Targeted gene therapy for dm-1 myotonic dystrophy

Abstract

Provided herein are RNAi molecules for treating myotonic dystrophy type 1 (DM1). Further provided herein are expression cassettes, vectors (e.g., rAAV), viral particles, and pharmaceutical compositions containing the RNAi. Yet further provided herein are methods and kits related to the use of the RNAi, for example, to treat DM1.

Description

This article provides an RNAi molecule for the treatment of myotonic dystrophy type 1 (DM1). It also provides expression cassettes, vectors (e.g., rAAV), viral particles, and pharmaceutical compositions containing this RNAi. Furthermore, it provides methods and kits related to using this RNAi, for example, to treat DM1. Abstract

Claims

CLAIMS What is claimed is 1. A recombinant adeno-associated virus (rAAV) particle comprising: a) an RNAi comprising a first strand and a second strand, wherein: i) the first strand and the second strand form a duplex; ii) the first strand comprises a guide region, wherein the guide region comprises nucleic acid with the sequence 5’- AGUCGAAGACAGUUCUAGGGU-3’ (SEQ ID NO:1) or with a sequence with about 90% identity to the sequence of SEQ ID NO:1; and iii) the second strand comprises a non-guide region; and b) an AAV capsid comprising an amino acid sequence with about 90% identity to a wildtype AAVrh74 capsid.

2. The rAAV particle of claim 1, wherein the non-guide region comprises nucleic acid with the sequence 5’-ACCCUAGAUGUCUUCGAUU-3’ (SEQ ID NO:2) or a with a sequence with about 90% identity to the sequence of SEQ ID NO:

2.

3. The rAAV particle of claim 1 or 2, wherein the first strand comprises nucleic acid with the sequence of SEQ ID NO:1 and the non-guide region comprises nucleic acid with the sequence of SEQ ID NO:

2.

4. The rAAV particle of any one of claims 1-3, wherein the first strand and the second strand are linked by means of an RNA linker capable of forming a loop structure.

5. The rAAV particle of claim 4, wherein the RNA linker comprises from about 4 to about 50 nucleotides.

6. The rAAV particle of claim 4 or 5, wherein the loop structure comprises from about 4 to about 20 nucleotides.

7. The rAAV particle of any one of claims 4-6, wherein the loop structure comprises nucleic sequence with of SEQ ID NO:3 or with a sequence with about 90% identity to the sequence of SEQ ID NO:

3.

8. The rAAV particle of any one of claims 4-7, wherein the RNAi comprises 5’ to 3’ the second strand, the RNA linker, and the first strand.

9. The rAAV particle of any one of claims 4-7, wherein the RNAi comprises 5’ to 3’ the first strand, the RNA linker, and the second strand.

10. The rAAV particle of any one of claims 1-8, wherein the RNAi comprises nucleic acid with the sequence of SEQ ID NO:7 or with a sequence with about 90% identity to the sequence of SEQ ID NO:

7.

11. The rAAV particle of any one of claims 1-10, wherein the RNAi is a small inhibitory RNA (siRNA), a microRNA (miRNA), or a small hairpin RNA (shRNA).

12. The rAAV particle of any one of claim 1-11, wherein the RNAi further comprises a scaffold.

13. The rAAV particle of claim 12, wherein the scaffold comprises all or a portion of the nucleic acid of SEQ ID No:

11.

14. The rAAV particle of claim 13, wherein the miRNA is embedded within the scaffold.

15. The rAAV particle of claims 14, wherein the scaffold has a 5’arm, wherein the 5’ arm is located 5’ to the nucleic acid encoding the RNAi, and a 3’arm, wherein the 3’ arm is located 3’ to the nucleic acid encoding the RNAi.

16. The rAAV particle of any one of claims 12-15, wherein the scaffold is a miR-155 scaffold.

17. The rAAV particle of any one of claims 12-16, wherein the miR-155 scaffold comprises the nucleic acid of SEQ ID NO: 9 or a sequence with about 90% identity to the sequence of SEQ ID NO: 9 located 5’ to the RNAi.

18. The rAAV particle of any one of claims 12-17, wherein the miR-155 scaffold comprises the nucleic acid of SEQ ID NO: 10 or a sequence with about 90% identity to the sequence of SEQ ID NO: 10 located 3’ to the RNAi.

19. The rAAV particle of any one of claims 1-18, wherein the RNAi targets RNA encoding a polypeptide associated with myotonic dystrophy-1 (DM1).

20. The rAAV particle of claim 19, wherein the polypeptide is dystrophia myotonica protein kinase (DMPK).

21. The rAAV particle of claim 20, wherein the DMPK comprises a mutation associated with DM-1.

22. The rAAV particle of claim 20 or 21, wherein the gene encoding DMPK comprises five or more CTG trinucleotide repeats.

23. An expression cassette comprising a nucleic acid sequence encoding the RNAi of any one of claims 1-22.

24. The expression cassette of claim 23, wherein the nucleic acid encoding the RNAi is operably linked to a promoter.

25. The expression cassette of claim 24, wherein the promoter is a muscle-specific promotor.

26. The expression cassette of claim 24 or 25, wherein the promoter is a desmin promoter or variant thereof.

27. The expression cassette of claim 26, wherein the desmin promoter comprises one or more enhancer elements and the promoter for the human desmin gene.

28. The expression cassette of claim 26 or 27, wherein the desmin promoter comprises two enhancer elements and the promoter for the human desmin gene.

29. The expression cassette of any one of claims 26-28, wherein the desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.

30. The expression cassette of any one of claims 26-29, wherein the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21 or a nucleotide sequence with about 90% identity to the sequence of SEQ ID NO:21 and / or one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:22 or a nucleotide sequence with about 90% identity to the sequence of SEQ ID NO:22.

31. The expression cassette of any one of claims 26-30, wherein the desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence with about 90% identity to the nucleotide sequence of SEQ ID NO: 12.

32. The expression cassette of any one of claims 23-31, wherein the expression cassette further comprises an intron.

33. The expression cassette of claim 32, wherein the intron is a rabbit β-globin intron.

34. The expression cassette of claim 32 or 33, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 13 or a sequence with about 90% identity to the sequence of SEQ ID NO:13.

35. The expression cassette of any one of claims 32-34, wherein the nucleic acid encoding the RNAi is embedded in the intron.

36. The expression cassette of claim 35, wherein the intron comprises a 5’ arm and a 3’ arm, wherein the 5’ arm is located 5’ to the nucleic acid encoding the RNAi and the 3’ arm is located 3’ to the nucleic acid encoding the RNAi.

37. The expression cassette of claim 36, wherein the 5’ arm of the intron comprises the nucleotide sequence of SEQ ID NO:14 or a sequence with about 90% identity to the sequence of SEQ ID NO:

14.

38. The expression cassette of claim 36 or 37, wherein the 3’ arm of the intron comprises the nucleotide sequence of SEQ ID NO:15 or a sequence with about 90% identity to the sequence of SEQ ID NO:

15.

39. The expression cassette of any one of claims 23-38, wherein the expression cassette further comprises a polyadenylation signal.

40. The expression cassette of claim 39 wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or a HSV TK pA.

41. The expression cassette of claim 40, wherein the polyadenylation signal is a minimal bovine growth hormone polyadenylation signal.

42. The expression cassette of any one of claims 39-41, wherein the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO:16 or a sequence with about 90% identity to the sequence of SEQ ID NO:

16.

43. The expression cassette of any one of claims 23-42, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO:17 or a sequence with about 90% identity to the sequence of SEQ ID NO:17.

44. An expression cassette, wherein the expression cassette comprises a modified desmin promoter, wherein the modified desmin promoter comprises one or more enhancer elements and the promoter for the human desmin gene.

45. The expression cassette of claim 44, wherein the modified desmin promoter comprises two enhancer elements and the promoter for the human desmin gene.

46. The expression cassette of claim 44 or 45, wherein the modified desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.

47. The expression cassette of any one of claims 44-46, wherein the modified desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21 or a nucleotide sequence with about 90% identity to the sequence of SEQ ID NO:21 and / or one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:22 or a nucleotide sequence with about 90% identity to the sequence of SEQ ID NO:

22.

48. The expression cassette of any one of claims 44-47, wherein the desmin promoter comprises the nucleotide sequence of SEQ ID NO:12 or a sequence with about 90% identity to the nucleotide sequence of SEQ ID NO:

12.

49. The expression cassette of any one of claims 44-48, wherein the expression cassette further comprises an intron.

50. The expression cassette of claim 49, wherein the intron is a rabbit β-globin intron.

51. The expression cassette of claim 49 or 50, wherein the intron comprises the nucleotide sequence of SEQ ID NO:13 or a sequence with about 90% identity to the sequence of SEQ ID NO:

13.

52. The expression cassette of any one of claims 44-51, wherein the nucleic acid encoding the transgene is embedded in the intron.

53. The expression cassette of claim 52, wherein the intron comprises a 5’ arm and a 3’ arm, wherein the 5’ arm is located 5’ to the nucleic acid encoding the transgene and the 3’ arm is located 3’ to the nucleic acid encoding the transgene.

54. The expression cassette of claim 53, wherein the 5’ arm of the intron comprises the nucleotide sequence of SEQ ID NO:14 or a sequence with about 90% identity to the sequence of SEQ ID NO:

14.

55. The expression cassette of claim 53 or 54, wherein the 3’ arm of the intron comprises the nucleotide sequence of SEQ ID NO:15 or a sequence with about 90% identity to the sequence of SEQ ID NO:

15.

56. The expression cassette of any one of claims 44-55, wherein the expression cassette further comprises a polyadenylation signal.

57. The expression cassette of claim 56, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or a HSV TK pA.

58. The expression cassette of claim 57, wherein the polyadenylation signal is a minimal bovine growth hormone polyadenylation signal.

59. The expression cassette of any one of claims 56-58, wherein the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO:16 or a sequence with about 90% identity to the sequence of SEQ ID NO:

16.

60. The expression cassette of any one of claims 44-59, wherein the transgene encodes a polypeptide or a nucleic acid.

61. The expression cassette of any one of claims 44-60, wherein the transgene encodes an RNAi.

62. A vector comprising the expression cassette of any one of claims 23-61.

63. The vector of claim 62, wherein the expression cassette is flanked by one or more stuffer nucleic acid sequences.

64. The vector of claim 63, wherein the one or more stuffer nucleic acid sequences is derived from the human SerpinAl gene.

65. The vector of claim 63 or 64, wherein a stuffer nucleic acid sequence located 5’ to the expression cassette is derived from the human SerpinAl gene.

66. The vector of any one of claims 63-65, wherein a stuffer sequence located 5’ to the expression cassette comprises the nucleotide sequence of SEQ ID NO: 18 or a sequence with about 90% identity to the sequence of SEQ ID NO: 18.

67. The vector of any one of claims 63-66, wherein a stuffer nucleic acid sequence located 3’ to the expression cassette is derived from the human SerpinAl gene.

68. The vector of any one of claims 63-67, wherein a stuffer sequence located 3’ to the expression cassette comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence with about 90% identity to the sequence of SEQ ID NO: 19.

69. The vector of any one of claims 62-68, wherein the vector is a recombinant adeno- associated virus (rAAV) vector.

70. The rAAV vector of claim 69, wherein the expression cassette is flanked by one or more AAV inverted terminal repeat (ITR) sequences.

71. The rAAV vector of claim 70, wherein the expression cassette is flanked by two AAV ITRs.

72. The rAAV vector of claim 70 or 71, wherein the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV1 1, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs.

73. The rAAV vector of any one of claims 70-72, wherein the AAV ITRs are AAV2 ITRs.

74. The rAAV vector of any one of claims 69-73, wherein the rAAV vector comprises the nucleotide sequence of SEQ ID NO:20 or a sequence with about 90% identity to the sequence of SEQ ID NO:20.

75. The rAAV vector of any one of claims 69-74, wherein the vector is a self-complementary rAAV vector.

76. A cell comprising the expression cassette of any one of claims 23-61, the vector of any one of claims 62-68, or the rAAV vector of any one of claims 69-75.

77. The rAAV particle of any one of claims 1-22, wherein the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 502.

78. The rAAV particle of claim 77, wherein the amino acid substitution at position 502 is an isoleucine (I).

79. The rAAV particle of claim 77 or 78, wherein the rAAV particle comprises a AAVrh74 N502I serotype capsid.

80. The rAAV particle of claim , wherein the ITR is an AAV2 ITR and the capsid of the rAAV particle is an AAVrh74 N502I serotype capsid.

81. The rAAV particle of any one of claims 1-22, wherein the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 505.

82. The rAAV particle of claim 81, wherein the amino acid substitution at position 505 is an arginine (R).

83. The rAAV particle of any one of claims 1-22, wherein the rAAV particle comprises a AAVrh74 W505R serotype capsid.

84. The rAAV particle of claim 83, wherein the ITR is an AAV2 ITR and the capsid of the rAAV particle is an AAVrh74 W505R serotype capsid.

85. An rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises the following nucleic acids 5’ to 3’, an AAV2 ITR, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5’ arm of a rabbit β-globin intron, a 5’ miR155 scaffold sequence, a DMPK204miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204miRNA passenger sequence, a 3’ miR155 scaffold sequence, a 3’ arm of a rabbit β-globin intron, a minimal bovine growth hormone polyadenylation sequence, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, and an AAV2 ITR; and wherein the capsid is an AAVrh74 N502I capsid.

86. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids 5’ to 3’, an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:18, a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:21, a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:22, a desmin promoter comprising the polynucleotide sequence of SEQ ID NO:23, a 5’ arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:14, a 5’ miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:40, a DMPK204miRNA guide sequence comprising the polynucleotide sequence of SEQ ID NO:4, a miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO:6, a DMPK204miRNA passenger sequence comprising the polynucleotide sequence of SEQ ID NO:5, a 3’ miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, a 3’ arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO:16, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is an AAVrh74 N502I capsid.

87. The rAAV particle of claim 85 or 86, wherein the AAVrh74 N502I capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO:

50.

88. An rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises the following nucleic acids 5’ to 3’, an AAV2 ITR, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5’ arm of a rabbit β-globin intron, a 5’ miR155 scaffold sequence, a DMPK204miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204miRNA passenger sequence, a 3’ miR155 scaffold sequence, a 3’ arm of a rabbit β-globin intron, a minimal bovine growth hormone polyadenylation sequence, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, and an AAV2 ITR; and wherein the capsid is an AAVrh74 W505R capsid.

89. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids 5’ to 3’, an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:18, a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:21, a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:22, a desmin promoter comprising the polynucleotide sequence of SEQ ID NO:23, a 5’ arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:14, a 5’ miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:40, a DMPK204miRNA guide sequence comprising the polynucleotide sequence of SEQ ID NO:4, a miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO:6, a DMPK204miRNA passenger sequence comprising the polynucleotide sequence of SEQ ID NO:5, a 3’ miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, a 3’ arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO:16, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is an AAVrh74 W505R capsid.

90. The rAAV particle of claim 88 or 89, wherein the AAVrh74 W505R capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 52.

91. A composition comprising the rAAV particle of any one of claims 1-22 and 77-90.

92. A pharmaceutical composition comprising the rAAV particle of any one of claims 1-22 and 77-90.

93. The composition of claim 91 or 92, wherein the composition further comprises a pharmaceutically acceptable carrier.

94. A kit comprising the rAAV particle of any one of claims 1-22 and 77-90.

95. A kit comprising the rAAV particle of any one of claims 1-22 and 77-90.

96. A kit comprising the composition of any one of claims 91-93.

97. The kit of any one of claims 94-96, further comprising instructions for use.

98. A method for treating myotonic dystrophy- 1 (DM1) in a mammal in need thereof comprising administering to the mammal an effective amount of the rAAV particle of any one of claims 1-22 and 77-90.

99. A method for inhibiting the expression of dystrophia myotonica protein kinase (DMPK) in a mammal with DM-1 in need thereof comprising administering to the mammal an effective amount of the rAAV particle of any one of claims 1-22 and 77-90.

100. A method for inhibiting the accumulation of DMPK RNA in a cell of a mammal with DM-1 in need thereof comprising administering to the mammal an effective amount of the RNAi of any one of claims 1-22 and 77-90.

101. A method for treating myotonic dystrophy-1 (DM1) in a mammal in need thereof comprising administering to the mammal an effective amount of the rAAV particle of any one of claims 1-22 and 77-90.

102. A method for inhibiting the expression of dystrophia myotonica protein kinase (DMPK) in a mammal with DM-1 in need thereof comprising administering to the mammal an effective amount of the rAAV particle of any one of claims 1-22 and 77-90.

103. A method for inhibiting the accumulation of DMPK RNA in a cell of a mammal with DM-1 in need thereof comprising administering to the mammal an effective amount of the rAAV particle of any one of claims 1-22 and 77-90.

104. The method of any one of claims 100-103, wherein the effective amount of the rAAV particle is a dose of about 1 x 108to about 2 x 1013genome copies / mL.

105. The method of claim 104, wherein the dose is about 5 x 1012genome copies / mL.

106. The method of claim 104, wherein the dose is about 1 x 1013genome copies / mL.

107. The method of claim 104, wherein the dose is about 2 x 1013genome copies / mL.

108. The method of any one of claims 101-103, wherein the effective amount of the rAAV particle is a dose of about 1 x 108to about 2 x 1014genome copies / kg of body weight.

109. The method of claim 108, wherein the dose is about 5 x 1013genome copies / kg of body weight.

110. The method of claim 108, wherein the dose is about 1 x 1014genome copies / kg of body weight.

111. The method of claim 108, wherein the dose is about 2 x 1014genome copies / kg of body weight.

112. A method for treating myotonic dystrophy-1 (DM1) in a mammal in need thereof comprising administering to the mammal an effective amount of the composition of any one of claims 91-93.

113. A method for inhibiting the expression of dystrophia myotonica protein kinase (DMPK) in a mammal with DM-1 in need thereof comprising administering to the mammal an effective amount of the composition of any one of claims 91-93.

114. A method for inhibiting the accumulation of DMPK RNA in a cell of a mammal with DM-1 in need thereof comprising administering to the mammal an effective amount of the composition of any one of claims 91-93.

115. The method of any one of claims 98-100, wherein the RNAi is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time, and / or after administration of the RNAi.

116. The method of any one of claims 101-103, wherein the viral particle or the rAAV particle is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time, and / or after administration of the viral particle or the rAAV particle.

117. The method of any one of claims 112-114, wherein the composition is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time, and / or after administration of the composition.

118. The method of any one of claims 98-117, wherein administration of the rAAV particle of any one of claims 1-22 and 77-90, the expression cassette of any one of claims 23-61, the vectorof any one of claims 75, the composition of any one of claims 91-93, or the kit of any one of claims 94-97, wherein the splicing of gene transcripts is measured after administration.

119. The method of claim 118, wherein the splicing of gene transcripts is a measurement of treatment efficacy.

120. The method of claim 118 or 119, wherein splicing of the gene transcripts is about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% compared to comparable healthy tissue.

121. The method of any one of claims 118-119, wherein the gene transcripts measured comprise one or more of genes selected from MBNL1, SOS1, PKM, zTTN, GOGLA4, CLASP1, LDB3, MBNL2, SPAG9, DNAseI, TNNT3 and ZBTB49.

122. A recombinant adeno-associated virus (rAAV) particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises the following nucleic acids 5’ to 3’, an AAV2 ITR, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5’ arm of a rabbit β-globin intron, a 5’ miR155 scaffold sequence, a DMPK204miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204miRNA passenger sequence, a 3’ miR155 scaffold sequence, a 3’ arm of a rabbit β-globin intron, a minimal bovine growth hormone polyadenylation sequence, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, and an AAV2 ITR;and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 50 123. A recombinant adeno-associated virus (rAAV) particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises the following nucleic acids 5’ to 3’, an AAV2 ITR, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5’ arm of a rabbit β-globin intron, a 5’ miR155 scaffold sequence, a DMPK204miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204miRNA passenger sequence, a 3’ miR155 scaffold sequence, a 3’ arm of a rabbit β-globin intron, a minimal bovine growth hormone polyadenylation sequence, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, and an AAV2 ITR; and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO:

52.

124. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids 5’ to 3’, an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:18, a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:21,a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:22, a desmin promoter comprising the polynucleotide sequence of SEQ ID NO:23, a 5’ arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:14, a 5’ miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:40, a DMPK204miRNA guide sequence comprising the polynucleotide sequence of SEQ ID NO:4, a miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO:6, a DMPK204miRNA passenger sequence comprising the polynucleotide sequence of SEQ ID NO:5, a 3’ miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, a 3’ arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO:16, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO:

50.

125. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids 5’ to 3’, an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:18,a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:21, a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:22, a desmin promoter comprising the polynucleotide sequence of SEQ ID NO:23, a 5’ arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:14, a 5’ miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:40, a DMPK204miRNA guide sequence comprising the polynucleotide sequence of SEQ ID NO:4, a miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO:6, a DMPK204miRNA passenger sequence comprising the polynucleotide sequence of SEQ ID NO:5, a 3’ miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, a 3’ arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO:16, nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 52.