Adeno-associated virus particles and methods of use thereof

Abstract

The present invention provides intrathecal compositions comprising AAV particles and their use for treating monogenic muscle disorders such as dystrophinopathies, including Duchenne muscular dystrophy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 229,936, filed August 5, 2021, and U.S. Provisional Patent Application No. 63 / 239,881, filed September 1, 2021, the contents of each of which are incorporated herein by reference in their entirety. FIELD OF THEINVENTION

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

[0003] Incorporating sequence tables The sequence listing associated with this application is provided in XML format in lieu of a paper copy and is incorporated herein by reference. The XML file containing the sequence listing is named INMD_166_02WO_SeqList_ST26.xml. The XML file is approximately 40,532 bytes, was created on August 3, 2022, and has been submitted electronically via the USPTO Patent Center. [Background technology]

[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 need to function properly. DMD is characterized in part by progressive muscle degeneration. As the disease progresses, initially affecting the muscles of the thighs, pelvis, and arms, DMD eventually affects all voluntary muscles and, at later stages, involves the cardiac and respiratory muscles. In Europe and North America, the prevalence of DMD is approximately 1 in 3,600 male births. DMD is the most frequent childhood form of muscular dystrophy and affects almost exclusively males. There is no known cure for DMD, and the current standard of care is primarily aimed at managing symptoms, including steroids, immunosuppressants, anticonvulsants, braces, corrective surgery, and assisted ventilation. Aggressive management of the dilated cardiomyopathy associated with DMD includes anticongestive medications and heart transplants in severe cases.

[0005] Gene therapy is a rapidly accelerating therapeutic approach in which nucleic acids are delivered to cells harboring mutant or non-functional genes to correct the mutant cell's defects. In one particular gene therapy, the nucleic acid is packaged within an adeno-associated virus (AAV), which delivers the nucleic acid to the cell. Once inside the cell nucleus, the nucleic acid then induces appropriate protein production and the virus is safely degraded. Gene therapy has been proposed for the treatment of DMD, but requires the delivery of high systemic viral titers that lead to patient toxicity and has high manufacturing costs due to the large amount of virus required per patient.

[0006] Delivery of microdystrophin (μDys), a modified but functional truncated dystrophin nucleic acid sequence, in animal models and humans has been reported to promote muscle function. μDys transgenes have been designed to encode various combinations of unique functional domains of the 427 kDa dystrophin protein. μDys sequences, generally less than 5 kilobases in length, have been tested using AAV to deliver μDys transgenes in mouse models 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 abolished full-length dystrophin expression (Sicinski et al. (1989) Science 244, pp. 1578-1580, incorporated herein by reference in its entirety). Despite the promise that delivery of μDys has shown, new therapeutic approaches are needed for the treatment of DMD. The present invention addresses this and other needs. Summary of the Invention

[0007] The present invention relates, in part, to adeno-associated virus (AAV) particles comprising capsids that package (i.e., encapsidate) a microdystrophin (μDys) transgene, and methods for treating various dystrophinopathies using the same, e.g., by intrathecal administration. In one embodiment, the μDys transgene encodes a μDys polypeptide comprising (i) an N-terminal region (NTD) that comprises an actin-binding site, (ii) a domain that comprises three hinge regions and four spectrin repeats, and (iii) a cysteine-rich domain. The μDys 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 that includes a capsid that encapsidates a vector genome. The vector genome, in one embodiment, includes, from 5' to 3', a 5' inverted terminal repeat (ITR), a promoter, a μDys transgene, an SV40 poly(A) tail, and a 3' ITR. The μDys transgene, in one embodiment, encodes a polypeptide including (i) an N-terminal region (NTD) that includes an actin-binding site, (ii) a central rod domain that includes 2-4 hinge regions and 4-6 spectrin repeats, and (iii) a cysteine-rich domain. In a further embodiment, the μDys transgene encodes a μDys polypeptide that includes an NTD, hinge regions 1, 2, and 4, spectrin repeats 1, 2, 3, and 24, and a cysteine-rich domain. In yet a further embodiment, the μDys transgene comprises the nucleic acid sequence set forth in SEQ ID NO:5. The AAV particles, in one embodiment, are AAV9 particles, and are present in the intrathecal composition in an effective amount, which in one embodiment comprises about 90% or less of the effective vector genome amount of an intravenous (IV) composition comprising AAV particles encapsidating a μDys transgene, e.g., the same μDys transgene, present in the intrathecal composition.

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

[0010] The promoter, in one embodiment, is the MHCK7 or chicken β-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. In one embodiment, the capsid of the AAV9 particle consists of the AAV9 capsid protein. In yet another embodiment, the AAV particle is an AAVrh74 particle.

[0012] In some embodiments, a recombinant AAV vector genome of the invention comprises, from 5' to 3', a 5' ITR, an SK-CRM4 enhancer, a promoter, a μDys 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 μDys coding sequence encodes a μDys protein comprising an actin-binding domain and at least four spectrin repeats, e.g., between four and six spectrin repeats. In some embodiments, the μDys transgene comprises or consists of the nucleic acid sequence of SEQ ID NO:5.

[0013] In some embodiments, the encapsidated vector genome of the 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, the 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 β-actin hybrid promoter. In a further embodiment, the chicken β-actin hybrid promoter has the nucleic acid sequence set forth in SEQ ID NO:3.

[0015] In some embodiments, an encapsidated vector genome of the 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 an 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 preferred embodiments, the capsid is an AAV9 capsid, which 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 administering a single dose of a composition comprising one of an effective amount of AAV particles encapsidating a vector genome comprising a μDys 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 yet a further embodiment, the dystrophinopathy is DMD. The effective amount of AAV particles, in a further embodiment, comprises about 90% or less vector genome than a corresponding IV composition (e.g., an intravenously administered composition) comprising an AAV particle encapsidating a vector genome comprising a μDys transgene, e.g., the same μDys transgene present in the intrathecally administered composition.

[0019] In one embodiment, the subject is administered the composition while in Trendelenburg position, hi a further embodiment, administration is in the absence of a non-ionic low osmolarity contrast agent.

[0020] In one embodiment of the methods of treating dystrophinopathy described herein, an effective dose of intrathecally administered AAV particles provides a superior therapeutic response than the same dose of intravenously administered AAV particles that encapsidate a vector genome that contains a μDys transgene, e.g., the same μDys transgene present in the intrathecally administered AAV particles. 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 AAV particles encapsulating a vector genome comprising a μDys transgene results in a reduction in the number of side effects or a reduction in the severity of one or more side effects in the subject compared to the number of side effects or the severity of side effects experienced by a second subject when the second subject is administered intravenously an effective amount of corresponding AAV particles encapsulating a vector genome comprising a μDys transgene. In a further embodiment, the dystrophinopathy is DMD. In yet a further embodiment, the AAV particles are AAV9 particles.

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

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

[0024] In some embodiments of the methods described herein, expression of the μDys transgene delivered by the AAV particles described herein in a subject is significantly less in the subject's liver tissue compared to the subject's skeletal muscle and / or cardiac muscle. In further embodiments, μDys 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 subject's skeletal muscle and / or cardiac muscle compared to the amount of μDys transgene expression in liver tissue. In another embodiment, μDys 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 subject's liver compared to the amount of μDys transgene expression in skeletal muscle and / or cardiac muscle. [Brief description of the drawings]

[0025] [Figure 1A] A schematic diagram of an exemplary μDys-encoding gene construct (INS1201) is shown. [Figure 1B] 1 shows a schematic diagram of an alternative μDys-encoding gene construct (INS1212). [Figure 1C] Agarose gel electrophoresis of the INS1201 gene construct cloned into a psZ01 vector backbone (pSZ01-INS1201) restriction site digested with HindIII / BsaI and SmaI (lanes 1 and 2, respectively) and the INS1212 gene construct cloned into a psZ01 vector backbone (pSZ01-INS1212) restriction site digested with HindIII / BsaI and SmaI (lanes 3 and 4, respectively). [Diagram 2] Silver stained SDS polyacrylamide gel electrophoresis (PAGE) of 1 μl of the INS1201-AAV9 preparation (lane 1), 1 μl of the INS1212-AAV9 preparation (lane 2), and 0.5 μl, 1 μl, 2 μl, and 4 μl of a 1 x 10 vg / ml AAV2 standard (lanes 3, 4, 5, and 6, respectively). [Figure 3A] 1 shows gastrocnemius muscle sections obtained from mdx mice 21 days after intramuscular injection with 2.7×10 vg of INS1201-AAV9 (iii) or INS1212-AAV9 (iv) and immunofluorescently stained for dystrophin. Gastrocnemius muscle sections obtained from uninjected mdx mice (i) and wild-type C57 / Bl mice (ii) and immunofluorescently stained for dystrophin are shown for comparison. [Figure 3B] Shown are gastrocnemius muscle sections obtained from mdx mice 21 days after intramuscular injection with 2.7×10 vector genomes (vg) of INS1212-AAV9 and stained with DAPI (i) and for dystrophin (ii). A merged image is shown in (iii). [Figure 4A] Shown are gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteus (iv), triceps (v), diaphragm (vi), heart (vii), and liver (viii) sections obtained from mdx mice 21 days after intracerebroventricular (ICV) injection with 2.7x1011vg of INS1201-AAV9 and immunofluorescently stained for dystrophin. [Figure 4B] Shown are gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteus (iv), triceps (v), diaphragm (vi), heart (vii), and liver (viii) sections obtained from mdx mice 21 days after intracerebroventricular (ICV) injection with 9x1010 vg of INS1201-AAV9 and immunofluorescently stained for dystrophin. [Diagram 5] Shown are gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteus (iv), triceps (v), diaphragm (vi), heart (vii), and liver (viii) sections obtained from mdx mice 21 days after intracerebroventricular (ICV) injection with 9x1010vg of INS1212-AAV9 and immunofluorescently stained for dystrophin. [Figure 6A]Shown are hematoxylin and eosin (H&E) stained gastrocnemius muscle sections obtained from mdx mice 80 days after intracerebroventricular (ICV) injection with 9x10vg (ii) or 2.7x10vg (iii) of INS1201-AAV9. H&E stained gastrocnemius muscle sections obtained from wild-type C57 / Bl mice (i) and non-injected mdx mice (iv) are shown for comparison. [Figure 6B] 1 shows gastrocnemius muscle sections taken from mdx mice 80 days after intracerebroventricular (ICV) injection with 9x10vg (ii) or 2.7x10vg (iii) of INS1201-AAV9 and stained for dystrophin. Gastrocnemius muscle sections taken from wild-type C57 / Bl mice (i) and non-injected mdx mice (iv) and stained for dystrophin are shown for comparison. [Figure 7A] Shown are hematoxylin and eosin (H&E) stained gastrocnemius muscle sections taken from mdx mice 80 days after intracerebroventricular (ICV) injection with 9x1010 vg (ii). H&E stained gastrocnemius muscle sections taken from wild-type C57 / Bl mice (i) and non-injected mdx mice (iii) are shown for comparison. [Figure 7B] Shown are gastrocnemius muscle sections taken from mdx mice 80 days after intracerebroventricular (ICV) injection with 9x10vg (ii) of INS1212-AAV9 and stained for dystrophin. Gastrocnemius muscle sections taken from wild-type C57 / Bl mice (i) and non-injected mdx mice (iii) and stained for dystrophin are shown for comparison. [Figure 8A] 1 shows a bar graph of the mean fiber diameter (μm) of gastrocnemius muscle cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg or 2.7×10 vg of INS1201-AAV9. The mean fiber diameter (μm) of gastrocnemius muscle cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 8B]A line graph of the relative frequency (%) of cell diameter (μm) of gastrocnemius muscle cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg or 2.7×10 vg of INS1201-AAV9 is shown. The relative frequency of cell diameter of gastrocnemius muscle cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 8C] 1 shows a bar graph of the mean fiber diameter (μm) of triceps cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg or 2.7×10 vg of INS1201-AAV9. The mean fiber diameter (μm) of triceps cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 8D] A line graph of the relative frequency (%) of cell diameter (μm) of triceps cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg or 2.7×10 vg of INS1201-AAV9 is shown. The relative frequency of cell diameter of triceps cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 8E] 1 shows a bar graph of the mean fiber diameter (μm) of tibialis anterior muscle cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg or 2.7×10 vg of INS1201-AAV9. The mean fiber diameter (μm) of tibialis anterior muscle cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 8F] 1 shows a line graph of the relative frequency (%) of cell diameter (μm) of tibialis anterior muscle cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg or 2.7×10 vg of INS1201-AAV9. The relative frequency of cell diameter of tibialis anterior muscle cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 8G]1 shows a bar graph of the mean fiber diameter (μm) of diaphragm muscle cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg or 2.7×10 vg of INS1201-AAV9. The mean fiber diameter (μm) of diaphragm muscle cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 8H] A line graph of the relative frequency (%) of cell diameter (μm) of diaphragm muscle cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg or 2.7×10 vg of INS1201-AAV9 is shown. The relative frequency of cell diameter of diaphragm muscle cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 9A] A bar graph of the mean fiber diameter (μm) of gastrocnemius muscle cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg of INS1212-AAV9 is shown. The mean fiber diameter (μm) of diaphragm muscle cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 9B] A line graph of the relative frequency (%) of cell diameter (μm) of gastrocnemius muscle cells in mdx mice 80 days after intracerebroventricular (ICV) injection with 9×10 vg of INS1212-AAV9. The relative frequency of cell diameter of diaphragm muscle cells in wild-type C57 / Bl mice and non-injected mdx mice is shown for comparison. [Figure 10A] FIG. 13 is a line graph of the percentage of EDL muscle contractile force resulting from eccentric contraction (EC) in wild-type C57 / Bl mice, mdx mice that received an intracerebroventricular (ICV) injection with vehicle on postnatal day 1 (p1), mdx mice that received an ICV injection of 2.7×10 vg of INS1201-AAV9 on postnatal day 1 p1, and mdx mice that received an ICV injection of 9×10 vg of INS1201-AAV9 on p1. [Figure 10B]Line graphs of the ratio of eccentric contraction (EC) post-stress to EC pre-stress in EDL muscle in (i) wild-type C57 / Bl mice and mdx mice that received intracerebroventricular (ICV) injections on postnatal day 1 (p1) with (ii) 9x109 vg of INS1201-AAV9, (iii) 9x1010 vg of INS1201-AAV9, (iv) 2.7x1011 vg of INS1201-AAV9, or (v) vehicle control. [Figure 10C] Graph showing percentage of contractile force of EDL muscle as a function of eccentric contraction (EC) number (eccentric contraction 1 (EC1)%) in (i) wild-type C57 / Bl mice and mdx mice that received intracerebroventricular (ICV) injection of either (ii) 9×10 vg of INS1201-AAV9, (iii) 2.7×10 vg of INS1201-AAV9, (iv) 5.4×10 vg of INS1201-AAV9, (v) 1.2×10 vg of INS1201-AAV9, or (vi) vehicle control at postnatal day 28 (p28). [Figure 10D] 1 is a bar graph showing the percentage of force ([post-EC5 / post-EC1]) of EDL muscle in (i) wild-type C57 / Bl mice and mdx mice that received an intracerebroventricular (ICV) injection of either (ii) 9×1010 vg of INS1201-AAV9, (iii) 2.7×1011 vg of INS1201-AAV9, (iv) 5.4×1011 vg of INS1201-AAV9, (v) 1.2×1012 vg of INS1201-AAV9, or (vi) vehicle control at postnatal day 28 (p28). [Figure 10E] Graph of maximum tension (kPa) of EDL muscle resulting from eccentric contraction (EC) in (i) wild-type C57 / Bl mice and mdx mice that received an intracerebroventricular (ICV) injection of either (ii) 9×109 vg of INS1201-AAV9, (iii) 9×1010 vg of INS1201-AAV9, (iv) 2.7×1011 vg of INS1201-AAV9, (v) 5.4×1011 vg of INS1201-AAV9, (vi) 1.2×1012 vg of INS1201-AAV9, or (vii) vehicle control at postnatal day 28 (p28). [Figure 10F]Graph of peak stress (kPa) resulting from eccentric contractions (EC) at various frequencies (Hz) in (i) wild-type C57 / Bl mice and mdx mice that received an intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9×109 vg of INS1201-AAV9, (iii) 9×1010 vg of INS1201-AAV9, (iv) 2.7×1011 vg of INS1201-AAV9, (v) 5.4×1011 vg of INS1201-AAV9, (vi) 1.2×1012 vg of INS1201-AAV9, or (vii) vehicle control. [Figure 11A] Shown are gastrocnemius muscle sections obtained from uninjected cynomolgus monkeys (i), cynomolgus monkeys 21 days after intravenous (IV) injection with 5×10 vg (ii) or 1×10 vg (iii) AAV9 CBA-GFP, or cynomolgus monkeys 21 days after intrathecal (IT) injection with 2.5×10 vg (iv), 5×10 vg (v), or 1×10 vg (vi) AAV9 CBA-GFP, and immunostained with NovaRed™ for detection of GFP expression. [Figure 11B] Quadriceps muscle sections obtained from uninjected cynomolgus monkeys (i), cynomolgus monkeys 21 days after intravenous (IV) injection with 5×10 vg (ii) or 1×10 vg (iii) AAV9 CBA-GFP, or cynomolgus monkeys 21 days after intrathecal (IT) injection with 2.5×10 vg (iv), 5×10 vg (v), or 1×10 vg (vi) AAV9 CBA-GFP, and immunostained with NovaRed™ for detection of GFP expression are shown. [Figure 11C] Shown are deltoid muscle sections obtained from uninjected cynomolgus monkeys (i), cynomolgus monkeys 21 days after intravenous (IV) injection with 5×10 vg (ii) or 1×10 vg (iii) AAV9 CBA-GFP, or cynomolgus monkeys 21 days after intrathecal (IT) injection with 2.5×10 vg (iv), 5×10 vg (v), or 1×10 vg (vi) AAV9 CBA-GFP, and immunostained with NovaRed™ for detection of GFP expression. [Figure 11D]Triceps muscle sections obtained from uninjected cynomolgus monkeys (i), cynomolgus monkeys 21 days after intravenous (IV) injection with 5×10 vg (ii) or 1×10 vg (iii) AAV9 CBA-GFP, or cynomolgus monkeys 21 days after intrathecal (IT) injection with 2.5×10 vg (iv), 5×10 vg (v), or 1×10 vg (vi) AAV9 CBA-GFP, and immunostained with NovaRed™ for detection of GFP expression are shown. [Figure 11E] Shown are biceps sections obtained from uninjected cynomolgus monkeys (i), cynomolgus monkeys 21 days after intravenous (IV) injection with 5×10 vg (ii) or 1×10 vg (iii) AAV9 CBA-GFP, or cynomolgus monkeys 21 days after intrathecal (IT) injection with 2.5×10 vg (iv), 5×10 vg (v), or 1×10 vg (vi) AAV9 CBA-GFP, and immunostained with NovaRed™ for detection of GFP expression. [Figure 11F] Shown are tibialis anterior muscle sections obtained from cynomolgus monkeys 21 days after intravenous (IV) injection with 5×10 vg (i) or 1×10 vg (ii) AAV9 CBA-GFP, or from cynomolgus monkeys 21 days after intrathecal (IT) injection with 2.5×10 vg (iii), 5×10 vg (iv), or 1×10 vg (v) AAV9 CBA-GFP, and immunostained with NovaRed™ for detection of GFP expression. [Figure 11G] Diaphragm muscle sections obtained from cynomolgus monkeys 21 days after intravenous (IV) injection with 5×10 vg (i) or 1×10 vg (ii) AAV9 CBA-GFP, or from cynomolgus monkeys 21 days after intrathecal (IT) injection with 2.5×10 vg (iii), 5×10 vg (iv), or 1×10 vg (v) AAV9 CBA-GFP, and immunostained with NovaRed™ for detection of GFP expression are shown. [Figure 11H]Shown are myocardial sections obtained from cynomolgus monkeys 21 days after intravenous (IV) injection with 5×10 vg (i) or 1×10 vg (ii) AAV9 CBA-GFP, or from cynomolgus monkeys 21 days after intrathecal (IT) injection with 2.5×10 vg (iii), 5×10 vg (iv), or 1×10 vg (v) AAV9 CBA-GFP, and immunostained with NovaRed™ for detection of GFP expression. [Figure 11I] Shown are liver sections obtained from cynomolgus monkeys 21 days after intravenous (IV) injection with 5×10 vg (i) or 1×10 vg (ii) AAV9 CBA-GFP, or from cynomolgus monkeys 21 days after intrathecal (IT) injection with 2.5×10 vg (iii), 5×10 vg (iv), or 1×10 vg (v) AAV9 CBA-GFP, and immunostained with NovaRed™ for detection of GFP expression. [Figure 12A] Ponceau staining (upper panels) or Western blots (lower panels) of muscle protein samples from biceps (1), triceps (2), deltoid (3), quadriceps (4), gastrocnemius (5), tibialis anterior (6), diaphragm (7), and heart (8) obtained from a cynomolgus monkey 21 days after intrathecal (IT) injection with 2.5 x 10 vg of AAV9 CBA-GFP and probed with anti-GFP antibodies are shown. [Figure 12B] Ponceau staining (upper panels) or Western blots (lower panels) of muscle protein samples from biceps (1), triceps (2), deltoid (3), quadriceps (4), gastrocnemius (5), tibialis anterior (6), diaphragm (7), and heart (8) obtained from a cynomolgus monkey 21 days after intrathecal (IT) injection with 5 x 1013 vg of AAV9 CBA-GFP and probed with anti-GFP antibodies are shown. [Figure 12C]Ponceau staining (upper panels) or Western blots (lower panels) of muscle protein samples from biceps (1), triceps (2), deltoid (3), quadriceps (4), gastrocnemius (5), tibialis anterior (6), diaphragm (7), and heart (8) obtained from a cynomolgus monkey 21 days after intrathecal (IT) injection with 1 x 1014 vg of AAV9 CBA-GFP and probed with anti-GFP antibodies are shown. [Figure 12D] Shown are Ponceau staining (upper panel) or Western blots of biceps (1) and triceps (2) muscles obtained from a non-injected cynomolgus monkey and probed with anti-GFP antibody. [Figure 13] Agarose gel electrophoresis of biceps (1), triceps (2), deltoid (3), tibialis anterior (4), gastrocnemius (5), quadriceps vastus lateralis (6), diaphragm (7), and cardiac muscle (8), as well as liver (9) tissue samples obtained from a cynomolgus monkey 21 days after intrathecal (IT) injection with 2.5x10vg 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 a non-injected cynomolgus monkey and subjected to RT-PCR in the presence (+) or absence (-) of reverse transcriptase are shown for comparison. [Figure 14] 1 shows various μDys protein domains encoded by the μDys transgenes provided herein. [Figure 15] FIG. 11 is a graph showing the number of INS1201 DNA copies per diploid genome in mdx mice that received an intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either (ii) 9×109 vg of INS1201-AAV9, (iii) 9×1010 vg of INS1201-AAV9, (iv) 2.7×1011 vg of INS1201-AAV9, (v) 5.4×1011 vg of INS1201-AAV9, (vi) 1.2×1012 vg of INS1201-AAV9, or (vii) vehicle control. [Figure 16]FIG. 13 is a graph showing the number of INS1201 RNA transcript copies normalized to the copy number of RPP30 in mdx mice that received an intracerebroventricular (ICV) injection at postnatal day 28 (p28) of either: (ii) 9×109 vg of INS1201-AAV9, (iii) 9×1010 vg of INS1201-AAV9, (iv) 2.7×1011 vg of INS1201-AAV9, (v) 5.4×1011 vg of INS1201-AAV9, (vi) 1.2×1012 vg of INS1201-AAV9, or (vii) vehicle control. [Figure 17] Graph of mean fiber diameter (μm) of EDL myocytes at postnatal day 120 (p120) in (i) wild-type C57 / Bl mice and mdx mice that received intracerebroventricular (ICV) injection of either (ii) 9×109 vg of INS1201-AAV9, (iii) 9×1010 vg of INS1201-AAV9, (iv) 2.7×1011 vg of INS1201-AAV9, (v) 5.4×1011 vg of INS1201-AAV9, (vi) 1.2×1012 vg of INS1201-AAV9, or (vii) vehicle control at postnatal day 28 (p28). [Figure 18] Graph of mean fiber diameter (μm) of the tibialis anterior muscle at postnatal day 120 (p120) in (i) wild-type C57 / Bl mice and mdx mice that received an intracerebroventricular (ICV) injection of either (ii) 9×109 vg of INS1201-AAV9, (iii) 9×1010 vg of INS1201-AAV9, (iv) 2.7×1011 vg of INS1201-AAV9, (v) 5.4×1011 vg of INS1201-AAV9, (vi) 1.2×1012 vg of INS1201-AAV9, or (vii) vehicle control at postnatal day 28 (p28). [Figure 19] Diaphragm muscle sections stained with picrosirius red after intracerebroventricular (ICV) injection with various doses of INS1201-AAV9. Diaphragm sections obtained from wild-type C57 / Bl mice and mdx mice receiving vehicle are shown in the upper section for comparison. [Figure 20]Graph showing collagen percentage in diaphragm muscle at postnatal day 120 (p120) in (i) wild-type C57 / Bl mice and mdx mice that received an intracerebroventricular (ICV) injection of either (ii) vehicle control, (iii) 9×10 vg of INS1201-AAV9, (iv) 9×10 vg of INS1201-AAV9, (v) 2.7×10 vg of INS1201-AAV9, (v) 5.4×10 vg of INS1201-AAV9, or (vi) 1.2×10 vg of INS1201-AAV9 on postnatal day 28 (p28). [Figure 21] The top shows extensor digitorum longus (EDL) muscle sections taken from mdx mice at p120 after intracerebroventricular (ICV) injection with 5.4×10 vg of INS1201-AAV9 at postnatal day 28 (p28) and stained with hematoxylin and eosin (H&E) (far left), laminin / dapi (second from left), dystrophin (second from right), and merged images (far right). The bottom of FIG. 21 shows EDL muscle sections taken from mdx mice at p120 after intracerebroventricular (ICV) injection with 5.4×10 vg of INS1201-AAV9 at postnatal day 28 (p28) and stained with hematoxylin and eosin (H&E) (far left), laminin / dapi (second from left), dystrophin (second from right), and merged images (far right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention relates, in part, to adeno-associated virus (AAV) particles and methods for preferentially delivering the same to cardiac and / or skeletal muscle in a subject in need of treatment for a monogenic muscle disease, e.g., 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 same to a subject in need thereof, for example to treat a monogenic muscle disease such as a dystrophinopathy, provide advantages over known AAV particles and methods of treatment because at least the present invention (i) allows for significantly lower dosages than IV delivery to achieve substantially the same or better therapeutic benefit, thereby reducing viral load and toxicity as well as 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 and providing a superior therapeutic benefit compared to intravenously delivered AAV vectors, and (iii) can benefit a larger patient population compared to IV formulations due to the required lower dosages, corresponding to a reduced manufacturing burden.

[0027] Aspects of the invention relate to AAV particles, methods for producing the same, and methods for delivering AAV particles to a subject in need of treatment. The AAV particles, e.g., AAV9 particles, include a capsid that includes one or more AAV9 capsid proteins and a vector genome encapsidated by the AAV9 capsid. The vector genome includes a transgene and a regulatory element that drives gene expression of the transgene when delivered into muscle cells, e.g., skeletal and / or cardiac muscle cells. In one embodiment, the transgene is a micro-dystrophin (μDys) transgene.

[0028] The method described herein comprises administering a single dose of a composition comprising an effective amount of the AAV particles described herein to a subject in need of treatment for dystrophinopathy. In one embodiment, the dystrophinopathy is DMD, Becker muscular dystrophy, or DCM. In a preferred embodiment, the dystrophinopathy is DMD. In an embodiment described herein, intrathecal delivery of the AAV particles of the invention to muscle cells can result in robust expression of μDys, significantly improving muscle health and function. The present invention also provides methods and cells for producing the AAV particles described herein. In one embodiment of the method described herein, following intrathecal administration of the AAV particles, the transgene is expressed at a higher level in the skeletal and / or cardiac muscle of the subject compared to transgene expression in liver tissue of the subject.

[0029] To facilitate the understanding of this invention, several terms and phrases are defined below.

[0030] As used herein, the terms "a" and "an" mean "one or more" and include plurals unless the context is inappropriate.

[0031] The term "nucleic acid", "nucleotide", or "oligonucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized similarly 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 can 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)).

[0032] The term "gene" may refer to a segment of DNA involved in producing or encoding a polypeptide chain. It may include the coding region (leader and trailer) as well as the regions before and after the intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" may refer to a segment of DNA involved in producing or encoding a non-translated RNA, such as rRNA, tRNA, guide RNA (gRNA), short interfering RNA (siRNA), or microRNA (miRNA).

[0033] As used herein, the term "transgene" refers to an exogenous gene present in a vector genome that is artificially introduced into the genome of a cell, or an endogenous gene that is artificially introduced into a non-native locus in the genome of a cell. A transgene may refer to a segment of DNA responsible for producing or encoding a polypeptide chain. A transgene may include coding regions (leader and trailer) as well as regions before and after the intervening sequences (introns) between individual coding segments (exons). A transgene according to embodiments described herein is μDys. The μDys transgene, in one embodiment, encodes a μDys polypeptide that includes an N-terminal region, about 2-3 hinge regions, about 4-6 spectrin repeats, and a cysteine-rich domain.

[0034] A "vector genome" as used herein is a nucleic acid genome that comprises one or more heterologous nucleic acid sequences. The one or more heterologous nucleic acid sequences comprise a transgene. In some embodiments of the invention, the vector genome comprises at least one ITR sequence (e.g., an AAV ITR sequence), optionally two ITRs (e.g., two AAV ITRs), which are typically at the 5' and 3' ends of the vector genome and will flank one or more heterologous nucleic acids. The ITRs can be the same as each other or different.

[0035] As used herein, the term "endogenous" with respect to a nucleic acid, e.g., a gene, or a protein in a cell, is a nucleic acid or protein that occurs in that particular cell as found in nature, e.g., at its native gene location or locus. Moreover, a cell that "endogenously expresses" a nucleic acid or protein expresses that nucleic acid or protein as found in nature.

[0036] A "promoter" is defined as one or more nucleic acid control sequences that induce transcription of a nucleic acid, e.g., a transgene, and may be present in a vector genome. As used herein, a promoter includes a nucleic acid sequence near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which may be located as many as several thousand base pairs from the start site of transcription.

[0037] "Regulatory element" as used herein refers to a nucleic acid sequence capable of regulating the transcription of a gene (e.g., a transgene) and / or regulating the stability or translation of a transcribed mRNA product and may be present in a vector genome. In some embodiments, a regulatory element may regulate tissue-specific transcription of a gene. A regulatory element may include at least one transcription factor binding site, e.g., a transcription factor binding site for a muscle-specific transcription factor. A regulatory element as used herein increases or enhances promoter-driven gene expression compared to 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 include a portion of a larger sequence involved in transcriptional control, e.g., a portion of a promoter sequence. However, a regulatory element alone is typically not sufficient to initiate transcription by itself and requires the presence of a promoter.

[0038] 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.

[0039] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the term encompasses amino acid chains of any length, including full-length proteins, and functional fragments thereof, in which the amino acid residues are linked by covalent peptide bonds.

[0040] 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. Guide RNAs (gRNAs) described herein can include sequences that are fully complementary or substantially complementary (e.g., with only a few mismatched bases) to a genomic sequence, e.g., a DNA targeting sequence.

[0041] As used herein, the term "introducing" or "delivering" in the context of a nucleic acid, e.g., an AAV vector, refers to the translocation of a nucleic acid from outside a cell to inside a cell, e.g., a muscle cell. In some cases, introducing refers to the translocation of a nucleic acid from outside a cell to inside the nucleus of a cell. Various methods of such translocation are contemplated, including, but not limited to, electroporation, contact with a nanowire or nanotube, receptor-mediated internalization, translocation via a cell-penetrating peptide, liposome-mediated translocation, and the like.

[0042] As used herein, the terms "packaged" or "encapsidated" refer to the inclusion of a vector genome within a capsid that contains viral capsid proteins to form an AAV particle.

[0043] The term "substantial identity" or "substantially identical" when used in the context of a polynucleotide or polypeptide sequence refers to a sequence having at least 60% sequence identity with a reference sequence. Alternatively, the percent identity can be any integer between 60% and 100%. Exemplary embodiments include at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% when compared to a reference sequence using a program described herein, preferably BLAST using standard parameters described below. One of skill in the art will recognize that these values ​​may be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0044] For sequence comparison, typically, one sequence acts as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into 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 identity of test sequence to reference sequence based on program parameters.

[0045] 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) J. Mol. Biol. 215:403-410, and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or meet a certain positive threshold score T when aligned with words 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 act as seeds to initiate 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. The cumulative score is calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hits in each direction is stopped when the cumulative alignment score falls off its maximum achieved value by an amount X, the cumulative score falls below zero due to the accumulation of one or more negative-scoring 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=1, 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)).

[0046] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. 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 that a match between two nucleotide or amino acid sequences would occur by chance. For example, the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.01, more preferably less than about 10 -5 less than about 10 -20 A nucleic acid is considered to be similar to a reference sequence if it is less than

[0047] 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, monkeys, mice, horses, cows, pigs, dogs, cats, and the like. In some embodiments, the subject or patient is a human. The subject in the methods of treatment provided herein, in one embodiment, is a male subject.

[0048] In embodiments where the subject is a male human, the male human subject is about 4 years old to about 7 years old, a newborn, about 1 year old to about 7 years old, about 2 years old to about 7 years old, about 2 years old to about 6 years old, about 2 years old to about 5 years old, about 2 years old to about 4 years old, about 3 years old to about 7 years old, about 3 years old to about 6 years old, about 1 month old to about 6 years old, about 1 month old to about 5 years old, about 1 month old to about 4 years old, about 1 month old to about 3 years old, about 1 month old to about 2 years old, or about 1 month old to about 12 months old.

[0049] In one embodiment, the subject is a male human patient between about 4 and about 7 years of age. In one embodiment, the subject is a male human patient between about 3 and about 7 years of age. In one embodiment, the subject is a male human patient between about 2 and about 7 years of age.

[0050] As used herein, the term "efficient delivery" or "efficiently delivering" refers to administration of AAV particles that contain AAV capsids that encapsidate a vector genome encoding a transgene that results in expression of the transgene in a desired cell or tissue.

[0051] As used herein, the term "effective amount" or "effective dose" refers to an amount of a substance (e.g., an AAV particle of the invention) sufficient to produce a beneficial or desired result (e.g., expression of a protein, or a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. When a dose is provided in a "vector genome," an "effective dose" may be referred to herein as an "effective vector genome dose."

[0052] As used herein, the term "treating" includes any effect that results in the improvement of a condition, disease, disorder, or the like, such as reducing, alleviating, modulating, ameliorating, or eliminating, or ameliorating a symptom thereof.

[0053] Throughout the description, where compositions are described as having, including, or comprising specific components, or processes and methods are described as having, including, or comprising specific steps, it is contemplated that in addition there are compositions of the invention that consist essentially of or consist of the recited components, and there are processes and methods of the invention that consist essentially of or consist of the recited processing steps.

[0054] Adeno-associated virus (AAV) particles As described herein, one aspect of the invention relates to AAV particles, comprising one or more AAV capsid proteins and a vector genome encapsidated by 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 transgene, an SV40 poly(A) tail, and a 3' ITR. When administered intrathecally to a subject in need of treatment, in one embodiment, the transgene is expressed at a higher level in the subject's skeletal muscle and / or cardiac muscle compared to the transgene expression in the subject's liver tissue. In another embodiment, the ratio of [(skeletal muscle and / or cardiac muscle transgene expression)] / (liver transgene expression)] provided by the AAV particles described herein is greater than the same ratio when the same dose of the same AAV particles is administered intravenously. In one preferred embodiment, the AAV particles are AAV9 particles comprising one or more AAV9 capsid proteins.

[0055] As used herein, "adeno-associated virus (AAV) particle" refers to an AAV virion that includes an AAV capsid and a vector genome enclosed by the AAV capsid. The vector genome typically includes a promoter and one or more transgenes flanked by AAV ITR sequences. The AAV capsid includes 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. The vector genomes described herein can be replicated and packaged into viral vectors (particles) when introduced into a host cell that also contains one or more plasmids encoding rep and cap gene products. In one embodiment, a helper plasmid is also transfected into the host cell to assist in vector production by the host cell. In one embodiment, the AAV vector used herein is an AAV9 vector described, for example, in U.S. Pat. No. 7,906,111, the disclosure of which is incorporated herein by reference in its entirety for all purposes. In one embodiment, the AAV capsid is an AAV9 capsid.

[0056] The terms "empty capsid," "empty vial particle," and "empty AAV" refer to an AAV capsid shell that lacks a vector genome packaged therein.

[0057] The encapsidated vector genome described herein can include one or more regulatory elements, for example, one or more regulatory elements upstream of a transgene. The encapsidated genome, in one embodiment, includes, 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 includes, from 5' to 3', a 5'ITR, an enhancer, a promoter, a transgene, an SV40 poly(A) tail, and a 3'ITR. The encapsidated genome, in yet another embodiment, includes, from 5' to 3', a 5'ITR, a promoter, an SV40 intron, a transgene, an SV40 poly(A) tail, and a 3'ITR. In yet 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.

[0058] Inverted terminal repeats Inverted terminal repeats (ITRs) are palindromic 145 nucleotide sequences that flank the transgene. The 5' and 3' ITRs of the AAV vector genome are necessary for both integration of the transgene into the host cell genome (e.g., chromosome 19 in humans) and encapsidation of the transgene into AAV particles.

[0059] In some embodiments, the AAV vector genome of the invention comprises ITR sequences from any one of the AAV serotypes, e.g., AAVrh.74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV 13. In some embodiments, the AAV vector genome disclosed herein comprises 5' and 3' AAV2 ITR sequences.

[0060] In some embodiments, the AAV vector genome described herein comprises 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.

[0061] In some embodiments, the AAV vector genome described herein comprises 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.

[0062] promoter Promoters drive the expression of transgenes and are typically positioned upstream (or 5') of the transgenes whose expression they regulate.

[0063] In some embodiments, the AAV vector genome of the present invention comprises a mammalian promoter, such as a human, non-human primate (e.g., cynomolgus monkey), mouse, equine, bovine, porcine, feline, and canine promoter. In some embodiments, the recombinant AAV vector genome disclosed herein comprises a strong, constitutively active promoter to drive high-level expression of the transgene. For example, in some embodiments, the promoter is a cytomegalovirus (CMV) promoter / enhancer, an elongation factor 1 alpha (EF1 alpha) promoter, a simian virus 40 (SV40) promoter, a chicken β-actin hybrid promoter, or a CAG promoter.

[0064] The promoter, in one embodiment, is the MHCK7 or chicken β-actin hybrid promoter.

[0065] In certain embodiments, the AAV vector genome of the invention comprises a muscle-specific promoter operably linked to a transgene to confer high-level and tissue-specific expression in muscle cells. For example, muscle-specific promoters of the invention include the desmin (DES, also known as CSM1 or CSM2) promoter, the alpha 2 actinin (ACTN2, also known as CMD1AA) 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 (SAR ...alpha 2 actinin (ACTN2, also known as CMD1AA) 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 alpha 2 actinin (ACTN2, also known as CMD1AA) promoter, the alpha 2 1 (also known as ATP2A1, ATP2A or SERCA1) promoter, troponin type I 1 (also known as TNNI1, SSTNI or 25TTNI) promoter, myosin-1 (MYH1) promoter, phosphorylatable fast skeletal myosin light chain (MYLPF) promoter, myosin 1 (also known as MYH1, MYHSA1, MYHa, MyC-2X / D or MyHC-2x) promoter, alpha-3 chain tropomyosin (also known as TPM3, CFTD, NEM1, OK / Scl.5, TM-5, TM3, TM30, TM30nm, TM5, TPMsk3, TRK, h TM5 or hscp30) promoter, ankyrin repeat domain-containing protein 2 (ANKRD2, also known as ARPP) promoter, myosin heavy chain (MHC) promoter, myosin light chain (MLC) promoter, muscle creatine kinase (MCK) promoter, Li et al. (1999. Nat Synthetic muscle promoters described in Wang et al. (2008. Gene Ther. 17:241-245), such as the SPc5-12 promoter, the muscle creatine kinase (MCK) promoter, the dMCK promoter (each consisting of a double or triple tandem of 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), Wang et al. (2008. Gene Ther.15:1489-1499), the MCK-C5-12 promoter described in, and the cardiac and skeletal muscle specific myosin chaperone Unc45b (195 bp) promoter described in Rudeck S et al. (2016, Genesis. 54(8):431-8), but are not limited to these. Non-limiting examples of cardiac specific promoters include calsequestrin 2 (PDIB2, also known as FLJ26321, FLJ93514, or CASQ2 (GeneID 845 for the human gene) promoter, ankyrin repeat domain 1 (also known as cardiac ankyrin repeat protein) promoter, cytokine inducible nuclear protein promoter, hepatic ankyrin repeat domain 1 (ANKRD1, GeneID 27063 for the human gene), myosin, light chain 2, regulatory, cardiac, slow (MYL2, GeneID 4633 for the human gene) promoter, myosin, light chain 3, alkaline, ventricular, skeletal 10 slow (MYL3, GeneID 4634 for the human gene) promoter, bromodomain-containing 7 (BP75, CELTIX1, NAG4 (BRD7, GeneID 4635 for the human gene) promoter, These include the 29117 (also known as 29117) promoter, the alpha myosin heavy chain (αMHC) promoter, the cardiac troponin C promoter, and the cardiac sodium-calcium exchanger (NCX1) promoter, which confers cardiac specificity.

[0066] In some embodiments, the AAV vector genome described herein comprises an 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.

[0067] SV40 intron In some embodiments, the AAV vector genome of the invention comprises an SV40 intron, a commonly used regulatory element in gene therapy vectors that enhances translation and stability of the expressed RNA transcript.

[0068] In certain embodiments, the SV40 intron is downstream (i.e., 3') of the promoter and upstream (i.e., 5') of the transgene. In other embodiments, the SV40 intron can be downstream (i.e., 3') of the transgene.

[0069] 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.

[0070] SV40-poly(A) tail In some embodiments, the AAV vector genome of the invention comprises a nucleic acid sequence encoding the SV40 poly(A) tail, a commonly used nucleic acid element in gene therapy vectors that aids in RNA export from the nucleus, RNA translation, and RNA stability.

[0071] In some embodiments, an AAV vector genome of the invention comprises 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.

[0072] Enhancer In some embodiments, the AAV vector genome of the invention comprises one or more enhancer sequences, which in one embodiment can increase transcription levels of a transgene by, for example, functioning as binding sites for transcription factors and coregulatory factors that assist in DNA looping and recruitment of the transcription machinery to the promoter.

[0073] 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.

[0074] In some embodiments, a recombinant AAV vector genome of the invention comprises an enhancer that significantly promotes transcription of a transgene in muscle cells, e.g., skeletal and / or cardiac muscle cells.

[0075] In some embodiments, the recombinant AAV vector described herein comprises 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.

[0076] In yet another embodiment, the AAV vector genome of the present invention comprises a cytomegalovirus (CMV) enhancer nucleic acid sequence. In further embodiments, the CMV enhancer is upstream of the promoter sequence. For example, in one embodiment, the CMV enhancer has the nucleic acid sequence of SEQ ID NO:9. 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 NO:9 (see Table 1). In some embodiments, the CMV enhancer comprises SEQ ID NO:9. In some embodiments, the CMV enhancer consists of SEQ ID NO:9.

[0077] Transgene A transgene for use with the present invention is a nucleic acid sequence that encodes a polypeptide or a functional fragment thereof that is expressed in a cell (eg, a muscle cell) into which the transgene is delivered.

[0078] In some embodiments, the transgene may be integrated into the genome of the host cell to which it is delivered, or may be expressed episomally.

[0079] In some embodiments, the transgene can encode a polypeptide or a functional fragment thereof that is not endogenously expressed by the cell to which the transgene is delivered. In some embodiments, the transgene encodes a mutant form of a polypeptide or a functional fragment thereof that is endogenously expressed by the cell to which the transgene is delivered. In some embodiments, the transgene encodes a protein or a functional fragment thereof that is endogenously expressed by the cell to which the transgene is delivered, but at a low level, and expression of the transgene results in a higher expression level of the protein or functional fragment thereof. In some embodiments, the cell to which the transgene is delivered carries one or more mutations that result in a reduced expression level of the endogenous protein and / or a functionally defective protein, and expression of the transgene results in the restoration of expression of the endogenous protein and / or functional replacement of the defective protein. In some embodiments, the transgene is silent when introduced into the cell to which the transgene is delivered and expression can be induced.

[0080] In some embodiments, the transgene can 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 can be heterologous or homologous to the cell to which the transgene is delivered.

[0081] In some embodiments, the transgene may be a full-length cDNA or genomic DNA sequence, or a fragment or variant thereof having functional activity. In some embodiments, the transgene may be a minigene, i.e., a gene sequence lacking some, most, or all of its intronic sequences, or may include all of its intronic 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 compared to the wild-type sequence.

[0082] In some embodiments, a transgene of the invention encodes a therapeutic protein. In certain embodiments, the transgene may encode a structural protein.

[0083] In some embodiments, the transgene is a microdystrophin (μDys) transgene encoding a μDys polypeptide. In further embodiments, the μDys polypeptide encoded by the transgene comprises (i) an N-terminal region (NTD) comprising an actin binding site, and (ii) a central rod domain comprising 2-4 hinge regions and 4-6 spectrin repeats. In further embodiments, the μDys transgene comprises the nucleic acid sequence set forth in SEQ ID NO:5. In yet further embodiments, the μDys transgene consists of the nucleic acid sequence set forth in SEQ ID NO:5. In another embodiment, the sequence encoding the μDys 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.

[0084] In one embodiment, the μDys transgene comprises the nucleic acid sequence set forth in SEQ ID NO:4 of U.S. Patent No. 10,351,611, which is incorporated by reference in its entirety for all purposes. In another embodiment, the sequence encoding the μDys 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.

[0085] In one embodiment, the μDys transgene encodes a μDys 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. In a further embodiment, the μDys transgene encodes a μDys 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 (iii) a cysteine-rich domain.

[0086] The μDys 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 μDys transgene encodes dystrophin spectrin repeats 1 and 24. In another embodiment, the μDys transgene encodes dystrophin spectrin repeats 1, 16 and 17, 23 and 24. In yet another embodiment, the μDys transgene encodes dystrophin spectrin repeats 1, 2, 3 and 24. In yet yet another embodiment, the μDys transgene encodes dystrophin spectrin repeats 1, 2, 22, 23 and 24.

[0087] The μDys transgene, in one embodiment, encodes dystrophin hinge regions 1 and 4. In another embodiment, the μDys transgene encodes dystrophin hinge regions 1, 3 and 4.

[0088] In one embodiment, the μDys transgene encodes a μDys protein that includes one of the μDys domain combinations set forth in FIG.

[0089] The AAV vector genome described herein comprises a μDys transgene encoding a μDys protein comprising (i) an NTD comprising an actin-binding site, (ii) a central rod domain comprising 2-4 hinge regions and 4-6 spectrin repeats, and (iii) a cysteine-rich domain. For example, in one embodiment, the μDys transgene encodes dystrophin spectrin repeats 16 and 17. In a further embodiment, the μDys transgene encodes dystrophin spectrin repeats 1 and 24. In another embodiment, the μDys transgene encodes dystrophin spectrin repeats 1, 16 and 17, 23 and 24. In yet another embodiment, the μDys transgene encodes dystrophin spectrin repeats 1, 2, 3 and 24. In yet yet another embodiment, the μDys transgene encodes dystrophin spectrin repeats 1, 2, 22, 23 and 24.

[0090] In one embodiment, the μDys transgene encodes dystrophin hinge regions 1 and 4. In another embodiment, the μDys transgene encodes dystrophin hinge regions 1, 3 and 4.

[0091] The AAV particles described herein, in one embodiment, comprise an encapsidated transgene encoding a μDys protein. For example, in some embodiments, the sequence encoding the μDys 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 the μDys protein comprises SEQ ID NO:5. In some embodiments, the sequence encoding the μDys protein consists of SEQ ID NO:5. [ka] [ka] [ka]

[0092] The nucleic acid elements disclosed herein can be ligated together using standard molecular biology techniques to form a genetic construct (see, e.g., "Molecular Cloning: A Laboratory Manual, 2nd Ed." (Sambrook et al., 1989); "Current Protocols in Molecular Biology" (Ausubel et al., 1987)).

[0093] The genetic constructs described herein minimally comprise (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 μDys. In further embodiments, the genetic constructs can comprise one or more additional regulatory elements. In one embodiment, the genetic construct comprises, from 5' to 3', a promoter, a transgene, and an SV40 poly(A) tail. In another embodiment, the genetic construct comprises, from 5' to 3', an enhancer, a promoter, a transgene, an SV40 intron, and an SV40 poly(A) tail.

[0094] In some embodiments, the genetic construct comprises (from 5' to 3'): (i) a promoter; (ii) an 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 μDys. In further embodiments, the genetic construct can comprise one or more additional regulatory elements. In some embodiments, the genetic construct described herein comprises a contiguous 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 genetic construct comprises SEQ ID NO:10. In some embodiments, the genetic construct consists of SEQ ID NO:10. [ka] [ka]

[0095] In some embodiments, the genetic 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 μDys. In further embodiments, the genetic construct can comprise one or more additional regulatory elements. In some embodiments, the genetic construct described herein comprises a contiguous 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 genetic construct comprises the nucleic acid sequence of SEQ ID NO:11. In some embodiments, the genetic construct consists of the nucleic acid sequence of SEQ ID NO:11.

[0096] In some embodiments, the 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 genetic construct consists of SEQ ID NO: 12. In some embodiments, the vector genome comprises the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the vector genome consists of the nucleic acid sequence of SEQ ID NO: 12. [ka] [ka] [ka] [ka]

[0097] AAV vector backbone In some embodiments, the AAV vector genomes of the invention can be assembled using standard molecular biology techniques by inserting the genetic constructs described herein into an appropriate adenoviral plasmid backbone (see, e.g., Sambrook et al. (1989). "Molecular Cloning: A Laboratory Manual, 2nd Ed."; Ausubel et al. (1987). "Current Protocols in Molecular Biology"). The adenoviral plasmid backbone, in one embodiment, comprises the 5'ITR and 3'ITR sequences described herein. The genetic constructs are inserted into the adenoviral plasmid backbone between the ITR sequences, downstream of the 5'ITR sequence, and upstream of the 3'ITR sequence.

[0098] For example, in one embodiment, an AAV vector genome of the invention can be assembled by inserting a genetic construct comprising the sequence of SEQ ID NO:10 into an adenovirus plasmid backbone comprising the sequence of SEQ ID NO:13.

[0099] In another embodiment, an AAV vector genome of the invention can be assembled by inserting a genetic construct comprising the sequence of SEQ ID NO:11 into an adenovirus plasmid backbone comprising the sequence of SEQ ID NO:13.

[0100] In some embodiments, an adenoviral plasmid backbone of the invention 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: 13. In some embodiments, an adenoviral plasmid backbone comprises the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, an adenoviral plasmid backbone consists of the nucleic acid sequence of SEQ ID NO: 13, provided below. [ka] [ka] [ka]

[0101] Adeno-associated virus (AAV) particle production AAV particles can be produced by any standard method (e.g., WO2001 / 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, all of which are incorporated by reference in their entireties), 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., Mol. Cell. Biol. 5:3251 (1985), all of which are incorporated by reference in their entireties. al, J. Virol, 62:1963 (1988), and Lebkowski et al, Mol. Cell. Biol, 7:349 (1988). Samulski et al. al, J. Virol., 63: 3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365, U.S. Pat. No. 5,658.776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. Vaccine 13: 1244-1250 (1995), Paul et al. Human Gene (See, e.g., Clark et al. 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).For example, in some embodiments, the AAV vector genomes described herein can be transformed into Escherichia coli for scaled DNA production, purified using any standard method (e.g., Maxi-Prep K, Thermo Scientific), and verified by restriction digestion or sequencing. The purified AAV vector genomes can then be combined with a plasmid containing the AAV rep and AAV cap genes, as well as an AAV helper plasmid, and transfected into an appropriate packaging cell line (e.g., HEK293, HeLa, or PerC.6, MRC-5, WI-38, Vera, and FRhL-2 cells) using standard methods (e.g., calcium phosphate transfection, polyethylenimine, electroporation, and the like). The AAV rep and cap genes can be from any AAV serotype and can be the same or different from those 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, the AAV particles described herein comprise AAV rep and cap genes from AAV2 and AAV9, respectively.

[0102] In some embodiments, the AAV particles described herein can be harvested from the 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, which are incorporated by reference in their entireties), such as by cesium chloride ultracentrifugation gradients or column chromatography.

[0103] Pharmaceutical Compositions The pharmaceutical compositions provided herein are intrathecal pharmaceutical compositions, i.e., intended for delivery via an intrathecal route. The intrathecal composition comprises an effective amount of AAV particles encapsidating a μDys transgene, as described herein. In some embodiments, the pharmaceutical compositions disclosed herein comprise the AAV particles of the present invention, a pharma- ceutically acceptable carrier, and optionally other agents, pharmaceuticals, stabilizers, buffers, carriers, adjuvants, diluents, and the like. By "pharmaceutically acceptable" is meant a material that is not toxic or otherwise harmful, i.e., the material can be administered to a subject without causing any undesirable biological effects. The pharmaceutical composition is an intrathecal pharmaceutical composition. An effective amount of AAV particles comprises a lower dose of vector genome compared to an IV AAV pharmaceutical composition comprising the same vector genome components or the same transgene. For example, in one embodiment, an effective amount of AAV particles described herein is about 90% or less of the vector genome than an effective amount of an IV composition comprising the same AAV particles or the same micro-dystrophin transgene.

[0104] In some embodiments, the pharmaceutical compositions provided herein include sterile aqueous and non-aqueous injection solutions that are optionally isotonic with the blood of the subject to which the pharmaceutical composition is delivered. The pharmaceutical compositions can contain antioxidants, buffers, bacteriostats, and solutes that render the composition isotonic with the blood of the intended subject to which it is 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, the pharmaceutical compositions include a pharma- ceutically acceptable vehicle and can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Preservatives and other additives can also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0105] In some embodiments, the pharmaceutical compositions can be presented in unit / dose or multi-dose containers, for example, sealed ampoules and vials and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, saline or water for injection, immediately prior to use.

[0106] In some embodiments, the pharmaceutical compositions disclosed herein may alternatively be formulated for IV, intramuscular, or intracerebroventricular (ICV) administration.

[0107] Treatment methods One aspect of the invention relates to a method of treating a dystrophinopathy in a subject in need thereof comprising intrathecally administering a single dose of an intrathecal composition comprising an effective amount of the AAV particles described herein. The method can be employed to preferentially deliver AAV particles to the heart and / or skeleton of a subject to treat a dystrophinopathy, such as, for example, 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 a subject in need thereof to treat a dystrophinopathy provide advantages over known AAV particles and methods of treatment because at least the present invention (i) allows for significantly lower dosages than IV delivery to achieve substantially the same or better therapeutic benefit, thereby reducing viral load and toxicity as well as 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 and providing a superior therapeutic benefit compared to intravenously delivered AAV vectors, and (iii) can benefit a larger patient population compared to IV formulations due to the lower dosages required, corresponding to a reduced manufacturing burden.

[0108] In one embodiment, a method of treating a subject in need of treatment is provided, comprising intrathecally administering to the subject a single dose of a composition comprising an effective amount of AAV particles comprising AAV capsids encapsidating a vector genome comprising a μDys transgene. In a further embodiment, the subject is positioned in Trendelenburg position prior to intrathecal administration. In one embodiment, the intrathecal administration of the composition is in the absence of a non-ionic low osmolality contrast agent. In another embodiment, the intrathecal administration is in the presence of a non-ionic low osmolality contrast agent.

[0109] The present invention is based, in part, on the finding that intrathecal administration of an effective amount of AAV particles described herein allows for a higher level of μDys transgene expression in the skeletal muscle and / or cardiac muscle of a subject compared to transgene expression in the liver tissue of a subject. For example, in one embodiment, after administration of an effective amount of AAV particles, e.g., AAV9 particles, the μDys transgene is expressed at a higher level in the skeletal muscle of a subject compared to the level of μDys transgene expression in the liver tissue. In another embodiment, after administration of an effective amount of AAV particles, e.g., recombinant AAV9 particles, the μDys transgene is expressed at a higher level in the cardiac muscle of a subject compared to the level of μDys transgene expression in the liver tissue. In yet another embodiment, after administration of an effective amount of AAV particles, e.g., recombinant AAV9 particles, the μDys transgene is expressed at a higher level in the skeletal muscle and cardiac muscle of a subject compared to the level of μDys transgene expression in the liver tissue.

[0110] 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 skilled in the art that to determine the level 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 level of μDys transgene expression in skeletal and / or cardiac muscle is, in one embodiment, 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 than the amount of μDys transgene expression in liver tissue. In a further embodiment, the μDys transgene comprises the nucleic acid sequence set forth in SEQ ID NO:5.

[0111] In one embodiment of the methods described herein, the method of delivering an effective dose of AAV particles encapsulating a μDys transgene provides greater μ transgene expression in skeletal and / or cardiac muscle compared to a substantially identical dose of AAV particles encapsulating a μDys transgene administered intravenously. In another embodiment, the method of delivering an effective dose of AAV particles provides greater μDys transgene expression in skeletal and / or cardiac muscle compared to an effective dose of AAV particles encapsulating a μDys transgene delivered 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 after administration of a composition comprising an effective amount of AAV particles. The transgene, in one embodiment, comprises the nucleic acid sequence set forth in SEQ ID NO:5.

[0112] In another embodiment, AAV particles encapsidating the μDys transgene of the invention, when administered intrathecally, provide 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 skeletal and / or cardiac muscle compared to transgene expression in the same tissue type when the same vector genome dose is administered intravenously. The same vector genome need not contain the same regulatory elements and / or the same transgene sequence or the same AAV capsid. However, the transgenes administered intrathecally and intravenously encode a μDys polypeptide.

[0113] In yet another embodiment, the ratio of [(skeletal and / or cardiac muscle μDys transgene expression)] / (liver μDys transgene expression)] measured after administration of AAV particles administered intrathecally is greater than the same ratio when the same dose of AAV particles encapsidating the μDys 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 after administration of a composition comprising an effective amount of AAV particles.

[0114] In one embodiment, an effective amount of AAV particles encapsulating a μDys transgene, when administered intrathecally, provides greater efficacy or greater therapeutic benefit compared to the same dose of AAV particles encapsulating a μDys transgene administered intravenously.

[0115] In some embodiments, an intrathecal (IT) vector genome (vg) dose sufficient to provide a therapeutic response for one of the therapeutic 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 of an intravenous (IV) vg dose of a vector genome encoding a μDys transgene sufficient to provide the same or substantially the same therapeutic response. The IT vector genome comprises a μDys transgene. The IT and IV μDys transgenes need not comprise the same nucleic acid sequence. In some embodiments, an IT vg dose sufficient to provide a therapeutic response for one of the therapeutic methods described herein is about 90% or less than an IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, an IT vg dose sufficient to provide a therapeutic response for one of the therapeutic methods described herein is about 75% or less than an IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, an IT vg dose sufficient to provide a therapeutic response for one of the therapeutic methods described herein is about 50% or less than an IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, an IT vg dose sufficient to provide a therapeutic response for one of the therapeutic methods described herein is about 25% or less than an IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, an IT vg dose sufficient to provide a therapeutic response for one of the therapeutic methods described herein is about 10% or less than an 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.In one embodiment, the therapeutic response is μDys transgene expression in muscle tissue, e.g., cardiac and / or skeletal muscle tissue. In another embodiment, the therapeutic response is an increase in the subject's score from baseline (i.e., pre-treatment) on the North Star Ambulatory Assessment (NSAA). In a further embodiment, the transgene encodes a μDys polypeptide.

[0116] In some embodiments, an intrathecal (IT) vector genome (vg) dose sufficient to provide a therapeutic response for one of the therapeutic methods described herein is lower than an intravenous (IV) vg dose sufficient to provide the same or substantially the same therapeutic response, and the IT vector genome and the IV vector genome each comprise a transgene encoding a μDys polypeptide, although each transgene need not comprise the same nucleic acid sequence. In some embodiments, an 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 an IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, an IT vg dose sufficient to provide a therapeutic response for one of the therapeutic 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 an IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, an IT vg dose sufficient to provide a therapeutic response for one of the therapeutic 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 an IV vg dose sufficient to provide the same or substantially the same therapeutic response. In some embodiments, an 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 an 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 therapeutic 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 therapeutic methods described herein is about 10-40-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 therapeutic methods described herein is about 25-40-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 therapeutic 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 therapeutic methods described herein is about 25 times 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 therapeutic methods described herein is about 40 times 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 therapeutic methods described herein is about 50 times 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 therapeutic methods described herein is about 100 times 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.

[0117] In some embodiments, the effective dose of an intrathecal (IT) composition comprising an AAV particle encapsidating a μDys transgene described herein is lower than the effective dose of an intravenous (IV) composition comprising an AAV particle encapsidating a μDys transgene. In some embodiments, the effective dose of an IT composition comprising an 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 of the vector genome than the effective amount of the IV composition. In some embodiments, the effective amount of an IT composition comprising an AAV particle is about 90% or less of the vector genome than the effective amount of the IV composition. In some embodiments, the effective amount of the IT composition comprising AAV particles is about 75% or less of the vector genome than the effective amount of the IV composition. In some embodiments, the effective amount of the IT composition comprising AAV particles is about 50% or less of the vector genome than the effective amount of the IV composition. In some embodiments, the effective amount of the IT composition comprising AAV particles is about 25% or less of the vector genome than the effective amount of the IV composition. In some embodiments, the effective amount of the IT composition comprising AAV particles is about 10% or less of the vector genome than the effective amount of the IV composition.

[0118] In some embodiments, the effective dose of the AAV particles in the IT composition is lower than the effective dose of the AAV particles in the IV composition, and each AAV particle encapsidates a μDys transgene. In some embodiments, the effective dose of the AAV particles in the 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 than the effective dose of the AAV particles in the IV composition. In some embodiments, the effective amount (effective dose) of an IT composition comprising AAV particles 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 effective amount (effective dose) of the same AAV particles, or an IV composition comprising AAV particles that encapsidate a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particles in the IT composition. In some embodiments, the effective amount of an intrathecal composition comprising AAV particles 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 effective amount of the same AAV particles or an IV composition comprising AAV particles that encapsidate a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particles in the IT composition. In some embodiments, an effective amount of an intrathecal composition comprising AAV particles 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 effective amount of the same AAV particles, or an IV composition comprising AAV particles that encapsidate a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particles in the IT composition. In some embodiments, an effective amount of an IT composition comprising AAV particles 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 effective amount of an IV composition comprising the same AAV particles.In some embodiments, the effective amount of an IT composition comprising AAV particles is about 25-fold to about 40-fold lower than the effective amount of the same AAV particles, or an IV composition comprising AAV particles that encapsidate a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particles in the IT composition. In some embodiments, the effective amount of an IT composition comprising AAV particles is about 10-fold lower than the effective amount of the same AAV particles, or an IV composition comprising AAV particles that encapsidate a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particles in the IT composition. In some embodiments, the effective amount of an IT composition comprising AAV particles is about 25-fold lower than the effective amount of the same AAV particles, or an IV composition comprising AAV particles that encapsidate a transgene encoding the same polypeptide as the transgene encapsidated by the AAV particles in the IT composition. In some embodiments, the effective amount of an IT composition comprising AAV particles is about 40 times lower than the effective amount of an IV composition comprising AAV particles that encapsidate the same AAV particles or a transgene that encodes the same polypeptide as the transgene encapsidated by the AAV particles in the IT composition. In some embodiments, the effective amount of an IT composition comprising AAV particles is about 50 times lower than the effective amount of an IV composition comprising AAV particles that encapsidate the same AAV particles or a transgene that encodes the same polypeptide as the transgene encapsidated by the AAV particles in the IT composition. In some embodiments, the effective amount of an IT composition comprising AAV particles is about 100 times lower than the effective amount of an IV composition comprising AAV particles that encapsidate the same AAV particles or a transgene that encodes the same polypeptide as the transgene encapsidated by the AAV particles in the IT composition. In some embodiments, the transgene is a μDys transgene.

[0119] In one embodiment of the methods of treatment provided herein, treating comprises decreasing serum creatine kinase (CK) levels in the subject compared to serum CK levels before treatment. In some embodiments, 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 before treatment. In further embodiments, serum CK levels are assessed before treatment with the AAV particles and about 12 months, about 18 months, about 24 months, or about 30 months after administration of the AAV particles. In further embodiments, the AAV particles comprise AAV9 capsids encapsulating a μDys transgene.

[0120] In another embodiment of the intrathecal treatment methods provided herein, treating comprises reducing the number of side effects or reducing the severity of one or more side effects in a subject being treated compared to a subject being treated via IV administration of an effective amount of the same AAV particles, or a different AAV particle that encapsidates a μDys transgene. The AAV particles administered intrathecally, in one embodiment, are AAV9 particles.

[0121] In some embodiments, the AAV particles of the invention, or pharmaceutical compositions comprising the same, can be used to treat dystrophinopathies, including, but not limited to, DMD, Becker muscular dystrophy, and DCM.

[0122] In some embodiments, the AAV particles of the invention are administered once or multiple times to a subject in need of treatment, such as a subject with DMD. In some embodiments, the AAV particles are administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more times to a subject in need of treatment. In a preferred embodiment, the intrathecal composition provided herein comprising AAV particles is administered once to a subject in need of treatment. In a further embodiment, the AAV particles are AAV9 particles.

[0123] In one embodiment, an effective amount of AAV particles comprising an AAV capsid encapsidating a μDys 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 administered a single dose of a composition comprising AAV particles intrathecally. In a further embodiment, the AAV particles are AAV9 particles. In yet a further embodiment, the subject is positioned in Trendelenburg position prior to administration.

[0124] In one embodiment of a method for treating DMD, a subject in need of treatment is administered a single dose intrathecally of a composition comprising an effective amount of AAV particles comprising an AAV capsid encapsulating a μDys transgene. The μDys transgene, in one embodiment, comprises a combination of dystrophin elements as set forth in FIG. 14. In one embodiment, the vector genome comprises a nucleic acid sequence as set forth in SEQ ID NO: 5, 10, 11, or 12.

[0125] In one embodiment of the method for treating DMD, treating comprises increasing the subject's score from baseline (i.e., before treatment) on the North Star Ambulatory Assessment (NSAA). The NSAA is a 17-item rating scale used to measure functional motor skills in ambulatory DMD subjects. The scale is ordinal, with 34 as the maximum score indicating fully independent function. Each activity is graded as either 0 (unable to accomplish independently), 1 (achieving a goal in a modified manner but without physical assistance from another person), or 2 (normal - no obvious modification of activity). See, for example, Mazonne et al. (2009). Neuromuscular Disorders 19, pp. 458-461, and researchrom.com / masterlist / view / 18#form2, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes. The change from baseline is measured, in one embodiment, 12 months after administration of the intrathecal composition. In another embodiment, the change from baseline is measured 18 months after administration of the intrathecal composition. In another embodiment, the change from baseline is measured 24 months after administration of the intrathecal composition. In yet another embodiment, the increase in the subject's score from baseline in the NSAA measured 12 months after administration is substantially unchanged or increases 18 months after administration. In yet another embodiment, the increase in the subject's score from baseline in the NSAA measured 12 months after administration is substantially unchanged or increases 24 months after administration. In yet another embodiment, the increase in the subject's score from baseline in the NSAA measured 12 months after administration is substantially unchanged or increases 60 months after administration.

[0126] Increasing the NSAA score, in one embodiment, includes increasing the NSAA score by about 5 to about 25, about 5 to about 20, about 5 to about 15, or about 5 to about 10. In another embodiment, increasing the NSAA score includes increasing the NSAA score by about 2 points to about 12 points. In another embodiment, increasing the NSAA score includes increasing the NSAA score by about 2 points to about 10 points. In yet another embodiment, increasing the NSAA score includes increasing the NSAA score by about 3 points to about 10 points. In yet another embodiment, increasing the NSAA score includes increasing the score by about 4 points to about 10 points. In yet another embodiment, increasing the NSAA score includes increasing the score by about 2 points to about 8 points. In another embodiment, increasing the NSAA score includes increasing the NSAA score by about 2 points to about 6 points.

[0127] In one embodiment of a method for treating DMD, a subject in need of treatment is administered a single dose intrathecally an effective amount of an AAV vector encapsulating a μDys transgene. The μDys transgene, in one embodiment, comprises a combination of dystrophin elements as set forth in FIG. 14. In one embodiment, the vector genome comprises a nucleic acid sequence as set forth in SEQ ID NO: 5, 10, 11, or 12. The effective amount of the AAV vector encapsulating a μDys 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 before treatment.

[0128] The AAV particles of the invention, or pharmaceutical compositions comprising the same, are administered as a single dose or as divided doses. In some embodiments, the dose is greater than or equal to 1×10 delivered as a single dose or as divided doses. 9 ~1×10 16 Vector genome (vg), 2.5 × 10 13 ~1×10 15 vg, 5×10 13 ~1×10 15 vg, 7.5×10 13 ~1×10 15 vg, 1×1014 ~1×10 15 vg, 2.5×10 14 ~1×10 15 vg, 5×10 14 ~1×10 15 vg, 7.5×10 14 ~1×10 15 vg, 1×10 13 ~7.5×10 14 vg, 2.5×10 13 ~7.5×10 14 vg, 5×10 13 ~7.5×10 14 vg, 7.5×10 13 ~7.5×10 14 vg, 1×10 13 ~5.0×10 14 vg, 2.5×10 13 ~5.0×10 14 vg, 5×10 13 ~5.0×10 14 vg, 7.5×10 13 ~5.0×10 14 vg, 1×10 13 ~2.5×10 14 vg, 2.5×10 13 ~2.5×10 14 vg, 5×10 13 ~2.5×10 14 vg, 7.5×10 13 ~2.5×10 14 vg, 1×10 13 ~1×10 14 vg, 2.5×10 13 ~1×10 14 vg, 5×10 13 ~1×10 14 vg, 7.5×10 13 ~1×10 14 For example, in some embodiments, the AAV particles of the invention or pharmaceutical compositions comprising the same are 2.5×10 13 In another embodiment, the AAV particles of the invention or pharmaceutical compositions comprising the same are administered as a single dose of 5×10 13 In yet another embodiment, the AAV particles of the invention or pharmaceutical compositions comprising the same are administered as a single dose of 1×10 14It is administered as a single dose of vg.

[0129] The AAV particles of the invention, or pharmaceutical compositions comprising the same, are administered as a single intrathecal dose. In some embodiments, the dose for intrathecal delivery is 1×10 delivered as a single dose or divided doses. 9 ~1×10 16 Vector genome (vg), 1 × 10 10 ~1×10 16 vg, 1×10 11 ~1×10 16 vg, 1×10 12 ~1×10 16 vg, 1×10 13 ~1×10 16 vg, 1×10 14 ~1×10 16 vg, 1×10 15 ~1×10 16 vg, 1×10 9 ~1×10 15 vg, 1×10 9 ~1×10 14 vg, 1×10 9 ~1×10 13 vg, 1×10 9 ~1×10 12 vg, 1×10 9 ~1×10 11 vg, 1×10 9 ~1×10 10 vg, 1×10 13 ~1×10 15 Vector vg, 2.5×10 13 ~1×10 15 vg, 5×10 13 ~1×10 15 vg, 7.5×10 13 ~1×10 15 vg, 1×10 14 ~1×10 15 vg, 2.5×10 14 ~1×10 15 vg, 5×10 14 ~1×10 15 vg, 7.5×10 14 ~1×10 15 vg, 1×10 13 ~7.5×10 14vg, 2.5×10 13 ~7.5×10 14 vg, 5×10 13 ~7.5×10 14 vg, 7.5×10 13 ~7.5×10 14 vg, 1×10 13 ~5.0×10 14 vg, 2.5×10 13 ~5.0×10 14 vg, 5×10 13 ~5.0×10 14 vg, 7.5×10 13 ~5.0×10 14 vg, 1×10 13 ~2.5×10 14 vg, 2.5×10 13 ~2.5×10 14 vg, 5×10 13 ~2.5×10 14 vg, 7.5×10 13 ~2.5×10 14 vg, 1×10 13 ~1×10 14 vg, 2.5×10 13 ~1×10 14 vg, 5×10 13 ~1×10 14 vg, 7.5×10 13 ~1×10 14 For example, in some embodiments, the AAV particles of the invention or pharmaceutical compositions comprising the same can contain up to 2.5×10 13 In another embodiment, the AAV particles of the invention or pharmaceutical compositions comprising the same can be administered as a single intrathecal dose of 5×10 vg. 13 In yet another embodiment, the AAV particles of the invention or pharmaceutical compositions comprising the same can be administered as a single intrathecal dose of 1×10 vg. 14 It can be administered as a single intrathecal dose of 100 mg / kg.

[0130] In other embodiments, the AAV particles of the invention or pharmaceutical compositions comprising the same can be administered as a single or divided intraventricular dose. In some embodiments, the dose for intraventricular delivery is 1×10 13 ~1×1015 vg, 2.5×10 13 ~1×10 15 vg, 5×10 13 ~1×10 15 vg, 7.5×10 13 ~1×10 15 vg, 1×10 14 ~1×10 15 vg, 2.5×10 14 ~1×10 15 vg, 5×10 14 ~1×10 15 vg, 7.5×10 14 ~1×10 15 vg, 1×10 13 ~7.5×10 14 vg, 2.5×10 13 ~7.5×10 14 vg, 5×10 13 ~7.5×10 14 vg, 7.5×10 13 ~7.5×10 14 vg, 1×10 13 ~5.0×10 14 vg, 2.5×10 13 ~5.0×10 14 vg, 5×10 13 ~5.0×10 14 vg, 7.5×10 13 ~5.0×10 14 vg, 1×10 13 ~2.5×10 14 vg, 2.5×10 13 ~2.5×10 14 vg, 5×10 13 ~2.5×10 14 vg, 7.5×10 13 ~2.5×10 14 vg, 1×10 13 ~1×10 14 vg, 2.5×10 13 ~1×10 14 vg, 5×10 13 ~1×10 14 vg, 7.5×10 13 ~1×10 14 For example, in some embodiments, the AAV particles of the invention or pharmaceutical compositions comprising the same can contain up to 2.5×10 13In another embodiment, the AAV particles of the invention or pharmaceutical compositions comprising the same can be administered as a single intracerebroventricular dose of 5×10 vg. 13 In yet another embodiment, the AAV particles of the invention or pharmaceutical compositions comprising the same can be administered as a single intracerebroventricular dose of 1×10 vg. 14 It can be administered as a single intracerebroventricular dose of 100 mg / kg. EXAMPLES

[0131] The invention generally described herein may be more readily understood by reference to the following examples, which are included solely for purposes of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0132] Selected Methods Intracerebroventricular (ICV) injection on postnatal day 1 (p1) Perform intracerebroventricular injections (ICV) of newborn mice (p1) via the cerebral hemisphere. Newborn mice are injected fully awake. Ensure that the needle is marked for an injection depth of 2 mm. 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 to the neonatal anterior). P0 is designated as the day of birth (DoB) of the mouse. Animals can be injected within 36 h of finding the littermates.

[0133] If an excessive amount of dosing solution is determined to have leaked from the injection site or if the injection site is completely missed, the animal will be removed from the study.

[0134] Intracerebroventricular (ICV) injection at postnatal day 28 (p28) A Hamilton syringe is loaded with the desired volume of dosing solution. Standard volume is 8 μl per injection site. Animals are individually removed from their cages and placed into the anesthesia chamber. Once in the chamber, the animals are anesthetized. Tubing is connected from the anesthesia machine to the stereotaxic apparatus to allow for continued anesthesia during injection. Each animal remains in the chamber for approximately 2 minutes before being removed and placed on the stereotaxic apparatus. Once the animals are placed on the stereotaxic apparatus, the coordinates for injection are set as follows: medial / lateral (M / L): + / - 1.00 mm, anterior / posterior (A / P): -0.5 to -0.8 mm, dorsal / ventral (D / F): -2.5 mm.

[0135] An iodine swab is applied to the incision site on the animal's scalp for sterilization purposes. Using a scalpel, a small incision is made in the animal's scalp and the scalp is gently peeled back to expose the cranial region. Once the needle is in the desired location, the syringe plunger is slowly depressed to inject the dosing solution into the cranial cavity. The injection site is monitored during and immediately after injection to ensure the quality of the injection.

[0136] Muscle preparation When animals reach the appropriate age, they are weighed and anesthetized via intraperitoneal (ip) injection (ketamine [80 mg / kg], acepromazine [0.5 mg / kg], and xylazine [16 mg / kg]). Tissue dissection is then performed. Scissors are used to cut the skin at the ankle, and then the skin on both legs is pulled back to expose the calf and talus muscles. One side of the tibialis anterior muscle is dissected near its insertion point, weighed to the nearest 0.1 mg, and then discarded. A 4.0 suture is then tied at the myotendinous junction of the proximal and distal ends of the extensor digitorum longus (EDL), which is then released and placed in Ringer's solution (137 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgSO4, 1 mm NaH2PO4, 24 mm NaHCO3, 11 mm glucose with 10 mg / liter curare) which is kept at room temperature.

[0137] After dissection of the EDL, the abdomen is opened and blood is collected from the inferior vena cava (approximately 300-500 μL) using a 1 cc syringe. Blood is allowed to sit at room temperature for 25-35 minutes and then centrifuged at 3,500 x g for 10 minutes at 4°C. After spinning down the supernatant, serum is isolated, placed in microcentrifuge tubes, and frozen at -80°C.

[0138] The Achilles tendon is then cut and pulled posteriorly to expose the soleus, plantaris, and gastrocnemius muscles. The soleus muscle is dissected, tubed, and frozen in liquid nitrogen-cooled isopentane. The plantaris muscle is bluntly dissected free of the gastrocnemius muscle, then cut as proximally as possible, tubed, frozen in liquid nitrogen-cooled isopentane, and stored at -80°C.

[0139] The gastrocnemius muscle is cut as proximally as possible, weighed to the nearest 0.1 mg, placed in a tube, and frozen. The tibialis anterior muscle from the opposite side is dissected free, weighed to the nearest 0.1 mg, and pinned on a cork at resting length. The EDL from the same side is also 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, wrapped in foil, and stored at -80°C. The quadriceps muscle is dissected, placed in a tube, frozen in liquid nitrogen-cooled isopentane, and stored at -80°C. A small piece of liver is dissected, placed in a tube, frozen in liquid nitrogen-cooled isopentane, and stored at -80°C. The diaphragm is dissected, folded in half, then folded again, then placed on a cork, and pinned. The cork is then immersed in liquid nitrogen-cooled isopentane for 30-45 seconds, after which the cork is placed on dry ice, wrapped in foil, and stored at -80°C.

[0140] Whole hearts are dissected, weighed to the nearest 0.1 mg, placed into tubes, and frozen in liquid nitrogen-cooled isopentane. The tubes are capped and placed into liquid nitrogen until storage at -80°C.

[0141] Example 1: Generation of recombinant adeno-associated virus particles Molecular cloning of microdystrophin gene constructs A gene construct encoding microdystrophin (μDys), referred to herein as INS1201 and previously known as MTS-001, was synthesized by operably linking the MHCK7 promoter and SV40 intron to a polynucleotide encoding μDys and an SV40 poly(A) signal (FIG. 1A). An alternative gene construct encoding μDys (referred to herein as INS1212 and previously known as MTS-003) was synthesized by operably linking the SK-CRM4 enhancer containing the MHCK7 promoter to a polynucleotide encoding an SV40 poly(A) signal (FIG. 1B). The INS1201 and INS1212 gene constructs were generated in a Puc57 vector backbone. The INS1201 and INS1212 constructs were confirmed by DNA sequencing, and the 4542bp and 4714 constructs, respectively, were isolated by NruI restriction digestion and gel purified for subsequent cloning into the appropriate AAV backbone.

[0142] The isolated INS1201 and INS1212 gene constructs were each independently blunt cloned into gel purified pSZ01 vector backbone containing the ITR sites and the kanamycin resistance gene linearized / isolated by NruI restriction digestion. After T4 ligation of the INS1212 construct with the pSZ01 vector backbone and the INS1212 construct with the pSZ01 vector backbone, the DNA was transformed into E. coli, grown, and purified using the NEB Monarch® Plasmid Miniprep kit. Gene constructs that were successfully ligated to produce complete pSZ01-INS1201 or psZ01-INS1212 plasmid clones were identified by HindIII / BsaI restriction digestion.

[0143] The pSZ01-INS1201 and psZ01-INS1212 clones were expanded by bacterial transformation in E. coli using a Maxi-Prep kit (GeneJET Endo-free Plasmid Maxiprep Kit, ThermoScientific). The correct plasmid sequence was reconfirmed by BsaI / HindIII digestion (Figure 1C, lanes 1 and 3). In addition, restriction digestion with SmaI (Figure 1C, lanes 2 and 4) was performed as further confirmation of correct integration of the ITR sites containing INS1201 and INS1212 into the pSZ01 vector backbone. Transient transfection and viral packaging

[0144] Validated pSZ01-INS1201 and psZ01-INS1212 AAV vectors were transiently transfected into HEK293 cells using standard calcium phosphate transfection methods (e.g., as described in Vandendriessche et al. (2007. J Thromb Haemost 5:16-24), incorporated herein by reference in its entirety) in combination with adenovirus helper plasmids and a chimeric packaging construct delivering the AAV2 rep gene along with the AAV9 cap gene. Two days after transfection, AAV particles were harvested and purified using two successive rounds of cesium chloride density gradient ultracentrifugation. 1×10 6 cells were resolved by SDS-PAGE and silver stained. 13 1 μl each of purified INS1201-AAV9 (Figure 2, lane 1) and INS1212-AAV9 (Figure 2, lane 2) were titrated by comparison with vg AAV2 standards (Figure 2, lane 3 (0.5 μl), lane 4 (1 μl), lane 5 (2 μl), lane 6 (4 μl)).

[0145] Example 2: Intramuscular delivery of AAV9 μDys (INS1201-AAV9 and INS1212-AAV9) results in increased μDys expression in MTX mice. INS1201-AAV9 and INS1212-AAV9 were injected intramuscularly into the gastrocnemius muscle of mdx mice, a common mouse model of Duchenne muscular dystrophy (see, e.g., Rodino-Klapac et al. (2013) Hum Mol Genet. 22(24):4929-37, incorporated herein by reference). As shown in Figure 3A, 21 days after intramuscular injection, 2.7 x 10 11 Gastrocnemius muscles injected with INS1201-AAV9 (iii) or INS1212-AAV9 (iv) in the vg showed widespread expression of μDy at levels significantly higher than uninjected mdx mice (i) and at levels comparable to wild-type C57 / Bl mice (ii). Figure 3B also shows that 2.7 × 10 11 13 shows high level expression of μDys in mdx mice 21 days after intramuscular injection with INS1212-AAV9 of vg.

[0146] Example 3: Intraventricular delivery of AAV9 μDys (INS1201-AAV9 and INS1212-AAV9) results in increased μDys expression in MDX mice. INS1201-AAV9 was injected intracerebroventricularly into mdx mice on postnatal day 1 (p1), and tissue samples were collected and analyzed for dystrophin. As shown in Figure 4A, 1.8 × 10 11 Twenty-one days after intracerebroventricular (ICV) injection with vg AAV, INS1201-AAV9 efficiently targeted expression of μDys in gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteal (iv), triceps (v), diaphragm (vi), and heart (vii) myocytes of mdx animals, with little or no expression in liver (viii). Similarly, as shown in Figure 4B, 9 × 10 10 Twenty-one days after ICV injection with vg AAV, INS1201-AAV9 efficiently targeted expression of μDys in myocytes of the gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteal (iv), triceps (v), diaphragm (vi), and heart (vii), with little or no expression in the liver (viii).

[0147] INS1212-AAV9 was also injected intracerebroventricularly into mdx mice at p1, and tissue samples were collected and immunofluorescently stained for dystrophin. As shown in Figure 5, 9 × 10 10 Twenty-one days after ICV injection with vg AAV, INS1212-AAV9 efficiently targeted expression of μDys in myocytes of the gastrocnemius (i), tibialis anterior (ii), quadriceps (iii), gluteal (iv), triceps (v), diaphragm (vi), and heart (vii), with little or no expression in the liver (viii).

[0148] As shown in the hematoxylin and eosin stained sample in Figure 6A, 9 x 10 10 vg(ii) and 2.7 × 10 11 Eighty days after ICV injection with INS1201-AAV9 in vg (iii), gastrocnemius tissue showed restoration of normal tissue architecture and correction of histopathological features of Duchenne muscular dystrophy compared to wild-type C57 / Bl (i) and non-injected mdx mouse controls (iv). As shown in Figure 6B, 9 × 10 10 vg(ii) and 2.7 × 10 11 At 80 days after ICV injection with INS1201-AAV9 in vg (iii), gastrocnemius muscle tissue showed levels of μDys that were comparable to dystrophin levels in wild-type C57 / Bl mice (i) and significantly higher than dystrophin levels in uninjected mdx mice (iv).

[0149] Similarly, as shown in the hematoxylin and eosin stained sample in FIG. 7A(ii), 9×10 10 Eighty days after ICV injection with INS1212-AAV9 vg, gastrocnemius tissue showed restoration of normal tissue architecture and correction of histopathological features of Duchenne muscular dystrophy compared to wild-type C57 / Bl (Figure 7A(i)) and non-injected mdx mouse controls (Figure 7A(iii)). As shown in Figure 7B(ii), 9 × 10 10At 80 days after ICV injection with INS1212-AAV9 in vg, gastrocnemius muscle tissue showed levels of μDys that were comparable to dystrophin levels in wild-type C57 / Bl mice (Figure 7B(i)) and significantly higher than dystrophin levels in uninjected mdx mice (Figure 7A(iii)).

[0150] As shown in Figure 8, 9 × 10 10 vg or 2.7×10 11 Gastrocnemius (Figure 8A), triceps (Figure 8C), tibialis anterior (Figure 8E), and diaphragm (Figure 8F) muscle tissue in mdx mice 80 days after ICV injection with INS1201-AAV9 in vg showed an increase in mean fiber diameter compared to non-injected mdx mice. 9 × 10 10 vg or 2.7×10 11 Mdx mice intracerebroventricularly injected with INS1201-AAV9 in vg also showed an increased frequency of cells with larger diameters (e.g., 25 µm to 60 µm) 80 days after injection in the gastrocnemius (Figure 8B), triceps (Figure 8D), tibialis anterior (Figure 8F), and diaphragm (Figure 8H) compared to non-injected mice.

[0151] Similarly, as shown in FIG. 9A, gastrocnemius tissue was cultured at 9×10 10 Gastrocnemius tissue in mdx mice 80 days after ICV injection with INS1212-AAV9 in vg mice showed an increase in mean fiber diameter compared to non-injected mdx mice, with a concomitant increase in the frequency of cells with larger diameters (e.g., 25 µm to 60 µm) (Figure 9B).

[0152] Example 4: Intraventricular delivery of INS1201-AAV9 results in increased μDys expression, improved muscle histology, and reduced fibrosis in mdx mice. Postnatal day 28 (p28) mdx mice (with a time window of -1 and +7 days such that no animal was younger than P27 and no animal older than P35 at the time of injection) were administered one of the following treatments via intracerebroventricular (ICV) injection: (i) 9 × 10 9 vg INS1201-AAV9 (n = 6), (ii) 9 × 10 10vg INS1201-AAV9 (n = 7), (iii) 2.7 × 10 11 vg INS1201-AAV9 (n = 10), (iv) 5.4 × 10 11 vg INS1201-AAV9 (n = 7), (v) 1.2 × 10 12 vg INS1201-AAV9 (n=8), or (vi) vehicle control (TFF formulation buffer, n=11). C57 / BL1 age-matched mice were used as wild-type (WT) controls (n=10).

[0153] Tissues were dissociated as described above at approximately postnatal day 120.

[0154] INS1201 copies per diploid genome were measured via droplet digital polymerase chain reaction (ddPCR) using primers specific for the INS1201 transgene. INS1201 DNA copies 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.

[0155] As shown in Figure 17, mdx mice administered INS1201-AAV9 at all doses tested showed an increase in mean EDL fiber diameter compared to non-injected mdx mice. Consistent with these findings, mdx mice intracerebroventricularly injected with INS1201-AAV9 at all doses also showed an increased frequency of cells with larger diameters (e.g., 25 μm to 60 μm) in the EDL muscle compared to non-injected mice.

[0156] As shown in Figure 18, mdx mice administered INS1201-AAV9 at the four highest doses tested showed an increase in mean TA fiber diameter compared to non-injected mdx mice. Consistent with these findings, the highest dose of the four tested (9x10 10 vg, 2.7×10 11 vg, 5.4×10 11 vg, 1.2×10 12vg), mdx mice intracerebroventricularly injected with INS1201-AAV9 showed an increased frequency of cells with larger diameters in the TA muscle compared to non-injected mice.

[0157] FIG. 19 shows diaphragm muscle sections stained with picrosirius red after intracerebroventricular (ICV) injection with various doses of INS1201-AAV9. Picrosirius red is used to visualize collagen content. Diaphragm sections obtained from wild-type C57 / Bl mice and mdx mice administered vehicle are shown for comparison (top panel). As shown in the bottom panel of FIG. 19, the five doses tested (9×10 9 vg, 9×10 10 vg, 2.7×10 11 vg, 5.4×10 11 vg, 1.2×10 12 Mdx mice administered INS1201-AAV9 (vg) showed reduced fibrosis as measured by collagen content compared to mdx mice administered vehicle. Collagen percentage in diaphragm muscle is also provided in FIG. 20.

[0158] The upper part of Figure 21 shows the 5.4 × 10 11Shown are EDL sections taken from mdx mice at p120 after intracerebroventricular (ICV) injection with INS1201-AAV9 at 100 mg / kg and stained with hematoxylin and eosin (H&E) (far left), laminin / dapi (second from left), dystrophin (second from right), and merged images (far right). The bottom of FIG. 21 shows EDL muscle sections taken from mdx mice at p120 after intracerebroventricular (ICV) injection with vehicle control at postnatal day 28 (p28) and stained with hematoxylin and eosin (H&E) (far left), laminin / dapi (second from left), dystrophin (second from right), and merged images (far right). Staining shows greater dystrophin expression in samples taken from mice treated with INS1201-AAV9 compared to samples taken from control mice. Muscles from INS1201-AAV9 treated mice also appear healthier, as evident from H&E and laminin / dapi staining.

[0159] Example 5: ICV delivery of INS-1201 AAV9 results in improved muscle physiology in mdx mice. Postnatal day 1 (p1) mdx mice were administered one of the following treatments via intracerebroventricular (ICV) injection: (i) 9 × 10 9 (ii) 9 × 10 INS1201-AAV9 in vg 10 vg INS-1201-AAV9, (iii) 2.7 × 10 11 vg INS1201-AAV9, (iv) vehicle control (TFF formulation buffer). C57 / BL10 age-matched mice (WT) were used as controls.

[0160] Postnatal day 28 (p28) mdx mice (with a time window of -1 and +7 days such that no animal was younger than P27 and no animal older than P35 at the time of injection) were administered one of the following treatments via intracerebroventricular (ICV) injection: (i) 9 × 10 9 (ii) 9 × 10 INS1201-AAV9 in vg 10 vg INS1201-AAV9, (iii) 2.7 × 10 11vg INS1201-AAV9, (iv) 5.4 × 10 11 INS1201-AAV9 in vg, (v) 1.2 × 10 12 vg of INS1201-AAV9, or (vi) vehicle control (TFF formulation buffer). C57 / BL1 age-matched mice were used as wild-type (WT) controls.

[0161] When the animals were 120-135 days old, muscle dissection and preparation were performed as described above.

[0162] Muscle Physiology Experimental Design Muscle mechanics in the EDL The EDL is mounted in a specialized chamber containing Ringer's solution (137 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgSO4, 1 mm NaH2PO4, 24 mm NaHCO3, 11 mm glucose with 10 mg / liter curare) at room temperature. The muscle origin is tethered to a rigid post and the attachment is fixed to the arm of a dual-mode ergometer (model 300B, Aurora Scientific, ON, Canada), which allows precise control of muscle length.

[0163] Muscle activation is provided via an electrical stimulator using parallel platinum plate electrodes that lengthen the muscle. Supramaximal stimulation conditions are established for each experiment by single 0.3 ms contraction pulses of increasing voltage using a value +50% greater than the value at which force reached a plateau for the experimental trial. The optimal muscle length for assessing force characteristics is determined using a series of contractions with progressively increasing muscle length (10% increments from the relaxed length) and is defined as the length at which supramaximal stimulation produces maximum contraction force. After establishing the optimal length, muscle fiber length (L f ) is measured and the muscle is allowed to rest for 2 minutes.

[0164] The muscle is then stimulated with two contractions 60 seconds apart (0.3 ms pulse duration) to assess the contractile properties (contractile tension, half-relaxation time, and time to peak tension).

[0165] Muscles are stimulated at increasing frequencies (pulse train duration of 400 ms and pulse duration of 0.3 ms; (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 (FF) relationship.

[0166] After the FF test, the optimal length is re-verified by experimentally testing the maximum contractile force up to ±20% of the length at the end of the test.

[0167] Eccentric Contraction After completing the FF curve, all stretches or contractions were performed at 2 min intervals, and stimulation was performed at 100 Hz with a pulse train duration of 400 ms and a pulse duration of 0.3 ms for isometric and eccentric contractions. Specifically, after a 2 min rest period from the end of the FF assessment, stimulation was performed at 10% L at 0.7 Lf / s with a 500 ms hold before returning to the starting muscle length. f The passive mechanical properties of the muscle are measured by applying two stretches, then two isometric contractions are taken as a measure of maximum isometric force before the eccentric contraction (EC).

[0168] Each muscle is then subjected to an EC bout of 10 contractions during which the muscle is isometrically stimulated for the first 200 ms, after which a 15% Lf length change is applied at a rate of 2 Lf / sec. The muscle is then held at this length for 500 ms before returning to the starting muscle length. After the 10 EC contractions, two isometric contractions are taken to determine the maximum isometric force after EC. Upon completion of the final isometric measurement, two additional passive stretches are taken to determine the passive mechanical measurement after EC.

[0169] After contraction testing, muscles are dissected from the outer tendons, blotted dry, and weighed to the nearest 0.01 mg. Muscle physiological cross-sectional area is calculated as a function of fiber length and muscle mass. For all analyses, force is normalized to muscle physiological cross-sectional area and reported as stress.

[0170] All doses tested (9 × 10 9 vg, 9×10 10 vg, 2.7×10 11 Mdx mice receiving ICV injections of INS1201-AAV9 at 100 mg / kg / day (p1) showed an attenuated decrease in contractile force rate emanating from EC compared to vehicle-treated mdx mice (Figure 10A). Figure 10B similarly shows that INS1201-AAV9 at the three doses tested at p1 (9 × 10 9 vg, 9×10 10 vg, 2.7×10 11 vg) mice receiving ICV injections of INS1201-AAV9 showed increased muscle physiology as indicated by an increased ratio of post-eccentric stress to pre-eccentric stress compared to vehicle-treated mice.

[0171] The highest dose of the three tested (2.7 × 10 11 vg, 5.4×10 11 vg, 1.2×10 12 vg) mdx mice receiving ICV injections of INS1201-AAV9 showed an attenuated decrease in contractile force rate emanating from ECs compared to vehicle-treated mdx mice (Figures 10C, 10D).

[0172] The highest dose of the three tested (2.7 × 10 11 vg, 5.4×10 11 vg, 1.2×10 12 Mdx mice receiving ICV injections of INS1201-AAV9 at 100-200 ng / mL (vg) showed improved and stabilised muscle function as indicated by increased peak muscle force following stimulation at various frequencies compared to vehicle-treated mdx mice (Figures 10E, 10F).

[0173] Example 6: Single-dose intrathecal administration of AAV9 targets transgene delivery to skeletal and cardiac tissues AAV9-CBA-GFP was administered intrathecally to cynomolgus monkeys that were screened for anti-AAV9 antibodies using a highly specific anti-AAV9 ELISA. Cynomolgus monkey subjects received 2.5 × 10 13 vg, 5×10 13 vg, or 1×10 14 vg were treated with AAV9-CBA-GFP, and GFP expression was determined by immunohistochemistry using NovaRed GFP immunostaining and Vector® HRP substrate, RT-PCR, and Western blot analysis.

[0174] As shown in Figure 11, 2.5 x 10 13 vg(iv), 5 × 10 13 vg(v), or 1×10 14 Cynomolgus monkeys that received a single intrathecal dose of AAV9-CBA-GFP in vg(vi) were administered 5 × 10 13 vg(ii) or 1 × 10 14 Compared to cynomolgus controls receiving AAV9-CBA-GFP in vg(iii), the muscle tissues showed increased GFP staining in the gastrocnemius ( FIG. 11A ), quadriceps ( FIG. 11B ), deltoid ( FIG. 11C ), triceps ( FIG. 11D ), and biceps ( FIG. 11E ). 13 vg(iii), 5 × 10 13 vg(iv), or 1 × 10 14 Cynomolgus monkeys receiving a single intrathecal injection of AAV9-CBA-GFP in vg(v) were treated with 5 × 10 13 vg(i) or 1×10 14 Compared to cynomolgus subjects that received AAV9-CBA-GFP in vg(ii), they showed increased GFP staining in muscle tissues of the diaphragm (FIG. 11F), tibialis anterior (FIG. 11G), and heart (FIG. 11H). As shown in FIGS. 11A-E(i), non-injected cynomolgus subjects showed little or no GFP staining.

[0175] As shown in Figure 11I, 5 × 1013 vg(i), or 1×10 14 Cynomolgus monkeys receiving a single intravenous dose of AAV9-CBA-GFP in vg(ii) showed high levels of GFP staining in liver tissue, whereas 2.5 × 10 13 vg(iii), 5 × 10 13 vg(iv), or 1 × 10 14 Subjects who received a single intrathecal dose of AAV9-CBA-GFP in vg(v) showed significantly less GFP staining.

[0176] As shown in Figure 12, the immunohistochemical staining of GFP shown in Figure 11 corresponded to the protein levels detected by anti-GFP Western blot. 13 vg (Figure 12A), 5 × 10 13 vg (Figure 12B), or 1 x 10 14 Cynomolgus monkeys receiving a single intrathecal injection of AAV9-CBA-GFP in the vg (Figure 12C) showed increased GFP levels in biceps (1), triceps (2), deltoid (3), quadriceps (4), gastrocnemius (5), tibialis anterior (6), diaphragm (7), and heart (8) muscle tissues, whereas no GFP protein was detected by Western blot in the biceps (Figure 12D, 1) or triceps (Figure 12D, 2) of uninjected controls.

[0177] As shown in Figure 13, GFP protein levels shown in Figures 11 and 12 correspond to GFP mRNA levels, and cynomolgus monkeys receiving a single intrathecal dose of AAV9-CBA-GFP showed detectable GFP mRNA expression in biceps (1), triceps (2), deltoid (3), tibialis anterior (4), gastrocnemius (5), vastus lateralis of quadriceps (6), diaphragm (7), and heart (8) muscle tissues analyzed by RT-PCR. Minimal GFP mRNA was detected in liver tissue (9), and GFP mRNA was not detectable in biceps (10), triceps (11), deltoid (12), and quadriceps (13) muscle tissues collected from non-injected subjects.

[0178] Example 7: Toxicity and biodistribution studies of INS1201-AAV9 following a single dose of intracerebroventricular injection in young C57BL / 6J mice Testing Systems Species: Mus musculus Strain: C57BL / 6J Gender: Male Age: Approximately 4 weeks old on day 1 Weight: appropriate for age · Number: 192 (+60 extra) Caging: Compliant with ASC SOP · Minimum acclimatization: 5 days

[0179] Species / strain, number, sex. Up to 252 male C57BL / 6J (strain no. 000664) mice approximately 4 weeks old at the start of the study will be obtained for this study. Only male mice will be used in the study as the intended patient population is exclusively male.

[0180] Starting age and weight range. Animals selected for use in this study will be as uniform in age and weight as possible at the start of the study. Animals will be approximately 3 weeks old at delivery and approximately 4 weeks old on day 1 of the study.

[0181] The animals will be fed a species-specific diet and 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.

[0182] The test and control articles used in this study are provided in Tables 2 and 3, respectively. [Table 1] [Table 2]

[0183] Experimental design research design The study consists of three cohorts: animals assigned to cohort 1 will be sacrificed 85 ± 10 days after the injection procedure, animals assigned to cohort 2 will be sacrificed 43 ± 7 days after the injection procedure, and cohort 3 will be sacrificed 22 ± 3 days after the injection procedure. The overall study design is presented in Table 4. Extra animals will be dosed and may be used to replace any unscheduled deaths of study group animals that occur as a direct result of the procedure activities (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 before day 4 (3 days after injection) may be replaced. These animals will not undergo gross necropsy. Animals that do not meet the age inclusion criteria may be replaced as necessary. All animal deaths will be reported regardless of the timing of death after injection. For groups 1-4 at each given time point, 5 animals are used for hematology, blood ddPCR, and tissue ddPCR, 5 animals are used for clinical chemistry and histopathology, and 5 animals are used for coagulation testing and histopathology (n=15 / group / time point). All group 5 animals are euthanized at week 12 for hematology, clinical chemistry, and coagulation (n=4 for each test). Five animals from group 5 are used for histopathology, while tissues from the remaining animals are collected and stored. [Table 3]

[0184] End of Cohort 1 - Day 85 ± 10. For cohort 1 of the study, 60 animals designated as groups 1-4 will receive an intracerebroventricular (ICV) injection on day 1 based on their group assignment (Table 1). An additional 12 animals in cohort 1 are part of group 5 animals ("Naive / Untreated", Table 1) that will not be injected. On day 85 ± 10 of the study, animals within all groups 1-5 will be sacrificed and blood and tissue samples will be collected as described in Table 5. [Table 4]

[0185] Cohort 2 - Termination on Day 43±7. For cohort 2, 60 animals will receive an ICV injection on day 1 based on their group assignment (Table 1). On day 43±7 of the study, animals will be sacrificed and blood and tissue samples will be collected as described in Table 6. [Table 5]

[0186] End of Cohort 3 - Day 22±3. For Cohort 3, 60 animals will receive an ICV injection on day 1 based on their group assignment (Table 1). On day 22±3 of the study, animals will be sacrificed and blood and tissue samples will be collected as described in Table 7. [Table 6]

[0187] Group Assignment. Animals are ordered and assigned to test groups in three separate cohorts per Tables 5, 6, and 7. Each cohort of animals, including up to 12 extra animals per group, is weighed and assigned a numerical rank from 1 to X in decreasing order according to body weight (heaviest animals are assigned rank = 1). Animals are then assigned sequentially to each test group based on the termination cohort, per study design (see Tables 5, 6, 7).

[0188] Controls for bias. All trials will be conducted to minimize potential study bias, including (a) inclusion of an appropriate control group, (b) randomized assignment of animals to study groups, and (c) appropriate alternating dosing of animals across groups.

[0189] Test substance administration and study procedures Anesthesia and surgical preparation. Animals are anesthetized using inhalation anesthesia (1-5% isoflurane with 100% oxygen for induction, 1-3% for maintenance during the surgical procedure). Alternatively, animals are anesthetized with a ketamine (up to 80 mg / kg) and xylazine (up to 12 mg / kg) cocktail administered intraperitoneally.

[0190] 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), vehicle and test article will be administered to all animals via intracerebroventricular stereotaxic injection based on their group assignment. Test and control articles will be kept on ice or at 2-8 °C until ready for use.

[0191] Cohort injection strategy. Injections for each termination date cohort are performed over a 3-4 day period. On each individual injection day, approximately 15-30 mice are injected. Given the test article dose formulation, on day 1 of each cohort, injections of all mice at dose 1 are completed, followed by vehicle injections to a subset of animals in the first group, as time permits. On day 2 of each cohort, injections of all dose 2 mice are completed, followed by vehicle injections to a second subset of animals, as time permits to the injection team. On day 3 of each cohort, injections of all dose 3 mice are completed, followed by vehicle injections to a third subset of animals, as time permits to the injection team. If not all vehicle-injected mice have been injected over the first 3 days, the remaining vehicle injections are performed on day 4.

[0192] Treatment. Treatment is administered to the animals by direct injection of treatment into the lateral ventricles according to Tables 5, 6, and 7. Each animal receives either one unilateral injection or two injections (one injection per side / bilateral) in a single surgical procedure depending on the dosing group. Surgical procedures are performed using stereotaxic equipment. Injection equipment, surgical instruments, drapes, and gowns are sterilized where appropriate. Modifications to the procedures described below may be performed at the surgeon's discretion. A single dose of meloxicam (1 mg / kg, SC / IM) and / or buprenorphine (0.01-0.05 mg / kg SC) is administered after induction of anesthesia prior to skull incision to help alleviate pain from surgery. Once the animal is anesthetized, the skin over the skull is scraped (if necessary) and the animal is mounted in a stereotaxic frame and maintained on a nose cone for anesthesia with the head positioned by use of ear bars and incisor bars as appropriate. Aseptic technique is used for all surgical procedures. The skin is disinfected with a betadine solution followed by a 70% alcohol wipe or equivalent.

[0193] An incision of approximately 2 cm is made in the midline on the skull. Electrocautery may be used to achieve hemostasis of any slight bleeding from the incision site. A Hamilton 2 inch, 26 gauge (or smaller), Hamilton syringe with a 12 degree bevel stainless steel needle is attached to the Z axis of the stereotaxic apparatus. The following approximate stereotaxic coordinates based on the previous section are used to target the lateral ventricles on either one or both sides. Injection coordinates: o Front and back (AP): -0.5~-0.8mm o Medial and lateral (ML):+ / -1.0mm* o When administering a unilateral injection, the injection is into the right side of the brain. When administering a bilateral injection, the first injection is into the right side and the second injection into the left side. o Dorsal ventral (DV):-2.5mm

[0194] The actual location of the injection may be adjusted by the surgeon if necessary. If the coordinates differ from those listed above, they will be recorded in the raw data surgical record.

[0195] Once the needle is in the desired location, the syringe plunger is slowly depressed to inject the dosing solution into the cranial cavity. The injection site is monitored during and immediately after injection to ensure the quality of the injection.

[0196] The desired injection volume is 8 μL per injection site. Injections target the lateral ventricle and include one or two injections total. The needle is left in place for approximately one minute after delivering the treatment to prevent backflow. Any injection abnormalities (leakage, backflow, etc.) are noted on the injection form of the animal being injected. After completion of the injection and removal of the needle, the incision may be closed using VetBond™ surgical adhesive and reinforced with sutures. Minor modifications of the surgical procedure based on the real-time condition of the animal may be made and will not be considered deviations from the study protocol.

[0197] Post-operative care. Following the surgical procedure, animals are maintained on heat support throughout recovery, positioned in alternating lateral recumbency as necessary. Mice are administered sterile saline subcutaneously (approximately 0.5-1 mL). Animals may also be provided with their chow and Diet-Gel on the cage floor as supplemental feed during their surgical recovery. In-life observations and measurements

[0198] Routine General Health Observations. Animals will be observed by animal care staff for changes in general appearance, behavior, and signs of disease during the acclimation period 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 its designated end date. Any animals exhibiting non-normal clinical signs prior to Day 1 will be excluded from the study.

[0199] Pre-Study Clinical Observations All animals will undergo a thorough examination for clinical signs prior to Day 1. Clinical signs indicating ill health, stress, or other abnormalities will be noted and animals may be removed from the study at the discretion of the Investigator / Attending Veterinarian and Sponsor Representative.

[0200] Clinical Observations. All study animals undergo detailed clinical observations along with body weights for 6-10 days post-surgery and then weekly thereafter until scheduled termination. Observations noted outside of scheduled observations will be entered as unscheduled. Evaluations include, but are not limited to, locomotor activity, neurological observations, posture, respiration, hydration status, surgical site, stereotypic behavior, bladder and bowel (feces) observations, and evaluation of overall physical condition.

[0201] The absence or presence of findings during scheduled clinical observations will be recorded. Clinical signs indicating ill health, stress, and pain will be noted and reported to the attending veterinarian. A final detailed examination will be performed by the attending veterinarian on any animals that are euthanized emergently.

[0202] Body Weights. Individual body weights are recorded for initial group assignment immediately upon arrival of the animals. Baseline body weights are obtained within 4 days prior to dosing. Body weights are performed weekly until scheduled termination (occurring simultaneously with clinical observations) with final body weights taken prior to scheduled termination. At the discretion of the Investigator, body weights may be taken more frequently if signs of adverse health or weight loss (i.e., 10% or more of the previous week's body weight) are observed.

[0203] Scheduled Sacrifice. Animals surviving to their scheduled termination date, study days 22±3, 43±7, and 85±10 will be humanely euthanized, necropsied, and blood and tissue samples collected as described below. Animals will be euthanized according to the AVMA Guidelines for Euthanasia of Animals: 2020 Edition.

[0204] Sample / Specimen Collection Blood Collection. Prior to necropsy, animals may be transferred to an anesthesia box and anesthetized using isoflurane (1-2%) for blood collection for hematology and droplet digital polymerase chain reaction (ddPCR), serum chemistry, and coagulation. An appropriate amount of whole blood will be collected from each animal via cardiac puncture or other appropriate vein using a 25 g needle and 1 cc syringe, or similar. Blood samples will be collected from all study animals on their scheduled termination date. Blood will be processed per ASC SOP and sent to QVL for analysis.

[0205] Samples for Hematology and ddPCR For hematology, approximately 250-500 µL of whole blood is placed into a vial containing K2EDTA as an anticoagulant, gently inverted several times to mix, and placed on wet ice until storage in a refrigerator set to maintain 2 °C-8 °C.

[0206] hematology Collection volume: Approximately 250-500μL Anticoagulant: K2EDTA

[0207] The hematology parameters analyzed are provided in Table 8. [Table 7]

[0208] Biodistribution by digital droplet polymerase chain reaction (ddPCR). In addition, approximately 250-500 μL of whole blood is collected for biodistribution by ddPCR analysis. Whole blood is placed into K2EDTA as an anticoagulant, gently inverted several times to mix, and placed on dry ice until storage at -80°C ± 10°C.

[0209] Samples for Clinical Chemistry. For clinical chemistry, approximately 500-1000 μL of whole blood is collected into tubes without anticoagulant and allowed to clot for at least 30 minutes at room temperature before centrifugation. The clotted whole blood is centrifuged at approximately 3000×g for 5 minutes at a temperature of 4° C. to produce serum. Serum samples are separated after centrifugation, frozen on dry ice immediately after collection, and stored frozen at -80° C.±10° C.

[0210] The serum chemistry parameters analyzed are provided in Table 9. [Table 8]

[0211] Samples for clotting: Plasma samples are separated after centrifugation and stored in a freezer set to maintain -10°C to -30°C.

[0212] Clotting. Volume collected: approximately 300-800 μL, anticoagulant: 3.2% sodium citrate, processing: up to plasma. Parameters analyzed: prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen.

[0213] Gross Necropsy. Necropsy consists of a systematic macroscopic external and internal examination of the animal's general physical condition and tissues (respiratory, cardiovascular, digestive, and genitourinary systems). It is performed on all scheduled and unscheduled sacrifices. Any gross lesions are documented and preserved. Details regarding tissue collection are described in detail below. Qualified trained research personnel will perform the gross necropsy and tissue collection.

[0214] Tissue Collection. Tissues collected at termination and analyzed by either histopathology or ddPCR are presented in Table 10. [Table 9]

[0215] Tissue Collection: Histopathology. Tissues for histopathology are collected from animals on their designated termination dates per Tables 2, 3, and 4. All tissues marked in Table 6 for histopathology (except eyes), including gross lesions / abnormal tissues, are collected and fixed in 10% neutral buffered formalin (NBF). Eyes are fixed in Davidson's solution for 24-48 hours and then transferred to 70% ethanol.

[0216] Tissues are excised, processed, embedded in paraffin, cut on a microtome, stained with H&E, and cover slipped. When possible, muscles are sectioned both transversely and longitudinally. The resulting slides are quality checked by microscope. All prepared slides are evaluated by an ACVP veterinary pathologist. A draft pathology report is issued, consisting of tabulated microscopic data and a discussion of notable changes. Photomicrographs are taken and annotated.

[0217] ddPCR Analysis. Representative samples of the tissues listed in Table 10 are collected and retained for ddPCR analysis. Specimens collected as one large piece are placed into a tube and placed in a cooler containing dry ice until flash frozen in liquid nitrogen and placed in a freezer set to maintain -60°C to 80°C.

[0218] Example 8: Biodistribution of INS1201-AAV9 following intrathecal delivery in non-human primates (NHPs) Testing Systems Genus: Macaque Species: Cynomolgus monkey Gender: Male Weight: Approx. 2-5kg

[0219] INS1201-AAV9 biodistribution in the periphery and skeletal muscle is measured in response to intrathecal administration.

[0220] All injections are performed intrathecally into the lumbar space of the spinal cord. A total of 12 animals are used as provided in Table 11. [Table 10]

[0221] Animals selected for the study are juvenile or adult monkeys aged 2 months to 5 years weighing approximately 3 kg. Animals are pre-screened and negative for AAV9 antibodies. A pre-screening blood draw is performed within 2 months prior to the injection procedure. Animals for screening are sedated and blood samples are collected.

[0222] Selected animals are brought to the hospital area several weeks prior to injection. On injection day (d0), subjects are sedated and blood samples are collected for chemical analysis and cytoplasmic blood count (CBC). Subjects are then injected with a single dose of INS1201--AAV9 according to the dosing table above. All animals under 9 months of age are housed with dams. Animals over 9 months of age are housed in small groups.

[0223] Injections are performed by lumbar puncture into the subarachnoid space of the lumbar theca. For intrathecal (IT) injections, the subject is placed in a lateral position and a posterior midline injection site (below the conus of the spinal cord) at approximately the L4 / 5 level is identified. Under sterile conditions, a spinal needle with a stylet is inserted and subarachnoid intubation is confirmed by clear CSF flow from the needle as well as injection of a small amount of iohexol followed by intraprocedural myelography. Approximately 1 ml of CSF is withdrawn, collected and frozen as a baseline sample. This is to mitigate the increase in pressure generated by subsequent injections of the test substance. To improve rostral flow distribution of the test substance, the subject is then tilted in Trendenberg position (slight head-down position). This is a routine procedure when performing CT myelograms in human subjects. For CSF tap / IT infusion, a hypodermic needle (22G 3 / 4 or 1 1 / 2") can also be used for this purpose.

[0224] In-life observations: Treated NHPs are kept in isolation to reduce exposure to confounding sources of toxicity. Subjects are observed twice daily by veterinary staff for activity, relative skin color, and general health. Animals are kept for approximately 21 days after injection. Biodistribution and clinical chemistry tests are performed on samples collected at the time of euthanasia.

[0225] The following segments are collected from the spinal cord for biodistribution studies: cervical, thoracic, lumbar, sacral, dorsal root ganglion (DRG) root cervical level, DRG root thoracic level, DRG root lumbar level. A total of two fragments are collected for each spinal cord segment. One fragment is stored in 4% paraformaldehyde (PFA) and one is flash frozen.

[0226] For muscles and organs, four samples of each are collected; two are placed in 4% PFA and two are flash frozen. The following muscle samples are collected: diaphragm, 6th / 7th ribs including intercostal muscles and nerves, psoas, deltoid, pectoralis major, biceps brachii, triceps brachii, rectus femoris, vastus medialis, vastus lateralis, gastrocnemius, tibialis anterior, soleus, tongue, masseter, extensor digitorum, rectus abdominis. Samples of the following organs are collected: heart, liver, lungs, kidneys, spleen.

[0227] Clinical chemistry tests were as follows: AST, ALT, GGT, Alk Phos, potassium, sodium, chloride, creatinine, blood urea nitrogen, CBC.

[0228] Brain samples are collected as follows: The cerebellum is separated from the rest of the brain. The cerebellum is cut into four quadrants, the right two quadrants are kept in 4% PFA and the left two quadrants are snap frozen. The brain is divided into right and left hemispheres. The brain is then cut into four quadrants (coronal cuts), the right two quadrants are kept in 4% PFA and the left two quadrants are snap frozen.

[0229] Six samples are taken from the quadriceps for subsequent muscle biopsy: three are stored in 4% PFA and three are flash frozen.

[0230] Cerebrospinal fluid (CSF) and serum are also collected for biodistribution studies.

[0231] Biodistribution studies are performed on the aforementioned samples by droplet digital polymerase chain reaction (ddPCR).

[0232] Incorporation by Reference The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0233] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. 1. An intrathecal composition comprising an effective amount of adeno-associated virus (AAV) particles and a pharmaceutically acceptable carrier, wherein the AAV particles comprise capsids that encapsidate a vector genome, the vector genome comprising, from 5' to 3': a 5' inverted terminal repeat (ITR); The promoter and SV40 intron, a microdystrophin (μDys) transgene; SV40 poly(A) tail; and a 3' ITR.

2. 1. An intrathecal composition comprising an effective amount of adeno-associated virus (AAV) particles and a pharmaceutically acceptable carrier, wherein the AAV particles comprise capsids that encapsidate a vector genome, the vector genome comprising, from 5' to 3': a 5' inverted terminal repeat (ITR); An enhancer, The promoter and a microdystrophin (μDys) transgene; SV40 poly(A) tail; and a 3' ITR.

3. An intrathecal composition as described in claim 1 or 2, wherein the effective amount of AAV particles contains approximately 90% or less of the vector genome than the effective amount of AAV particles that encapsidate the μDys transgene in the intravenous composition.

4. 3. The intrathecal composition of claim 1 or 2, wherein the μDys transgene comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:

5.

5. 3. The intrathecal composition of claim 1 or 2, wherein the 5' ITR is an AAV2 ITR.

6. 3. The intrathecal composition of claim 1 or 2, wherein the 3' ITR is an AAV2 ITR.

7. The intrathecal composition of claim 1 or 2, wherein the promoter is the MHCK7 promoter.

8. The intrathecal composition of claim 1 or 2, wherein the promoter is a chicken β-actin hybrid promoter.

9. 3. The intrathecal composition of claim 1 or 2, wherein the AAV particles are AAV9 particles and the capsid comprises AAV9 capsid proteins.

10. 4. The intrathecal composition of claim 3, wherein the effective amount of the AAV particles contains about 10 to 40 times fewer vector genomes than the effective amount of the intravenous composition.

11. An intrathecal composition as described in claim 1 or 2, for use in the treatment of Duchenne muscular dystrophy (DMD) in a subject in need thereof, said use comprising intrathecally administering the intrathecal composition to the subject in a single dose.

12. 12. The intrathecal composition of claim 11, wherein the effective amount of the AAV particles in the intrathecal composition provides a superior therapeutic response than the same AAV particles of the same vector genome dose administered intravenously.

13. The intrathecal composition of claim 11, wherein the use comprises increasing the subject's North Star Ambulatory Assessment (NSAA) score after the use compared to the subject's baseline NSAA score.

14. 14. The intrathecal composition of claim 13, wherein the NSAA score increases by about 5 to about 25 points, or about 5 to about 20 points, about 5 to about 15 points, or about 5 to about 10 points.

15. 14. The intrathecal composition of claim 13, wherein the NSAA score increases by about 2 to about 12 points.

16. The intrathecal composition of claim 11, wherein the use comprises increasing the number of meters walked by the subject in a 6-minute walk test (6MWT) after use compared to the baseline number of meters walked by the subject in the 6MWT.

17. 15. The intrathecal composition of claim 14, wherein the number of meters walked by the subject in the 6MWT increases by about 5 meters to about 50 meters, about 5 meters to about 45 meters, about 5 meters to about 40 meters, about 5 meters to about 35 meters, about 5 meters to about 30 meters, about 5 meters to about 25 meters, about 5 meters to about 20 meters, about 5 meters to about 15 meters, or about 5 meters to about 10 meters.

18. The effective amount of the AAV particles in the intrathecal composition is about 1.0 x 10 ９ ~Approx. 1×10 １６ The intrathecal composition of claim 11, wherein the vector genome is a vector genome of the

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