CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/800,484, filed February 2, 2019, which is incorporated by reference.

SEQUENCE LISTING

[0002] Incorporated by reference in its entirety herein is a nucleotide/amino acid sequence listing submitted concurrently herewith.

BACKGROUND OF THE INVENTION

[0003] Duchenne muscular dystrophy (DMD) is a lethal X-linked inherited disease due to dystrophin gene mutation. It is the most common inherited muscular disorder. Heart failure is the leading cause of premature death in DMD patients. Inflammation is a secondary pathological mechanism of DMD in progressive muscle degeneration while the inhibition of NF-kappaB (NF-KB) reduces muscle inflammation, ameliorates muscle pathology, and improves muscle physiological function in DMD mice (Yang, 2012 and Yin, 2017).

[0004] Gene therapy-based genetic replacement is a potential treatment for DMD (Wang, 2000), although there is no cure yet for DMD. Chronic inflammation, caused by the up- regulated NF-KB signaling, challenges DMD gene therapy (Mendell JR, 2010 The NEJM). The universal promoters such as the CMV promoter often cause unwished toxicity and immune response in muscle gene therapy. Therefore, a specific muscle-targeted gene delivery system is important for the safety of DMD clinical trial (Wang, 2008). Moreover, the muscle-specific expression mini-dystrophin for DMD therapy should lead to robust expression of mini-dystrophin both in skeletal muscle and cardiac muscle, given that, as noted congestive heart failure is the major complication arising from DMD.

[0005] It has been demonstrated that reduction of NF-KB via shRNA technology ameliorates such pathological process in mdx mice (Yang, 2012). Other preliminary results also showed that when two recombinant adeno-associated viral (AAV) vectors respectively carrying mini-dystrophin and NF-KB/p65-shRNA were simultaneously injected into DMD mouse model, expression of mini-dystrophin via reduction of NF-KB increased remarkably. However, the injection of two kinds of AAV is, in many applications, too complex to apply in clinic.

[0006] Therefore, a more practical vehicle for introducing both the mini-dystrophin gene and NF-KB/p65-shRNA in both skeletal muscle and cardiac muscle is desired.

BRIEF SUMMARY OF THE INVENTION

[0007] In one embodiment, the invention provides a dual-cassette gene vehicle comprising cassettes for expression of both a mini-dystrophin gene and NF-KB/p65-shRNA gene in cardiac muscle tissue and skeletal muscle tissue, which is an adeno-associated viral (AAV) vector, wherein the mini-dystrophin gene is operably linked to a construct comprising a muscle-specific first promoter and a modified Mcken (MCK) enhancer and wherein the NF- KB/p65-shRNA gene is under the control of a second promoter. Also are provided pharmaceutical compositions comprising such gene vehicles and a method for ameliorating Duchenne muscular dystrophy (DMD) employing such gene delivery vehicles and pharmaceutical compositions.

[0008] No similar technology exists. The up-regulated NF-kB pathway in DMD, not only plays a key role in the downstream pathogenesis (Acharyya 2007J Clin Invest), but also clearly affects the efficiency of dystrophin gene replacement (Jayandharan, 2011 PNAS). Dual-therapeutic gene therapy strategy is innovative, should have more benefits than single mini-dystrophin replacement.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0009] Figure 1, panel A depicts schematic diagrams of single- and dual-cassette AAV constructs. Figure 1, panel B depicts Western blot of dystrophin tested in cardiac tissues. Figure 1, panel C depicts Immunofluorescence (IF) staining of dystrophin in cardiac and liver tissues. Figure 1, panel D depicts IF staining of mini-dystrophin in heart tissue, skeletal muscle tissue (DIA is diaphragm, GAS is gastrocnemius muscle, TA is tibialis anterior muscle,) and liver tissue from mdx/utrn ⁷ mice systemically treated by dual-cassette AAV and single-cassette AAV treatment. Mini-dystrophin was expressed successfully in heart, diaphragm, GAS, and TA muscle in both singe-homo and dual-homo group. Moreover, there was no expression of mini-dystrophin in liver tissue, indicating the tissue specificity. Figure 1, panel E is a graph showing the mini-dystrophin positive cell ratio in different kinds of tissue. There was no difference in heart and DIA tissue between single-homo and dual-homo group. But, the dual-homo group have much more mini-dystrophin expressed in GAS and TA muscle. (****means p<0.0001)

[0010] Figure 2, panel A, depicts IF staining of CD68, CD4, CD8 and p-P65 in gastrocnemius muscles. Figure 2, panel B, depicts IF staining of p-P65 in heart and gastrocnemius muscles. WT: wild-type, Dual-homo: Dual-cassette AAV treated dKO-homo, Single-homo: Single-cassette AAV treated dKO-homo, Ct-homo: Untreated dKO-homo. Magnification: 200x. (*means p<0.05, **means p<0.01, ***means p<0.001, ****means p<0.0001)

[0011] Figure 3, panel A, represents characterization of the transgenic and knockdown mice discussed in Example 2. Figure 3, panel B depicts Western blot analysis of expressions of dystrophin and p-P65 in GAS muscles at the ages of 2 months. Figure 3, panel C depicts Hematoxylin and Eosin (H&E) staining, and IF staining of dystrophin and b-sarcoglycan performed in GAS muscles at the same age.

[0012] Figure 4A depicts Masson’s Trichrome staining and IF staining of mlgG, CD68, CD4, and CD8 in gastrocnemius muscles of the transgenic and knockdown mice at the ages of 2 months. Figure 4B presents statistical analysis revealing that CD68, CD4, and CD8 showed significant differences between groups of different transgenic and knockdown mice. *p <0.01, **p<0.001, and ***p<0.0001. Figure 4C depicts the results of IF staining of CD8, CD4, and CD68 in rAAV-treated heart and/or gastrocnemius muscles (there are almost no CD8 positive in heart tissue). In the graphs in the lower panel, the Y axis is the positive cell number per visual frame. Single -homo has less immune cell infiltration than Ct-homo group, and dual-homo less than single-homo group. (n=3, *means p<0.05, **means p<0.01, ***means p<0.001, ****means p<0.0001). Figure 4D depicts the overall health of AAV- treated mice, wherein (1) denotes WT, (2) denotes dual-homo, (3) denotes single-homo, and (4) denotes Ct-homo. The muscle function at 1 month and 2 month of age with 0 to 200 HZ frequency is shown in the top graphs. The body weight from different groups at 1 month and 2 months of age are shown in the lower graph (*means p<0.05, **means p<0.01, ***means p<0.001, ****means p<0.0001). [0013] Figure 5A is a schematic vector diagram representing embodiments of the present invention: vector pAAV-Mlp65EnsynOpti3978 (SEQ ID NO: 1) and pAAV- M2p65EnsynOpti3978 (SEQ ID NO: 23). Figure 5B graphically indicates the location of certain genetic elements within the pAAV-Mlp65EnsynOpti3978 and pAAV- M2p65EnsynOpti3978 vectors.

[0014] Figure 6A is a schematic vector diagram representing embodiments of the present invention: vector pAAV-Mlp65EnsynOpti3837 (SEQ ID NO: 25) and pAAV- M2p65EnsynOpti3837 (SEQ ID NO: 26). Figure 6B graphically indicates the location of certain genetic elements within the pAAV-Mlp65EnsynOpti3837 and pAAV- M2p65EnsynOpti3837 vectors.

DETAILED DESCRIPTION OF THE INVENTION

[0015] In one embodiment, the invention provides a dual-cassette gene vehicle comprising cassettes for expression of both a mini-dystrophin gene and NF-KB/p65-shRNA gene in cardiac muscle tissue and skeletal muscle tissue, which is an adeno-associated viral (AAV) vector, wherein the mini-dystrophin gene is operably linked to a construct comprising a muscle-specific first promoter and a modified Mcken (MCK) promoter enhancer and wherein the NF-KB/p65-shRNA gene is operably linked to a second promoter.

[0016] The AAV vector can be derived from any strain of AAV suitable for use as a gene therapy vector, such as are known to persons of ordinary skill in the art. Purely for example, the parent AAV strain can be an AAV1, AAV2, AAV6, or AAV9, although others can be employed.

[0017] Exemplary AAV vectors in the context of the present invention (or plasmids for generating the vectors encoding, such from which the viral sequences would be understood by a person of ordinary skill in the art) are described herein as AAV-Mlp65EnsynOpti3978 or AAV- M2p65EnsynOpti3978 (including a plasmid for generating such vectors). The sequences of these exemplary AAVs for use in the context of the present invention comprise the sequence of SEQ ID NO:l, which is AAV-Mlp65EnsynOpti3978; SEQ ID NO: 23, which is AAV-M2p65EnsynOpti3978; SEQ ID NO: 25, which is AAV- Mlp65EnsynOpti3837; and SEQ ID NO: 26, which is AAV-M2p65EnsynOpti3837. [0018] AAV-M2p65EnsynOpti3978 differs from AAV- Mlp65EnsynOpti3978 in that it comprises the sequence agtccctgtctgcacctgtctcgagacaggtgcagacagggactttttttt (encoding m2p65 shRNA, SEQ ID NO:22 at bp 1701-1751 in place of the sequence encoding mlp65 present in SEQ ID NO:l (tgtgtccattgtctcactcctcgaggagtgagacaatggacacattttttt (SEQ ID NO: 17)).

[0019] Similarly, AAV-M2p65EnsynOpti3837 differs from AAV- Mlp65EnsynOpti3837 in that it comprises the sequence agtccctgtctgcacctgtctcgagacaggtgcagacagggactttttttt (encoding m2p65 shRNA, SEQ ID NO:22 at bp 1701-1751 in place of the sequence encoding mlp65 present in SEQ ID NO: 25 (tgtgtccattgtctcactcctcgaggagtgagacaatggacacattttttt (SEQ ID NO: 17)).

[0020] As noted, an AAV for use in the context of the present invention comprises a cassette for expression of a mini-dystrophin gene in cardiac muscle and in skeletal muscle. Preferably, the mini-dystrophin gene is human-codon optimized. For example, a human optimized mini-dystrophin gene including 5 rods (1,2,22,23,24) and 3 hinges (1, 3, 4), and CR domain can be used to treat DMD animal models and for human clinical trial and ultimately for treatment of CDM. See, for example, Komegay JN, et al.. Molecular Therapy. 2010, 18(8): 1501-1508, PMID: 20517298 (incorporated herein by reference in its entirety). The sequence of one such human optimized mini-dystrophin (also referred to as micro dystrophin) is opti-DysA3978 (comprising 3978 base pairs) and is set forth at sequence ID NO:2:

atggtgtggtgggaggaagtggaggactgctacgagagagaggacgtgcagaagaaa accttcaccaagtgggtgaacgcccagt tcagcaagttcggcaagcagcacatcgagaacctgttcagcgacctgcaggatggcagga gactgctggacctgctggagggcctg accggccagaagctgcccaaggagaagggcagcaccagagtgcacgccctgaacaacgtg aacaaggccctgagagtgctgca gaacaacaacgtggacctggtgaacatcggcagcaccgacatcgtggacggcaaccacaa gctgaccctgggcctgatctggaac atcatcctgcactggcaggtgaagaacgtgatgaagaacatcatggccggcctgcagcag accaacagcgagaagatcctgctgag ctgggtgaggcagagcaccagaaactacccccaggtgaacgtgatcaacttcaccacctc ctggagcgacggcctggccctgaacg ccctgatccacagccacagacccgacctgttcgactggaacagcgtggtgtgtcagcaga gcgccacccagagactggagcacgc cttcaacatcgccagataccagctgggcatcgagaagctgctggaccccgaggacgtgga caccacctaccccgacaagaaaagc atcctcatgtacattaccagcctgttccaggtgctgccccagcaggtgtccatcgaggcc atccaggaagtggaaatgctgcccaggc cccccaaagtgaccaaggaggagcacttccagctgcaccaccagatgcactacagccagc agatcacagtgagcctggcccaggg ctatgagagaaccagcagccccaagcccagattcaagagctacgcctacacccaggccgc ctacgtgaccacctccgaccccacca gaagccccttccccagccagcacctggaggcccccgaggacaagagcttcggcagcagcc tgatggagagcgaagtgaacctgg acagataccagaccgccctggaggaagtgctgtcctggctgctgagcgccgaggacaccc tgcaggcccagggcgagatcagca acgacgtggaagtggtgaaggaccagttccacacccacgagggctacatgatggatctga ccgcccaccagggcagagtgggcaa tatcctgcagctgggcagcaagctgatcggcaccggcaagctgagcgaggacgaggagac cgaagtgcaggagcagatgaacct gctgaacagcagatgggagtgcctgagagtggccagcatggagaagcagagcaacctgca cagagtgctgatggacctgcagaac cagaagctgaaggagctgaacgactggctgaccaagaccgaggagcggaccagaaagatg gaggaggagcccctgggccccga cctggaggacctgaagagacaggtgcagcagcacaaagtgctgcaggaggacctggagca ggagcaggtgcgcgtgaacagcct gacccacatggtggtggtcgtggacgagagcagcggcgaccacgccacagccgccctgga agagcagctgaaagtgctgggcga cagatgggccaatatttgtaggtggaccgaggacagatgggtgctgctgcaggaccagcc cgacctggcccctggcctgaccacca tcggcgccagccccacccagaccgtgaccctggtgacccagcccgtggtgacaaaggaga ccgccatcagcaagctggagatgc ccagctccctgatgctggaagtgcccacccaccgcctgctccagcagttccccctggacc tggagaagttcctggcctggctgaccg aggccgaaaccaccgccaatgtgctccaggacgccactagaaaggagaggctgctggagg acagcaagggcgtgaaagagctga tgaagcagtggcaggatctgcagggcgaaatcgaggcccacaccgacgtgtaccacaacc tggacgagaacagccagaagattct gaggagcctggagggcagcgacgacgccgtcctgctccagaggaggctggacaacatgaa cttcaagtggagcgagctgcggaa gaagagcctgaacatccggagccacctggaagccagcagcgaccagtggaagagactgca cctgagcctgcaggagctgctggt gtggctgcagctgaaggacgacgagctgagcagacaggcccccatcggcggcgacttccc cgccgtgcagaagcagaacgacgt gcaccgggccttcaagagggagctgaaaaccaaggaacccgtgatcatgagcaccctgga gacagtgcggatcttcctgaccgag cagcccctggagggactggagaagctgtaccaggagcccagagagctgccccccgaggag agagcccagaacgtgaccaggct gctgagaaagcaggccgaggaagtgaataccgagtgggagaagctgaatctgcacagcgc cgactggcagagaaagatcgacga gaccctggagagactccaggaactgcaggaagccaccgacgagctggacctgaagctgag acaggccgaagtgatcaagggcag ctggcagcctgtgggcgatctgctgatcgactccctgcaggatcacctggagaaagtgaa ggccctgcggggcgagatcgcccccc tgaaggagaatgtgagccacgtgaacgacctggccagacagctgaccaccctgggcatcc agctgagcccctacaacctgagcac actggaggatctgaacacccggtggaaactgctgcaggtggccgtggaggatagagtgag gcagctgcacgaagcccacagaga cttcggccctgcctcccagcacttcctgagcaccagcgtgcagggcccctgggagagagc catctcccccaacaaagtgccctacta catcaaccacgagacccagaccacctgctgggaccaccctaagatgaccgagctgtatca gagcctggccgacctgaacaatgtgc ggttcagcgcctacagaaccgccatgaagctgcggagactgcagaaggccctgtgcctgg atctgctgagcctgagcgccgcctgc gacgccctggaccagcacaacctgaagcagaatgaccagcccatggacatcctgcagatc atcaactgcctgaccacaatctacgac cggctggaacaggagcacaacaacctggtgaatgtgcccctgtgcgtggacatgtgcctg aattggctgctgaacgtgtacgacacc ggcaggaccggcagaatccgcgtgctgagcttcaagaccggcatcatcagcctgtgcaag gcccacctggaggataagtaccgcta cctgttcaagcaggtggccagcagcaccggcttctgcgatcagaggagactgggcctgct gctgcacgatagcatccagatccctag gcagctgggcgaagtggccagctttggcggcagcaacatcgagccctctgtgaggagctg cttccagttcgccaacaacaagcccg agatcgaggccgccctgttcctggactggatgaggctggagcctcagagcatggtgtggc tgcctgtgctgcacagagtggccgcc gccgagaccgccaagcaccaggccaagtgcaatatctgcaaggagtgccccatcatcggc ttccggtacaggagcctgaagcactt caactacgacatctgccagagctgctttttcagcggcagagtggccaagggccacaaaat gcactaccccatggtggagtactgcac ccccaccacctccggcgaggatgtgagagacttcgccaaagtgctgaagaataagttccg gaccaagcggtactttgccaagcaccc caggatgggctacctgcccgtgcagaccgtgctggaaggcgacaacatggagacctga. This sequence is included within SEQ ID N0: 1 and SEQ ID NO: 23 as base-pairs 2365-6342.

[0021] Within SEQ ID NO: 2, the following domains of the human optimized mini dystrophin can be identified. Their nucleotide position within the coding sequence for the human optimized mini-dystrophin gene is indicated in Figure 5B:

[0022] N-TERMINUS OF HUMAN DYSTROPHIN (756 base pairs); SEQ ID NO:3: atggtgtggtgggaggaagtggaggactgctacgagagagaggacgtgcagaagaaaacc ttcaccaagtgggtgaacgcccagt tcagcaagttcggcaagcagcacatcgagaacctgttcagcgacctgcaggatggcagga gactgctggacctgctggagggcctg accggccagaagctgcccaaggagaagggcagcaccagagtgcacgccctgaacaacgtg aacaaggccctgagagtgctgca gaacaacaacgtggacctggtgaacatcggcagcaccgacatcgtggacggcaaccacaa gctgaccctgggcctgatctggaac atcatcctgcactggcaggtgaagaacgtgatgaagaacatcatggccggcctgcagcag accaacagcgagaagatcctgctgag ctgggtgaggcagagcaccagaaactacccccaggtgaacgtgatcaacttcaccacctc ctggagcgacggcctggccctgaacg ccctgatccacagccacagacccgacctgttcgactggaacagcgtggtgtgtcagcaga gcgccacccagagactggagcacgc cttcaacatcgccagataccagctgggcatcgagaagctgctggaccccgaggacgtgga caccacctaccccgacaagaaaagc atcctcatgtacattaccagcctgttccaggtgctgccccagcaggtgtccatcgaggcc atccaggaagtggaa;

[0023] HINGE 1 (225 base pairs); SEQ ID NO:4:

atgctgcccaggccccccaaagtgaccaaggaggagcacttccagctgcaccaccag atgcactacagccagcagatcacagtga gcctggcccagggctatgagagaaccagcagccccaagcccagattcaagagctacgcct acacccaggccgcctacgtgaccac ctccgaccccaccagaagccccttccccagccagcacctggaggcccccgaggac;

[0024] ROD1 (333 base pairs); SEQ ID NO:5:

agcgaagtgaacctggacagataccagaccgccctggaggaagtgctgtcctggctg ctgagcgccgaggacaccctgcaggccc agggcgagatcagcaacgacgtggaagtggtgaaggaccagttccacacccacgagggct acatgatggatctgaccgcccacca gggcagagtgggcaatatcctgcagctgggcagcaagctgatcggcaccggcaagctgag cgaggacgaggagaccgaagtgc aggagcagatgaacctgctgaacagcagatgggagtgcctgagagtggccagcatggaga agcagagcaacctgcacaga;

[0025] ROD2 (327 base pairs) ; SEQ ID NO : 6 :

gtgctgatggacctgcagaaccagaagctgaaggagctgaacgactggctgaccaag accgaggagcggaccagaaagatggag gaggagcccctgggccccgacctggaggacctgaagagacaggtgcagcagcacaaagtg ctgcaggaggacctggagcagga gcaggtgcgcgtgaacagcctgacccacatggtggtggtcgtggacgagagcagcggcga ccacgccacagccgccctggaaga gcagctgaaagtgctgggcgacagatgggccaatatttgtaggtggaccgaggacagatg ggtgctgctgcaggac;

[0026] HINGE3 (141 base pairs); SEQ ID N0:7:

cagcccgacctggcccctggcctgaccaccatcggcgccagccccacccagaccgtg accctggtgacccagcccgtggtgacaa aggagaccgccatcagcaagctggagatgcccagctccctgatgctggaagtgccc;

[0027] ROD22 (348 base pairs); SEQ ID N0:8;

acccaccgcctgctccagcagttccccctggacctggagaagttcctggcctggctg accgaggccgaaaccaccgccaatgtgctc caggacgccactagaaaggagaggctgctggaggacagcaagggcgtgaaagagctgatg aagcagtggcaggatctgcaggg cgaaatcgaggcccacaccgacgtgtaccacaacctggacgagaacagccagaagattct gaggagcctggagggcagcgacga cgccgtcctgctccagaggaggctggacaacatgaacttcaagtggagcgagctgcggaa gaagagcctgaacatccggagccac ctggaagcc;

[0028] ROD23 (387 base pairs); SEQ ID N0:9:

agcagcgaccagtggaagagactgcacctgagcctgcaggagctgctggtgtggctg cagctgaaggacgacgagctgagcaga caggcccccatcggcggcgacttccccgccgtgcagaagcagaacgacgtgcaccgggcc ttcaagagggagctgaaaaccaag gaacccgtgatcatgagcaccctggagacagtgcggatcttcctgaccgagcagcccctg gagggactggagaagctgtaccagga gcccagagagctgccccccgaggagagagcccagaacgtgaccaggctgctgagaaagca ggccgaggaagtgaataccgagt gggagaagctgaatctgcacagcgccgactggcagagaaagatcgacgag;

[0029] ROD24 (327 base pairs); SEQ ID NO: 10:

accctggagagactccaggaactgcaggaagccaccgacgagctggacctgaagctg agacaggccgaagtgatcaagggcagc tggcagcctgtgggcgatctgctgatcgactccctgcaggatcacctggagaaagtgaag gccctgcggggcgagatcgcccccct gaaggagaatgtgagccacgtgaacgacctggccagacagctgaccaccctgggcatcca gctgagcccctacaacctgagcaca ctggaggatctgaacacccggtggaaactgctgcaggtggccgtggaggatagagtgagg cagctgcacgaa;

[0030] HINGE4 (216 base pairs) ; SEQ ID NO : 11 :

gcccacagagacttcggccctgcctcccagcacttcctgagcaccagcgtgcagggc ccctgggagagagccatctcccccaacaa agtgccctactacatcaaccacgagacccagaccacctgctgggaccaccctaagatgac cgagctgtatcagagcctggccgacct gaacaatgtgcggttcagcgcctacagaaccgccatgaagctg;

[0031] CYSTEINE-RICH (CR)-DOMAIN (888 base pairs); SEQ ID NO: 12:

cggagactgcagaaggccctgtgcctggatctgctgagcctgagcgccgcctgcgac gccctggaccagcacaacctgaagcaga atgaccagcccatggacatcctgcagatcatcaactgcctgaccacaatctacgaccggc tggaacaggagcacaacaacctggtga atgtgcccctgtgcgtggacatgtgcctgaattggctgctgaacgtgtacgacaccggca ggaccggcagaatccgcgtgctgagctt caagaccggcatcatcagcctgtgcaaggcccacctggaggataagtaccgctacctgtt caagcaggtggccagcagcaccggct tctgcgatcagaggagactgggcctgctgctgcacgatagcatccagatccctaggcagc tgggcgaagtggccagctttggcggc agcaacatcgagccctctgtgaggagctgcttccagttcgccaacaacaagcccgagatc gaggccgccctgttcctggactggatg aggctggagcctcagagcatggtgtggctgcctgtgctgcacagagtggccgccgccgag accgccaagcaccaggccaagtgca atatctgcaaggagtgccccatcatcggcttccggtacaggagcctgaagcacttcaact acgacatctgccagagctgctttttcagc ggcagagtggccaagggccacaaaatgcactaccccatggtggagtactgcacccccacc acctccggcgaggatgtgagagactt cgccaaagtgctgaagaataagttccggaccaagcggtactttgccaagcaccccaggat gggctacctgcccgtgcagaccgtgct ggaaggcgacaacatggagacc; and

[0032] STOP CODON (3 base pairs); SEQ ID NO: 13: tga

[0033] Thus, a suitable human optimized mini-dystrophin gene for use in the context of the present invention can comprise, consist essentially of, or consist of SEQ ID NO: 2 and can comprise a domain represented by a sequence selected from the group of sequences consisting of any of SEQ ID NOs:3-13, although such domain(s) can comprise, consist essentially of, or consist of such sequence. Of course it will be apparent that the human optimized mini-dystrophin gene can comprise a functional variant of SEQ ID NOs: 2-13. A functional variant, in this context, indicates that a variant of a given sequence can be employed so long as the encoded gene product retains the function of the reference encoded molecule. Thus, sequence variants from any of SEQ ID NOs: 2-13 can be employed for encoding a suitable human optimized mini-dystrophin in the context of the present invention. Such variants can vary from the exemplary sequences set forth herein by retaining from about 75% to about 99% to one or more of SEQ ID Nos:2-13, such as at least about 75%, such as at least about 80%, or at least about 90%, at least about 95%, or at least about 99% sequence identity to one or more of SEQ ID Nos:2-13.

[0034] Other mini-dystrophins (micro-dystrophins) with a smaller size for use in the invention include mini-dystrophin genes (human or canine) containing an N-terminus, 5 rods (Rods 1,2,22,23,24), 2 hinges (Hinge 1 and 4), and a cysteine-rich domain, such as hADys3849 and hOptiADys3837. hOptiADys3837 differs from hADys3849 in that (i) in that hOptiADys3837 is codon-optimized and (ii) 12 bases of full exon 79 have been removed.

The essential functional domains are the same. The sequence of hOptiADys3837 (also referred to as opti-DysA3837), which comprises 3837 base pairs, is set forth in SEQ ID NO: 27. This sequence is included within SEQ ID NOs:25 and 26 as base-pairs 2365-6201 (Fig. 6B). Exemplary mini-dystrophins are described in Wang et al., PNAS USA, 97(25): 13714- 13719 (2000), PMID 11095710; Wang et al., Gene Therapy, 15(15): 1099-1106 (2008),

PMID 18432277; Wang et al., Gene Therapy, 15(22): 1489-1499 (2008), PMID 18563184; Romesh et al., Journal of Muscle Research and Cell Motility, 27(1): 53-67 (2006), PMID 16496225; and Koppanati et al., Gene Therapy, 17(11): 1355-1362 (2010), PMID 20535217, all of which are incorporated by reference.

[0035] Thus, a suitable human optimized mini-dystrophin gene for use in the context of the present invention can comprise, consist essentially of, or consist of SEQ ID NO: 27 and can comprise a domain represented by a sequence selected from the group of sequences consisting of any of SEQ ID NOs: 3-6 and 8-13, although such domain(s) can comprise, consist essentially of, or consist of such sequence. Of course it will be apparent that the human optimized mini-dystrophin gene can comprise a functional variant of SEQ ID NOs: 3- 6, 8-13, and 27. Such variants can vary from the exemplary sequences set forth herein by retaining from about 75% to about 99% to one or more of SEQ ID NOs: 3-6, 8-13, and 27, such as at least about 75%, such as at least about 80%, or at least about 90%, at least about 95%, or at least about 99% sequence identity to one or more of SEQ ID NOs: 3-6, 8-13, and 27.

[0036] As noted, within the AAV useful in the context of the present invention, the cassette for expression of the mini-dystrophin gene in cardiac muscle and in skeletal muscle comprises muscle-specific first promoter and a modified MCK promoter enhancer. Within this cassette, the mini-dystrophin gene is operably linked to the muscle- specific first promoter and the modified MCK promoter enhancer such that the expression of the mini-dystrophin is under the control of the muscle-specific first promoter and the modified MCK promoter enhancer.

[0037] For use in the context of the present invention, the modified muscle MCK promoter enhancer which permits muscle-specific expression. Preferably, the modified muscle MCK promoter enhancer truncated version of the MCK promoter, which is useful given the size limitations of the AAV genome. For one proffered modified muscle MCK promoter enhancer, in addition to muscle-specific cis-elements, mef-2, right e-box (mefl) and a/t-rich elements can be maintained in the enhancer region for the tissue-specificity in differentiated muscle, including two right e-boxes and one s5 modified region. See Bing Wang, et al., Gene Ther., 2008, 15:1489-1499. PMID: 18563184 (incorporated herein by reference in its entirety). The sequence of one preferred modified muscle MCK promoter enhancer (comprising 216 base pairs) is set forth at nucleotides 1757-1972 of SEQ ID NO:l (Fig. 5B), SEQ ID NO: 23 (Fig. 5B), SEQ ID NO: 25 (Fig. 6B), and SEQ ID NO: 26 (Fig.

6B) and is represented by sequence ID NO: 14:

ccactacgggtctaggctgcccatgtaaggaggcaaggcctggggacacccgagatg cctggttataattaaccccaacacctgctg cccccccccccccaacacctgctgcctgagcctgagcggttaccccaccccggtgcctgg gtcttaggctctgtacaccatggagga gaagctcgctctaaaaataaccctgtccctggtggatcccct.

[0038] Thus, a suitable modified muscle MCK promoter enhancer for use in the context of the present invention can comprise, consist essentially of, or consist of SEQ ID NO: 14. Of course it will be apparent that the modified muscle MCK promoter can comprise a functional variant of SEQ ID NO: 14. A functional variant, in this context, indicates that a variant of a given sequence can be employed so long as the variant retains the function of the reference sequence. Thus, sequence variants from SEQ ID NO: 14 can be employed as modified muscle MCK promoter enhancer in the context of the present invention. Such variants can vary from the exemplary sequence set forth herein by retaining from about 75% to about 99% sequence identity to SEQ ID NO: 14, such as at least about 75%, such as at least about 80%, or at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 14.

[0039] For use in the context of the present invention, the muscle-specific first promoter permits muscle-specific expression. One preferred modified muscle-specific first promoter is a synthetic promoter (Syn), which, in addition to muscle-specific cis-elements, comprises a MEF-2 and a right e-box. See Bing Wang, et al., Gene Ther., 2008, 15:1489-1499. PMID: 18563184 (incorporated herein by reference in its entirety). The sequence of one preferred muscle-specific first promoter (comprising 317 base pairs) is set forth at nucleotides 1973- 2289 of SEQ ID NO:l (Fig. 5B), SEQ ID NO: 23 (Fig. 5B), SEQ ID NO: 25 (Fig. 6B), and SEQ ID NO: 26 (Fig. 6B) and is represented by sequence ID NO: 15:

gcatgcggccgtccgccctcggcaccattcctcacgacaccgaaatatggcgacggg tgaggaatggtggggagttatttttagagc ggtgaggaatggtgggcaggcagcaggtgttgggggagttatttttagagcggggagtta tttttagagcggtgaggaatggtggaca ccgaaatatggcgacgggtgaggaatggtgccgtcgccatatttgggtgtcccgtccgcc ctcggccggggccgcattcctggggg ccgggcggtgctcccgcccgcctcgataaaaggctccggggccggcggcggcccac.

[0040] Thus, a suitable muscle-specific first promoter for use in the context of the present invention can comprise, consist essentially of, or consist of SEQ ID NO: 15. Of course it will be apparent that the muscle-specific first promoter can comprise a functional variant of SEQ ID NO: 15. A functional variant, in this context, indicates that a variant of a given sequence can be employed so long as the variant retains the function of the reference sequence. Thus, sequence variants from SEQ ID NO: 15 can be employed as muscle-specific first promoter in the context of the present invention. Such variants can vary from the exemplary sequence set forth herein by retaining from about 75% to about 99% sequence identity to SEQ ID NO: 15, such as at least about 75%, such as at least about 80%, or at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 15.

[0041] The cassette for expression of the mini-dystrophin gene in cardiac muscle and in skeletal muscle also can contain other desired elements for enhancing expression of the mini dystrophin gene. For example, the exemplary AAV sequence set forth in SEQ ID NO:l includes a Kozak consensus sequence (GCCACC (SEQ ID NO: 16)), which serves as an enhancer for translation of the mini-dystrophin gene. See Bing Wang, et al., Journal of Orthopaedic Research, 2009, 27:4,421-42,. PMID: 18973234 (incorporated herein by reference in its entirety).

[0042] As noted, an AAV for use in the context of the present invention comprises a cassette in which the NF-KB/p65-shRNA gene is operably linked to a second promoter. The mouse NF-KB/p65-shRNA cassette specifically silences subunit 65 of NF-icB/p65. See Qing Yang, et al. Gene Ther. 2012, 19: 1196-1204, PubMed PMID: 22278411 (incorporated herein by reference in its entirety). Without being bound by theory, it is believed that the specific silencing of subunit 65 of NF-icB/p65 reduces inflammation, which can lead to more robust expression of the mini-dystrophin gene. A mouse specific NF-KB/p65-shRNA includes 51 base pairs and is included at nucleotides 1701-1751 of SEQ ID NO: l (Fig 5B) and SEQ ID NO: 25 (Fig. 6B). Its sequence is tgtgtccattgtctcactcctcgaggagtgagacaatggacacattttttt (SEQ ID NO: 17). Another mouse specific NF-KB/p65-shRNA includes 51 base pairs and is included at nucleotides 1701-1751 of SEQ ID NO:23 (Fig 5B) and SEQ ID NO: 26 (Fig. 6B). Its sequence is agtccctgtctgcacctgtctcgagacaggtgcagacagggactttttttt (SEQ ID NO:22).

Furthermore, for use in human clinical trials or in treatment of DMD, an shRNA sequence based on the human NF-KB sequence preferably is employed. Human siRNA oligo sequence, named (REL1096 (gattgaggagaaacgtaaatt SEQ ID NO:24)) to inhibit the NF-kB the induction of inflammatory cytokine in human synoviocytes (Lee, Ui Jin et al, . Mol Biol Rep (2008) 35:291-298 (incorporated herein in its entirety by reference)) and its inhibition of constitutive or tumor necrosis factor-induced NF-kB in cancer cells (Jutooru, Indira et al.. J. Biol. Chem. (2010), 285: 25332-25344 (incorporated herein in its entirety by reference)) is known to persons of ordinary skill.

[0043] Thus, a suitable NF-KB/p65-shRNA for use in the context of the present invention can comprise, consist essentially of, or consist of SEQ ID NO: 17, SEQ ID NO:22, or SEQ ID NO:24. Of course it will be apparent that the NF-KB/p65-shRNA can comprise a functional variant of SEQ ID NO: 17 or SEQ ID NO:22. A functional variant, in this context, indicates that a variant of a given sequence can be employed so long as the variant retains the function of the reference sequence. Thus, sequence variants from SEQ ID NO: 17, SEQ ID NO:22, or SEQ ID NO:24 can be employed, which inhibit in the context of the present invention. Such variants can vary from the exemplary sequence set forth herein by retaining from about 75% to about 99% sequence identity to SEQ ID NO: 17, such as at least about 75%, such as at least about 80%, or at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 17, SEQ ID NO:22, or SEQ ID NO:24.

[0044] As noted, the NF-KB/p65-shRNA is operably linked to (under transcriptional control) of a second promoter. A preferred promoter for this purpose is the U6 promoter, most preferably the human U6 promoter. See Qing Yang, et al. Gene Ther. 2012, 19:1196- 1204, PubMed PMID: 22278411 (incorporated herein by reference in its entirety). The sequence of the human U6 promoter includes 241 base pairs and is included at nucleotides 1452-1692 of SEQ ID NO: l (Fig 5B), SEQ ID NO: 23 (Fig. 5B), SEQ ID NO: 25 (Fig. 6B), and SEQ ID NO: 26 (Fig. 6B). Its sequence is (SEQ ID NO: 18) is:

gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataattagaattaatttgactgtaaacacaaag atattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttta aaattatgttttaaaatggactatcatatgcttacc gtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggac.

[0045] Thus, a suitable second promoter for use in the context of the present invention can comprise, consist essentially of, or consist of SEQ ID NO: 18. Of course it will be apparent that the second promoter a functional variant of SEQ ID NO: 18. A functional variant, in this context, indicates that a variant of a given sequence can be employed so long as the variant retains the function of the reference sequence. Thus, sequence variants from SEQ ID NO: 18 can be employed as the second promoter in the context of the present invention. Such variants can vary from the exemplary sequence set forth herein by retaining from about 75% to about 99% sequence identity to SEQ ID NO: 18, such as at least about 75%, such as at least about 80%, or at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 18.

[0046] Furthermore, while the U6 promoter can be used to drive expression of the NF- KB/p65-shRNA, other promoters can suitably be used as the second promoter. Many promoters are known to persons of ordinary skill in the art. Preferably a promoter operably linked to drive expression of the KB/p65-shRNA should have activity in both immune cells and dytrophic muscle cells. Examples of some suitable promoters are Polymerase III promoters such as human HI and U6.

[0047] Aside from dual cassettes for expression of both a mini-dystrophin gene and NF- KB/p65-shRNA gene in cardiac muscle tissue and skeletal muscle tissue, an AAV for use in the context of the present can comprise other elements. For example, 5’ and 3’ inverted terminal repeats (ITRs) facilitate packaging the genome into the vector package. See Xiao, et ak, J. Virol. 1998;72(3):2224-32. PubMed PMTD: 9499080 (incorporated herein by reference in its entirety). Sequences of such 3’ and 5’ ITRs are known to persons of ordinary skill in the art, but examples include:

ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtc gcccgacgcccgggctttgcccgggcggc ctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct (SEQ ID NO: 19 - 5’ ITR and)

aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactg aggccgcccgggcaaagcccgggcgtcgg gcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa (SEQ ID NO:20 - 3’ITR). Furthermore, inclusion of a poly(A) signal sequence can enhance gene expression from the vector. See Bing Wang, et ak, Proc. Natl. Acad. Sci. U S A., 2000, 97(25):13714-13719, PMTD: 11095710 (incorporated herein by reference in its entirety). PolyA sequences are well known to persons of ordinary skill in the art, but a non- limiting example of one suitable poly(A) is tcgaggcctaataaagagctcagatgcatcgatcagagtgtgttggttttttgtgtgaga (SEQ ID NO:21). The location of these sequences within SEQ ID NOs: 1, 23, 25, and 26 is depicted in Figs. 5B and 6B). Other useful genetic elements for inclusion include genes conferring resistance to antibiotics, such as ampicillin, which is commonly used during culture of viral packaging cells to select for infected cells to increase viral titer. [0048] The AAV for use as the dual-cassette gene vehicle comprising cassettes for expression of both a mini-dystrophin gene and NF-KB/p65-shRNA gene in cardiac muscle tissue and skeletal muscle tissue in the context of the present invention can be generated by standard molecular and cellular techniques known to persons of ordinary skill. For example, a plasmid comprising the dual cassettes and other AAV sequences can be can engineered by standard molecular techniques. The plasmid then can be employed to generate a viral vector, for example by transfecting it into a suitable packaging cell line (such as HEK 293 cells or HEK 293T cells, but other cell lines can be employed). Transformants can be selected from the population, for example by exposing the culture to compound for which the AAV vector genome confers resistance (e.g., ampicillin). Once the cells have produced AAV packages, they can be purified (for example, using CsCl gradients or via other methods known to persons of ordinary skill). Thereafter they can be stored (e.g., cryopreserved) or formulated for use.

[0049] In an embodiment, the invention provides a pharmaceutical composition comprising the inventive gene vehicle comprising cassettes for expression of both a mini dystrophin gene and NF-KB/p65-shRNA gene in cardiac muscle tissue and skeletal muscle tissue (the AAV described herein) and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can include any carrier known to persons of ordinary skill. The carrier of the composition can be any suitable carrier for the vector. The carrier typically will be liquid and formulated for injection, but also can be solid, or a combination of liquid and solid components. The carrier desirably is a pharmaceutically acceptable (e.g., a physiologically or pharmacologically acceptable) carrier (e.g., excipient or diluent).

Pharmaceutically acceptable carriers are well known and are readily available. The choice of carrier will be determined, at least in part, by the particular vector and the particular method used to administer the composition.

[0050] The composition can further comprise any other suitable components, especially for enhancing the stability of the composition and/or its end-use. Accordingly, there is a wide variety of suitable formulations of the composition of the invention. The following formulations and methods are merely exemplary and are in no way limiting.

[0051] Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood or other tissue of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water, for injections, immediately prior to use.

[0052] In addition, the composition can comprise additional therapeutic or biologically- active agents. For example, therapeutic factors useful in the treatment of a particular indication can be present. Factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the vector and physiological distress. Immune system suppressors can be administered with the composition method to reduce any immune response to the vector itself or associated with a disorder. Alternatively, immune enhancers can be included in the composition to upregulate the body's natural defenses against disease. Antibiotics, i.e., microbicides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders.

[0053] In an embodiment, the invention provides a method for ameliorating Duchenne muscular dystrophy (DMD), the method comprising administrating the gene vehicle (AAV) or the pharmaceutical composition as described herein a patient or subject who is suffering from or at risk of developing DMD in an amount and at a location to ameliorate DMD. In this context the patient or subject typically is a human (e.g., a patient suffering from and being treated for DMD or a subject of a clinical trial). However, the method also can be applied to non-human animals for assessment of gene expression or animal models of DMD. Such non-human animals typically are those commonly employed in laboratory studies (e.g., mice, rats, dogs, non-human primates, etc.).

[0054] In performance of the method, the gene vehicle or the pharmaceutical composition typically is administered by parenteral (e.g., intraperitoneal, intravenous, intramuscular, etc.) injection. However other routes of delivery may be suitable and employed by a treating physician or for a clinical trial protocol. [0055] The amount of viral particles to deliver in performing the inventive method can be determined by a physician or laboratory researcher and may depend on the size of the subject or species of the subject or patient. However enough of the gene vehicle (AAV) should be delivered to infect cardiac and skeletal muscles to result in the expression from the dual cassettes within the inventive gene vehicle. For laboratory studies, as reported in the Examples below, 10 ¹¹ AAV genomes were injected intraperitoneally, although somewhat less than this may be sufficient. However, for human patients or subjects, a greater number of AAV genomes may be necessary or desirable. For example, in carrying out the inventive method, at least 10 ⁸ AAV genomes, or at least 10 ⁹ AAV genomes, or at least 10 ¹⁰ AAV genomes, or at least 10 ¹¹ AAV genomes, or at least at least 10 ¹² AAV genomes, or at least 10 ¹³ AAV genomes, or at least 10 ¹⁴ AAV genomes (or at least“about” such numbers of AAV genomes may be employed. While the upper limit of AAV genomes to administer to the patient or subject may be, in part, depending on how concentrated a pharmaceutical composition can be formulated, up to 10 ²⁰ AAV genomes, or up to 10 ¹⁹ AAV genomes, or up to 10 ¹⁸ AAV genomes, or up to 10 ¹⁷ AAV genomes, or up to 10 ¹⁶ AAV genomes, or up to 10 AAV genomes (or up to“about” such numbers of AAV genomes) may suitably be administered. Greater or lesser amounts of AAV genomes can be employed to; the dose ultimately decided by a physician, laboratory researcher, or clinical trial protocol. Typical doses are 2xl0 ¹⁰ -lxlO ¹¹ viral genome/kg (patient body) (see Duan, DS, Mol. Ther. 2018, 26:2337-2356, Table 4 (incorporated herein by reference in its entirety)

[0056] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES

[0057] In the Examples below, a such dual-cassette AAV vector having a modified MCK enhancer (Mcken) and a muscle- specific synthetic promoter (Syn) driving human-codon optimized mini-dystrophin gene (Wang, 2008 Gene Therapy, and Kornegay, 2010 Molecular Therapy) was generated. This ensured efficient and specific expression of mini-dystrophin gene in whole-body muscles of a severe DMD murine model, the dystrophin/utrophin double knockout (dys-/-:utro-/-, dKO-homo) mouse as demonstrated by high level expression in cardiac muscle. Such dual-therapeutic approach also resulted in efficient inhibition of chronic inflammation, especially in skeletal muscle of this severe DMD model.

[0058] Next, through cross-breeding of Tg.dKO-het (dys-/-:utro+/- ).p65+/- mice, both Tg.dKO-homo.p65+/+ and Tg.dKO-homo.p65+/- mice were obtained. It also was observed that synergistic therapeutic effects were beneficial from the genetic ablation of the p65 subunit of NF-KB (p65+/-) accompanied with a transgenic human mini-dystrophin gene in a severe DMD murine model, without toxicity.

[0059] In summary, a novel AAV vector with a dual-cassette containing a compact promoter for muscle-specific dystrophin gene expression and specific shRNA of NF-KB for anti-inflammation may enhance therapeutic efficiency and safety of DMD gene therapy.

EXAMPLE 1

[0060] This Example demonstrates the optimization of a dual-cassette AAV vector via the design of a modified MCK enhancer (Mcken) and a muscle- specific synthetic promoter (Syn) driving human-codon optimized mini-dystrophin gene (Wang, 2008 and Komegay, 2010). This ensured efficient and specific expression of mini-dystrophin gene in whole-body muscles of a severe DMD murine model, especially in cardiac muscle. Such dual-therapeutic approach also resulted in efficient inhibition of chronic inflammation, especially in skeletal muscle.

METHODS

[0061] IACUC protocol was approved for all strains of mice in this study. Wild-type (WT, C57/BL10) mice were purchased from Jackson Laboratory. The dystrophin/utrophin double knockout (dys-/-:utro-/-, dKO-homo) mice were derived from in-house colony through breeding of heterozygous dystrophin/utrophin double knockout mice (dys-/-:utro+/-). As shown in Figure 1, panel A, the single cassette of mini-dystrophin AAV vector was generated that a computer-codon optimized mini-dystrophin gene, including N-terminus, 5 Rods (1 ,2, 22, 23, 24), 3 Hinges (1, 3, 4), and CR terminus, was cloned into a single AAV vector and driven by a MCK modified enhancer regulated muscle synthetic promoter. Based on published functional mouse NF-KB/p65 mRNA sequences (Yang, 2012), two versions (ml and m2) of mouse p65/shRNA driven by a U6 promoter that were designed and respectively cloned into the single cassette pAAV(ss)-Mcken-Syn-hOptiDys3978 (Kornegay, 2010). The plasmids were purified through CsCl gradient ultracentrifugation. One such plasmid, pAAV- Mlp65EnsynOpti3978 is schematically depicted in Figures 5A and 5B, and its sequence is set forth in SEQ ID NO: 1.

[0062] The AAV9 vectors were packaged by co-transfection in 293 cells and purified by twice CsCl gradient ultracentrifugation according to Yang, 2012. A single I.P. injection with 50pl (lxlO ¹¹ viral genome particles) virus was performed to 5-day-old dKO-homo pups, and the cryostat sections of cardiac and skeletal muscle, as well as liver tissue were analyzed at the ages of 8 weeks. Sections were applied for immunofluorescence (IF) staining with antibodies against human dystrophin (Rods 1 & 2), phosphorylated NF-KB/p65 (#8242, Cell Signaling), CD4 (BD550280, BD Biosciences), CD 8 (ab22378, Abeam), CD68 (#9936, Cell Signaling), ColIV (ab6586, Abeam). Nuclei were also stained with DAPI (in blue). Images were taken at 200X magnification.

RESULTS

[0063] As shown in Figure 1, panel B, cardiac muscles were tested by western blot assay, efficient expressions of full-length dystrophin (450 kDa) in WT mice and mini-dystrophin (150 kDa) in dKO-homo mice treated with either single or dual-cassette vector were observed. IF staining of dystrophin also confirmed the specificity of muscle-targeted gene delivery in heart and skeletal muscle tissue (diaphragm, gastrocnemius muscle, and tibialis anterior muscle), as demonstrated by no expression of dystrophin in liver tissue (Figure 1, panels C and D). There was no difference the dystrophin positive cell ratio in heart and diaphragm tissue between mice treated with either single or dual-cassette vector (Figure 1, panel E). But, the mice treated with dual-cassette vector have much more mini-dystrophin expressed in gastrocnemius muscle and tibialis anterior muscle (Figure 1, panel E).

[0064] The results demonstrated reduced inflammation in skeletal muscle treated by the dual-cassette AAV vector, which shows synergistic effects can be achieved by a single AAV vector combining dystrophin gene replacement and anti-inflammation.

[0065] Gastrocnemius muscles (GAS) were IF stained by p-P65 to determine the level of active NF-KB, CD4 and CD8 to evaluate immune cell infiltration, and CD68 to detect inflammatory macrophage. As shown in Figure 2A, GAS muscle treated with dual-cassette AAV represented the less inflammatory markers such as CD4, CD8 and CD68 via the reduction of NF-KB, comparing the treatment with a single-cassette AAV. WT GAS did not show inflammation in muscle. However, the non-treated GAS of dKO-homo mice had remarkable inflammation· Figure 2B demonstrates the IF staining of p-P65 in heart and GAS muscle. In heart tissue, there are almost no positive cells in all groups. But, in GAS muscle, the dual-homo group showed much less p-P65 than single -homo and untreated homo mice group (Ct-homo).

[0066] These results demonstrate that there is a remarkable reduction of active NF-KB (p- P65) observed in skeletal muscle treated by dual-cassette AAV compared to single cassette AAV or no treatment. Unlike in skeletal muscle in mdx/utm-/- mice, there was no significant difference of active NF-KB (p-P65) in cardiac muscle, no matter whether the mice were treated with dual-cassette AAV or single-cassette AAV or no treatment.

DISCUSSION

[0067] The systemic efficacy of muscle-targeted gene replacement combining inhibition of NF-KB via a single AAV vehicle was investigated, which ensures efficient expression of computer-codon optimized human mini-dystrophin and reduction of inflammation in whole body muscles. These results demonstrate that a novel AAV vector with a dual-cassette containing a compact promoter for muscle-specific dystrophin gene expression and specific shRNA of NF-KB for anti-inflammation may enhance therapeutic efficiency and safety of DMD gene therapy, in clinical trials or otherwise.

EXAMPLE 2

[0068] This Example demonstrates that synergistic therapeutic effects may be beneficial from the genetic ablation of the p65 subunit of NF-KB (p65+/-) accompanied with a transgenic human mini-dystrophin gene in a severe DMD murine model (dys-/-:utro-/-, dKO- homo). METHODS

[0069] IACUC protocol was approved for all strains of mice in this study. Wild-type (WT, C57/BL10) and mdx (dys-/-) mice were purchased from Jackson Laboratory. The mdx.p65+/- and human mini-dystrophin (Dys \3990).mdx (Tg.mdx) mice (Yin, 2017 and Wang, 2000) were derived from an in-house colony. Through cross-breeding of Tg.dKO-het (dys-/-:utro+/-).p65+/- mice, both Tg.dKO-homo.p65+/+ and Tg.dKO-homo.p65+/- mice were obtained (Figure 3, panel A).

[0070] Cryostat sections were prepared using gastrocnemius muscle (GAS) of mice at the ages of 2 months. Sections were applied for Hematoxylin and Eosin (H&E) and Masson's trichrome staining. Immunofluorescence (IF) staining was also performed by the use of antibodies against human dystrophin (Rods 1 & 2), b-sarcoglycan (NCL-a-SARC, Leica), phosphorylated NF-KB/p65 (p-P65) (#8242, Cell Signaling), CD68 (#9936, Cell Signaling), CD4 (BD550280, BD Biosciences), CD8 (ab22378, Abeam), ColIV (ab6586, Abeam), and mouse IgG (mlgG) (C2181, Sigma). Nuclei were stained with DAPI. Images were taken at 200X magnification. Protein expressions were detected by Western blot analysis with antibody against GAPDH as an endogenous control. Statistical analysis was performed using GraphiPad Prism 7 software. All results were given as the mean ±SD (n > 4 per group). Differences were considered statistically significant when the P-value was <0.05.

RESULTS

[0071] As shown in Figure 3, panel B, high level of expression of full-length dystrophin (450 kDa) in WT or mini-dystrophin (150 kDa) in Tg mice was detected. Phosphorylated NF-KB (65 kDa) was found to be significantly reduced in p65 knockout background

(Tg.dKO-homo.p65+/+ vs Tg.dKO-homo.p65+/- ; dKO-homo.p65+/+ vs dKO-homo.p65+/-). In Figure 3, panel C, high levels of dystrophin or dystrophin associated protein (b- sarcoglycan) was also found in mini-dystrophin transgenic DMD mice accompanied with improved morphology of muscle cells in H&E staining.

[0072] Moreover, Masson’s Trichrome staining and IF staining of mlgG, CD68, CD4, and CD8 were applied to analyze muscle fibrosis, necrosis, and inflammation. Results in Figure 4A demonstrated the improvement in cell morphology, and reduction in fibrosis, necrosis and inflammation at background with p65 knockdown, especially Tg.dKO- homo.p65+/- vs Tg.dKO-homo.p65+/+, indicating that genetic ablation of NF-KB/p65 subunit could reduce inflammation levels.

[0073] As shown in Figure 4B, statistical analysis from IF staining of CD68, CD4 and CD8 in GAS muscles (Tg.dKO-homo.p65+/+ vs Tg.dKO-homo.p65+/-; dKO-homo.p65+/+ vs dKO-homo.p65+/-) at the ages of 2 months demonstrated that genetic reduction of p65 subunit could significantly reduce levels of fibrosis, necrosis and infiltration of immune cells, counted from more than 200 total cells per group. The inflammation was not observed in WT mice, and untreated dKO-homo GAS muscle showed higher level of inflammation comparing mdx mice (Figures 4A and 4B), consistent with previous observations (Mu, 2015).

[0074] As shown in Figure 4C, unlike in skeletal muscle in mdx/utm ⁷ mice, there was no significant difference of CD68+ macrophages in cardiac muscle, no matter whether the mice were treated with dual-cassette AAV or single-cassette AAV or no treatment. In contrast, there was a remarkable reduction of CD4+ and CD8+ immune cells and CD68+ macrophages observed in skeletal muscle treated with dual-cassette AAV compared to single-cassette AAV.

[0075] Figure 4D demonstrates a comparison of overall health and muscle function of mdx/utrn ⁷ mice systemically treated by dual-cassette AAV and single-cassette AAV treatment. When the muscle function at 1 -month and 2-month age with 0 to 200 HZ frequency was tested, both treatment groups had significant improvement at 200 HZ at 1- month age, but no difference as compared dual-cassette AAV and single-cassette AAV treatment. However, the dual-cassette AAV treatment showed a synergistic effect compared to single-cassette AAV at 200 HZ at late age, such as 2-month.

[0076] Additionally, early onset (at 1 -month age) of improvement in body weight was observed in the dual-cassette AAV treatment, and improvement of body weight of mdx/utrn- /- mice treated by dual-cassette AAV and single-cassette AAV treatment was observed at 2- month age.

DISCUSSION

[0077] This Example demonstrates the efficacy of gene replacement and anti

inflammation in transgenic and NF-KB/p65 knockdown in a severe DMD mouse model. The results reveal that the mini-dystrophin expression accompanied with NF-kB/p65 knockout in dKO-homo mice background could ameliorate muscle morphology, reduce fibrosis, necrosis, and inflammation. These results imply that genetic reduction of NF-KB might enhance efficacy of mini-dystrophin gene therapy in large animals and clinical trials, highlighting its application for gene therapy to DMD patients through the combinational dystrophin gene replacement and anti-inflammation·

[0078] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. These include, but are not limited to, the following references:

• Acharyya, et al. J. Clin. Invest., 2007, Apr.;117(4):889-901

• Bing Wang, et al., Journal of Orthopaedic Research, 2009, 27:4,421-42,. PMID:

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[0079] The use of the terms“a” and“an” and“the” and“at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term“at least one” followed by a list of one or more items (for example,“at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms“comprising,”“having,”“including,” and“containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0080] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.