ADENO-ASSOCIATED VIRUS PARTICLES AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 63/229,936, filed on August 5, 2021, and U.S. Provisional Application No. 63/239,881, filed on September 1, 2021, the content of each of which is incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

[0002] The invention generally relates to adeno-associated virus (AAV) particles for delivering a micro-dystrophin transgene, methods of producing the AAV particles, cells producing the AAV particles, and methods of using the AAV particles for the delivery of a micro-dystrophin transgene to skeletal and/or cardiac muscle for the treatment of dystrophinopathies, for example, Duchenne muscular dystrophy.

INCORPORATION OF THE SEQUENCE LISTING

[0003] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is INMD_166_02WO_SeqList_ST26.xml. The XML file is approximately 40,532 bytes, was created on August 3, 2022, and is being submitted electronically via USPTO Patent Center.

BACKGROUND OF THE INVENTION

[0004] Duchenne muscular dystrophy (DMD) is inherited in an X-linked recessive pattern and is caused by a genetic mutation that prevents the body from producing dystrophin, a protein that muscles require to work properly. DMD is characterized in part by progressive muscle degeneration. As the disease progresses, initially affecting muscles in the thigh, pelvis, and arms, DMD eventually affects all voluntary muscles and involves the heart and breathing muscles in later stages. In Europe and North America, the prevalence of DMD is approximately 1 in 3,600 male births. DMD is the most common childhood onset form of muscular dystrophy and affects males almost exclusively. There is no known cure for DMD and current standards of treatment are primarily aimed at management of symptoms including: steroids, immunosuppressants, anticonvulsants, braces, corrective surgery, and assisted ventilation. Aggressive management of dilated cardiomyopathy associated with DMD includes anti-congestive medications and cardiac transplantation in severe cases.

[0005] Gene therapy is a rapidly accelerating therapeutic approach wherein nucleic acids are delivered to cells harboring mutated or non-functional genes to correct the defect in the mutated cells. In certain gene therapies, nucleic acids are packaged within adeno-associated viruses (AAV) that deliver the nucleic acids to the cells. Once inside the cell nucleus, the nucleic acid then directs appropriate protein production and the virus is safely degraded. Gene therapies for the treatment of DMD have been proposed but require delivery of high systemic viral titers leading to patient toxicity, and have high manufacturing costs due to the large quantities of virus required per patient.

[0006] Delivery of micro-dystrophin (pDys), modified but functional shortened dystrophin nucleic acid sequences, in animal models and humans has been reported to promote muscle function. pDys transgenes are designed to encode various combinations of the unique functional domains of the 427 kDa dystrophin protein. pDys sequences, generally less than 5 kilobases in length, have previously been tested using AAV to deliver pDys transgenes in the murine model of DMD using the mdx mouse, the most widely used animal model for DMD research. The mutation in the mdx mouse is a nonsense point mutation (C-to-T transition) in exon 23 that aborted full-length dystrophin expression (Sicinski et al. (1989) Science 244, pp. 1578-1580, incorporated by reference herein in its entirety). Despite the promise that delivery of pDys has shown, novel therapies are needed for the treatment of DMD. The present invention addresses this and other needs.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention relates in part to adeno-associated virus (AAV) particles comprising a capsid that packages (i.e., encapsidates) a micro-dystrophin (pDys) transgene and methods for treating various dystrophinopathies with the same, for example, by intrathecal administration. In one embodiment, the pDys transgene encodes a pDys polypeptide comprising (i) an N-terminal region (NTD) comprising an actin binding site, (ii) a domain comprising three hinge regions and four spectrin repeats, and (iii) a cysteine rich domain. The pDys transgene, in one embodiment, comprises the nucleic acid sequence set forth in SEQ ID NO:5.

[0008] In one aspect, an AAV particle is provided, comprising a capsid encapsidating a vector genome. The vector genome, in one embodiment, comprises from 5’ to 3’: 5’ inverted terminal repeat (ITR); a promoter; a pDys transgene, an SV40 poly (A) tail; and a 3’ ITR. The pDys transgene, in one embodiment, encodes a polypeptide comprising (i) an N-terminal region (NTD) comprising an actin binding site, (ii) a central rod domain comprising from two to four hinge regions and from four to six spectrin repeats, and (iii) a cysteine rich domain. In a further embodiment, the pDys transgene encodes a pDys polypeptide comprising an NTD, hinge regions 1, 2 and 4, and spectrin repeats 1, 2, 3 and 24, and a cysteine rich domain. In even a further embodiment, the pDys transgene comprises the nucleic acid sequence set forth in SEQ ID NO:5. The AAV particle, in one embodiment, is an AAV9 particle, and is present in an effective amount in an intrathecal composition. The effective amount of the AAV particle, in one embodiment, comprises about 90% or less vector genomes than the effective vector genome amount of an intravenous (IV) composition comprising an AAV particle encapsidating a pDys transgene, e.g., the same pDys transgene that is present in the intrathecal composition.

[0009] In one embodiment, the vector genome further comprises an SV40 intron 5’ (upstream) of the pDys transgene and 3’ (downstream) of the promoter. In another embodiment, the vector genome further comprises an enhancer is 3 ’(downstream) of the 5’ ITR and 5’ (upstream) of the promoter.

[0010] The promoter, in one embodiment, is an MHCK7 or chicken [3-actin hybrid promoter.

[0011] In a preferred embodiment, the AAV particle is an AAV9 particle, i.e., the AAV particle comprises one or more AAV9 capsid proteins. The AAV9 particle’s capsid, in one embodiment, consists of AAV9 capsid proteins. In yet another embodiment, the AAV particle is an AAVrh74 particle.

[0012] In some embodiments, a recombinant AAV vector genome of the present invention comprises from 5’ to 3’: a 5’ ITR; an SK-CRM4 enhancer; a promoter; a pDys transgene; an SV40 poly (A) tail; and a 3’ ITR. In some embodiments, the SK-CRM4 enhancer has a sequence comprising or consisting of SEQ ID NO: 8. In some embodiments, the pDys coding sequence encodes a pDys protein comprising an actin-binding domain and at least four spectrin repeats, e.g., from four to six spectrin repeats. In some embodiments, the pDys transgene comprises or consists of the nucleic acid sequence of SEQ ID NO:5.

[0013] In some embodiments, the encapsidated vector genome of the present invention comprises a 5’ AAV2 ITR and a 3’ AAV2 ITR. In some embodiments, the 5’ AAV2 ITR has a sequence comprising or consisting of SEQ ID NO:1. In some embodiments, the 3’ AAV2 ITR has a sequence comprising or consisting of SEQ ID NO:7.

[0014] In some embodiments, encapsidated vector genome comprises an MHCK7 promoter. In some embodiments, the MHCK7 promoter has a sequence comprising or consisting of SEQ ID NO:2. In another embodiment, the promoter is a chicken [3-actin hybrid promoter. In a further embodiment, the chicken [3-actin hybrid promoter has the nucleic acid sequence set forth in SEQ ID NO:3.

[0015] In some embodiments, the encapsidated vector genome of the present invention comprises an SV40 intron having a sequence comprising or consisting of SEQ ID NO:4.

[0016] In some embodiments, the encapsidated vector genome comprises a SV40 poly(A) tail having a sequence comprising or consisting of SEQ ID NO:6.

[0017] In some embodiments, the capsid of the AAV particle comprises one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 capsid proteins. In a preferred embodiment, the capsid is an AAV9 capsid and the AAV9 capsid consists of AAV9 capsid proteins.

[0018] In another aspect, the present invention relates to a method of treating a dystrophinopathy in a subject in need thereof, comprising, intrathecally administering to the subject in a single dose, a composition comprising an effective amount of one of the AAV particles encapsidating a vector genome comprising a pDys transgene, as further described herein. In one embodiment, the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, or DMD-associated dilated cardiomyopathy (DCM). In even a further embodiment, the dystrophinopathy is DMD. The effective amount of the AAV particle, in a further embodiment, comprises about 90% or less vector genomes than the effective amount of a counterpart IV composition (e.g, an intravenously administered composition) comprising an AAV particle encapsidating a vector genome comprising a pDys transgene, e.g., the same pDys transgene that is present in the intrathecally administered composition.

[0019] In one embodiment, the subject is administered the composition when in the Trendelenburg position. In a further embodiment, administration is in the absence of a nonionic, low-osmolar contrast agent.

[0020] In one embodiment of the method of treating a dystrophinopathy described herein, the effective dose of the intrathecally administered AAV particle provides a greater therapeutic response than the identical dose of an intravenously administered AAV particle encapsidating a vector genome comprising a pDys transgene, e.g., the same pDys transgene that is present in the intrathecally administered AAV particle. The therapeutic response, in one embodiment, is an increase from baseline on the North Star Ambulatory Assessment (NSAA).

[0021] In another embodiment of the method of treating a dystrophinopathy described herein, intrathecal administration of an effective amount of the AAV particle encapsidating a vector genome comprising a pDys transgene, results in a decreased number of side effects, or a reduced severity of one or more side effects in the subject, compared to the number of side effects, or severity of side effects experienced by a second subject, when the second subject is intravenously administered an effective amount of a counterpart AAV particle encapsidating a vector genome comprising a pDys transgene. In a further embodiment, the dystrophinopathy is DMD. In even a further embodiment, the AAV particle is an AAV9 particle.

[0022] In even another embodiment of the method of treating a dystrophinopathy, the effective amount of the intrathecally administered AAV particle provides greater pDys transgene expression in skeletal and/or cardiac muscle, compared to the amount of pDys transgene expression in liver tissue. In a further embodiment, the dystrophinopathy is DMD. In even a further embodiment, the AAV particle is an AAV9 particle. In yet even a further embodiment, the AAV particle encapsidates a pDys transgene having the nucleic acid sequence set forth in SEQ ID NO:5.

[0023] In another aspect of the present invention, a method for preferentially delivering a pDys transgene to skeletal and/or cardiac muscle of a subject is provided. The method entails intrathecally administering to a subject in a single dose, a composition comprising an effective amount of an AAV9 particle comprising an AAV9 capsid and a vector genome comprising a pDys transgene, encapsidated by the AAV9 capsid. The encapsidated genome comprises from 5’ to 3’: a 5’ ITR; a promoter; a pDys transgene; an SV40 poly (A) tail; and a 3’ ITR. Subsequent to administration, the pDys transgene is expressed at higher levels in the skeletal and/or cardiac muscle of the subject, compared to the transgene expression in liver tissue of the subject.

[0024] In some embodiments of a method described herein, expression of a pDys transgene delivered by an AAV particle described herein in a subject is significantly less in liver tissue of the subject as compared to skeletal and/or cardiac muscle of the subject. In a further embodiment, pDys transgene expression is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% greater in the skeletal and/or cardiac muscle of the subject compared to the amount of pDys transgene expression in the liver tissue. In another embodiment, pDys transgene expression is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% less in the liver of the subject compared to the amount of pDys transgene expression in skeletal and/or cardiac muscle.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1A shows a schematic illustration of an exemplary pDys encoding gene construct (INS 1201).

[0026] FIG. IB shows a schematic illustration of an alternative pDys encoding gene construct (INS 1212).

[0027] FIG. 1C shows an agarose gel electrophoresis of the INS 1201 gene construct cloned into the psZOl vector backbone (pSZOl -INS 1201) restriction digested with Hindlll/Bsal, and Smal (lanes 1 and 2, respectively), and the INS1212 gene construct cloned into the psZOl vector backbone (pSZ01-INS1212) restriction digested with Hindlll/Bsal, and Smal (lanes 3 and 4, respectively)

[0028] FIG. 2 shows a silver-stained SDS polyacrylamide gel electrophoresis (PAGE) of 1 pl of INS 1201 -AAV 9 preparation (lane 1), 1 pl of INS1212-AAV9 preparation (lane 2), and 0.5 pl, 1 pl, 2 pl, and 4 pl of a 1 xlO ¹³ vg/ml AAV2 standard (lanes 3, 4, 5, and 6, respectively).

[0029] FIG. 3A shows gastrocnemius muscle sections obtained from mdx mice 21 days post intramuscular injection with 2.7X 10 ¹¹ vg of INS1201-AAV9 (iii) or INS1212-AAV9 (iv) and immunofluorescently stained for dystrophin. Gastrocnemius sections obtained from a noninjected mdx mouse (i) and wildtype C57/B1 mouse (ii) and immunofluorescently stained for dystrophin are shown for comparison.

[0030] FIG. 3B shows a gastrocnemius muscle section obtained from an mdx mouse 21 days post intramuscular injection with 2.7/ I 0 ^{1 1} vector genomes (vg) of INS1212-AAV9 and stained with DAPI (i) and for dystrophin (ii). Merged image shown in (iii).

[0031] FIG. 4A shows gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteus (iv), triceps (v), diaphragm (vi), heart (vii), and liver (viii) sections obtained from an mdx mouse 21 days post intracerebroventricular (ICV) injection with 2.7/ I O ^{1 1} vg of INS1201-AAV9 and immunofluorescently stained for dystrophin. [0032] FIG. 4B shows gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteus (iv), triceps (v), diaphragm (vi), heart (vii), and liver (viii) sections obtained from an mdx mouse 21 days post intracerebroventricular (ICV) injection with 9*1O ¹⁰ vg of INS1201-AAV9 and immunofluorescently stained for dystrophin.

[0033] FIG. 5 shows gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteus (iv), triceps (v), diaphragm (vi), heart (vii), and liver (viii) sections obtained from an mdx mouse 21 days post intracerebroventricular (ICV) injection with 9*1O ¹⁰ vg of INS1212-AAV9 and immunofluorescently stained for dystrophin.

[0034] FIG. 6A shows gastrocnemius muscle sections obtained from mdx mice 80 days post intracerebroventricular (ICV) injection with 9* 10 ¹⁰ vg (ii) or 2.7* 10 ¹¹ vg (iii) of INS 1201- AAV9 and stained with hematoxylin and eosin (H&E). Gastrocnemius sections obtained from a wildtype C57/B1 mouse (i) and non-injected mdx mouse (iv) and H&E stained are shown for comparison.

[0035] FIG. 6B shows gastrocnemius muscle sections obtained from mdx mice 80 days post intracerebroventricular (ICV) injection with 9* 10 ¹⁰ vg (ii) or 2.7* 10 ¹¹ vg (iii) of INS 1201- AAV9 and stained for dystrophin. Gastrocnemius sections obtained from a wildtype C57/B1 mouse (i) and non-injected mdx mouse (iv) and stained for dystrophin are shown for comparison.

[0036] FIG. 7A shows a gastrocnemius muscle section obtained from an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9*1O ¹⁰ vg (ii) of INS1212-AAV9 and stained with hematoxylin and eosin (H&E). Gastrocnemius sections obtained from a wildtype C57/B1 mouse (i) and non-injected mdx mouse (iii) and H&E stained are shown for comparison.

[0037] FIG. 7B shows a gastrocnemius muscle section obtained from an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9*1O ¹⁰ vg (ii) of INS1212-AAV9 and stained for dystrophin. Gastrocnemius sections obtained from a wildtype C57/B1 mouse (i) and non-injected mdx mouse (iii) and stained for dystrophin are shown for comparison.

[0038] FIG. 8A shows a bar graph of mean fiber diameter (pm) in gastrocnemius muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9*1O ¹⁰ vg or 2.7X10 ¹¹ vg of INS1201-AAV9. Mean fiber diameters (pm) in gastrocnemius muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0039] FIG. 8B shows a line graph of relative frequencies (%) of cell diameters (pm) in gastrocnemius muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9*1O ¹⁰ vg or 2.7X10 ¹¹ vg of INS1201-AAV9. Relative frequencies of cell diameters in gastrocnemius muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0040] FIG. 8C shows a bar graph of mean fiber diameter (pm) in triceps muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9*1O ¹⁰ vg or 2.7* 10 ¹¹ vg of INS1201-AAV9. Mean fiber diameters (pm) in triceps muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0041] FIG. 8D shows a line graph of relative frequencies (%) of cell diameters (pm) in triceps muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9xio ¹⁰ vg or 2.7X10 ¹¹ vg of INS1201-AAV9. Relative frequencies of cell diameters in triceps muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0042] FIG. 8E shows a bar graph of mean fiber diameter (pm) in tibialis anterior muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9xlO ¹⁰ vg or 2.7X10 ¹¹ vg of INS1201-AAV9. Mean fiber diameters (pm) in tibialis anterior muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0043] FIG. 8F shows a line graph of relative frequencies (%) of cell diameters (pm) in tibialis anterior muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9xlO ¹⁰ vg or 2.7X10 ¹¹ vg of INS1201-AAV9. Relative frequencies of cell diameters in tibialis anterior muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0044] FIG. 8G shows a bar graph of mean fiber diameter (pm) in diaphragm muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9xlO ¹⁰ vg or 2.7X10 ¹¹ vg of INS1201-AAV9. Mean fiber diameters (pm) in diaphragm muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0045] FIG. 8H shows a line graph of relative frequencies (%) of cell diameters (in pm) in diaphragm muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9xlO ¹⁰ vg or 2.7X10 ¹¹ vg of INS1201-AAV9. Relative frequencies of cell diameters in diaphragm muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0046] FIG. 9A shows a bar graph of mean fiber diameter (pm) in gastrocnemius muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9xlO ¹⁰ vg of INS1212-AAV9. Mean fiber diameters (pm) in diaphragm muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0047] FIG. 9B shows a line graph of relative frequencies (%) of cell diameters (pm) in gastrocnemius muscle cells in an mdx mouse 80 days post intracerebroventricular (ICV) injection with 9*1O ¹⁰ vg of INS1212-AAV9. Relative frequencies of cell diameters in diaphragm muscle cells in wildtype C57/B1 and non-injected mdx mice are shown for comparison.

[0048] FIG. 10A is a line graph of percent contractile force in EDL muscle resulting from eccentric contractions (EC) in a wildtype C57/B1 mouse, an mdx mouse receiving intracerebroventricular (ICV) injection at postnatal day 1 (pl) with vehicle, an mdx mouse receiving ICV injection at postnatal day 1 pl of 2.7X10 ¹¹ vg of INS1201-AAV9, and an mdx mouse receiving ICV injection at pl of 9*1O ¹⁰ vg of INS1201-AAV9.

[0049] FIG 10B is a bar graph of percent post-eccentric contraction (EC) stress in EDL muscle relative to pre-EC stress in (i) a wildtype C57/B1 mouse, and mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 1 (pl) of (ii) 9*10 ⁹ vg of INS1201- AAV9; (iii) 9xlO ¹⁰ vg of INS1201-AAV9; (iv) 2.7X10 ¹¹ vg of INS1201-AAV9 or (v) vehicle control.

[0050] FIG. 10C is a graph showing the percent of contractile force in EDL muscle (% eccentric contraction 1 (ECI)) as a function of the eccentric contraction (EC) number in (i) a wildtype C57/B1 mouse, and mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9xlO ¹⁰ vg ofINS1201-AAV9; (iii) 2.7x 10 ¹¹ vg of INS 1201- AAV9; (iv) 5.4X10 ¹¹ vg of INS1201-AAV9; (v) 1.2xl0 ¹² vg ofINS1201-AAV9; or (vi) vehicle control.

[0051] FIG. 10D is a bar graph showing the percent of force in EDL muscle ([post EC5/post EC 1) in (i) a wildtype C57/B1 mouse, and mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9xlO ¹⁰ vg of INS1201-AAV9; (iii) 2.7X10 ¹¹ vg of INS1201-AAV9; (iv) 5.4xlO ⁿ vg of INS1201-AAV9; (v) 1.2xl0 ¹² vg of INS1201-AAV9; or (vi) vehicle control.

[0052] FIG. 10E is a graph of maximum tension (kPa) in EDL muscle resulting from eccentric contractions (EC), in (i) a wildtype C57/B1 mouse, and mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9xl0 ⁹ vg of INS1201-AAV9; (iii) 9xlO ¹⁰ vg of INS1201-AAV9; (iv) 2.7X10 ¹¹ vg of INS1201-AAV9; (v) 5.4X10 ¹¹ vg of INS1201-AAV9; (vi) 1.2xl0 ¹² vg of INS1201-AAV9; or (vii) vehicle control.

[0053] FIG. 10F is a graph of peak stress (kPa) resulting from eccentric contractions (EC) at various frequencies (Hz), in (i) a wildtype C57/B1 mouse, and mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9xl0 ⁹ vg of INS1201-AAV9; (iii) 9xlO ¹⁰ vg of INS1201-AAV9; (iv) 2.7X10 ¹¹ vg of INS1201-AAV9; (v) 5.4X10 ¹¹ vg of INS1201-AAV9; (vi) 1.2xl0 ¹² vg of INS1201-AAV9; or (vii) vehicle control.

[0054] FIG. 11A shows gastrocnemius muscle sections obtained from non-injected cynomolgus macaques (i), cynomolgus macaques 21 days post intravenous (IV) injections with 5xl0 ¹³ vg (ii) or I xlO ¹⁴ vg (iii) of AAV9 CBA-GFP; or cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5xl0 ¹³ vg (iv), 5xl0 ¹³ vg (v), or I xlO ¹⁴ vg (vi) of AAV9 CBA-GFP and immunostained with NovaRed™ for detection of GFP expression.

[0055] FIG. 11B shows quadriceps muscle sections obtained from non-injected cynomolgus macaques (i), cynomolgus macaques 21 days post intravenous (IV) injections with 5xl0 ¹³ vg (ii) or I xlO ¹⁴ vg (iii) of AAV9 CBA-GFP; or cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5xl0 ¹³ vg (iv), 5xl0 ¹³ vg (v), or I xlO ¹⁴ vg (vi) of AAV9 CBA-GFP and immunostained with NovaRed™ for detection of GFP expression.

[0056] FIG. 11C shows deltoid muscle sections obtained from non-injected cynomolgus macaques (i); cynomolgus macaques 21 days post intravenous (IV) injections with 5 x 10 ¹³ vg (ii) or IxlO ¹⁴ vg (iii) of AAV9 CBA-GFP; or cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5xl0 ¹³ vg (iv), 5xl0 ¹³ vg (v), or IxlO ¹⁴ vg (vi) of AAV9 CBA-GFP and immunostained with NovaRed™ for detection of GFP expression.

[0057] FIG. 11D shows triceps muscle sections obtained from non-injected cynomolgus macaques (i); cynomolgus macaques 21 days post intravenous (IV) injections with 5xl0 ¹³ vg (ii) or IxlO ¹⁴ vg (iii) of AAV9 CBA-GFP; or cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5xl0 ¹³ vg (iv), 5xl0 ¹³ vg (v), or IxlO ¹⁴ vg (vi) of AAV9 CBA-GFP and immunostained with NovaRed™ for detection of GFP expression.

[0058] FIG. HE shows biceps muscle sections obtained from non-injected cynomolgus macaques (i); cynomolgus macaques 21 days post intravenous (IV) injections with 5xl0 ¹³ vg (ii) or IxlO ¹⁴ vg (iii) of AAV9 CBA-GFP; or cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5xl0 ¹³ vg (iv), 5xl0 ¹³ vg (v), or IxlO ¹⁴ vg (vi) of AAV9 CBA-GFP and immunostained with NovaRed™ for detection of GFP expression. [0059] FIG. 11F shows tibialis anterior muscle sections obtained from cynomolgus macaques 21 days post intravenous (IV) injections with 5*10 ¹³ vg (i) or l *10 ¹⁴ vg (ii) of AAV9 CBA-GFP; or cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5* 10 ¹³ vg

(iii), 5*10 ¹³ vg (iv), or l*10 ¹⁴ vg (v) of AAV9 CBA-GFP and immunostained with NovaRed™ for detection of GFP expression.

[0060] FIG. 11G shows diaphragm muscle sections obtained from cynomolgus macaques 21 days post intravenous (IV) injections with 5 *10 ¹³ vg (i) or 1 *10 ¹⁴ vg (ii) of AAV9 CBA- GFP; or cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5 *10 ¹³ vg (iii), 5 *10 ¹³ vg (iv), or 1 *10 ¹⁴ vg (v) of AAV9 CBA-GFP and immunostained with NovaRed™ for detection of GFP expression.

[0061] FIG. 11H shows heart muscle sections obtained from cynomolgus macaques 21 days post intravenous (IV) injections with 5 *10 ¹³ vg (i) or 1 *10 ¹⁴ vg (ii) of AAV9 CBA-GFP; or cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5* 10 ¹³ vg (iii), 5* 10 ¹³ vg (iv), or l *10 ¹⁴ vg (v) of AAV9 CBA-GFP and immunostained with NovaRed™ for detection of GFP expression.

[0062] FIG. Ill shows liver sections obtained from cynomolgus macaques 21 days post intravenous (IV) injections with 5*10 ¹³ vg (i) or l*10 ¹⁴ vg (ii) of AAV9 CBA-GFP; or cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5 *10 ¹³ vg (iii), 5*10 ¹³ vg

(iv), or l*10 ¹⁴ vg (v) of AAV9 CBA-GFP and immunostained with NovaRed™ for detection of GFP expression.

[0063] FIG. 12A shows a Ponceau stain (top panel) or Western Blot (bottom panel) of biceps (1), triceps (2), deltoid (3), quadriceps (4), gastrocnemius (5), tibialis anterior (6), diaphragm (7), and heart (8) muscle protein samples obtained from cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5*10 ¹³ vg of AAV9 CBA-GFP and probed with anti-GFP antibody.

[0064] FIG. 12B shows a Ponceau stain (top panel) or Western Blot (bottom panel) of biceps (1), triceps (2), deltoid (3), quadriceps (4), gastrocnemius (5), tibialis anterior (6), diaphragm (7), and heart (8) muscle protein samples obtained from cynomolgus macaques 21 days post intrathecal (IT) injections with 5*10 ¹³ vg of AAV9 CBA-GFP and probed with anti- GFP antibody.

[0065] FIG. 12C shows a Ponceau stain (top panel) or Western Blot (bottom panel) of biceps (1), triceps (2), deltoid (3), quadriceps (4), gastrocnemius (5), tibialis anterior (6), diaphragm (7), and heart (8) muscle protein samples obtained from cynomolgus macaques 21 days post intrathecal (IT) injections with 1 / 10 ¹⁴ vg of AAV9 CBA-GFP and probed with anti- GFP antibody.

[0066] FIG. 12D shows a Ponceau stain (top panel) or Western Blot of biceps (1), and triceps (2) muscle protein samples obtained from non-injected cynomolgus macaques and probed with anti-GFP antibody.

[0067] FIG. 13 shows an agarose gel electrophoresis of biceps (1), triceps (2), deltoid (3), tibialis anterior (4), gastrocnemius (5), quadriceps vastus lateralis (6), diaphragm (7), and heart (8) muscle and liver (9) tissue samples obtained from cynomolgus macaques 21 days post intrathecal (IT) injections with 2.5/ 10 ¹³ vg of AAV9 CBA-GFP and subjected to RT-PCR in the presence (+) or absence (-) of reverse transcriptase. Biceps (10), triceps (11), deltoid (12), and quadriceps (13) muscle tissue samples obtained from non-injected cynomolgous macaques and subjected to RT-PCR in the presence (+) or absence (-) of reverse transcriptase are shown for comparison.

[0068] FIG. 14 shows various pDys protein domains encoded by pDys transgenes provided herein.

[0069] FIG. 15 is a graph showing the number of INS1201 DNA copies per diploid genome in mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9xl0 ⁹ vg of INS1201-AAV9; (iii) 9xlO ¹⁰ vg of INS1201-AAV9; (iv) 2.7X10 ¹¹ vg of INS1201-AAV9; (v) 5.4X10 ¹¹ vg of INS1201-AAV9; (vi) 1.2xl0 ¹² vg of INS1201-AAV9; or (vii) vehicle control.

[0070] FIG. 16 is a graph showing the number of INS1201 RNA transcript copies normalized to the number of copies of RPP30 in mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9xl0 ⁹ vg ofINS1201-AAV9; (iii) 9xlO ¹⁰ vg of INS1201-AAV9; (iv) 2.7xlO ⁿ vg of INS1201-AAV9; (v) 5.4X10 ¹¹ vg of INS1201- AAV9; (vi) 1.2xl0 ¹² vg of INS1201-AAV9; or (vii) vehicle control.

[0071] FIG. 17 is a graph of mean fiber diameter (pm) in EDL muscle cells at postnatal day 120 (pl 20) in (i) wildtype C57/B1 mice, and mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9xl0 ⁹ vg of INS1201-AAV9; (iii) 9xlO ¹⁰ vg of INS1201-AAV9; (iv) 2.7xlO ⁿ vg of INS1201-AAV9; (v) 5.4X10 ¹¹ vg of INS1201- AAV9; (vi) 1.2xl0 ¹² vg of INS1201-AAV9; or (vii) vehicle control. [0072] FIG. 18 is a graph of mean fiber diameter (pm) in tibialis anterior muscle cells at postnatal day 120 (pl20) in (i) wildtype C57/B1 mice, and mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9*10 ⁹ vg of INS1201-AAV9; (iii) 9xlO ¹⁰ vg of INS1201-AAV9; (iv) 2.7* 10 ¹¹ vg of INS1201-AAV9; (v) 5.4X10 ¹¹ vg of INS1201-AAV9; (vi) 1.2xl0 ¹² vg of INS1201-AAV9; or (vii) vehicle control.

[0073] FIG. 19 shows diaphragm muscle sections stained with picrosirius red, post intracerebroventricular (ICV) injection with various doses of INS1201-AAV9. Diaphragm sections obtained from a wildtype C57/B1 mouse and mdx mouse administered vehicle are shown for comparison in the top section.

[0074] FIG. 20 is a graph showing the percent collagen in diaphragm muscle at postnatal day 120 (pl20), in (i) wildtype C57/B1 mice, and mdx mice receiving intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) vehicle control; (iii) 9xl0 ⁹ vg of INS1201-AAV9; (iv) 9xlO ¹⁰ vg of INS1201-AAV9; (v) 2.7X10 ¹¹ vg of INS1201-AAV9; (v) 5.4X10 ¹¹ vg of INS1201-AAV9 or (vi) 1.2xl0 ¹² vg of INS1201-AAV9.

[0075] FIG. 21, top, shows an extensor digitorum longus (EDL) muscle section obtained from an mdx mouse at postnatal day 120 (p!20) subsequent to intracerebroventricular (ICV) injection at p28 with 5.4xlO ⁿ vg of INS1201-AAV9 and stained with hematoxylin and eosin (H&E) (far left); laminin/dapi (second from left); dystrophin (second from right); and a merged image (far right). FIG. 21, bottom, shows a EDL muscle section obtained from an mdx mouse at postnatal day 120 (pl 20) subsequent to intracerebroventricular (ICV) injection at p28 with 5 vehicle control, and stained with hematoxylin and eosin (H&E) (far left); laminin/dapi (second from left); dystrophin (second from right); and a merged image (far right).

DETAILED DESCRIPTION OF THE INVENTION

[0076] The present invention relates in part to adeno-associated viral (AAV) particles and methods for preferentially delivering the same to cardiac and/or skeletal muscle to a subject in need of treatment of a monogenic muscle disease, for example, a dystrophinopathy such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, or DMD-associated dilated cardiomyopathy (DCM). Without wishing to be bound by theory, the particles and methods for delivering the same to subjects in need thereof, for example to treat a monogenic muscle disease such as a dystrophinopathy, provide a superior benefit to known AAV particles and treatment methods at least because the present invention: (i) allows for significantly lower dosages than IV delivery, in order to achieve substantially the same or a better therapeutic benefit, thereby reducing viral load and toxicity and other side effects; and/or (ii) allows for preferential transgene targeting and expression in cardiac and/or skeletal muscle tissue compared to liver tissue, thereby targeting the transgene to cells of interest to provide a greater therapeutic benefit compared to AAV vectors delivered intravenously; (iii) can benefit greater patient populations compared to IV formulations, because of the lower dosages needed, corresponding to a decreased manufacturing burden.

[0077] Aspects of the invention relate to AAV particles, methods for producing the same, and methods for delivering the AAV particles to a subject in need of treatment. The AAV particle for example, an AAV9 particle, comprises a capsid comprising one or more AAV9 capsid proteins and a vector genome encapsidated by the AAV9 capsid. The vector genome comprises a transgene and regulatory elements that promote gene expression of the transgene when delivered into muscle cells, for example skeletal and/or cardiac muscle cells. In one embodiment, the transgene is a micro-dystrophin (pDys) transgene.

[0078] Methods described herein comprise intrathecally administering to a subject in need of treatment of a dystrophinopathy, in a single dose, a composition comprising an effective amount of an AAV particle as described herein. The dystrophinopathy in one embodiment, is DMD, Becker muscular dystrophy, or DCM. In a preferred embodiment, the dystrophinopathy is DMD. In embodiments described herein, intrathecal delivery of AAV particles of the present invention to muscle cells can result in robust expression of pDys and can significantly improve muscle health and function. The present invention also provides methods and cells for producing the AAV particles described herein. In one embodiment of the methods described herein, subsequent to intrathecal administration of the AAV particle, the transgene is expressed at higher levels in the skeletal and/or cardiac muscle of the subject, compared to the transgene expression in liver tissue of the subject.

[0079] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0080] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0081] The term “nucleic acid,” “nucleotide,” or “oligonucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0082] The term “gene” can refer to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, guide RNA (gRNA), short-interfering RNA (siRNA), or micro RNA (miRNA).

[0083] As used herein, the term “transgene” refers to an exogenous gene present in a vector genome, artificially introduced into the genome of a cell, or an endogenous gene artificially introduced into a non-natural locus in the genome of a cell. A transgene can refer to a segment of DNA involved in producing or encoding a polypeptide chain. Transgenes may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The transgene according to embodiments described herein, is pDys. The pDys transgene, in one embodiment, encodes a pDys polypeptide comprising an N-terminal region, from about two to three hinge regions, from about four to six spectrin repeats, and a cysteine rich domain.

[0084] “Vector genome” as used herein, is a nucleic acid genome comprising one or more heterologous nucleic acid sequences. The one or more heterologous nucleic acid sequences comprises a transgene. In some embodiments of the invention, the vector genome comprises at least one ITR sequence (e.g., AAV ITR sequence), optionally two ITRs (e.g., two AAV ITRs), which typically will be at the 5’ and 3’ ends of the vector genome and flank the one or more heterologous nucleic acids. The ITRs can be the same or different from each other.

[0085] As used herein, the term “endogenous” with reference to a nucleic acid, for example, a gene, or a protein in a cell, is a nucleic acid or protein that occurs in that particular cell as it is found in nature, for example, at its natural genomic location or locus. Moreover, a cell “endogenously expressing” a nucleic acid or protein expresses that nucleic acid or protein as it is found in nature.

[0086] A “promoter” is defined as one or more nucleic acid control sequence(s) that direct transcription of a nucleic acid, e.g., a transgene, and can be present within a vector genome. As used herein, a promoter includes nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0087] A “regulatory element” as used herein refers to a nucleic acid sequence capable of regulating transcription of a gene (e.g., a trans gene), and/or regulate the stability or translation of a transcribed mRNA product, and can be present within a vector genome. In some embodiments, regulatory elements can regulate tissue-specific transcription of a gene. Regulatory elements can comprise at least one transcription factor binding site, for example, a transcription factor binding site for a muscle-specific transcription factor. Regulatory elements as used herein increase or enhance promoter-driven gene expression when compared to the transcription of the gene from the promoter alone in the absence of the regulatory element. Regulatory elements as used herein may occur at any distance (i. e. , proximal or distal) to the transgene they regulate. Regulatory elements as used herein may comprise part of a larger sequence involved in transcriptional control, e.g., part of a promoter sequence. However, regulatory elements alone are typically not sufficient to initiate transcription on its own and require the presence of a promoter.

[0088] A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0089] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, and functional fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.

[0090] As used herein, the term “complementary” or “complementarity” refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are, generally, A and T (or A and U), and G and C. The guide RNAs (gRNAs) described herein can comprise sequences, for example, DNA targeting sequences that are perfectly complementary or substantially complementary (e.g., having a small fraction of mismatched bases) to a genomic sequence.

[0091] As used herein, the terms “introducing” or “delivering” in the context of nucleic acids, for example, AAV vectors, refers to the translocation of the nucleic acid from outside a cell to inside the cell, for example, a muscle cell. In some cases, introducing refers to translocation of the nucleic acid from outside the cell to inside the nucleus of the cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome-mediated translocation, and the like.

[0092] As used herein, the terms “packaged” or “encapsidated” refers to the inclusion of a vector genome in a capsid comprising viral capsid proteins to form an AAV particle.

[0093] The term “substantial identity” or “substantially identical,” as used in the context of polynucleotide or polypeptide sequences, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0094] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0095] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) Afo/. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0096] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat ’I. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10' ⁵ , and most preferably less than about IO' ²⁰ .

[0097] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms include, but are not limited to, mammals such as humans, simians, murines, equines, bovines, porcines, canines, felines, and the like. In some embodiments, the subject or patient is human. The subject in the methods of treatment provided herein, in one embodiment, is a male subject. [0098] In embodiments where the subject is a male human, the male human subject is from about 4 years old to about 7 years old, a newborn, about 1 year to about 7 years old, about 2 years to about 7 years old, about 2 years to about 6 years old, about 2 years to about 5 years old, about 2 years to about 4 years old, about 3 years to about 7 years old, about 3 years to about 6 years old, about 1 month to about 6 years old, about 1 month to about 5 years old, about

1 month to about 4 years old, about 1 month to about 3 years old, about 1 month to about 2 years old, or about 1 month to about 12 months old.

[0099] In one embodiment, the subject is a male human patient from about 4 years old to about 7 years old. In one embodiment, the subject is a male human patient from about 3 years old to about 7 years old. In one embodiment, the subject is a male human patient from about

2 years old to about 7 years old.

[0100] As used herein, the term “efficient delivery” or “efficiently delivering” refers to administration of an AAV particle comprising an AAV capsid encapsidating a vector genome encoding a transgene that results in expression of the transgene in a desired cell or tissue.

[0101] As used herein, the term “effective amount” or “effective dose”, refers to the amount of a substance (e.g., an AAV particle of the present invention) sufficient to effect beneficial or desired results (e.g., expression of a protein, or a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administration(s), application(s) or dosage(s) and is not intended to be limited to a particular formulation or administration route. Where a dose is provided in “vector genomes”, an “effective dose” may be referred to herein as an “effective vector genome dose”.

[0102] As used herein, the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.

[0103] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps. Adeno-Associated Virus (AAV) Particles

[0104] As described herein, one aspect of the present invention relates to an AAV particle comprising one or more AAV capsid proteins and a vector genome encapsidated by the one or more capsid proteins; and intrathecal compositions comprising the same. The genome comprises from 5’ to 3’, a 5’ inverted terminal repeat (ITR), a promoter, a trans gene, an SV40 poly (A) tail; and a 3’ ITR. When intrathecally administered to subjects in need of treatment, in one embodiment, the transgene is expressed at higher levels in the skeletal and/or cardiac muscle of the subject, compared to the transgene expression in liver tissue of the subject. In another embodiment, the ratio of [(skeletal and/or cardiac muscle transgene expression)] / (liver transgene expression)] provided by an AAV particle described herein, is greater than the same ratio when the identical dose of the same AAV particle is administered intravenously. In one preferred embodiment, the AAV particle is an AAV9 particle comprising one or more AAV9 capsid proteins.

[0105] As used herein, an “adeno-associated virus (AAV) particle”, refers to an AAV virion comprising an AAV capsid and a vector genome encapsidated by the AAV capsid. The vector genome typically comprises a promoter and one or more transgenes, that are flanked by AAV ITR sequences. The AAV capsid comprises one or more AAV capsid proteins. The AAV capsid proteins can be from the same or different AAV serotypes and can be wild-type or engineered. Vector genomes described herein can be replicated, and packaged into viral vectors (particles) when introduced into a host cell also comprising one or more plasmids encoding rep and cap gene products. In one embodiment, a helper plasmid is also transfected into the host cell to aid in vector production by the host cell. In one embodiment, the AAV vector used herein is an AAV9 vector, for example, as described in U.S. Patent No. 7,906,111, the disclosure of which is incorporated by reference herein in its entirety for all purposes. In one embodiment, the AAV capsid is an AAV9 capsid.

[0106] The terms “empty capsid,” “empty vial particle,” and “empty AA ’ refer to an AAV capsid shell lacking a vector genome packaged within.

[0107] Encapsidated vector genomes described herein can include one or more regulatory elements, for example one or more regulatory elements upstream of the transgene. The encapsidated genome, in one embodiment, comprises from 5’ to 3’, a 5’ ITR, a promoter, a transgene, an SV40 poly (A) tail; and a 3’ ITR. In another embodiment, the encapsidated genome comprises from 5’ to 3’, a 5’ ITR, an enhancer, a promoter, a trans gene, an SV40 poly (A) tail; and a 3’ ITR. The encapsidated genome, in even another embodiment, comprises from 5’ to 3’, a 5’ ITR, a promoter, a SV40 intron, a transgene, an SV40 poly (A) tail; and a 3’ ITR. In yet even another embodiment, the encapsidated genome comprises from 5’ to 3’, a 5’ ITR, an enhancer, a promoter, an SV40 intron, a transgene, an SV40 poly (A) tail; and a 3’ ITR.

Inverted Terminal Repeats

[0108] Inverted terminal repeats (ITR) are palindromic 145 nucleotide sequences that flank a transgene. The 5’ and 3’ ITRs of an AAV vector genome are necessary for both the integration of the transgene into the host cell genome (e.g. , chromosome 19 in humans) and for encapsidation of the transgene into the AAV particle.

[0109] In some embodiments, AAV vector genomes of the present invention comprise ITR sequences from any one AAV serotype, for example, AAVrh.74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. In some embodiments, the AAV vector genomes disclosed herein comprise a 5’ AAV2 ITR and a 3’ AAV2 ITR sequence.

[0110] In some embodiments, AAV vector genomes described herein comprise a 5’ AAV2 ITR having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1 (see Table 1). In some embodiments, the 5’ AAV2 ITR comprises SEQ ID NO:1. In some embodiments, the 5’ AAV2 ITR consists of SEQ ID NO:1.

[oni] In some embodiments, AAV vector genomes described herein comprise a 3’ AAV2 ITR having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7 (see Table 1). In some embodiments, the 3’ AAV2 ITR comprises SEQ ID NO:7. In some embodiments, the 3’ AAV2 ITR consists of SEQ ID NO:7.

Promoters

[0112] Promoters drive the expression of the transgene and are typically located upstream (or 5’) of the transgene whose expression they regulate.

[0113] In some embodiments, AAV vector genomes of the present invention comprise a mammalian promoter, for example, human, non-human primate (e.g., cynomolgous macaque), mouse, horse, cow, pig, cat, and dog promoters. In some embodiments, recombinant AAV vector genomes disclosed herein comprise strong, constitutively active promoters to drive high- level expression of the transgene. For example, in some embodiments, the promoter is a cytomegalovirus (CMV) promoter/enhancer, an elongation factor la (EFla) promoter, a simian virus 40 (SV40) promoter, a chicken [3-actin hybrid promoter, or a CAG promoter.

[0114] The promoter, in one embodiment, is an MHCK7 or chicken P-actin hybrid promoter.

[0115] In certain embodiments, an AAV vector genome of the present invention comprise a muscle-specific promoter that is operably linked to the transgene to drive high-level and tissue-specific expression in muscle cells. For example, muscle specific promoters of the present invention include, but are not limited to, promoters selected from: desmin (DES, also known as CSM1 or CSM2) promoter, the alpha 2 actinin (ACTN2, also known as CMD1 AA) promoter, the filamin-C (FLNC, also known as actin-binding-like protein (ABLP), filamin-2 (FLN2), ABP-280, ABP280A, ABPA, ABPL, MFM5 or MPD4) promoter, the sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (ATP2A1, also known as ATP2A or SERCA1) promoter, the troponin I type 1 (TNNI1, also known as SSTNI or 25TTNI) promoter, the myosin-1 (MYH1) promoter, the phosphorylatable, fast skeletal muscle myosin light chain (MYLPF) promoter, myosin 1 (MYH1, also known as MYHSA1, MYHa, MyC-2X/D or MyHC-2x) promoter, the alpha-3 chain tropomyosin (TPM3, also known as CFTD, NEM1, OK/Scl.5, TM-5, TM3, TM30, TM30nm, TM5, TPMsk3, TRK, h TM5 or hscp30) promoter, the ankyrin repeat domain-containing protein 2 (ANKRD2, also known as ARPP) promoter, the myosin heavy-chain (MHC) promoter, the myosin light-chain (MLC) promoter, the muscle creatine kinase (MCK) promoter, synthetic muscle promoters as described in Li et al. (1999. Nat Biotechnol. 17:241-245), such as the SPc5-12 promoter, the muscle creatine kinase (MCK) promoter, the dMCK promoter, the tMCK promoter consisting of respectively, a double or triple tandem of the MCK enhancer to the MCK basal promoter as described in Wang et al. (2008. Gene Ther. 15: 1489-1499) and hybrid promoters such as the hybrid alpha-myosin heavy chain enhancer/MCK enhancer (MHCK7; 770 bp); the MCK-C5-12 promoter as described in Wang et al. (2008. Gene Ther. 15:1489-1499) and the cardiac and skeletal muscle-specific myosin chaperone Unc45b (195 bp) promoter as described in Rudeck S et al. (2016, Genesis. 54(8): 431-8). Non-limiting examples of heart-specific promoters include the calsequestrin 2 (also known as PDIB2, FLJ26321, FLJ93514 or CASQ2 (GenelD 845 for the human gene)) promoter, the ankyrin repeat domain 1 (also known as cardiac ankyrin repeat protein) promoter, the cytokine inducible nuclear protein promoter; the liver ankyrin repeat domain 1 (ANKRD1; GenelD 27063 for the human gene) promoter; the myosin, light chain 2, regulatory, cardiac, slow (MYL2; GenelD 4633 for the human gene) promoter; the myosin, light chain 3, alkali; ventricular, skeletal 10 slow (MYL3; GenelD 4634 for the human gene) promoter; the bromodomain containing 7 (also known as BP75, CELTIX1, NAG4(BRD7; GenelD 29117 for the human gene)) promoter; the alpha myosin heavy chain (aMHC) promoter; the cardiac troponin C promoter and the promoter of the cardiac sodium-calcium exchanger (NCX1), which confers cardiac specificity.

[0116] In some embodiments, AAV vector genomes described herein comprise a MHCK7 promoter. For example, in some embodiments the MHCK7 promoter has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2 (see Table 1). In some embodiments, the MHCK7 promoter comprises SEQ ID NO:2. In some embodiments, the MHCK7 promoter consists of SEQ ID NO:2.

SV40 Intron

[0117] In some embodiments, AAV vector genomes of the present invention comprise an SV40 intron. The SV40 intron is a commonly used regulatory element in gene therapy vectors and enhances translation and stability of the expressed RNA transcript.

[0118] In certain embodiments, the SV40 intron is downstream (i.e., 3’) of the promoter and upstream (i.e., 5’) of the trans gene. In other embodiments, the SV40 intron can be downstream (i.e., 3’) of the transgene.

[0119] In some embodiments, the SV40 intron has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4 (see Table 1). In some embodiments, the SV40 intron comprises SEQ ID NO:4. In some embodiments, the SV40 intron consists of SEQ ID NO:4.

SV40-poly(A) Tail

[0120] In some embodiments, AAV vector genomes of the present invention comprise a nucleic acid sequence encoding an SV40 poly(A) tail. The SV40-poly(A) tail sequence is a commonly used nucleic acid element in gene therapy vectors that assists in RNA export from the nucleus, translation of RNA, and RNA stability.

[0121] In some embodiments, AAV vector genomes of the present invention comprise a sequence encoding an SV40 poly(A) tail having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6 (see Table 1). In some embodiments, the sequence encoding the SV40 poly(A) tail comprises SEQ ID NO:6. In some embodiments, the sequence encoding the SV40 poly(A) tail consists of SEQ ID NO:6.

Enhancers

[0122] In some embodiments, AAV vector genomes of the present invention comprise one or more enhancer sequence(s). An enhancer sequence, in one embodiment, can increase the level of transcription of the transgene, for example, by serving as a binding site for transcription factors and co-regulators that assist in DNA looping and recruitment of the transcriptional machinery to promoters.

[0123] In some embodiments, the enhancer is downstream (i.e., 3’) of the 5’ ITR and upstream (i.e., 5’) of the promoter. In some embodiments, the enhancer is downstream (i.e., 3’) of the promoter and upstream (i.e., 5’) of the transgene. In some embodiments, the enhancer is downstream (i.e., 3’) of the transgene and upstream (i.e., 5’) of the 3’ UTR.

[0124] In some embodiments, recombinant AAV vector genomes of the present invention comprise an enhancer that significantly promotes the transcription of a transgene in muscle cells, e.g., skeletal and/or cardiac muscle cells.

[0125] In some embodiments, recombinant AAV vectors described herein comprise a skeletal-cis-regulatory module 4 (SK-CRM4) enhancer. For example, in some embodiments the SK-CRM4 enhancer has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8 (see Table 1). In some embodiments, the SK-CRM4 enhancer comprises SEQ ID NO: 8. In some embodiments, the SK-CRM4 enhancer consists of SEQ ID NO: 8.

[0126] In yet another embodiment, an AAV vector genome of the present invention comprises a cytomegalovirus (CMV) enhancer nucleic acid sequence. In a further embodiment, the CMV enhancer is upstream of a promoter sequence. For example, in one embodiment, the CMV enhancer has the nucleic acid sequence of SEQ ID NOV. In some embodiments, the CMV enhancer has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOV (see Table 1). In some embodiments, the CMV enhancer comprises SEQ ID NOV. In some embodiments, the CMV enhancer consists of SEQ ID NOV. Transgene

[0127] Transgenes for use with the present invention are nucleic acid sequences encoding a polypeptide, or functional fragment thereof, to be expressed in a cell (e.g. , a muscle cell) into which the transgene is delivered.

[0128] In some embodiments, the transgene may be incorporated into the genome of the host cell to which it is delivered or may be expressed episomally.

[0129] In some embodiments, a trans gene can encode a polypeptide, or functional fragment thereof, that is not endogenously expressed by the cell in which the transgene is delivered. In some embodiments, the transgene encodes a mutant form of a polypeptide, or functional fragment thereof, that is endogenously expressed by the cell in which the transgene is delivered. In some embodiments, the transgene encodes a protein, or functional fragment thereof, that is endogenously expressed by the cell in which the transgene is delivered but is expressed at low levels, and wherein expression of the transgene results in higher expression levels of the protein, or functional fragment thereof. In some embodiments, the cell in which the transgene is delivered harbors one or more mutation(s) that results in lowered levels of expression of an endogenous protein and/or a functionally defective protein, and wherein expression of the transgene results in restored expression of the endogenous protein and/or functional supplementation of the defective protein. In some embodiments, the transgene is silent when introduced into the cell in which the transgene is delivered and expression may be induced.

[0130] In some embodiments, the transgene may be heterologous (i.e., from a different species) or homologous (i.e., from the same species) to the promoter and/or other regulatory elements present in the recombinant AAV vectors described herein. In some embodiments, the transgene may be heterologous or homologous to the cell into which the transgene is delivered.

[0131] In some embodiments, the transgene may be a full length cDNA or genomic DNA sequence, or a fragment or mutant thereof having functional activity. In some embodiments, the transgene may be a minigene, i.e., a gene sequence lacking part, most or all of its intronic sequences or may contain all of its intron sequences. In some embodiments, the transgene may be a hybrid nucleic acid sequence comprising homologous and/or heterologous cDNA and/or genomic DNA fragments. In some embodiments, the transgene may comprise one or more nucleotide substitutions, deletions, and/or insertions as compared to a wildtype sequence. [0132] In some embodiments, transgenes of the present invention encode a therapeutic protein. In certain embodiments, the transgene may encode a structural protein.

[0133] In some embodiments, the transgene is a micro-dystrophin (pDys) transgene that encodes a pDys polypeptide. In a further embodiment, the pDys polypeptide encoded by the transgene comprises (i) an N-terminal region (NTD) comprising an actin binding site, (ii) a central rod domain comprising from two to four hinge regions and from four to six spectrin repeats. In a further embodiment, the pDys transgene comprises the nucleic acid sequence set forth in SEQ ID NO:5. In even a further embodiment, the pDys transgene consists of the nucleic acid sequence set forth in SEQ ID NO:5. In another embodiment, the sequence encoding a pDys protein has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5.

[0134] The pDys transgene in one embodiment, comprises the nucleic acid sequence set forth at SEQ ID NO:4 of U.S. Patent No. 10,351,611, incorporated by reference herein in its entirety for all purposes. In another embodiment, the sequence encoding a pDys protein has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4 of U.S. Patent No. 10,351,611.

[0135] In one embodiment, the pDys transgene encodes a pDys polypeptide comprising (i) an N-terminal region (NTD) comprising an actin binding site, (ii) a domain comprising three hinge regions and four spectrin repeats, and (iii) a cysteine rich domain. The pDys transgene, in a further embodiment, encodes a pDys polypeptide comprising an N-terminal region (NTD) comprising an actin binding site, (ii) a central rod domain comprising hinge regions 1, 2 and 4, and spectrin repeats 1, 2, 3 and 24, and a (iii) cysteine rich domain.

[0136] The pDys transgene, in one embodiment, encodes dystrophin spectrin repeats 16 and 17, which have been reported as a scaffold for sarcolemmal neuronal nitric oxide synthase (nNOS) targeting. In a further embodiment, the pDys transgene encodes dystrophin spectrin repeats 1 and 24. In another embodiment, the pDys transgene encodes dystrophin spectrin repeats 1, 16 and 17, 23 and 24. In yet another embodiment, the pDys transgene encodes dystrophin spectrin repeats 1, 2, 3 and 24. In yet even another embodiment, the pDys transgene encodes dystrophin spectrin repeats 1, 2, 22, 23 and 24.

[0137] The pDys transgene, in one embodiment, encodes dystrophin hinge regions 1 and 4. In another embodiment, the pDys transgene encodes dystrophin hinge regions 1, 3 and 4. [0138] In one embodiment, the pDys transgene encodes a pDys protein comprising one of the combinations of pDys domains set forth in FIG. 14.

[0139] The AAV vector genomes described herein comprise a pDys transgene encoding a pDys protein comprising (i) an NTD comprising an actin binding site, (ii) a central rod domain comprising from two to four hinge regions and from four to six spectrin repeats, and (iii) a cysteine rich domain. For example, in one embodiment, the pDys transgene encodes dystrophin spectrin repeats 16 and 17. In a further embodiment, the pDys transgene encodes dystrophin spectrin repeats 1 and 24. In another embodiment, the pDys transgene encodes dystrophin spectrin repeats 1, 16 and 17, 23 and 24. In yet another embodiment, the pDys transgene encodes dystrophin spectrin repeats 1, 2, 3 and 24. In yet even another embodiment, the pDys transgene encodes dystrophin spectrin repeats 1, 2, 22, 23 and 24.

[0140] The pDys transgene in one embodiment, encodes dystrophin hinge regions 1 and 4. In another embodiment, the pDys transgene encodes dystrophin hinge regions 1, 3 and 4.

[0141] AAV particles described herein, in one embodiment, comprise an encapsidated transgene encoding a pDys protein. For example, in some embodiments the sequence encoding a pDys protein has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5 (see Table 1). In some embodiments, the sequence encoding a pDys protein comprises SEQ ID NO: 5. In some embodiments, the sequence encoding a pDys protein consists of SEQ ID NO:5.

[0142] Nucleic acid elements disclosed herein can be ligated together using standard molecular biology techniques to form gene constructs (see, for example, “Molecular Cloning: A Laboratory Manual, 2nd Ed.” (Sambrook et al., 1989), “Current Protocols in Molecular Biology” (Ausubel et al., 1987)).

[0143] Gene constructs described herein minimally comprise (from 5’ to 3’): (i) a promoter, and (ii) a transgene. For example, in some embodiments the promoter is an MHCK7 promoter. In certain embodiments, the transgene comprises a nucleic acid encoding pDys. In further embodiments, said gene constructs can comprise one or more additional regulatory element. In one embodiment, agene construct comprises from 5’ to 3’, apromoter, atransgene, and a SV40 poly (A) tail. In another embodiment, a gene construct comprises from 5’ to 3’, an enhancer, a promoter, atransgene, an SV40 intron and a SV40 poly (A) tail.

[0144] In some embodiments, a gene construct comprises (from 5’ to 3’): (i) a promoter, (ii) and SV40 intron, and (iii) a transgene. In certain embodiments, the promoter is an MHCK7 promoter. In certain embodiments, the transgene is a nucleic acid encoding pDys. In further embodiments, said gene constructs can comprise one or more additional regulatory element. In some embodiments, gene constructs described herein comprise a continuous nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the gene construct comprises SEQ ID NO: 10. In some embodiments, the gene construct consists of SEQ ID NO:10. [0145] In some embodiments, a gene construct comprises (from 5’ to 3’): (i) an enhancer, (ii) a promoter, (iii) a transgene, and (iv) a sequence encoding an SV40 poly(A) tail. In certain embodiments, the enhancer is an SK-CRM4 enhancer. In certain embodiments, the promoter is an MHCK7 promoter. In certain embodiments the transgene is a nucleic acid encoding a pDys. In further embodiments, said gene constructs can comprise one or more additional regulatory element. In some embodiments, a gene construct described herein comprises a continuous nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the gene construct comprises a nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the gene construct consists of a nucleic acid sequence of SEQ ID NO: 11.

[0146] In some embodiments, a vector genome described herein comprises a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the gene construct consists of SEQ ID NO: 12. In some embodiments, the vector genome comprises a nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the vector genome consists of a nucleic acid sequence of SEQ ID NO: 12.

AAV Vector Backbones

[0147] In some embodiments, an AAV vector genome of the present invention can be assembled by inserting a gene construct, as described herein, into an appropriate adenovirus plasmid backbone using standard molecular biology techniques (see, for example, Sambrook et al. (1989). “Molecular Cloning: A Laboratory Manual, 2nd Ed.”; Ausubel et al. (1987). “Current Protocols in Molecular Biology”). The adenovirus plasmid backbone, in one embodiment, comprises the 5’ ITR and 3’ ITR sequences described herein. The gene construct is inserted into the adenovirus plasmid backbone between the ITR sequences, downstream of the 5’ ITR sequence and upstream of the 3’ ITR sequence.

[0148] For example, in one embodiment, an AAV vector genome of the present invention can be assembled by inserting a gene construct comprising the sequence of SEQ ID NO: 10 into an adenovirus plasmid backbone comprising the sequence of SEQ ID NO: 13. [0149] In another embodiment, an AAV vector genome of the present invention can be assembled by inserting a gene construct comprising the sequence of SEQ ID NO: 11 into an adenovirus plasmid backbone comprising the sequence of SEQ ID NO: 13.

[0150] In some embodiments, the adenovirus plasmid backbone of the present invention comprises anucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the adenovirus plasmid backbone comprises a nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the adenovirus plasmid backbone consists of a nucleic acid sequence of SEQ ID NO: 13, provided below.

Adeno-Associated Viruses (AAV) Particle Production

[0151] AAV particles can be produced by any standard method (see, for example, WO 2001/083692; Masic et al. 2014. Molecular Therapy, 22(11): 1900-1909; Carter, 1992, Current Opinions in Biotechnology, 1533-539; Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97- 129); Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. 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. Patent No. 5,173,414; WO 95/13365; U.S. Patent No. 5,658.776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US 96/ 14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. Vaccine 13: 1244-1250 (1995); Paul et al. Human Gene Therapy 4:609-615 (1993); Clark et al. Gene Therapy 3: 1124- 1132 (1996); U.S. Patent. No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent. No. 6,258,595, herein incorporated by reference in their entireties). For example, in some embodiments, AAV vector genomes described herein can be transformed into Escherichia coli to scale-up DNA production, purified using any standard method (for example, a Maxi-Prep K, Thermo Scientific), and verified by restriction digest or sequencing. Purified AAV vector genomes can then be transfected using a standard method (e.g., calcium phosphate transfection, polyethyleneimine, electroporation, and the like) into an appropriate packaging cell line (e.g., HEK293, HeLa, or PerC.6, MRC-5, WI-38, Vera, and FRhL-2 cells) in combination with a plasmid comprising AAV rep and AAV cap genes, and an AAV helper plasmid. The AAV rep and cap genes may be from any AAV serotype and may be the same or different from that of the recombinant AAV vector ITRs including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In certain embodiments, AAV particles described herein comprise AAV rep and cap genes derived from AAV2 and AAV9, respectively.

[0152] In some embodiments, AAV particles described herein can be harvested from packaging cells and purified by methods standard in the art (e.g., Clark et al, Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Patent No. 6,566,118 and WO 98/09657, incorporated herein in their entirety by reference) such as by cesium chloride ultracentrifugation gradient or column chromatography.

Pharmaceutical Compositions

[0153] The pharmaceutical compositions provided herein are intrathecal pharmaceutical compositions, i.e., are intended for delivery via the intrathecal route. The intrathecal composition comprises an effective amount of an AAV particle encapsidating a pDys transgene, as described herein. In some embodiments, pharmaceutical compositions disclosed herein comprise an AAV particle of the present invention and a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. By “pharmaceutically acceptable” it is meant a material that is not toxic or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects. The pharmaceutical composition is an intrathecal pharmaceutical composition. The effective amount of the AAV particle comprises a lower dose of vector genomes compared to an IV AAV pharmaceutical composition comprising the same vector genome components, or the same transgene. For example, in one embodiment, the effective amount of the AAV particle described herein is about 90% or less vector genomes than the effective amount of an IV composition comprising the same AAV particle or the same micro-dystrophin transgene.

[0154] In some embodiments, pharmaceutical compositions provided herein comprise sterile aqueous and non-aqueous injection solutions, which are optionally isotonic with the blood of the subject to whom the pharmaceutical composition is to be delivered. Pharmaceutical compositions can contain antioxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended subject to be administered. Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In some embodiments pharmaceutical compositions comprise pharmaceutically acceptable vehicles and can include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. [0155] In some embodiments, pharmaceutical compositions can be presented in unit/dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.

[0156] In some embodiments, pharmaceutical compositions disclosed herein can be alternatively formulated for IV, intramuscular, or intracerebroventricular (ICV) administration.

Methods of Treatment

[0157] One aspect of the present invention relates to a method of treating a dystrophinopathy in a subject in need thereof, comprising intrathecally administering in a single dose, an intrathecal composition comprising an effective amount of an AAV particle described herein. The methods can be employed for preferentially delivering the AAV particle to cardiac and/or skeletal of the subject, for example, to treat a dystrophinopathy such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, or DMD-associated dilated cardiomyopathy (DCM). Without wishing to be bound by theory, the AAV particles and methods for delivering the same to subjects in need thereof, to treat a dystrophinopathy, provide a superior benefit to known AAV particles and treatment methods at least because the present invention: (i) allows for significantly lower dosages than IV delivery, in order to achieve substantially the same or a better therapeutic benefit, thereby reducing viral load and toxicity and other side effects; and/or (ii) allows for preferential transgene targeting and expression in cardiac and/or skeletal muscle tissue compared to liver tissue, thereby targeting the transgene to cells of interest to provide a greater therapeutic benefit compared to AAV vectors delivered intravenously; (iii) can benefit greater patient populations compared to IV formulations, because of the lower dosages needed which corresponds to a decreased manufacturing burden.

[0158] In one embodiment, a method of treating a subject in need thereof is provided comprising intrathecally administering to the subject in a single dose, a composition comprising an effective amount of an AAV particle comprising an AAV capsid encapsidating a vector genome comprising a pDys transgene. In a further embodiment, the subject is positioned in the Trendelenburg position prior to the intrathecal administration. In one embodiment, intrathecal administration of the composition is in the absence of a non-ionic, low-osmolar contrast agent. In another embodiment, intrathecal administration is in the presence of a non-ionic, low-osmolar contrast agent. [0159] The present invention is based in part on the finding that intrathecal administration of an effective amount of an AAV particle described herein allows for pDys transgene expression at higher levels in skeletal and/or cardiac muscle of the subject compared to transgene expression in liver tissue of the subject. For example, in one embodiment, subsequent to administration of the effective amount of the AAV particle, e.g., an AAV9 particle, the pDys transgene is expressed at higher levels in skeletal muscle of the subject, compared to the levels of pDys transgene expression in liver tissue. In another embodiment, subsequent to administration of the effective amount of the AAV particle, e.g., a recombinant AAV9 particle, the pDys transgene is expressed at higher levels in cardiac muscle of the subject, compared to the levels of pDys transgene expression in liver tissue. In yet another embodiment, subsequent to administration of the effective amount of the AAV particle, e.g., an AAV9 particle, the pDys transgene is expressed at higher levels in skeletal and cardiac muscle of the subject, compared to the levels of pDys transgene expression in liver tissue.

[0160] According to the embodiments described herein, transgene expression may refer to gene expression (i.e., by measuring mRNA levels) or expression of the corresponding protein. It will be understood by those of ordinary skill in the art that in order to determine levels of transgene expression in different tissue types, substantially the same amount of tissue, or substantially the same number of cells should be compared for gene expression levels. The higher levels of pDys transgene expression in the skeletal and/or cardiac muscle, in one embodiment, is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% higher, compared to the amount of pDys transgene expression in the liver tissue. In a further embodiment, the pDys transgene comprises the nucleic acid sequence set forth in SEQ ID NO:5.

[0161] In one embodiment of the methods described herein, the method of delivering an effective dose of an AAV particle encapsidating a pDys transgene provides greater pDys transgene expression in skeletal and/or cardiac muscle, compared to a substantially identical dose of an AAV particle encapsidating a pDys transgene administered intravenously. In another embodiment, the method of delivering an effective dose of an AAV particle provides greater pDys transgene expression in skeletal and/or cardiac muscle, compared to when an effective amount of an AAV particle encapsidating a pDys transgene is administered intravenously. Transgene expression, in one embodiment, is measured about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months or about 24 months subsequent to administration of the composition comprising the effective amount of the AAV particle. The transgene, in one embodiment, comprises the nucleic acid sequence set forth in SEQ ID NO:5.

[0162] In another embodiment, an AAV particle encapsidating a pDys transgene of the present invention, when administered intrathecally, provides at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% greater transgene expression in the skeletal and/or cardiac muscle compared to the transgene expression in the same tissue type when the identical vector genome dose is administered intravenously. The identical vector genome need not contain the same regulatory elements and/or an identical transgene sequence or the same AAV capsid. However, the transgene administered intrathecally and intravenously encode for a pDys polypeptide.

[0163] In yet another embodiment, the ratio of [(skeletal and/or cardiac muscle pDys transgene expression)] / (liver pDys transgene expression)] measured subsequent to administration of the intrathecally administered AAV particle, is greater than the same ratio when the identical dose of an AAV particle encapsidating a pDys transgene, is administered intravenously. Transgene expression, in one embodiment, is measured about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months or about 60 months, subsequent to administration of the composition comprising the effective amount of the AAV particle.

[0164] In one embodiment, the effective amount of the AAV particle encapsidating a pDys transgene, when intrathecally administered, provides a greater efficacy or a greater therapeutic benefit, compared to an AAV particle encapsidating a pDys transgene administered intravenously, at the same dose.

[0165] In some embodiments, the intrathecal (IT) vector genome (vg) dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 90%, about 90% or less, about 85%, about 85% or less, about 80%, about 80% or less, about 75%, about 75% or less, about 70%, about 70% or less, about 60%, about 60% or less, about 50%, about 50% or less, about 40%, about 40% or less, about 30%, about 30% or less, about 25%, about 25% or less, about 10%, or about 10% or less, an intravenous (IV) vg dose of a vector genome encoding a pDys transgene sufficient to provide the same or substantially the same therapeutic response. The IT vector genome comprises a pDys transgene. The IT and IV pDys transgenes need not include the same nucleic acid sequence. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 90% or less than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 75% or less than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 50% or less than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 25% or less than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 10% or less than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 10-40 times less than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. The therapeutic response in one embodiment, is pDys transgene expression in muscle tissue, e.g., cardiac and/or skeletal muscle tissue. In another embodiment, the therapeutic response is an increase of the subject’s score from baseline (i.e. , prior to treatment) in the NorthStar Ambulatory Assessment (NSAA). In a further embodiment, the transgene encodes a pDys polypeptide.

[0166] In some embodiments, the intrathecal (IT) vector genome (vg) dose sufficient to provide a therapeutic response for one of the treatment methods described herein is lower than an intravenous (IV) vg dose sufficient to provide the same or substantially the same therapeutic response, wherein the IT and IV vector genomes each includes a transgene that encodes for a pDys polypeptide. However, the respective transgenes need not include the same nucleic acid sequence. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75- fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150- fold, about 200-fold, about 250-fold, about 500-fold, or about 1000-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 10-fold to about 20-fold, about 10-fold to about 30-fold, about 10-fold to about 40-fold, about 10-fold to about 50-fold, about 10-fold to about 75-fold, about 10-fold to about 100-fold, or about 10-fold to about 1000-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 25-fold to about 30-fold, about 25-fold to about 40-fold, about 25-fold to about 50-fold, about 25-fold to about 75-fold, about 25-fold to about 100-fold, about 25-fold to about 500-fold, or about 25-fold to about 1000-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 50-fold to about 75 -fold, about 50-fold to about 100-fold, about 50-fold to about 250-fold, about 50-fold to about 500-fold, or about 50-fold to about 1000-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 100-fold to about 200-fold, about 100-fold to about 250-fold, about 100-fold to about 500-fold, or about 100-fold to about 1000-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 10- fold to about 40-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 25-fold to about 40-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 10-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 25-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 40-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 50-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, the IT vg dose sufficient to provide a therapeutic response for one of the treatment methods described herein is about 100-fold lower than the IV vg dose sufficient to provide the same or substantially the same therapeutic response. The therapeutic response in one embodiment, is transgene expression in muscle tissue, e.g., cardiac and/or skeletal muscle tissue.

[0167] In some embodiments, an effective dose of an intrathecal (IT) composition comprising the AAV particles encapsidating a pDys transgene described herein is lower than the effective dose of an intravenous (IV) composition comprising an AAV particle encapsidating a pDys transgene. In some embodiments, the effective dose of the IT composition comprising the AAV particle is about 90%, about 90% or less, about 85%, about 85% or less, about 80%, about 80% or less, about 75%, about 75% or less, about 70%, about 70% or less, about 60%, about 60% or less, about 50%, about 50% or less, about 40%, about 40% or less, about 30%, about 30% or less, about 25%, about 25% or less, about 10%, or about 10% or less vector genomes than the effective amount of the IV composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 90% or less vector genomes than the effective amount of the IV composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 75% or less vector genomes than the effective amount of the IV composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 50% or less vector genomes than the effective amount of the IV composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 25% or less vector genomes than the effective amount of the IV composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 10% or less vector genomes than the effective amount of the IV composition.

[0168] In some embodiments, an effective dose of an AAV particle in an IT composition is a lower dose than the effective dose of an AAV particle in an IV composition, wherein each AAV particle encapsidates a pDys transgene. In some embodiments, the effective dose of an AAV particle in an IT composition is about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80- fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200- fold, about 250-fold, about 500-fold, or about 1000-fold lower dose than the effective dose of the AAV particle in the IV composition. In some embodiments, the effective amount (effective dose) of the IT composition comprising the AAV particle is about 10-fold to about 20-fold, about 10-fold to about 30-fold, about 10-fold to about 40-fold, about 10-fold to about 50-fold, about 10-fold to about 75 -fold, about 10-fold to about 100-fold, or about 10-fold to about 1000- fold lower dose than the effective amount (effective dose) of the IV composition comprising the same AAV particle, or an AAV particle encapsidating a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particle in the IT composition. In some embodiments, the effective amount of the intrathecal composition comprising the AAV particle is about 25-fold to about 30-fold, about 25-fold to about 40-fold, about 25-fold to about 50- fold, about 25-fold to about 75-fold, about 25-fold to about 100-fold, about 25-fold to about 500-fold, or about 25-fold to about 1000-fold lower dose than the effective amount of the IV composition comprising the same AAV particle, or an AAV particle encapsidating a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particle in the IT composition. In some embodiments, the effective amount of the intrathecal composition comprising the AAV particle is about 50-fold to about 75-fold, about 50-fold to about 100- fold, about 50-fold to about 250-fold, about 50-fold to about 500-fold, or about 50-fold to about 1000-fold lower dose than the effective amount of the IV composition comprising the same AAV particle, or an AAV particle encapsidating a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particle in the IT composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 100-fold to about 200-fold, about 100-fold to about 250-fold, about 100-fold to about 500-fold, or about 100-fold to about 1000-fold lower dose than the effective amount of the IV composition comprising the same AAV particle. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 25 -fold to about 40-fold lower than the effective amount of the IV composition comprising the same AAV particle, or an AAV particle encapsidating a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particle in the IT composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 10-fold lower dose than the effective amount of the IV composition comprising the same AAV particle, or an AAV particle encapsidating a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particle in the IT composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 25 -fold lower dose than the effective amount of the IV composition comprising the same AAV particle, or an AAV particle encapsidating a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particle in the IT composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 40-fold lower dose than the effective amount of the IV composition comprising the same AAV particle, or an AAV particle encapsidating a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particle in the IT composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 50-fold lower dose than the effective amount of the IV composition comprising the same AAV particle, or an AAV particle encapsidating a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particle in the IT composition. In some embodiments, the effective amount of the IT composition comprising the AAV particle is about 100-fold lower dose than the effective amount of the IV composition comprising the same AAV particle, or an AAV particle encapsidating a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particle in the IT composition. In some embodiments, the transgene is a pDys transgene.

[0169] In one embodiment of the treatment methods provided herein, treating comprises decreasing in serum creatine kinase (CK) levels in a subject compared to the serum CK levels prior to treatment. In some embodiments, the serum CK levels are decreased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more compared to serum CK levels prior to treatment. In a further embodiment, serum CK levels are assessed prior to treatment with an AAV particle and at about 12 months, about 18 months, about 24 months, or about 30 months subsequent to administration of the AAV particle. In a further embodiment, the AAV particle comprises an AAV9 capsid encapsidating a pDys transgene.

[0170] In another embodiment of the intrathecal treatment methods provided herein, treating comprises decreasing the number of side effects, or a reducing the severity of one or more side effects in the subject being treated, compared to a subject treated via IV administration of an effective amount of the same AAV particle, or different AAV particle encapsidating a pDys transgene. The AAV particle administered intrathecally, in one embodiment, is an AAV9 particle. [0171] In some embodiments, an AAV particle of the present invention or a pharmaceutical composition comprising the same can be used to treat a dystrophinopathy including, but not limited to, DMD, Becker muscular dystrophy, and DCM.

[0172] In some embodiments, the AAV particle of the present invention is administered once or multiple times to a subject in need of treatment, e.g., a subject with DMD. In some embodiments, the AAV particle is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more to a subject in need of treatment. In a preferred embodiment, an intrathecal composition provided herein comprising the AAV particle is administered once to a subject in need of treatment. In a further embodiment, the AAV particle is an AAV9 particle.

[0173] In one embodiment, an effective amount of an AAV particle comprising an AAV capsid encapsidating a pDys transgene as described herein is used in a method for treating DMD in a subject in need of treatment. In a further embodiment, the subject is intrathecally administered a composition comprising the AAV particle in a single dose. In a further embodiment, the AAV particle is an AAV9 particle. In even a further embodiment, the subject is positioned in the Trendelenburg position prior to the administration.

[0174] In one embodiment of a method for treating DMD, a subject in need of treatment is intrathecally administered in a single dose, a composition comprising an effective amount of an AAV particle comprising an AAV capsid encapsidating a pDys transgene. The pDys transgene, in one embodiment, includes a combination of dystrophin elements set forth in FIG. 14. In one embodiment, the vector genome comprises a nucleic acid sequence set forth in SEQ ID NO:5, 10, 11 or 12.

[0175] In one embodiment of a method for treating DMD, treating comprises increasing the subject’s score from baseline (i.e., prior to treatment) in the NorthStar Ambulatory Assessment (NSAA). The NSAA is a 17-item rating scale that is used to measure functional motor abilities in ambulatory DMD subjects. The scale is ordinal with 34 as the maximum score indicating fully independent function. Each activity is graded as either a 0 (unable to achieve independently), 1 (modified method but achieves goal independent of physical assistance from another), or 2 (normal - no obvious modification of activity). See, e.g., Mazonne et al. (2009). Neuromuscular Disorders 19, pp. 458-461 and researchrom.eom/masterlist/view/18#form2, the disclosure of each of which is incorporated by reference in its entirety for all purposes. The change from baseline, in one embodiment, is measured 12 months subsequent to administration of the intrathecal composition. In another embodiment, the change from baseline is measured 18 months subsequent to administration of the intrathecal composition. In another embodiment, the change from baseline is measure 24 months subsequent to administration of the intrathecal composition. In even another embodiment, the subject’s increased score from baseline in the NSAA as measured 12 months subsequent to administration is substantially unchanged or increased at 18 months subsequent to administration. In yet even another embodiment, the subject’s increased score from baseline in the NSAA as measured 12 months subsequent to administration is substantially unchanged or increased at 24 months subsequent to administration. In yet even another embodiment, the subject’s increased score from baseline in the NSAA as measured 12 months subsequent to administration is substantially unchanged or increased at 60 months subsequent to administration.

[0176] Increasing the NSAA score, in one embodiment, comprises increasing the NSAA score by from about 5 to about 25, from about 5 to about 20, from about 5 to about 15 or from about 5 to about 10. In another embodiment, increasing the NSAA score, comprises increasing the NSAA score by from about 2 to about 12 points. In another embodiment, increasing the NSAA score, comprises increasing the NSAA score by from about 2 to about 10 points. In yet another embodiment, increasing the NSAA score, comprises increasing the NSAA score by from about 3 to about 10 points. In even another embodiment, increasing the NSAA score comprises increasing the score by from about 4 to about 10 points. In yet even another embodiment, increasing the NSAA score comprises increasing the score by from about 2 to about 8 points. I n another embodiment, increasing the NSAA score, comprises increasing the NSAA score by from about 2 to about 6 points.

[0177] In one embodiment of a method for treating DMD, a subject in need of treatment is intrathecally administered in a single dose, an effective amount of an AAV vector encapsidating a pDys transgene. The pDys transgene, in one embodiment, includes the combination of dystrophin elements set forth in FIG. 14. In one embodiment, the vector genome comprises the nucleic acid sequence set forth in SEQ ID NO:5, 10, 11 or 12. An effective amount of the AAV vector encapsidating a pDys transgene, in one embodiment, is an amount sufficient to increase the number of meters walked in a 6-minute walk test (6MWT), compared to the number of meters walked prior to treatment.

[0178] The AAV particles of the present invention, or pharmaceutical compositions comprising the same, are administered as a single dose or as divided doses. In some embodiments, the dose is l*10 ⁹ to l*10 ¹⁶ vector genomes (vg), 2.5*10 ¹³ to l*10 ¹⁵ vg, 5*10 ¹³ to IxlO ¹⁵ vg, 7.5X10 ¹³ to 1X10 ¹⁵ vg, lxl0 ¹⁴ to IxlO ¹⁵ vg, 2.5xl0 ¹⁴ to IxlO ¹⁵ vg, 5xl0 ¹⁴ to IxlO ¹⁵ vg, 7.5xl0 ¹⁴ to IxlO ¹⁵ vg, lxl0 ¹³ to 7.5xl0 ¹⁴ vg, 2.5xl0 ¹³ to 7.5xl0 ¹⁴ vg, 5xl0 ¹³ to 7.5xl0 ¹⁴ vg, 7.5xl0 ¹³ to 7.5xl0 ¹⁴ vg, lxl0 ¹³ to 5.0xl0 ¹⁴ vg, 2.5xl0 ¹³ to 5.0xl0 ¹⁴ vg, 5xl0 ¹³ to 5.0 x 10 ¹⁴ vg, 7.5xl0 ¹³ to 5.0xl0 ¹⁴ vg, IxlO ¹³ to 2.5xl0 ¹⁴ vg, 2.5xl0 ¹³ to 2.5xl0 ¹⁴ vg, 5xl0 ¹³ to 2.5xl0 ¹⁴ vg, 7.5xl0 ¹³ to 2.5xl0 ¹⁴ vg, IxlO ¹³ to lxl0 ¹⁴ vg, 2.5xl0 ¹³ to lxl0 ¹⁴ vg, 5xl0 ¹³ to IxlO ¹⁴ vg, 7.5xl0 ¹³ to IxlO ¹⁴ vg delivered as a single dose or as divided doses. For example, in some embodiments, AAV particles of the present invention or pharmaceutical compositions comprising the same, is administered as a single dose of 2.5x 10 ¹³ vg. In another embodiment, AAV particles of the present invention or pharmaceutical compositions comprising the same, is administered as a single dose of 5xl0 ¹³ vg. In yet another embodiment, AAV particles of the present invention or pharmaceutical compositions comprising the same, is administered as a single dose of 1 x 10 ¹⁴ vg.

[0179] The AAV particles of the present invention, or pharmaceutical compositions comprising the same, are administered as a single intrathecal dose. In some embodiments, doses for intrathecal delivery can be IxlO ⁹ to IxlO ¹⁶ vector genomes (vg), lxl0 ¹⁰ to lxl0 ¹⁶ vg, lxlO ⁿ tO lxio ¹⁶ vg, lxl0 ¹² tO lxio ¹⁶ vg, IxlO ¹³ to lxio ¹⁶ vg, lxl0 ¹⁴ tO lxio ¹⁶ vg, IxlO ¹⁵ to lxio ¹⁶ vg, IxlO ⁹ to lxio ¹⁵ vg, IxlO ⁹ to lxio ¹⁴ vg, IxlO ⁹ to 1 X 10 ¹³ vg, IxlO ⁹ to lxl0 ¹² vg, IxlO ⁹ to lxl0 ⁿ vg, IxlO ⁹ to IxlO ¹⁰ vg, IxlO ¹³ to IxlO ¹⁵ vector vg, 2.5xl0 ¹³ to lxl0 ¹⁵ vg, 5xl0 ¹³ to lxio ¹⁵ vg, 7.5X10 ¹³ to lxio ¹⁵ vg, lxl0 ¹⁴ to lxl0 ¹⁵ vg, 2.5xl0 ¹⁴ to IxlO ¹⁵ vg, 5xl0 ¹⁴ to lxl0 ¹⁵ vg, 7.5xl0 ¹⁴ to lxl0 ¹⁵ vg, IxlO ¹³ to 7.5xl0 ¹⁴ vg, 2.5xl0 ¹³ to 7.5xl0 ¹⁴ vg, 5xl0 ¹³ to 7.5xl0 ¹⁴ vg, 7.5xl0 ¹³ to 7.5xl0 ¹⁴ vg, IxlO ¹³ to 5.0xl0 ¹⁴ vg, 2.5xl0 ¹³ to 5.0xl0 ¹⁴ vg, 5xl0 ¹³ to 5.0xl0 ¹⁴ vg, 7.5xl0 ¹³ to 5.0xl0 ¹⁴ vg, IxlO ¹³ to 2.5xl0 ¹⁴ vg, 2.5xl0 ¹³ to 2.5xl0 ¹⁴ vg, 5xl0 ¹³ to 2.5xl0 ¹⁴ vg, 7.5xl0 ¹³ to 2.5xl0 ¹⁴ vg, IxlO ¹³ to lxl0 ¹⁴ vg, 2.5xl0 ¹³ to lxl0 ¹⁴ vg, 5xl0 ¹³ to IxlO ¹⁴ vg, 7.5xl0 ¹³ to IxlO ¹⁴ vg delivered as a single or as divided doses. For example, in some embodiments, AAV particles of the present invention or pharmaceutical compositions comprising the same, can be administered as a single intrathecal dose of 2.5 xlO ¹³ vg. In another embodiment, AAV particles of the present invention or pharmaceutical compositions comprising the same, can be administered as a single intrathecal dose of 5xl0 ¹³ vg. In yet another embodiment, AAV particles of the present invention or pharmaceutical compositions comprising the same, can be administered as a single intrathecal dose of IxlO ¹⁴ vg.

[0180] In other embodiments, AAV particles of the present invention or pharmaceutical compositions comprising the same, can be administered as a single or divided intracerebroventricular doses. In some embodiments, doses for intracerebroventricular delivery can be l*10 ¹³ to l *10 ¹⁵ vg, 2.5*10 ¹³ to l *10 ¹⁵ vg, 5*10 ¹³ to l*10 ¹⁵ vg, 7.5*10 ¹³ to lxio ¹⁵ vg, l xl0 ¹⁴ to l xio ¹⁵ vg, 2.5xl0 ¹⁴ to l xio ¹⁵ vg, 5xl0 ¹⁴ to l xl0 ¹⁵ vg, 7.5xl0 ¹⁴ to I xlO ¹⁵ vg, l xl0 ¹³ to 7.5xl0 ¹⁴ vg, 2.5xl0 ¹³ to 7.5xl0 ¹⁴ vg, 5xl0 ¹³ to 7.5xl0 ¹⁴ vg, 7.5xl0 ¹³ to 7.5xl0 ¹⁴ vg, l xl0 ¹³ to 5.0xl0 ¹⁴ vg, 2.5xl0 ¹³ to 5.0xl0 ¹⁴ vg, 5xl0 ¹³ to 5.0xl0 ¹⁴ vg, 7.5xl0 ¹³ to 5.0xl0 ¹⁴ vg, l xl0 ¹³ to 2.5xl0 ¹⁴ vg, 2.5xl0 ¹³ to 2.5xl0 ¹⁴ vg, 5xl0 ¹³ to 2.5xl0 ¹⁴ vg, 7.5xl0 ¹³ to 2.5xl0 ¹⁴ vg, l xl0 ¹³ to IxlO ¹⁴ vg, 2.5xl0 ¹³ to lxl0 ¹⁴ vg, 5xl0 ¹³ to l xl0 ¹⁴ vg, 7.5xl0 ¹³ to lxl0 ¹⁴ vg. For example, in some embodiments, AAV particles of the present invention or pharmaceutical compositions comprising the same, can be administered as a single intracerebroventricular dose of 2.5 xlO ¹³ vg. In another embodiment, AAV particles of the present invention or pharmaceutical compositions comprising the same, can be administered as a single intracerebroventricular dose of 5xl0 ¹³ vg. In yet another embodiment, AAV particles of the present invention or pharmaceutical compositions comprising the same, can be administered as a single intracerebroventricular dose of 1 x 10 ¹⁴ vg.

EXAMPLES

[0181] The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and is not intended to limit the invention.

Select Methods

Intracerebroventricular (ICV) injections at postnatal day 1 (pl)

[0182] Perform Intracerebroventricular injection (ICV) of neonatal pups (pl) via the Cerebral Hemisphere. Neonatals are injected fully awake. Ensure needles are marked for an injection depth of 2mm. The injection site is the midpoint between the ear and eye (location is approximately 0.7 -1.0 mm lateral to the sagittal suture and 0.7 -1.0 mm caudal from the neonatal bregma). P0 is designated as the mouse Date of Birth (DoB). Animals may be injected within 36 hours of discovering the litter.

[0183] If an excessive amount of Dosing solution is determined to have leaked out of the injection site, or the injection site was missed entirely, the animal is excluded from the study.

Intracerebroventricular (ICV) injections at postnatal day 28 (p28) [0184] Hamilton syringe re loaded with desired volume of dosing solution. Standard volume is 8pl per injection site. Animals are removed individually from cage and placed in anesthesia chamber. Once in chamber, the animal is anaesthetized. Tubing is connected from anesthesia machine to stereotaxic device to allow for continued anaesthetization during injection. The respective animal remains in chamber for about two minutes before being removed and placed on the stereotaxic device. Once the animal is on the stereotaxic device, coordinates for injection are set at medial/lateral (M/L): +/- 1.00 mm, anterior/posterior (A/P): -0.5— 0.8 mm, dorsal/ventral (D/F): -2.5 mm.

[0185] An iodine swab is applied at incision site on scalp of animal for sterility purposes. Using a scalpel, a small incision is made into the scalp of the animal and the scalp is gently peeled back to expose cranial region. Once needle is in desired location, syringe plunger is slowly pushed down to inject dosing solution into cranial space. The site of injection is monitored during injection and immediately post injection to confirm quality of injection.

Muscle preparation

[0186] When animals reach the appropriate age, they are weighed and anesthetized (ketamine [80 mg/kg], acepromazine [0.5 mg/kg], and xylazine [16 mg/kg]) via intraperitoneal (i.p.) injection. Dissection of tissues is then carried out. Scissors are used to cut the skin at the ankle and then the skin on both lower legs is pulled back to expose the lower and upper leg muscles. The tibialis anterior on one side is dissected as close to its insertion point and weighed to the nearest 0. Img and then discarded. Then, 4.0 suture is tied at the my otendinous junction of the proximal and distal ends of the extensor digitorum longus (EDL) and then dissected and placed into Ringer’s solution (137 mm NaCl, 5 mm KC1, 2 mm CaCh, 1 mm MgSCh, 1 mm NaH2PO4, 24 mm NaHCOs. 11 mm glucose containing 10 mg/liter curare) that is kept at room temperature.

[0187] After dissecting the EDL, the abdomen is opened, and blood is drawn using a 1 cc syringe from the inferior vena cava (about 300-500 pL). The blood sits at room temperature for 25-35 minutes and then is centrifuged at 3.500/g for 10 minutes at 4 °C. After spinning the supernatant, serum is isolated and placed into a microcentrifuge tube and frozen at -80 °C.

[0188] The Achilles tendon is then cut and pulled back to expose the soleus, plantaris and gastrocnemius. The soleus is dissected out and placed in a tube and frozen in liquid nitrogen- cooled isopentane. The plantaris is blunt dissected free of the gastrocnemius and then cut as proximally as possible and placed in a tube and frozen in liquid nitrogen-cooled isopentane and stored at -80 °C.

[0189] The gastrocnemius is cut as proximally as possible, weighed to the nearest 0.1 mg and placed in a tube and frozen. The tibialis anterior from the contralateral side is dissected free, weighed to the nearest 0.1 mg and pinned on cork at resting length. The EDL from the same side will also be dissected free and pinned on the same cork at resting length. The cork is then immersed in liquid nitrogen-cooled isopentane. After about 30 to about 45 seconds, the cork is placed on dry ice, and wrapped in foil and stored at -80 °C. The quadriceps muscle is dissected and placed in a tube and frozen in liquid nitrogen-cooled isopentane and stored at - 80 °C. A small piece of liver is dissected and placed in a tube and frozen in liquid nitrogen- cooled isopentane and stored at -80 °C. The diaphragm is dissected, folded in half, and then folded again, and then placed on cork and pinned. Then, the cork is immersed in liquid nitrogen-cooled isopentane. After 30-45 seconds the cork is placed on dry ice, wrapped in foil, and stored at -80 °C.

[0190] The whole heart is dissected, weighed to the nearest 0. Img and placed in a tube and frozen in liquid nitrogen-cooled isopentane. The tube is capped and placed in liquid nitrogen until storage at -80 °C.

Example 1: Generation of recombinant adeno-associated virus particles

Molecular Cloning of Micro-Dystrophin Gene Constructs

[0191] A micro-dystrophin (pDys) encoding gene construct, referred to herein as INS1201 and formerly known as MTS-001, was synthesized by operably linking an MHCK7 promoter and an SV40 intron to a polynucleotide encoding pDys and an SV40 poly(A) signal (FIG. 1A). An alternative pDys encoding gene construct (referred to herein as INS 1212 and formerly known as MTS-003) was synthesized by operably linking an SK-CRM4 enhancer with an MHCK7 promoter to a polynucleotide encoding and an SV40 poly(A) signal (FIG. IB). The INS 1201 and INS 1212 gene constructs were generated in a Puc57 vector backbone. The INS1201 and INS1212 constructs were confirmed by DNA sequencing and the 4542 bp and 4714 constructs, respectively, were isolated by Nrul restriction digest and gel-purified for subsequent cloning into an appropriate AAV backbone.

[0192] The isolated INS1201 and INS1212 gene constructs were each independently blunt cloned into a gel-purified pSZOl vector backbone containing ITR sites and a kanamycin resistance gene that was linearized/isolated by Nrul restriction digest. Following T4 ligation of the INS1212 construct with the pSZOl vector backbone and INS1212 construct with the pSZOl vector backbone, DNA was transformed into E. coli, grown and purified using a NEB Monarch® Plasmid Miniprep kit. Gene constructs that had undergone successful ligation to produce complete pSZ01-INS1201 or psZ01-INS1212 plasmid clones were identified by Hindlll/Bsal restriction digest.

[0193] pSZ01-INS1201 and psZ01-INS1212 clones were scaled-up by bacterial transformation in E. coli using a Maxi-Prep Kit (GeneJET Endo-free Plasmid Maxiprep Kit, ThermoScientific). Correct plasmid sequences were re-confirmed by Bsal/Hindlll digest (FIG. 1C, lanes 1 and 3). Additionally, restriction digest using Smal (FIG. 1C, lanes 2 and 4) was performed as further confirmation of correct incorporation of ITR sites containing INS 1201 and INS 1212 within the pSZOl vector backbone.

Transient Transfection and Viral Packaging

[0194] The confirmed pSZ01-INS1201 and psZ01-INS1212 AAV vectors were transiently transfected into HEK293 cells in combination with adenoviral helper plasmid and a chimeric packaging construct that delivers the AAV2 rep gene together with the AAV9 cap gene, using a standard calcium phosphate transfection method (for example, as described in Vandendriessche et al. (2007. J Thromb Haemost 5:16-24), incorporated by reference herein in its entirety). Two days post transfection, AAV particles were harvested and purified using two successive rounds of cesium chloride density gradient ultracentrifugation. 1 pl each of purified INS1201-AAV9 (FIG. 2, lane 1) and INS1212-AAV9 (FIG. 2, lane 2) were titered by comparing to 1 x 10 ¹³ vg AAV2 standard (FIG. 2, lane 3 (0.5pl), lane 4 (1 pl), lane 5 (2 pl), lane 6 (4 pl)) resolved by SDS-PAGE and silver-stained.

Example 2: Intramuscular Delivery of AAV9 uDys (INS1201-AAV9 and INS1212-AAV9) results in increased uDvs expression in MTX mice.

[0195] INS1201-AAV9 and INS1212-AAV9 were intramuscularly injected into the gastrocnemius muscle of mdx mice, a common murine model of Duchenne muscular dystrophy (see, for example Rodino-Klapac et al. (2013) Hum Mol Genet. 22(24):4929-37, incorporated herein by reference). As shown in FIG. 3A, 21 days-post intramuscular injection, gastrocnemius muscle injected with 2.7X10 ¹¹ vg of INS1201-AAV9 (iii) or INS1212-AAV9 (iv) exhibited widespread expression of pDys at significantly higher levels than non-injected mdx mice (i) and at comparable levels to wildtype C57/B1 mice (ii). FIG. 3B similarly shows high level expression of pDys in mdx mice 21 days post intramuscular injection with 2.7 * 10 ¹¹ vg of INS1212-AAV9.

Example 3: Intracerebroventricular Delivery of AAV9 (INS1201-AAV9 and INS 1212-AAV9) results in increased uDys expression in MDX mice.

[0196] INS1201-AAV9 was intracerebroventicularly injected into mdx mice on postnatal day 1 (pl) and tissue samples were collected and analyzed for dystrophin. As shown in FIG. 4A, 21 days-post intracerebroventricular (ICV) injection with 1 .8/ IO ^{1 1} vg of AAV, INS1201- AAV9 efficiently targeted and resulted in the expression of pDys in gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteus (iv), triceps (v), diaphragm (vi), and heart (vii) muscle cells with little to no expression in the liver (viii) of mdx animals. Similarly, as shown in FIG. 4B, 21 days-post ICV injection with 9xlO ¹⁰ vg of AAV, INS1201-AAV9 efficiently targeted and resulted in the expression of pDys in gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteus (iv), triceps (v), diaphragm (vi), and heart (vii) muscle cells with little to no expression in the liver (viii).

[0197] INS1212-AAV9 was also intracerebroventicularly injected into mdx mice on pl and tissue samples were collected and immunofluorescently stained for dystrophin. As shown in FIG. 5, 21 days-post ICV injection with 9*1O ¹⁰ vg of AAV, INS1212-AAV9 efficiently targeted and resulted in the expression of pDys in gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteus (iv), triceps (v), diaphragm (vi), and heart (vii) muscle cells with little to no expression in the liver (viii).

[0198] As shown in the hematoxylin and eosin-stained samples of FIG. 6A, 80 days post ICV injection with 9*1O ¹⁰ vg (ii) and 2.7X10 ¹¹ vg (iii) of INS1201-AAV9, gastrocnemius muscle tissue exhibited restoration of normal tissue architecture and correction of histopathological features of Duchenne muscular dystrophy as compared to wildtype C57/B1 (i) and non-injected mdx mouse controls (iv). As shown in FIG. 6B, at 80 days post ICV injection with 9*1O ¹⁰ vg (ii) and 2.7X10 ¹¹ vg (iii) of INS1201-AAV9, gastrocnemius muscle tissue exhibited levels of pDys comparable to dystrophin levels in wildtype C57/B1 mice (i), and significantly more than dystrophin levels in non-injected mdx mice (iv).

[0199] Similarly, as shown in the hematoxylin and eosin-stained samples of FIG. 7A(ii), 80 days post ICV injection with 9xlO ¹⁰ vg of INS1212-AAV9, gastrocnemius muscle tissue exhibited restoration of normal tissue architecture and correction of histopathological features of Duchenne muscular dystrophy as compared to wildtype C57/B1 (FIG. 7A(i)) and non- injected mdx mouse controls (FIG. 7A(iii)). As shown in FIG. 7B(ii), at 80 days post ICV injection with 9xlO ¹⁰ vg of INS1212-AAV9, gastrocnemius muscle tissue exhibited levels of pDys comparable to dystrophin levels in wildtype C57/B1 mice (FIG. 7B(i)), and significantly more than dystrophin levels in non-injected mdx mice (FIG. 7A(iii)).

[0200] As shown in FIG. 8, gastrocnemius (FIG. 8A), triceps (FIG. 8C), tibialis anterior (FIG. 8E), and diaphragm (FIG. 8F) muscle tissue in mdx mice 80 days post ICV injection with 9*1O ¹⁰ vg or 2.7x10 ¹¹ vg of INS 1201 -AAV 9 exhibited an increase in mean fiber diameter as compared to non-injected mdx mice. Mdx mice intracerebroventricularly injected with 9xio ¹⁰ vg or 2.7X10 ¹¹ vg ofINS1201-AAV9 also exhibited increased frequency of cells having larger diameters (e.g, 25 pm to 60 pm) 80 days post injection in gastrocnemius (FIG. 8B), triceps (FIG. 8D), tibialis anterior (FIG. 8F), and diaphragm (FIG. 8H), as compared to noninjected mice.

[0201] Similarly, as shown in FIG. 9A, gastrocnemius muscle tissue in mdx mice 80 days post ICV injection with 9xlO ¹⁰ vg of INS1212-AAV9 exhibited an increase in mean fiber diameter as compared to non-injected mdx mice with a concomitant increase in the frequency of cells having larger diameters (e.g, 25 pm to 60 pm) (FIG. 9B).

Example 4: Intracerebroventricular Delivery of INS1201-AAV9 results in Increased uDys expression, Improved Muscle Histology and Reduced Fibrosis in mdx Mice.

[0202] Mdx mice at post-natal day 28 (p28) (with a window of -1 and +7 days such that no animal is less P27 and no animal greater than P35 at time of injection) were administered via intracerebroventricular (ICV) injection one of the following treatments: (i) 9xl0 ⁹ vg of INS1201-AAV9 (n=6); (ii) 9xlO ¹⁰ vg of INS1201-AAV9 (n=7); (iii) 2.7xlO ⁿ vg of INS1201- AAV9 (n=10); (iv) 5.4X10 ¹¹ vg of INS1201-AAV9 (n=7); (v) 1.2xl0 ¹² vg of INS1201-AAV9 (n=8) or (vi) vehicle control (TFF formulation buffer, n=l 1). C57/BL1 age-matched mice were used as a wild type (WT) comparator (n=10).

[0203] Tissues were dissected as described above at approximately postnatal day 120.

[0204] INS1201 copies were measured per diploid genome via droplet digital polymerase chain reaction (ddPCR) using primers specific for the INS 1201 trans gene. DNA copies of INS1201 are provided in FIG. 15. RNA transcript copies are provided in FIG. 16. TA: tibialis anterior; EDL: extensor digitorum longus; GAS: gastrocnemius; DIA: diaphragm. RPP30: ribonuclease P/MRP Subunit P30. [0205] As shown in FIG. 17, mdx mice administered INS1201-AAV9 at all doses tested exhibited an increase in mean EDL fiber diameter, as compared to non-injected mdx mice. Consistent with these findings, mdx mice intracerebroventricularly injected with INS1201- AAV9 at all doses also exhibited increased frequency of cells having larger diameters (e.g., 25 pm to 60 pm) in EDL muscle, as compared to non-injected mice.

[0206] As shown in FIG. 18, mdx mice administered INS1201-AAV9 at the four highest doses tested exhibited an increase in mean TA fiber diameter, as compared to non-injected mdx mice. Consistent with these findings, mdx mice intracerebroventricularly injected with INS1201-AAV9 at the four highest doses tested (9x lO ¹⁰ vg; 2.7 xlO ¹¹ vg; 5.4X10 ¹¹ vg; 1.2* 10 ¹² vg) exhibited increased frequency of cells having larger diameters in TA muscle, as compared to non-injected mice.

[0207] FIG. 19 shows diaphragm muscle sections stained with picrosirius red, post intracerebroventricular (ICV) injection with various doses of INS1201-AAV9. Picrosirius red is used to visualize collagen content. Diaphragm sections obtained from a wildtype C57/B1 mouse and mdx mouse administered vehicle are shown for comparison (top panels). As shown in the lower panels of FIG. 19, the mdx mice administered INS1201-AAV9 at the five doses tested (9* 10 ⁹ vg; 9x lO ¹⁰ vg; 2.7X10 ¹¹ vg; 5.4X 10 ¹¹ vg; 1.2xl0 ¹² vg) exhibited decreased fibrosis, compared to a mdx mouse administered vehicle, as measured by collagen content. Percent collagen in diaphragm muscle is also provided in FIG. 20.

[0208] FIG. 21, top, are microphraphs of EDL sections obtained from an mdx mouse at postnatal day 120 (pl20) subsequent to intracerebroventricular (ICV) injection at p28 with 5.4* 10 ¹¹ vg of INS1201-AAV9 and stained with hematoxylin and eosin (H&E) (far left); laminin/dapi (second from left); dystrophin (second from right); and a merged image (far right). FIG. 21, bottom, shows a EDL muscle section obtained from an mdx mouse at postnatal day 120 (p!20) subsequent to intracerebroventricular (ICV) injection at p28 with vehicle control, and stained with hematoxylin and eosin (H&E) (far left); laminin/dapi (second from left); dystrophin (second from right); and a merged image (far right). The staining shows greater dystrophin expression in the sample obtained from the mouse treated with INS1201-AAV9 compared to the sample taken from the control mouse. Muscle from the INS1201-AAV9 treated mouse also appear healthier, as evident from the H&E and laminin/dapi stains.

Example 5: ICV Delivery of INS- 1201 AAV9 results in improved muscle physiology in mdx mice. [0209] Mdx mice at post-natal day 1 (pl) were administered via intracerebroventricular (ICV) injection one of the following treatments: (i) 9* 10 ⁹ vg of INS1201-AAV9; (ii) 9*1O ¹⁰ vg of INS-1201-AAV9; (iii) 2.7X 10 ¹¹ vg of INS1201-AAV9; (iv) vehicle control (TFF formulation buffer). C57/BL10 age-matched mice (WT) were used as a comparator.

[0210] Mdx mice at post-natal day 28 (p28) (with a window of -1 and +7 days such that no animal is less P27 and no animal greater than P35 at time of injection) were administered via intracerebroventricular (ICV) injection one of the following treatments: (i) 9x l0 ⁹ vg of INS1201-AAV9; (ii) 9x lO ¹⁰ vg of INS1201-AAV9; (iii) 2.7xlO ⁿ vg of INS1201-AAV9; (iv) 5.4X 10 ¹¹ vg of INS1201-AAV9; (v) 1.2x l0 ¹² vg of INS1201-AAV9 or (vi) vehicle control (TFF formulation buffer). C57/BL1 age-matched mice were used as a wild type (WT) comparator.

[0211] Muscle dissection and preparation was carried out as described above when animals were from 120 to 135 days old.

Muscle Physiology Experimental Design

Muscle mechanics in EDL

[0212] The EDL is mounted in a specialized chamber containing Ringer’s solution (137 mM NaCl, 5 mM KC1, 2 mM CaCh, 1 mM MgSCU, 1 mM NaFLPCL, 24 mM NaHCOs, 11 mM glucose containing 10 mg/liter curare) at room temperature. The muscle origin is tied to a rigid post, and the insertion is secured to the arm of a dual-mode ergometer (model 300B; Aurora Scientific, ON, Canada), which allows precise control of muscle length.

[0213] Muscle activation is provided via an electrical stimulator using parallel platinum plate electrodes that extend the length of the muscle. Supramaximal stimulation conditions are established for each experiment by single 0.3 ms twitch pulses of increasing voltage, using +50% more than the value where the force plateaued for experimental testing. Optimal muscle length for assessing force characteristics is determined using a series of twitches while incrementally increasing muscle length (10% increments from slack length) and are defined as the length at which supramaximal stimulation produces maximal twitch force. After establishing optimal length, muscle fiber length (Lf) is measured and the muscle is rested for 2 min. [0214] The muscle is then stimulated with two twitches spaced 60 s apart (0.3 ms pulse duration) to assess twitch characteristics (twitch tension, half-relaxation time, and time-to-peak tension).

[0215] The muscle is stimulated at increasing frequencies (400 ms train duration and 0.3 ms pulse duration; (i) EDL: 1 Hz, 10 Hz, 30 Hz, 50 Hz, 70 Hz, 120 Hz; (ii) soleus: 1 Hz, 5 Hz, 10 Hz, 20 Hz, 40 Hz, 60 Hz, 80 Hz, and 100 Hz) with 120 s intervals between contractions to determine the force-frequency (F-F) relationship.

[0216] Following the F-F testing, optimal length is re-verified by empirically testing maximal twitch force up to ±20% of the length at the end of testing.

Eccentric contractions

[0217] After completing the F-F curve, all stretches or contractions are performed with two- minute intervals, and stimulation is performed at 100Hz with 400ms train duration and 0.3ms pulse duration for isometric and eccentric contractions. Specifically, following a two-minute rest period from the end of the F-F assessment, the passive mechanical properties of the muscle are measured by imposing a 10% Lf stretch at 0.7Lf/s twice with a 500ms hold before returning to the starting muscle length. Then, two isometric contractions are obtained as a measurement of pre-eccentric contraction (EC) maximal isometric force.

[0218] Each muscle then undergoes an EC bout of ten contractions, during which the muscle is stimulated isometrically for the first 200 ms, after which a 15% Lf length change is imposed at a rate of 2 Lf/s. The muscle is then held at this length for 500ms before returning to the starting muscle length. After 10 EC contractions, two isometric contractions are obtained to determine the post-EC maximal isometric force. At the completion of the final isometric measurements, two additional passive stretches are obtained to determine post-EC passive mechanical measurements.

[0219] Following contraction testing, muscles are trimmed of external tendons, blotted dry and weighed to the nearest O.Olmg. Physiological cross-sectional areas of the muscles are calculated as function of fiber length and muscle mass. For all analysis, force is normalized to the muscle physiological cross-sectional area and reported as stress.

[0220] Mdx mice receiving ICV injection on postnatal day 1 (pl) of INS 1201 -AAV 9 at all doses tested (9xl0 ⁹ vg; 9xlO ¹⁰ vg; 2.7X10 ¹¹ vg) exhibited an attenuated decrease in percent contractile force resulting from EC as compared to mdx mice treated with vehicle (FIG. 10A). FIG. 10B similarly shows that mice receiving ICV injection of INS 1201 -AAV 9 on pl, at the three doses tested (9xl0 ⁹ vg; 9xlO ¹⁰ vg; 2.7X10 ¹¹ vg), exhibited improved muscle physiology as indicated by increased percent post-eccentric contraction stress relative to pre-eccentric contraction stress as compared to vehicle treated mice.

[0221] Mdx mice receiving ICV injection on postnatal day 28 (p28) of INS1201-AAV9 at three highest doses tested (2.7X10 ¹¹ vg; 5.4xlO ⁿ vg; 1.2xl0 ¹² vg) exhibited an attenuated decrease in percent contractile force resulting from EC as compared to mdx mice treated with vehicle (FIG. 10C, 10D).

[0222] Mdx mice receiving ICV injection on postnatal day 28 (p28) of INS1201-AAV9 at three highest doses tested (2.7X10 ¹¹ vg; 5.4X10 ¹¹ vg; 1.2xl0 ¹² vg) exhibited improved and stabilized muscle function as shown by an increase in peak muscle force following stimulation at various frequencies as compared to mdx mice treated with vehicle (FIG. 10E, FIG. 10F).

Example 6: One-dose Intrathecal Administration of AAV9 Targets Transgene Delivery to Skeletal and Cardiac Muscle Tissue

[0223] AAV9-CBA-GFP was intrathecally administered to cynomolgus macaques who were screened for anti-AAV9 antibodies using a highly specific anti-AAV9 ELISA. Cynomolgus subjects were treated with 2.5*10 ¹³ vg, 5*10 ¹³ vg, or l*10 ¹⁴ vg of AAV9-CBA- GFP via a single intrathecal dose and GFP expression was determined by immunohistochemical analysis with NovaRed GFP immunostain and Vector® HRP substrate, RT-PCR, and western blot analysis.

[0224] As shown in FIG. 11, cynomolgus macaques receiving a single intrathecal dose of [0225] As shown in FIG. Ill, cynomolgus macaques receiving a single intravenous dose of AAV9-CBA-GFP at 5*10 ¹³ vg (i), or l*10 ¹⁴ vg (ii), exhibited high levels of GFP staining in liver tissue, whereas subjects receiving a single intrathecal dose of AAV9-CBA-GFP at 2.5* 10 ¹³ vg (iii), 5*10 ¹³ vg (iv), or l *10 ¹⁴ vg (v), exhibited significantly lower GFP staining.

[0226] As shown in FIG. 12, immunohistochemical staining for GFP shown in FIG. 11 corresponded to protein levels as detected by anti-GFP western blot. Cynomolgus macaques receiving a single intrathecal dose of AAV9-CBA-GFP at 2.5*10 ¹³ vg (FIG. 12A), 5*10 ¹³ vg (FIG. 12B), or 1 xlO ¹⁴ vg (FIG. 12C), exhibited increased GFP levels in biceps (1), triceps (2), deltoid (3), quadriceps (4), gastrocnemius (5), tibialis anterior (6), diaphragm (7) and heart (8) muscle tissue, whereas no GFP protein was detected by western blot in the biceps (FIG. 12D, 1) or triceps (FIG. 12D, 2) of non-injected subjects.

[0227] As shown in FIG. 13, GFP protein levels shown in FIGs. 11 and 12 corresponded to GFP mRNA levels, wherein cynomolgus macaques receiving a single intrathecal dose of AAV9-CBA-GFP exhibited detectable GFP mRNA expression in biceps (1), triceps (2), deltoid (3), tibialis anterior (4), gastrocnemius (5), quadriceps vastus lateralis (6), diaphragm (7), and heart (8) muscle tissue as analyzed by RT-PCR. Minimal GFP mRNA was detected in liver tissue (9) and no GFP mRNA was detectable in biceps (10), triceps (11), deltoid (12) and quadriceps (13) muscle tissue collected from non-injected subjects.

Example 7: Toxicity and Biodistribution Study of INS1201-AAV9 Following Single Dose Administration of Intracerebro ventricular Injection in Juvenile C57BL/6 J Mice

[0228] Test System

• Species: Mus mus cuius

• Strain: C57BL/6J

• Sex: Male

• Age: Approximately 4 weeks on Day 1

• Weight: Commensurate with age

• Number: 192 (+ 60 extra)

• Caging: As per ASC SOPs

• Minimum Acclimation: 5 days [0229] Species/Strain, Number and Sex. Up to 252 male C57BL/6J (strain# 000664) mice, approximately 4 weeks old at study start, are acquired for this study. Only male mice are used in the study, as the intended patient population is male only.

[0230] Starting Age and Weight Range. Animals selected for use in this study will be as uniform in age and weight as possible at start of study. Animals will be approximately 3 weeks of age upon delivery and approximately 4 weeks old on day 1 of study.

[0231] Animals will be fed a species-specific diet, fed ad libitum. No contaminants are known to be present in the diet at levels that would interfere with the results of this study. Irradiated water will be available ad libitum to each animal.

[0232] The test article and control articles used in this study are provided in Table 2 and Table 3, respectively.

[0233] Experimental Design

[0234] Study Design

[0235] The study consists of three cohorts: animals assigned to cohort 1 will be terminated on Day 85±10 following injection procedure, animals assigned to cohort 2 will be terminated on Day 43±7 following injection procedure and cohort 3 will be terminated on Day 22±3 following injection procedure. The overall study design is presented in Table 4. Extra animals may be dosed and be used to replace any unscheduled deaths of study group animals that occur as a direct result of procedural activity (i.e., injection, restraint, and handling during injection and/or blood collection). Animals that do not survive the test article administration procedure, die or require early termination prior to Day 4 (3 days post-injection) may be replaced. These animals will not be subject to a gross necropsy. Animals that do not meet the inclusion criteria for age may be replaced as necessary. All animal deaths will be reported, regardless of timing of death postinjection. For Groups 1-4 at each given time point, 5 animals will be used for hematology, blood ddPCR and tissue ddPCR; 5 animals will be used for clinical chemistry and histopathology; and 5 animals for coagulation test and histopathology (n=15/group/time point). All Group 5 animals will be euthanized in Week 12 for hematology, clinical chemistry and coagulation (n = 4 for each test). Five (5) animals from Group 5 will be used for histopathology while tissues from the rest of the animals will be collected and saved.

[0236] Cohort 1 - Day 85±10 Termination. For Cohort 1 of this study, 60 animals designated to groups 1-4 will receive intracerebroventricular (ICV) injections on Day 1, based on their group allocation (Table 1). An additional 12 animals in Cohort 1 are part of Group 5 animals (“Naive/untreated”, Table 1), which will not be injected. At Study Day 85±10, animals in all groups, 1-5 will be terminated, and blood and tissue samples will be collected as described in Table 5.

[0237] Cohort 2 - Day 43±7 termination. For Cohort 2, 60 animals will receive ICV injections on Day 1, based on their group allocation (Table 1). At Study Day 43±7, animals will be terminated, and blood and tissue samples will be collected as described in Table 6.

[0238] Cohort 3 - Day 22±3 Termination. For Cohort 3, 60 animals will receive ICV injections on Day 1, based on their group allocation (Table 1). At Study Day 22±3, animals will be terminated, and blood and tissue samples will be collected as described in Table 7. [0239] Group Assignment. Animals will be ordered and assigned to the study groups in three separate cohorts per Tables 5, 6 and 7. Each cohort of animals, including extras, up to 12 animals per group, will be weighed and assigned a numeric rank from 1 to X according to body weight in a decreasing order (the heaviest animal will be assigned rank = 1). The animals will then be assigned sequentially to each study group based on termination cohort per the study design (see Tables 5, 6, 7).

[0240] Control for Bias. All attempts will be made to minimize potential study bias, including: (a) inclusion of appropriate control groups; (b) randomized assignment of animals to study groups; and (c) appropriate staggered dosing of animals across groups.

[0241] Test Article Administration and Study Procedures

[0242] Anesthesia and Surgical Preparation. Animals are anesthetized using an inhalation anesthetic (1-5% isofl urane with 100% oxygen for induction; 1-3% for maintenance during the surgical procedures). Alternatively, animals are anesthetized with a ketamine (up to 80 mg/kg) and xylazine (up to 12 mg/kg) cocktail administered intraperitoneally.

[0243] Test Article Preparation and Delivery. All test article preparation and administration will be performed by Sponsor designated personnel. On Day 1, for each cohort (Tables 5-7), the vehicle and test article will be administered to all animals, via stereotaxic injections into ventricle, based on their group assignment. Test and control article will be kept on ice or between 2-8 °C until ready for use.

[0244] Cohort injection strategy. Injections for each termination day cohort will take place over 3-4 days. On each individual injection day, approximately 15-30 mice will be injected. In consideration of test article dose formulations, on the first day of each cohort, injections of all mice of Dose 1 will be completed, followed by vehicle injections to a subset of animals in Group 1 as time allows. On the second day of each cohort, injections of all Dose

2 mice will be completed, followed by vehicle injections to the second subset of animals as time allows for the injection team. On the third day of each cohort, injections of all Dose 3 mice will be completed, followed by vehicle injections to the third subset of animals as time allows for the inj ection team. If not all vehicle-inj ected mice have been inj ected across the first

3 days, the remainder of vehicle injections will take place on the fourth day.

[0245] Treatment. Treatments will be administered to animals, in accordance with Tables 5, 6 and 7, by direct injection of treatments into the lateral ventricle. Each animal, depending on dosing group, will receive either 1 unilateral injection, or 2 injections (bilateral/one injection per side) in a single surgical procedure. Surgical procedures will be performed using stereotaxic instrumentation. Injection devices, surgical instruments, drapes and gowns will be sterile where appropriate. Modifications to the procedures as described below may be performed at the discretion of the surgeon. A single dose of mel oxicam (1 mg/kg, SC/IM) and/or buprenorphine (0.01-0.05 mg/kg SC) will be administered to help relieve pain from surgery after induction of anesthesia prior to skull incision. With the animal anesthetized, the skin over the cranium will be shaved (as needed) and the animal mounted in a stereotaxic frame, maintaining the animal on a nose cone for anesthesia with the head positioned by the use of ear bars and the incisor bar as applicable. Aseptic techniques will be used for all surgical procedures. The skin is disinfected with betadine solution followed by 70% alcohol wipe, or equivalent.

[0246] An approximately 2 cm incision will be made at the midline over the skull. Electrocautery may be used to achieve hemostasis of any small hemorrhages from the incision site. A Hamilton syringe with a Hamilton 2-inch, 26-gauge (or smaller), 12-degree bevel stainless steel needle will be attached to the Z-axis of the stereotactic device. The following approximate stereotaxic coordinates, based on bregma, will be used to target the lateral ventricle, either unilaterally or bilaterally:

• Injection Coordinates: o Anterior-Posterior (AP): -0.5 to -0.8 mm o Medio-Lateral (ML): +/- 1.0 mm* o When administering unilateral injections, injection is on right side of the brain. When administering bilateral injections, first injection is on the right, second injection is on the left. o Dorso-Ventral (DV): -2.5 mm

[0247] The actual locations of the injections may be adjusted by the surgeon as needed. If the coordinates differ from the ones described above, they will be recorded within the surgical records of the raw data.

[0248] Once needle is in desired location, syringe plunger is slowly pushed down to inject dosing solution into cranial space. The site of injection is monitored during the injection and immediately post injection to confirm quality of injection. [0249] The desired injection volume is 8 pL per injection site. Injections will target the lateral ventricle and will include 1 or 2 total injections. The needle will be left in place for approximately 1 minute after delivering the treatment to prevent reflux. Any injection abnormalities (leakage, backflow, etc.) will be noted on the Injection sheet for the animal being injected. Following completion of injection(s) and needle removal, the incision is closed using VetBond™ surgical glue, and may be reinforced with a suture. Slight alterations in surgical procedure based on real time status of the animal may be made and will not be considered a deviation to the study protocol.

[0250] Post-Operative Care. Following the surgical procedures, animals are maintained on thermal support through recovery and will be placed in lateral recumbence alternating sides as needed. Mice are administered sterile saline subcutaneously (approximately 0.5 to 1 mL). Animals may also be provided their feed and Diet-Gel on the cage floor as supplemental feed during their surgical recovery.

In-Life Observations and Measurements

[0251] Routine General Health Observations. Animals will be observed for changes in general appearance, behavior, and signs of illness during the acclimation period by the husbandry staff, and records will be kept on file as part of the facility records. Daily observations will be recorded beginning on Day 1 and throughout the course of the study until their designated termination day. Any animals exhibiting non-normal clinical signs prior to Day 1 will be excluded from the study.

[0252] Pre-Study Clinical Observations. All animals will receive detailed examinations for clinical signs prior to Day 1. Clinical signs indicative of poor health, stress, or other abnormalities will be noted and animals may be excluded from the study at the discretion of the Study Director/ Attending Veterinarian and Sponsor Representative.

[0253] Clinical Observations. All study animals will receive a detailed clinical observation 6-10 days’ post-operatively then once weekly thereafter, in conjunction with body weight, until scheduled termination. Observations noted outside of scheduled observations will be entered as unscheduled. Assessments will include, but will not be limited to: assessment of locomotor activity, neurological observations, posture, respiration, hydration status, surgical site, stereotypic behavior, bladder and bowel (stool) observations, as well as overall body condition. [0254] The absence or presence of findings during the scheduled clinical observations will be recorded. Clinical signs indicative of poor health, stress, and pain will be noted and will be reported to the Attending Veterinarian. A final detailed examination will be conducted by the Attending Veterinarian for any animal euthanized in extremis.

[0255] Body Weights Individual body weights will be recorded for initial group assignment shortly after animal arrival. Baseline body weights will be taken no more than 4 days prior to dosing. Body weights will be performed once weekly (coinciding with clinical observations) until scheduled termination, and a final body weight will be measured prior to scheduled termination. Per the Study Director’s discretion, body weights may be measured more frequently if signs of adverse health or weight loss (i. e. , greater than or equal to 10% of the previous week’s weight) are observed.

[0256] Scheduled Sacrifice. Animals that survive until their scheduled termination date, Study Day 22±3, Day 43±7, Day 85±10, will be humanely euthanized, necropsied, and blood and tissue samples will be collected as described below. Animals will be euthanized in accordance with the AVMA Guidelines for Euthanasia of Animals: 2020 Edition.

Sample/Specimen Collection

[0257] Blood Collection. Prior to necropsy, animals may be transferred to an anesthesia box and anesthetized using isoflurane (1-2%) for blood collections for hematology and droplet digital polymerase chain reaction (ddPCR), serum chemistry, and coagulation. Appropriate volume of whole blood will be collected using a 25g needle and 1 cc syringe, or similar, from each animal via cardiac puncture, or other appropriate vessel. Blood samples will be collected from all study animals at their scheduled termination day. Blood will be processed per ASC SOPs and sent to QVL for analysis.

[0258] Samples for Hematology and ddPCR

[0259] For hematology, approximately 250-500 pL of whole blood will be placed into vials containing K ₂ EDTA as the anticoagulant, inverted gently several times to mix, and placed on wet ice until storage in a refrigerator set to maintain 2 °C to 8 °C.

[0260] Hematology

[0261] Collection Volume: Approximately 250-500 pL

[0262] Anticoagulant: K ₂ EDTA [0263] Hematology parameters to be analyzed are provided in Table 8.

[0264] Biodistribution by Digital Droplet Polymerase Chain Reaction (ddPCR). In addition, approximately 250-500 pL of whole blood will be collected for biodistribution by ddPCR analysis. The whole blood will be placed into K2EDTA as the anticoagulant, inverted gently several times to mix, and placed on dry ice until storage at -80 °C±10 °C.

[0265] Samples for Clinical Chemistry. For clinical chemistry, approximately 500-1000 pL of whole blood will be collected into tubes containing no anticoagulant and allowed to clot at room temperature for at least 30 minutes prior to centrifugation. Clotted whole blood will be centrifuged to yield serum at a temperature of 4 °C, at approximately 3000 / g for 5 minutes. The serum samples will be separated following centrifugation, frozen on dry ice immediately after collection and stored frozen at -80 °C±10 °C.

[0266] Serum chemistry parameters to be analyzed are provided in Table 9.

[0267] Samples for Coagulation. The plasma samples will be separated following centrifugation, and stored in a freezer, set to maintain -10 °C to - 30 °C.

[0268] Coagulation. Collection Volume: Approximately 300-800 pL; Anticoagulant: 3.2 % Sodium citrate; Processing: To plasma. Parameters Analyzed: prothrombin time (PT); activated partial thromboplastin time (APTT); fibrinogen.

[0269] Gross Necropsy. The necropsy will consist of a systematic, macroscopic external and internal examination of the animal’s general physical condition and tissues (respiratory, cardiovascular, digestive and urogenital systems). This will be performed for all scheduled and unscheduled sacrifices. Any gross lesions will be documented and preserved. Details on tissue collection are described in detail below. Trained and qualified study personnel will conduct gross necropsies and tissue collection.

[0270] Tissue Collection. Tissues to be collected at termination and analyzed by either histopathology or ddPCR are presented in Table 10.

[0271] Tissue Collection: Histopathology. Tissues for histopathology will be collected from animals at their designated termination days per Tables 2, 3 and 4. All tissues marked in Table 6 for histopathology (except eyes), including gross lesions/abnormal tissues, will be collected and fixed in 10% neutral buffered formalin (NBF). Eyes will be fixed in Davidson Solution for 24 to 48 hours then transferred to 70% ethanol.

[0272] Tissues will be trimmed, processed, embedded in paraffin, microtomed, stained with H&E and coverslipped. When possible, muscles will be sectioned both transversely and longitudinally. The resulting slides will be microscopically quality checked. All prepared slides will be evaluated by an ACVP veterinary pathologist. A draft pathology report, consisting of tabulated microscopic data and a discussion of noteworthy changes, will be issued. Photomicrographs will be taken and annotated

[0273] ddPCR analysis. Representative samples of tissues listed in Table 10, will be collected and retained for ddPCR analysis. Specimens collected as one large portion will be placed in tubes, snap frozen in liquid nitrogen, placed in a cooler containing dry ice until placed in a freezer set to maintain -60 °C-80 °C.

Example 8: Biodistribution of INS1201-AAV9 Following Intrathecal Delivery in Nonhuman Primates (NHPs)

[0274] Test System

• Genus: Macaca

• Species: fascicularis

• Sex: Male

• Weight: from about 2-5 kg

[0275] INS1201-AAV9 biodistribution in the periphery and skeletal muscle is measured in response to intrathecal administration. [0276] All injections are performed intrathecally into the lumbar space of the spinal cord.

Twelve (12) total animals are used, as provided in Table 11:

[0277] Animals selected for study will be juvenile or adult monkeys between 2 months and 5 years of age weighing approximately 3kg. Animals are pre-screened and negative for AAV9 antibodies. Pre-screening blood collection will take place within 2 months prior to injection procedure. Animals for screening will be sedated and a blood sample collected.

[0278] Selected animals will be brought to the hospital area a few weeks prior infusion. On the day of infusion (dO), the subjects are sedated and a blood sample is collected for chemistry analysis and cell blood count (CBC). The subjects are then injected with a single dose of INS1201-AAV9 according to the dosing table above. All animals less than 9 months old will be housed with their dams. Animals older than 9 months will be housed in small groups.

[0279] The injection is performed by lumbar puncture into the subarachnoid space of the lumbar thecal sac. For intrathecal (IT) injections, subjects are placed in the lateral decubitus position and the posterior midline injection site at -L4/5 level identified (below the conus of the spinal cord). Under sterile conditions, a spinal needle with stylet is inserted and subarachnoid cannulation is confirmed with the flow of clear CSF from the needle, as well as injection of a small volume of iohexol followed by intra-procedural myelography. Approximately 1ml CSF will be drained, collected and frozen as baseline sample. This is to alleviate the increase in pressure that is created by the subsequent injection of test article. To improve rostral flow distribution of the test article, the subject will then be tilted in the trendelenberg position (mild head down position). This is a routine procedure when performing CT myelograms in human subjects. For the CSF taps/IT infusions - hypodermic needles (22G % or 1 !4”) can also be used for this purpose

[0280] In-Life Observations: Treated NHPs will be kept in isolation to reduce exposure to confounding sources of toxicity. Subjects are observed twice daily for activity, relative skin color and general health by the veterinary staff. The animals will be kept for approximately 21 days post injection. Biodistribution and clinical chemistry tests are performed on samples collected at euthanasia.

[0281] The following segments are collected from the spinal cord for biodistribution studies: cervical spinal cord, thoracic spinal cord, lumbar spinal cord, sacral spinal cord, dorsal root ganglion (DRG) root cervical level, DRG root thoracic level, DRG root lumbar level. For each spinal cord segment, two total pieces are collected. One piece is stored in 4% paraformaldehyde (PF A) and one is flash frozen.

[0282] For muscles and organs, four samples of each are collected. Two are placed in 4% PFA and two are flash frozen. Samples of the following muscles are collected: diaphragm, #6/#7 Rib with intercostal muscle and nerve, psoas muscle, deltoid, pectoralis major, biceps brachii, triceps brachii, rectus femoris, vastus medialis, vastus lateralis, gastrocnemius, tibialis anterior, soleus, tongue, masseter, extensor digitorium, rectus abdominus. Samples of the following organs are collected: heart, liver, lung, kidney, spleen.

[0283] Clinical chemistry tests are as follows: AST, ALT, GGT, Aik Phos, Potassium, Sodium, Chloride, Creatinine, Blood urea nitrogen, CBC.

[0284] Brain samples are collected as follows. The cerebellum is separated from the rest of the brain. The cerebellum is cut into four quadrants with right two quadrants stored in 4% PFA and the left two quadrants flash frozen. The brain is split into right and left hemispheres. The brain is then cut into four quadrants (coronal cuts), with the right two quadrants stored in 4% PFA and the left two quadrants flash frozen.

[0285] Six samples are taken from the quadriceps for subsequent muscle biopsy. Three are stored in 4% PFA and three are flash frozen.

[0286] Cerebral spinal fluid (CSF) and serum are also collected for biodistribution studies.

[0287] Biodistribution studies are carried out on the aforementioned samples by droplet digital polymerase chain reaction (ddPCR). INCORPORATION BY REFERENCE

[0288] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0289] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.