Cpg-free codon-optimized human microdystrophin for duchenne muscular dystrophy gene therapy

Abstract

The present disclosure is directed to compositions and methods for treating dystrophinopathies. In particular, the present disclosure provides microdystrophins for treating dystrophinopathies by administering compositions encoding microdystrophins.

Description

CPG-FREE CODON-OPTIMIZED HUMAN MICRODYSTROPHIN FORDUCHENNE MUSCULAR DYSTROPHY GENE THERAPYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority’ to U.S. Provisional Patent Application Serial No. 63 / 595,428, filed on November 2, 2023, the disclosure of which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under NS090634 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.INCORPORATION OF SEQUENCE LISTING

[0003] A computer readable form of a Sequence Listing containing the file named "21UMC070PCT.xml", which is 85,859 bytes in size as measured in MICROSOFT WINDOWS® EXPLORER) and created on October 21, 2024, is provided herein and is herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs:l-14.BACKGROUND

[0004] The present disclosure relates to compositions and methods for treating disease. In particular, the present disclosure provides CpG-free codon-optimized human microdystrophin for Duchenne muscular dystrophy gene therapy.

[0005] Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease caused by the lack of dystrophin. The full-length dystrophin coding sequence is ~12 kb. The full-length dystrophin protein contains four major domains, including the N-terminal (NT) actin-binding domain, a large center rod domain with 24 spectrin-like repeats and 4 proline-rich hinges, a cysteine-rich (CR) beta-dystroglycan binding domain, and a C-terminal (CT) domain. Dystrophin and its interacting proteins form the dystrophin-associated protein complex (DAPC). The basic function of dystrophin can be accomplished with the NT domain, a shortened rod domain with at least 4 repeats and 2 hinges, and the CR domain.

[0006] DMD gene therapy requires body wide delivery'. Adeno-associated virus (AAV) is the only vector capable of efficient systemic gene delivery. But the ~12 kb dystrophin coding sequence greatly exceeds the 5 kb maximal packaging capacity of the AAV vector. For this reason, highly abbreviated micro-dystrophin genes have been developed. A micro-dystrophin gene contains approximately one-third of the dystrophin coding sequence. It encodes only the most critical domains in the dystrophin protein including the NT domain, a shortened rod domain, and the CR domain. More than 30 micro-dystrophin constructs have been developed by different groups. The primary difference of these micro-dystrophins is the rod domain composition. Certain compositions yield minimum or no muscle protection while others can more effectively ameliorate muscle disease. The present disclosure provides rationally designed novel synthetic micro-dystrophins with superior therapeutic efficacy.

[0007] Among more than 30 engineered micro-dystrophins, three are currently being tested in DMD patients by Pfizer, Sarepta, and Solid Biosciences. Preliminary results from these trials showed abundant micro-dystrophin expression in biopsied muscles. There is also suggestive evidence of motor function improvement in some patients. The rod domain of all three micro-dystrophins contains hinges 1 and 4 (Hl and H4), and repeats 1 and 24 (R1 and R24). Solid micro-dystrophin carries the R16 / 17 nNOS-binding domain which has been shown to yield superior muscle protection in animal studies and is associated with milder clinical presentations in human patients. Sarepta microdystrophin carries hinge 2 (H2) which has been shown to negatively impact muscle function in mouse studies. Besides these common and unique features, three micro-dystrophins differ in the number of hinges and repeats. A central hinge is included in the Pfizer construct (hinge 3, H3) and Sarepta construct (H2) but not in the Solid construct. Pfizer and Solid constructs have five repeats while Sarepta construct has four repeats. It is currently unclear whether inclusion (or exclusion) of a central hinge and whether four (paired) or five (odd number) repeats make a difference in the functional outcome.BRIEF DESCRIPTION

[0008] In one aspect, the present disclosure is directed to a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; four (4) spectrin-like repeat domains: a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain, wherein the nucleic acid does not encode a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 16 (R1 ) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-likerepeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain or a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain.

[0009] In one aspect, the present disclosure is directed to a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 1 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain.

[0010] In one aspect, the present disclosure is directed to a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a hinge 4 (H4) domain; a cysteine- rich (CR) domain; and lacking a central hinge 3 (H3) domain.

[0011] In one aspect, the present disclosure is directed to a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; four (4) spectrin-like repeat domains; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain, wherein the nucleic acid does not encode a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl): a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain or a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain.

[0012] In one aspect, the present disclosure is directed to a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain.

[0013] In one aspect, the present disclosure is directed to a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 2 (R2) domain; a spectrinlike repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain.

[0014] In one aspect, the present disclosure is directed to a method for treating dystrophinopathy in a subject in need thereof, the method comprising: administering to the subject in need thereof a vector selected from the group consisting of a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; four (4) spectrin-like repeat domains: a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain, wherein the nucleic acid does not encode a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine- rich (CR) domain or a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (Rl 6) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain; a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge domain and a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a hinge 4 (H4) domain; a cysteine- rich (CR) domain; and lacking a central hinge 3 (H3) domain.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0016] FIG. 1 is a schematic illustration depicting the protein domain structures of full- length dystrophin and four representative microdystrophins (pDys).

[0017] FIGS. 2A-2E depict results of the combined use of the muscle-specific promoter and the miR142-3p target site showing attenuation of T cell infiltration following intramuscular injection of an AAV microdystrophin vector in the canine DMD model. Three pDys constructs were packaged in AAV 8 and delivered to adult affected dogs via intramuscular injection at the dose of IxlO13vg / muscle. Injected muscles were harvested at two months later. FIG. 2A, is a cartoon illustration of three microdystrophin (pDys) constructs used in the study. AR2 pDys carries dystrophin N-terminal actin-binding domain, hinges 1, 3 and 4, spectrin-like repeats 1, 16, 17. and 24, the cysteine-rich domain, and syntrophin / dystrobrevin binding motif. In the first construct, AR2 pDys is expressed from the ubiquitous CMV promoter. In the second construct, AR2 pDys is expressed from the muscle-specific Spc5-12 promoter. The third construct is similar to the second construct except it also carries the miR142-3p target site. FIG. 2B depicts representative pDys and CD4 immunostaining photomicrographs from muscles that received three different AAV vectors. The CMV pDys vector induced robust CD4+ T cell infiltration and limited pDys expression. The use of the muscle-specific Spc5-12 promoter improved pDys expression and reduced CD4+ T cell infiltration. The combined use of the muscle-specific Spc5-12 promoter and the miR142-3p target site further reduced CD4+ T cell infiltration. FIG. 2C depicts representative HE staining, pDys, CD4, and CD8 immunostaining photomicrographs of muscles that received the CMV pDys vector and the Spc5-12 / miR142- 3p pDys vector. FIG. 2D and FIG. 2E depict bar graphs showing quantification of CD4+ (FIG. 2D) and CD8+ (FIG. 2E) T cell infiltration. Uninjected normal and affected dog muscles were included as controls.

[0018] FIG. 3 is a cartoon illustration of four-repeat (XP41) and five-repeat (XP42) pDys constructs. In both constructs, pDys was expressed from the muscle-specific CK8 promoter. XP41 pDys carries the N-terminal domain (N), hinges 1 and 4 (Hl and H4). spectrinlike repeats 1, 16, 17, and 24 (Rl, R16, R17, R24), and the cysteine-rich (CR) domain. XP42 is identical to XP41 except an additional spectrin-repeat (R23).

[0019] FIG. 4 is a graph depicting the evaluation of AAV pDys vector biodistribution in the tibialis anterior (TA), diaphragm, and heart. The XP41 and XP42 pDys constructs were packaged in AAV9 and delivered to 6-week-old mdx4cv mice at the dose of 4x1014vg / kg via tail vein injection. Tissues were harv ested at 10 weeks after AAV injection. AAV vectorgenome copy number was quantified by qPCR. There was no significant difference between the two vectors.

[0020] FIG. 5 depicts the evaluation of pDys expression in the tibialis anterior (TA), diaphragm, and heart by immunofluorescence staining. The XP41 and XP42 pDys constructs were packaged in AAV9 and delivered to 6-week-old mdx4cv mice at the dose of 4xl014vg / kg via tail vein injection. Tissues were harvested at 10 weeks after AAV injection. Dystrophin expression was determined by immunostaining. Representative pDys immunostaining photomicrographs are shown. Both vectors resulted in saturated pDys expression. There were no apparent differences between the two vectors. Uninjected mdx4cv and normal BL6 (WT) were included as controls.

[0021] FIGS. 6A-6D depict the evaluation of pDys expression in the tibialis anterior (FIGS. 6 A and 6C) and heart (FIGS. 6B and 6D) by western blot. The XP41 and XP42 pDys constructs were packaged in AAV9 and delivered to 6-week-old mdx4cv mice at the dose of 4xl014vg / kg via tail vein injection. Tissues were harvested at 10 weeks after AAV injection. Dystrophin expression was determined by western blot (FIGS. 6A and 6B). Representative pDys western blot (FIGS. A and 6B) and densitometry quantification (FIGS. 6C and 6D) are shown. The bottom images in western blot are loading controls. Both vectors resulted in supraphysiological pDys expression. There were no apparent differences between the two vectors. Uninjected mdx4cv and normal BL6 (WT) were included as controls. Lane 1, uninjected normal control; Lane 2, uninjected mdx4cv control; Lanes 3 to 5, mice that received XP41 injection; Lanes 6 to 8, mice that received XP42 injection.

[0022] FIGS. 7A and 7B depict the evaluation of centronucleation (CN) in the tibialis anterior (TA) (FIG. 7A) and diaphragm (FIG. 7B). The XP41 and XP42 pDys constructs were packaged in AAV9 and delivered to 6-week-old mdx4cv mice at the dose of 4xl014vg / kg via tail vein injection. Tissues were harvested at 10 weeks after AAV injection. The percentage of myofibers that had centrally localized myonuclei was quantified in HE-stained images. Uninjected mdx4cv and normal BL6 (WT) were included as controls. XP41 and XP42 resulted in similar levels of reduction of CN%.

[0023] FIGS. 8A and 8B are graphs depicting the evaluation of contractility of the tibialis anterior muscle. The XP41 and XP42 pDys constructs were packaged in AAV9 and delivered to 6-week-old mdx4cv mice at the dose of 4x1014vg / kg via tail vein injection. TAmuscle force was evaluated at 10 weeks after AAV injection. Uninjected mdx4cv and normal BL6 (WT) were included as controls. FIG. 8A depicts specific tetanic force (Po). FIG. 8B depicts force-frequency. Both vectors significantly improved muscle force. However, only XP41 normalized muscle force.

[0024] FIGS. 9 A and 9B are graphs depicting the evaluation of electrocardiography (ECG). The XP41 and XP42 pDys constructs were packaged in AAV9 and delivered to 6- week-old mdx4cv mice at the dose of 4xl014vg / kg via tail vein injection. ECG was evaluated at 10 weeks after AAV injection. Uninjected mdx4cv and normal BL6 (WT) were included as controls. FIG. 9A depicts the Q amplitude. FIG. 9B depicts the cardiomyopathy index.

[0025] FIG. 10 depicts cartoon illustrations of pDys with (XP16) or without (XP23) a central hinge (H3). In both constructs, pDys was expressed from the muscle-specific Spc5-12 promoter. XP16 pDys carries the N-terminal domain (N), hinges 1, 3, and 4 (Hl, H3 and H4), spectrin-like repeats 1, 16, 17, and 24 (Rl. R16, R17, R24), the cysteine-rich (CR) domain, the syntrophin / dystrobrevin binding motif (syn / dbr), and the miR142-3p target site. XP23 is identical to XP16 except without hinge 3.

[0026] FIG. 11 are immunofluorescent micrographs depicting the evaluation of pDys expression in the tibialis anterior (TA) muscle by immunofluorescence staining. The XP16 and XP23 pDys constructs were packaged in AAV9 and delivered to 10-week-old mdx mice at the dose of 4xl014vg / kg via tail vein injection. Tissues were harvested at 18 weeks after AAV injection. pDys expression was determined by immunostaining. Representative pDys immunostaining photomicrographs are shown. Both vectors resulted in saturated pDys expression. There were no apparent differences between the two vectors. Uninjected mdx and normal BL 10 (WT) were included as controls.

[0027] FIGS. 12A and 12B depict the evaluation of pDys expression in the tibialis anterior (TA) (FIG. 12A) and heart (FIG. 12B) by western blot. The XP16 and XP23 pDys constructs were packaged in AAV9 and delivered to 10-week-old mdx mice at the dose of 4xl014vg / kg via tail vein injection. Tissues were harvested at 18 weeks after AAV injection. Dystrophin expression was determined by western blot. Both vectors resulted in supraphysiological pDys expression. There were no apparent differences between the two vectors. Uninjected mdx and normal BL 10 (Wild type) were included as controls. Myosinheavy chain (MHC) was used as the loading control in TA muscle western blot. Vinculin was used as the loading control in heart western blot.

[0028] FIGS. 13A and 13B are graphs depicting the evaluation of forelimb grip force (FIG. 13 A) and the specific tetanic force (Po) of the tibialis anterior (TA) muscle (FIG. 13B). The XP16 and XP23 pDys constructs were packaged in AAV9 and delivered to 10-week-old mdx mice at the dose of 4x1014vg / kg via tail vein injection. Grip force (FIG. 13 A) and TA force (FIG. 13B) were examined at 18 weeks after AAV injection. Both vectors resulted in significant improvements in grip force and specific tetanic force. There were no apparent differences between the two vectors. Uninjected mdx and normal BL10 (Wild type) were included as controls.

[0029] FIG. 14 is a graph depicting the evaluation of the eccentric contraction profile of the tibialis anterior (TA) muscle. The XP16 and XP23 pDys constructs were packaged in AAV9 and delivered to 10-week-old mdx mice at the dose of 4xl014vg / kg via tail vein injection. TA muscle function was examined at 18 weeks after AAV injection. Although both vectors protected eccentric contraction induced force drop, XP23 resulted in better protection than XP16. Uninjected mdx and normal BL10 (Wild type) were included as controls.

[0030] FIG. 15 is a graph depicting the evaluation of serum creatine kinase (CK). The XP16 and XP23 pDys constructs were packaged in AAV9 and delivered to 10-week-old mdx mice at the dose of 4xl014vg / kg via tail vein injection. Serum creatine kinase levels were examined at 18 weeks after AAV injection. Although both vectors reduced the CK levels, only XP23 resulted in statistically significant reduction. Uninjected mdx and normal BU10 (Wild type) were included as controls.

[0031] FIGS. 16A-16C are graphs depicting the evaluation of electrocardiography (ECG). The XP16 and XP23 pDys constructs were packaged in AAV9 and delivered to 10- week-old mdx mice at the dose of 4xl014vg / kg via tail vein injection. ECG was examined at 18 weeks after AAV injection. Uninjected mdx and normal BL 10 (WT) were included as controls. Mice that were treated with XP16 were significantly different from WT in three parameters (QRS duration. QTc interval, and the cardiomyopathy index).

[0032] FIG. 17 depicts a cartoon illustration of XP49pDys. XP49 pDys was expressed from the muscle-specific CK8 promoter. XP49 is a four-repeat pDys that does not carry a central hinge. XP49 pDys carries the N-terminal domain (N), hinges 1, and 4 (Hl and H4),spectrin-like repeats 1, 16, 17, and 24 (Rl, R16, R17, R24), the cysteine-rich (CR) domain, the syntrophin / dystrobrevin binding motif (syn / dbr), a dystrophin-2 epitope (Dys-2-epitope), and the miR142-3p target site. XP49 pDys is codon-optimized and does not carry CpG motifs.

[0033] FIGS. 18A-18V are immunofluorescence micrographs evaluating XP49 pDys expression by immunofluorescence staining following systemic delivery in affected dogs. XP49 pDys was packaged in AAV8 and delivered to ~3-m-old affected dogs at the dose of IxlO14vg / kg by intravenous injection. FIG. 18A and 18B depict representative dystrophin immunofluorescence staining of the biceps femoris muscle at 6 (FIG. 18 A) and 12 weeks (FIG. 18B) post-inj ection. FIGS . 18C and 18D depict representative dy strophin immunofluorescence staining of the biceps femoris muscle at 24 (FIG. 18C) and 72 weeks (FIG. 18D) post-injection. FIGS. 18E-18H depict representative dystrophin immunofluorescence staining of the biceps femoris (FIG. 18E), thoracic longissimus (FIG. 18F), flexor carpi ulnaris (FCU) (FIG. 18G), and dorsi latissimus (FIG. 18H) muscles at 21.57 months post-injection from injected dog Mina. FIGS. 181 and 18J depict representative dystrophin immunofluorescence staining of the tongue (FIG. 181) and sternohyoid muscles (FIG. 18J) at 10.7 months post-injection from injected dog Ulric. FIGS. 18K and 18L depict representative dystrophin immunofluorescence staining of the extensor carpi ulnaris (ECU) (FIG. 18K) and flexor carpi ulnaris (FCU) (FIG. 18L) muscles at 10.8 months post-injection from injected dog Vanessa. FIGS. 18M and 18N depict representative dystrophin immunofluorescence staining of the gastrocnemius (lateral head) (FIG. 18M) and rectus abdominus (FIG. 18N) muscles at 13.03 months post-injection from injected dog Xtra. FIGS. 180 and 18P depict representative dystrophin immunofluorescence staining of the superficial pectoralis (FIG. 180) and teres major muscles (FIG. 18P) at 13.63 months post-injection from injected dog Ink. FIGS. 18Q and 18R depict representative dystrophin immunofluorescence staining of the extensor carpi ulnaris (ECU) (FIG. 18Q) and gracilis muscles (FIG. 18R) at 13.7 months post-injection from injected dog Issac. FIGS. 18S and 18T depict representative dystrophin immunofluorescence staining of the extensor carpi ulnaris (ECU) (FIG. 18S) and biceps femoris (BF) (FIG. 18T) muscles at 13.33 months post-injection from injected dog Joey. FIG. 18U and 18V depict representative dystrophin immunofluorescence staining of the extensor carpi ulnaris (ECU) (FIG. 18U) and semitendinous (FIG. 18V) muscles at 13.53 months post-injection from injected dog Josh.

[0034] FIGS. 19A and 19B are graphs depicting the evaluation of the extensor carpi ulnaris (ECU) muscle function in affected dogs that received systemic XP49 pDys genetherapy. XP49 pDys was packaged in AAV8 and delivered to ~3-mo old affected dogs at the dose of IxlO14vg / kg by intravenous injection. ECU muscle force was evaluated at the age of 15.50±4.35 (mean ± SD) months (—15 months post-injection). Uninjected normal dogs (14.86±1.79 months) and affected dogs (14.00±2.88 months) were included as controls. FIG. 19A depicts absolute twitch force (Pt) specific twitch force (sPt) and muscle force vs stimulation frequency. FIG. 19B depicts absolute tetanic force (Po), specific tetanic force (sPo), and muscle force vs eccentric contraction cycle. XP49 therapy significantly improved protection against eccentric contraction-induced force drop.

[0035] FIGS. 20 A and 20B depicts acetylcholine-induced vasodilation in a normal dog femoral arterial. FIG. 20 depicts a cartoon illustration of femoral arterial wall. The femoral arterial wall has three layers including tunica intima (endothelium), tunica media (smooth muscle layer), and tunica adventitia. Dystrophin is expressed in tunica intima and tunica media. M3 and M5 muscarinic acetylcholine receptors are present in tunica intima. Dystrophin anchors eNOS to tunica intima. When acetylcholine binds to its receptors, it will activate eNOS to produce nitric oxide (NO) and generate cGMP. It will also activate cyclooxygenase (COX) to produce prostaglandins and generate cAMP. As a result, it will lead to vasorelaxation in a dosedependent manner (FIG. 20B).

[0036] FIGS. 21 A and 21B depict compromised acetylcholine-induced vasodilation in the affected dog femoral arterial. FIG. 21 depicts a cartoon illustration of femoral arterial wall. The femoral arterial wall has three layers including tunica intima (endothelium), tunica media (smooth muscle layer), and tunica adventitia. Affected dogs do not have dystrophin in tunica intima and tunica media. As a result, eNOS level is reduced in tunica intima in affected dog femoral artery. Hence, upon acetylcholine stimulation, it will generate less NO and less cGMP, and less vasorelaxation (FIG. 2 IB).

[0037] FIGS. 22A and 22B are graphs depicting the systemic XP49 pDys gene therapy normalized acetylcholine and sodium nitroprusside (SNP)-induced vasodilation in the affected dog femoral arterial. Acetylcholine-induced vasodilation (left panels) and SNP-induced vasodilation (right panels) were examined in the femoral artery of normal dogs, affected dogs, and two XP49-treated affected dogs, Dog U Ulric (FIG. 22A) and Dog X Xtra (FIG. 22B). Absence of dystrophin compromised acetylcholine-induced vasodilation in affected dogs. XP49 pDys therapy restored acetylcholine-induced vasodilation in affected dogs. SNP is a potent NO donor and it induces endothelium-independent vasodilation by activating cGMP invascular smooth muscle. In DMD, smooth muscle in the artery w all is sensitized to NO. Hence, SNP induced more pronounced vasodilation in the affected dog femoral artery. This was reversed in the femoral artery of XP49 treated dogs.

[0038] FIG. 23 depicts cartoon illustration for deimmunizing dystrophin Hl. Bioinformatics analysis of dystrophin Hl and utrophin Hl identified an 11-mer peptide in dystrophin Hl that is not within utrophin Hl. The NetMHCpan-4. 1 software predicted a 9-mer peptide that binds to MHC class I. Dystrophin Hl (boxed region) can be (i) abbreviated to remove 9-mer or 11-mer sequences of dystrophin Hl, (ii) substituted with dystrophin H2, (iii) substituted with dystrophin H3, and (iv) substituted with utrophin Hl.DETAILED DESCRIPTION

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below;

[0040] A reference human dystrophin amino acid sequence is provided in Accession No. GI:M18533.1. A reference mouse dystrophin amino acid sequence is provided in Accession No. GI: M68859.1.

[0041] In one aspect, the present disclosure is directed to a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; four (4) spectrin-like repeat domains; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain, wherein the nucleic acid does not encode a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 16 (R1 ) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain or a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain.

[0042] In one aspect, the present disclosure is directed to a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-hke repeat 1 (Rl) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain. The microdystrophin encoded by the nucleic acid would then have a protein domain structure of: NT-H1-R1-R16- R17-R24-H4-CR.

[0043] Suitably, the hinge 1 (Hl) domain is selected from an unmodified hinge 1 (Hl) domain and a modified hinge 1 (Hl) domain. Suitable modified hinge 1 (Hl) domains include one of a deletion of an amino acid sequence HQMHYSQQI (SEQ ID NO: 1) in the Hl domain; a deletion of an amino acid sequence HFQLHHQMHYS (SEQ ID NO:2) in the Hl domain; a deletion of an amino acid sequence HFQLHHQMHYS QQI (SEQ ID NO:3) in the Hl domain; a substitution of the Hl domain with a hinge 2 (H2) domain, a substitution of the Hl domain with a hinge 3 (H3) domain, or a substitution of the Hl domain with a utrophin hinge 1 (Hl) domain. Without being bound by theory, an immunogenic region was mapped to the Hl domain encoded by exons 8 and 9. An 11-mer peptide was identified in dystrophin Hl, but not in utrophin Hl. Dystrophin Hl was screened with NetMHCpan-4.1, a software that predicts peptide binding to MHC class I, which identified a 9-mer peptide overlapping with the 11-mer sequence that was predicted to bind HLA-A*02:01. Thus, a deletion of the amino acid sequence HQMHYSQQI (SEQ ID NO:1 ) of the Hl domain, a deletion of the amino acid sequence HFQLHHQMHYS (SEQ ID NO:2) of the Hl domain, a deletion of the amino acid sequence HFQLHHQMHYSQQI (SEQ ID NO:3) of the Hl domain, a substitution of the Hl domain with a hinge 2 (H2) domain, a substitution of the Hl domain with a hinge 3 (H3) domain, or a substitution of the Hl domain with a utrophin hinge 1 (Hl) domain deimmunizes the microdystrophin by eliminating or substituting the immunogenic amino acid sequence contained in the Hl domain.

[0044] The nucleic acid can further encode a syntrophin / dystrobrevin binding motif. The microdystrophin encoded by the nucleic acid would then have a protein domain structure of: NT-Hl-Rl-R16-R17-R24-H4-CR-syn / dbr.

[0045] The microdystrophin can further include a dystrophin epitope. The dystrophin epitope (referred to herein as "Dys2 epitope") allows for detection of the microdystrophin using an anti-Dys2 epitope antibody (commonly known as “Dys-2 antibody”). The microdystrophinencoded by the nucleic acid would then have a protein domain structure of: NT-H1-R1-R16- R17-R24-H4-CR-Dys2 epitope. Embodiments that include both the syn / dbr binding motif and the Dys2 epitope would then have a protein domain structure of: NT-H1-R1-R16-R17-R24- H4-CR-syn / dbr-Dys2 epitope.

[0046] The microdystrophin can further include a miRNA site to restrict expression to muscle cells. Without being bound by theory, the miRNA avoids expression of the microdystrophin in antigen-presenting cells and reduces immunogenicity to the microdystrophin. A particularly suitable miRNA is Mirl42-3pT. Embodiments that include the miRNA, the syn / dbr binding motif and the Dys2 epitope would then have a protein domain structure of: NT-Hl-Rl-R16-R17-R24-H4-CR-syn / dbr-Dys2 epitope-miRNA.

[0047] In particularly suitable embodiments, the nucleic acid sequence is codon- optimized to remove CpG motifs.

[0048] In one aspect, the present disclosure is directed to a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a hinge 4 (H4) domain; a cysteine- rich (CR) domain; and lacking a central hinge 3 (H3) domain. The microdystrophin encoded by the nucleic acid would have a protein domain structure of: NT-H1-R1-R-2-R16-R17-H4- CR.

[0049] Suitably, the hinge 1 (Hl) domain is selected from an unmodified hinge 1 (Hl) domain and a modified hinge 1 (Hl) domain. Suitable modified hinge 1 (Hl) domains include one of a deletion of an amino acid sequence HQMHYSQQI (SEQ ID NO: 1) in the Hl domain; a deletion of an amino acid sequence HFQLHHQMHYS (SEQ ID NO:2) in the Hl domain; a deletion of an amino acid sequence HFQLHHQMHYS QQI (SEQ ID NO:3) in the Hl domain; a substitution of the Hl domain with a hinge 2 (H2) domain, a substitution of the Hl domain with a hinge 3 (H3) domain, or a substitution of the Hl domain with a utrophin hinge 1 (Hl) domain. Without being bound by theory, an immunogenic region was mapped to the Hl domain encoded by exons 8 and 9. An 11-mer peptide was identified in dystrophin Hl, but not in utrophin Hl. Dystrophin Hl was screened with NetMHCpan-4. 1, a software that predicts peptide binding to MHC class I, which identified a 9-mer peptide overlapping with the 11-mer sequence that was predicted to bind HLA- A* 02: 01. Thus, a deletion of the amino acid sequenceHQMHYSQQI (SEQ ID NO:1) of the Hl domain, a deletion of the amino acid sequence HFQLHHQMEIYS (SEQ ID NO:2) of the Hl domain, a deletion of the amino acid sequence HFQLHHQMHYSQQI (SEQ ID NO:3) of the Hl domain, a substitution of the Hl domain with a hinge 2 (H2) domain, a substitution of the Hl domain with a hinge 3 (H3) domain, or a substitution of the Hl domain with a utrophin hinge 1 (Hl) domain deimmunizes the microdystrophin by eliminating or substituting the immunogenic amino acid sequence contained in the Hl domain.

[0050] The nucleic acid can further encode a syntrophin / dystrobrevin binding motif. The microdystrophin encoded by the nucleic acid would then have a protein domain structure of: NT-H1-R1-R2-R16-R17- H4-CR-syn / dbr.

[0051] The microdystrophin can further include a dystrophin epitope. The dystrophin epitope (referred to herein as "Dys2 epitope") allows for detection of the microdystrophin using an anti -dystrophin antibody. The microdystrophin encoded by the nucleic acid would then have a protein domain structure of: NT-H1-R1- R2-R16-R17-H4-CR-Dys2 epitope. Embodiments that include both the syn / dbr binding motif and the D s2 epitope would then have a protein domain structure of: NT-Hl-Rl-R2-R16-R17-H4-CR-syn / dbr-Dys2 epitope.

[0052] The microdystrophin can further include a miRNA site to restrict expression to muscle cells. Without being bound by theory, the miRNA avoid expression of the microdystrophin in antigen-presenting cells and reduces immunogenicity7to the microdystrophin. A particularly suitable miRNA is Mirl42-3pT. Embodiments that include the miRNA, the syn / dbr binding motif and the Dys2 epitope would then have a protein domain structure of: NT-Hl-Rl-R2-R16-R17-H4-CR-syn / dbr-Dys2 epitope-miRNA.

[0053] In particularly suitable embodiments, the nucleic acid sequence is codon- optimized to remove CpG motifs.

[0054] In one aspect, the present disclosure is directed to a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; four (4) spectrin-like repeat domains; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain, wherein the nucleic acid does not encode a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl): a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich(CR) domain or a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain.

[0055] In one aspect, the present disclosure is directed to a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain.

[0056] In one aspect, the present disclosure is directed to a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 2 (R2) domain; a spectrinlike repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge domain.

[0057] Suitable vector constructs are expression vector constructs. Suitable vectors include viral vectors and non-viral vectors. Suitable viral vectors are chosen (or selected) from lentiviral vectors and adeno-associated-virus vectors. Suitable vectors include AAV serotypes such as, for example, adeno-associated-virus serotype- 1 (AVV-1), adeno-associated-virus serotype-5 (AVV-5), adeno-associated-virus serotype-6 (AVV-6), adeno-associated-virus serotype-8 (AVV-8), adeno-associated-virus serotype-9 (AVV-9). adeno-associated-virus serotype-rh74 (AVV-rh74), adeno-associated-virus serotype-rhlO (AVV-rhlO). adeno- associated-virus-2i8 (AVV-2i8), adeno-associated-virus-Bl (AVV-B 1) , adeno-associated- virus-CAM130 (AVV-CAM130), adeno-associated-virus-M41 (AVV-M41), adeno- associated-virus MTP (AAV587MTP and AAV588MTP), adeno-associated-virus NP22 (AAV-NP22), adeno-associated-virus NP66 (AAV-NP66), adeno-associated-virus MYO (AAVMYO) and its variants), Myotropic adeno-associated-virus (MyoAAV-1, 2, 3, and 4) and their variants, linked-integrin-complex adeno-associated-virus 1 (LICA1), adeno-associated- virus tyrosine mutants, and ancestral adeno-associated-virus (ancAVV). Suitable vectors also include plasmid, liposome, exosome, and nanoparticles.

[0058] The vectors of the present disclosure are suitably codon optimized to remove substantially all CpG. In a particularly suitable embodiment, the vectors do not contain any CpG.

[0059] The exact details of the vector construct vary according to the particular host cell that is to be used as well as to the desired characteristics of the expression system, as is well known in the art. For example, promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing one or more convenient restriction sites for insertion of a contemplated nucleic acid segment. Suitable promoters and vectors include the Rec 7 promoter that is inducible by exogenously supplied nalidixic acid, JHEX25 (commercially available from Promega, Madison, WI) that is inducible by exogenously supplied isopropyl-P-D-thiogalacto-pyranoside (IPTG), tac (a hybrid of the trp and lac promoter / operator) present in plasmid vector pKK223-3 (commercially available from Pharmacia, Piscataway, N.J.) and is also inducible by exogenously supplied IPTG. Other suitable promoters and promoter / operators include the araB, trp, lac, gal, T7, and the like. For production in S. cerevisiae, the nucleic acid encoding a thrombin precursor of the disclosure is placed into operable linkage with a promoter that is operable in S. cerevisiae and which has the desired characteristics (e.g., inducible / derepressible or constitutive), such as GALI -10, PHOS5, PGK1, GDP1, PMA1, MET3, CUP1, GAP, TPI, MFal and MFa2, as well as the hybrid promoters PGK / a2, TPI / a2. GAP / GAL. PGK / GAL, GAP / ADH2, GAP / PHO5, ADH2 / PHO5, CYC1 / GRE, and PGK / ARE and other promoters known in the art. For a mammalian cell line, the promoter can be a viral promoter / enhancer (e.g., the herpes virus thymidine kinase (TK) promoter or a simian virus promoter (e.g., the SV40 early or late promoter) or the Adenovirus major late promoter, a long terminal repeat (LTR), such as the LTR from cytomegalovirus-(CMV), Rous sarcoma virus (RSV) or mouse mammary7tumor virus (MMTV)) or a mammalian promoter, suitably7an inducible promoter such as the metallothionein or glucocorticoid receptor promoters and the like. For muscle, suitable promoters include an endogenous and synthetic heart-specific or muscle-specific promoters (e.g.. SPc5-12, muscle creatine kinase (see e.g.. Wang et al., Gene Ther. 2008 Nov;15(22): 1489-99, which is incorporated by reference), miniMCK, HLH, Myoglobin, Pitx3, SK-CRM-desmin, desmin, MYODI, and MYLK2.

[0060] Constructs can include additional nucleic acids appropriate for the intended host cell. For example, expression constructs for use in higher eukaryotic cell lines (e.g., vertebrateand insect cell lines) include a polyadenylation site and can include an intron (including signals for processing the intron), as the presence of an intron appears to increase mRNA export from the nucleus in many systems. Additionally, a secretion signal sequence operable in the host cell can be included as part of the construct. Other suitable secretion signal sequences can be obtained from human serum albumin, human prothrombin, human tissue plasminogen activator, and preproins ulin. Expression constructs may also contain other commonly used regulator elements such as microRNA target sites, to reduce expression in non-targeted tissues / cells. Expression constructs may also contain elements that are used to express two transgenes, such as IRES and 2A. Where the expression construct is intended for use in a prokaryotic cell, the expression construct can include a signal sequence that directs transport of the synthesized polypeptide into the periplasmic space or expression can be directed intracellularly. Constructs can also selectable markers for selecting host cells that contain the construct. Selectable markers are well known in the art. Marker genes contained in the expression vector for a microorganism can be, for example, an ampicillin resistance gene, tetracycline resistance gene for E. coli as a host; Leu2 gene for yeast as a host, and the like. Marker genes contained in the expression vector for an animal cell can be, for example, aminoglycoside 3'phosphotransferase (neo) gene, dihydrofolate reductase (dhfr) gene, glutamine synthetase (GS) gene, and the like.

[0061] Nucleic acids and vectors of the present disclosure desirably include musclespecific regulatory cassettes. Muscle-specific regulatory cassettes can minimize off-target effects. Suitable muscle-specific regulatory' cassettes include those derived from the MCK (such as miniMCK, CKS, CK6, CK7, CK8, CK8e, CK9, HLH, and MHCK7) gene, myoglobin gene and desmin genes, cardiac promoters (such as cTNl. NCX1. MLC-2v, alpha- 1c, mini- alphaMHC), Pitx3, skeletal muscle alpha-actin, and synthetic promoters (such as SPc5-12, SK- CRM1, SK-CRM2, SK-CRM3, SK-CRM4, SK-CRMS, SK-CRM6, SK-CRM7, SK-CRM- Des, SK-CRM-SPc5-12, SK448, and SP1-28). Gene expression can be enhanced using stronger regulatory cassettes and using codon-optimized, functionally enhanced cDNAs.

[0062] In one aspect, the present disclosure is directed to a method for treating dystrophinopathy in a subject in need thereof. The method includes: administering to the subject in need thereof a vector selected from the group consisting of a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; four (4) spectrin-like repeat domains; a hinge 4 (H4) domain; a cysteine-rich(CR) domain; and lacking a central hinge 3 (H3) domain, wherein the nucleic acid does not encode a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin -like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine- rich (CR) domain or a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain; a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge domain and a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a hinge 4 (H4) domain; a cysteine- rich (CR) domain; and lacking a central hinge domain.

[0063] As used herein, “a subject in need thereof’ (also used interchangeably herein with “a patient in need thereof’) refers to a subject susceptible to or at risk of a specified disease, disorder, or condition. The methods disclosed herein can be used with a subset of subjects who are susceptible to or at elevated risk for dystrophinopathies. Because some of the method embodiments of the present disclosure are directed to specific subsets or subclasses of identified subjects (that is, the subset or subclass of subjects “in need” of assistance in addressing one or more specific conditions noted herein), not all subjects will fall within the subset or subclass of subjects as described herein for certain diseases, disorders or conditions. In one embodiment, the subject has or is suspected of having a dystrophinopathy. In one embodiment, the subject is a carrier of a dystrophinopathy. As used herein, a “carrier” (or “hereditary carrier”) of a dystrophinopathy refers to a subject that has inherited a recessive allele for a genetic trait or mutation known or believed to cause a dystrophinopathy. A carrier may not show any symptoms of the dystrophinopathy or may show mild symptoms such as muscle weakness, cramps, cardiomyopathy, and combinations thereof. Dystrophinopathies include diseases caused by or resulting from a mutation in the dystrophin gene. Suitable dystrophinopathies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dialated cardiomyopathy (XLDC) and their carriers.

[0064] The vector can be administered in a pharmaceutically acceptable carrier. As understood by those skilled in the art, pharmaceutically acceptable carriers, and, optionally, other therapeutic and / or prophylactic ingredients must be ‘'acceptable” in the sense of being compatible with the other ingredients of the formulation and not be harmful to the recipient thereof. Suitable pharmaceutically acceptable carriers include water, saline, isotonic saline, phosphate buffered saline, Ringer's lactate, polymers (such as poloxamers). and the like.

[0065] Formulations for delivering the modified dystrophin can also include other components such as surfactants, preservatives, and excipients. Surfactants can reduce or prevent surface-induced aggregation of the dystrophin microgenes. Suitable surfactants fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range from about 0.001 and about 4% by weight of the formulation. Pharmaceutically acceptable preservatives include, for example, phenol, o-cresol, m-cresol, p-cresol, methyl p- hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, but l p-hydroxybenzoate, 2- phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal. bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesin (3p-chlorphenoxypropane-l,2-diol) and mixtures thereof. The preservative can be present in concentrations ranging from about 0. 1 mg / ml to about 20 mg / ml, including from about 0.1 mg / ml to about 10 mg / ml. The use of a preservative in pharmaceutical compositions is well-known to those skilled in the art. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995. Formulations can include suitable buffers such as sodium acetate, glycylglycine, HEPES (4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid) and sodium phosphate. Excipients include components for tonicity adjustment, antioxidants, and stabilizers as commonly used in the preparation of pharmaceutical formulations. Other inactive ingredients include, for example, L-histidine, L-histidine monohydrochloride monohydrate, sorbitol, polysorbate 80, sodium citrate, sodium chloride, and EDTA disodium.

[0066] The compositions of the present disclosure can be administered to animals, preferably to mammals, and in particular to humans as therapeutics per se, as mixtures with one another or in the form of pharmaceutical preparations, and which as active constituent contains an effective dose of the active agent, in addition to customary pharmaceutically innocuous excipients and additives.

[0067] Suitable methods for administration of formulations of the present disclosure are by parenteral (e.g.. intravenous (IV), intramuscular (IM), subcutaneous (SC), intrathecal (IC), or intraperitoneal (IP)) routes and the formulations administered ordinarily include effective amounts of product in combination with acceptable diluents, carriers and / or adjuvants. Standard diluents such as human serum albumin are contemplated for pharmaceutical compositions of the disclosure, as are standard carriers as described herein.

[0068] As used herein, an '‘effective amount”, a “therapeutically effective amount”, a “prophylactically effective amount” and a “diagnostically effective amount” is the amount of the modified dystrophin of the present disclosure needed to elicit the desired biological response following administration. The amount of the modified dystrophin will depend on the form the modified dystrophin is in such as whether it is administered as a nucleic acid encoding the modified dystrophin (including being packaged in an expression construct and / or vector) or as a modified dystrophin protein.

[0069] Effective dosages are expected to vary substantially depending upon the modified dystrophin used and the specific disease, disorder, or condition treated. Dosages can range from about 1010vector genomes per kilogram to about 1014vector genomes per kilogram. Suitable dosage for use in the methods of the present disclosure will depend upon a number of factors including, for example, age and weight of an individual, the specific dystrophinopathy. severity of the dystrophinopathy, nature of a composition, route of administration and combinations thereof. Ultimately, a suitable dosage can be readily determined by one skilled in the art such as, for example, a physician, a veterinarian, a scientist, and other medical and research professionals. For example, one skilled in the art can begin with a low dosage that can be increased until reaching the desired treatment outcome or result. Alternatively, one skilled in the art can begin with a high dosage that can be decreased until reaching a minimum dosage needed to achieve the desired treatment outcome or result.

[0070] The disclosure will be more fully understood upon consideration of the following Examples.EXAMPLES

[0071] To study the importance of the central hinge, two microdystrophin vectors were engineered that were identical except one had aH3 and one did not. AAV stocks were produced using the same method and injected intravenously to 10-week-old mdx mice. Vectorbiodistribution and microdystrophin expression in muscle and heart were evaluated at 18 weeks after AAV injection. No significant difference was detected between two constructs. Muscle pathology was determined by quantifying myofibers with centrally localized nuclei. No difference was seen between two constructs. Muscle function was evaluated by measuring the grip strength, and the tetanic force and eccentric contraction profile of the tibialis anterior muscle. Two microdystrophins resulted in similar improvement in grip strength and tetanic force. However, the construct without H3 was more effective in preventing eccentric contraction induced force drop. Cardiac function was evaluated by electrocardiography. The construct without H3 outperformed the construct with H3. Collectively, the data suggested that a central hinge w as not necessary in the context of microdystrophin. Removal of the central hinge improves microdystrophin function.

[0072] To determine whether four and five repeats make a difference, two microdystrophin vectors were engineered that were identical except one had four repeats and the other had five repeats. AAV stocks were produced using the same method and injected intravenously to 6-week-old mdx4cv mice. Vector biodistribution and microdystrophin expression in muscle and heart were evaluated at 10 weeks after AAV injection. No significant difference was detected between two constructs. Muscle pathology was determined by quantifying myofibers with centrally localized nuclei. No difference was seen between two constructs. Muscle function was evaluated by measuring the tetanic force and eccentric contraction profile of the tibialis anterior muscle. The four-repeat microdystrophin significantly outperformed the five-repeat microdystrophin in tetanic force, force-frequency relationship, and eccentric contraction. Cardiac function was evaluated by electrocardiography. The four-repeat microdystrophin also outperformed the five-repeat microdystrophin in ECG analysis. Collectively, the data suggested that four repeats was a better design for microdystrophin.

[0073] Of the four domains of the full-length dystrophin protein, the CT domain was the only domain completely eliminated in three microdystrophins currently in clinical trials. A primary function of the CT domain is to recruit syntrophin and dystrobrevin to the dystrophin- associated protein complex (DAPC). In the absence of the CT domain, syntrophin and dystrobrevin are recruited indirectly to the DAPC via the sarcoglycan-sarcospan complex. Since syntrophin and dystrobrevin are important signaling and scaffolding proteins, a directrecruitment strategy7was investigated. Hence, the syntrophin / dystrobrevin binding motif was included in the microdystrophin.

[0074] At the end of human dystrophin, there is a 17 amino acid residue sequence (encoded by 51 nucleotides). This sequence is the epitope for Dys-2 antibody. Since Dys-2 is highly robust and widely used antibody for dystrophin detection (immunostaining and western blot), this region was included in the human microdystrophin.

[0075] In summary7, a new microdystrophin was prepared. The microdystrophin contains the NT domain, Hl, Rl, R16. R17, R24, CR and the syntrophin / dystrobrevin binding motif (NT-Hl-Rl-R16-R17-R24-H4-CR-Syn / Dbr). Including R16 / 17 improved muscle perfusion during contraction and prevented functional ischemia in exercise. Paired design of repeats (four repeats) and elimination of central hinge (H3) improved microdystrophin function. The syntrophin / dystrobrevin binding motif allowed direct interaction of microdystrophin with syntrophin and dystrobrevin.

[0076] Systemic AAV gene therapy clinical trials have been conducted for a number of neuromuscular diseases (DMD, spinal muscular atrophy, and X-linked myotubular myopathy) using several AAV serotypes (AAV8, AAV9, and AAV-rh74). Immune response- associated toxicity has emerged as a primary' safety concern in these studies. To determine whether the muscle-specific promoter is sufficient to reduce T cell infiltration, two AAV microdystrophin vectors were compared, one with the ubiquitous CMV promoter and the other with a muscle-specific promoter, in the canine DMD model by intramuscular injection. The use of the muscle-specific promoter greatly reduced T cell infiltration. However, infiltrating T cells were still readily detectable. Several recent studies suggest that microRNA 142-3p target site (mirl42-3pT) can lessen the transgene product-associated immune response by reducing untoward expression in antigen-presenting cells. To further decrease T cell infiltration, we engineered a third AAV microdystrophin vector that contained the muscle-specific promoter and the mir!42-3pT site. Following intramuscular injection in affected dogs, nominal T cell infiltration was detected.

[0077] The CpG-motif in the AAV vector genome can be detected by the Toll-like receptor-9 (TLR-9) to prime the adaptive immune response. The human microdystrophin (NT- Hl-Rl-R16-R17-R24-H4-CR-Syn / Dbr) of the present disclosure contained 62 CpG motifs. After codon-optimization, the number of CpG motifs increased to 129. To eliminate the CpGmotifs in the microdystrophin, every CpG was manually edited using degenerative codons. The CpG-free sequence was further modified by removing antiviral motifs, polymerase slippage sites, alternative splicing sites, and the destabilizing sequence. The modified sequence was then manually edited based on the human codon usage preference. The final human microdystrophin contained no CpG motifs. The gene was then cloned into an expression cassette containing the CK8 promoter and miR142-3pT. The entire expression cassette was cloned between two inverted terminal repeats of AAV2 for vector production. The genome size of the human microdystrophin vector is 4,841 bp, which is particularly suitable for AAV packaging. In summary', there are a total of 42 CpG motifs in the vector of the present disclosure, including 32 CpG from ITR and 10 CpG from the promoter. An unmodified human microdystrophin contains a total of 104 (62 in human microdystrophin, 10 in promoter, and 32 in ITR) CpG motif in the vector. Thus, the modification described herein reduced the total CpG motifs in the vector by 60%. Removing -60% of CpG motifs from the vector genome improved the immune profile of the vector.

[0078] The canine model provides a large animal model for preclinical evaluation of candidate AAV microdystrophin vectors. However, the human microdystrophin vector cannot be tested in the canine model because the human protein elicits a strong cellular immune response in canines. Consequently, the human protein is rapidly eliminated from the dog muscle. For this reason, the canine version of the human protein is used in canine studies. With the muscle-specific promoter and the miRl 32-3pT site in the expression cassette, and also with the removal of CpG motifs from human microdystrophin coding sequence, it was hypothesized that a clinical trial compatible human microdystrophin AAV vector can be tested in the canine DMD model. To test this hypothesis, a highly purified AAV8 human microdystrophin vector stock was prepared and delivered to ten ~3-month old affected dogs via intravenous injection. Microdystrophin expression was evaluated in biopsied muscle tissues at various times postinjection (from 1 month to 16 months post-injection). AAV microdystrophin treated dogs were euthanized at -15 months post-injection (range: from 10.5 months to 24 months post-injection). Robust, persistent body wide muscle and heart human microdystrophin expression was detected in all treated dogs. In situ muscle force measurement at the termination showed improvement in twitch and tetanic force. Human microdystrophin treatment significantly prevented eccentric contraction-induced force drop. Dystrophin was not only expressed in skeletal and cardiac muscle, but also expressed in smooth muscle and non-muscle tissues / cells. Loss of dystrophin significantly compromises dog femoral artery function. Preliminaryanalysis suggests that treatment with the rationally designed AAV microdystrophin vector significantly improved femoral artery function.

Claims

CLAIMSWhat is claimed is:

1. A nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; four (4) spectrin-like repeat domains; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain, wherein the nucleic acid does not encode a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (Rl 7) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain or a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain.

2. The nucleic acid of claim 1, wherein the hinge 1 (Hl) domain is selected from an unmodified Hl domain and a modified Hl domain.

3. The nucleic acid of claim 2, wherein modified Hl domain is selected from the group consisting of a truncated Hl domain; a substitution of the Hl domain with a hinge 2 (H2) domain; a substitution of the Hl domain with a hinge 3 (H3) domain; and a substitution of the Hl domain with a utrophin hinge 1 (Hl) domain.

4. The nucleic acid of claim 3, wherein the truncated Hl domain is selected from the group consisting of a deletion of an amino acid sequence HQMHYSQQI (SEQ ID NO: 1) in the Hl domain; a deletion of an amino acid sequence HFQLHHQMHYS (SEQ ID NO:2) in the Hl domain; and a deletion of an amino acid sequence HFQLHHQMHYSQQI (SEQ ID NO:3) in the Hl domain.

5. The nucleic acid of claim 1, further encoding a syntrophin / dystrobrevin binding motif.

6. The nucleic acid of claim 1, further encoding an anti-dystrophin antibody epitope.

7. The nucleic acid of claim 1, further encoding a miRNA-binding site.

8. The nucleic acid of claim 1, wherein the nucleic acid is substantially free ofCpG motifs.

9. A nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain: a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain.

10. The nucleic acid of claim 9, wherein the hinge 1 (Hl) domain is selected from an unmodified Hl domain and a modified Hl domain.

11. The nucleic acid of claim 10, wherein modified H 1 domain is selected from the group consisting of a deletion of an amino acid sequence HQMHYSQQI (SEQ ID NO:1) in the Hl domain; a deletion of an amino acid sequence HFQLHHQMHYS (SEQ ID NO:2) in the Hl domain; a deletion of an amino acid sequence HFQLHHQMHYSQQI (SEQ ID NO:3) in the Hl domain; a substitution of the Hl domain with a hinge 2 (H2) domain, a substitution of the Hl domain with a hinge 3 (H3) domain, and a substitution of the Hl domain with a utrophin hinge 1 (Hl) domain.

12. The nucleic acid of claim 9, further encoding a syntrophin / dystrobrevin binding motif.

13. The nucleic acid of claim 9, further encoding an anti-dystrophin antibody epitope.

14. The nucleic acid of claim 9, further encoding a miRNA-binding site.

15. The nucleic acid of claim 9, wherein the nucleic acid is substantially free of CpG motifs.

16. A nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a hinge 4 (H4) domain: a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain.

17. The nucleic acid of claim 16, wherein the hinge 1 (Hl) domain is selected from an unmodified Hl domain and a modified Hl domain.

18. The nucleic acid of claim 17, wherein modified Hl domain is selected from the group consisting of a deletion of an amino acid sequence HQMHYSQQI (SEQ ID NO: 1) in the Hl domain; a deletion of an amino acid sequence HFQLHHQMHYS (SEQ ID NO: 2) in the Hl domain; a deletion of an amino acid sequence HFQLHHQMHYSQQI (SEQ ID NO:3) in the Hl domain; a substitution of the Hl domain with a hinge 2 (H2) domain, a substitution of the Hl domain with a hinge 3 (H3) domain, and a substitution of the Hl domain with a utrophin hinge 1 (Hl) domain19. The nucleic acid of claim 16, further encoding a syntrophin / dystrobrevin binding motif.

20. The nucleic acid of claim 16, further encoding an anti-dystrophin antibody epitope.

21. The nucleic acid of claim 16, further encoding a miRNA-binding site.

22. The nucleic acid of claim 16, wherein the nucleic acid is substantially free of CpG motifs.

23. A method for treating a dystrophinopathy in a subject in need thereof, the method comprising: administering to the subject in need thereof a vector selected from the group consisting of: a vector comprising a nucleic acid encoding a microdystrophin comprising:; an amino terminal (NT) domain; a hinge 1 (Hl) domain; four (4) spectrin-like repeat domains; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain, wherein the nucleic acid does not encode a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain or a microdystrophin having an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin like repeat 1 (Rl); a spectrin-like repeat 2 (R2) domain; a spectrin-like repeat 16 (R16) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; and a cysteine-rich (CR) domain;a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrinlike repeat 16 (R16) domain; a spectrin-like repeat 17 (R17) domain; a spectrin-like repeat 24 (R24) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain; and a vector comprising a nucleic acid encoding a microdystrophin comprising: an amino terminal (NT) domain; a hinge 1 (Hl) domain; a spectrin-like repeat 1 (Rl) domain; a spectrinlike repeat 2 (R2) domain; a spectrin-like repeat 1 (Rl 6) domain; a spectrin-like repeat 17 (R17) domain; a hinge 4 (H4) domain; a cysteine-rich (CR) domain; and lacking a central hinge 3 (H3) domain.

24. The method of claim 23, wherein the subject has or is suspected of having a dystrophinopathy selected from the group consisting of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and X-linked dilated cardiomyopathy (XLDC).

25. The method of claim 23, wherein the nucleic acid further encodes a syntrophin / dystrobrevin binding motif.

26. The method of claim 23, wherein the nucleic acid further encodes an antidystrophin antibody epitope.

27. The method of claim 23, wherein the nucleic acid further encodes a miRNA- binding site.

28. The method of claim 23, wherein the vector is substantially free of CpG motifs.

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