the nucleotide has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically at least 90%, 91%, 92%, 93% or 94%, even more typically at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 1, wherein the protein retains minor anti-dystrophin activity. The micro-dystrophin protein provides stability to the myomembrane during muscle contraction, e.g., the micro-dystrophin protein acts as a shock absorber during muscle contraction.

In one aspect, the rAAV is raavrh74.mhck7. A mini-dystrophin protein, i.e., the rAAV serotype rh74 capsid encapsulates a viral particle form comprising a nucleic acid expression cassette or genome of a mini-dystrophin transgene driven by an MHCK7 promoter/enhancer, also referred to under the non-proprietary drug name delandistrogene moxeparvovec in the context of administration to a subject. In some aspects, the data (e.g., bar graphs) may be more simply represented as "treated" when referring to a study subject administered delandistrogene moxeparvovec in the examples.

In one aspect, the raavrh74.mhck7. Minor anti-muscular dystrophin protein is raavrh74.mhck7. Minor anti-muscular dystrophin of SEQ ID No. 9, or raavrh74.mhck7. Minor anti-muscular dystrophin of nucleotides 55-5021 of SEQ ID No. 3, nucleotides 1-4977 of SEQ ID No. 8, or nucleotides 56-5022 of SEQ ID No. 6, wherein rAAV is produced in adherent cells, and wherein adherent cells are cultured in N-1 containers under suspension conditions. In one aspect, the rAAV is aavrh74.mck. In one aspect, the raavrh74.mck. Minor anti-muscular dystrophy protein is raavrh74.mck. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID No. 5.

The disclosure also provides rAAV wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID No. 1 or a complement thereof and encodes a functional minor anti-dystrophin protein.

In one aspect, the rAAV is a non-replicating recombinant adeno-associated virus (AAV), which is designated as rAAV having nucleotide numbers 55-5021 of SEQ ID NO:9, nucleotide numbers 55-4977 of SEQ ID NO:8, or nucleotide numbers 56-5022 of SEQ ID NO: 6. MHC 74.MHCK7. Mini-dystrophin, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured in N-1 containers under suspension conditions. The vector genome contains minimal elements required for gene expression, including AAV2 Inverted Terminal Repeats (ITRs), mini-dystrophin, SV40 introns (SD/SA), and synthetic polyadenylation (poly a) signals, all of which are under the control of the MHCK7 promoter/enhancer. A schematic representation of the vector genome and expression cassette is shown in FIG. 1. AAVrh74 serotypes can be used to achieve high efficiency gene transfer in skeletal muscle and cardiac muscle following IV administration.

In one aspect, the disclosure provides a rAAV, wherein the muscle-specific control element is a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer binding factor (MEF), a Muscle Creatine Kinase (MCK), a truncated MCK (tMCK), a Myosin Heavy Chain (MHC), a hybrid α -myosin heavy chain enhancer-/MCK enhancer-promoter (MHCK 7), a C5-12, a murine creatine kinase enhancer element, a skeletal contractile troponin C gene element, a contractile cardiac troponin C gene element, a contractile troponin i gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a Glucocorticoid Response Element (GRE).

For example, the muscle-specific control element is the MHCK7 promoter nucleotide sequence SEQ ID NO. 2 or SEQ ID NO. 7, or the muscle-specific control element is the MCK nucleotide sequence SEQ ID NO. 4. Furthermore, in any rAAV vector of the disclosure, a muscle-specific control element nucleotide sequence (e.g., MHCK7 or MCK nucleotide sequence) is operably linked to a nucleotide sequence encoding a minor anti-dystrophin protein. For example, the MHCK7 promoter nucleotide sequence (SEQ ID NO:2 or SEQ ID NO: 7) is operably linked to a human microdystrophin coding sequence (SEQ ID NO: 1) as set forth in the constructs provided in FIG. 1 or FIG. 2 (SEQ ID NO: 3) or FIG. 13 (SEQ ID NO: 9). In another example, the MCK promoter (SEQ ID NO: 4) is operably linked to a human microdystrophin coding sequence (SEQ ID NO: 1) as shown in the constructs provided in FIG. 5 or FIG. 6 (SEQ ID NO: 5). In another aspect, the disclosure provides rAAV vectors comprising the nucleotide sequences of SEQ ID NO. 1 and SEQ ID NO. 2 or SEQ ID NO. 1 and SEQ ID NO. 7. The disclosure also provides rAAV vectors comprising the nucleotide sequences of SEQ ID NO. 1 and SEQ ID NO. 4.

In yet another aspect, the present disclosure provides a rAAV construct comprised within a plasmid, comprising the nucleotide sequence of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, or SEQ ID NO. 8. For example, AAVrh74.MHCK7. Mini-dystrophin vector comprises the nucleotide sequence within and including the ITR of SEQ ID NO. 3, as shown in FIG. 2. In another aspect, the rAAV vector comprises a 5'itr, an MHCK7 promoter, a chimeric intron sequence, a coding sequence for a human microdystrophin gene, a poly a, and a 3' itr. In one aspect, the vector comprises nucleotides 55-5021 of SEQ ID NO. 3. The plasmid shown in SEQ ID NO. 3 further comprises ampicillin resistance and pGEX plasmid backbone with pBR322 origin of replication.

In another aspect, the disclosure provides a rAAV comprising the nucleotide sequence of SEQ ID NO. 9, wherein the rAAV is produced in an adherent cell, and wherein the adherent cell is cultured in an N-1 vessel under suspension conditions. For example, AAVrh74.MHCK7. Mini-dystrophin vector constructs comprise the nucleotide sequence of SEQ ID NO. 9, as shown in FIG. 13. The rAAV vector construct comprises an MHCK7 promoter, a chimeric intron sequence, a coding sequence of a human micro-dystrophin gene and a poly A. In one aspect, the rAAV vector construct further comprises an ITR at the 5 'end of the promoter and an ITR at the 3' end of the poly a. In one aspect, the rAAV is AAVrh74.

In another aspect, raavrh74.mhck7. The mini-dystrophin vector (i.e., viral vector) comprises a nucleotide sequence within and including the ITR of SEQ ID No. 8, as shown in fig. 15. The rAAV vector comprises a 5'ITR, an MHCK7 promoter, a chimeric intron sequence, a coding sequence of a human micro-dystrophin gene, a poly A and a 3' ITR. In one aspect, the vector comprises nucleotides 1-4977 of SEQ ID NO. 9. The plasmid shown in SEQ ID No. 3 further comprises kanamycin resistance and pGEX plasmid backbone with pBR322 origin of replication.

In another aspect, the present disclosure provides a plasmid comprising an aavrh74.Mhck7. Mini-dystrophin construct. In one aspect, the plasmid comprises a 5'itr, an MHCK7 promoter, a chimeric intron sequence, a coding sequence for a human microdystrophin gene, a poly a, and a 3' itr. In one aspect, the plasmid comprises kanamycin resistance, and optionally comprises a pGEX plasmid backbone with a pBR322 origin of replication. In a particular aspect, the plasmid is as set forth in SEQ ID NO. 8, as shown in FIGS. 14 and 15.

The present disclosure provides recombinant AAV vectors comprising a human microdystrophin nucleotide sequence of SEQ ID No. 1 and a MHCK7 promoter nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 7, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured in an N-1 vessel under suspension conditions. The rAAV vector is AAV serotype AAVrh.74.

The present disclosure also provides a rAAV comprising an AAVrh74.MHCK7. Mini-anti-dystrophin construct nucleotide sequence within and including the ITR of SEQ ID NO. 3, a nucleotide sequence within and including the ITR of SEQ ID NO. 8, or a nucleotide sequence set forth in SEQ ID NO. 9, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured in an N-1 vessel under suspension conditions. The rAAV vector is AAV serotype AAVrh.74.

The rAAV vectors of the present disclosure can be of any AAV serotype, such as serotype aavrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

The present disclosure also provides pharmaceutical compositions (or sometimes referred to herein simply as "compositions") comprising any of the rAAV vectors of the present disclosure.

In another aspect, the disclosure provides a method of producing a rAAV vector particle comprising culturing a cell transfected with any of the rAAV vectors of the disclosure, and recovering the rAAV particle from the supernatant of the transfected cell. The disclosure also provides viral particles comprising any of the recombinant AAV vectors of the disclosure.

Compositions comprising rAAV and administration thereof

In another aspect, the present disclosure contemplates compositions comprising the rAAV of the present disclosure. The compositions of the present disclosure comprise a rAAV and a pharmaceutically acceptable carrier. The composition may also contain other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are non-toxic to the receptor and are preferably inert at the dosages and concentrations employed, including buffers and surfactants such as pluronics.

The present disclosure provides compositions for treating muscular dystrophy (e.g., DMD) in a subject in need thereof comprising recombinant adeno-associated virus (rAAV) raavrh74.mhck7. Mini-anti-muscular dystrophy protein, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured in an N-1 vessel under suspension conditions.

In some aspects, the present disclosure provides compositions comprising recombinant adeno-associated virus (rAAV) raavrh74.mhck7. Mini-dystrophin, wherein the rAAV is produced in adherent mammalian cells by the suspension seeding process described herein. In some aspects, the composition comprises: a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO. 9; b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO. 3; and/or c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO. 8.

The titer of the rAAV administered in the methods of the present disclosure will vary depending on, for example, the particular rAAV, mode of administration, therapeutic target, individual, and cell type targeted, and can be determined by standard methods in the art. The range of rAAV titers may be about 1x10 per milliliter ⁶ About 1x10 ⁷ About 1x10 ⁸ About 1x10 ⁹ About 1x10 ¹⁰ About 1x10 ¹¹ About 1x10 ¹² About 1x10 ¹³ Up to about 1x10 ¹⁴ Or more Dnase Resistant Particles (DRP). The dose may also be expressed in viral genome (vg) units. One exemplary method of determining the genomic titre of an encapsulated vector is to use quantitative PCR, such as that described in (Pozsgai et al, mol. Ther.25:855-869 (2017)).

The present disclosure contemplates methods of transducing target cells with rAAV in vivo or in vitro. The in vivo methods comprise the step of administering to an animal (including a human) in need thereof an effective dose or an effective multiple dose of a composition comprising a rAAV of the present disclosure. If the dosage administered is prior to the occurrence of the condition/disorder, the administration is prophylactic. If the dosage administered is after the condition/disease has occurred, the administration is therapeutic. In various aspects of the disclosure, an effective dose refers to a dose that is capable of alleviating (eliminating or alleviating) at least one symptom associated with the disorder/disease state being treated, slowing or preventing the progression of the disorder/disease state, reducing the extent of the disease, resulting in remission (partial or total) of the disease, and/or prolonging survival. One example of a disease contemplated for prevention or treatment with the methods of the present disclosure is DMD.

The present disclosure also contemplates combination therapies. Combinations as used herein include concurrent and sequential treatments. Combinations of the methods of the present disclosure with standard drug therapies (e.g., corticosteroids) and with new therapies are specifically contemplated.

The effective dose of the composition may be administered by standard routes in the art including, but not limited to, intramuscular, parenteral, intravenous, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal or vaginal. One of skill in the art can select and/or match the route of administration of the rAAV of the present disclosure and the serotypes of AAV components (particularly AAV ITRs and capsid proteins) depending on the infection and/or disease state being treated and the target cells/tissues expressing the mini-dystrophin.

The present disclosure provides methods of locally and systemically administering an effective dose of the rAAV and compositions of the present disclosure. For example, systemic administration refers to administration into the circulatory system, which affects the whole body. Systemic administration includes enteral administration, such as by gastrointestinal absorption, and parenteral administration by injection, infusion or implantation.

In particular, actual administration of the rAAV of the present disclosure may be accomplished by using any physical method that can deliver the rAAV recombinant vector to animal target tissue. Methods of administration according to the present disclosure include, but are not limited to, intramuscular injection and blood injection. Simple resuspension of the rAAV in phosphate buffered saline has proven to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations to the carrier or other components that can be co-administered with the rAAV (although the use of DNA degrading compositions with the rAAV in the normal manner should be avoided). The capsid protein of the rAAV can be modified to enable the rAAV to target a particular target tissue of interest (e.g., muscle). See, for example, WO02/053703, incorporated herein by reference. The pharmaceutical composition may be prepared in injection or topical form for delivery to muscle by transdermal delivery. Many dosage forms for intramuscular injection and transdermal delivery have been previously developed and may be used in the practice of the present disclosure. For ease of administration and manipulation, the rAAV may be used with any pharmaceutically acceptable carrier.

In one aspect of the disclosure, the aavrh74.mhck7. Mini-dystrophin proteins described herein are formulated in a solution containing 20mM Tris (ph 8.0), 1mM magnesium chloride (MgCl) ₂ ) 200mM sodium chloride (NaCl) and 0.001%poloxamer 188.

The dose of rAAV administered in the methods disclosed herein will vary depending upon, for example, the particular rAAV, mode of administration, therapeutic target, individual, and cell type targeted, and can be determined by standard methods in the art. The titer range of each rAAV administered can be about 1x10 per milliliter ⁶ About 1x10 ⁷ About 1x10 ⁸ About 1x10 ⁹ About 1x10 ¹⁰ About 1x10 ¹¹ About 1x10 ¹² About 1x10 ¹³ About 1x10 ¹⁴ About 2x10 ¹⁴ Or about 1x10 ¹⁵ Or more Dnase Resistant Particles (DRP). The dose can also be expressed in viral genome (vg) units (i.e., 1x10, respectively ⁷ vg、1x10 ⁸ vg、1x10 ⁹ vg、1x10 ¹⁰ vg、1x10 ¹¹ vg、1x10 ¹² vg、1x10 ¹³ vg、1x10 ¹⁴ vg、2x10 ¹⁴ vg、1x10 ¹⁵ vg). The dose can also be expressed in units of viral genome (vg) per kilogram (kg) body weight (i.e., 1x10, respectively) ¹⁰ vg/kg、1x10 ¹¹ vg/kg、1x10 ¹² vg/kg、1x10 ¹³ vg/kg、1x10 ¹⁴ vg/kg、1.25x10 ¹⁴ vg/kg、1.5x10 ¹⁴ vg/kg、1.75x10 ¹⁴ vg/kg、2.0x10 ¹⁴ vg/kg、2.25x10 ¹⁴ vg/kg、2.5x10 ¹⁴ vg/kg、2.75x10 ¹⁴ vg/kg、3.0x10 ¹⁴ vg/kg、3.25x10 ¹⁴ vg/kg、3.5x10 ¹⁴ vg/kg、3.75x10 ¹⁴ vg/kg、4.0x10 ¹⁴ vg/kg、1x10 ¹⁵ vg/kg). Methods for AAV titer determinations are described in Clark et al, hum. Gene Ther.,10:1031-1039 (1999).

In particular, actual administration of the rAAV of the present disclosure may be accomplished by using any physical method that can deliver the rAAV recombinant vector to animal target tissue. Administration according to the present disclosure includes, but is not limited to, intramuscular injection and blood injection. Simple resuspension of the rAAV in phosphate buffered saline has proven to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations to the carrier or other components that can be co-administered with the rAAV (although the use of DNA degrading compositions with the rAAV in the normal manner should be avoided). The capsid protein of the rAAV can be modified to enable the rAAV to target a particular target tissue of interest (e.g., muscle). See, for example, WO02/053703, incorporated herein by reference. The pharmaceutical composition may be prepared in injection or topical form for delivery to muscle by transdermal delivery. Many dosage forms for intramuscular injection and transdermal delivery have been previously developed and may be used in the practice of the present disclosure. For ease of administration and manipulation, the rAAV may be used with any pharmaceutically acceptable carrier.

For intramuscular injection purposes, solutions in adjuvants (e.g., sesame or peanut oil) or aqueous propylene glycol, as well as sterile aqueous solutions, may be used. If desired, these aqueous solutions may be buffered and the liquid diluent rendered isotonic with physiological saline or glucose. Solutions of rAAV as free acid (DNA containing acidic phosphate groups) or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant (e.g. hydroxypropylcellulose). The rAAV dispersants may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and oils. Under ordinary conditions of storage and use, these formulations contain a preservative to prevent the growth of microorganisms. In this respect, the sterile aqueous medium used is readily available through standard techniques well known to those skilled in the art.

Pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must have a degree of flow that is easy to inject. It must remain stable under the conditions of manufacture and storage and must be able to prevent the contaminating action of microorganisms (such as bacteria and fungi). The carrier may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size for the dispersion and by the use of surfactants. The action of microorganisms can be prevented by a variety of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. The absorption of the injectable composition may be prolonged by the use of delayed absorbents, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the rAAV in the required amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, the dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Transduction of rAAV can also be performed in vitro. In one aspect, desired target muscle cells are removed from the subject, transduced with rAAV, and then reintroduced into the subject. Alternatively, synthetic or xenogeneic muscle cells may also be used, wherein these cells do not generate undue immune responses in the subject.

Suitable methods of transduction and reintroduction of transduced cells into a subject are known in the art. In one aspect, cells can be transduced by combining rAAV with muscle cells (e.g., in an appropriate medium) and selecting cells that carry DNA of interest using conventional techniques such as Southern blotting and/or PCR or using selectable markers. The transduced cells can then be formulated into pharmaceutical compositions and the compositions introduced into a subject by a variety of techniques, such as by intramuscular injection, intravenous injection, subcutaneous injection, and intraperitoneal injection, or by injection into smooth muscle and myocardium using, for example, catheters.

Transduction of cells with the rAAV of the present disclosure results in sustained expression of tiny dystrophin proteins. Thus, the present disclosure provides methods of administering/delivering rAAV expressing a small anti-dystrophin protein to an animal (preferably a human). These methods include transducing tissue (including, but not limited to, tissues such as muscles, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAV of the present disclosure. Transduction can be performed with a gene cassette comprising a tissue specific control element. For example, one aspect of the present disclosure provides methods of transducing muscle cells and muscle tissue directed by muscle-specific control elements, including but not limited to control elements from the actin and myosin gene families, such as control elements from the myoD gene family (see Weintraub et al, science,251:761-766 (1991)), the muscle Cell-specific enhancer binding factor MEF-2 (Cserjesi and Olson, mol Cell Biol 11:4854-4862 (1991)), control elements from the human skeletal actin gene (Muscat et al, mol Cell Biol,7:4089-4099 (1987)), the cardiac actin gene, the muscle creatine kinase sequence elements (Johnson et al, mol Cell Biol9:3393-3399 (1989)), and the murine creatine kinase enhancer (mCK)), control elements from the skeletal fast troponin C gene, slow cardiac troponin C gene and slow nuclear factor I (1998), and the inducing elements (F-88:563, and the like (1998:563, and the human glucocorticoid-563 (1997, and the like).

Muscle tissue is an attractive target for DNA delivery in vivo because it is not an important organ and is readily accessible. The present disclosure contemplates sustained expression of minute anti-dystrophin proteins from transduced muscle fibers.

Accordingly, the present disclosure provides methods of administering an effective dose (or multiple doses administered substantially simultaneously or at intervals) of a rAAV encoding a mini-anti-dystrophin to a subject in need thereof (e.g., a subject having a muscular dystrophy).

The present disclosure provides nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO 3, 8 or 9. The disclosure also provides a rAAV comprising the nucleic acid sequence of SEQ ID NO:9 or nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 55-5021 of SEQ ID NO:3, and a rAAV particle comprising the nucleic acid sequence of SEQ ID NO:9 or nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 55-5021 of SEQ ID NO:3, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured in a N-1 vessel under suspension conditions.

Another aspect of the disclosure provides a composition comprising a nucleic acid molecule comprising a nucleotide sequence of SEQ ID No. 3, 8, or 9, a rAAV comprising a nucleotide sequence of SEQ ID No. 9 or nucleotides 1-4977 of SEQ ID No. 8 or nucleotides 55-5021 of SEQ ID No. 3, and a rAAV particle comprising a nucleotide sequence of SEQ ID No. 9 or nucleotides 1-4977 of SEQ ID No. 8 or nucleotides 55-5021 of SEQ ID No. 3, wherein the rAAV is produced in an adherent cell, and wherein the adherent cell is cultured in an N-1 vessel under suspension conditions. Any of the methods disclosed herein can be practiced with these compositions.

Mixed inoculation training expansion of adherent cells

Some aspects of the disclosure relate to a cell expansion method comprising: (a) Culturing cells in an N-2 vessel with a first growth medium comprising serum; (b) removing the cells from the first medium; (c) Inoculating the cells from step (b) into a second medium in the N-1 vessel that does not contain serum or has a serum concentration lower than the first medium; (d) culturing the cells in the N-1 vessel under suspension conditions; and (e) inoculating the third medium in the bioreactor with cells from step (d). In one aspect, the second medium is a serum-free medium. In another aspect, the second medium comprises a lower serum concentration than the first medium.

Some aspects of the disclosure relate to an inoculating training expansion method comprising (a) culturing cells in an N-3 container with a first medium comprising serum; (b) removing the cells from the first medium; (c) Inoculating the cells from step (b) in an N-2 vessel to a second medium that is free of serum or has a lower serum concentration than the first medium; (d) culturing the cells in an N-2 vessel under suspension conditions; (e) And inoculating the cells from step (d) into a second medium in an N-1 vessel; and (f) inoculating the third medium in the bioreactor with cells from step (d). In one aspect, the second medium is a serum-free medium. In another aspect, the second medium comprises a lower serum concentration than the first medium.

Some aspects of the present disclosure relate to a method of cell expansion of adherent cells, comprising (a) culturing adherent cells in a first medium comprising serum under adherent conditions; (b) removing adherent cells from the first culture medium; (c) Suspending adherent cells in a second medium that does not contain serum or has a serum concentration lower than the first medium; (d) culturing the adherent cells under suspension conditions; and (e) inoculating the third medium with adherent cells from step (d) in the bioreactor. In some aspects, the method may further comprise passaging the adherent cells from step (a) at least once under adherent conditions. In some aspects, the method may further comprise passaging the adherent cells from step (d) at least once under suspension conditions. In one aspect, the second medium is a serum-free medium. In another aspect, the second medium comprises a serum concentration that is lower than the serum concentration in the first medium in the N-1 container.

The first medium, the second medium, and the third medium may be any medium suitable for the particular cells being cultured. In some aspects, the culture medium contains, for example, inorganic salts, carbohydrates (e.g., sugars such as glucose, galactose, maltose, or fructose), amino acids, vitamins (e.g., B12), vitamin a, vitamin E, riboflavin, thiamine, and biotin), fatty acids and lipids (e.g., cholesterol and steroids), proteins and peptides (e.g., albumin, transferrin, fibronectin, and fetuin), serum (e.g., compositions comprising albumin, growth factors, and growth-inhibitory factors, such as fetal bovine serum, neonatal bovine serum, and horse serum), trace elements (e.g., zinc, copper, selenium, and tricarboxylic acid intermediates), hydrolysates (hydrolyzed proteins from plant or animal sources), and combinations thereof. The growth medium may be a commercially available medium such as 5x concentrated DMEM/F12 (Invitrogen), CD OptiCHO feed (Invitrogen), CD Effect feed (Invitrogen), cell Boost (HyClone), balanCD CHO feed (Irvine Scientific), BD Recharge (Becton Dickinson), cellvento feed (EMD Millipore), ex-Cell CHOZN feed (Sigma-Aldrich), CHO feed bioreactor feed (Sigma-Aldrich), sheffCHO (Kerry), zap-CHO (Invitroa), actiCHO (PAA/GE Healthcare), ham's F10 (Sigma), minimal essential media ([ MEM ], sigma), RPMI-1640 (Sigma) and Dulbecco modified Eagle Medium ([ DMEM ].

In some aspects, the serum-free growth second medium that is free of serum or has a lower serum concentration than the first serum is substantially free (contains no more than trace levels) of calcium ions, fetal Bovine Serum (FBS), fibronectin, collagen, laminin, or proteoglycans or non-proteoglycans that can support cell anchoring in the extracellular matrix. In one aspect, the second medium is a serum-free medium. In another aspect, the second medium comprises a lower serum concentration than the first medium.

In some aspects of the present invention, the pH of the growth medium may be between about 6.5 and about 7.5, between about 6.5 and about 7.4, between about 6.5 and about 7.3, between about 6.5 and about 7.2, between about 6.5 and about 7.1, between about 6.5 and about 7.0, between about 6.5 and about 6.9, between about 6.5 and about 6.8, between about 6.5 and about 6.7, between about 6.6 and about 7.5, between about 6.6 and about 7.4, between about 6.6 and about 7.3, between about 6.6 and about 7.2, between about 6.6 and about 7.1, between about 6.6 and about 7.0, between about 6.6 and about 6.9, between about 6.6 and about 6.8, between about 6.7 and about 7.5, between about 6.7 and about 7.2, between about 6.7 and about 7.1, between about 6.6.6 and about 7.7, between about 7.5 and about 7.2, between about 6.7.7.7, between about 6.5 and about 7.8, between about 6.7.7 and about 7.2, between about 6.6.6.6 and about 7.5, between about 7.8, between about 7.7.5 and about 7.8, between about 7.5 and about 7.5, between about 7.1, between about 6.5 and about 7.5, between about 7.8, between about 6.5 and about 7.8.8, between about 6.8, between about 6.5 and about 7.8.8, between about 6.5 and about 6.8.5, between about 6.5 and about 6.5.

In some aspects, the cells may be cultured at a temperature of between 32 ℃ and about 39 ℃, between about 32 ℃ and about 37 ℃, between about 32 ℃ and about 37.5 ℃, between about 34 ℃ and about 37 ℃, between about 35 ℃ and about 37 ℃, between about 35.5 ℃ and about 37.5 ℃, between about 36 ℃ and about 37 ℃, or about 36.5 ℃. In some aspects, from the beginning to the end of the incubation period, the cells may be incubated at a temperature of about 37 ℃. In some aspects, the temperature of the incubation period may be changed or slightly changed, for example on an hour or day basis. In some aspects, the temperature may be changed or shifted (e.g., increased or decreased) about one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, fourteen, or fifteen days after the start of the incubation period or at any point in time during the incubation period. In some aspects, the temperature may be up-converted by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 ℃. In some aspects, the temperature may be shifted down by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 ℃.

In some aspects, the cell culture may be in a medium containing about 1% to about 15%CO ₂ Is carried out in an atmosphere of (2). In some aspects, a composition containing about 14% CO may be used ₂ 、12％CO ₂ 、10％CO ₂ 、8％CO ₂ 、6％CO ₂ 、5％CO ₂ 、4％CO ₂ 、3％CO ₂ 、2％CO ₂ Or about 1% CO ₂ Is used for culturing cells in the atmosphere.

In some aspects of the present invention, cell culture may be accomplished by maintaining dissolved oxygen (dO 2) in the cell culture between about 3% and about 55%, between about 3% and about 50%, between about 3% and about 45%, between about 3% and about 40%, between about 3% and about 35%, between about 3% and about 30%, between about 3% and about 25%, between about 3% and about 20%, between about 3% and about 15%, between about 5% and about 55%, between about 5% and about 50%, between about 5% and about 45%, between about 5% and about 40%, between about 5% and about 35%, between about 5% and about 30%, between about 5% and about 25%, between about 5% and about 20%, between about 5% and about 15%, between about 5% and about 10%, between about 10% and about 55%, between about 10% and about 50%, between about 10% and about 45%, between about 10% and about 40%, between about between about 10% and about 35%, between about 10% and about 30%, between about 10% and about 25%, between about 10% and about 20%, between about 15% and about 55%, between about 15% and about 50%, between about 15% and about 45%, between about 15% and about 40%, between about 15% and about 35%, between about 15% and about 30%, between about 15% and about 25%, between about 15% and about 20%, between about 20% and about 55%, between about 20% and about 50%, between about 20% and about 45%, between about 20% and about 40%, between about 20% and about 35%, between about 20% and about 30%, between about 20% and about 25%, between about 25% and about 55%, between about 25% and about 50%, between about 25% and about 45%, between about 25% and about 40%, between about 25% and about 35%, between about 25% and about 30%, between about 30% and about 25%, between about, between about 30% and about 55%, between about 30% and about 50%, between about 30% and about 45%, between about 30% and about 40%, between about 30% and about 35%, between about 35% and about 55%, between about 35% and about 50%, between about 35% and about 45%, between about 35% and about 40%, between about 40% and about 55%, between about 40% and about 50%, between about 40% and about 45%, between about 45% and about 55%, between about 45% and about 50%, or between about 50% and about 55%.

In some aspects, the pH of the cell culture may be maintained at a particular pH value by adding an alkaline solution (e.g., an alkaline solution of a base). The pH of the cell culture may be maintained between about 6.5 and about 7.5, between about 6.5 and about 7.4, between about 6.5 and about 7.3, between about 6.5 and about 7.2, between about 6.5 and about 7.1, between about 6.5 and about 7.0, between about 6.5 and about 6.9, between about 6.5 and about 6.8, between about 6.5 and about 6.7, between about 6.6 and about 7.5, between about 6.6 and about 7.4, between about 6.6 and about 7.2, between about 6.6 and about 7.1, between about 6.6 and about 7.0, between about 6.6 and about 6.9, between about 6.6 and about 6.8, between about 6.7 and about 7.4, between about 6.7 and about 7.3, between about 6.7 and about 7.2, between about 6.7.7 and about 7.3, between about 6.6.6 and about 7.2, between about 7.1, between about 6.6.6 and about 7.7.3, between about 6.7.7, between about 7.7.7 and about 7.2, between about 6.6.6 and about 7.8, between about 7.7.7.7, between about 6.7 and about 7.5, between about 7.7.7, between about 6.7 and about 7.8, between about 7.7 and about 7.8, between about 7.6.7, between about 7.5 and about 7.3, between about 7.6.6 and about 7.5, between about 7.6.5 and about 7.8, between about 7.5, between about 6.7 and about 7.5, between about 7.5 and about 7.8.3, between about 7.5 and about 7.8.5, between about and about 7.8.5.8.5.5, between about and about 6.5.5.5.5.5.5.5.5.

In some aspects, cell culture under suspension conditions may be performed in any type of cell culture flask suitable for stable or mixed/shake suspension cell expansion using, for example, a T-flask, roller bottle, or shake bottle; or a combination thereof. In some aspects, the N-1 container is a shake flask.

In some aspects, the suspension conditions may include some form of agitation. In some aspects, the stirring may be rotary stirring. In some aspects of the present invention, the agitation may be between about 25 to about 500, between about 25 and about 480, between about 25 and about 460, between about 25 and about 440, between about 25 and about 420, between about 25 and about 400, between about 25 and about 380, between about 25 and about 360, between about 25 and about 340, between about 25 and about 320, between about 25 and about 300, between about 25 and about 280, between about 25 and about 260, between about 25 and about 240, between about 25 and about 220, between about 25 and about 200, between about 25 and about 180, between about 25 and about 160, between about 25 and about 140, between about 25 and about 120, between about 25 and about 100, between about 25 and about 80, between about 25 and about 60, between about 25 and about 40, between about 25 and about 35, between about 25 and about 30, between about 50 and about 500, between about 50 and about 50, between about 460, between about 50 and about 440, between about 50 and about 420. Between about 50 and about 400, between about 50 and about 380, between about 50 and about 360, between about 50 and about 340, between about 50 and about 320, between about 75 and about 480, between about 75 and about 440, between about 75 and about 420, between about 75 and about 400, between about 75 and about 360, between about 50 and about 200, between about 50 and about 180, between about 50 and about 160, between about 50 and about 140, between about 50 and about 120, between about 50 and about 100, between about 50 and about 80, between about 50 and about 60, between about 75 and about 500, between about 75 and about 480, between about 75 and about 460, between about 75 and about 440, between about 75 and about 420, between about 75 and about 400, between about 75 and about 360, between about 340, between about 75 and about 320, between about 75 and about 280, between about 75 and about 40, between about 75 and about 260 A run-time test is performed between the two steps of the method and the method, between about 240 and about 420, between about 240 and about 400, between about 240 and about 380, between about 240 and about 360, between about 240 and about 340, between about 240 and about 320, between about 240 and about 300, between about 240 and about 280, between about 240 and about 260, between about 260 and about 500, between about 260 and about 480, between about 260 and about 460, between about 260 and about 440, between about 260 and about 420, between about 260 and about 400, between about 260 and about 380, between about 260 and about 360, between about 260 and about 340, between about 260 and about 320, between about 260 and about 300, between about 260 and about 280, between about 280 and about 500, between about 260 and about between about 280RPM and about 480RPM, between about 280RPM and about 460RPM, between about 280RPM and about 440RPM, between about 280RPM and about 420RPM, between about 280RPM and about 400RPM, between about 280RPM and about 380RPM, between about 280RPM and about 360RPM, between about 280RPM and about 340RPM, between about 280RPM and about 320RPM, between about 280RPM and about 280RPM, between about 300RPM and about 500RPM, between about 380RPM and about 480, between about 380RPM and about 460RPM, between about 380RPM and about 440RPM, between about 380RPM and about 420RPM, between about 380RPM and about 400RPM, between about 400RPM and about 500RPM, between about 400RPM and about 480RPM, between about 400RPM and about 460RPM, between about 400RPM and about 440RPM, or between about 400RPM and about 420 RPM. The stirring may be carried out continuously or periodically.

In some aspects, the cells are passaged no more than twice under suspension conditions.

In some aspects, the cells are cultured in suspension culture for about 24 to about 96 hours. In some aspects, the cells are cultured in suspension culture for about 36 to about 84 hours. In some aspects, the cells are cultured in suspension culture for about 48 to 72 hours. In some aspects, the cells are cultured in suspension culture for about 54 to about 66 hours. In some aspects, the cells are cultured in suspension culture for about 24, about 30, about 36, about 42, about 48, about 54, about 60, about 66, about 72, about 78, about 84, about 90, or about 96 hours.

In some aspects of the disclosure, the cell is an adherent cell. In some aspects, the adherent cells are HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, or COS cells. In some aspects, the adherent cells are human cells. In some aspects, the adherent cells are HeLa or HEK-293 cells. In some aspects, the adherent cells are HEK-293 cells.

In some aspects, the adherent cells are not suspension adapted. In some aspects, culturing a cell under suspension conditions does not alter the adherence dependence of the cell. In some aspects, the method does not alter the cells to create a new cell line. The methods disclosed herein do not alter the genomic or transcriptomic characteristics of the cell. The methods disclosed herein do not alter the phenotype of the cell.

In some aspects, cells are passaged multiple times under adherent conditions in serum-supplemented growth medium prior to seeding the N-1 container. In some aspects, cells are cultured in N-2, N-3, N-4, N-5, N-6, N-7, N-8, N-9, or N-10 vessels prior to seeding the N-1 vessels. In some aspects, cells are cultured in N-3 and N-2 vessels. In some aspects, cells are cultured in N-4, N-3, and N-2 vessels.

In some aspects, the bioreactor comprises at least one, more preferably a plurality of carriers to which adherent cells are attached, which carriers may float or be immobilized in the bioreactor. Preferably, the support may be made by using, for example, polyethylene terephthalate, polystyrene, polyester, polypropylene, DEAE-dextran, collagen, glass, alginate or acrylamide. In some aspects, the bioreactor may be a bioreactor containing a bead-type microcarrier (e.g.Brand bead carriers, commercially available from GE Healthcare Inc. of General Electric Corp.) or matrix carriers (e.g., fiber-Cell) ^TM Tablet-shaped carrier, commercially available from Eppendorf corp.). In some aspects, the bioreactor uses a polyester fiber carrier, e.g.)>Nano or->Polyester fiber carriers (commercially available from Advanced Technology Materials inc. (Brussels, belgium) and Pall corporation (Fall River, mass)) used in the 500 bioreactor.

In some aspects, the third medium in the bioreactor comprises at least one factor that promotes cell adhesion. In some aspects, the at least one factor that promotes cell adhesion is selected from proteoglycans or non-proteoglycans polysaccharides of FBS, fibronectin, collagen, laminin, calcium ions, extracellular matrix; and combinations thereof. In some aspects, the at least one factor that promotes cell adhesion may be added to the third medium before, during, or after seeding the suspended cells into the bioreactor.

In some aspects, the growth medium comprises DMEM and about 10% by weight FBS. In some aspects, the growth medium comprises about 2% to about 20% by weight FBS. In some aspects, the growth medium comprises about 3% to about 19% by weight FBS. In some aspects, the growth medium comprises about 4% to about 18% FBS by weight. In some aspects, the growth medium comprises about 5% to about 17% by weight FBS. In some aspects, the growth medium comprises about 6% to about 16% by weight FBS. In some aspects, the growth medium comprises about 7% to about 15% by weight FBS. In some aspects, the growth medium comprises about 8% to about 14% FBS by weight. In some aspects, the growth medium comprises about 9% to about 13% by weight FBS. In some aspects, the growth medium comprises about 10% to about 12% FBS by weight. In some aspects, the growth medium comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% FBS by weight.

In some aspects, suspension expanded cells from step (d) may be inoculated directly into the bioreactor. In some aspects, the number of cells seeded into the bioreactor varies depending on the size of the bioreactor. In some aspects, a 4m2 bioreactor (e.gA nano bioreactor). In some aspects, the 4m2 bioreactor is seeded with about 1x10 ⁸ Up to 1x10 ⁹ Individual cells.In some aspects, the 4m2 bioreactor is inoculated with about 3x10 ⁸ To 7x10 ⁸ Individual cells. In some aspects, the 4m2 bioreactor is seeded with about 4x10 ⁸ To 6x10 ⁸ Individual cells. In some aspects, the 4m2 bioreactor is inoculated with about 5x10 ⁸ Individual cells. In some aspects, equivalent cell densities are used for other sizes of bioreactors.

In some aspects, the methods of the present disclosure may further comprise culturing the cells in a bioreactor. In some aspects, the cell culture comprises batch culture. In some aspects, the cell culture comprises a fed-batch culture. In some aspects, the cell culture comprises perfusion culture.

Fed-batch culture involves the incremental (periodic) or continuous addition of feed medium to the initial cell culture without substantial or significant removal of growth medium from the cell culture. The cell culture in fed-batch culture may be placed in a bioreactor (e.g., a production bioreactor, such as a 10,000-L production bioreactor). In some aspects, the feed medium may be the same as the growth medium. The feed medium may be in liquid or dry powder form. In some aspects, the feed medium is a concentrated form of the growth medium and/or is added in the form of a dry powder. In some aspects, both the first liquid feed medium and the second, different liquid feed medium can be added (e.g., added continuously) to the growth medium. In some aspects, the addition of the first liquid feed medium and the addition of the second liquid feed medium to the culture may begin at about the same time. In some aspects, the total volume of the first liquid feed medium and the second liquid feed medium added to the medium throughout the culturing period may be about the same.

When the feed medium is continuously added, the addition rate of the feed medium may be kept constant or increased (e.g., steadily increased) during the culture period. Continuous addition of feed medium during the incubation period may begin at a particular point in time (e.g., when the cells reach a target viable cell density, e.g., a viable cell density of about 1 x 10) ⁶ Individual cells/mL, about 1.1X10 ⁶ Individual cells/mL, about 1.2X10 ⁶ Individual cells/mL, about 1.3X10 ⁶ Individual cells/mL, about 1.4X10 ⁶ Individual cells/mL, about 1.5X10 ⁶ Individual cells/mL, about 1.6X10 ⁶ Individual cells/mL, about 1.7X10 ⁶ Individual cells/mL, about 1.8X10 ⁶ Individual cells/mL, about 1.9X10 ⁶ Individual cells/mL or about 2.0X10 ⁶ Individual cells/mL). In some aspects, continuous addition of feed medium may begin on day 2, day 3, day 4, or day 5 of the culture period.

In some aspects, when the cells reach a target cell density (e.g., about 1 x 10 ⁶ Individual cells/mL, about 1.1X10 ⁶ Individual cells/mL, about 1.2X10 ⁶ Individual cells/mL, about 1.3X10 ⁶ Individual cells/mL, about 1.4X10 ⁶ Individual cells/mL, about 1.5X10 ⁶ Individual cells/mL, about 1.6X10 ⁶ Individual cells/mL, about 1.7X10 ⁶ Individual cells/mL, about 1.8X10 ⁶ Individual cells/mL, about 1.9X10 ⁶ Individual cells or about 2.0X10 ⁶ Individual cells/mL) may begin to be added incrementally (periodically). In some aspects, the addition of the incremental feed medium may be performed at regular intervals (e.g., daily, every other day, or every third day), or may be performed when the cells reach a particular target cell density (e.g., a target cell density that increases during the culture period). In some aspects, the amount of feed medium added may be gradually increased between a first incremental addition and a subsequent addition of feed medium. In some aspects, the volume of liquid culture feed medium added to the initial cell culture during any 24 hour period of the culture period may be a portion of the initial volume of the bioreactor containing the culture or a portion of the volume of the initial culture.

In some aspects, the addition of liquid feed medium (continuous or periodic) can occur at the following points in time after the start of the incubation period: between 6 hours and 7 days, between about 6 hours and about 6 days, between about 6 hours and about 5 days, between about 6 hours and about 4 days, between about 6 hours and about 3 days, between about 6 hours and about 2 days, between about 6 hours and about 1 day, between about 12 hours and about 7 days, between about 12 hours and about 6 days, between about 12 hours and about 5 days, between about 12 hours and about 4 days, between about 12 hours and about 3 days, between about 12 hours and about 2 days, between about 1 day and about 7 days, between about 1 day and about 6 days, between about 1 day and about 5 days, between about 1 day and about 4 days, between about 1 day and about 3 days, between about 1 day and about 2 days, between about 2 days and about 7 days, between about 2 days and about 6 days, between about 2 days and about 4 days, between about 2 days and about 3 days, between about 3 days and about 6 days, between about 3 days and about 5 days, between about 1 day and about 4 days, between about 4 days and about 5 days.

In some aspects, the volume of liquid feed medium added (continuously or periodically) to the initial cell culture over any 24 hour period can be between 0.01 x and about 0.3 x the capacity of the bioreactor. The portion may be between about 0.01 x and about 0.28 x, between about 0.01 x and about 0.26 x, between about 0.01 x and about 0.24 x, between about 0.01 x and about 0.22 x, between about 0.01 x and about 0.20 x, between about 0.01 x and about 0.18 x, between about 0.01 x and about 0.16 x, between about 0.01 x and about 0.14 x, between about 0.01 x and about 0.12 x, between about 0.01 x and about 0.10 x, between about 0.01 x and about 0.08 x, between about 0.01 x and about 0.06 x about 0.01X and about 0.04X, about 0.02X and about 0.3X, about 0.02X and about 0.28X, about 0.02X and about 0.26X, about 0.02X and about 0.24X, about 0.02X and about 0.22X, about 0.02X and about 0.20X, about 0.02X and about 0.18X, about 0.02X and about 0.16X, about 0.02X and about 0.14X, about 0.02X and about 0.12X, about 0.02X and about 0.10X, about 0.02X and about 0.08X about 0.02X and about 0.06X, about 0.02X and about 0.05X, about 0.02X and about 0.04X, about 0.02X and about 0.03X, about 0.025X and about 0.3X, about 0.025X and about 0.28X, about 0.025X and about 0.26X, about 0.025X and about 0.24X, about 0.025X and about 0.22X, about 0.025X and about 0.20X, about 0.025X and about 0.18X, about 0.025X and about 0.16X, about 0.025X and about 0.14X, about 0.025X and about 0.12X, about 0.025X and about 0.10X, about 0.025X and about 0.08X, about 0.025X and about 0.06X, about 0.025X and about 0.04X, about 0.05X and about 0.3X, about 0.05X and about 0.28X, about 0.05X and about 0.26X and about 0.05X and about 0.26X, about 0.05X and about 0.12X About 0.05 x and about 0.16 x, about 0.05 x and about 0.14 x, about 0.05 x and about 0.12 x, about 0.05 x and about 0.10 x, about 0.1 x and about 0.3 x, about 0.1 x and about 0.28 x, about 0.1 x and about 0.26 x, about 0.1 x and about 0.24 x, about 0.1 x and about 0.22 x, about 0.1 x and about 0.20 x, about 0.1 x and about 0.18 x, about 0.1 x and about 0.16 x, about 0.1 x and about 0.14 x, about 0.1 x, about 0.15 x and about 0.3 x, about 0.15 x and about 0.2 x, about 0.2 x and about 0.3 x, or about 0.25 x and about 0.3 x.

In some aspects, the volume of liquid feed medium added (continuous or periodic) to the initial cell culture during any 24 hour period within the culture period may be between 0.02 x and about 1.0 x of the initial cell culture, between about 0.02 x and about 0.9 x of the initial cell culture, between about 0.02 x and about 0.8 x of the initial cell culture, between about 0.02 x and about 0.7 x of the initial cell culture, between about 0.02 x and about 0.6 x of the initial cell culture, between about 0.02 x and about 0.5 x of the initial cell culture, between about 0.02 x and about 0.4 x of the initial cell culture, between about 0.02 x and about 0.3 x of the initial cell culture, between about 0.02 x and about 0.2 x of the initial cell culture, between about 0.02 x and about 0.06 x of the initial cell culture, between about 0.02 x and about 0.05 x of the initial cell culture, between about 0.02 x and about 0.04 x of the initial cell culture, between about 0.02 x and about 0.03 x of the initial cell culture, between about 0.02 x and about 0.0.0.0.0.0.4 x and about 0.2 x of the initial cell culture, between about 0.02 x and about 0.05 x of the initial cell culture, between about 0.05 x and about 0.0.0.0.0.0.0 x and about 0.0.0 x of the initial cell culture, between about 0.05 x and about 0.0.0.0.0.0 x and about 0.0.0 x of the initial cell culture, between about 0.0.0.0.0.0.0.0.0 x and about 0.0 x and about 0.0.0.0.0.0 x and about.0.0.0.0.0 x of the 0.0 x of the 0.0.0 x of the sample.

In some aspects of the present invention, the total amount of feed medium added (continuously or periodically) throughout the culture period may be between about 1% and about 40% of the volume of the initial culture (e.g., between about 1% and about 35%, between about 1% and about 30%, between about 1% and about 25%, between about 1% and about 20%, between about 1% and about 15%, between about 1% and about 10%, between about 1% and about 5%, between about 1% and about 4%, between about 2% and about 40%, between about 2% and about 35%, between about 2% and about 30%, between about 2% and about 25%, between about 2% and about 20%, between about 2% and about 15%, between about 2% and about 10%, between about 2% and about 5%, between about 3% and about 40%, between about 3% and about 35%, between about 3% and about 30%, between about 3% and about 25%, between about 3% and about 20%, between about 2% and about 35%, between about 2% and about 25%, between about 2% and about 20% >, between about 3% and about 15%, between about 3% and about 10%, between about 3% and about 5%, between about 4% and about 40%, between about 4% and about 35%, between about 4% and about 30%, between about 4% and about 25%, between about 4% and about 20%, between about 4% and about 15%, between about 4% and about 10%, between about 4% and about 8%, between about 5% and about 40%, between about 5% and about 35%, between about 5% and about 30%, between about 5% and about 25%, between about 5% and about 20%, between about 5% and about 15%, between about 5% and about 10%, between about 10% and about 40%, between about 10% and about 35%, between about 10% and about 30%, between about 10% and about 25%, between about 10% and about 20%, between about 10% and about 15%, between about 15% and about 40%, between about 5% and about 30%, between about 25%, between about 5% and about 20%, between about 10% and about 35%, between about 10% and about 10%, between about 10% and about 30%, between about, between about 15% and about 35%, between about 15% and about 30%, between about 15% and about 25%, between about 15% and about 20%, between about 20% and about 40%, between about 20% and about 35%, between about 20% and about 30%, between about 20% and about 25%, between about 25% and about 40%, between about 25% and about 35%, between about 25% and about 30%, between about 30% and about 40%, between about 30% and about 35%, or between about 35% and about 40%.

In some aspects, two different feed media are added (continuously or incrementally) during fed-batch culture. In some aspects, the amount or volume of the first and second feed media added may be substantially the same or may be different. In some aspects, the first feed medium can be in liquid form and the second feed medium can be in solid form. In some aspects, the first feed medium and the second feed medium can be liquid feed media.

Perfusion culturing includes removing a first volume of growth medium from the bioreactor and adding a second volume of second growth medium to the production bioreactor, wherein the first and second volumes are approximately equal. The cells are retained in the bioreactor by cell retention means or by techniques such as cell sedimentation in a sedimentation cone. In some aspects, the removal and addition of growth medium may be performed simultaneously or sequentially, or some combination of the two. In some aspects, the removing and adding may be performed continuously, for example at a rate of between 0.1% and 800%, between 1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%, between 1% and 350%, between 1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and 50%, between 15% and 40%, between 8% and 80%, or between 4% and 30% of the volume of the capacity of the removal and replacement bioreactor.

In some aspects, the first volume of the first growth medium removed and the second volume of the second growth medium added during each 24 hour period may remain approximately the same. In some aspects, the rate at which the first volume of the first growth medium is removed (volume/unit time) and the rate at which the second volume of the second growth medium is added (volume/unit time) can vary and depend on the conditions of the particular cell culture system. In some aspects, the rate of removal of the first volume of the first growth medium (volume/unit time) and the rate of addition of the second volume of the second growth medium (volume/unit time) may be about the same or may be different.

In some aspects, the volume removed and added during each 24 hour period may be changed by gradual increases. In some aspects, during the incubation period, the volume of the first growth medium removed and the volume of the second growth medium added may be increased during each 24 hour period. In some aspects, the volume may be increased by a volume of 0.5% to 20% of the capacity of the bioreactor over a 24 hour period. In some aspects, the volume may be increased to a volume of 25% to 150% of the volume of the bioreactor during the incubation period or to the volume of the first liquid medium during the 24 hour period.

In some aspects, after the first 48 to 96 hours of the incubation period, the first volume of the first growth medium removed and the second volume of the second growth medium added during each 24 hour period is about 10% to about 95%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 85% to about 95%, about 60% to about 80%, or about 70% of the volume of the first growth medium.

In some aspects, the first growth medium and the second growth medium may be the same type of medium. In some aspects, the first growth medium and the second growth medium may be different. In some aspects, the second liquid medium may be more concentrated in one or more medium components.

In some aspects, the first volume of the first growth medium may be removed by using any automated system. In some aspects, alternating tangential flow filtration may be used. In some aspects, the first volume of the first growth medium may be removed by permeation or gravity flow of the first volume of the first growth medium through a sterile membrane having a molecular weight cutoff that excludes cells. In some aspects, the first volume of the first growth medium can be removed by stopping or significantly reducing the agitation rate for at least 1 minute, at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour, and removing or aspirating the first volume of the first growth medium from the top of the production bioreactor.

In some aspects, the second volume of the second liquid medium may be added to the first liquid medium by a pump. In some aspects, the second liquid medium can be added to the first liquid medium manually (e.g., by pipetting or injecting the second volume of the second liquid medium directly into the first liquid medium) or in an automated fashion.

In some aspects, the method further comprises contacting the cell with a first polynucleotide sequence. In some aspects, the method further comprises transfecting the cell with a polynucleotide sequence. In some aspects, the polynucleotide sequence is a plasmid. In some aspects, the plasmid encodes a capsid of a recombinant viral particle selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vaccine virus. In some aspects, the cells are transfected prior to seeding them into the bioreactor. In some aspects, the cells are transfected after they are inoculated into the bioreactor. In some aspects, the cell is contacted or transfected with a second polynucleotide comprising a nucleic acid encoding the transgene. In some aspects, the cells are cultured under conditions that produce the viral vector. In some aspects, the method further comprises isolating the viral vector produced.

In some aspects, the polynucleotide is a viral vector. In some aspects, the viral vectors are adenovirus and adeno-associated virus (AAV) vectors. These vectors can infect a variety of dividing and non-dividing cell types, including synovial cells and liver cells. The episomal nature of adenovirus and AAV vectors after entry into cells makes these vectors suitable for therapeutic use (Russell, j.gen. Virol.81:2573-2604 (2000)); goncalves, virol J.2 (1): 43 2005)), as described above. AAV vectors can achieve very stable long-term expression of transgene expression (up to 9 years in dogs (Niemeyer et al, blood 113 (4): 797-806 (2009)), and up to 2 years in humans (nahwani et al, N Engl J med.365 (25): 2357-2365 (2011); simonelli et al Mol ter.18 (3): 643-50 (2010), epub 2009 dec.1)). In some aspects, the adenovirus vector is modified to reduce host response, as reviewed by Russell (2000, supra). Methods of gene therapy using AAV vectors are described in Wang et al, 2005,J Gene Med.March 9 (Epub prepublished); mandel et al, curr Opin Mol Ther.6:482-90 (2004); martin et al, eye 18:1049-55 (2004); nathwani et al, N Engl J Med.22;365:2357-65 (2011) and Aparailly et al, hum Gene Ther.16 (4): 426-34 (2005).

In some aspects, the first polynucleotide sequence comprises one or more of an inverted terminal repeat, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, a nucleic acid encoding at least one AAV structural capsid protein, or a combination thereof.

In some aspects, the cells are cultured under conditions that produce recombinant viral particles. In some aspects, the method further comprises isolating the recombinant viral particles produced.

In some aspects, the viral vector comprises a transgene operably linked to appropriate regulatory sequences. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of a protein. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, methods in Enzymology 185,Academic Press,San Diego,CA (1990)). In some aspects, the regulatory sequence may comprise a promoter sequence. In some aspects, the promoter sequence may be a Cytomegalovirus (CMV) immediate early promoter, a viral long terminal repeat promoter (LTR) (e.g., a promoter from Murine Moloney Leukemia Virus (MMLV), rous sarcoma virus, or HTLV-1), a simian cavitation virus 40 (SV 40) early promoter, or a herpes simplex virus thymidine kinase promoter.

In some aspects, the viral vector comprises an additional nucleotide sequence encoding an additional polypeptide. In some aspects, the additional polypeptide may be a (selectable) marker polypeptide that can be used to identify, select and/or screen for cells containing a viral vector. In some aspects, the marker polypeptide may be a fluorescent protein GFP, a selectable marker gene HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), tn5 aminoglycoside phosphotransferase (for selection on G418), and dihydrofolate reductase (DHFR) (for selection on methotrexate), CD20, a low affinity nerve growth factor gene. Sources of these marker genes and methods for their use are provided in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3 rd edition), cold Spring Harbor Laboratory, cold Spring Harbor Laboratory Press, new York.

Method for producing viral vectors (e.g., AAV)

Some aspects of the disclosure relate to methods of producing a viral vector (e.g., a rAAV as disclosed herein) comprising expanding cells according to any of the seeding training expansion methods disclosed herein, seeding a growth medium with cells in a bioreactor, transfecting the cells with a polynucleotide sequence encoding a viral particle, and culturing the cells in the bioreactor under conditions that produce the viral particle. In some aspects, the generation of viral vectors is disclosed in U.S. application Ser. No. 63/123,602, which is expressly incorporated herein by reference.

Methods for introducing exogenous nucleic acids into host cells are well known in the art and vary with the host cell used. These techniques include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polyethylenimine-mediated transfection, polygel-mediated transfection, protoplast fusion, electroporation, viral or phage infection, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Transfection may be transient or stable.

In some aspects, the polynucleotide sequence is a plasmid. In some aspects, the plasmid encodes a viral particle from AAV.

In some aspects, the polynucleotide sequence is a viral vector. In some aspects, the viral vector encodes a viral particle. In a preferred aspect, the viral particles are from AAV. In some aspects, the rAAV comprises the nucleic acid sequence of SEQ ID NO. 9. In some aspects, the disclosure provides rAAV particles comprising a nucleic acid sequence of SEQ ID NO. 9. In some aspects, the disclosure provides rAAV comprising nucleotides 55-5021 of SEQ ID NO. 3. In some aspects, the disclosure provides rAAV particles comprising nucleotides 55-5021 of SEQ ID NO. 3. In some aspects, the rAAV comprises nucleotides 1 to 4988 of SEQ ID NO. 8. In some aspects, the disclosure provides rAAV particles comprising nucleotides 1-4977 of SEQ ID NO. 8.

In some aspects, the viral vector is an AAV vector. In some aspects, the AAV vector may comprise a recombinant AAV vector (rAAV). As used herein, "rAAV vector" refers to a recombinant vector comprising a portion of an AAV genome encapsulated in a protein envelope from a capsid protein of an AAV serotype as disclosed herein. In some aspects, an AAV vector can comprise an Inverted Terminal Repeat (ITR) from an adeno-associated virus serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, etc.).

Typically, the vector genome requires the use of flanking 5 'and 3' itr sequences in order to efficiently package the vector genome into the rAAV capsid. In some aspects, the rAAV genome present in the rAAV vector comprises at least the nucleotide sequence of, or substantially the same as, an inverted terminal repeat region (ITR) of one of the AAV serotypes, and a nucleic acid sequence encoding a transgene under the control of a suitable regulatory element (e.g., a promoter), wherein the regulatory element and the modified nucleic acid sequence are interposed between the two ITRs.

The complete genomes of several AAV serotypes and corresponding ITRs have been sequenced (Chiorini et al, J.of Virology 73:1309-1319 (1999)). They can be cloned or made by chemical synthesis methods known in the art, for example using an oligonucleotide synthesizer supplied by, for example, applied Biosystems inc (Fosters, calif., USA) or by standard molecular biology techniques. The ITRs can be cloned from the AAV viral genome or excised from a vector comprising the AAV ITRs. The ITR nucleotide sequence can be ligated to either end of the nucleotide sequence encoding one or more therapeutic proteins using standard molecular biology techniques, or the wild-type AAV sequence between ITRs can be replaced with the desired nucleotide sequence.

In some aspects, the viral capsid component of the packaged viral vector can be a parvoviral capsid, such as an AAV Cap and/or chimeric capsid. Examples of suitable parvoviral viral capsid components are capsid components from the parvoviridae family, such as autonomous parvoviruses or dependent viruses. For example, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAV9, AAV10, AAVRH10, AAV11, or AAV12 capsids; one of skill in the art will recognize that there may be other unidentified variants with the same or similar function), or may comprise components from two or more AAV capsids. All AAV capsid proteins comprise VP1, VP2 and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein may comprise less than a full set of AAV Cap proteins, or a full set of AAV Cap proteins may be provided.

In some aspects, one or more AAV Cap proteins may be chimeric proteins, including the amino acid sequences of AAV caps from two or more viruses (preferably two or more AAV). For example, a chimeric viral capsid can comprise an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. In some aspects, the rAAV genome present in the rAAV vector does not comprise any nucleotide sequence encoding a viral protein, such as the rep (replication) or cap (capsid) genes of AAV. In some aspects, the rAAV genome may further comprise a marker or reporter gene, such as a gene encoding an antibiotic resistance gene, a fluorescent protein (e.g., gfp), or a gene encoding a chemical, enzymatic, or other detectable and/or selectable product known in the art (e.g., lacZ, aph, etc.).

In some aspects, the rAAV genome present in the rAAV vector may further comprise a promoter sequence operably linked to the nucleotide sequence encoding the transgene.

In some aspects, suitable 3' untranslated sequences may also be operably linked to a modified nucleic acid sequence that encodes a transgene. Suitable 3 'untranslated regions may be naturally associated with the nucleotide sequence or may be derived from different genes, such as bovine growth hormone 3' untranslated regions (e.g., bGH polyadenylation signals, SV40 polyadenylation signals, and enhancer sequences).

Unless otherwise indicated, methods known to those of skill in the art can be used to construct recombinant parvoviral and AAV (rAAV) constructs, to package vectors expressing parvoviral Rep and/or Cap sequences, and to transiently and stably transfect packaging cells. These techniques are well known to those skilled in the art. See, e.g., SAMBROOK et al MOLECULAR CLONING: ALABORATORY MANUAL, version 2 (Cold Spring Harbor, n.y., 1989); AUSUBEL et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, inc. and John Wiley Sons, inc., new York).

In some aspects, suitable 3' untranslated sequences may also be operably linked to a nucleic acid sequence that encodes a transgene. Suitable 3 'untranslated regions may be naturally associated with the nucleotide sequence or may be derived from different genes, such as bovine growth hormone 3' untranslated regions (e.g., bGH polyadenylation signals, SV40 polyadenylation signals, and enhancer sequences).

In some aspects, additional nucleotide sequences may be operably linked to the nucleic acid sequence encoding the transgene, e.g., a nucleotide sequence encoding a signal sequence, a nuclear localization signal, an expression enhancer, and the like.

Unless otherwise indicated, methods known to those of skill in the art can be used to construct lentiviral constructs, vectors, and transiently and stably transfected packaging cells. These techniques are well known to those skilled in the art. See, e.g., SAMBROOK et al MOLECULAR CLONING: A LABORATORY MANUAL, version 2 (Cold Spring Harbor, n.y., 1989); AUSUBEL et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, inc. and John Wiley Sons, inc., new York).

In some aspects, methods according to the present disclosure include transfecting cells with a transgenic plasmid comprising raavrh74.mhck7. Mini-dystrophin construct, a plasmid comprising AAV rep gene and AAV cap gene, and an adenovirus helper plasmid. In some aspects, the transgenic plasmid comprising raavrh74.mhck7. The mini-dystrophin construct comprises: the nucleic acid sequence of SEQ ID NO. 9, nucleotides 55-5021 of SEQ ID NO. 3 or nucleotides 1-4977 of SEQ ID NO. 8. In some aspects, the plasmid comprising an AAV rep gene and an AAV cap gene comprises an AAV2 rep gene and a rAAVrh74 cap gene. In some aspects, the adenovirus helper plasmid comprises adenovirus 5E2A, E4ORF6 and a VA RNA gene.

In some aspects, the method further comprises isolating the produced viral particles. Viral vectors replicate in cells, thereby amplifying and producing viral particles. Viral infection results in lysis of transfected cells. Thus, the lytic nature of viral vectors (e.g., AAV) allows for the use of two different modes of viral particle generation and isolation. The first mode is to harvest the virus particles before cell lysis, and to lyse the cells using external factors. The second mode is to harvest virus particles from the supernatant after almost complete lysis of the cells by the virus produced.

Methods which can be used for active cell lysis are known to the person skilled in the art. In some aspects, the cells may be lysed by freeze-thawing, solid shearing, hypertonic and/or hypotonic lysis, liquid shearing, sonication, high pressure extrusion, detergent lysis, combinations of the above, and the like.

In some aspects, the cells may be lysed using at least one detergent. In some aspects, detergents may include anionic, cationic, zwitterionic, and nonionic detergents. In some aspects, the concentration of detergent may be about 0.1% -5% (w/w). In some aspects, the detergent may be Triton X-100.

In some aspects, nucleases can be used to remove contaminating nucleic acids, i.e., native nucleic acids in transfected cells. In some aspects, the nuclease may be Or any other dnase and/or rnase commonly used in the art.

Methods for harvesting or isolating viral vectors from transfected cells have been widely disclosed in WO2005/080556, which is incorporated herein by reference in its entirety.

In some aspects, the time to harvest or isolate the viral vector is between about 24 hours and 120 hours, between about 36 hours and 108 hours, between about 48 hours and about 96 hours, between about 60 hours and about 84 hours after transfection. In some aspects, the time to harvest or isolate the vector is about 72 hours after transfection.

In some aspects, the isolated viral particles may be further purified. In some aspects, purification of the viral particles may be performed in several steps including clarification, ultrafiltration, diafiltration, or chromatographic separation. These methods are described in WO2005/080556, which is incorporated herein by reference in its entirety. In some aspects, clarification may be accomplished by a filtration step to remove cell debris and other impurities from the cell lysate. In some aspects, ultrafiltration is used to concentrate the virus solution. In some aspects, diafiltration, buffer exchange or ultrafiltration may be used to remove and exchange salts, sugars, and the like. The person skilled in the art knows how to find the best conditions for each purification step.

In some aspects, purification can be achieved by density gradient centrifugation. In some aspects, the purification employs at least one chromatography step. In some aspects, the viral vector may be purified by anion exchange chromatography, size exclusion chromatography, or a combination thereof.

Method of treating muscular dystrophy (e.g., DMD)

The present disclosure provides a method of treating muscular dystrophy in a human subject in need thereof comprising the step of administering a recombinant adeno-associated virus (rAAV) raav.mhck7. Mini-anti-muscular dystrophy protein wherein the rAAV is at about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁵ The dose of vg/kg is administered by a systemic route of administration, and wherein the rAAV is produced in mammalian adherent cells, and wherein the adherent cells are cultured in an N-1 vessel under suspension conditions. In some aspects, the muscular dystrophy is duchenne muscular dystrophy or becker muscular dystrophy. In some aspects, the muscular dystrophy is duchenne muscular dystrophy.

The present disclosure also provides a method of treating muscular dystrophy in a human subject in need thereof comprising administering to the humanThe subject administers a composition comprising a rAAV as described herein. In some aspects, the rAAV is at about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁵ The dose of vg/kg is administered by the systemic route of administration. In some aspects, the systemic route of administration is intravenous and the dose of rAAV administered is about 2x10 ¹⁴ vg/kg。

In some aspects, the dose of rAAV administered is at a concentration of about 10 mL/kg. In some aspects, the rAAV is administered by injection, infusion, or implantation. In some aspects, the rAAV is administered by infusion within about one hour. In some aspects, the rAAV is administered via the intravenous route via the external Zhou Zhiti vein.

In some aspects, in methods of treating patients with DMD, the composition is administered to a genotyped patient. In some aspects, the patient is genotyped for the human anti-dystrophin (DMD) gene. In some aspects, the genotyped patient is genotyped for at least one mutation in exons 18-79 of the human anti-dystrophin (DMD) gene.

In some aspects, detection of a frameshift deletion, frameshift repeat, premature termination, or other pathogenic variation that results in non-expression of human anti-dystrophin protein can determine that a subject is suitable for administration of a composition disclosed herein.

In one aspect, at least one mutation in the DMD gene is a mutation in exons 1-17, an in-frame deletion, an in-frame repeat, a vague Variation (VUS), or a mutation entirely contained in exon 45, thereby determining that the subject is unsuitable for administration of the compositions disclosed herein.

The present disclosure also provides for the use of the compositions described herein for treating muscular dystrophy in a human subject in need thereof. In some aspects, the disclosure. The present disclosure also provides the use of a composition as described herein in the manufacture of a medicament for the treatment of muscular dystrophy.

For example, the dose of rAAV administered is about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to about 5.0x10 ¹³ vg/kg, or about 5.0x10 ¹² vg/kg to about 2.0x10 ¹³ vg/kg, or about 5.0x10 ¹² vg/kg to about 1.0x10 ¹³ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 1.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to 1.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 5.0x10 ¹³ vg/kg, or about 1.0x10 ¹³ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 5.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 6.0x10 ¹⁴ vg/kg, or 1.0x10 ¹³ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 5.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 5.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 6.0x10 ¹⁴ vg/kg, or 5.0x10 ¹³ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 6.0x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 5.0x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 4.0x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 1.25x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 1.25x10 ¹⁴ vg/kg to 4.0x10 ¹⁴ Or about 1.25x10 ¹⁴ vg/kg to 1.0x10 ¹⁵ Or about 1.25x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 2.75x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or 1.25x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or 1.5x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or about 1.5x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 1.5x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 1.5x10 ¹⁴ vg/kg to 4.0x10 ¹⁴ Or about 1.5x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 3.25x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 2.75x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or 1.75x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or about 1.75x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 1.75x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 1.75x10 ¹⁴ vg/kg to 4.0x10 ¹⁴ Or about 1.75x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 3.25x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.75x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.25x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to 1.0x10 ¹⁵ Or about 2.0x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 2.0x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 2.0x10 ¹⁴ vg/kg to about 4.0x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to about 3.25x10 ¹⁴ vg/kg。

In some aspects, the human subject is between about 4 years old and less than 8 years old. In some aspects, the human subject is aged about 4, about 4.25, about 4.5, about 4.75, about 5, about 5.25, about 5.5, about 5.75, about 6, about 6.25, about 6.5, about 6.75, about 7, about 7.25, about 7.5, or about 7.75 years old.

In some aspects, the human subject is between about 8 years old and less than 18 years old. In some aspects, the human subject is aged about 8, about 8.25, about 8.5, about 8.75, about 9, about 9.25, about 9.5, about 9.75, about 10, about 10.25, about 10.5, about 10.75, about 11, about 11.25, about 11.5, about 11.75, about 12, about 12.25, about 12.5, about 12.75, about 13, about 13.25, about 13.5, about 13.75, about 14, about 14.5, about 14.75, about 15, about 15.25, about 15.5, about 15.75, about 16, about 16.25, about 16.5, about 16.75, about 17, about 17.25, about 17.5, or about 17.75.

In one aspect, the methods of the disclosure comprise systemically administering a rAAV, wherein the systemic route of administration is intravenous and the dose of the rAAV administered is about 2.0x10 ¹⁴ vg/kg. In another aspect, the methods of the disclosure comprise systemically administering the rAAV, wherein the systemic route of administration is intravenous and the dose of the rAAV administered is about 5.0x10 ¹² vg/kg, or about 6.0x10 ¹² vg/kg, or about 7.0x10 ¹² vg/kg, or about 8.0x10 ¹² vg/kg, or about 9.0x10 ¹² vg/kg, or about 1.0x10 ¹³ vg/kg, or about 1.25x10 ¹³ vg/kg, or about 1.5x10 ¹³ vg/kg, or about 1.75x10 ¹³ vg/kg, or about 2.25x10 ¹³ vg/kg, or about 2.5x10 ¹³ vg/kg, or about 2.75x10 ¹³ vg/kg, or about 3.0x10 ¹³ vg/kg, or about 3.25x10 ¹³ vg/kg, or about 3.5x10 ¹³ vg/kg, or about 3.75x10 ¹³ vg/kg, or about 4.0x10 ¹³ vg/kg, or about 5.0x10 ¹³ vg/kg, or about 6.0x10 ¹³ vg/kg, or about 7.0x10 ¹³ vg/kg, or about 8.0x10 ¹³ vg/kg, or about 9.0x10 ¹³ vg/kg, or about 1.0x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg, or about 2.25x10 ¹⁴ vg/kg, or about 2.5x10 ¹⁴ vg/kg, or about 2.75x10 ¹⁴ vg/kg, or about 3.0x10 ¹⁴ vg/kg, or about 3.25x10 ¹⁴ vg/kg, or about 3.5x10 ¹⁴ vg/kg, or about 3.75x10 ¹⁴ vg/kg, or about 4.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹⁴ vg/kg, or about 6.0x10 ¹⁴ vg/kg, or about 1x10 ¹⁵ vg/kg. In one aspect, the rAAV is aavrh74.mhck7. Mini-dystrophin or aavrh74.mck. Mini-dystrophin. In one aspect, the rAAV is AAVrh74.MHCK7. Mini-dystrophin of nucleotide 55-5021 of SEQ ID NO 9, SEQ ID NO 3, nucleotide 1-4977 of SEQ ID NO 8 or nucleotide 56-5022 of SEQ ID NO 6. In one aspect, the rAAV is AAVrh74.MCK. Mini-dystrophin of nucleotide 56-4820 of SEQ ID NO. 5.

In any of the methods of the disclosure, a dose of rAAV can be administered at about 5mL/kg to about 15mL/kg, or about 8mL/kg to about 12mL/kg, or 8mL/kg to about 10mL/kg, or 5mL/kg to about 10mL/kg, or about 10mL/kg to about 12mL/kg, or about 10mL/kg to 15mL/kg, or 10mL/kg to about 20 mL/kg. In a particular aspect, the dose of rAAV is administered at about 10 mL/kg. In one aspect, the rAAV is aavrh74.mhck7. Mini-dystrophin or aavrh74.mck. Mini-dystrophin. In one aspect, the rAAV is AAVrh74.MHCK7. Mini-dystrophin of nucleotide 55-5021 of SEQ ID NO 9, SEQ ID NO 3, nucleotide 1-4977 of SEQ ID NO 8 or nucleotide 56-5022 of SEQ ID NO 6. In one aspect, the rAAV is AAVrh74.MCK. Mini-dystrophin of nucleotide 56-4820 of SEQ ID NO. 5.

In any of the methods of the disclosure, the dose of rAAV can be administered by injection, infusion, or implantation. For example, a dose of rAAV is administered by infusion within about one hour. In addition, the dose of rAAV is administered via the intravenous route via an external Zhou Zhiti vein (e.g., an external Zhou Beibu vein or an external Zhou Tuibu vein). Alternatively, the infusion may be administered within about 30 minutes, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6. In one aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

The rAAV administered by any method of the disclosure can comprise the human microdystrophin nucleotide sequence of SEQ ID NO. 1, the MHCK7 promoter sequence of SEQ ID NO. 2 or SEQ ID NO. 7. In addition, the rAAV administered by any method of the disclosure includes the human microdystrophin nucleotide sequence of SEQ ID NO. 1 and the MHCK7 promoter sequence of SEQ ID NO. 2 or SEQ ID NO. 7. For example, the rAAV can comprise the AAVrh74.MHCK7. Mini-dystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID NO:9, nucleotide 55-5021 of SEQ ID NO:3, nucleotide 1-4977 of SEQ ID NO:8, or nucleotide 56-5022 of SEQ ID NO: 6. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6.

In one aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

In any of the methods of the disclosure, the rAAV administered is of serotype AAVrh7.4.

In some aspects, the methods of the present disclosure treat duchenne muscular dystrophy or becker muscular dystrophy. An exemplary aspect is the treatment of duchenne muscular dystrophy in a human subject in need thereofA method of malor becker muscular dystrophy comprising the step of administering a dose of recombinant adeno-associated virus (rAAV) raav.mhck7. Mini-anti-muscular dystrophy protein wherein the route of administration is intravenous infusion and the dose of rAAV administered is about 2x10 within about one hour ¹⁴ vg/kg, and wherein the rAAV vector comprises the aavrh74.mhck7. Mini-dystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID No. 9 or SEQ ID No. 3, nucleotide 1-4977 of SEQ ID No. 8, or nucleotide 56-5022 of SEQ ID No. 6. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9 or SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6. In one aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

In any method of treating muscular dystrophy, the level of expression of a minute anti-muscular dystrophy protein gene in cells of the subject is increased following administration of the rAAV. The expression of the microdystrophin genes in cells is detected by measuring the microdystrophin levels by western blotting in muscle biopsies before and after administration of rAAV. In particular, the level of the mini-anti-dystrophin protein after administration of the rAAV is increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90%, as compared to the level of the mini-anti-dystrophin protein prior to administration of the rAAV. For example, the level of the microdystrophin protein after administration of the rAAV is increased by at least about 70% or at least about 71% or at least about 72% or at least about 73% or at least about 74% or at least about 75% or at least about 76% or at least about 77%, or at least about 78% or at least about 79% or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% as compared to the level of the microdystrophin protein prior to administration of the rAAV.

Furthermore, the expression of the microdystrophin gene in cells is detected by immunohistochemical measurement of microdystrophin levels in muscle biopsies before and after administration of rAAV. The level of the mini-anti-dystrophin protein after administration of the rAAV is increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90%, as compared to the level of the mini-anti-dystrophin protein prior to administration of the rAAV. For example, the level of the microdystrophin protein after administration of the rAAV is increased by at least about 70% or at least about 71% or at least about 72% or at least about 73% or at least about 74% or at least about 75% or at least about 76% or at least about 77% or at least about 78% or at least about 79% or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% as compared to the level of the microdystrophin protein prior to administration of the rAAV.

In any method of treating muscular dystrophy, the subject's serum CK levels are reduced after administration of the rAAV as compared to the serum CK levels prior to administration of the rAAV. For example, the subject's serum CK level is reduced by about 65% to about 90% or about 65% to about 95% or about 75% to about 90% or about 80% to about 90% or about 85% to about 95% or about 87% to about 90% compared to the serum CK level prior to administration of the rAAV 60 days after administration of the rAAV. In particular, in any of the methods of the disclosure for treating muscular dystrophy, the serum CK level of the subject is reduced by about 87% compared to the serum CK level of the subject 60 days after administration of the rAAV, or in any of the methods of the disclosure for treating muscular dystrophy, the serum CK level of the subject 60 days after administration of the rAAV is reduced by about 72% compared to the serum CK level of the subject prior to administration of the rAAV, or in any of the methods of the disclosure for treating muscular dystrophy, the serum CK level of the subject 60 days after administration of the rAAV is reduced by about 73% compared to the serum CK level of the subject prior to administration of the rAAV, or in any of the methods of the disclosure for treating muscular dystrophy, the serum CK level of the subject 60 days after administration of the rAAV is reduced by about 78% compared to the serum CK level of the subject 60 days after administration of the rAAV. In any method of treating muscular dystrophy, the amount of micro-dystrophin positive fibers in the muscle tissue of the subject is increased after administration of the rAAV as compared to the amount of micro-dystrophin prior to administration of the rAAV. For example, the number of micro-dystrophin positive fibers is detected by measuring micro-dystrophin levels by western blotting or immunohistochemistry on muscle biopsies before and after administration of rAAV.

In any of the methods of treating muscular dystrophy described herein, the administration of rAAV upregulates the expression of DAPC protein (e.g., α -or β -proteoglycan). For example, the level of α -actin of the subject is increased after administration of the rAAV as compared to the level of α -actin prior to administration of the rAAV. Furthermore, the subject's level of β -actin is increased after administration of the rAAV as compared to the level of β -actin prior to administration of the rAAV. The level of alpha-or beta-proteoglycans is detected by measuring the alpha-or beta-proteoglycan protein level by western blotting or immunohistochemistry on muscle biopsies before and after administration of rAAV.

In any method of treating muscular dystrophy, the disease progression in the subject is delayed following administration of the rAAV, as measured by any of the following methods: six minute walk test, rise time, up 4 steps, up and down 4 steps, arctic walk assessment (North Star Ambulatory Assessment, NSAA), 10 meter time test, 100 meter time test, hand Held Dynamometer (HHD), time up walk and/or gross motor minute test scale (Bayley-III) score.

For example, in any of the methods, the NSAA score of the subject is improved by at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 score compared to the NSAA score of the subject at least 270 days after administration of the rAAV. Moreover, in any of the methods, the rise time of the subject is improved by at least about 0.8 seconds at least 270 days after administration of the rAAV as compared to the rise time prior to administration of the rAAV. Furthermore, in any of the methods, the time of the ascending 4-stage step test of the subject at least 270 days after administration of the rAAV is improved by at least about 1.2 seconds as compared to the time of the ascending 4-stage step test prior to administration of the rAAV. Furthermore, in any of the methods, the 100 meter timing test of the subject is improved by at least about 7 seconds at least 270 days after administration of the rAAV as compared to the 100 meter timing test prior to administration of the rAAV.

In another aspect, the present disclosure provides a method of expressing a microdystrophin gene in cells of a patient comprising administering to the patient the aavrh74.Mhck7 microdystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID No. 9, nucleotide 55-5021 of SEQ ID No. 3, nucleotide 1-4977 of SEQ ID No. 8 or nucleotide 56-5022 of SEQ ID No. 6. For example, the expression of a microdystrophin gene in a patient's cells is detected by measuring the microdystrophin level by western blot or immunohistochemistry in muscle biopsies before and after administration of the raav.mhck7. Microdystrophin construct. In addition, the patient's expression of the microdystrophin gene is measured by detecting a greater number of vector genomes in each cell nucleus, where 1 vector genome in each cell nucleus is about 50% of the microdystrophin expression and greater than 1 copy in each cell nucleus is consistent with the level of microdystrophin expression. For example, a cell has 1.2 vector copies per cell nucleus, or 1.3 vector copies per cell nucleus, or 1.4 vector copies per cell nucleus, or 1.5 vector copies per cell nucleus, or 1.6 vector copies per cell nucleus, or 1.7 vector copies per cell nucleus, or 1.8 vector copies per cell nucleus, or 1.9 vector copies per cell nucleus.

In some aspects, the disclosure provides methods of reducing serum CK levels in a subject in need thereof, the methods comprising administering to the patient the aavrh74.mhck7. Mini-dystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID No. 9, SEQ ID No. 3, nucleotide 1-4977 of SEQ ID No. 8, or nucleotide 56-5022 of SEQ ID No. 6. For example, the serum CK level of a patient is reduced by at least about 65% to about 90% or about 65% to about 95% or about 75% to about 90% or about 80% to about 90% or about 85% to about 95% or about 87% to about 90% 60 days after administration of the rAAV as compared to the serum CK level prior to administration of the rAAV. In particular, the serum CK level of the subject 60 days after administration of the rAAV is reduced by about 87% compared to the serum CK level of the subject prior to administration of the rAAV, or in any method of the disclosure for treating muscular dystrophy, the serum CK level of the subject 60 days after administration of the rAAV is reduced by about 72% compared to the serum CK level prior to administration of the rAAV, or in any method of the disclosure for treating muscular dystrophy, the serum CK level of the subject 60 days after administration of the rAAV is reduced by about 73% compared to the serum CK level of the subject 60 days after administration of the rAAV, or in any method of the disclosure for treating muscular dystrophy, the serum CK level of the subject 60 days after administration of the rAAV is reduced by about 78% compared to the serum CK level of the subject prior to administration of the rAAV.

The present disclosure also provides a method of increasing a microdystrophin positive fiber in muscle tissue of a patient comprising administering to the patient the aavrh74.mhck7 microdystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID No. 9, nucleotide 55-5021 of SEQ ID No. 3, nucleotide 1-4977 of SEQ ID No. 8 or nucleotide 56-5022 of SEQ ID No. 6. For example, the number of tiny anti-dystrophin positive fibers is detected by measuring the level of anti-dystrophin protein by western blot or immunohistochemistry on a muscle biopsy before and after administration of rAAV. In addition, the patient's expression of the microdystrophin gene is measured by detecting a greater number of vector genomes in each cell nucleus, where 1 vector genome in each cell nucleus is about 50% of the microdystrophin expression and greater than 1 copy in each cell nucleus is consistent with the level of microdystrophin expression. For example, a cell has 1.2 vector copies per cell nucleus, or 1.3 vector copies per cell nucleus, or 1.4 vector copies per cell nucleus, or 1.5 vector copies per cell nucleus, or 1.6 vector copies per cell nucleus, or 1.7 vector copies per cell nucleus, or 1.8 vector copies per cell nucleus, or 1.9 vector copies per cell nucleus.

In another aspect, the present disclosure provides a method of increasing expression of α -actin glycans in a patient in need thereof, comprising administering to the patient the aavrh74.mhck7. Mini-dystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID No. 9, SEQ ID No. 3, nucleotide 1-4977 of SEQ ID No. 8, or nucleotide 56-5022 of SEQ ID No. 6. For example, the level of alpha-actin can be detected by measuring the level of alpha-actin protein by western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV.

Furthermore, the present disclosure provides a method of increasing expression of β -actin in a patient in need thereof, comprising administering to the patient the aavrh74.mhck7. Mini-dystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID No. 9, SEQ ID No. 3, nucleotide 1-4977 of SEQ ID No. 8, or nucleotide 56-5022 of SEQ ID No. 6. For example, the level of β -actin can be detected by measuring β -actin protein levels by western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV.

The present disclosure also provides a method of treating a patient suffering from duchenne muscular dystrophy or becker muscular dystrophy comprising administering to the patient the aavrh74.Mhck7. Mini-anti-muscular dystrophy protein construct nucleotide sequence of nucleotides 55-5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 56-5022 of SEQ ID NO:6 such that the disease progression of the patient is delayed as measured by any of the following methods: six minute walk test, rise time, up 4 steps, up and down 4 steps, arctic walk assessment (NSAA), 10 meter time test, 100 meter time test, hand Held Dynamometer (HHD), time to rise walk and/or gross motor minute test scale (Bayley-III) score.

For example, in any of the methods, the NSAA score of the subject is improved by at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 score compared to the NSAA score of the subject at least 270 days after administration of the rAAV. The rise time of the subject is improved by at least about 0.8 seconds at least 270 days after administration of the rAAV as compared to the rise time prior to administration of the rAAV. Furthermore, in any of the methods, the time of the ascending 4-stage step test of the subject at least 270 days after administration of the rAAV is improved by at least about 1.2 seconds as compared to the time of the ascending 4-stage step test prior to administration of the rAAV. Furthermore, in any of the methods, the 100 meter timing test of the subject is improved by at least about 7 seconds at least 270 days after administration of the rAAV as compared to the 100 meter timing test prior to administration of the rAAV.

"fibrosis" refers to the process of abnormal repair of tissues (including skeletal muscle, cardiac muscle, liver, lung, kidney and pancreas) following injury by excessive or uncontrolled deposition of extracellular matrix (ECM) components. The ECM components deposited include fibronectin and collagen, such as collagen 1, collagen 2, or collagen 3.

The disclosure also provides a method of reducing or preventing fibrosis in a subject having a muscular dystrophy comprising administering a therapeutically effective amount of a rAAV comprising a human microdystrophin nucleotide sequence of SEQ ID No. 1 and an MHCK7 promoter nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 7; or an AAVrh74.MHCK7. A rAAV vector comprising the nucleotide sequence of the microdystrophin construct of nucleotide 55-5021 of SEQ ID NO 9, nucleotide 55-4977 of SEQ ID NO 3, nucleotide 1-4977 of SEQ ID NO 8 or nucleotide 56-5022 of SEQ ID NO 6. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-dystrophin protein is AAVrh74.MHCK7. Minor anti-dystrophin protein of nucleotides 55-5021 of SEQ ID NO. 3. In another aspect, the aavrh74.mhck7. Minor anti-muscular dystrophy protein is aavrh74.mhck7. Minor anti-muscular dystrophy protein of SEQ ID No. 9. In another aspect, AAVrh74.MHCK7. Microdystrophin is AAVrh74.MHCK7. Microdystrophin of nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5066 of SEQ ID NO. 6. In yet another aspect, the rAAV is aavrh74.Mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

In another aspect, the present disclosure provides a method of preventing fibrosis in a subject in need thereof comprising administering a therapeutically effective amount of the human microdystrophin nucleotide sequence of SEQ ID No. 1 and the MHCK7 promoter nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 7; or an AAV74.MHCK7. Mini-dystrophin construct nucleotide sequence comprising nucleotide 55-5021 of SEQ ID NO 9, nucleotide 55-4977 of SEQ ID NO 8 or nucleotide 56-5022 of SEQ ID NO 6. For example, any rAAV of the present disclosure may be administered to a subject with muscular dystrophy to prevent fibrosis, e.g., the rAAV of the present disclosure that expresses human minor anti-muscular dystrophy protein is administered before fibrosis in the subject is observed. Furthermore, rAAV of the present disclosure that express human microdystrophin genes can be administered to subjects at risk of developing fibrosis, e.g., subjects suffering from or diagnosed with muscular dystrophy (e.g., DMD). The rAAV of the present disclosure can be administered to subjects suffering from muscular dystrophy to prevent new fibrosis in these subjects.

The present disclosure contemplates administration of rAAV prior to the observation of fibrosis in a subject. Furthermore, the rAAV can be administered to a subject at risk of developing fibrosis, e.g., a subject suffering from or diagnosed with muscular dystrophy (e.g., DMD). rAAV can be administered to patients with muscular dystrophy who have developed fibrosis to prevent new fibrosis in these patients.

The present disclosure also provides a method of increasing muscle strength and/or muscle mass in a subject suffering from muscular dystrophy comprising administering a therapeutically effective amount of the human microdystrophin nucleotide sequence of SEQ ID No. 1 and the MHCK7 promoter nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 7; or AAVrh74.MHCK7 comprising nucleotide sequences of SEQ ID NO 9, nucleotide sequences 55-5021 of SEQ ID NO 3, nucleotide sequences 1-4977 of SEQ ID NO 8 or nucleotide sequences 56-5022 of SEQ ID NO 6.

The present disclosure contemplates administering the rAAV vector to a subject diagnosed with DMD prior to observing fibrosis in the subject or prior to a decrease in muscle strength or prior to a decrease in muscle mass.

The present disclosure also contemplates administering to a subject having muscular dystrophy who has developed fibrosis a human microdystrophin nucleotide sequence of SEQ ID No. 1 and a MHCK7 promoter nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 7; or an rAAV comprising the nucleotide sequence of the aavrh74.mhck7. Mini-dystrophin construct of nucleotide 55-5021 of SEQ ID NO 9, SEQ ID NO 3, nucleotide 1-4977 of SEQ ID NO 8, or nucleotide 56-5022 of SEQ ID NO 6, to prevent new fibrosis or reduce fibrosis in these patients. The present disclosure also provides for administering to a subject with muscular dystrophy having reduced muscle strength or reduced muscle mass a human microanti-muscular dystrophy protein nucleotide sequence of SEQ ID No. 1 and an MHCK7 promoter nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 7; or an rAAV vector comprising the nucleotide sequence of the microdystrophin construct of AAVrh74.MHCK7 of nucleotide 55-5021 of SEQ ID NO 9, SEQ ID NO 3, nucleotide 1-4977 of SEQ ID NO 8 or nucleotide 56-5022 of SEQ ID NO 6 to protect the muscle from further injury.

The present disclosure also provides methods of treating cardiomyopathy in a human subject having muscular dystrophy (e.g., DMD) comprising administering to the human subject any of the compositions (e.g., delandistrogene moxeparvovec) described herein. In some aspects, the methods are for improving cardiac function in a subject with DMD. In some aspects, cardiac function is improved by: for example, increasing Ejection Fraction (EF); increasing the Fractional Shortening (FS); decreasing left ventricular diastolic inner diameter (LVIDd); decreasing Left Ventricular End Systole Diameter (LVESD); and/or maintaining or reducing serum troponin blood levels. In any of the methods of the disclosure, the subject may have muscular dystrophy (e.g., DMD) or any other muscular dystrophy associated anti-muscular dystrophy protein.

In other aspects of any of the methods of the disclosure described herein, the percent level of decrease in serum CK levels in the subject after administration of the rAAV as compared to the serum CK levels prior to administration of the rAAV is selected from the group consisting of:

a) At least 78% 90 days, 180 days, or 270 days after administration;

b) At least 46, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 85% 270 days after administration;

c) At least 72, 73, 74 or 95% 180 days after administration;

d) At least 87%, 88%, 93% or 95% 90 days after administration;

e) At least 70% 270 days after administration;

f) 70% to 95% 90 days, 180 days or 270 days after administration;

g) 90, 180, or 270 days after administration is at least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%; and

h) 90, 180 or 270 days after administration is at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95%.

In another aspect, the present disclosure provides a composition for treating muscular dystrophy in a human subject in need thereof, wherein the composition comprises a dose of recombinant adeno-associated virus (rAAV) raav.mhck7. Mini-anti-muscular dystrophy protein, wherein the composition is formulated for systemic administration route and the dose of rAAV is about 1x10 ¹⁴ vg/kg to about 4x10 ¹⁴ vg/kg. In one aspect, the rAAV is aavrh74.mhck7.In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6. In one aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

For example, compositions of the present disclosure comprise a dose of rAAV of about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to about 5.0x10 ¹³ vg/kg, or about 5.0x10 ¹² vg/kg to about 2.0x10 ¹³ vg/kg, or about 5.0x10 ¹² vg/kg to about 1.0x10 ¹³ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 1.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to 1.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 5.0x10 ¹³ vg/kg, or about 1.0x10 ¹³ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 5.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 6.0x10 ¹⁴ vg/kg, or 1.0x10 ¹³ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 5.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 5.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 6.0x10 ¹⁴ vg/kg, or 5.0x10 ¹³ vg/kg toAbout 1.0x10 ¹⁵ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 6.0x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 5.0x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 4.0x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 1.25x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 1.25x10 ¹⁴ vg/kg to 4.0x10 ¹⁴ Or about 1.25x10 ¹⁴ vg/kg to 1.0x10 ¹⁵ Or about 1.25x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 2.75x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or 1.25x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or 1.5x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or about 1.5x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 1.5x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 1.5x10 ¹⁴ vg/kg to 4.0x10 ¹⁴ Or about 1.5x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 3.25x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 2.75x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or 1.75x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or about 1.75x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 1.75x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 1.75x10 ¹⁴ vg/kg to 4.0x10 ¹⁴ Or about 1.75x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 3.25x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.75x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.25x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to 1.0x10 ¹⁵ Or about 2.0x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 2.0x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 2.0x10 ¹⁴ vg/kg to about 4.0x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to about 3.25x10 ¹⁴ vg/kg. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6. In one aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

In one aspect, the compositions of the present disclosure are formulated for intravenous administration and comprise a dose of rAAV of about 2.0x10 ¹⁴ vg/kg. In another aspect, the compositions of the present disclosure are formulated for intravenous administration and comprise a dose of rAAV of about 5.0x10 ¹² vg/kg, or about 6.0x10 ¹² vg/kg, or about 7.0x10 ¹² vg/kg, or about 8.0x10 ¹² vg/kg, or about 9.0x10 ¹² vg/kg, or about 1.0x10 ¹³ vg/kg, or about 1.25x10 ¹³ vg/kg, or about 1.5x10 ¹³ vg/kg, or about 1.75x10 ¹³ vg/kg, or about 2.25x10 ¹³ vg/kg, or about 2.5x10 ¹³ vg/kg, or about 2.75x10 ¹³ vg/kg, or about 3.0x10 ¹³ vg/kg, or about 3.25x10 ¹³ vg/kg, or about 3.5x10 ¹³ vg/kg, or about 3.75x10 ¹³ vg/kg, or about 4.0x10 ¹³ vg/kg, or about 5.0x10 ¹³ vg/kg, or about 6.0x10 ¹³ vg/kg, or about 7.0x10 ¹³ vg/kg, or about 8.0x10 ¹³ vg/kg, or about 9.0x10 ¹³ vg/kg, or about 1.0x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg, or about 2.25x10 ¹⁴ vg/kg, or about 2.5x10 ¹⁴ vg/kg, or about 2.75x10 ¹⁴ vg/kg, or about 3.0x10 ¹⁴ vg/kg, or about 3.25x10 ¹⁴ vg/kg, or about 3.5x10 ¹⁴ vg/kg, or about 3.75x10 ¹⁴ vg/kg, or about 4.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹⁴ vg/kg, or about 6.0x10 ¹⁴ vg/kg, or about 1x10 ¹⁵ . In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8, nucleotides 56-5022 of SEQ ID NO. 6. In another aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

In any composition of the disclosure, the dose of rAAV is delivered at about 5mL/kg to about 15mL/kg, or about 8mL/kg to about 12mL/kg, or 8mL/kg to about 10mL/kg, or 5mL/kg to about 10mL/kg, or about 10mL/kg to 12mL/kg, or about 10mL/kg to 15mL/kg, or 10mL/kg to about 20 mL/kg. In a particular aspect, the composition comprises a rAAV delivered at a dose of about 10 mL/kg. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6. In another aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

The compositions of the present disclosure are formulated for administration by injection, infusion or implantation. For example, the composition is formulated for administration by infusion within about one hour. Further, the compositions of the present disclosure are formulated for intravenous administration via an external Zhou Zhiti vein (e.g., an external Zhou Beibu vein or an external Zhou Tuibu vein). Alternatively, the infusion may be administered within about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

Any composition of the disclosure comprises a rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID No. 1 and the MHCK7 promoter sequence of SEQ ID No. 2 or SEQ ID No. 7 or a rAAV vector comprising the aavrh74.MHCK7. Microdystrophin construct nucleotide sequence of nucleotides 55-5021 of SEQ ID No. 9, SEQ ID No. 3, nucleotides 1-4977 of SEQ ID No. 8 or nucleotides 56-5022 of SEQ ID No. 6.

In particular, the compositions of the present disclosure are useful for treating duchenne muscular dystrophy or becker muscular dystrophy. For example, the present disclosure provides a composition for treating duchenne muscular dystrophy or becker muscular dystrophy in a human subject in need thereof, wherein the composition comprises a dose of recombinant adeno-associated virus (rAAV) raav.mhck7. Mini-anti-muscular dystrophy protein, wherein the composition is formulated for administration by intravenous infusion within about one hour, and the dose of rAAV administered is about 2x10 ¹⁴ vg/kg, and wherein the rAAV comprises the AAVrh74.MHCK7. Mini-dystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotide 1-4977 of SEQ ID NO:8, or nucleotide 56-5022 of SEQ ID NO: 6.

In another aspect, the disclosure also provides a composition comprising a rAAV for use in reducing fibrosis in a subject in need thereof. In addition, the present disclosure also provides compositions comprising rAAV vectors for preventing fibrosis in a subject having muscular dystrophy.

The present disclosure also provides compositions comprising rAAV for increasing muscle strength and/or muscle mass in a subject with muscular dystrophy. In yet another aspect, the disclosure provides a composition comprising any rAAV of the disclosure for use in treating muscular dystrophy.

In other aspects of any of the compositions of the present disclosure, the percent level of decrease in serum CK levels in a subject after administration of the composition to a human subject in need of treatment for muscular dystrophy, as compared to the serum CK levels prior to administration of the composition, is selected from the group consisting of:

a) At least 78% 90 days, 180 days, or 270 days after administration;

b) At least 46%, 55%, 70% or 85% 270 days after administration;

c) At least 72%, 73%, 74% or 95% 180 days after administration;

d) At least 87%, 99%, 93% or 95% 90 days after administration;

e) At least 70% 270 days after administration;

f) 70% to 95% 90 days, 180 days or 270 days after administration;

g) 90, 180, or 270 days after administration is at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%; and

h) The amount of the compound is 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% 90, 180 or 270 days after administration.

In another aspect, the present disclosure provides the use of a dose of recombinant adeno-associated virus (rAAV) raav.mhck7. Mini-anti-dystrophin for the manufacture of a medicament for treating muscular dystrophy in a human subject in need thereof, wherein the medicamentFormulated for systemic administration route and dose of rAAV is about 1x10 ¹⁴ vg/kg to about 4x10 ¹⁴ vg/kg. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6. In one aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

For example, the drug comprises a dose of rAAV of about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹² vg/kg to about 5.0x10 ¹³ vg/kg, or about 5.0x10 ¹² vg/kg to about 2.0x10 ¹³ vg/kg, or about 5.0x10 ¹² vg/kg to about 1.0x10 ¹³ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 1.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to 1.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 5.0x10 ¹³ vg/kg, or about 1.0x10 ¹³ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 5.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹³ vg/kg to about 6.0x10 ¹⁴ vg/kg, or 1.0x10 ¹³ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 5.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 1.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 5.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹³ vg/kg to about 6.0x10 ¹⁴ vg/kg, or 5.0x10 ¹³ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 6.0x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 5.0x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 4.0x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.0x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or 1.0x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 1.25x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 1.25x10 ¹⁴ vg/kg to 4.0x10 ¹⁴ Or about 1.25x10 ¹⁴ vg/kg to 1.0x10 ¹⁵ Or about 1.25x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 2.75x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or 1.25x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or 1.5x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or about 1.5x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 1.5x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 1.5x10 ¹⁴ vg/kg to 4.0x10 ¹⁴ Or about 1.5x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 3.25x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 2.75x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or 1.75x10 ¹⁴ vg/kg to about 1.0x10 ¹⁵ vg/kg, or about 1.75x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 1.75x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 1.75x10 ¹⁴ vg/kg to 4.0x10 ¹⁴ Or about 1.75x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 3.25x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 3.0x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.75x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.5x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.25x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg to about 2.0x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to 1.0x10 ¹⁵ Or about 2.0x10 ¹⁴ vg/kg to 6.0x10 ¹⁴ Or about 2.0x10 ¹⁴ vg/kg to 5.0x10 ¹⁴ Or about 2.0x10 ¹⁴ vg/kg to about 4.0x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to about 3.75x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to about 3.5x10 ¹⁴ vg/kg, or about 2.0x10 ¹⁴ vg/kg to about 3.25x10 ¹⁴ vg/kg. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6. In one aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

In one aspect, the medicament of the present disclosure is formulated for systemic administration of a dose of rAAV, wherein the systemic route of administration is intravenous route and the dose of rAAV administered is about 2.0x10 ¹⁴ vg/kg. In another aspect, the medicament of the present disclosure is formulated for systemic administration of a dose of rAAV, wherein the systemic route of administration is intravenous route and the dose of rAAV is about5.0x10 ¹² vg/kg, or about 6.0x10 ¹² vg/kg, or about 7.0x10 ¹² vg/kg, or about 8.0x10 ¹² vg/kg, or about 9.0x10 ¹² vg/kg, or about 1.0x10 ¹³ vg/kg, or about 1.25x10 ¹³ vg/kg, or about 1.5x10 ¹³ vg/kg, or about 1.75x10 ¹³ vg/kg, or about 2.25x10 ¹³ vg/kg, or about 2.5x10 ¹³ vg/kg, or about 2.75x10 ¹³ vg/kg, or about 3.0x10 ¹³ vg/kg, or about 3.25x10 ¹³ vg/kg, or about 3.5x10 ¹³ vg/kg, or about 3.75x10 ¹³ vg/kg, or about 4.0x10 ¹³ vg/kg, or about 5.0x10 ¹³ vg/kg, or about 6.0x10 ¹³ vg/kg, or about 7.0x10 ¹³ vg/kg, or about 8.0x10 ¹³ vg/kg, or about 9.0x10 ¹³ vg/kg, or about 1.0x10 ¹⁴ vg/kg, or about 1.25x10 ¹⁴ vg/kg, or about 1.5x10 ¹⁴ vg/kg, or about 1.75x10 ¹⁴ vg/kg, or about 2.25x10 ¹⁴ vg/kg, or about 2.5x10 ¹⁴ vg/kg, or about 2.75x10 ¹⁴ vg/kg, or about 3.0x10 ¹⁴ vg/kg, or about 3.25x10 ¹⁴ vg/kg, or about 3.5x10 ¹⁴ vg/kg, or about 3.75x10 ¹⁴ vg/kg, or about 4.0x10 ¹⁴ vg/kg, or about 5.0x10 ¹⁴ vg/kg, or about 6.0x10 ¹⁴ vg/kg, or about 1x10 ¹⁵ vg/kg. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6. In one aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

In any use of the disclosure, the medicament comprises a dose of rAAV of about 5mL/kg to about 15mL/kg, or about 8mL/kg to about 12mL/kg, or 8mL/kg to about 10mL/kg, or 5mL/kg to about 10mL/kg, or about 10mL/kg to about 12mL/kg, or about 10mL/kg to about 15mL/kg, or 10mL/kg to about 20mL/kg. In a particular aspect, the dose or rAAV is about 10mL/kg. In one aspect, the rAAV is aavrh74.mhck7. In one aspect, the AAVrh74.MHCK7. Minor anti-muscular dystrophy protein is AAVrh74.MHCK7. Minor anti-muscular dystrophy protein of nucleotides 55-5021 of SEQ ID NO. 9, SEQ ID NO. 3, nucleotides 1-4977 of SEQ ID NO. 8 or nucleotides 56-5022 of SEQ ID NO. 6. In one aspect, the rAAV is aavrh74.mck. In one aspect, the AAVrh74.MCK. Minor anti-muscular dystrophy protein is AAVrh74.MCK. Minor anti-muscular dystrophy protein of nucleotides 56-4820 of SEQ ID NO. 5.

In any use of the present disclosure, the medicament is formulated for administration by injection, infusion or implantation. For example, the medicament is formulated for administration by infusion within about one hour. In addition, the medicament is formulated for intravenous administration via an external Zhou Zhiti vein (e.g., an external Zhou Beibu vein or an external Zhou Tuibu vein). Alternatively, the infusion may be administered within about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

In any use of the disclosure, the medicament comprises a rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID No. 1 and the MHCK7 promoter sequence of SEQ ID No. 2 or SEQ ID No. 7 or the aavrh74.mhck7. Microdystrophin construct nucleotide sequence of SEQ ID No. 9, nucleotides 55-5021 of SEQ ID No. 3, nucleotides 1-4977 of SEQ ID No. 8 or nucleotides 56-5022 of SEQ ID No. 6.

A particular use of the present disclosure is in the manufacture of a medicament for the treatment of duchenne muscular dystrophy or becker muscular dystrophy. For example, the present disclosure provides the use of a dose of recombinant adeno-associated virus (rAAV) raav.mhck7. Mini-anti-dystrophin for the manufacture of a medicament for treating duchenne muscular dystrophy or becker muscular dystrophy in a human subject in need thereof, wherein the medicament is formulated for administration by intravenous infusion within about one hour, and the dose of rAAV administered is about 2x10 ¹⁴ vg/kg, and wherein the rAAV comprises AAVrh74.MHCK7 of nucleotides 55-5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 56-5022 of SEQ ID NO: 6.A mini-dystrophin construct nucleotide sequence.

In yet another aspect, the present disclosure provides use of a rAAV for the manufacture of a medicament for reducing fibrosis in a subject in need thereof. For example, a subject in need thereof may have muscular dystrophy, such as DMD or any other anti-muscular dystrophy protein-associated muscular dystrophy.

In another aspect, the present disclosure provides use of a rAAV for the manufacture of a medicament for preventing fibrosis in a subject having muscular dystrophy.

Furthermore, the present disclosure also provides the use of a rAAV for the manufacture of a medicament for increasing muscle strength and/or muscle mass in a subject suffering from muscular dystrophy.

The disclosure also provides for the use of a rAAV for the preparation of a medicament for the treatment of muscular dystrophy.

The present disclosure provides the use of a rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID No. 1 and the MHCK7 promoter nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 7 for the manufacture of a medicament for treating muscular dystrophy, or the use of a rAAV vector comprising the nucleotide 55-5021 of SEQ ID No. 9, SEQ ID No. 3, nucleotide 1-4977 of SEQ ID No. 8 or nucleotide 56-5022 of SEQ ID No. 6 for the manufacture of a rAAV vector of the microdystrophin construct nucleotide sequence for treating muscular dystrophy.

In other aspects of any use of the disclosure, the percent level of decrease in serum CK levels in a subject after administration of an rAAV to the subject as compared to the serum CK levels prior to administration of the rAAV is selected from the group consisting of:

a) At least 78% 90 days, 180 days, or 270 days after administration;

b) At least 46%, 55%, 70% or 95% 270 days after administration;

c) At least 72%, 73%, 74% or 95% 180 days after administration;

d) At least 87%, 88%, 93% or 95% 90 days after administration;

e) At least 70% 270 days after administration;

f) 70% to 95% 90 days, 180 days or 270 days after administration;

h) The amount of the composition is 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% at 90 days, 180 days or 270 days after administration.

In any composition for treating muscular dystrophy or any use of a medicament for treating muscular dystrophy, the level of micro-dystrophy protein gene expression in the cells of the subject increases after administration of the composition or medicament. The expression of the microdystrophin gene in the cells is detected by measuring the microdystrophin level by western blot in muscle biopsies before and after administration of the composition or drug. In particular, the level of the mini-dystrophin protein after administration of the composition or the drug is increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90%, as compared to the level of the mini-dystrophin protein prior to administration of the composition or the drug. For example, the level of the microdystrophin protein is increased by at least about 70% or at least about 71% or at least about 72% or at least about 73% or at least about 74% or at least about 75% or at least about 76% or at least about 77% or at least about 78% or at least about 79% or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the composition as compared to the level of the microdystrophin protein prior to administration of the composition or the drug.

Furthermore, the expression of the microdystrophin gene in the cells is detected by measuring the microdystrophin level by immunohistochemistry in a muscle biopsy before or after administration of the composition or drug. The level of the mini-anti-dystrophin protein after administration of the rAAV is increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90%, as compared to the level of the mini-anti-dystrophin protein prior to administration of the composition or drug. For example, the level of the microdystrophin protein is increased by at least about 70% or at least about 71% or at least about 72% or at least about 73% or at least about 74% or at least about 75% or at least about 76% or at least about 77% or at least about 78% or at least about 79% or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the composition or the drug as compared to the level of the microdystrophin protein prior to administration of the composition or the drug.

In any composition for treating muscular dystrophy, the subject's serum CK level is reduced after administration of the rAAV as compared to the serum CK level prior to administration of the composition or drug. For example, the subject's serum CK level is reduced by about 65% to about 90% or about 65% to about 95% or about 75% to about 90% or about 80% to about 90% or about 85% to about 95% or about 87% to about 90% 60 days after administration of the composition or drug as compared to the serum CK level prior to administration of the composition or drug. In particular, in any composition of the present disclosure in which the serum CK level of the subject is reduced by about 87% after administration of the composition or drug compared to the serum CK level of the subject 60 days after administration of the composition or drug, or in any use of the composition of the present disclosure in which the serum CK level of the subject 60 days after administration of the composition or drug is reduced by about 72% after administration of the composition or drug compared to the serum CK level of the subject 60 days after administration of the composition or drug, or in any composition of the present disclosure in which the serum CK level of the subject 60 days after administration of the composition or drug is reduced by about 73% after administration of the composition or drug compared to the serum CK level of the subject 60 days before administration of the composition or drug. In any use of any composition or medicament for treating muscular dystrophy, the number of tiny anti-dystrophin positive fibers in the muscle tissue of a subject after administration of the composition or medicament is increased as compared to the number of tiny anti-dystrophin positive fibers prior to administration of the composition or medicament. For example, the number of micro-dystrophin positive fibers is detected by measuring the micro-dystrophin level by western blot or immunohistochemistry on a muscle biopsy before and after administration of the composition or drug.

In any composition for treating muscular dystrophy or any use of a medicament for treating muscular dystrophy, administration of the composition or medicament upregulates the expression of DAPC protein (e.g., alpha-or beta-proteoglycans). For example, the level of alpha-actin is increased in the subject after administration of the composition or the drug as compared to the level of alpha-actin prior to administration of the composition or the drug. Furthermore, the subject's level of β -actin is increased after administration of the composition or medicament as compared to the level of β -actin prior to administration of the composition or medicament. The level of alpha-or beta-actin-glycans is detected by measuring the alpha-or beta-actin-glycan protein level by western blot or immunohistochemistry on muscle biopsies before and after administration of the composition or drug.

In any use of any composition or medicament for treating muscular dystrophy, the disease progression in a subject is delayed after administration of the composition or medicament, as measured by any of the following methods: six minute walk test, rise time, up 4 steps, up and down 4 steps, arctic walk assessment (NSAA), 10 meter time test, 100 meter time test, hand Held Dynamometer (HHD), time to rise walk and/or gross motor minute test scale (Bayley-III) score.

For example, the NSAA score of a subject is improved by at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 score after administration of any composition for treating muscular dystrophy or any use of a medicament for treating muscular dystrophy compared to the NSAA score prior to administration of the rAAV for at least 270 days after administration of the composition or medicament. Moreover, in any of the methods, the subject's rise time is improved by at least about 0.8 seconds at least 270 days after administration of the composition or drug, as compared to the rise time prior to administration of the composition or drug. Furthermore, in any method or use of the present disclosure, the time of the ascending level 4 step test of a subject is improved by at least about 1.2 seconds at least 270 days after administration of the composition or drug as compared to the time of the ascending level 4 step test prior to administration of the composition or drug. Furthermore, in any method or use of the present disclosure, the 100 meter timing test of the subject is improved by at least about 7 seconds at least 270 days after administration of the composition or drug as compared to the 100 meter timing test prior to administration of the composition or drug.

In another aspect, the present disclosure provides a composition for expressing a microdystrophin gene in a patient's cells, the composition comprising aavrh74.mhck7. Microdystrophin construct nucleotide sequences of nucleotides 55-5021 of SEQ ID No. 9, SEQ ID No. 3, nucleotides 1-4977 of SEQ ID No. 8, or nucleotides 56-5022 of SEQ ID No. 6. In yet another aspect, the present disclosure provides the use of a dose of the aavrh74.mhck7. Mini-anti-dystrophin construct nucleotide sequence of SEQ ID NO 9, nucleotides 55-5021 of SEQ ID NO 3, nucleotides 1-4977 of SEQ ID NO 8 or nucleotides 56-5022 of SEQ ID NO 6 for the preparation of a medicament for expressing a mini-anti-dystrophin gene in a patient cell. For example, the expression of a microdystrophin gene in a patient's cells is detected by measuring the microdystrophin level by western blot or immunohistochemistry in muscle biopsies before and after administration of the raav.mhck7. Microdystrophin construct. In addition, the patient's expression of the microdystrophin gene is measured by detecting a greater number of vector genomes in each cell nucleus, where 1 vector genome in each cell nucleus is about 50% of the microdystrophin expression and greater than 1 copy in each cell nucleus is consistent with the level of microdystrophin expression. For example, a cell has 1.2 vector copies per cell nucleus, or 1.3 vector copies per cell nucleus, or 1.4 vector copies per cell nucleus, or 1.5 vector copies per cell nucleus, or 1.6 vector copies per cell nucleus, or 1.7 vector copies per cell nucleus, or 1.8 vector copies per cell nucleus, or 1.9 vector copies per cell nucleus.

In yet another aspect, the present disclosure provides a composition for reducing serum CK levels in a patient in need thereof, the composition comprising aavrh74.mhck7. Mini-dystrophin construct nucleotide sequences of nucleotides 55-5021 of SEQ ID No. 9, SEQ ID No. 3, nucleotides 1-4977 of SEQ ID No. 8, or nucleotides 56-5022 of SEQ ID No. 6. Furthermore, the present disclosure provides the use of a dose of the aavrh74.mhck7. Mini-dystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID NO 9, SEQ ID NO 3, nucleotide 1-4977 of SEQ ID NO 8 or nucleotide 56-5022 of SEQ ID NO 6 for the preparation of a medicament for reducing serum CK levels in a patient in need thereof. For example, the patient's serum CK level is reduced by at least about 65% to about 90% or about 65% to about 95% or about 75% to about 90% or about 80% to about 90% or about 85% to about 95% or about 87% to about 90% after 60 days of administration of the composition or drug as compared to the serum CK level prior to administration of the composition or drug. In particular, the subject's serum CK level is reduced by about 87% 60 days after administration of the composition or drug compared to the serum CK level prior to administration of the composition or drug, or by about 72% 60 days after administration of the composition or drug compared to the serum CK level prior to administration of the composition or drug, or by about 73% 60 days after administration of the composition or drug, or by about 78% 60 days after administration of the composition or drug compared to the serum CK level prior to administration of the composition or drug, or by about 95% 60 days after administration of the composition or drug compared to the serum CK level prior to administration of the composition or drug.

The present disclosure also provides a composition for increasing a microdystrophin positive fiber in muscle tissue of a patient comprising aavrh74.mhck7. Microdystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID NO 9, SEQ ID NO 3, nucleotide 1-4977 of SEQ ID NO 8 or nucleotide 56-5022 of SEQ ID NO 6. Furthermore, the present disclosure provides the use of a dose of the aavrh74.mhck7. Mini-dystrophin construct nucleotide sequence of nucleotides 55-5021 of SEQ ID NO 9, SEQ ID NO 3, nucleotides 1-4977 of SEQ ID NO 8 or nucleotides 56-5022 of SEQ ID NO 6 for the preparation of a medicament for increasing mini-dystrophin positive fibers in patient muscle tissue. For example, the number of tiny anti-dystrophin positive fibers is detected by measuring the level of anti-dystrophin protein by western blotting or immunohistochemistry on a muscle biopsy before and after administration of the composition or drug. In addition, the patient's expression of the microdystrophin gene is measured by detecting a greater number of vector genomes in each cell nucleus, where 1 vector genome in each cell nucleus is about 50% of the microdystrophin expression and greater than 1 copy in each cell nucleus is consistent with the level of microdystrophin expression. For example, a cell has 1.2 vector copies per cell nucleus, or 1.3 vector copies per cell nucleus, or 1.4 vector copies per cell nucleus, or 1.5 vector copies per cell nucleus, or 1.6 vector copies per cell nucleus, or 1.7 vector copies per cell nucleus, or 1.8 vector copies per cell nucleus, or 1.9 vector copies per cell nucleus.

In another aspect, the present disclosure provides a composition for increasing expression of α -myoproteoglycans in a patient in need thereof, the composition comprising aavrh74.mhck7. Mini-dystrophin construct nucleotide sequences of nucleotides 55-5021 of SEQ ID No. 9, SEQ ID No. 3, nucleotides 1-4977 of SEQ ID No. 8, or nucleotides 56-5022 of SEQ ID No. 6. The present disclosure also provides the use of a dose of the aavrh74.mhck7. Mini-anti-dystrophin construct nucleotide sequence of nucleotide 55-5021 of SEQ ID NO 9, SEQ ID NO 3, nucleotide 1-4977 of SEQ ID NO 8 or nucleotide 56-5022 of SEQ ID NO 6 for the preparation of a medicament for increasing expression of alpha-actin glycans in a patient in need thereof. For example, the level of alpha-actin can be detected by measuring the level of alpha-actin protein by western blotting or immunohistochemistry on a muscle biopsy before or after administration of the composition or drug.

Furthermore, the present disclosure provides a composition for increasing expression of β -actin glycans in a patient in need thereof, comprising aavrh74.mhck7. Mini-dystrophin construct nucleotide sequences of nucleotides 55-5021 of SEQ ID No. 9, SEQ ID No. 3, nucleotides 1-4977 of SEQ ID No. 8 or nucleotides 56-5022 of SEQ ID No. 6. The present disclosure also provides the use of aavrh74.mhck7. Mini-dystrophin construct nucleotide sequences of nucleotide 55-5021 of SEQ ID No. 9, nucleotide 55-5021 of SEQ ID No. 3, nucleotide 1-4977 of SEQ ID No. 8 or nucleotide 56-5022 of SEQ ID No. 6 for the preparation of a medicament for increasing expression of β -actin in a patient in need thereof. For example, the level of beta-actin can be detected by measuring the level of beta-actin protein by western blotting or immunohistochemistry on muscle biopsies before and after administration of the composition or drug.

The present disclosure also provides the use of a dose of aavrh74.mhck7 of nucleotide 55-5021 of SEQ ID NO 9, SEQ ID NO 3, nucleotide 1-4977 of SEQ ID NO 8 or nucleotide 56-5022 of SEQ ID NO 6 for the preparation of a medicament for treating a patient suffering from duchenne muscular dystrophy or becker muscular dystrophy such that administration of the medicament results in the disease progression of the patient being delayed as measured by any of the following methods: six minute walk test, rise time, up 4 steps, up and down 4 steps, arctic walk assessment (NSAA), 10 meter time test, 100 meter time test, hand Held Dynamometer (HHD), time to rise walk and/or gross motor minute test scale (Bayley-III) score.

For example, the NSAA score of the subject is improved by at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 score compared to the NSAA score of the subject at least 270 days after administration of the composition or drug. Moreover, the rise time of the subject is improved by at least about 0.8 seconds at least 270 days after administration of the composition or drug, as compared to the rise time prior to administration of the composition or drug. Furthermore, the time of the up-going level 4 step test for the subject is improved by at least about 1.2 seconds at least 270 days after administration of the composition or drug as compared to the time of the up-going level 4 step test prior to administration of the composition or drug. Furthermore, the 100 meter time test of the subject is improved by at least about 7 seconds at least 270 days after administration of the composition or drug as compared to the 100 meter time test prior to administration of the composition or drug.

It is to be understood that the detailed description section (and not the summary and abstract sections) is intended to be used to interpret the claims. The summary and abstract sections may set forth one or more, but not all exemplary aspects of the disclosure as contemplated by the inventors, and thus are not intended to limit the disclosure and appended claims in any way.

The following examples are provided for purposes of illustration and not limitation. The numerical ranges recited include each integer value within each range and include the lowest and highest integers recited.

Examples

Example 1

A) AAVrh74.MHCK7. Generation of micro anti-dystrophin protein constructs

Aavrh74.mhck7. Mini-dystrophin plasmid contains a human mini-dystrophin cDNA expression cassette flanked by AAV2 Inverted Terminal Repeats (ITRs) (see fig. 1). The mini-dystrophin construct is characterized by an in-frame rod deletion (R4-R23), while hinges 1, 2 and 4 and the cysteine-rich domain still produce a 138kDa protein. Expression of the mini-dystrophin protein (3579 bp) is directed by the MHCK7 promoter (792 bp). The plasmid was constructed from a raav.mck. Mini-dystrophin plasmid by removing the MCK promoter and inserting the MHCK7 promoter. Following the core promoter, there is 53bp endogenous mouse MCK exon 1 (untranslated) for efficient transcription initiation, followed by SV40 late 16S/19S splicing signal (150 bp) and small 5' UTR (61 bp). Introns and 5' UTRs are derived from plasmid pCMV beta (Clontech). The mini-dystrophin cassette has a consensus Kozak immediately preceding the ATG start and a small 53bp synthetic poly a signal for mRNA termination. The human mini-dystrophin cassette comprises the (R4-R23/Δ71-78) domain as previously described by Harper et al (Nature Medicine 8,253-261 (2002)). Complementary DNA was codon optimized for human use and synthesized by GenScript (Piscataway, NJ) (Mol ter 18,109-117 (2010)). The only viral sequence contained in the vector is the inverted terminal repeat of AAV2, which is necessary for viral DNA replication and packaging. The mini-dystrophin cassette has a small 53bp synthetic poly-a signal for mRNA termination.

Previous studies have demonstrated cardiac expression using the MHCK7 promoter (Salva et al, mol ter 15,320-329 (2007) and skeletal, diaphragmatic and myocardial expression using AAVrh74 (Sondergaard et al, annals of Clinical and Transl Neurology 2,256-270 (2015)). The sequences of the construct of fig. 1 were packaged into aavrh.74 virions. Molecular cloning of the aavrh.74 serotype was cloned from rhesus lymph nodes and discussed in rodno-Klapac et al, journal of Translational Medicine 5,45 (2007).

Table 1 shows the molecular characteristics of the plasmid AAVrh74.MHCK7. Mini-anti-dystrophin protein (SEQ ID NO: 3)

TABLE 1 molecular characterization of plasmid rAAV.MHCK7. Mini-dystrophin

B) Generation of AAVrh74.MHCK7. Micro anti-dystrophin constructs from a plasmid encoding kanamycin (Kan) resistance

Cloning of mhck7. Mu. Dys.kan was achieved by isolating mhck7. Mu. Dys fragments from the mhck7. Mu. Dys.amp plasmid and kanamycin backbone and annealing them using the nebulider cloning workflow. The mhck7. Mu Dys fragment was isolated by restriction enzyme digestion using SnaBI. Digestion was performed in 50 μl total reaction in 1xCutSmart buffer (NEB) and 1 μl SnaBI at 37 ℃ for 1 hour. The resulting fragments were separated by electrophoresis using a 1% agarose gel at 105 volts for 1.5 hours. The band corresponding to the mhck7. Mu. Dys insert was excised and purified using a gel purification kit (Macherey-Nagel). The DNA concentration of the resulting fragment was 10 ng/. Mu.L. Kan backbone fragments were isolated by XbaI restriction enzyme digestion in 50. Mu.L reactions using 1xCutSmart buffer (NEB) and 1. Mu.L XbaI digested for 1 hour at 37 ℃. The resulting fragments were separated by electrophoresis using a 1% agarose gel at 105 volts for 1.5 hours. The band corresponding to Kan backbone was excised and purified by gel purification kit (Macherey-Nagel). The DNA concentration of the resulting fragment was 8.1 ng/. Mu.L. The two fragments were annealed using the NEB Builder cloning workflow, which was able to join two fragments with overlapping sequences. NEBuilder cloning reactions were performed at 50℃for 15 minutes using a 1:1 ratio of MHCK7. Mu.Dys to kanamycin backbone in 1xNEBuilder HiFi DNA Assembly Master Mix in a total reaction volume of 20. Mu.L according to the manufacturer's protocol. The resulting clone was transformed by adding 2.5. Mu.L of clone product to the cells, then placing on ice for 30 minutes, then placing at 42℃for 30 seconds, and then placing on ice for 5 minutes In stable competent E.coli (C3040). After transformation, 950 μl of growth medium was added to the cells and allowed to grow at 30 ℃ for 1.5 hours with shaking at 225 rpm. After growth, 450. Mu.L of these cells were plated on 50. Mu.g/mL kanamycin LB agar plates and incubated overnight at 30℃in a dry incubator. Picking from the plateColonies were taken and cultured overnight in LB containing 50. Mu.g/mL kanamycin. Use->The Spin Miniprep kit (Qiagen) isolated DNA from 3mL of this culture. This DNA was used to confirm the cloned product. The clone product was confirmed by restriction enzyme digestion with PmeI, mscI and SmaI followed by gel electrophoresis. The cloned product was also confirmed additionally by sequencing. The resulting plasmid is shown in SEQ ID NO. 8 and is shown in FIGS. 14 and 15. The sequence of the construct of FIG. 13 (which corresponds to the sequence of SEQ ID NO: 9) and nucleotides 1-4977 of SEQ ID NO:8 are packaged into AAVrh.74 virions as described above.

Example 2

Systemic gene delivery clinical trial for Du's muscular dystrophy

This is a single dose control test for DMD subjects using raavrh74.mhck7 of SEQ ID No. 3, nucleotides 55-5021. Cohort a included six subjects aged 3 months to 3 years, and cohort B included six subjects aged 4 years to 7 years. All subjects received intravenous mini-dystrophin carrier (2 x10 in 10mL/kg ¹⁴ vg/kg). rAAV rh74.MHCK7. Micro anti-muscular dystrophy protein in a solution containing 20mM Tris (pH 8.0), 1mM magnesium chloride (MgCl) ₂ ) Formulated in 200mM sodium chloride (NaCl) and 0.001% poloxamer 188 buffer.

In this study raavrh74.mhck7. Mini-dystrophin was infused intravenously through the peripheral arm so that it could reach all muscles in the body. DMD subjects aged 3 months to 3 years were enrolled in cohort a, and DMD subjects aged 4 years to 7 years were enrolled in cohort B. All subjects received intravenous mini-dystrophin carrier (2 x10 in 10mL/kg ¹⁴ vg/kg). Vector genome titres encapsulated at the doses administered compared to supercoiled DNA plasmid standards were determined using quantitative PCR using primers directed against the MHCK7 promoter using Prism 7500Taqman detector system (PE Applied Biosystems) (Pozsgai et al mol. Ther.25 (4): 855-869 (2017)).

Subjects received infusion for more than 1 hour in the Pediatric Intensive Care Unit (PICU) of the national pediatric hospital. Prior to gene therapy, a muscle biopsy was performed at the screening visit. The subject will take a second muscle biopsy 90 days after delivery to determine if the gene achieves replacement of the deleted anti-dystrophin protein. Following gene transfer, the patient is carefully monitored for any therapeutic side effects. The monitoring included blood and urine tests and physical examination during screening visits and on days 0, 1, 7, 14, 30, 60, 90 and 180 and on months 9, 12, 18, 24, 30 and 36 to ensure that there were no side effects from gene injection.

Subjects in cohort a (n=6) were aged 3 months to 3 years and received intravenous raavrh74.mhck7 a mini-dystrophin vector (2 x10 in 10mL/kg ¹⁴ vg/kg). The day before gene transfer, subjects in cohort a began taking prednisone or deflazacort 1mg/kg and remained there for 30 days while monitoring immune responses. If day 30 is negative, steroid use is stopped for 1 week. If T cells respond to AAV or minor anti-dystrophin proteins>125SFC/106PBMC, the steroid is maintained until the level falls below this threshold.

Subjects in cohort B (n=6) were aged 4 to 7 years and received intravenous raavrh74.mhck7. Mini-dystrophin vector (2 x10 in 10mL/kg ¹⁴ vg/kg). These subjects maintained a stable dose of corticosteroid throughout the course of the trial, but if T cells respond to AAV or mini-dystrophin>125SFC/106PBMC, it is possible to increase the corticosteroid dose in a short period of time.

Qualification criteria

The inclusion criteria for this study are as follows:

age of group entry: queue a:3 months to 7 years old (inclusive), cohort B:4 to 7 years old (inclusive).

Molecular characterization of DMD gene with frame shift (deletion or repetition) or premature stop codon mutation between exons 18 to 58.

CK rise >1000U/L

Cohort a subjects: bayley-III motion assessment for coarse motion was below average, defined as a scale score of 9 or less

Queue B:100 meters timing test below average, defined as < 80% of predicted value

Men of any ethnic group.

Can coordinate with the motion assessment test.

Cohort a subjects: no corticosteroid treatment has been received in the past.

Cohort B subjects: the stable dose equivalent of corticosteroid was administered orally at least 12 weeks prior to screening, and the dose was expected to remain constant throughout the study (except for modifications to accommodate changes in body weight).

The exclusion criteria for this study were as follows:

active viral infection based on clinical observations.

Myocardial disease signs, including echocardiography with ejection fraction below 40%.

Serological evidence of HIV infection, hepatitis B or hepatitis C infection.

Diagnosis (or continued treatment) of autoimmune diseases.

Abnormal laboratory values considered clinically significant

PI is believed to carry an unnecessary risk for gene transfer with disease or the need for long-term drug therapy.

Determination of AAVrh74 or AAV8 antibody titres >1:400 by ELISA immunoassay.

Researchers believe that it may be detrimental to the ability of a subject to follow a test or procedure required by a regimen or to a medical condition or scenario that compromises the health, safety, or clinical interpretability of a subject.

Severe infections (e.g. pneumonia, pyelonephritis or meningitis) occur within 4 weeks prior to gene transfer visit (group entry may be delayed).

Within the last 6 months prior to screening for the present study, any study drug (except corticosteroids) or exon skipping drugs (including ExonDys) were received experimentally or otherwise)。

Accept any type of gene therapy, cell-based therapy (e.g., stem cell transplantation) or CRISPR/Cas9 therapy.

The family does not want to disclose the patient's study participation to primary healthcare doctors and other medical providers.

Results index

The main outcome indicator is safety based on the number of participants who have had adverse events (time frame: 3 years). Adverse reactions were monitored and scored for severity and relevance to the study article.

The secondary outcome measures are as follows:

gross motor score test scale (Bayley-III) score (time frame: screening, day 30-3): gross motor scale scores measured motor development. From day 30 to 3, cohort A was scored on the Bayley-III gross motor score test at each follow-up. Any subjects with an age of 43-47 months (inclusive) at the time of screening had calculated scale scores compared to standard data for 42 month old children. Bayley-III provides specification data for children 1-42 months old.

Physical therapy assessment (time frame: screening, day 30-3 years) for 100 meter timing test (100 m): 100m is the primary motion result for queue B. The 100 meter timing test is a exploratory result initiated by the age of 3 for the child for queue a.

Physical therapy assessment of North Star Walking assessment (NSAA) (time frame: screening, day 30-3): the arctic walk assessment (NSAA) is an exploratory result initiated for queue a at the age of four children and for queue B. NSAA measures walking quality in young boys with duchenne muscular dystrophy.

Chrono-riser walking (TUG) physical therapy assessment for child modification (time frame: screening, day 30-3): the exploratory results for queue B included timed standing walks (TUGs) modified for children.

Physical therapy evaluation of 4 steps up and down (time frame: screening, day 30-3): the exploratory result of queue B would include 4 steps up and down.

Physical therapy assessment by hand held load cell (HHD) (time frame: screening, day 30-3): exploratory results for cohort B included a hand-held dynamometer (HHD) for the extensor and flexor of the knee and extensor of the elbow.

Quantification of micro-dystrophin gene expression by immunofluorescence (time frame: screen, day 90): the expression level of the micro anti-dystrophin gene was quantified by immunofluorescence and compared before and after muscle biopsy.

Quantification of micro-dystrophin gene expression by western blotting (time frame: screen, day 90): the expression level of the micro anti-dystrophin gene was quantified by western blot analysis and compared before and after muscle biopsy.

Decrease in CK after gene therapy (time frame: 3 years): reduction of CK levels in circulating blood.

Cardiac magnetic resonance imaging (1 year old).

Micro anti-muscular dystrophy protein gene expression

The change in micro-dystrophin expression from baseline was analyzed and quantified by Immunofluorescence (IF) fiber intensity. As shown in table 2, subject 1 (5 years old) showed 78% expression of micro-dystrophin in the myofibers of gastrocnemius biopsies after administration of raavrh74.mhck7; subject 2 (4 years old) showed 73.5% expression of microdystrophin in the muscle fibers of gastrocnemius biopsies following administration of raavrh74.mhck7; and subject 3 (6 years) showed 77.0% expression of the microdystrophin protein in the muscle fibers of gastrocnemius biopsies after administration of raavrh74.mhck7. Subject 4 (4 years) showed 96.2% expression of mini-dystrophin in the muscle fibers of gastrocnemius biopsies after administration of raavrh74.mhck7. All patients showed robust expression of transduced minute anti-dystrophin proteins correctly localized to the muscle myomembrane as measured by immunohistochemistry (fig. 7).

TABLE 2

The change in microdystrophin gene expression from baseline to day 60 was also assessed by quantifying microdystrophin expression as measured by western blot of biopsy muscle tissue. As shown in fig. 8A and 8B, western blot analysis detected minute anti-dystrophin expression in subject 1 (5 years), subject 2 (4 years), and subject 3 (6 years). Figure 8C provides a western blot analysis to detect expression of mini-dystrophin in subject 4 (4 years). As measured by western blot, all post-treatment biopsies showed robust levels of micro anti-dystrophin with an average value of 74.3 for subjects 1-4 compared to the normal case using method 1 and 95.8% compared to the normal case according to method 2 adjusted for fat and fibrotic tissue.

For each subject, vector genome copies were measured for each core of muscle fiber. As shown in table 3, the vector genome copy of each nuclease per subject was greater than 1 after administration of raavrh74.mhck7. One copy of the vector indicates about 50% expression of the mini-dystrophin gene. An average of 1.6 vector copies per nucleus were measured in subjects 1-3, consistent with the observed high levels of minimal dystrophin expression. Average vector copy number/. Mu.g DNA when the value of subject 4 was included >10 ⁵ Wherein each cell nucleus has an average of 3.3 copies of the vector.

TABLE 3 Table 3

A subject	Vector copy number/. Mu.g DNA	Copy number per core
			1	>10 ⁵	1.7
2	>10 ⁵	1.3
			3	>10 ⁵	1.9

Protein levels of alpha-and beta-glucan in muscle biopsies were measured by immunohistochemistry before and after administration of raavrh74.mhck7. Mini-dystrophin. Administration of raavrh74.mhck7 also resulted in up-regulation of DAPC protein in the subject. As shown in fig. 9, in subject 1 (fig. 9A), subject 2 (fig. 9B) and subject 3 (fig. 9C), the expression of α -and β -xylan in muscle biopsies was increased compared to the levels of these proteins in muscle biopsies prior to administration of raavrh74.mhck7.

Circulating serum CK levels

Intravenous rAAV rh74.MHCK7. Mini anti-dystrophin protein vehicle (2X 10 in 10 mL/kg) ¹⁴ vg/kg), blood samples were obtained every 30 days. CK levels were measured at each visit and compared to baseline levels obtained prior to administration of raavrh74.mhck7. Mini-dystrophin (day 0 of visit). Table 4 below provides baseline serum CK levels (units/liter). As shown in fig. 10, circulating serum CK levels decreased by about 87% after 2 months of administration of raavrh74.mhck7. Serum Creatine Kinase (CK) levels were significantly reduced in all subjects, 2 months after treatment The average drop in CK was more than 87% (n=3). CK is an enzyme associated with muscle damage, and DMD patients consistently exhibit high levels of CK. Indeed, CK that is significantly elevated is typically used as a primary diagnostic tool for DMD, followed by confirmatory gene testing.

The CK levels for each subject are provided in table 4 and fig. 10. Figure 11 provides the average CK levels over time and shows that the average CK levels decrease significantly over time following administration of raavrh74.mhck7. 27,064U/L the average baseline CK levels (average of Table 4) decreased by about 63% to an average of 9,982U/L (average, day 270, table 5).

TABLE 4 Table 4

Table 5: changes in CK levels from baseline to day 270

Efficacy assessment

In addition to minor anti-dystrophin and CK levels, efficacy was also measured by the following functional test: time to rise from floor, 4 steps up, arctic walk assessment (NSAA), rise time test, 4 stairs up test, 10 meter timing test (10 m) and 100 meter timing test (100 m). The data are provided in tables 6 and 7 below, and demonstrate consistent durable improvement at 9 months after administration of raavrh74.mhck7. NSAA improvement over time is also provided in fig. 12.

Table 6: NSAA change from baseline to day 270

Table 7: change from baseline to day 270

Security assessment

No Serious Adverse Events (SAE) were observed in the study. Three subjects had elevated gamma-glutamyl transferase (GGT) that was relieved and restored to baseline levels using increased steroids within one week. There were no other laboratory findings of clinical significance. Patients often have a brief nausea with increased steroid dosing during the first week of treatment. This is not associated with elevated liver enzymes or any other abnormalities.

Example 3

Randomized, double-blind, placebo-controlled, systemic gene delivery phase I/IIa clinical trial

This is a randomized, double-blind, single-dose trial with raavrh74.mhck7. Mini-dystrophin for DMD subjects. The study included 24 subjects aged 4 to 7 years. The subjects were randomized to receive treatment or placebo at the time of group entry. 12 subjects received intravenous rAAV rh74.MHCK7. Micro anti-dystrophin protein vehicle (about 10mL/kg 2X 10) ¹⁴ vg/kg), 13 subjects received 10mL/kg placebo (ringer lactate). Placebo subjects will receive treatment again one year after the last treated subject in the same manner as 12 previously treated subjects. The subject received an infusion of rAAV or ringer lactate carrying a minor anti-dystrophin protein within about 1 hour. Needle biopsies of the muscle were performed before and after treatment (90 days) of the gastrocnemius muscle.

The main objective of this study was to assess the safety of intravenous administration of raavrh74.mhck7, a minor anti-dystrophin protein, to DMD subjects via the external Zhou Zhiti vein. Safety endpoints were assessed by hematology, serum chemistry, urinalysis, changes in immune responses to rAAVrh74 and minute anti-dystrophin proteins, and reported medical history and symptomatic observations. The expression of the anti-dystrophin gene is used as a main result index together with safety. Quantification was performed using validated immunofluorescence and immunoblot assays. The decrease in CK after gene therapy was used as a secondary result. Efficacy was measured by the following functional tests: rise time, ascending 4 steps, polaroid walk assessment (NSAA), 10 meter timing test (10 m), 100 meter timing test (100 m). Exploratory measurements include hand held load cells (HHD) of the extensor and flexor of the knee, and the flexor and extensor of the elbow.

The inclusion criteria for this study are as follows:

age of group entry: 4-7 years old (inclusive).

Indication of symptomatic muscular dystrophy: CK rise >1000U/L and less than the average predicted time percentage for the 100 meter walk test

Men of any ethnic group are eligible.

Can coordinate with the motion assessment test.

Stable dose equivalent of oral corticosteroid at least 12 weeks prior to screening, and dose is expected to remain constant throughout the study (except for possible modifications to accommodate weight changes).

The exclusion criteria for this study were as follows:

active viral infection based on clinical observations.

Serological evidence of HIV infection, hepatitis B or hepatitis C infection.

Diagnosis (or continued treatment) of autoimmune diseases.

Abnormal laboratory values (GGT >3XULN, bilirubin. Gtoreq.3.0 mg/dL, creatinine. Gtoreq.1.8 mg/dL, hgb <8 or >18g/Dl; WBC >18,500/cm), platelets. Ltoreq.50,000.

Determination of AAVrh74 or AAV8 antibody titres >1:400 by ELISA immunoassay. If the endpoint titer is positive at the time of screening, the test can be repeated prior to exclusion.

Efficacy assessment

The expression of the anti-dystrophin gene is used as a main result index together with safety. Quantification was performed using validated immunofluorescence and immunoblot assays. The decrease in CK after gene therapy is a secondary result. Furthermore, efficacy was measured by the following functional tests: time to rise from floor, step 4 up, arctic walk assessment (NSAA), 10 meter timing test (10 m), 100 meter timing test (100 m) ]. Exploratory measurements include hand held load cells (HHD) of the extensor and flexor of the knee, and the flexor and extensor of the elbow.

Ultrasound guided muscle biopsies were used to quantitatively compare transgene expression at baseline and day 90. Biopsies are performed on the same muscle as the original biopsy but on the opposite leg. One year after dosing all subjects, placebo-crossed subjects will restart the study schedule at visit 1. Placebo subjects did not undergo the following at the second baseline screening: cardiac MRI and muscle biopsies. Placebo subjects received a muscle biopsy on day 90 (a total of 3 muscle biopsies). The frozen sections were stained for dystrophin using indirect Immunofluorescence (IF). Full slide scans were performed and the microdystrophin intensity and percent positive fibers were quantified using a validated image scan and a musclemasptm analysis algorithm. Muscle morphometry was performed unknowingly, including fiber size histograms. The blind frozen muscle biopsy sections were used for quantitative protein analysis of the mini-dystrophin protein using a validated western blot method.

Muscle puncture biopsies of gastrocnemius (unless PI deems contraindicated for a particular subject, in which case PI will select for biopsies of alternative muscles) are used to quantify mini-dystrophin expression.

Efficacy analysis

The primary efficacy endpoint was the change in the amount of micro-dystrophin expression from baseline to day 90 as measured by western blot of biopsy muscle tissue. Treatment group differences at the primary efficacy endpoint were assessed by an analysis of covariance (ANCOVA) model with treatment as a fixed factor and baseline value as a covariate. The Wilcoxon rank sum test was performed as a supportive analysis. A similar analysis was performed for changes in micro-dystrophin expression from baseline by Immunofluorescence (IF) fiber intensity.

The supportive efficacy endpoints included the rise time from floor, the ascending 4-step, NSAA, 10 meter timing test (10 m), the change from baseline to each predetermined assessment for 100 meter timing test (100 m), and the change in CK. Exploratory materials include HHD for the extensor and flexor of the knee and extensor of the elbow. Treatment group differences were assessed using the ANCOVA model with treatment as a fixed factor and baseline values as covariates. The Wilcoxon rank sum test was performed as a supportive analysis.

Example 4

The experiments and studies described in examples 2 and 3 above may alternatively be carried out using the sequence shown in SEQ ID NO. 9; as shown in SEQ ID NO. 8, nucleotide 1-4977; or rAAV 74.MHCK7 as shown in SEQ ID No. 6, nucleotide 56-5022.

Example 5

production of pAAV. MCK. Micro anti-dystrophin constructs

pAAV. MCK. Mini-dystrophin plasmid was constructed by inserting the MCK expression cassette driving the codon optimized human mini-dystrophin cDNA sequence into AAV cloning vector psub201 (Samulski, R.J. et al, J.Virol.61 (10): 3096-3101 (1987)). Muscle-specific regulatory elements are included in the construct to drive muscle-specific gene expression. The regulatory element comprises a mouse MCK core enhancer (206 bp) fused to a 351bp MCK core promoter (proximal). Following the core promoter, the construct contained 53bp endogenous mouse MCK exon 1 (untranslated) for efficient transcription initiation, followed by SV40 late 16S/19S splicing signal (97 bp) and small 5' utr (61 bp). Introns and 5' UTRs are derived from plasmid pCMV beta (Clontech). The mini-dystrophin cassette has a consensus Kozak immediately preceding the ATG start and a small 53bp synthetic poly a signal for mRNA termination. The human mini-dystrophin cassette comprises the (R4-R23/Δ71-78) domain as described previously in Harper et al, nat.Med.8 (3): 253-61 (2002).

The paav.mck. Mini-dystrophin plasmid contains a human mini-dystrophin cDNA expression cassette flanked by AAV2 Inverted Terminal Repeats (ITRs) (see fig. 5). This sequence was encapsulated into aavrh.74 virions. Molecular cloning of the AAVrh.74 serotype was cloned from rhesus lymph nodes and is described in Rodio-Klapac et al, J Transl. Med.5:45 (2007).

Example 6

rAAV production using mixed vaccination training amplification

The following procedure can be used to generate the rAAV constructs described herein.

HEK-293 cells were passaged four times under adherent conditions. Cells were harvested and centrifuged to wash off serum (@ 300g,5 min) before entering the penultimate expansion culture and resuspended in serum-free growth medium (EXPI 293) in suspension shake flasks at an seeding density of 0.5+e6 cells/mL. They were then allowed to grow and expand in number within 48-72 hours. The suspension cells are then collected and seeded in shake flasks or WAVE bags, depending on the number of living cells required for bioreactor seeding. At the end of 72 hours, the concentration of living cells was determined using a cell counting device. The necessary volume containing the preferred total living cells was then added to an adherent bioreactor containing DMEM and 10% fbs. Additional FBS was added appropriately to account for the added serum-free suspension culture volume such that the final FBS concentration was maintained at 10%.

As shown in fig. 18, the cell viability in the seeding training and the anchorage system was similar. Regarding Viable Cell Density (VCD), VCD after passage 6 was higher than VCD after passage 1 in the mixed inoculation training system (fig. 19A), which is comparable to the adherent system (fig. 19B).

In inoculation adherence bioreactorThe adherent cultures are then transiently transfected with a transgenic plasmid (including, for example, the construct shown in SEQ ID NO:9 contained in the transgenic plasmid of SEQ ID NO: 8) carrying the mini-dystrophin constructs described herein. In addition to transgenic plasmids, rep/cap plasmids (AAV 2 rep/rh74 cap) and helper plasmids are included. After the desired growth cycle, rAAV particles were harvested by cell lysis and column chromatography.

In some aspects, the rAAV is produced by a suspension seeding process comprising:

(b) Removing cells from the first medium;

(d) Culturing cells in an N-1 vessel under suspension conditions; and

(e) Inoculating the third medium in the bioreactor with cells from step (d).

In some aspects, the suspension seeding process further comprises:

(f) Cells were transfected with a transgenic plasmid containing raavrh74.mhck7. Mini-dystrophin construct, a plasmid containing AAV rep gene and AAV cap gene, and an adenovirus helper plasmid.

In some aspects, the transgenic plasmid comprising raavrh74.mhck7. The mini-dystrophin construct comprises: the nucleic acid sequence of SEQ ID NO. 9, nucleotides 55-5021 of SEQ ID NO. 3 or nucleotides 1-4977 of SEQ ID NO. 8. In some aspects, the plasmid comprising an AAV rep gene and an AAV cap gene comprises an AAV2 rep gene and a rAAVrh74 cap gene.

In some aspects, the adenovirus helper plasmid comprises adenovirus 5E2A, E4ORF6 and a VA RNA gene.

In some aspects, the suspension seeding process further comprises: (g) lysing the cells. In some aspects, the cells are lysed by freeze-thawing, solid shearing, hypertonic and/or hypotonic lysis, liquid shearing, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

In some aspects, the suspension seeding process further comprises: (h) purifying the rAAV by at least one column chromatography step. In some aspects, the at least one column chromatography step comprises anion exchange chromatography or size exclusion chromatography or a combination thereof.

Example 7

Open-label, systemic gene delivery studies to assess the safety and expression of raavrh74.mhck7. Mini-anti-dystrophin constructs in subjects with duchenne muscular dystrophy using commercially representative materials

This is a phase 1b open-label study performed in boys with duchenne muscular dystrophy using raavrh74.mhck7 a commercially representative material for the mini-anti-muscular dystrophy protein construct. Initially, 20 patients were enrolled and this example 7 provides data from the first 11 patients aged less than 8 years (e.g., 2 patients aged 4-5 years; 9 patients aged 6-7 years) (cohort 1; up to 20 subjects with DMD-walkable males aged ∈4 to <8 years) (table 7). The study was later expanded to include cohort 2 (about 6 subjects with DMD-walkable men between 8 and <18 years of age) and cohort 3 (about 6 subjects with DMD-walkable men), as further described in example 8.

Table 8: baseline demographics data: the first 11 patients in cohort 1

The main objective was to evaluate the expression of the microdystrophin protein from raavrh74.mhck7. Microdystrophin constructs (e.g., commercially representative materials) 12 weeks after infusion (part 1), with the corresponding endpoint being the change in the expression of the microdystrophin protein from baseline to week 12 (part 1) as measured by western blotting of biopsy muscle tissue. The secondary objective is to evaluate: (1) Expression of mini-dystrophin assessed by Immunofluorescence (IF) fiber intensity at week 12; (2) Minute dystrophin expression assessed by the IF Percentage of Dystrophin Positive Fibers (PDPF) at week 12; and (3) security.

The study inclusion criteria applicable to examples 7 and 8 are as follows:

the subject must meet all of the following criteria in order to be eligible for participation in the study:

1. only queue 1 (walking <8 years): male at birth, walkable, screening at 4 to <8 years old, and NSAA score >17 and 26 at screening visit.

2. Only queue 2 (walking for no less than 8 years): male at birth, walkable, at screening ages no less than 8 to no more than 18 years old, and NSAA score no less than 15 and no more than 26 at screening visit.

3. Queue 3 only (cannot walk): male at birth and unable to walk for at least 9 months, NSAA walk score of "0" and unable to perform 10MWR at screening visit and have a PUL entry score of ∈2. The definition of the onset of disability is the age of the continuously used wheelchair reported by the participant or caregiver, approximating the last month.

4. Prior to screening, definitive diagnosis of DMD was made based on clinical outcome records and confirmatory genetic tests previously performed using clinical diagnostic genetic tests.

5. Indication of symptomatic muscular dystrophy:

CK rise >1000U/L and

only queues 1 and 2 (walk): less than 95% of the predicted time for 100 MWR.

6. Can cooperate with a motion assessment test.

7. Weekly stable dose equivalents of oral corticosteroid for at least 12 weeks prior to screening, and doses were expected to remain constant throughout the first year of the study (except for modifications to accommodate body weight changes).

8. The rAAV rh74 antibody titer was less than or equal to 1:400 (i.e., not increased) as determined by ELISA.

9. Sexually active subjects must agree to use condoms throughout the duration of the study, and female sexual partners must also use medically acceptable contraceptive measures (e.g., oral contraceptives).

10. Having parents or legal guardians or being subjects with an age of 18 years or more, are able to understand and follow the study visit schedule and all other regimen requirements.

11. Is willing to provide informed consent or consent (if applicable) and has parents or legal guardians, or is a subject of age > 18 years old, willing to provide written informed consent to participate in the study.

The exclusion criteria for this study were as follows:

subjects meeting any of the following criteria will be excluded from the study:

1. has a left ventricular ejection fraction of <40% of screening ECHO or clinical signs and/or symptoms of cardiomyopathy.

2. Only queues 2 and 3 (walking for more than or equal to 8 years and no walk): FVC < 50% of predicted values at screening and/or nocturnal ventilation support is required.

3. Major surgery within 3 months prior to day 1 or surgery was planned at any time during the study.

4. There are any other significant genetic diseases other than DMD.

5. There is serological evidence of current, chronic or active human immunodeficiency virus, hepatitis c or hepatitis b infection.

6. Is diagnosed with an autoimmune disease.

7. With concomitant diseases or requiring long-term medication, researchers believe that this may present an unnecessary risk for gene transfer.

8. Researchers believe that it may be possible to impair the ability of a subject to follow a test or procedure required by a regimen or to impair the health, safety or clinical interpretable medical condition or disposition of a subject.

9. Symptomatic infections (e.g., upper respiratory tract infections, pneumonia, pyelonephritis, meningitis) appear within 4 weeks prior to day 1.

10. It appears that researchers believe that cognitive retardation or impairment may affect motor development.

11. Treatment was performed using any of the following therapies in the prescribed time frame:

anytime:

gene therapy

Cell-based therapies (e.g., stem cell transplantation)

CRISPR/Cas9, or any other form of gene editing

Within 12 weeks from the first day:

-using human growth factors or vamololone

Within 6 months from the first day:

-any study drug

-queue 1 only: any treatment aimed at increasing expression of anti-dystrophin protein (e.g. Translarna ^TM 、EXONDYS 51、VYONDYS 53、VILTEPSO ^TM ). And (3) injection: subjects in cohorts 2 and 3 receiving these treatments were expected to stop before day 1. Treatments aimed at increasing expression of anti-dystrophin protein may resume and/or begin after week 72

12. Live virus vaccine was received within 4 weeks after visit on day 1, or inactivated vaccine was received within 2 weeks after visit on day 1, or vaccination was expected to be received within the first 3 months after day 1.

13. Having abnormal laboratory values that are considered clinically significant, including but not limited to:

gamma-glutamyl transferase (GGT) > 2-fold upper normal limit (ULN)

Total bilirubin > ULN. Note that elevated total bilirubin, believed to be caused by gilbert syndrome, is not precluded.

White cell count >18,500/. Mu.l

Platelet ∈150,000/. Mu.L

14. The subject or family does not want to disclose study participation of the subject to the general practitioner/primary care physician and other medical providers.

Researchers believe that subjects are unlikely to follow the study regimen. All subjects received raavrh74.mhck7. Mini-dystrophin construct (1.33×10) by a single IV infusion ¹⁴ vg/kg) (e.g., commercially representative materials).

Efficacy evaluation:

muscle biopsies for assessing micro-dystrophin expression were collected from all subjects at baseline and week 12. Muscle biopsies were collected using either open biopsies or vacoa core biopsies. Biopsies require collection of muscle tissue from the medial gastrocnemius muscle. If medial gastrocnemius is not feasible, the sponsor is required to approve in advance to use the replacement muscle.

Biopsy samples were used to quantify transgene expression by western blot, IF intensity and pdgf.

The average vector genome copy number per core was determined to be 3.87 (+ -2.4) from baseline. The average percentage of normal minor dystrophin expression and the change from baseline was determined to be 55.4% (±43.4) as determined by western blot. The average percentage of anti-dystrophin positive fibers was determined by immunofluorescence assay to be 70.5% (57.7% (±22.2) change from baseline) and the average intensity was 116.9% (75.9% ±46.4 change from baseline). These results are consistent with placebo group patients of example 3 administered the material described in example 4 (e.g., average vector copy number per core-2.62; normally expressed% -51.7%; anti-dystrophin positive fibers% -79.2%;% intensity 100.6%).

Figure 20 graphically shows the average NSAA scores from cohort 1 (the first 11 patients treated with raavrh74.mhck7. Mini-dystrophin). The first 11 patients had an improvement of 3 points from baseline. Children aged 6 to 7 (n=9) increased 2.9 points from baseline. Each time point represents 11 patients.

The safety of the commercial representative material is consistent with previous experience with raavrh74.mhck7. Mini-dystrophin constructs. Adverse events caused by 79 treatments were observed in 11 patients. The most common adverse event is vomiting, usually occurring within the first week, with lighter symptoms and receiving standard anti-emetic therapy. The increase in liver enzymes is transient and is responsive to steroids, wherein no patient has signs of impaired liver function. Serious adverse events were observed for both patients and were completely resolved. One patient had elevated transaminase and received intravenous steroid therapy. Nausea and vomiting occurred in one patient. No adverse events suggest complement-mediated events.

Overall, the characteristics of raavrh74.mhck7. Mini-dystrophin construct were confirmed by commercial representative materials. Robust transduction was observed (e.g., an average vector genome copy number per core of 3.87). Average robust expression was observed and correctly localized to the myomembrane (e.g., western blot-55.4%; positive fiber-70.5%; intensity-116.9%). By applying materials that are commercially representative, safety, well tolerated and consistent safety characteristics are observed. No clinical manifestation of complement was observed. Overall, these results confirm the manufacturing process and analysis and are sufficient to supply the duchenne's dystrophy population.

Example 8

The trial and study described in example 7 was extended to approximately 32 subjects in 3 cohorts: cohort 1 consists of up to 20 subjects with DMD-walkable males between 4 and <8 years of age; cohort 2 consisted of about 6 subjects with DMD-capable males aged no less than 8 years to <18 years old; cohort 3 consisted of about 6 subjects with DMD in males who were unable to walk.

The first 2 subjects in each cohort will be whistle subjects, with dosing intervals of at least 1 week. In combination cohorts 2 and 3, at least 3 subjects with a weight of <50kg will be included, and at least 3 subjects with a weight of > 50kg will be included. The study will consist of the following 4 phases:

1. up to about 3 weeks of screening period (pre-infusion) during which disease characteristics and baseline treatment will be assessed and pre-infusion assessment completed.

2. A baseline period (pre-infusion) starting at the time of qualification and ending the day before the first day of infusion, during which baseline assessment is completed.

3. An infusion period during which a single Intravenous (IV) infusion of the open label raavrh74.mhck7. Mini-dystrophin will be administered to all subjects within 31 days after obtaining the rAAVrh74 enzyme-linked immunosorbent assay (ELISA) sample. Heaviness of day 1 <70kg subjects will receive IVrAAV rh74.MHCK7. Micro-dystrophin (1.33X10) ¹⁴ vg/kg); subjects weighing more than or equal to 70kg on day 1 will receive 9.31x10 ¹⁵ vg raavrh74.mhck7. Total fixed dose of mini-dystrophin, equivalent to 1.33x10 in 70kg subjects ¹⁴ Dose of vg/kg. Starting from the day before infusion, subjects will receive at least 1mg/kg of glucocorticoid (prednisone equivalent) per day in addition to a baseline stable oral corticosteroid dose of at least 60 days after infusion; the 1 mg/kg/day added steroid will be administered followed by a total daily dose of 60 mg/day (except for the added steroid in the event of an associated increase in GGT and/or other clinically significant liver dysfunction). If GGT levels of 150U/L or other clinically significant liver dysfunction is confirmed, the glucocorticoid added for immunosuppression after infusion should be increased to 2mg/kg per day (or to 120 mg/day if the subject receives a fixed dose of 60 mg/day). The researcher may make subsequent adjustments to the immunosuppressive therapy to address subsequent acute liver injury or other adverse events. Liver disease specialists must be consulted if liver biochemical markers (including GGT, bilirubin and ALT relative to baseline) are elevated severely or very severely, or are not responsive to an elevation of 2 mg/kg/day or 120 mg/day, respectively. In this case, IV bolus steroids may be considered. The doses shown in examples 8 and 7 were determined using linearized DNA qPCR standards. In addition, as described herein The doses shown were determined using supercoiled DNAq PCR criteria. For example, 1.33X10 shown in examples 7 and 8 ¹⁴ The dose of vg/kg corresponds to 2X 10 as otherwise described herein ¹⁴ Dose of vg/kg. The experiments and studies described in examples 7 and 8 above utilize the one shown in SEQ ID NO: 9; as shown in SEQ ID NO. 8, nucleotide 1-4977; or is shown as SEQ ID NO. 6; rAAV rh74.MHCK7. Mini-dystrophin construct of nucleotides 56-5022.

A follow-up period of 4.260 weeks (post infusion) during which safety, efficacy and expression parameters will be assessed. The subject will be expected to attend remote and in-person visits to complete the required procedure/assessment. Part 1 of the follow-up period will start from post infusion (day 1) up to week 12. Part 2 of the follow-up period will start after week 12 until week 260. Frequent visits (almost once a week) are required for the first 12 weeks (part 1) after infusion. Additional unplanned visits are allowed at the discretion of the investigator. For subjects who completed the study, the last study visit will be at week 260. For subjects who follow-up after termination of infusion in advance, the follow-up will need to be terminated in advance; however, each subject should be strongly encouraged to continue study follow-up until 260 weeks after infusion.

The study was further expanded to include cohort 4 consisting of about 6 subjects with DMD-capable males from 3 years old to <4 years old. Subjects in cohort 4 who did not take oral corticosteroid for their DMD at the time of screening, began 1.5 mg/kg/day prednisolone 1 week prior to infusion, which would last at least 60 days after infusion.

The following exclusion criteria were specified only for queues 1 and 4: any treatment aimed at increasing expression of anti-dystrophin protein (e.g. Translarna ^TM 、EXONDYS 51、VYONDYS 53、VILTEPSO ^TM ). Treatments aimed at increasing expression of anti-dystrophin protein may resume and/or begin after week 72.

Example 9

Gene diagnosis

In additional studies, or in the above studies, if applicable, the subject must have a definitive diagnosis of DMD prior to screening based on a record of clinical findings and a prior validated genetic test using a clinical diagnostic genetic test.

Genetic testing is used to genotype patients for at least one mutation in the human dystrophin (DMD) gene. As used herein, "genotyping" refers to the process of determining the composition of a particular allele of a cell and/or subject at one or more locations within the genome by determining the nucleic acid sequence at that location. Genotyping refers to nucleic acid analysis and/or analysis at the nucleic acid level.

In one aspect, the method of treating DMD described herein further comprises genotyping the human dystrophin (DMD) gene of the human subject prior to administering the composition to the human subject.

In some aspects, the subject's human anti-dystrophin gene (DMD) is genotyped to characterize mutations in genes suitable for treatment with the compositions disclosed herein.

In some aspects, at least one mutation in exons 18 to 79 of the DMD gene of the subject is genotyped. In particular, subjects are genotyped for mutations that are expected to result in the loss of anti-dystrophin protein in the patient. For example, in some aspects, the subject is genotyped for a frameshift deletion, frameshift repeat, premature termination, or other pathogenic variation in the DMD gene that is entirely contained between exons 18 to 79. The identification of at least one of these mutations indicates that the patient is eligible to receive treatment according to the present disclosure.

In some aspects, patient genotyping of the DMD gene may provide results that indicate that the subject is unsuitable for treatment according to the present disclosure. For example, genotyping results showing mutations between exons 1-17 or comprising exons 1-17, in-frame deletions, in-frame duplications, ambiguous mutations ("VUS"), mutations completely contained within exon 45, indicate that the patient is unsuitable for treatment according to the present disclosure.

Many genotyping techniques are known to those skilled in the art.

Example 10

Phase 3 transnational, randomized, double-blind, placebo-controlled systemic gene delivery studies aimed at assessing the safety and efficacy of Delandistrogene Moxeparvovec in duchenne muscular dystrophy subjects.

This is a term that is between about 120 years of age of 4 or more and about 5 years of age<Study drug raavrh74.mhck7 in 8 year old male DMD-walkable subjects a randomized, double-blind, placebo-controlled two-part study of systemic gene delivery of mini-dystrophin (also as described in examples 6-8 above). Randomization is based on age group (. Gtoreq.4 to) at randomization<6 years of age vs. gtoreq.6 to<8 years old) and a NSAA total score (.ltoreq.22 vs) at screening.>22 Layering; at randomization, at least 50% of the subjects must be randomized into 4 or more to<Age group 6. All patients will have the opportunity to receive Intravenous (IV) study medication in either part 1 or part 2 (1.33x10 ¹⁴ vg/kg). In the treatment arm, the participants will receive a single Intravenous (IV) infusion of the study drug on day 1. The participants will then receive a single IV infusion of matched placebo at year 2. In the placebo arm, the participants will receive a matched placebo IV infusion on day 1. The participants will then have the opportunity to receive a single IV infusion of the study medication at year 2. The study will consist of the following 4 phases:

Screening period (pre-infusion) which starts at most 31 days before the first day of infusion and during which disease characteristics and baseline treatment will be assessed and pre-infusion assessment completed.

Baseline period (pre-infusion), which starts when qualification is confirmed, and ends the day before the first day of infusion, during which baseline assessment is completed.

Infusion period during which a single Intravenous (IV) infusion of blind study drug or placebo will be administered within 31 days after the rAAVrh74 enzyme-linked immunosorbent assay (ELISA) sample is obtained. During the infusion phase of part 1, approximately 60 subjects will receive IV study drug (1.33 x 10 ¹⁴ vg/kg; as determined using linear DNA qPCR standards), and approximately 60 subjects will receive placebo (normal saline, 0.9% sodium chloride solution). During the infusion phase of part 2, the subject receiving placebo in part 1 willSubjects receiving the IV study medication in part 1 will receive placebo. All subjects, parents/caregivers, researchers and on-site staff will be blinded to the subject treatment (study medication or placebo), except for the on-site pharmacist who is not blind. All subjects will receive additional glucocorticoid (prednisone equivalent) for at least 60 days from the day prior to infusion.

A 104 week follow-up period (after part 1 infusion) during which safety and efficacy parameters will be evaluated in parts 1 and 2. The subject will be expected to take part in remote and in-person visits to complete the required procedure/assessment. Additional unplanned visits are allowed at the discretion of the investigator. For subjects who completed the study, the last study visit will be at week 52 of part 2. For subjects following a follow-up after an early cessation of infusion, it will be necessary to end the study/terminate the follow-up early; however, each subject should be strongly encouraged to continue study follow-up until 52 weeks after each infusion.

Inclusion/exclusion criteria:

inclusion criteria:

1. male at birth, walking, and randomization at 4-8 years old.

2. Prior to screening, there was a definitive diagnosis of DMD based on clinical outcome records and confirmatory genetic tests previously performed using clinical diagnostic genetic tests. Genetic reports must describe frame shift deletions, frame shift repeats, premature termination ("nonsense"), typical splice site mutations, or other pathogenic variations in the DMD gene entirely contained between (inclusive of) exons 18 to 79, which are expected to result in loss of dystrophin protein.

a. Mutations between exons 1-17 or including exons 1-17 are disqualified.

b. In-frame deletions, in-frame duplicates, and ambiguous variations ("VUS") are disqualified.

c. Mutations contained entirely within exon 45 are not eligible.

3. Can cooperate with a motion assessment test.

4. NSAA score >16 and <29 at screening visit.

5. The time to rise from the floor at screening visit was <5 seconds.

6. Daily doses of oral corticosteroid were stable for at least 12 weeks prior to screening, and doses and treatment regimens were expected to remain unchanged throughout the study (except for modifications to accommodate changes in body weight).

7. The rAAVrh74 antibody titres were <1:400 (i.e. not elevated) as determined by ELISA.

8. Sexually active subjects must agree to use condoms throughout the duration of the study, and female sexual partners must also use medically acceptable contraceptive measures (e.g., oral contraceptives).

9. There are parents or legal guardians that can understand and follow the study visit schedule and all other regimen requirements.

10. A parent or legal guardian willing to provide informed consent (if applicable) and willing to provide informed consent to the subject to participate in the study.

Exclusion criteria

Subjects meeting any of the following criteria will be excluded from the study:

1. with <40% of left ventricular ejection fraction or clinical signs and/or symptoms of cardiomyopathy screened for ECHO.

2. Major surgery within 3 months prior to day 1, or surgery or procedure that would interfere with the study's planning at any time during the study.

3. There is an unnecessary risk that the researcher thinks will cause gene transfer or any other clinically significant disease that appears to the researcher to potentially impair the ability of the subject to follow the test or procedure required for the regimen or to impair the health, safety or clinical interpretable medical condition or situation of the subject, including heart, lung, liver, kidney, blood, immune or behavioral disease, or infection or malignancy or concomitant disease or need for long-term drug therapy.

4. There is serological evidence of current, chronic or active human immunodeficiency virus, hepatitis c or hepatitis b infection.

5. Symptomatic infections (e.g., upper respiratory tract infections, pneumonia, pyelonephritis, meningitis) appear within 4 weeks prior to day 1.

6. It appears that researchers believe that cognitive retardation or impairment may affect motor development.

7. Treatment was performed with any of the following therapies, according to the prescribed time frame:

Anytime:

gene therapy

Cell-based therapies (e.g., stem cell transplantation)

CRISPR/Cas9, or any other form of gene editing

Within 12 weeks after the first day and at any time during the study:

-using human growth factors or vamololone

Within 6 months after the first day and at any time during the study:

-any study drug

Any treatment aimed at increasing the expression of anti-dystrophin proteins (for example Translarna ^TM 、EXONDYS 51 ^TM 、VILTEPSO ^TM )

8. Live virus vaccine was received within 4 weeks after visit on day 1, or inactivated vaccine was received within 2 weeks after visit on day 1, or vaccination was expected to be received within the first 3 months after day 1.

9. Having abnormal laboratory values that are considered clinically significant, including but not limited to:

gamma-glutamyl transferase > 2-fold Upper Limit of Normal (ULN)

Glutamate dehydrogenase (GLDH) >15U/L

White cell count >18,500/. Mu.l

Platelet ∈150,000/. Mu.L

10. The family does not want to disclose study participation of the subject to the general practitioner/primary care physician and other medical providers.

11. Researchers believe that subjects are unlikely to follow the study regimen.

Gene diagnosis

Prior to screening, subjects must have a definitive diagnosis of DMD based on a record of clinical findings and a prior validated genetic test using a clinical diagnostic genetic test. Genetic reports must describe frame shift deletions, frame shift repeats, premature termination, or other pathogenic variations in the DMD gene entirely contained between exons 18 to 79 (inclusive) that are expected to result in loss of dystrophin.

a. Mutations between exons 1-17 or including exons 1-17 are disqualified.

c. Mutations contained entirely within exon 45 are not eligible.

The statistical method comprises the following steps:

sample amount:

the sample size of the study was based on the efficacy of the primary efficacy endpoint, i.e., the change in NSAA total score from baseline to week 52 (part 1).

It was assumed that a standard deviation of 3.5 and an exit rate of 10% at week 52 (part 1) (sample size with type 1 error of 0.05 (both sides), randomization ratio of 120 at 1:1) would provide approximately 90% efficacy to detect an average difference of 2.2 in NSAA total score change from baseline to week 52 (part 1) between study drug and placebo groups.

The study was also directed to age groups of ≡4 to <6 years. The study will enroll at least 60 subjects aged 4 years or more to <6 years. It is assumed that a standard deviation of ≡4 to < 3.2 for the primary endpoint in age groups of 6 years and an exit rate of 10% at week 52 (part 1) (sample size of 60 with a 0.05 type 1 error (both sides), randomization ratio of 1:1) will provide at least 80% efficacy to detect an average difference of 2.5 in the NSAA total score change from baseline to week 52 (part 1) between study drug and placebo groups.

The test procedure for the multiplex adjustment will be used to control the overall type 1 error to a two-sided level of 0.05, the details will be specified in SAP.

Randomization of

The subjects will be randomized at a 1:1 ratio to receive study drug or placebo through a single IV infusion. Subjects receiving study medication in study part 1 will receive placebo in part 2. Subjects receiving placebo in study part 1 will have an opportunity to receive study medication in part 2.

Randomization will be per age group at randomization (. Gtoreq.4 to)<Age 6 or greater than 6 to<8 years old) and NSAA total score (less than or equal to 22 or less) at screening>22 Layering; at randomization, at least 50% of the subjects must be randomized into 4 or more to <Age group 6. All patients will have the opportunity to receive Intravenous (IV) study medication in either part 1 or part 2 (1.33x10 ¹⁴ vg/kg)。

Corticosteroids

Subjects must take a stable dose of oral corticosteroid daily for at least 12 weeks prior to the primary screening visit, and the dose remains constant throughout the study (except for modifications to accommodate weight changes). All changes in corticosteroid type, dosing frequency, corticosteroid dose start and end dates and dose will be recorded in the subject's source files and eCRF.

Immunosuppressant prior to infusion

The day prior to infusion (study drug or placebo), subjects will begin additional glucocorticoids for immunosuppression (prednisone equivalent) in addition to the baseline stable oral corticosteroid for DMD. Subjects receiving baseline daily corticosteroid dosing for their DMD will take their usual dose of DMD corticosteroid in addition to the added immunosuppressive dose of 1 mg/kg/day. Administration of 1 mg/kg/day will then achieve a total daily dose of 60 mg/day.

Immunosuppressant after infusion

The subject will maintain its baseline daily stable oral corticosteroid dose for DMD for the first 60 days after infusion, and furthermore will take 1 mg/kg/day of glucocorticoid (prednisone equivalent) for immunosuppression. The advance taper may be allowed to manage Adverse Events (AEs) subject to approval by the medical monitor. Administration of 1 mg/kg/day will then reach a total daily dose of 60 mg/day, except for the added steroid in the event of elevated GGT and/or other clinically significant liver dysfunction.

If the subject is unable to tolerate the oral immunosuppressive glucocorticoid due to vomiting, the glucocorticoid should be administered intravenously.

If GGT levels of 150U/L or other clinically significant liver dysfunction is confirmed following infusion, the glucocorticoid added for immunosuppression following infusion should be increased. The researcher may make subsequent adjustments to the immunosuppressive therapy to address subsequent acute liver injury or other adverse events. Liver specialists must be consulted for severe or very severe elevations in liver biochemical markers (including GGT, bilirubin, GLDH and ALT relative to baseline), or elevations in response to 2 mg/kg/day or 120 mg/day. In this case, IV bolus steroids may be considered.

If the subject is taking 1mg/kg of steroid for immunosuppression addition in addition to his DMD steroid, the dose that should be taken in addition to his DMD steroid should be increased to 2mg/kg of steroid for immunosuppression addition.

If the subject receives a fixed dose of 60 mg/day, this should be increased to 120 mg/day.

Subjects with normal GGT values and no signs of acute liver injury on day 60 should gradually decrease their immunosuppressive glucocorticoids over 2 weeks. At the discretion of the investigator, the duration of the taper may be adjusted to manage AEs, but continued use of the steroid at doses exceeding the baseline daily treatment regimen after day 90 should be discussed with the medical monitor. Immunosuppressive glucocorticoids in subjects with elevated GGT values and/or signs of acute liver injury at day 60 should be managed as described above until GGT values are normalized (or obviously trended to normal) and all signs of acute liver injury subside, at which time they should gradually decrease their immunosuppressive glucocorticoids over 2 weeks. Once the additional steroids for immunosuppression are gradually reduced, they should maintain their baseline daily steroid treatment regimen for DMD and make any necessary adjustments based on body weight.

Efficacy analysis:

north Star walk evaluation (NSSA)

The primary endpoint and some secondary endpoints will be tested in a hierarchical fashion using an appropriate multiplex test method that provides strong control of the clan type 1 error rate at a double sided 0.05 level.

For the primary endpoint of change in NSAA total score from baseline to week 52 (part 1), summary statistics of NSAA total score at baseline, each post-baseline visit in part 1, and change from baseline to each post-baseline visit in part 1 will be provided by the treatment group. For baseline and week 52 (part 1) visits in which 2 NSAA scores will be collected, the average NSAA score will be used in the analysis.

As a primary analysis, the NSAA total score change from baseline to week 52 (part 1) for the 2 treatment groups will be compared using a mixed model repeat measurement analysis based on constrained maximum likelihood. In this model, the response vector consisted of the change from baseline in the NSAA total score for each post-baseline visit in part 1. The model will include covariates for treatment groups (classified), visit (classified), treatment groups interacted with per visit, age groups at randomization.

NSAA is a clinician-managed scale for rating the performance of various functional activities (Mazzone, E et al, neuroomuscul discord.20 (11): 712-716 (2010)). It is designed for DMD-enabled boys, and it has been used for DMD boys in the age range of the study (. Gtoreq.4 years and <8 years) (Connolly AM et al, "Motor and cognitive assessment of infants and young boys with Duchenne Muscular Dystrophy: results from the Muscular Dystrophy Association DMD Clinical Research Network," Neurokunul Disord.23 (7): 529-539 (2013)); mercury E et al, "Revised North Star Ambulatory Assessment for Young Boys with Duchenne Muscular Dystrophy," PLoS One 11 (8): E0160195 (2016)).

During this evaluation, the subject underwent 17 different functional activities including 10MWR, standing up from sitting, standing up with one leg, climbing up the box step, walking down the box step, sitting up from lying, lifting the head off the floor, standing up with the heel, and jumping.

The subjects will be ranked as follows: 2 = normal, no significant change in activity; 1 = modified approach, but without physical assistance of others to achieve the goal; and 0=no independent achievement of the objective.

Detailed information about NSAA management is provided in the clinical assessment manual.

Two NSAA scores will be collected on day 2 alone at baseline, day 2 alone at week 52 in part 1, and day 2 alone at week 52 in part 2.

Time to rise from floor

The time to rise from the floor test is part of NSAA (item 11) which quantifies the time required for a subject to stand in a double-armed upright position starting from a double-armed supine position (Henricson, EK et al, muscle Nerve 48 (1): 55-67 (2013)). The time required for the subject to complete the task will be recorded during NSAA management. As with NSAA, the time to rise from the floor will be collected at baseline and during 2 days of the 52 th week visit in part 1 and part 2.

10 meter walking/running

Timing 10MWR is part of NSAA (item 17) and quantifies the time required for a subject to run or walk 10 meters (on a straight walk) from a standing position (McDonald, CM et al, muscle Nerve 48 (3): 357-368 (2013)). Subjects were encouraged to run through the 10 meter mark. The time required for the subject to complete the distance will be recorded during NSAA management. As with NSAA, the timing 10MWR will be collected at baseline and 52 week visits at parts 1 and 2.

Time of ascending 4 steps

The timed 4 step test quantifies the time required for a subject to ascend 4 standard steps (6 inches in height per step) (Bushby, K et al, clin invest (lon) 1 (9): 1217-1235 (2011)). The time required for the subject to climb up to 4 standard-sized steps will be recorded.

100 meter walking/running

100MWR quantifies the time required for a subject to run or walk 100 meters (on a straight walk) from a standing position (Alfano, LN et al, neurokul Disord.27 (5): 452-457 (2017)). Subjects were encouraged to run through the 100 meter mark. The time required for the subject to complete the distance will be recorded.

Wearable device

A wearable device will be provided for the subject to collect daily physical activity. The purpose of the wearable device is to accurately measure the subject's locomotion and activity levels in normal daily life outside of the study site visit. The device consisted of 2 sensors, one of which was worn on each ankle. The sensor is not worn at night because the wearable device battery needs to be charged every day after use. The field personnel, subjects and parents/caregivers will receive training regarding proper use of the device.

During the pre-infusion, the wearable device will wear on both ankles for three weeks per day to capture the baseline value. During the follow-up, the wearable device will be worn on both ankles for three weeks per day before the 12 th, 24 th, 36 th and 52 th weeks in sections 1 and 2/prematurely terminate the office visit. The wearable device will not be worn at the visit of the clinic, but the subject will resume use immediately after the clinical test is completed.

Micro anti-muscular dystrophy protein expression

Muscle biopsies for assessing micro-dystrophin expression will be collected from a group of subjects at part 1, week 12 and part 2, week 12. Muscle biopsies should be collected using either open biopsies or VACORA core biopsies with sponsor approval. Biopsies must require collection of muscle tissue from the medial gastrocnemius muscle. If medial gastrocnemius is not feasible, the sponsor is required to approve in advance to use the replacement muscle.

The biopsy samples will be used to quantify the expression of the microdystrophin protein by western blotting adjusted for muscle content, and will use immunohistochemistry as IF intensity and PDPF to assess localization of the microdystrophin protein. For more details on the treatment and processing of biopsy tissue, see biopsy procedures and laboratory manuals.

Vector genome and quantification

Serum will be collected to assess vector quantification at different time points. On the first day, samples will be collected about 4 to 6 hours after the end of infusion.

Vector genome copies will be measured in muscle biopsy samples using polymerase chain reaction at part 1, week 12 and part 2, week 12.

Creatine kinase

The creatine kinase levels following drug infusion were studied to be used as exploratory efficacy metrics.

Patient report result measurement information system

A subset of subjects based on region availability will complete a proci measurement; the study protocol will outline the participating countries.

PROMIS is a series of tools developed and validated for assessing health-related quality of life (PROMIS Pediatric and parental agent profiling tools see http:// www.healthmeasures.net/images/PROMIS/agents/PROMIS_Pediatric_and_proxy_Profile_screening_Manual.pdf., 10October 2018,Bevans,KB et al Expert Rev Pharmacoecon Outcomes Res.10 (4): 385-396 (2010): PROMIS measurement has been developed according to methodological standards for assessing patient reported outcomes by the U.S. Food and Drug administration (Bevans, KB et al, food and Drug administration for Industry Patient-Reported Outcome Measures: use in Medical Product Development to Support Labeling Claims; htts:// www.fda.gov/downloads/keys/guide' 282.Pdf.;10October 2018), and specific measurements made by the human body, health and mental program of adults and children include the following specific measures for the present agent in the measurement:

PROMIS family Long agent project library V2.0-mobility

PROMIS family long proxy SF V2.0-upper limb-Profile 8a

PROMIS family long proxy SF V2.0-fatigue-Profile 10a

If the subject is over 8 years of age during the study, the subject will also perform and complete the following pediatric PROMIS measurements. The parent/caretaker will continue to complete the agent measurement.

PROMIS pediatric project library V2.0-mobility

PROMIS pediatric project library V2.0-upper limb-Profile 8a

PROMIS pediatric project library V2.0-fatigue-Profile 10a

Summary of key efficacy assessments in section 1:

main outcome measurement: change in NSAA total score from baseline at week 52.

Secondary outcome measure:

at week 12, the amount of mini-dystrophin expression of a subset of participants was measured by western blot (week 12).

-change from baseline of time to rise from floor at week 52, time to complete 100 and 10 meter walking/running, time to count 4 steps up stairs test (baseline, week 52)

Change from baseline (baseline to week 52) of pace 95 percentile (SV 95C) measured by wearable device

Changes from baseline in the results measurement information (PROMIS) score reported by each field patient at week 52 (baseline, week 52)

PROMIS is a series of tools developed and validated for assessing health-related quality of life. Parents will be asked "how do you evaluate changes in your clinical status since the beginning of the study given all aspects of your child's observable symptoms, physical ability, daily activity, and general health? The following scoring criteria were used: 1 = very much improved, 2 = very much improved, 3 = little improved, 4 = no change, 5 = slightly worse, 6 = much worse, 7 = very worse. "

-part 1: the amount of skill obtained or improved at week 52 (from baseline until week 52) as measured by NSAA.

Example 11

Post-systemic delivery Delandistrogene Moxeparvovec DMD ^mdx Assessment of cardiac function in rats

DMD ^mdx The rat model is a model withThe phenotypic properties of a small animal model of Duchenne Muscular Dystrophy (DMD) of value closely approximate human DMD pathology. See Larcher T. Et al, plos One 9 (10): e110371 (2014); szab, P.L. et al, diseases Models&Mechanisms 14:dmm047704.Doi:10.1242/dmm.047704 (2021). The purpose of this study was to evaluate male Sprague-Dawley DMD mutant rats (DMD) aged 21-28 days ^mdx Rats) deliver a single dose (1.33x10) throughout the body ¹⁴ vg/kg) delandistrogene moxeparvovec and compared to saline.

Study endpoints included safe pharmacological measurements, targeted mini-dystrophin expression in skeletal muscle and cardiac muscle, biodistribution, histology, and echocardiography to assess cardiac function. The treatment cohort included systemic delivery of a single dose (1.33x10 ¹⁴ vg/kg) rAAV rh74.MHCK7. DMD of a mini-anti-dystrophin protein vector (delandistrogene moxeparvovec) as described herein ^mdx Rats. The control queue included a DMD that received saline ^mdx Rats. Cardiac function between the two cohorts was assessed at 12 and 24 week time points as described below.

Targeted expression of tiny anti-muscular dystrophy proteins in skeletal and cardiac muscles

Systemic delivery of delandistrogene moxeparvovec has been demonstrated to target expression in both skeletal and cardiac muscles. DMD after treatment with delandistrogene moxeparvovec relative saline ^mdx Visualization of micro-dystrophin expression in the bones and myocardium of rats (immunofluorescence) is shown in fig. 21A-21C. Figures 21A-21C show DMD 12 weeks (figure 21B) and 24 weeks (figure 21C) compared to saline after treatment with delandistrogene moxeparvovec ^mdx Micro-dystrophin expression in skeletal muscle ("LTA") and cardiac muscle ("HRT") of rats (fig. 21A).

FIGS. 22A-22B depict DMDs at 12 weeks and 24 weeks after treatment with delandistrogene moxeparvovec ^mdx Quantification of mini-dystrophin expression (immunofluorescence) (fig. 22A) and vector transduction (vector genome copy) (fig. 22B) in some muscle tissues of rats. Legend: TA = tibial muscle; HRT = heart; MG = medial gastrocnemius; LG = lateral medial gastrocnemius; dia=Diaphragm muscle; TRI = triceps; PSO = PSO.

Reduction of muscle degeneration and fibrosis

In comparison with brine, in DMD ^mdx Significant reduction in muscle degeneration was observed in rats 12 and 24 weeks after administration delandistrogene moxeparvovec by skeletal muscle central nucleation analysis. Fig. 24A depicts H of the left medial gastrocnemius muscle&E staining, which illustrates DMD treated with saline ^mdx Delandistrogene moxeparvovec treated DMD compared to the more severe dystrophic phenotype exhibited in rats ^mdx Improvement of muscle pathology in rats.

Histological parameters in LMG were also quantified. Central nucleation and fiber diameter (an indication of muscle degeneration) were examined. See Pastret, C. Et al, J Neur Sci 129:97-105 (1995); rodio-Klapac et al Mol Genet.22:4929-4937 (2013), potter R.A. et al Hum Gene Ther.32:375-89 (2021). Average central nucleation showed an improvement in Central Nucleation (CN) of the treated animals, indicating less muscle degeneration in the treated animals. Saline-dosed rats exhibited an average positive central nucleation percentage of greater than 50% in both the 12-week and 24-week cohorts, while delandistrogene moxeparvovec central nucleation was less than 25% in both the 12-week and 24-week cohorts (fig. 24B). The differences between animals treated with saline and animals treated with delandistrogene moxeparvovec were statistically significant at both weeks 12 and 24 and showed improvement in the denaturation/regeneration process and reduction in myofiber injury with delandistrogene moxeparvovec treatment. At week 12 and 24, the fiber diameters treated with delandistrogene moxeparvovec showed normalization of the fiber size distribution and an increase in the average fiber diameter compared to normal saline, indicating a normalized muscle environment.

Fibrosis was analyzed by quantifying the percentage of collagen in tissue sections. Figures 25A-25B depict analysis of collagen deposition in skeletal and cardiac muscles. The image in fig. 25A depicts Ma Senmao chromosome staining 12 weeks after treatment. Representative images in fig. 25A show fibrosis of MG and HRT of delandistrogene moxeparvovec and saline treated animals at 12 weeks post-dose. Blue dyeingColor indicates fibrosis and red staining indicates muscle fiber. post-DMD treatment with delandistrogene moxeparvovec at 12 and 24 weeks compared to saline ^mdx Fibrosis was reduced in rats. Fig. 25B quantifies collagen deposition in various types of skeletal muscle and cardiac muscle at 12 and 24 weeks post-treatment. Quantification of fibrosis showed that the delandistrogene moxeparvovec treated cohort had a lower percentage of fibrotic area in each stained muscle section than the saline treated cohort in all tissues analyzed (fig. 25B). (HRT = heart; MG = medial gastrocnemius; DIA = diaphragm).

Cardiac function assessment

DMD was evaluated by echocardiography at 24 weeks after administration delandistrogene moxeparvovec, compared to saline ^mdx Ventricular contractility in rats. A bar graph depicting data for the "treatment" versus control ("saline") cohorts is shown in fig. 27A (for Left Ventricular End Systole Diameter (LVESD)), fig. 27B (for ejection fraction (%) (EF)) 1 and fig. 27C (for foreshortening fraction (%) (FS)).

DMD for relative saline treatment ^mdx Administration of rats, delandistrogene moxeparvovec resulted in modest improvements in Ejection Fraction (EF) (fig. 27B) and foreshortening Fraction (FS) (fig. 27B). The trend towards reverse diastole using delandistrogene moxeparvovec resulted in a slight decrease in LVIDd (left ventricular inside diameter, diastole) and LVIDs (left ventricular inside diameter, systole)/LVESD (left ventricular end systole diameter), as well as a decrease in End Diastole Volume (EDV). A slight decrease in end-systole diameter of the left ventricle 24 weeks after treatment with delandistrogene moxeparvovec versus saline is shown in the bar graph of fig. 27A.

The data shown in fig. 27A-27C (reflecting three echocardiographic measurements of cardiac function) show a positive trend toward durable cardiac function 24 weeks after treatment with delandistrogene moxeparvovec. Data support delandistrogene moxeparvovec at DMD ^mdx Improving cardiac function and reversing effects of adverse cardiac remodeling in rats.

Improved walking and vertical exercise

DMD ^mdx The movement of the rat is carried out in an open field moving cageMeasured by laser monitoring. Movement and vertical activity (measurement of vertical movement) were measured based on the number of beam breaks per hour. Over time, the diseased animal loses walking and vertical locomotion capabilities due to muscle damage. The moving cage was performed within two weeks of necropsy.

All treatment groups showed a significant increase in locomotion (including walking and vertical activity) at all time points. A statistically significant increase in walking and vertical activity was demonstrated 12 weeks after systemic delivery delandistrogene moxeparvovec (fig. 23A and 23B). The same trend appears at the 24-week time point.

Significant differences were observed in walking at 24 weeks in the treatment versus saline cohorts (fig. 23A). The improvement in vertical movement at 24 weeks was less pronounced but age-matched DMD with saline treatment ^mdx The queue phase still had statistical significance (fig. 23B).

In summary, the data show that DMD after treatment with delandistrogene moxeparvovec at 12 and 24 week time points compared to saline ^mdx The mobility of rats was significantly improved.

Serum troponin levels

At 1.33x10 ¹⁴ delandistrogene moxeparvovec (n=6) one week (7 days) after administration to saline (n=6), at DMD ^mdx Serum troponin I levels were assessed in a subset of rats. Troponin I was evaluated in different subgroups of animals at necropsy (n=5) at 12 weeks post-dose relative to physiological saline (n=5).

Troponin I measures the level of troponin I protein in serum. These proteins are released when the heart muscle is damaged, such as in a heart attack. The more severe the heart damage, the more troponin I content in the serum. Consistent with the DMD animal model, troponin I elevation in serum and heart disease was DMD ^mdx Characteristics of the rat model.

However, as shown in fig. 26, no significant differences in serum troponin 1 blood levels were observed between saline-treated animals and delandistrogene moxeparvovec-treated animals at 1 week post-dose and 12 weeks post-dose, indicating that gene therapy treatment did not cause cardiotoxicity.

Conclusion(s)

The study was aimed at testing delandistrogene moxeparvovec treatment on DMD ^mdx Efficacy and function improvement in rat models. DMD (digital micromirror device) ^mdx The rat model showed a higher DMD ratio ^mdx The mouse model is more severe phenotype and better mimics the cardiac and fibrotic phenotype of DMD patients.

After delandistrogene moxeparvovec treatment, the life function result of the movable cage is improved. Vertical locomotion and walking ability of treated animals were significantly improved compared to saline control at all time points. Sustained functional improvement at all time points indicated 1.33x10 of clinically expected dose ¹⁴ Persistence of expression and function of systemic delivery of vg/kg delandistrogene moxeparvovec.

Echocardiography showed improved cardiac performance using delandistrogene moxeparvovec systemic treatment at 12 and 24 weeks post-treatment. Overall, the data indicate that, in DMD ^mdx Improvement of cardiac function using delandistrogene moxeparvovec treatment during 24 weeks post-delivery in the rat model.

In DMD ^mdx In the rat model, tiny anti-dystrophin proteins are expressed in muscles including the heart, and this is associated with a reduction in dystrophic pathogenesis and improved walking ability. Ultrasound cardiography demonstrated that targeted expression of tiny anti-dystrophin proteins in the heart reduces cardiac fibrosis and improves cardiac function. In conclusion, delandistrogene moxeparvovec has shown targeted expression in skeletal muscle and cardiac muscle, which corresponds to improved functional outcomes (e.g. heart) in mdx rat models.

Reference material

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<223> adeno-associated virus

<400> 2

gtttaaacaa gcttgcatgt ctaagctaga cccttcagat taaaaataac tgaggtaagg 60

gcctgggtag gggaggtggt gtgagacgct cctgtctctc ctctatctgc ccatcggccc 120

tttggggagg aggaatgtgc ccaaggacta aaaaaaggcc atggagccag aggggcgagg 180

gcaacagacc tttcatgggc aaaccttggg gccctgctgt ctagcatgcc ccactacggg 240

tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct ggttataatt 300

aacccagaca tgtggctgcc cccccccccc caacacctgc tgcctctaaa aataaccctg 360

tccctggtgg atcccctgca tgcgaagatc ttcgaacaag gctgtggggg actgagggca 420

ggctgtaaca ggcttggggg ccagggctta tacgtgcctg ggactcccaa agtattactg 480

ttccatgttc ccggcgaagg gccagctgtc ccccgccagc tagactcagc acttagttta 540

ggaaccagtg agcaagtcag cccttggggc agcccataca aggccatggg gctgggcaag 600

ctgcacgcct gggtccgggg tgggcacggt gcccgggcaa cgagctgaaa gctcatctgc 660

tctcaggggc ccctccctgg ggacagcccc tcctggctag tcacaccctg taggctcctc 720

tatataaccc aggggcacag gggctgccct cattctacca ccacctccac agcacagaca 780

gacactcagg agccagccag cggcgcgccc 810

<210> 3

<211> 8562

<212> DNA

<213> artificial sequence

<220>

<223> adeno-associated virus

<400> 3

gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgcgcgc 60

tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc 120

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc 180

cttgtagtta atgattaacc cgccatgcta cttatctacg tagccatgct ctagagttta 240

aacaagcttg catgtctaag ctagaccctt cagattaaaa ataactgagg taagggcctg 300

ggtaggggag gtggtgtgag acgctcctgt ctctcctcta tctgcccatc ggccctttgg 360

ggaggaggaa tgtgcccaag gactaaaaaa aggccatgga gccagagggg cgagggcaac 420

agacctttca tgggcaaacc ttggggccct gctgtctagc atgccccact acgggtctag 480

gctgcccatg taaggaggca aggcctgggg acacccgaga tgcctggtta taattaaccc 540

agacatgtgg ctgccccccc ccccccaaca cctgctgcct ctaaaaataa ccctgtccct 600

ggtggatccc ctgcatgcga agatcttcga acaaggctgt gggggactga gggcaggctg 660

taacaggctt gggggccagg gcttatacgt gcctgggact cccaaagtat tactgttcca 720

tgttcccggc gaagggccag ctgtcccccg ccagctagac tcagcactta gtttaggaac 780

cagtgagcaa gtcagccctt ggggcagccc atacaaggcc atggggctgg gcaagctgca 840

cgcctgggtc cggggtgggc acggtgcccg ggcaacgagc tgaaagctca tctgctctca 900

ggggcccctc cctggggaca gcccctcctg gctagtcaca ccctgtaggc tcctctatat 960

aacccagggg cacaggggct gccctcattc taccaccacc tccacagcac agacagacac 1020

tcaggagcag ccagcggcgc gcccaggtaa gtttagtctt tttgtctttt atttcaggtc 1080

ccggatccgg tggtggtgca aatcaaagaa ctgctcctca gtggatgttg cctttacttc 1140

taggcctgta cggaagtgtt acttctgctc taaaagctgc ggaattgtac ccgcggccgc 1200

caccatgctg tggtgggagg aggtggagga ttgttatgaa agggaggacg tgcagaagaa 1260

gacttttacc aagtgggtga acgctcagtt cagcaaattt gggaagcagc acatcgagaa 1320

tctgttttcc gacctgcagg atgggagacg gctgctggat ctgctggaag gactgactgg 1380

ccagaagctg cccaaagaga aggggagcac tagggtgcac gccctgaaca acgtgaacaa 1440

agctctgaga gtgctgcaga acaacaacgt ggatctggtg aatattggca gtactgatat 1500

cgtggacggg aaccacaaac tgacactggg cctgatctgg aacattattc tgcactggca 1560

ggtgaaaaat gtgatgaaga acatcatggc cgggctgcag cagaccaatt ccgagaagat 1620

cctgctgtct tgggtgcggc agagcacccg caactatccc caggtgaacg tgattaactt 1680

cactacatcc tggagcgacg ggctggccct gaatgctctg attcacagcc acaggcctga 1740

tctgttcgac tggaatagcg tggtgtgcca gcagtctgcc acacagcgcc tggaacatgc 1800

cttcaatatc gctcggtacc agctggggat cgaaaaactg ctggacccag aggatgtgga 1860

cactacatac ccagataaaa agtctattct gatgtacatt actagcctgt tccaggtgct 1920

gccacagcag gtgtctattg aagccattca ggaggtggaa atgctgcccc gcccccccaa 1980

agtgactaaa gaggagcatt ttcagctgca tcatcagatg cattacagcc agcagattac 2040

cgtgagcctg gctcagggat atgagcgcac cagtagtcca aaaccacggt tcaagtccta 2100

cgcttatacc caggctgcct acgtgacaac tagcgaccct actagatccc cctttccatc 2160

ccagcacctg gaggccccag aggacaagag ctttgggtcc agcctgatgg aaagcgaggt 2220

gaatctggat cggtaccaga cagccctgga ggaggtgctg agctggctgc tgagtgctga 2280

agacacactg caggcccagg gcgaaatttc caatgacgtg gaagtggtga aggatcagtt 2340

ccacacacac gagggctata tgatggacct gacagctcac caggggcgcg tgggcaatat 2400

cctgcagctg ggctctaaac tgatcggcac cgggaaactg agtgaggacg aggaaacaga 2460

agtgcaggag cagatgaacc tgctgaacag ccgctgggag tgtctgagag tggctagtat 2520

ggagaagcag tccaacctgc accgggtgct gatggacctg cagaaccaga aactgaaaga 2580

gctgaacgac tggctgacaa agactgagga acgcacaagg aagatggagg aggagccact 2640

gggacccgac ctggaggatc tgaagagaca ggtgcagcag cataaggtgc tgcaggagga 2700

tctggaacag gagcaggtgc gggtgaactc cctgacacat atggtggtgg tggtggacga 2760

atctagtgga gatcacgcca ccgccgccct ggaggaacag ctgaaggtgc tgggggaccg 2820

gtgggccaac atttgccggt ggaccgagga caggtgggtg ctgctgcagg acatcctgct 2880

gaaatggcag aggctgaccg aggagcagtg tctgtttagt gcttggctga gcgagaaaga 2940

ggacgccgtg aacaagatcc acacaaccgg ctttaaggat cagaacgaaa tgctgtctag 3000

cctgcagaaa ctggctgtgc tgaaggccga tctggagaaa aagaagcaga gcatgggcaa 3060

actgtatagc ctgaaacagg acctgctgag caccctgaag aacaagagcg tgacccagaa 3120

gacagaagcc tggctggata actttgcccg ctgctgggac aacctggtgc agaaactgga 3180

gaaaagtaca gctcagatct ctcaggctgt gaccacaacc cagcctagcc tgacccagac 3240

aaccgtgatg gaaaccgtga ccaccgtgac aacccgcgaa cagatcctgg tgaaacatgc 3300

ccaggaagag ctgccacctc cacctcccca gaagaagaga accctggagc ggctgcagga 3360

gctgcaggaa gccactgacg aactggacct gaagctgagg caggccgaag tgattaaggg 3420

gtcttggcag cctgtgggcg atctgctgat tgattccctg caggaccacc tggaaaaggt 3480

gaaggctctg agaggcgaaa ttgctccact gaaggagaac gtgagtcatg tgaacgatct 3540

ggctagacag ctgacaacac tgggcatcca gctgagccca tacaatctga gcacactgga 3600

ggacctgaat accaggtgga agctgctgca ggtggctgtg gaagaccggg tgcggcagct 3660

gcatgaggcc catcgcgact tcggaccagc cagccagcac tttctgagca catccgtgca 3720

ggggccctgg gagagggcca tttctcccaa caaggtgccc tactatatta atcacgagac 3780

ccagaccact tgttgggacc atcccaagat gacagaactg taccagtccc tggccgatct 3840

gaacaacgtg aggtttagcg cttacagaac cgctatgaag ctgagacggc tgcagaaggc 3900

cctgtgcctg gatctgctgt ccctgtccgc cgcctgcgat gccctggatc agcataatct 3960

gaagcagaac gatcagccaa tggatatcct gcagatcatc aactgcctga ccactatcta 4020

cgacaggctg gagcaggagc acaacaacct ggtgaacgtg cctctgtgcg tggatatgtg 4080

cctgaactgg ctgctgaacg tgtatgacac tgggcgcacc ggccggatca gagtgctgag 4140

ttttaaaact gggattatct ccctgtgtaa ggcccacctg gaggacaagt acaggtacct 4200

gttcaagcag gtggctagta gcactggatt ttgtgaccag cgccgcctgg gactgctgct 4260

gcatgatagt atccagattc ctagacagct gggagaggtg gctagtttcg gaggatctaa 4320

catcgaaccc agcgtgcgca gctgtttcca gtttgccaat aacaaacctg aaatcgaggc 4380

tgctctgttc ctggattgga tgcgcctgga accacagagc atggtgtggc tgcctgtgct 4440

gcacagagtg gctgccgccg aaactgccaa gcaccaggct aaatgcaaca tctgcaagga 4500

atgtcccatt atcggctttc gctacaggag tctgaaacat tttaactacg atatttgcca 4560

gagctgcttc ttttccggaa gagtggccaa aggacacaag atgcactacc ctatggtgga 4620

atattgcacc ccaactacat ctggcgaaga tgtgcgcgat tttgccaagg tgctgaagaa 4680

taagtttcgg actaagaggt acttcgccaa gcacccccgc atggggtatc tgccagtgca 4740

gacagtgctg gaaggagaca atatggagac cgatacaatg tgagcggccg caataaaaga 4800

tctttatttt cattagatct gtgtgttggt tttttgtgtg tctagagcat ggctacgtag 4860

ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca 4920

ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 4980

cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctggcgt aatagcgaag 5040

aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgattcc 5100

gttgcaatgg ctggcggtaa tattgttctg gatattacca gcaaggccga tagtttgagt 5160

tcttctactc aggcaagtga tgttattact aatcaaagaa gtattgcgac aacggttaat 5220

ttgcgtgatg gacagactct tttactcggt ggcctcactg attataaaaa cacttctcag 5280

gattctggcg taccgttcct gtctaaaatc cctttaatcg gcctcctgtt tagctcccgc 5340

tctgattcta acgaggaaag cacgttatac gtgctcgtca aagcaaccat agtacgcgcc 5400

ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 5460

tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 5520

cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 5580

acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 5640

ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 5700

gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 5760

tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 5820

ttttaacaaa atattaacgc ttacaattta aatatttgct tatacaatct tcctgttttt 5880

ggggcttttc tgattatcaa ccggggtaca tatgattgac atgctagttt tacgattacc 5940

gttcatcgat tctcttgttt gctccagact ctcaggcaat gacctgatag cctttgtaga 6000

gacctctcaa aaatagctac cctctccggc atgaatttat cagctagaac ggttgaatat 6060

catattgatg gtgatttgac tgtctccggc ctttctcacc cgtttgaatc tttacctaca 6120

cattactcag gcattgcatt taaaatatat gagggttcta aaaattttta tccttgcgtt 6180

gaaataaagg cttctcccgc aaaagtatta cagggtcata atgtttttgg tacaaccgat 6240

ttagctttat gctctgaggc tttattgctt aattttgcta attctttgcc ttgcctgtat 6300

gatttattgg atgttggaat cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 6360

tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 6420

agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 6480

ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 6540

catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 6600

tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 6660

cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 6720

cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 6780

tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 6840

tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 6900

atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 6960

gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 7020

aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 7080

aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 7140

gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 7200

cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 7260

atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 7320

tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 7380

ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 7440

ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 7500

ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 7560

tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 7620

tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 7680

aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 7740

tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 7800

tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 7860

gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 7920

agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 7980

tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 8040

ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 8100

cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 8160

tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 8220

acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 8280

gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 8340

ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 8400

tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 8460

attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 8520

cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gc 8562

<210> 4

<211> 564

<212> DNA

<213> artificial sequence

<220>

<223> adeno-associated virus

<400> 4

cagccactat gggtctaggc tgcccatgta aggaggcaag gcctggggac acccgagatg 60

cctggttata attaacccag acatgtggct gctccccccc cccaacacct gctgcctgag 120

cctcaccccc accccggtgc ctgggtctta ggctctgtac accatggagg agaagctcgc 180

tctaaaaata accctgtccc tggtgggctg tgggggactg agggcaggct gtaacaggct 240

tgggggccag ggcttatacg tgcctgggac tcccaaagta ttactgttcc atgttcccgg 300

cgaagggcca gctgtccccc gccagctaga ctcagcactt agtttaggaa ccagtgagca 360

agtcagccct tggggcagcc catacaaggc catggggctg ggcaagctgc acgcctgggt 420

ccggggtggg cacggtgccc gggcaacgag ctgaaagctc atctgctctc aggggcccct 480

ccctggggac agcccctcct ggctagtcac accctgtagg ctcctctata taacccaggg 540

gcacaggggc tgcccccggg tcac 564

<210> 5

<211> 8409

<212> DNA

<213> artificial sequence

<220>

<223> adeno-associated virus

<400> 5

gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcgcg 60

ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc 120

cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt 180

ccttgtagtt aatgattaac ccgccatgct aattatctac gtagccatgt ctagacagcc 240

actatgggtc taggctgccc atgtaaggag gcaaggcctg gggacacccg agatgcctgg 300

ttataattaa cccagacatg tggctgctcc ccccccccaa cacctgctgc ctgagcctca 360

cccccacccc ggtgcctggg tcttaggctc tgtacaccat ggaggagaag ctcgctctaa 420

aaataaccct gtccctggtg ggctgtgggg gactgagggc aggctgtaac aggcttgggg 480

gccagggctt atacgtgcct gggactccca aagtattact gttccatgtt cccggcgaag 540

ggccagctgt cccccgccag ctagactcag cacttagttt aggaaccagt gagcaagtca 600

gcccttgggg cagcccatac aaggccatgg ggctgggcaa gctgcacgcc tgggtccggg 660

gtgggcacgg tgcccgggca acgagctgaa agctcatctg ctctcagggg cccctccctg 720

gggacagccc ctcctggcta gtcacaccct gtaggctcct ctatataacc caggggcaca 780

ggggctgccc ccgggtcacc accacctcca cagcacagac agacactcag gagccagcca 840

gccaggtaag tttagtcttt ttgtctttta tttcaggtcc cggatccggt ggtggtgcaa 900

atcaaagaac tgctcctcag tggatgttgc ctttacttct aggcctgtac ggaagtgtta 960

cttctgctct aaaagctgcg gaattgtacc cgcggccgcc accatgctgt ggtgggagga 1020

ggtggaggat tgttatgaaa gggaggacgt gcagaagaag acttttacca agtgggtgaa 1080

cgctcagttc agcaaatttg ggaagcagca catcgagaat ctgttttccg acctgcagga 1140

tgggagacgg ctgctggatc tgctggaagg actgactggc cagaagctgc ccaaagagaa 1200

ggggagcact agggtgcacg ccctgaacaa cgtgaacaaa gctctgagag tgctgcagaa 1260

caacaacgtg gatctggtga atattggcag tactgatatc gtggacggga accacaaact 1320

gacactgggc ctgatctgga acattattct gcactggcag gtgaaaaatg tgatgaagaa 1380

catcatggcc gggctgcagc agaccaattc cgagaagatc ctgctgtctt gggtgcggca 1440

gagcacccgc aactatcccc aggtgaacgt gattaacttc actacatcct ggagcgacgg 1500

gctggccctg aatgctctga ttcacagcca caggcctgat ctgttcgact ggaatagcgt 1560

ggtgtgccag cagtctgcca cacagcgcct ggaacatgcc ttcaatatcg ctcggtacca 1620

gctggggatc gaaaaactgc tggacccaga ggatgtggac actacatacc cagataaaaa 1680

gtctattctg atgtacatta ctagcctgtt ccaggtgctg ccacagcagg tgtctattga 1740

agccattcag gaggtggaaa tgctgccccg cccccccaaa gtgactaaag aggagcattt 1800

tcagctgcat catcagatgc attacagcca gcagattacc gtgagcctgg ctcagggata 1860

tgagcgcacc agtagtccaa aaccacggtt caagtcctac gcttataccc aggctgccta 1920

cgtgacaact agcgacccta ctagatcccc ctttccatcc cagcacctgg aggccccaga 1980

ggacaagagc tttgggtcca gcctgatgga aagcgaggtg aatctggatc ggtaccagac 2040

agccctggag gaggtgctga gctggctgct gagtgctgaa gacacactgc aggcccaggg 2100

cgaaatttcc aatgacgtgg aagtggtgaa ggatcagttc cacacacacg agggctatat 2160

gatggacctg acagctcacc aggggcgcgt gggcaatatc ctgcagctgg gctctaaact 2220

gatcggcacc gggaaactga gtgaggacga ggaaacagaa gtgcaggagc agatgaacct 2280

gctgaacagc cgctgggagt gtctgagagt ggctagtatg gagaagcagt ccaacctgca 2340

ccgggtgctg atggacctgc agaaccagaa actgaaagag ctgaacgact ggctgacaaa 2400

gactgaggaa cgcacaagga agatggagga ggagccactg ggacccgacc tggaggatct 2460

gaagagacag gtgcagcagc ataaggtgct gcaggaggat ctggaacagg agcaggtgcg 2520

ggtgaactcc ctgacacata tggtggtggt ggtggacgaa tctagtggag atcacgccac 2580

cgccgccctg gaggaacagc tgaaggtgct gggggaccgg tgggccaaca tttgccggtg 2640

gaccgaggac aggtgggtgc tgctgcagga catcctgctg aaatggcaga ggctgaccga 2700

ggagcagtgt ctgtttagtg cttggctgag cgagaaagag gacgccgtga acaagatcca 2760

cacaaccggc tttaaggatc agaacgaaat gctgtctagc ctgcagaaac tggctgtgct 2820

gaaggccgat ctggagaaaa agaagcagag catgggcaaa ctgtatagcc tgaaacagga 2880

cctgctgagc accctgaaga acaagagcgt gacccagaag acagaagcct ggctggataa 2940

ctttgcccgc tgctgggaca acctggtgca gaaactggag aaaagtacag ctcagatctc 3000

tcaggctgtg accacaaccc agcctagcct gacccagaca accgtgatgg aaaccgtgac 3060

caccgtgaca acccgcgaac agatcctggt gaaacatgcc caggaagagc tgccacctcc 3120

acctccccag aagaagagaa ccctggagcg gctgcaggag ctgcaggaag ccactgacga 3180

actggacctg aagctgaggc aggccgaagt gattaagggg tcttggcagc ctgtgggcga 3240

tctgctgatt gattccctgc aggaccacct ggaaaaggtg aaggctctga gaggcgaaat 3300

tgctccactg aaggagaacg tgagtcatgt gaacgatctg gctagacagc tgacaacact 3360

gggcatccag ctgagcccat acaatctgag cacactggag gacctgaata ccaggtggaa 3420

gctgctgcag gtggctgtgg aagaccgggt gcggcagctg catgaggccc atcgcgactt 3480

cggaccagcc agccagcact ttctgagcac atccgtgcag gggccctggg agagggccat 3540

ttctcccaac aaggtgccct actatattaa tcacgagacc cagaccactt gttgggacca 3600

tcccaagatg acagaactgt accagtccct ggccgatctg aacaacgtga ggtttagcgc 3660

ttacagaacc gctatgaagc tgagacggct gcagaaggcc ctgtgcctgg atctgctgtc 3720

cctgtccgcc gcctgcgatg ccctggatca gcataatctg aagcagaacg atcagccaat 3780

ggatatcctg cagatcatca actgcctgac cactatctac gacaggctgg agcaggagca 3840

caacaacctg gtgaacgtgc ctctgtgcgt ggatatgtgc ctgaactggc tgctgaacgt 3900

gtatgacact gggcgcaccg gccggatcag agtgctgagt tttaaaactg ggattatctc 3960

cctgtgtaag gcccacctgg aggacaagta caggtacctg ttcaagcagg tggctagtag 4020

cactggattt tgtgaccagc gccgcctggg actgctgctg catgatagta tccagattcc 4080

tagacagctg ggagaggtgg ctagtttcgg aggatctaac atcgaaccca gcgtgcgcag 4140

ctgtttccag tttgccaata acaaacctga aatcgaggct gctctgttcc tggattggat 4200

gcgcctggaa ccacagagca tggtgtggct gcctgtgctg cacagagtgg ctgccgccga 4260

aactgccaag caccaggcta aatgcaacat ctgcaaggaa tgtcccatta tcggctttcg 4320

ctacaggagt ctgaaacatt ttaactacga tatttgccag agctgcttct tttccggaag 4380

agtggccaaa ggacacaaga tgcactaccc tatggtggaa tattgcaccc caactacatc 4440

tggcgaagat gtgcgcgatt ttgccaaggt gctgaagaat aagtttcgga ctaagaggta 4500

cttcgccaag cacccccgca tggggtatct gccagtgcag acagtgctgg aaggagacaa 4560

tatggagacc gatacaatgt gagcggccgc aataaaagat ctttattttc attagatctg 4620

tgtgttggtt ttttgtgtgt ctagagcatg gctacgtaga taagtagcat ggcgggttaa 4680

tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 4740

cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 4800

cagtgagcga gcgagcgcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 4860

tcccaacagt tgcgcagcct gaatggcgaa tggaagttcc agacgattga gcgtcaaaat 4920

gtaggtattt ccatgagcgt ttttcctgtt gcaatggctg gcggtaatat tgttctggat 4980

attaccagca aggccgatag tttgagttct tctactcagg caagtgatgt tattactaat 5040

caaagaagta ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc 5100

ctcactgatt ataaaaacac ttctcaggat tctggcgtac cgttcctgtc taaaatccct 5160

ttaatcggcc tcctgtttag ctcccgctct gattctaacg aggaaagcac gttatacgtg 5220

ctcgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 5280

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 5340

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 5400

ccctttaggg ttccgattta gtgatttacg gcacctcgac cccaaaaaac ttgattaggg 5460

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 5520

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 5580

ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 5640

gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttaaat 5700

atttgcttat acaatcttcc tgtttttggg gcttttctga ttatcaaccg gggtacatat 5760

gattgacatg ctagttttac gattaccgtt catcgattct cttgtttgct ccagactctc 5820

aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg 5880

aatttatcag ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt 5940

tctcacccgt ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag 6000

ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag 6060

ggtcataatg tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat 6120

tttgctaatt ctttgccttg cctgtatgat ttattggatg ttggaagttc ctgatgcggt 6180

attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 6240

tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 6300

cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 6360

gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 6420

tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 6480

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 6540

atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 6600

agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 6660

ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 6720

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 6780

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 6840

tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 6900

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 6960

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 7020

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 7080

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 7140

cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 7200

tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 7260

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 7320

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 7380

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 7440

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 7500

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 7560

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 7620

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 7680

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 7740

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 7800

agttaggcca ccacttcaag aactctgtag caccgcgtac atacctcgct ctgctaatcc 7860

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 7920

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 7980

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 8040

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 8100

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 8160

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 8220

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 8280

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gggtttgagt 8340

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaccaag 8400

cggaagagc 8409

<210> 6

<211> 8611

<212> DNA

<213> artificial sequence

<220>

<223> adeno-associated virus

<400> 6

gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcgcg 60

ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc 120

cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt 180

ccttgtagtt aatgattaac ccgccatgct aattatctac gtagccatgt ctagagttta 240

aacaagcttg catgtctaag ctagaccctt cagattaaaa ataactgagg taagggcctg 300

ggtaggggag gtggtgtgag acgctcctgt ctctcctcta tctgcccatc ggccctttgg 360

ggaggaggaa tgtgcccaag gactaaaaaa aggccatgga gccagagggg cgagggcaac 420

agacctttca tgggcaaacc ttggggccct gctgtctagc atgccccact acgggtctag 480

gctgcccatg taaggaggca aggcctgggg acacccgaga tgcctggtta taattaaccc 540

agacatgtgg ctgccccccc ccccccaaca cctgctgcct ctaaaaataa ccctgtccct 600

ggtggatccc ctgcatgcga agatcttcga acaaggctgt gggggactga gggcaggctg 660

taacaggctt gggggccagg gcttatacgt gcctgggact cccaaagtat tactgttcca 720

tgttcccggc gaagggccag ctgtcccccg ccagctagac tcagcactta gtttaggaac 780

cagtgagcaa gtcagccctt ggggcagccc atacaaggcc atggggctgg gcaagctgca 840

cgcctgggtc cggggtgggc acggtgcccg ggcaacgagc tgaaagctca tctgctctca 900

ggggcccctc cctggggaca gcccctcctg gctagtcaca ccctgtaggc tcctctatat 960

aacccagggg cacaggggct gccctcattc taccaccacc tccacagcac agacagacac 1020

tcaggagcca gccagcggcg cgcccaggta agtttagtct ttttgtcttt tatttcaggt 1080

cccggatccg gtggtggtgc aaatcaaaga actgctcctc agtggatgtt gcctttactt 1140

ctaggcctgt acggaagtgt tacttctgct ctaaaagctg cggaattgta cccgcggccg 1200

ccaccatgct gtggtgggag gaggtggagg attgttatga aagggaggac gtgcagaaga 1260

agacttttac caagtgggtg aacgctcagt tcagcaaatt tgggaagcag cacatcgaga 1320

atctgttttc cgacctgcag gatgggagac ggctgctgga tctgctggaa ggactgactg 1380

gccagaagct gcccaaagag aaggggagca ctagggtgca cgccctgaac aacgtgaaca 1440

aagctctgag agtgctgcag aacaacaacg tggatctggt gaatattggc agtactgata 1500

tcgtggacgg gaaccacaaa ctgacactgg gcctgatctg gaacattatt ctgcactggc 1560

aggtgaaaaa tgtgatgaag aacatcatgg ccgggctgca gcagaccaat tccgagaaga 1620

tcctgctgtc ttgggtgcgg cagagcaccc gcaactatcc ccaggtgaac gtgattaact 1680

tcactacatc ctggagcgac gggctggccc tgaatgctct gattcacagc cacaggcctg 1740

atctgttcga ctggaatagc gtggtgtgcc agcagtctgc cacacagcgc ctggaacatg 1800

ccttcaatat cgctcggtac cagctgggga tcgaaaaact gctggaccca gaggatgtgg 1860

acactacata cccagataaa aagtctattc tgatgtacat tactagcctg ttccaggtgc 1920

tgccacagca ggtgtctatt gaagccattc aggaggtgga aatgctgccc cgccccccca 1980

aagtgactaa agaggagcat tttcagctgc atcatcagat gcattacagc cagcagatta 2040

ccgtgagcct ggctcaggga tatgagcgca ccagtagtcc aaaaccacgg ttcaagtcct 2100

acgcttatac ccaggctgcc tacgtgacaa ctagcgaccc tactagatcc ccctttccat 2160

cccagcacct ggaggcccca gaggacaaga gctttgggtc cagcctgatg gaaagcgagg 2220

tgaatctgga tcggtaccag acagccctgg aggaggtgct gagctggctg ctgagtgctg 2280

aagacacact gcaggcccag ggcgaaattt ccaatgacgt ggaagtggtg aaggatcagt 2340

tccacacaca cgagggctat atgatggacc tgacagctca ccaggggcgc gtgggcaata 2400

tcctgcagct gggctctaaa ctgatcggca ccgggaaact gagtgaggac gaggaaacag 2460

aagtgcagga gcagatgaac ctgctgaaca gccgctggga gtgtctgaga gtggctagta 2520

tggagaagca gtccaacctg caccgggtgc tgatggacct gcagaaccag aaactgaaag 2580

agctgaacga ctggctgaca aagactgagg aacgcacaag gaagatggag gaggagccac 2640

tgggacccga cctggaggat ctgaagagac aggtgcagca gcataaggtg ctgcaggagg 2700

atctggaaca ggagcaggtg cgggtgaact ccctgacaca tatggtggtg gtggtggacg 2760

aatctagtgg agatcacgcc accgccgccc tggaggaaca gctgaaggtg ctgggggacc 2820

ggtgggccaa catttgccgg tggaccgagg acaggtgggt gctgctgcag gacatcctgc 2880

tgaaatggca gaggctgacc gaggagcagt gtctgtttag tgcttggctg agcgagaaag 2940

aggacgccgt gaacaagatc cacacaaccg gctttaagga tcagaacgaa atgctgtcta 3000

gcctgcagaa actggctgtg ctgaaggccg atctggagaa aaagaagcag agcatgggca 3060

aactgtatag cctgaaacag gacctgctga gcaccctgaa gaacaagagc gtgacccaga 3120

agacagaagc ctggctggat aactttgccc gctgctggga caacctggtg cagaaactgg 3180

agaaaagtac agctcagatc tctcaggctg tgaccacaac ccagcctagc ctgacccaga 3240

caaccgtgat ggaaaccgtg accaccgtga caacccgcga acagatcctg gtgaaacatg 3300

cccaggaaga gctgccacct ccacctcccc agaagaagag aaccctggag cggctgcagg 3360

agctgcagga agccactgac gaactggacc tgaagctgag gcaggccgaa gtgattaagg 3420

ggtcttggca gcctgtgggc gatctgctga ttgattccct gcaggaccac ctggaaaagg 3480

tgaaggctct gagaggcgaa attgctccac tgaaggagaa cgtgagtcat gtgaacgatc 3540

tggctagaca gctgacaaca ctgggcatcc agctgagccc atacaatctg agcacactgg 3600

aggacctgaa taccaggtgg aagctgctgc aggtggctgt ggaagaccgg gtgcggcagc 3660

tgcatgaggc ccatcgcgac ttcggaccag ccagccagca ctttctgagc acatccgtgc 3720

aggggccctg ggagagggcc atttctccca acaaggtgcc ctactatatt aatcacgaga 3780

cccagaccac ttgttgggac catcccaaga tgacagaact gtaccagtcc ctggccgatc 3840

tgaacaacgt gaggtttagc gcttacagaa ccgctatgaa gctgagacgg ctgcagaagg 3900

ccctgtgcct ggatctgctg tccctgtccg ccgcctgcga tgccctggat cagcataatc 3960

tgaagcagaa cgatcagcca atggatatcc tgcagatcat caactgcctg accactatct 4020

acgacaggct ggagcaggag cacaacaacc tggtgaacgt gcctctgtgc gtggatatgt 4080

gcctgaactg gctgctgaac gtgtatgaca ctgggcgcac cggccggatc agagtgctga 4140

gttttaaaac tgggattatc tccctgtgta aggcccacct ggaggacaag tacaggtacc 4200

tgttcaagca ggtggctagt agcactggat tttgtgacca gcgccgcctg ggactgctgc 4260

tgcatgatag tatccagatt cctagacagc tgggagaggt ggctagtttc ggaggatcta 4320

acatcgaacc cagcgtgcgc agctgtttcc agtttgccaa taacaaacct gaaatcgagg 4380

ctgctctgtt cctggattgg atgcgcctgg aaccacagag catggtgtgg ctgcctgtgc 4440

tgcacagagt ggctgccgcc gaaactgcca agcaccaggc taaatgcaac atctgcaagg 4500

aatgtcccat tatcggcttt cgctacagga gtctgaaaca ttttaactac gatatttgcc 4560

agagctgctt cttttccgga agagtggcca aaggacacaa gatgcactac cctatggtgg 4620

aatattgcac cccaactaca tctggcgaag atgtgcgcga ttttgccaag gtgctgaaga 4680

ataagtttcg gactaagagg tacttcgcca agcacccccg catggggtat ctgccagtgc 4740

agacagtgct ggaaggagac aatatggaga ccgatacaat gtgagcggcc gcaataaaag 4800

atctttattt tcattagatc tgtgtgttgg ttttttgtgt gtctagagca tggctacgta 4860

gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc 4920

actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc 4980

ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gccagctggc gtaatagcga 5040

agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaagtt 5100

ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc gtttttcctg ttgcaatggc 5160

tggcggtaat attgttctgg atattaccag caaggccgat agtttgagtt cttctactca 5220

ggcaagtgat gttattacta atcaaagaag tattgcgaca acggttaatt tgcgtgatgg 5280

acagactctt ttactcggtg gcctcactga ttataaaaac acttctcagg attctggcgt 5340

accgttcctg tctaaaatcc ctttaatcgg cctcctgttt agctcccgct ctgattctaa 5400

cgaggaaagc acgttatacg tgctcgtcaa agcaaccata gtacgcgccc tgtagcggcg 5460

cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 5520

tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 5580

gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgattta cggcacctcg 5640

accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 5700

tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 5760

gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 5820

cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 5880

tattaacgtt tacaatttaa atatttgctt atacaatctt cctgtttttg gggcttttct 5940

gattatcaac cggggtacat atgattgaca tgctagtttt acgattaccg ttcatcgatt 6000

ctcttgtttg ctccagactc tcaggcaatg acctgatagc ctttgtagag acctctcaaa 6060

aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc atattgatgg 6120

tgatttgact gtctccggcc tttctcaccc gtttgaatct ttacctacac attactcagg 6180

cattgcattt aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc 6240

ttctcccgca aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg 6300

ctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga 6360

tgttggaagt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 6420

atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac 6480

ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 6540

caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 6600

cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata 6660

atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 6720

ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 6780

cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 6840

cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 6900

aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 6960

ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 7020

gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 7080

cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 7140

acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 7200

gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 7260

aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 7320

ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 7380

ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 7440

gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 7500

aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 7560

aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 7620

aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 7680

gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 7740

gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 7800

tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 7860

gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 7920

caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 7980

actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcgt 8040

acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 8100

cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 8160

gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 8220

cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 8280

gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 8340

tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 8400

tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 8460

gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 8520

aaccgtatta ccgggtttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 8580

agcgagtcag tgagcgacca agcggaagag c 8611

<210> 7

<211> 792

<212> DNA

<213> artificial sequence

<220>

<223> adeno-associated virus

<400> 7

aagcttgcat gtctaagcta gacccttcag attaaaaata actgaggtaa gggcctgggt 60

aggggaggtg gtgtgagacg ctcctgtctc tcctctatct gcccatcggc cctttgggga 120

ggaggaatgt gcccaaggac taaaaaaagg ccatggagcc agaggggcga gggcaacaga 180

cctttcatgg gcaaaccttg gggccctgct gtctagcatg ccccactacg ggtctaggct 240

gcccatgtaa ggaggcaagg cctggggaca cccgagatgc ctggttataa ttaacccaga 300

catgtggctg cccccccccc cccaacacct gctgcctcta aaaataaccc tgtccctggt 360

ggatcccctg catgcgaaga tcttcgaaca aggctgtggg ggactgaggg caggctgtaa 420

caggcttggg ggccagggct tatacgtgcc tgggactccc aaagtattac tgttccatgt 480

tcccggcgaa gggccagctg tcccccgcca gctagactca gcacttagtt taggaaccag 540

tgagcaagtc agcccttggg gcagcccata caaggccatg gggctgggca agctgcacgc 600

ctgggtccgg ggtgggcacg gtgcccgggc aacgagctga aagctcatct gctctcaggg 660

gcccctccct ggggacagcc cctcctggct agtcacaccc tgtaggctcc tctatataac 720

ccaggggcac aggggctgcc ctcattctac caccacctcc acagcacaga cagacactca 780

ggagcagcca gc 792

<210> 8

<211> 8629

<212> DNA

<213> artificial sequence

<220>

<223> kanamycin plasmid

<400> 8

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 180

agtttaaaca agcttgcatg tctaagctag acccttcaga ttaaaaataa ctgaggtaag 240

ggcctgggta ggggaggtgg tgtgagacgc tcctgtctct cctctatctg cccatcggcc 300

ctttggggag gaggaatgtg cccaaggact aaaaaaaggc catggagcca gaggggcgag 360

ggcaacagac ctttcatggg caaaccttgg ggccctgctg tctagcatgc cccactacgg 420

gtctaggctg cccatgtaag gaggcaaggc ctggggacac ccgagatgcc tggttataat 480

taacccagac atgtggctgc cccccccccc ccaacacctg ctgcctctaa aaataaccct 540

gtccctggtg gatcccctgc atgcgaagat cttcgaacaa ggctgtgggg gactgagggc 600

aggctgtaac aggcttgggg gccagggctt atacgtgcct gggactccca aagtattact 660

gttccatgtt cccggcgaag ggccagctgt cccccgccag ctagactcag cacttagttt 720

aggaaccagt gagcaagtca gcccttgggg cagcccatac aaggccatgg ggctgggcaa 780

gctgcacgcc tgggtccggg gtgggcacgg tgcccgggca acgagctgaa agctcatctg 840

ctctcagggg cccctccctg gggacagccc ctcctggcta gtcacaccct gtaggctcct 900

ctatataacc caggggcaca ggggctgccc tcattctacc accacctcca cagcacagac 960

agacactcag gagcagccag cggcgcgccc aggtaagttt agtctttttg tcttttattt 1020

caggtcccgg atccggtggt ggtgcaaatc aaagaactgc tcctcagtgg atgttgcctt 1080

tacttctagg cctgtacgga agtgttactt ctgctctaaa agctgcggaa ttgtacccgc 1140

ggccgccacc atgctgtggt gggaggaggt ggaggattgt tatgaaaggg aggacgtgca 1200

gaagaagact tttaccaagt gggtgaacgc tcagttcagc aaatttggga agcagcacat 1260

cgagaatctg ttttccgacc tgcaggatgg gagacggctg ctggatctgc tggaaggact 1320

gactggccag aagctgccca aagagaaggg gagcactagg gtgcacgccc tgaacaacgt 1380

gaacaaagct ctgagagtgc tgcagaacaa caacgtggat ctggtgaata ttggcagtac 1440

tgatatcgtg gacgggaacc acaaactgac actgggcctg atctggaaca ttattctgca 1500

ctggcaggtg aaaaatgtga tgaagaacat catggccggg ctgcagcaga ccaattccga 1560

gaagatcctg ctgtcttggg tgcggcagag cacccgcaac tatccccagg tgaacgtgat 1620

taacttcact acatcctgga gcgacgggct ggccctgaat gctctgattc acagccacag 1680

gcctgatctg ttcgactgga atagcgtggt gtgccagcag tctgccacac agcgcctgga 1740

acatgccttc aatatcgctc ggtaccagct ggggatcgaa aaactgctgg acccagagga 1800

tgtggacact acatacccag ataaaaagtc tattctgatg tacattacta gcctgttcca 1860

ggtgctgcca cagcaggtgt ctattgaagc cattcaggag gtggaaatgc tgccccgccc 1920

ccccaaagtg actaaagagg agcattttca gctgcatcat cagatgcatt acagccagca 1980

gattaccgtg agcctggctc agggatatga gcgcaccagt agtccaaaac cacggttcaa 2040

gtcctacgct tatacccagg ctgcctacgt gacaactagc gaccctacta gatccccctt 2100

tccatcccag cacctggagg ccccagagga caagagcttt gggtccagcc tgatggaaag 2160

cgaggtgaat ctggatcggt accagacagc cctggaggag gtgctgagct ggctgctgag 2220

tgctgaagac acactgcagg cccagggcga aatttccaat gacgtggaag tggtgaagga 2280

tcagttccac acacacgagg gctatatgat ggacctgaca gctcaccagg ggcgcgtggg 2340

caatatcctg cagctgggct ctaaactgat cggcaccggg aaactgagtg aggacgagga 2400

aacagaagtg caggagcaga tgaacctgct gaacagccgc tgggagtgtc tgagagtggc 2460

tagtatggag aagcagtcca acctgcaccg ggtgctgatg gacctgcaga accagaaact 2520

gaaagagctg aacgactggc tgacaaagac tgaggaacgc acaaggaaga tggaggagga 2580

gccactggga cccgacctgg aggatctgaa gagacaggtg cagcagcata aggtgctgca 2640

ggaggatctg gaacaggagc aggtgcgggt gaactccctg acacatatgg tggtggtggt 2700

ggacgaatct agtggagatc acgccaccgc cgccctggag gaacagctga aggtgctggg 2760

ggaccggtgg gccaacattt gccggtggac cgaggacagg tgggtgctgc tgcaggacat 2820

cctgctgaaa tggcagaggc tgaccgagga gcagtgtctg tttagtgctt ggctgagcga 2880

gaaagaggac gccgtgaaca agatccacac aaccggcttt aaggatcaga acgaaatgct 2940

gtctagcctg cagaaactgg ctgtgctgaa ggccgatctg gagaaaaaga agcagagcat 3000

gggcaaactg tatagcctga aacaggacct gctgagcacc ctgaagaaca agagcgtgac 3060

ccagaagaca gaagcctggc tggataactt tgcccgctgc tgggacaacc tggtgcagaa 3120

actggagaaa agtacagctc agatctctca ggctgtgacc acaacccagc ctagcctgac 3180

ccagacaacc gtgatggaaa ccgtgaccac cgtgacaacc cgcgaacaga tcctggtgaa 3240

acatgcccag gaagagctgc cacctccacc tccccagaag aagagaaccc tggagcggct 3300

gcaggagctg caggaagcca ctgacgaact ggacctgaag ctgaggcagg ccgaagtgat 3360

taaggggtct tggcagcctg tgggcgatct gctgattgat tccctgcagg accacctgga 3420

aaaggtgaag gctctgagag gcgaaattgc tccactgaag gagaacgtga gtcatgtgaa 3480

cgatctggct agacagctga caacactggg catccagctg agcccataca atctgagcac 3540

actggaggac ctgaatacca ggtggaagct gctgcaggtg gctgtggaag accgggtgcg 3600

gcagctgcat gaggcccatc gcgacttcgg accagccagc cagcactttc tgagcacatc 3660

cgtgcagggg ccctgggaga gggccatttc tcccaacaag gtgccctact atattaatca 3720

cgagacccag accacttgtt gggaccatcc caagatgaca gaactgtacc agtccctggc 3780

cgatctgaac aacgtgaggt ttagcgctta cagaaccgct atgaagctga gacggctgca 3840

gaaggccctg tgcctggatc tgctgtccct gtccgccgcc tgcgatgccc tggatcagca 3900

taatctgaag cagaacgatc agccaatgga tatcctgcag atcatcaact gcctgaccac 3960

tatctacgac aggctggagc aggagcacaa caacctggtg aacgtgcctc tgtgcgtgga 4020

tatgtgcctg aactggctgc tgaacgtgta tgacactggg cgcaccggcc ggatcagagt 4080

gctgagtttt aaaactggga ttatctccct gtgtaaggcc cacctggagg acaagtacag 4140

gtacctgttc aagcaggtgg ctagtagcac tggattttgt gaccagcgcc gcctgggact 4200

gctgctgcat gatagtatcc agattcctag acagctggga gaggtggcta gtttcggagg 4260

atctaacatc gaacccagcg tgcgcagctg tttccagttt gccaataaca aacctgaaat 4320

cgaggctgct ctgttcctgg attggatgcg cctggaacca cagagcatgg tgtggctgcc 4380

tgtgctgcac agagtggctg ccgccgaaac tgccaagcac caggctaaat gcaacatctg 4440

caaggaatgt cccattatcg gctttcgcta caggagtctg aaacatttta actacgatat 4500

ttgccagagc tgcttctttt ccggaagagt ggccaaagga cacaagatgc actaccctat 4560

ggtggaatat tgcaccccaa ctacatctgg cgaagatgtg cgcgattttg ccaaggtgct 4620

gaagaataag tttcggacta agaggtactt cgccaagcac ccccgcatgg ggtatctgcc 4680

agtgcagaca gtgctggaag gagacaatat ggagaccgat acaatgtgag cggccgcaat 4740

aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgtcta gagtcgacca 4800

gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta 4860

gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca 4920

aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc 4980

tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 5040

cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 5100

actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 5160

gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 5220

ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 5280

acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 5340

ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 5400

cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 5460

tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 5520

gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 5580

ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 5640

acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 5700

gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 5760

ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 5820

tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 5880

gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 5940

tctaaagtat atatgagtaa aaatattccg gaattgccag ctggggcgcc ctctggtaag 6000

gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc 6060

aggggatcaa gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 6120

ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 6180

caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 6240

gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg 6300

cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 6360

gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatcc 6420

caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 6480

cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 6540

actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 6600

gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc 6660

gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 6720

ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 6780

cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 6840

atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 6900

accggtaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 6960

aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 7020

ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 7080

ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc 7140

cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg 7200

cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg 7260

tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 7320

gcatcaggcg attccaacat ccaataaatc atacaggcaa ggcaaagaat tagcaaaatt 7380

aagcaataaa gcctcagagc ataaagctaa atcggttgta ccaaaaacat tatgaccctg 7440

taatactttt gcgggagaag cctttatttc aacgcaagga taaaaatttt tagaaccctc 7500

atatatttta aatgcaatgc ctgagtaatg tgtaggtaaa gattcaaacg ggtgagaaag 7560

gccggagaca gtcaaatcac catcaatatg atattcaacc gttctagctg ataaattcat 7620

gccggagagg gtagctattt ttgagaggtc tctacaaagg ctatcaggtc attgcctgag 7680

agtctggagc aaacaagaga atcgatgaac ggtaatcgta aaactagcat gtcaatcata 7740

tgtaccccgg ttgataatca gaaaagcccc aaaaacagga agattgtata agcaaatatt 7800

taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca 7860

ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag 7920

atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc 7980

aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc 8040

taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc 8100

ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa 8160

gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc 8220

acacccgccg cgcttaatgc gccgctacag ggcgcgtact atggttgctt tgacgagcac 8280

gtataacgtg ctttcctcgt tagaatcaga gcgggagcta aacaggaggc cgattaaagg 8340

gattttagac aggaacggta cgccagaatc ctgagaagtg tttttataat cagtgaggcc 8400

accgagtaaa agagtctgtc catcacgcaa attaaccgtt gtcgcaatac ttctttgatt 8460

agtaataaca tcacttgcct gagtagaaga actcaaacta tcggccttgc tggtaatatc 8520

cagaacaata ttaccgccag ccattgcaac ggaatcgcca ttcgccattc aggctgcgca 8580

actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagct 8629

<210> 9

<211> 4977

<212> DNA

<213> artificial sequence

<220>

<223> kanamycin cassette

<400> 9

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 180

agtttaaaca agcttgcatg tctaagctag acccttcaga ttaaaaataa ctgaggtaag 240

ggcctgggta ggggaggtgg tgtgagacgc tcctgtctct cctctatctg cccatcggcc 300

ctttggggag gaggaatgtg cccaaggact aaaaaaaggc catggagcca gaggggcgag 360

ggcaacagac ctttcatggg caaaccttgg ggccctgctg tctagcatgc cccactacgg 420

gtctaggctg cccatgtaag gaggcaaggc ctggggacac ccgagatgcc tggttataat 480

taacccagac atgtggctgc cccccccccc ccaacacctg ctgcctctaa aaataaccct 540

gtccctggtg gatcccctgc atgcgaagat cttcgaacaa ggctgtgggg gactgagggc 600

aggctgtaac aggcttgggg gccagggctt atacgtgcct gggactccca aagtattact 660

gttccatgtt cccggcgaag ggccagctgt cccccgccag ctagactcag cacttagttt 720

aggaaccagt gagcaagtca gcccttgggg cagcccatac aaggccatgg ggctgggcaa 780

gctgcacgcc tgggtccggg gtgggcacgg tgcccgggca acgagctgaa agctcatctg 840

ctctcagggg cccctccctg gggacagccc ctcctggcta gtcacaccct gtaggctcct 900

ctatataacc caggggcaca ggggctgccc tcattctacc accacctcca cagcacagac 960

agacactcag gagcagccag cggcgcgccc aggtaagttt agtctttttg tcttttattt 1020

caggtcccgg atccggtggt ggtgcaaatc aaagaactgc tcctcagtgg atgttgcctt 1080

tacttctagg cctgtacgga agtgttactt ctgctctaaa agctgcggaa ttgtacccgc 1140

ggccgccacc atgctgtggt gggaggaggt ggaggattgt tatgaaaggg aggacgtgca 1200

gaagaagact tttaccaagt gggtgaacgc tcagttcagc aaatttggga agcagcacat 1260

cgagaatctg ttttccgacc tgcaggatgg gagacggctg ctggatctgc tggaaggact 1320

gactggccag aagctgccca aagagaaggg gagcactagg gtgcacgccc tgaacaacgt 1380

gaacaaagct ctgagagtgc tgcagaacaa caacgtggat ctggtgaata ttggcagtac 1440

tgatatcgtg gacgggaacc acaaactgac actgggcctg atctggaaca ttattctgca 1500

ctggcaggtg aaaaatgtga tgaagaacat catggccggg ctgcagcaga ccaattccga 1560

gaagatcctg ctgtcttggg tgcggcagag cacccgcaac tatccccagg tgaacgtgat 1620

taacttcact acatcctgga gcgacgggct ggccctgaat gctctgattc acagccacag 1680

gcctgatctg ttcgactgga atagcgtggt gtgccagcag tctgccacac agcgcctgga 1740

acatgccttc aatatcgctc ggtaccagct ggggatcgaa aaactgctgg acccagagga 1800

tgtggacact acatacccag ataaaaagtc tattctgatg tacattacta gcctgttcca 1860

ggtgctgcca cagcaggtgt ctattgaagc cattcaggag gtggaaatgc tgccccgccc 1920

ccccaaagtg actaaagagg agcattttca gctgcatcat cagatgcatt acagccagca 1980

gattaccgtg agcctggctc agggatatga gcgcaccagt agtccaaaac cacggttcaa 2040

gtcctacgct tatacccagg ctgcctacgt gacaactagc gaccctacta gatccccctt 2100

tccatcccag cacctggagg ccccagagga caagagcttt gggtccagcc tgatggaaag 2160

cgaggtgaat ctggatcggt accagacagc cctggaggag gtgctgagct ggctgctgag 2220

tgctgaagac acactgcagg cccagggcga aatttccaat gacgtggaag tggtgaagga 2280

tcagttccac acacacgagg gctatatgat ggacctgaca gctcaccagg ggcgcgtggg 2340

caatatcctg cagctgggct ctaaactgat cggcaccggg aaactgagtg aggacgagga 2400

aacagaagtg caggagcaga tgaacctgct gaacagccgc tgggagtgtc tgagagtggc 2460

tagtatggag aagcagtcca acctgcaccg ggtgctgatg gacctgcaga accagaaact 2520

gaaagagctg aacgactggc tgacaaagac tgaggaacgc acaaggaaga tggaggagga 2580

gccactggga cccgacctgg aggatctgaa gagacaggtg cagcagcata aggtgctgca 2640

ggaggatctg gaacaggagc aggtgcgggt gaactccctg acacatatgg tggtggtggt 2700

ggacgaatct agtggagatc acgccaccgc cgccctggag gaacagctga aggtgctggg 2760

ggaccggtgg gccaacattt gccggtggac cgaggacagg tgggtgctgc tgcaggacat 2820

cctgctgaaa tggcagaggc tgaccgagga gcagtgtctg tttagtgctt ggctgagcga 2880

gaaagaggac gccgtgaaca agatccacac aaccggcttt aaggatcaga acgaaatgct 2940

gtctagcctg cagaaactgg ctgtgctgaa ggccgatctg gagaaaaaga agcagagcat 3000

gggcaaactg tatagcctga aacaggacct gctgagcacc ctgaagaaca agagcgtgac 3060

ccagaagaca gaagcctggc tggataactt tgcccgctgc tgggacaacc tggtgcagaa 3120

actggagaaa agtacagctc agatctctca ggctgtgacc acaacccagc ctagcctgac 3180

ccagacaacc gtgatggaaa ccgtgaccac cgtgacaacc cgcgaacaga tcctggtgaa 3240

acatgcccag gaagagctgc cacctccacc tccccagaag aagagaaccc tggagcggct 3300

gcaggagctg caggaagcca ctgacgaact ggacctgaag ctgaggcagg ccgaagtgat 3360

taaggggtct tggcagcctg tgggcgatct gctgattgat tccctgcagg accacctgga 3420

aaaggtgaag gctctgagag gcgaaattgc tccactgaag gagaacgtga gtcatgtgaa 3480

cgatctggct agacagctga caacactggg catccagctg agcccataca atctgagcac 3540

actggaggac ctgaatacca ggtggaagct gctgcaggtg gctgtggaag accgggtgcg 3600

gcagctgcat gaggcccatc gcgacttcgg accagccagc cagcactttc tgagcacatc 3660

cgtgcagggg ccctgggaga gggccatttc tcccaacaag gtgccctact atattaatca 3720

cgagacccag accacttgtt gggaccatcc caagatgaca gaactgtacc agtccctggc 3780

cgatctgaac aacgtgaggt ttagcgctta cagaaccgct atgaagctga gacggctgca 3840

gaaggccctg tgcctggatc tgctgtccct gtccgccgcc tgcgatgccc tggatcagca 3900

taatctgaag cagaacgatc agccaatgga tatcctgcag atcatcaact gcctgaccac 3960

tatctacgac aggctggagc aggagcacaa caacctggtg aacgtgcctc tgtgcgtgga 4020

tatgtgcctg aactggctgc tgaacgtgta tgacactggg cgcaccggcc ggatcagagt 4080

gctgagtttt aaaactggga ttatctccct gtgtaaggcc cacctggagg acaagtacag 4140

gtacctgttc aagcaggtgg ctagtagcac tggattttgt gaccagcgcc gcctgggact 4200

gctgctgcat gatagtatcc agattcctag acagctggga gaggtggcta gtttcggagg 4260

atctaacatc gaacccagcg tgcgcagctg tttccagttt gccaataaca aacctgaaat 4320

cgaggctgct ctgttcctgg attggatgcg cctggaacca cagagcatgg tgtggctgcc 4380

tgtgctgcac agagtggctg ccgccgaaac tgccaagcac caggctaaat gcaacatctg 4440

caaggaatgt cccattatcg gctttcgcta caggagtctg aaacatttta actacgatat 4500

ttgccagagc tgcttctttt ccggaagagt ggccaaagga cacaagatgc actaccctat 4560

ggtggaatat tgcaccccaa ctacatctgg cgaagatgtg cgcgattttg ccaaggtgct 4620

gaagaataag tttcggacta agaggtactt cgccaagcac ccccgcatgg ggtatctgcc 4680

agtgcagaca gtgctggaag gagacaatat ggagaccgat acaatgtgag cggccgcaat 4740

aaaagatctt tattttcatt agatctgtgt gttggttttt tgtgtgtcta gagtcgacca 4800

gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta 4860

gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca 4920

aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgc 4977

Claims

1. A method of producing recombinant adeno-associated virus (rAAV) raavrh74.mhck7. Mini-dystrophin in adherent mammalian cells by a suspension seeding process comprising:

(b) Removing cells from the first medium;

(d) Culturing cells in an N-1 vessel under suspension conditions; and

(e) Inoculating the third medium in the bioreactor with cells from step (d).

2. The method of claim 1, wherein the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID No. 1.

3. The method of claim 2, wherein the rAAV comprises the MHCK7 promoter sequence of SEQ ID No. 7.

4. The method of any one of claims 1 to 3, wherein the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID No. 1 and the MHCK7 promoter sequence of SEQ ID No. 7.

5. The method of any one of claims 1 to 4, wherein the suspension seeding process further comprises:

(f) Adherent cells were transfected with transgenic plasmids containing raavrh74.mhck7. Mini-dystrophin constructs, plasmids containing AAV rep genes and AAV cap genes, and adenovirus helper plasmids.

6. The method of claim 5, wherein the transgenic plasmid comprising raavrh74.mhck7. The mini-dystrophin construct comprises:

The nucleic acid sequence of SEQ ID NO. 9;

nucleotides 55-5021 of SEQ ID NO. 3; or alternatively

Nucleotides 1 to 4977 of SEQ ID NO. 8.

7. The method of claim 5 or 6, wherein the plasmid comprising an AAV rep gene and an AAV cap gene comprises an AAV2 rep gene and a rAAVrh74 cap gene.

8. The method of any one of claims 5 to 7, wherein the adenovirus helper plasmid comprises adenovirus 5E2A, E ORF6 and VA RNA genes.

9. The method of any one of claims 1 to 8, wherein the suspension seeding process further comprises:

(g) Adherent cells were lysed.

10. The method of claim 9, wherein the adherent cells are lysed by freeze-thawing, solid shearing, hypertonic and/or hypotonic lysis, liquid shearing, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

11. The method of any one of claims 1 to 10, wherein the suspension seeding process further comprises:

(h) The rAAV is purified by at least one column chromatography step.

12. The method of claim 11, wherein the at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

13. The method of any one of claims 1 to 12, wherein the suspension seeding process further comprises culturing cells with the first growth medium in an N-3 vessel.

14. The method of claim 13, wherein the suspension seeding process further comprises culturing cells with the first growth medium in an N-4 vessel.

15. The method of any one of claims 1 to 14, wherein the bioreactor is an adherent bioreactor.

16. The method of claim 15, wherein the rAAV is purified from a culture produced in an adherent bioreactor.

17. The method of claim 15 or 16, wherein the third medium in the bioreactor comprises at least one factor that promotes cell adhesion.

18. The method of claim 17, wherein the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycans polysaccharides of the extracellular matrix, and combinations thereof.

19. The method of claim 17 or 18, wherein the third medium in the bioreactor comprises DMEM and 10% fbs.

20. The method of any one of claims 1 to 19, wherein the adherent cells are cultured under suspension conditions for about 48-72 hours.

21. The method of any one of claims 1 to 20, wherein the N-1 vessel is a suspension shake flask.

22. The method of any one of claims 1 to 21, wherein the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells.

23. The method of claim 22, wherein the adherent cells are HeLa cells or HEK-293 cells.

24. The method of claim 23, wherein the adherent cells are HEK-293 cells.

25. The method of any one of claims 1 to 24, wherein the adherent cells are not suspension adapted.

26. The method of any one of claims 1 to 25, wherein culturing the cells under suspension conditions does not alter the adherence dependence of the cells.

27. The method of any one of claims 1 to 26, wherein the culturing does not alter the cells to produce a new cell line.

28. A composition comprising recombinant adeno-associated virus (rAAV) raavrh74.mhck7. A minor anti-dystrophin protein, wherein the rAAV is prepared by the method of any one of claims 1 to 27.

29. The composition of claim 28, wherein the composition comprises:

a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO. 9;

b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO. 3; and/or

c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO. 8.

30. A method of treating muscular dystrophy in a human subject in need thereof comprising administering to the human subject the composition of claim 29.

31. The method of claim 30, wherein the rAAV uses a systemic route of administration and is administered at about 5.0x10 ¹² vg/kg to about 1.0x10 ¹⁵ The dose of vg/kg.

32. The method of claim 31, wherein the systemic route of administration is intravenous and the dose of rAAV administered is about 2x 10 ¹⁴ vg/kg。

33. The method of any one of claims 30 to 32, wherein the dose of rAAV is administered at a concentration of about 10 mL/kg.

34. The method of any one of claims 30 to 33, wherein the rAAV is administered by injection, infusion, or implantation.

35. The method of claim 34, wherein the rAAV is administered by infusion within about one hour.

36. The method of any one of claims 30 to 35, wherein the rAAV is administered via the intravenous route via the external Zhou Zhiti vein.

37. The method of any one of claims 30 to 36, wherein the muscular dystrophy is duchenne muscular dystrophy or becker muscular dystrophy.

38. The method of claim 37, wherein the muscular dystrophy is duchenne muscular dystrophy.

39. The method of any one of claims 30 to 38, wherein the level of microdystrophin gene expression in cells of the subject after administration of the rAAV is increased compared to the level of microdystrophin gene expression prior to administration of the rAAV.

40. The method of claim 39, wherein the expression of a microdystrophin gene in the cell is detected by measuring the level of microdystrophin in a muscle biopsy by western blotting before and after administration of the rAAV.

41. The method of claim 40, wherein expression after administration of the rAAV is at least 55.4% as compared to before administration.

42. The method of any one of claims 30 to 41, wherein the average percentage of micro-dystrophin positive fibers in the muscle tissue of the subject after administration of the rAAV is increased compared to the number of micro-dystrophin positive fibers prior to administration of the rAAV.

43. The method of claim 42, wherein the average percentage of microdystrophin-positive fibers is at least 70.5% and the average intensity is at least 116.9% as detected by Immunofluorescence (IF) in muscle biopsies before and after administration of the rAAV.

44. The method of any one of claims 30 to 43, wherein the minute dystrophin protein transduction by vector genome count is at least 3.87 average vector genome copies per cell nucleus.

45. The method of any one of claims 30 to 44, wherein the composition is administered to a genotyped patient.

46. The method of claim 45, wherein the genotyped patient is genotyped for at least one mutation in exons 18-79 of the human anti-dystrophin (DMD) gene.

47. The method of claim 45 or 46, further comprising genotyping the DMD gene of the human subject prior to administering the composition to the human subject.

48. The method of claim 47, wherein the genotyping detects at least one mutation in exons 18 to 79 of the DMD gene.

49. The method of claim 48, wherein said at least one mutation is a frameshift deletion, frameshift repeat, premature termination, or other pathogenic variation resulting in a lack of expression of human anti-dystrophin protein.

50. Use of the composition of claim 28 or 29 for treating muscular dystrophy in a human subject in need thereof.

51. Use of a composition according to claim 28 or 29 in the manufacture of a medicament for the treatment of muscular dystrophy.

52. The use of claim 50 or 51, wherein the muscular dystrophy is duchenne muscular dystrophy or becker muscular dystrophy.

53. The use of claim 52, wherein the muscular dystrophy is duchenne muscular dystrophy.