Targeted gene therapy for DM-1 myotonic dystrophy

Abstract

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

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 494,453, filed April 5, 2023, and U.S. Provisional Patent Application Serial No. 63 / 589,417, filed October 11, 2023, the contents of which are incorporated herein by reference in their entirety. Submission of sequence list

[0002] The following content, submitted as an XML file, is incorporated herein by reference in its entirety: Computer-readable form of a sequence list (CRF) (filename: 752749-SA9-363BPC.xml, created: April 3, 2024, size: 84,754 bytes). Technical Field

[0003] This invention relates to variant RNAi molecules. In some aspects, this invention relates to variant RNAi molecules for the treatment of muscular dystrophy. Background Technology

[0004] In basic research on gene function, RNA interference (RNAi) has been confirmed as a useful tool for gene silencing, and RNAi shows great promise as a therapeutic agent to suppress genes associated with the development of many diseases. In nature, gene regulation by RNAi is carried out by small RNAs called microRNAs (miRNAs) (Ambros, (2004) Nature 431:350-355; Krol et al., (2010) Nat. Rev. Genet. 11:597-610). MicroRNAs have become powerful regulators of a variety of cellular processes, and when delivered by viral vectors, artificial miRNAs are continuously expressed, resulting in robust and sustained repression of target genes. Elucidation of the mechanisms involved in miRNA processing has allowed scientists to utilize endogenous cellular RNAi mechanisms and use artificial miRNAs to guide the degradation of target gene products (see, for example, US PG Pub. [Pre-authorization Publication] 2014 / 0163214 and Davidson et al., (2012) Cell [Cell] 150:873-875).

[0005] Myotonic dystrophy type 1 (DM1) is a monogenic, autosomal dominant, progressive disease caused by the amplification of CTG repeat sequences (>50) at the DMPK locus. The DMPK with the repeat sequence is transcribed into mRNA, which forms a hairpin and binds to RNA-binding proteins, preventing these proteins from functioning properly. This leads to the appearance of nuclear foci, missplicing of mRNA, and ultimately, myotonia. DM1 primarily affects skeletal, cardiac, and smooth muscle, causing significant physical, cognitive, and behavioral impairments and disability. Currently, there are no approved therapies for DM1. Therefore, there is a high unmet medical need for treatment of DM1.

[0006] All references cited in this article, including patent applications and publications, are incorporated herein by reference in their entirety. Summary of the Invention

[0007] In some aspects, the present invention provides a recombinant adeno-associated virus (rAAV) particle comprising: an RNAi comprising a first strand and a second strand, wherein the first and second strands form a double strand, the first strand comprising a guide region comprising nucleic acid having the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1) or having a sequence having about 90% identity with the sequence of SEQ ID NO:1, and the second strand comprising a non-guide region; and an AAV capsid comprising an amino acid sequence having about 90% identity with the wild-type AAVrh74 capsid. In some embodiments, the first strand comprises nucleic acid having the sequence of SEQ ID NO:1, and the non-guide region comprises nucleic acid having the sequence of SEQ ID NO:2. In some embodiments, the first strand and the second strand are joined by an RNA adapter capable of forming a loop structure. In some embodiments, the RNA adapter comprises about 4 to about 50 nucleotides. In some embodiments, the loop structure comprises about 4 to about 20 nucleotides. In some embodiments, the loop structure comprises a nucleic acid sequence having the sequence of SEQ ID NO:3 or having a sequence having approximately 90% identity with the sequence of SEQ ID NO:3. In some embodiments, the RNAi comprises the second strand, the RNA adapter, and the first strand from 5' to 3'. In some embodiments, the RNAi comprises the first strand, the RNA adapter, and the second strand from 5' to 3'. In some embodiments, the RNAi comprises a nucleic acid having the sequence of SEQ ID NO:7 or having a sequence having approximately 90% identity with the sequence of SEQ ID NO:7. In some embodiments, the RNAi is a small repressive RNA (siRNA), a microRNA (miRNA), or a small hairpin RNA (shRNA).

[0008] In some embodiments of the invention, the RNAi further comprises a scaffold. In some embodiments, the scaffold comprises all or a portion of the nucleic acid of SEQ ID No:11. In some embodiments, the miRNA is embedded within the scaffold. In some embodiments, the scaffold has a 5' arm and a 3' arm, wherein the 5' arm is located at the 5' of the nucleic acid encoding the RNAi, and wherein the 3' arm is located at the 3' of the nucleic acid encoding the RNAi. In some embodiments, the scaffold is a miR-155 scaffold. In some embodiments, the miR-155 scaffold comprises a nucleic acid located at the 5' of the RNAi having the sequence of SEQ ID NO:9 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:9. In some embodiments, the miR-155 scaffold comprises a nucleic acid located at the 3' of the RNAi having the sequence of SEQ ID NO:10 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:10.

[0009] In some embodiments of the invention, the RNAi targets RNA encoding a polypeptide associated with myotonic dystrophy type 1 (DM1). In some embodiments, the polypeptide is dystrophic myotonic protein kinase (DMPK). In some embodiments, the DMPK contains a mutation associated with DM1. In some embodiments, the gene encoding the DMPK contains five or more CTG trinucleotide repeat sequences.

[0010] In some aspects, the present invention provides an expression cassette comprising a nucleic acid encoding any of the RNAi described herein. In some embodiments, the nucleic acid encoding the RNAi is operatively linked to a promoter. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the promoter is a desmin promoter or a variant thereof. In some embodiments, the desmin promoter comprises one or more enhancer elements and a promoter of the human desmin gene. In some embodiments, the desmin promoter comprises two enhancer elements and a promoter of the human desmin gene. In some embodiments, the desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements. In some embodiments, the desmin promoter comprises: one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21 or a nucleotide sequence having about 90% identity with 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 having about 90% identity with the sequence of SEQ ID NO:22. In some embodiments, the desmin promoter comprises the nucleotide sequence of SEQ ID NO:12 or a sequence having approximately 90% identity with the nucleotide sequence of SEQ ID NO:12. In some embodiments, the expression cassette further comprises an intron. In some embodiments, the intron is a rabbit β-globin intron. In some embodiments, the intron comprises the nucleotide sequence of SEQ ID NO:13 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:13. In some embodiments, the nucleic acid encoding the RNAi is embedded in the intron. In some embodiments, the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at the 5' of the nucleic acid encoding the RNAi and the 3' arm is located at the 3' of the nucleic acid encoding the RNAi. In some embodiments, the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO:14 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:14. In some embodiments, the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO:15 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:15. In some embodiments, the expression cassette further includes a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or an HSV TK pA. In some embodiments, the polyadenylation signal is a minimal bovine growth hormone polyadenylation signal. In some embodiments, the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO:16 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:16.In some embodiments, the expression cassette contains the nucleotide sequence of SEQ ID NO:17 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:17.

[0011] In some aspects, the present invention provides an expression cassette comprising a modified desmin promoter, wherein the modified desmin promoter comprises one or more enhancer elements and a promoter of the human desmin gene. In some embodiments, the modified desmin promoter comprises two enhancer elements and a promoter of the human desmin gene. In some embodiments, the modified desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements. In some embodiments, the modified desmin promoter comprises: one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21 or a nucleotide sequence having about 90% identity with 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 having about 90% identity with the sequence of SEQ ID NO:22. In some embodiments, the desmin promoter comprises the nucleotide sequence of SEQ ID NO:12 or a sequence having about 90% identity with the nucleotide sequence of SEQ ID NO:12. In some embodiments, the expression cassette further comprises introns. In some embodiments, the intron is a rabbit β-globin intron. In some embodiments, the intron comprises the nucleotide sequence of SEQ ID NO:13 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:13. In some embodiments, a nucleic acid encoding a transgene is embedded in the intron. In some embodiments, the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at the 5' of the nucleic acid encoding the transgene and the 3' arm is located at the 3' of the nucleic acid encoding the transgene. In some embodiments, the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO:14 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:14. In some embodiments, the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO:15 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:15. In some embodiments, the expression cassette further comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or HSV TK pA. In some embodiments, the polyadenylation signal is the minimum bovine growth hormone polyadenylation signal. In some embodiments, the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO:16 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:16. In some embodiments, the transgene encodes a polypeptide or nucleic acid. In some embodiments, the transgene encodes RNAi.

[0012] In some aspects, the present invention provides a vector comprising any of the expression cassettes described herein. In some embodiments, the flanking portion of the expression cassette is one or more stuffer nucleic acid sequences. In some embodiments, the one or more stuffer nucleic acid sequences are derived from the human SerpinA1 gene. In some embodiments, the stuffer nucleic acid sequence located at 5' of the expression cassette is derived from the human SerpinA1 gene. In some embodiments, the stuffer sequence located at 5' of the expression cassette comprises the nucleotide sequence of SEQ ID NO:18 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:18. In some embodiments, the stuffer nucleic acid sequence located at 3' of the expression cassette is derived from the human SerpinA1 gene. In some embodiments, the stuffer sequence located at 3' of the expression cassette comprises the nucleotide sequence of SEQ ID NO:19 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:19.

[0013] In some embodiments of the invention, the vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the expression cassette is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the expression cassette is flanked by two AAV ITRs. In some embodiments, these AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVDJ, goat AAV, bovine AAV, or mouse AAV serotype ITRs. In some embodiments, these AAV ITRs are AAV2 ITRs. In some embodiments, the rAAV vector comprises the nucleotide sequence of SEQ ID NO:20 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:20. In some embodiments, the vector is a self-complementary rAAV vector.

[0014] In some embodiments, the present invention provides cells comprising any expression cassette, any vector, or any rAAV vector described herein.

[0015] In some aspects, the present invention provides viral particles comprising any of the vectors described herein. In some aspects, the present invention provides recombinant AAV particles comprising any rAAV vector described herein. In some embodiments, the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 502. In some embodiments, the amino acid substitution at position 502 is isoleucine (I). In some embodiments, the rAAV viral particle comprises an AAVrh74 N502I serotype capsid. In some embodiments, the ITR is AAV2 ITR and the capsid of the rAAV particle is an AAVrh74 N502I serotype capsid. In some embodiments, the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 505. In some embodiments, the amino acid substitution at position 505 is arginine (R). In some embodiments, the rAAV particle comprises an AAVrh74 W505R serotype capsid. In some embodiments, the ITR is AAV2 ITR and the capsid of the rAAV particle is an AAVrh74 W505R serotype capsid.

[0016] In some aspects, the present invention provides an rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: AAV2 ITR, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, the 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, and DMPK. 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 The miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimum bovine growth hormone polyadenylated sequence, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, and AAV2 ITR; and wherein the capsid is AAVrh74N502I capsid.

[0017] In some aspects, the present invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43; a nucleic acid encoding a filler 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; and a DMPK comprising the polynucleotide sequence of SEQ ID NO:4. 204 miRNA guide sequence, miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6, and DMPK containing the polynucleotide sequence of SEQ ID NO:5. 204 The miRNA passenger sequence comprises a 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is an AAVrh74 N502I capsid. In some embodiments, the AAVrh74 N502I capsid comprises a capsid protein containing the amino acid sequence of SEQ ID NO:50.

[0018] In some aspects, the present invention provides an rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: AAV2 ITR, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, the 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, and DMPK. 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204The miRNA passenger sequence, the 3' miR155 scaffold sequence, the 3' arm of the rabbit β-globin intron, the smallest bovine growth hormone polyadenylated sequence, the nucleic acid encoding the filler nucleic acid sequence from the human serpinA1 gene, and the AAV2 ITR; and wherein the capsid contains a capsid protein containing the amino acid sequence of SEQ ID NO:50.

[0019] In some aspects, the present invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43; a nucleic acid encoding a filler 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; and a DMPK comprising the polynucleotide sequence of SEQ ID NO:4. 204 miRNA guide sequence, miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6, and DMPK containing the polynucleotide sequence of SEQ ID NO:5. 204 The miRNA passenger sequence comprises a 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid comprises a capsid protein containing the amino acid sequence of SEQ ID NO:50.

[0020] In some aspects, the present invention provides an rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: AAV2 ITR, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, the 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, and DMPK.204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 The miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimum bovine growth hormone polyadenylated sequence, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, and AAV2 ITR; and wherein the capsid is AAVrh74W505R capsid.

[0021] In some embodiments, the present invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43; a nucleic acid encoding a filler 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; and a DMPK comprising the polynucleotide sequence of SEQ ID NO:4. 204 miRNA guide sequence, miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6, and DMPK containing the polynucleotide sequence of SEQ ID NO:5. 204 The miRNA passenger sequence comprises a 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is an AAVrh74 W505R capsid. In some embodiments, the AAVrh74 W505R capsid comprises a capsid protein containing the amino acid sequence of SEQ ID NO:52.

[0022] In some aspects, the present invention provides an rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: AAV2 ITR, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, the 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, and DMPK. 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 The miRNA passenger sequence, the 3' miR155 scaffold sequence, the 3' arm of the rabbit β-globin intron, the smallest bovine growth hormone polyadenylated sequence, the nucleic acid encoding the filler nucleic acid sequence from the human serpinA1 gene, and the AAV2 ITR; and wherein the capsid contains a capsid protein containing the amino acid sequence of SEQ ID NO:52.

[0023] In some embodiments, the present invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43; a nucleic acid encoding a filler 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; and a DMPK comprising the polynucleotide sequence of SEQ ID NO:4. 204 miRNA guide sequence, miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6, and DMPK containing the polynucleotide sequence of SEQ ID NO:5. 204The miRNA passenger sequence comprises a 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid comprises a capsid protein containing the amino acid sequence of SEQ ID NO:52.

[0024] In some aspects, the present invention provides compositions comprising any viral particles or rAAV particles described herein. In some embodiments, the present invention provides pharmaceutical compositions comprising any viral particles or rAAV particles described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0025] In some aspects, the present invention provides kits comprising one or more of the following: RNAi, viral particles, AAV particles, or compositions as described herein. In some embodiments, the kit further comprises instructions for use.

[0026] In some aspects, the present invention provides methods for treating myotonic dystrophy type 1 (DM1) in mammals in need, methods comprising administering an effective amount of any RNAi described herein to the mammal. In some aspects, the present invention provides methods for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1, methods comprising administering an effective amount of any RNAi described herein to the mammal. In some aspects, the present invention provides methods for inhibiting the accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1, methods comprising administering an effective amount of any RNAi described herein to the mammal.

[0027] In some aspects, the present invention provides methods for treating myotonic dystrophy type 1 (DM1) in mammals in need, methods comprising administering to the mammal an effective amount of any viral particles as described herein (e.g., rAAV particles). In some aspects, the present invention provides methods for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1, methods comprising administering to the mammal an effective amount of any viral particles as described herein (e.g., rAAV particles). In some aspects, the present invention provides methods for inhibiting the accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1, methods comprising administering to the mammal an effective amount of any viral particles as described herein (e.g., rAAV particles).

[0028] In some embodiments of the present invention, the effective amount of viral particles (e.g., rAAV particles) is about 1 × 10⁻⁶. 8 Approximately 2×10 13 A dose of one genome copy / mL. In some embodiments of the invention, this dose is approximately 5 × 10⁻⁶. 12 One genome copy / mL. In some embodiments of the invention, this dose is approximately 1 × 10⁻⁶. 13 One genome copy / mL. In some embodiments of the invention, this dose is approximately 2 × 10⁻⁶. 13 One genome copy / mL.

[0029] In some embodiments of the present invention, the effective amount of viral particles (e.g., rAAV particles) is about 1 × 10⁻⁶. 8 Approximately 2×10 14 A dose of one genome copy per kg of body weight. In some embodiments of the invention, this dose is approximately 5 × 10⁻⁶. 13 One genome copy per kg body weight. In some embodiments of the invention, this dose is approximately 1 × 10⁻⁶. 14 One genome copy per kg body weight. In some embodiments of the invention, this dose is approximately 2 × 10⁻⁶. 14 One genome copy per kg of body weight.

[0030] In some aspects, the present invention provides methods for treating myotonic dystrophy type 1 (DM1) in mammals in need, methods comprising administering to the mammal an effective amount of any composition as described herein. In some aspects, the present invention provides methods for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1, methods comprising administering to the mammal an effective amount of any composition as described herein. In some aspects, the present invention provides methods for inhibiting the accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1, methods comprising administering to the mammal an effective amount of any composition as described herein.

[0031] In some embodiments of the invention, the RNAi is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, concurrently with, and / or after the RNAi administration. In some embodiments, the viral particles or the rAAV particles are administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, concurrently with, and / or after the viral particles or the rAAV particles. In some embodiments, the composition is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, concurrently with, and / or after the composition administration. Attached Figure Description

[0032] This application can be understood by referring to the following description in conjunction with the accompanying drawings.

[0033] Figure 1A The nDes-miR155-amiR-DMPK was described. 204 Schematic diagram of the gene cassette sequence. The heterozygous muscle promoter is located at miR155-amiR-DMPK. 204 Upstream of the sequence. Downstream of the miRNA is the bovine growth hormone polyadenylated sequence (minBGHpA). Filler sequences from the A1AT introns flank either side of this box. All of these are flanked by two AAV2ITRs, resulting in a combined vector genome size of 3739 bp.

[0034] Figure 1B The description of the method for cloning nDes-miR155-amiR-DMPK 204 The gene cassette contains an ITR plasmid. The ITR plasmid contains an A1AT filler sequence flanking the AAV2 5' and 3' ITRs. The A1AT filler sequence contains NcoI and SphI restriction sites for cloning.

[0035] Figure 1C The study described a small-scale packaging assay in HEK 293 cells to confirm the DC969-nDes-miR155-amiR-DMPK. 204Results of plasmid packaging. Small-scale production was performed using the AAV rep / cap plasmid. The y-axis shows the amount of vector produced per HEK 293 cell compared to the standard EGFP plasmid gene cassette (CD627-CBA-GFP). DRP: DNase-resistant particles.

[0036] Figures 2A-2C The study described the effect of tail vein injection on DMSXL mice containing AAV, namely nDes-miR155-amiR-DMPK. 204 The (amiR155-204) expression box received a low rating for DMPK. Figure 2A The transduction efficiency and biodistribution of AAV, evaluated by quantifying transgene copy numbers in different organs, are shown. qPCR results are expressed as the average ratio of AAV copy number to cell nuclei. Figure 2B The transconverted tissue amiR-DMPK was shown 204 The microRNA input level was normalized to U6 small nuclear RNA and set relative to cells treated with BSS (balanced salt solution). Figure 2C The silencing of DMPK in transduced tissues is shown. Total DMPK was determined by qRT-PCR. mRNA input was normalized against tata box-binding protein (TBP) and relative to a BSS-treated cell setting. Dashed lines indicate 50% DMPK expression relative to TBP expression. Figures 2A-2C The paired Student's T-test was used to evaluate the data: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. n=13 (BSS), n=12 (amiR155-204).

[0037] Figures 3A-3B The AAV nDes-miR155-amiR-DMPK was described. 204 Inhibition of human DMPK. DMSXL mice were administered a dose-dependent systemic injection of AAV nDes-miR155-amiR-DMPK. 204 Eight weeks later, the mice were euthanized, their organs were harvested, and amiR-DMPK was measured. 204 and DMPK transcript levels. Figure 3A The amiR-DMPK for U6 normalization was depicted in various organizations. 204 The abundance of. Figure 3B The abundance of mTBP-normalized hDMPK transcripts in various tissues was depicted. Data were evaluated using ANOVA and multiple comparison tests: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. (n = wt-10, BSS-7, low-dose 5, medium-dose 13, and high-dose 7).

[0038] Figure 4 The description uses AAV nDes-miR155-amiR-DMPK 204 Correction for splicing abnormalities in DMSXL mice after systemic treatment. After 8 weeks of treatment, RT-PCR was used to assess the splicing of selective exon 11 in LDB3 of gastrocnemius muscle. Data were evaluated using ANOVA and multiple comparison tests: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. (n = wt-10, BSS-7, low-dose 5, medium-dose 13, and high-dose 7).

[0039] Figure 5A and Figure 5B The use of AAV nDes-miR155-amiR-DMPK is shown. 204 Female DMSXL mice treated in a dose-dependent manner showed increased survival and body weight. Figure 5A Kaplan-Meier survival curves are shown, demonstrating improved survival rates after 8 weeks of treatment with a medium dose compared to animals treated with low doses or BSS. Figure 5B The results showed that, compared with BSS-treated or low-dose-treated animals, the effects of AAV nDes-miR155-amiR-DMPK were significant. 204 Increased body weight was observed in the treated DMSXL animals.

[0040] Figure 6 The description uses AAV nDes-miR155-amiR-DMPK 204 Reversal of electrophysiological features of DM1 disease in a dose-dependent systemic treatment model of DMSXLDM1 mouse. Data were evaluated using ANOVA and multiple comparison tests: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. (n = wt-10, BSS-7, low-dose 5, medium-dose 13, and high-dose 7).

[0041] Figure 7A A schematic diagram of the experimental protocol is shown. Blood was collected on day -1. To reconfirm the neutralizing antibody. On day 0, AAV encoding the GFP reporter factor was administered to NHP at a dose of 1e13 vg / kg, and an autopsy was performed on day 21, with multiple tissues collected to evaluate biodistribution. Figures 7B-7E Quantification of GFP expression in various tissues from animals injected with AAV9, AAVrh74, and AAVrh74 N502I (rh74M) is shown. The bar graph represents the tibialis anterior muscle (TA). Figure 7B ), biceps femoris ( Figure 7Cquadriceps ( Figure 7D ),heart( Figure 7E ) and liver ( Figure 7F The amount of GFP measured by ELISA in the sample. Data were evaluated using ANOVA and multiple comparison tests: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0042] Figure 8A and Figure 8B The AAV rh74 N502I nDes-miR155-amiR-DMPK is depicted. 204 Inhibition of human DMPK. DMSXL mice were administered a dose-dependent systemic injection of AAV rh74 N502I nDes-miR155-amiR-DMPK. 204 Eight weeks later, the mice were euthanized, their organs were harvested, and amiR-DMPK was measured. 204 and DMPK transcript levels. Figure 8A The amiR-DMPK for U6 normalization was depicted in various organizations. 204 The abundance of. Figure 8B The abundance of mTBP-normalized hDMPK transcripts in various tissues was depicted. Data were evaluated using ANOVA and multiple comparison tests: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0043] Figure 9 The AAVrh74N502I nDes-miR155-amiR-DMPK is shown. 204 Silencing of endogenous DMPK. Total DMPK levels were determined by qRT-PCR using RNA extracted from human DM1 cardiomyocytes differentiated from iPSCs. These cardiomyocytes were silencing endogenous DMPK. 204 Transduction. mRNA input levels were normalized to TBP mRNA. miRCTL3 was used as a negative control and set to 1. Paired Student's t-test: **p<0.01.

[0044] Figure 10 It displays amiR-DMPK 204 Volcano plot of genome-wide gene expression changes in treated HEK293 cells (Benjamini Hochberg FDR < 1%). The table shows the top 4 differentially expressed (DE) genes, where 3′UTR seed complementation is used, FDR < 1% and FDR < 5%.

[0045] Figure 11The amiRDMPK sequence is shown in multiple sequences. 204 Conservation of target sequences. The human target is SEQ ID NO:28, the macaque target is SEQ ID NO:29, the mouse target is SEQ ID NO:30, the rat target is SEQ ID NO:31, and the dog target is SEQ ID NO:32.

[0046] Figure 12 This describes the biodistribution of viral genome copy number / cell in various tissues in the treatment groups shown.

[0047] Figure 13 The dose-dependent expression of amiR-DMPK in different muscle tissues in the treatment groups shown is illustrated.

[0048] Figure 14 The levels of DMPK expression in different muscles of each treatment group after treatment are shown.

[0049] Figures 15A-15F The study explained the splicing changes of six genes (MBNL1, SOS1, PKM, zTTN, GOLGA4, and CLASP1), and compared healthy myotubes, untreated DM1 myotubes, and those treated with amiR-DMPK. 204 The DM1 myotube was treated.

[0050] Figures 16A-16B This describes the method used to identify skeletal muscle (human muscle tubes); Figure 16A It has high activity in hepatocytes (Huh7 cells); Figure 16B In vitro screening of promoters with low activity was conducted. Promoters S1-S9 were compared with publicly available muscle promoters and ubiquitous CAG promoters.

[0051] Figures 17A-17F This data is derived from immortalized human DM1 myotubes transduced with AAV2 carrying miRNAs targeting DMPK expressed under the desmin promoter. PCR quantification of miRNAs was performed. Figure 17A ) and DMPK mRNA levels ( Figure 17B Nuclear CTG RNA foci were imaged using fluorescence in situ hybridization (FISH) and in untreated ( Figure 17C ) and processed ( Figure 17D Quantification within cells. The quantification of foci can be performed... Figures 17E-17F I saw it in the middle.

[0052] Figure 18 This explains the use of 9×10 13 vg / kg AAV.amiR155-DMPK 204DMPK levels in DMSXL-treated mice. DMPK expression was measured by PCR 8 weeks after administration. Compared with untreated mice, treated mice showed a significant reduction in DMPK in several muscle groups, while no significant reduction in DMPK mRNA was detected in the liver.

[0053] Figure 19 This demonstrates a significant reduction in RNA foci size in the hearts of treated (DMSXL+) mice compared to the control (buffered; DMSXL-).

[0054] Figure 20 The splicing of various genes (Ldb3, Mbnl2, Spag9, DNase1, Tnnt3, and Zbtb49) was demonstrated in wild-type mice, buffer-treated DMSXL mice (DMSXL-), and treated DMSXL mice (DMSXL+).

[0055] Figure 21 The description uses AAV.amiR155-DMPK 204 Functional benefits in treated DMSXL mice. Myotonia in three different muscle groups—gastrocnemius, quadriceps, and tibialis anterior (TA)—was measured using electromyography (EMG) in wild-type mice, untreated DMSXL mice (DMSXL-), and treated DMSXL mice (DMSXL+). The number of grade 0 events (no myotonia) was compared with that of grade 1–3 events in each group.

[0056] Figures 22A-22B The study described the effects of using or not using AAV.amiR155-DMPK compared to WT mice. 204 Cardiac changes in treated DMSXL mice. Figure 22A It is a graph depicting the changes in the diameter (mm) of the aorta. Figure 22B The variation in cardiac output (μL / s) was depicted. Detailed Implementation

[0057] In some aspects, the present invention provides an RNAi comprising a first strand and a second strand, wherein a) the first strand and the second strand form a double strand; b) the first strand comprises a guiding region, wherein the guiding region comprises a nucleic acid having the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1) or having a sequence having approximately 90% identity with the sequence of SEQ ID NO:1; and c) the second strand comprises a non-guiding region, wherein the non-guiding region comprises a nucleic acid having the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2) or having a sequence having approximately 90% identity with the sequence of SEQ ID NO:2. In some embodiments, the present invention provides for expressing the nucleic acid encoding the RNAi; for example, an expression cassette for expressing the RNAi in the muscle of a mammal. In some embodiments, the expression cassette is in an rAAV vector.

[0058] In some aspects, the present invention provides a method for treating myotonic dystrophy type 1 (DM-1) in a mammal by administering the RNAi of the present invention to that mammal. In some embodiments, the administered RNAi inhibits the expression of dystrophic myotonic protein kinase (DMPK) in the mammal, thereby improving DM-1 in the mammal. General Technology

[0059] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are typically employed using conventional methods, such as those widely used in the following literature: Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (edited by F.M. Usubel et al., 2003); Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (edited by M.J. MacPherson, B.D. Hames, and G.R. Taylor, 1995); Antibodies: A Laboratory Manual (edited by Harlow and Lane, 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RIFreshney, 6th edition, J. Wiley and Sons, 2010); Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J.E.C. Liss, Academic Press, 1998); Introduction to Cell and Tissue Culture (J.M. Pather and P.E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A.Edited by Doyle, J.B. Griffiths, and D.G. Newell, J. Wiley and Sons (John Wiley & Sons Publishing, 1993-8); Handbook of Experimental Immunology (edited by D.M. Weir and C.C. Blackwell, 1996); Gene Transfer Vectors for Mammalian Cells (edited by J.M. Miller and M.C. Calos, 1987); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); Current Protocols in Immunology (edited by J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (edited by Ausubel et al., J. Wiley and...). Sons (John Willie & Son Publishing, 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty, IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic). Publishers (Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by V.T. DeVita et al., JB).Lippincott Company (2011). definition

[0060] As used in this article, “vector” refers to a recombinant plasmid or virus containing nucleic acid to be delivered to a host cell in vitro or in vivo.

[0061] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (as commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may contain polymers of synthetic subunits such as aminophosphates, and thus may be oligodeoxynucleotide aminophosphates (P-NH2) or mixed aminophosphate-phosphodiester oligomers. Furthermore, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing the complementary strand and annealing it under appropriate conditions, or by using a DNA polymerase to synthesize the complementary strand de novo with appropriate primers.

[0062] The terms "peptide" and "protein" are used interchangeably and refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues can contain native or non-native amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Full-length proteins and fragments thereof are included in this definition. These terms also include post-expression modifications of peptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of this invention, "peptide" refers to a protein that includes modifications to its native sequence, such as deletions, additions, and substitutions (generally conserved in nature), provided that the protein maintains the desired activity. These modifications can be intentional, such as through site-directed mutagenesis, or can be accidental, such as by mutations in the host producing the protein or due to errors in PCR amplification.

[0063] "Recombinant viral vector" refers to a recombinant polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not derived from viruses). In the case of recombinant AAV vectors, the recombinant nucleic acid has at least one flanking position, and in some embodiments, it is two inverted terminal repeat (ITR) sequences.

[0064] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not derived from AAV), flanked by at least one, and in some embodiments, by two AAV inverted terminal repeat (ITR) sequences. Such rAAV vectors can replicate and be packaged into infectious viral particles when present in host cells that have been infected with a suitable helper virus (or are expressing a suitable helper function) and are expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When rAAV vectors are incorporated into larger polynucleotides (e.g., in chromosomes or in another vector such as plasmids used for cloning or transfection), they can be referred to as "pre-vectors," which can be "rescued" through replication and capsidation in the presence of AAV packaging function and a suitable helper function. rAAV vectors can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and capsidated in viral particles, particularly AAV particles. The rAAV vector can be packaged into the capsid of an AAV virus to produce "recombinant adeno-associated virus particles (rAAV particles)".

[0065] "Heterologous" means originating from an entity that is genotype different from the entity being compared to, introduced into, or incorporated into. For example, a polynucleotide introduced into a different cell type through genetic engineering is a heterologous polynucleotide (and when expressed, it can encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or a portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0066] The term "transgenic" refers to a polynucleotide introduced into a cell that is capable of being transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In some respects, it endows the introduced cell with desired properties or otherwise produces desired therapeutic or diagnostic outcomes. In other respects, it can be transcribed into molecules that mediate RNA interference, such as miRNA, siRNA, or shRNA.

[0067] The terms “genomic particles (gp),” “genomic equivalents,” or “genomic copies (gc)” used in relation to viral titers refer to the number of virions containing the recombinant AAV DNA genome, and are unrelated to infectivity or functionality. The number of genomic particles in a particular vector formulation can be measured using procedures such as those described in the examples presented herein or, for example, Clark et al. (1999) Hum. Gene Ther. [Human Gene Therapy], 10:1031-1039; Veldwijk et al. (2002) Mol. Ther. [Molecular Therapy], 6:272-278.

[0068] As used herein, the term "vector genome (vg)" can refer to one or more polynucleotides comprising a set of polynucleotide sequences of a vector (e.g., a viral vector). The vector genome may be capsidated within a viral particle. Depending on the specific viral vector, the vector genome may comprise single-stranded DNA, double-stranded DNA, or single-stranded or double-stranded RNA. The vector genome may include endogenous sequences associated with the specific viral vector and / or any heterologous sequences inserted into the specific viral vector via recombination techniques. For example, a recombinant AAV vector genome may include at least one ITR sequence flanking the promoter, filler sequence, target sequence (e.g., RNAi), and polyadenylated sequence. A complete vector genome may comprise a set of polynucleotide sequences of the vector. In some embodiments, the nucleic acid titer of the viral vector may be measured in vg / mL. Suitable methods for measuring such titers are known in the art (e.g., quantitative PCR).

[0069] As used herein, the term "inhibition" can refer to an action that blocks, reduces, eliminates, or otherwise antagonizes the presence or activity of a specific target. Inhibition can refer to partial or complete inhibition. For example, inhibiting gene expression can refer to any action that results in the arrest, reduction, elimination, or any other antagonism of gene expression, including a decrease in mRNA abundance (e.g., silencing mRNA transcription), mRNA degradation, inhibition of mRNA translation, etc. In some embodiments, inhibiting DMPK expression can refer to the arrest, reduction, elimination, or any other antagonism of DMPK expression, including a decrease in DMPK mRNA abundance (e.g., silencing DMPK mRNA transcription), DMPK mRNA degradation, inhibition of DMPK mRNA translation, etc. For example, inhibiting protein accumulation in cells can refer to any action that results in the arrest, reduction, elimination, or any other antagonism of protein expression, including a decrease in mRNA abundance (e.g., silencing mRNA transcription), mRNA degradation, inhibition of mRNA translation, protein degradation, etc. In some embodiments, inhibiting the accumulation of DMPK proteins in cells refers to the blocking, reduction, elimination, or other antagonism of DMPK protein expression in cells, including a decrease in DMPK mRNA abundance (e.g., silencing DMPK mRNA transcription), degradation of DMPK mRNA, inhibition of DMPK mRNA translation, and degradation of DMPK proteins.

[0070] The terms “infectious unit (iU)”, “infectious particle”, or “replication unit” used in relation to viral titers refer to the number of infectious and replicable recombinant AAV vector particles measured by infection center assay (also known as replication center assay), as described, for example, in McLaughlin et al. (1988) J. Virol. [Journal of Virology], 62: 1963-1973.

[0071] The term “transduction unit (tu)” used in relation to viral titers refers to the number of infectious recombinant AAV vector particles that cause the production of functional transgenic products, as measured in functional assays such as those described in the examples presented herein or, for example, in Xiao et al. (1997) Exp. Neurobiol. [Experimental Neurobiology], 144:113-124; or Fisher et al. (1996) J. Virol. [Journal of Virology], 70:520-532.

[0072] "Inverted terminal repeat" or "ITR" sequence is a well-known term in the field, referring to a relatively short sequence with opposite orientation found at the end of a viral genome.

[0073] The term "AAV inverted terminal repeat (ITR)" is well-known in the art and refers to a sequence of approximately 145 nucleotides located at both ends of a naturally occurring single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two optional orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (named A, A', B, B', C, C', and D regions) that allow intrastrand base pairing to occur within this portion of the ITR.

[0074] The "terminal parsing sequence" or "trs" is the sequence in the D region of AAVITR that is cleaved by the AAV rep protein during viral DNA replication. The mutant terminal parsing sequence is resistant to AAV rep protein cleavage.

[0075] "AAV helper functions" refer to the functions that allow AAV to be replicated and packaged by the host cell. AAV helper functions can be provided in any of a variety of forms, including but not limited to helper viruses or helper virus genes that facilitate AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0076] A "helper virus" in AAV refers to a virus that allows AAV (a defective parvovirus) to be replicated and packaged by host cells. Helper viruses provide the "accessory function" that allows AAV replication. Many such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses such as vaccinia and baculoviruses. Adenoviruses encompass many different subgroups, but subgroup C5 adenovirus (Ad5) is the most commonly used. Many adenoviruses of human, non-human mammalian, and avian origin are known and available from collections such as the ATCC. Herpes family viruses also available from collections such as the ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Examples of adenovirus helper functions used for AAV replication include E1A, E1B, E2A, VA, and E4orf6 functions. Baculoviruses available from depository institutions include the Autographacalifornica nuclear polyhedrosis virus.

[0077] A rAAV formulation is said to be "substantially free" of helper virus if the ratio of infectious AAV particles to infectious helper virus particles is at least about 10²:1, at least about 10⁴:1, at least about 10⁶:1, or at least about 10⁸:1 or greater. In some embodiments, the formulation also does not contain an equal amount of helper virus proteins (i.e., proteins present due to such levels of helper virus if the aforementioned helper virus particle impurities are present in a disrupted form). Viral and / or cellular protein contamination can typically be observed on an SDS gel as the presence of Coomassie staining bands (e.g., the appearance of bands other than those corresponding to AAV capsid proteins VP1, VP2, and VP3).

[0078] The "percentage (%) sequence identity" of a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to those in a reference polypeptide or nucleic acid sequence after sequence alignment and the introduction of vacancies (if necessary) to obtain the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignments used to determine the percentage identity of amino acid or nucleic acid sequences can be performed in various ways within the scope of the art, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (edited by Ausubel et al., 1987), Supplement 30, Part 7.7.18, Table 7.7.1, and including BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. For the purposes of this document, the % amino acid sequence identity of a given amino acid sequence A with, and or against, a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity with, and or against, a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues that are scored as identical matches in the alignment of A and B by the sequence alignment program, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A with B will not be equal to the % amino acid sequence identity of B with A. For the purposes of this paper, the % nucleotide sequence identity of a given nucleic acid sequence C with, and or against, a given nucleic acid sequence D (which can be alternatively expressed as a given nucleic acid sequence C having or containing a certain % nucleotide sequence identity with, and or against, a given nucleic acid sequence D) is calculated as follows: 100 multiplied by the fraction W / Z, where W is the number of nucleotides that are scored as identical matches in the alignment of C and D by the sequence alignment program, and Z is the total number of nucleotides in D. It should be understood that when the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleotide sequence identity of C with D will not be equal to the % nucleotide sequence identity of D with C.

[0079] "Isolated" molecules (e.g., nucleic acids or proteins) or cells mean that they have been identified and isolated and / or recovered from components of their natural environment.

[0080] An "effective dose" is a dose sufficient to achieve a beneficial or desired outcome, including clinical outcomes (e.g., improvement of symptoms, achievement of clinical endpoints, etc.). An effective dose can be administered once or multiple times. In terms of disease state, an effective dose is a dose sufficient to improve, stabilize, or delay disease progression.

[0081] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0082] As used herein, “treatment” is a method for achieving a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of disease state (e.g., no worsening), prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, improvement or mitigation of disease state, and detectable or undetectable remission (partial or complete). “Treatment” may also mean prolonged survival compared to expected survival without treatment.

[0083] As used herein, the term "preventive treatment" refers to treatment in which an individual knows or suspects they have a condition or is at risk of having a condition, but has not yet shown symptoms of the condition or has shown the minimum symptoms of the condition. Individuals undergoing preventive treatment may receive treatment before symptoms develop.

[0084] As used herein, the term "myotonic dystrophy type 1" or "DM1" refers to a multisystem disorder affecting skeletal and smooth muscles, as well as the eye, heart, endocrine system, and central nervous system. There are three overlapping categories of DM-1 (Bird, TD, Myotonic Dystrophy Type 1. [Myotonic Dystrophy Type 1] September 17, 1999 [Updated March 25, 2021]. See: Adam MP, Ardinger HH, Pagon RA et al., eds. [Internet]. Seattle (WA): University of Washington, Seattle (1993–2022). Mild DM1 is characterized by cataracts and mild myotonia, while classic DM1 is characterized by muscle weakness and wasting, myotonia, cataracts, and often cardiac conduction abnormalities. Congenital DM1 is characterized by hypotonia and severe generalized weakness at birth, often accompanied by respiratory failure and early death; intellectual disability is common.

[0085] As used herein, the terms “dystrophic myotonic kinase,” “DMPK,” “myotonic protein-kinase,” “MT-PK,” “myotonic dystrophy protein kinase,” or “MDPK” can refer to the gene or its polypeptide product associated with most cases of DM1. The 3' untranslated region of the DMPK gene contains 5–37 copies of the CTG trinucleotide repeat sequence. This unstable motif extends to 50–1,000 copies, resulting in myotonic dystrophy type I, the severity of which increases with the copy number of the repeat element.

[0086] As used herein, “RNAi” can refer to any RNA molecule that induces RNA interference in cells. Examples of RNAi include, but are not limited to, small repressive RNAs (siRNAs), microRNAs (miRNAs), and small hairpin RNAs (shRNAs).

[0087] “miRNA” can refer to a polynucleotide containing: (i) a double-stranded sequence that targets a target gene to knock down via RNAi and (ii) an additional sequence that forms a stem-loop structure similar to that of an endogenous miRNA. In some embodiments, the miRNA includes nucleic acids flanking the stem-loop structure. These flanking sequences are referred to as “miRNA scaffolds.” The sequence of the target gene targeting RNAi (e.g., a short sequence of ~20-nt) can be linked to the sequence that produces the miRNA-like stem-loop and the sequence that pairs with the target sequence bases to form a double strand when the polynucleotide is assembled into a miRNA-like secondary structure. As described herein, the double strand may not be fully hybridized; for example, it may contain one or more unpaired or mismatched bases. When the polynucleotide is cleaved by Dicer, the double strand containing the target gene sequence can unfold and be incorporated into the RISC complex. The miRNA scaffold can refer to the miRNA itself or to the DNA polynucleotide encoding the miRNA. Examples of miRNA scaffolds include the miR-155 sequence (Lagos-Quintana, M. et al. (2002) Curr. Biol. [Current Biology] 12:735-9) and the mirGE scaffold (WO2014016817A2). Commercially available kits for cloning sequences into miRNA scaffolds are known in the art (e.g., from Life Technologies, Thermo Fisher Scientific, and Invitrogen). TM BLOCK-iT TM Pol II miR RNAi expression vector kit; Waltham, Massachusetts (MA)).

[0088] As used herein, the term "DMPK-mediated splicing defect" refers to dysregulation of alternative splicing occurring in tissues affected by DM1. These splicing defects contribute to core symptoms of the disease, such as insulin resistance, myotonia, myasthenia, and cardiac arrhythmias. Amplification of CUG repetitive sequences in DMPK transcripts accumulates in the cell nucleus, thereby impairing the physiological function of proteins involved in transcription, splicing, or RNA export. These accumulations lead to dysregulation of alternative splicing in different transcripts due to alterations in splicing mechanisms. In some embodiments, gene transcripts known to have splicing dysregulation in DM1 can be used to measure the effect of treatment with constructs as described herein. In some embodiments, splice rescue can be measured. In some embodiments, splice rescue can be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to equivalent healthy tissue. In some embodiments, the gene transcript being measured may be any one or more of MBNL1, SOS1, PKM, zTTN, GOGLA4, and CLASP1. Splicing can be measured by any one or more techniques known in the art, such as RNA sequencing (RNA-seq) or platform technologies such as the Nanostring platform.

[0089] As used herein, the term "sense" nucleic acid is a nucleic acid that contains all or part of the sequence encoding a transgene. In some instances, the mRNA of a transgene is a sense nucleic acid.

[0090] As used in this article, "antisense" nucleic acid is a nucleic acid sequence that is complementary to "sense" nucleic acid. For example, antisense nucleic acid can be complementary to mRNA encoding transgenes.

[0091] As used in this article, the “guide region” of RNAi is typically the RNAi strand that binds to the target mRNA based on complementarity. The complementarity region may encompass all or part of the guide region. Usually, the complementarity region includes at least the seed region. In many cases, the antisense region of RNAi is the guide region.

[0092] As used herein, the “passenger region” or “non-director region” of RNAi, which may be used interchangeably, is the region of RNAi that is complementary to the director region. In many cases, the meaningful region of RNAi is the passenger region.

[0093] As used herein, the “seed region” of RNAi (e.g., miRNA) is a region of approximately 1–8 nucleotides in length. In some instances, the seed region and its 3'-UTR of the target mRNA can be key determinants in RNAi recognition.

[0094] As used in this article, "off-target gene silencing" refers to the pairing of the seed region of RNAi with a sequence in the 3′-UTR of an unintended mRNA and directing translational repression and destabilization of those transcripts (e.g., reducing the expression of the unintended mRNA).

[0095] The “about” value or parameter mentioned herein includes (and describes) embodiments relating to that value or parameter itself. For example, a description referring to “about X” includes a description of “X”.

[0096] As used herein, unless otherwise indicated, the singular forms of the article “a,” “one,” and “the” include plural references.

[0097] It should be understood that the aspects and embodiments of the present invention described herein include “comprising aspects and embodiments,” “consisting of aspects and embodiments,” and / or “consisting substantially of aspects and embodiments.” RNAi

[0098] In some aspects, the present invention provides modified RNAi targeting DMPK RNA for the treatment of myotonic dystrophy type 1 (DM1). In some embodiments, the RNAi is a small repressive RNA (siRNA), microRNA (miRNA), or small hairpin RNA (shRNA). Small repressive or interfering RNAs (siRNAs) are known in the art as double-stranded RNA molecules of about 19-25 (e.g., 19-23) base pairs in length that induce RNAi in cells. miRNAs are generally smaller than siRNAs, can have multiple targets, and function to repress translation, degrade mRNA, and in some cases cleave mRNA by endonucleonucleotides. Small hairpin RNAs (shRNAs) are known in the art as RNA molecules of about 19-25 (e.g., 19-23) base pairs comprising double-stranded RNA linked by short loops (e.g., ~4-11 nucleotides) that induce RNAi in cells. In some embodiments, RNAi comprises a first strand and a second strand, wherein a) the first and second strands form a double strand; b) the first strand comprises a guiding region, wherein the guiding region comprises the nucleic acid sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1); and c) the second strand comprises a non-guiding region. In some embodiments, the guiding region comprises the nucleic acid sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1), and the non-guiding region comprises the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2).

[0099] In some embodiments, the first strand includes a guide region containing a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1). In some embodiments, the first strand includes a guide region containing a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1), but retaining at least one CpG motif. In some embodiments, the second strand includes a non-guided region comprising a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2). In some embodiments, the second strand includes a non-guided region comprising a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2), but retaining at least one CpG motif.

[0100] In some embodiments, RNAi comprises the nucleic acid sequence of SEQ ID NO:7. In some embodiments, RNAi comprises a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:7. In some embodiments, RNAi comprises a nucleic acid sequence having more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:7, but retaining at least one sequence (e.g., in the seed sequence).

[0101] In some embodiments, the present invention provides a nucleic acid encoding RNAi, the RNAi comprising a first strand and a second strand, wherein a) the first and second strands form a double strand; b) the first strand comprises a guiding region; and c) the second strand comprises a non-guiding region. In some embodiments, the nucleic acid encoding RNAi comprises the nucleic acid sequence of SEQ ID NO:4 and / or the nucleic acid of SEQ ID NO:5. In some embodiments, the nucleic acid encoding RNAi comprises a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:4 and / or a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:5. In some embodiments, the RNAi is encoded by the nucleic acid sequence of SEQ ID NO:8. In some embodiments, RNAi is encoded by a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:8.

[0102] MicroRNAs (miRNAs) are known in the art as RNA molecules that induce RNAi in cells, comprising a short (e.g., 19-25 base pairs) sequence of a double-stranded RNA linked by a loop and one or more additional sequences containing one or more bumps (e.g., mispaired or unpaired base pairs) of the double-stranded RNA. As used herein, the term “miRNA” encompasses both endogenous and exogenous or heterologous miRNAs. In some embodiments, “miRNA” may refer to pri-miRNA or pre-miRNA. During miRNA processing, pri-miRNA transcripts are produced. pri-miRNA is processed by Drosha-DGCR8 to produce pre-miRNA by removing one or more sequences, leaving a pre-miRNA with a 5' flanking region, a guiding strand, a loop region, an unguided strand, and a 3' flanking region; or a pre-miRNA with a 5' flanking region, an unguided strand, a loop region, a guiding strand, and a 3' flanking region. The pre-miRNA is then exported to the cytoplasm and processed by Dicer to produce siRNA with a guiding strand and an unguided (or passenger) strand. The RISC complex then uses a guide strand to catalyze gene silencing, for example by recognizing a target RNA sequence complementary to the guide strand. Further descriptions of miRNAs can be found, for example, in WO 2008 / 150897. The recognition of a target sequence by a miRNA is primarily determined by the pairing between the target and the miRNA seed sequence (e.g., nucleotides 1–8 (5' to 3') of the guide strand) (see, for example, Boudreau, RL et al. (2013) Nucleic Acids Res. [Nucleic Acid Research] 41:e9).

[0103] In the pri / pre-miRNA structure, the guide strand:non-guide strand interface in the duplex is partially formed by complementary base pairing (e.g., Watson-Crick base pairing). However, in some embodiments, this complementary base pairing does not extend throughout the entire duplex. In some embodiments, a protrusion in the interface may be present at one or more nucleotide sites. As used herein, the term "protrusion" may refer to a nucleic acid region that is not complementary to the nucleic acid opposite it in the duplex. In some embodiments, a protrusion is formed when regions of complementary nucleic acids bind to each other, while regions of the central non-complementary region do not bind. In some embodiments, a protrusion is formed when the two nucleic acid strands located between two complementary regions have different lengths. As described below, a protrusion may contain one or more nucleotides.

[0104] During miRNA processing, the miRNA is cleaved at a cleavage site near the guide strand:non-guide strand interface, thereby releasing the guide strand and non-guide strand siRNA duplexes. In some embodiments, the miRNA includes a protrusion in the sense strand or antisense strand near the cleavage site. In other words, in some embodiments, the miRNA includes a protrusion in the guide strand or non-guide strand near the seed sequence.

[0105] In some embodiments, the miRNA includes a protrusion on the guiding strand opposite to the 5' cleavage site of the mature non-guiding strand. In some embodiments, the miRNA includes a protrusion opposite to the 5' nucleotide of the non-guiding strand. In some embodiments, the miRNA includes a protrusion on the sense strand opposite to the 3' cleavage site of the mature guiding strand. In some embodiments, the miRNA includes a protrusion opposite to the 3' nucleotide of the guiding strand.

[0106] The safety of RNAi-based therapies can be hampered by the ability of small repressive RNAs (siRNAs) to bind to and reduce the expression of unwanted mRNAs (an effect known as off-target gene silencing). Off-target effects primarily occur when the seed region (nucleotides 2–8 of the siRNA) pairs with a sequence in the 3′-UTR of the unwanted mRNA and directs translational repression and destabilization of those transcripts. Reduced off-target RNAi can be designed by substituting bases within both the directing and undirecting sequences; for example, by generating CpG motifs. Potential substitutions that produce significantly lower off-target scores can be evaluated using the SiSPOTR algorithm, a specific-focused siRNA design algorithm that identifies candidate sequences with the lowest off-target potential and effective silencing ability (Boudreau et al., Nucleic Acids Res. 2013 Jan; 41(1)e9). Reduced SiSPOTR scores predict a lower number of potential human off-target sequences compared to the parental RNAi molecule. In some embodiments of the present invention, RNAi is modified to reduce off-target gene silencing.

[0107] In some embodiments, the first and second strands are joined by an RNA capable of forming a loop structure (e.g., an RNA adapter). As is generally known in the art, when an RNA molecule contains two RNA sequences, an RNA loop structure (e.g., a stem-loop or hairpin) is formed, where the two RNA sequences are base-paired together and separated by an unpaired RNA sequence. For example, a loop structure can be formed in RNA molecule ABC if sequences A and C are complementary or partially complementary, causing them to pair together, but the bases in sequence B are unpaired.

[0108] In some embodiments, the RNA capable of forming a loop structure comprises 4 to 50 nucleotides. In some embodiments, the RNA capable of forming a loop structure comprises 13 nucleotides. In some embodiments, the number of nucleotides in the RNA capable of forming a loop is 4 to 50 nucleotides or any integer between them. In some embodiments, 0-50% of the loop may be complementary to another portion of the loop. As used herein, the term "loop structure" is a sequence connecting two complementary strands of a nucleic acid. In some embodiments, 1-3 nucleotides of the loop structure are adjacent to the complementary strand of the nucleic acid and may be complementary to 1-3 nucleotides of the distal portion of the loop structure. For example, the three nucleotides at the 5' end of the loop structure may be complementary to the three nucleotides at the 3' end of the loop structure.

[0109] In some embodiments, the nucleic acid encoding the RNAi disclosed herein comprises a heterologous miRNA scaffold. In some embodiments, the heterologous miRNA scaffold is used to regulate miRNA expression; for example, to increase or decrease miRNA expression. Any miRNA scaffold known in the art can be used. In some embodiments, the miRNA scaffold is derived from the miR-155 scaffold (see, for example, Lagos-Quintana, M. et al. (2002) Curr. Biol. [Current Biology] 12:735-9) and Invitrogen from Thermo Fisher Scientific, a life sciences company. TM BLOCK-iT TM Pol II miR RNAi expression vector kit; Waltham, Massachusetts) or mirGE scaffold (WO 2014 / 016817). Treatment methods for myotonic dystrophy type 1 (DM-1)

[0110] Myotonic dystrophy type 1 (DM1) is a monogenic, autosomal dominant, progressive disease caused by the expansion of CTG repeat sequences (>50) at the DMPK locus (dystrophic myotonic protein kinase). The DMPK with the repeat sequence is transcribed into mRNA, which forms hairpins and binds to RNA-binding proteins, blocking their normal function. This leads to nuclear foci, missplicing, and ultimately myotonia. DM1 primarily affects skeletal, cardiac, and smooth muscle, causing significant physical, cognitive, and behavioral impairments and disability.

[0111] In some aspects, the present invention provides methods and compositions for treating myotonic dystrophy type 1 (DM1) in mammals, the methods comprising administering to the mammal a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition comprising rAAV particles disclosed herein). In some aspects, the present invention provides methods and compositions for inhibiting the expression of DMPK in mammals suffering from DM-1, the methods comprising administering to the mammal a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition comprising rAAV particles disclosed herein). In some aspects, the present invention provides methods and compositions for inhibiting the accumulation of DMPK in the cells of mammals suffering from DM1, the methods comprising administering to the mammal a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition comprising rAAV particles disclosed herein). In some aspects, the present invention provides methods and compositions for improving symptoms of DM1, the methods comprising administering to the mammalian muscle brain an effective amount of rAAV particles comprising a vector encoding an RNAi disclosed herein.

[0112] In some aspects, the present invention provides RNAi for targeting DMPK mRNA in mammals suffering from DM1. In some embodiments, the RNAi comprises a first strand and a second strand, the first strand comprising a first nucleic acid containing the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1), and the second strand comprising a second nucleic acid containing the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2). The RNAi described herein (e.g., as part of an rAAV vector) is particularly useful for the treatment of DM1.

[0113] In some embodiments, the RNAi is a small repressive RNA (siRNA), a microRNA (miRNA), or a small hairpin RNA (shRNA). Small repressive or interfering RNAs (siRNAs) are known in the art as double-stranded RNA molecules of about 19-25 (e.g., 19-23) base pairs in length that induce RNAi in cells. miRNAs are generally smaller than siRNAs, can have multiple targets, and function to repress translation, degrade mRNA, and in some cases cleave mRNA by endonucleonucleotides. Small hairpin RNAs (shRNAs) are known in the art as RNA molecules of about 19-25 (e.g., 19-23) base pairs comprising double-stranded RNA linked by short loops (e.g., ~4-11 nucleotides) that induce RNAi in cells.

[0114] In some embodiments, the miRNA comprises a guide sequence that is approximately 90% identical to that of SEQ ID NO:1. In some embodiments, the miRNA comprises a guide sequence that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:1.

[0115] In some embodiments, the miRNA comprises a non-guided sequence (passenger strand) that is approximately 90% identical to SEQ ID NO:2. In some embodiments, the miRNA comprises a non-guided sequence that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2.

[0116] In some embodiments, the first strand and the second strand are linked by RNA capable of forming a loop structure. As is generally known in the art, when an RNA molecule contains two RNA sequences, an RNA loop structure (e.g., stem-loop or hairpin) is formed, in which the two RNA sequences are base-paired together and separated by an unpaired RNA sequence. For example, a loop structure can be formed in RNA molecule ABC if sequences A and C are complementary or partially complementary such that they are base-paired, but the bases in sequence B are unpaired.

[0117] In some embodiments, the RNA capable of forming a loop structure comprises 4 to 50 nucleotides. In some embodiments, the RNA capable of forming a loop structure comprises 13 nucleotides. In some embodiments, the RNA capable of forming a loop structure comprises the nucleotide sequence GUUUUGGCCACUGACUGAC (SEQ ID NO:3). In some embodiments, the vector genome comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:3.

[0118] In some aspects, the present invention provides a method of administering RNAi comprising a first strand and a second strand to a mammal (e.g., a mammal with DM1), the first strand comprising a first nucleic acid containing the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1), and the second strand comprising a second nucleic acid containing the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2). In some embodiments, the recombinant viral particle comprises RNAi. In some embodiments, the recombinant viral particle is an AAV particle capsidated with an rAAV vector, wherein the rAAV vector encodes RNAi.

[0119] In some embodiments, rAAV particles are delivered by systemic injection of rAAV particles into a mammal. In some embodiments, systemic injection is intravenous injection, intra-arterial injection, intramuscular injection, intraperitoneal injection, intradermal or subcutaneous injection, intra-CSF administration, and intrathecal administration (IT).

[0120] In some aspects, the present invention provides methods for treating DM1 in mammals, methods comprising administering the pharmaceutical composition disclosed herein to the mammal. In some aspects, the present invention provides methods for inhibiting the accumulation of DMPK in the cells of mammals suffering from DM1, methods comprising administering the pharmaceutical composition disclosed herein to the mammal. In some aspects, the present invention provides methods for inhibiting the expression of DMPK in mammals suffering from DM1, methods comprising administering the pharmaceutical composition disclosed herein to the mammal. In some embodiments, the DMPK is a mutant DMPK (e.g., a DMPK containing more than 37 or more than 50 CTG repeat sequences).

[0121] In some embodiments, the present invention provides a method for treating a person with DM1 by administering an effective amount of a pharmaceutical composition comprising an rAAV vector encoding the RNAi disclosed herein to inhibit the activity of a mutant DMPK. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0122] In some embodiments, these methods include administering an effective amount of a pharmaceutical composition comprising an rAAV vector encoding the RNAi disclosed herein to inhibit the activity of mutant DMPK. In some embodiments, the viral titer of these rAAV particles is at least about 5 × 10⁻⁶. 12 6×10 12 7×10 12 8×10 12 9×10 12 10×10 12 11×10 12 15×10 12 20×10 12 25×10 12 30×10 12 Or 50×10 12 Any of the following: genome copies / mL. In some embodiments, the viral titer of these rAAV particles is approximately 5 × 10⁻⁶. 12 Up to 6×10 12 6×10 12 Up to 7×10 12 7×10 12 Up to 8×10 12 8×10 12 Up to 9×10 12 9×10 12 Up to 10×10 12 10×10 12 Up to 11×10 12 11×10 12 Up to 15×10 12 15×10 12 Up to 20×10 12 20×10 12 Up to 25×10 12 25×10 12 Up to 30×10 12 30×10 12 Up to 50×10 12 Or 50×10 12 Up to 100×10 12 Any of the following: genome copies / mL. In some embodiments, the viral titer of these rAAV particles is approximately 5 × 10⁻⁶. 12 Up to 10×10 12 10×10 12 Up to 25×10 12 Or 25×10 12 Up to 50×10 12 Any of the following: genome copies / mL. In some embodiments, the viral titer of these rAAV particles is at least about 5 × 10⁻⁶. 96×10 9 7×10 9 8×10 9 9×10 9 10×10 9 11×10 9 15×10 9 20×10 9 25×10 9 30×10 9 Or 50×10 9 Any of the following: transduction units / mL. In some embodiments, the viral titer of these rAAV particles is approximately 5 × 10⁻⁶. 9 Up to 6×10 9 6×10 9 Up to 7×10 9 7×10 9 Up to 8×10 9 8×10 9 Up to 9×10 9 9×10 9 Up to 10×10 9 10×10 9 Up to 11×10 9 11×10 9 Up to 15×10 9 15×10 9 Up to 20×10 9 20×10 9 Up to 25×10 9 25×10 9 Up to 30×10 9 30×10 9 Up to 50×10 9 Or 50×10 9 Up to 100×10 9 Any of the following: transduction units / mL. In some embodiments, the viral titer of these rAAV particles is approximately 5 × 10⁻⁶. 9 Up to 10×10 9 10×10 9 Up to 15×10 9 15×10 9 Up to 25×10 9 Or 25×10 9 Up to 50×10 9 Any of the following: transduction units / mL. In some embodiments, the viral titer of these rAAV particles is at least about 5 × 10⁻⁶. 10 6×10 10 7×10 10 8×10 10 9×1010 10×10 10 11×10 10 15×10 10 20×10 10 25×10 10 30×10 10 40×10 10 Or 50×10 10 Any of the following: infection units / mL. In some embodiments, the viral titer of these rAAV particles is at least about 5 × 10⁻⁶. 10 Up to 6×10 10 6×10 10 Up to 7×10 10 7×10 10 Up to 8×10 10 8×10 10 Up to 9×10 10 9×10 10 Up to 10×10 10 10×10 10 Up to 11×10 10 11×10 10 Up to 15×10 10 15×10 10 Up to 20×10 10 20×10 10 Up to 25×10 10 25×10 10 Up to 30×10 10 30×10 10 Up to 40×10 10 40×10 10 Up to 50×10 10 Or 50×10 10 Up to 100×10 10 Any of the following: infection units / mL. In some embodiments, the viral titer of these rAAV particles is at least about 5 × 10⁻⁶. 10 Up to 10×10 10 10×10 10 Up to 15×10 10 15×10 10 Up to 25×10 10 Or 25×10 10 Up to 50×10 10 Any of the following: infection units / mL.

[0123] In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 8 Approximately 2×10 13Any of the following: genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 8 Approximately 5×10 8 Approximately 5×10 8 Approximately 10×10 8 Approximately 10×10 8 Approximately 20×10 8 Approximately 20×10 8 Approximately 30×10 8 Approximately 30×10 8 Approximately 40×10 8 Approximately 40×10 8 Approximately 50×10 8 Or approximately 50×10 8 Approximately 100×10 8 Any of the following: genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 9 Approximately 5×10 9 Approximately 5×10 9 Approximately 10×10 9 Approximately 10×10 9 Approximately 20×10 9 Approximately 20×10 9 Approximately 30×10 9 Approximately 30×10 9 Approximately 40×10 9 Approximately 40×10 9 Approximately 50×10 9 Or approximately 50×10 9 Approximately 100×10 9 Any of the following: genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 10 Approximately 5×10 10 Approximately 5×10 10 Approximately 10×10 10 Approximately 10×10 10 Approximately 20×10 10 Approximately 20×10 10 Approximately 30×10 10 Approximately 30×10 10 Approximately 40×10 10 Approximately 40×10 10 Approximately 50×10 10 Or approximately 50×10 10 Approximately 100×10 10 Any of the following: genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 1 × 10⁻⁶.11 Approximately 5×10 11 Approximately 5×10 11 Approximately 10×10 11 Approximately 10×10 11 Approximately 20×10 11 Approximately 20×10 11 Approximately 30×10 11 Approximately 30×10 11 Approximately 40×10 11 Approximately 40×10 11 Approximately 50×10 11 Or approximately 50×10 11 Approximately 100×10 11 Any of the following: genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 12 Approximately 5×10 12 Approximately 5×10 12 Approximately 10×10 12 Approximately 10×10 12 Approximately 20×10 12 Approximately 20×10 12 Approximately 30×10 12 Approximately 30×10 12 Approximately 40×10 12 Approximately 40×10 12 Approximately 50×10 12 Or approximately 50×10 12 Approximately 100×10 12 Any of the following: genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 13 Approximately 2×10 13 Any of the following: genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 8 Approximately 5×10 8 Approximately 1×10 9 Approximately 5×10 9 Approximately 1×10 10 Approximately 5×10 10 Approximately 1×10 11 Approximately 5×10 11 Approximately 1×10 12 Approximately 5×10 12 Approximately 1×10 13 Or approximately 2×10 13 One genome copy / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 5 × 10⁻⁶. 12One genome copy / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 13 One genome copy / mL. In some embodiments, the dose concentration of rAAV particles administered to an individual is approximately 2 × 10⁻⁶. 13 One genome copy / mL.

[0124] In some embodiments, the dose of rAAV particles administered to an individual is at least about 1 × 10⁻⁶. 8 Approximately 2×10 14 Any of the following: genome copies / kg body weight. In some embodiments, the dose of rAAV particles administered to an individual is between about 1 × 10⁻⁶. 8 Approximately 2×10 14 Between one genome copy per kg of body weight. In some embodiments, the dose of rAAV particles administered to an individual is between approximately 1 × 10⁻⁶. 8 To approximately 1×10 14 5×10 8 To approximately 1×10 14 1×10 9 To approximately 1×10 14 5×10 9 To approximately 1×10 14 1×10 10 To approximately 1×10 14 5×10 10 To approximately 1×10 14 1×10 11 To approximately 1×10 14 5×10 11 To approximately 1×10 14 1×10 12 To approximately 1×10 14 5×10 12 To approximately 1×10 14 1×10 13 To approximately 1×10 14 5×10 13 To approximately 1×10 14 1×10 8 Approximately 5×10 13 5×10 8 Approximately 5×10 13 1×10 9 Approximately 5×10 13 5×10 9 Approximately 5×10 13 1×10 10 Approximately 5×10 13 5×10 10 Approximately 5×10 13 1×1011 Approximately 5×10 13 5×10 11 Approximately 5×10 13 1×10 12 Approximately 5×10 13 5×10 12 Approximately 5×10 13 1×10 13 Approximately 5×10 13 1×10 8 To approximately 1×10 13 5×10 8 To approximately 1×10 13 1×10 9 To approximately 1×10 13 5×10 9 To approximately 1×10 13 1×10 10 To approximately 1×10 13 5×10 10 To approximately 1×10 13 1×10 11 To approximately 1×10 13 5×10 11 To approximately 1×10 13 1×10 12 To approximately 1×10 13 5×10 12 To approximately 1×10 13 1×10 8 Approximately 5×10 12 5×10 8 Approximately 5×10 12 1×10 9 Approximately 5×10 12 5×10 9 Approximately 5×10 12 1×10 10 Approximately 5×10 12 5×10 10 Approximately 5×10 12 1×10 11 Approximately 5×10 12 5×10 11 Approximately 5×10 12 1×10 12 Approximately 5×10 12 1×10 8 To approximately 1×10 12 5×10 8 To approximately 1×10 12 1×10 9 To approximately 1×10 12 5×109 To approximately 1×10 12 1×10 10 To approximately 1×10 12 5×10 10 To approximately 1×10 12 1×10 11 To approximately 1×10 12 5×10 11 To approximately 1×10 12 1×10 8 Approximately 5×10 11 5×10 8 Approximately 5×10 11 1×10 9 Approximately 5×10 11 5×10 9 Approximately 5×10 11 1×10 10 Approximately 5×10 11 5×10 10 Approximately 5×10 11 1×10 11 Approximately 5×10 11 1×10 8 To approximately 1×10 11 5×10 8 To approximately 1×10 11 1×10 9 To approximately 1×10 11 5×10 9 To approximately 1×10 11 1×10 10 To approximately 1×10 11 5×10 10 To approximately 1×10 11 1×10 8 Approximately 5×10 10 5×10 8 Approximately 5×10 10 1×10 9 Approximately 5×10 10 5×10 9 Approximately 5×10 10 1×10 10 Approximately 5×10 10 1×10 8 To approximately 1×10 10 5×10 8 To approximately 1×10 10 1×10 9 To approximately 1×10 10 5×10 9 To approximately 1×10 10 1×108 Approximately 5×10 9 5×10 8 Approximately 5×10 9 1×10 9 Approximately 5×10 9 1×10 8 To approximately 1×10 9 5×10 8 To approximately 1×10 9 Or 1×10 8 Approximately 5×10 8 Between any of the values ​​of gc / kg body weight. In some embodiments, the dose of rAAV particles administered to an individual is approximately 1 × 10⁻⁶ g / kg body weight. 9 Approximately 5×10 9 Approximately 1×10 10 Approximately 5×10 10 Approximately 1×10 11 Approximately 5×10 11 Approximately 1×10 12 Approximately 5×10 12 Approximately 1×10 13 Approximately 5×10 13 Approximately 1×10 14 Or approximately 2×10 14 One genome copy per kg body weight. In some embodiments, the dose of rAAV particles administered to an individual is approximately 5 × 10⁻⁶. 13 One genome copy per kg body weight. In some embodiments, the dose of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 14 One genome copy per kg body weight. In some embodiments, the dose of rAAV particles administered to an individual is approximately 2 × 10⁻⁶. 14 One genome copy per kg of body weight.

[0125] In some embodiments, the total amount of rAAV particles administered to an individual is at least about 1 × 10⁻⁶. 9 Approximately 2×10 14 Any of the following: genome copies / kg body weight. In some embodiments, the total amount of rAAV particles administered to an individual is approximately 1 × 10⁻⁶. 9 Approximately 2×10 14 Any of the following: genome copies / kg body weight. In some embodiments of the invention, the volume of the composition injected into the striatum is greater than any of or between about 10 μl, 25 μl, 50 μl, 75 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, 1 mL, 5 mL, 10 mL, 25 mL, 50 mL, 75 mL, or 100 mL.

[0126] The compositions of the present invention (e.g., rAAV particles comprising a vector encoding the RNAi disclosed herein) can be used alone or in combination with one or more other therapeutic agents for treating DM1. The interval between sequential administrations can be at least (or alternatively, less than) minutes, hours, or days.

[0127] In some embodiments, RNAi for treating DM1 is administered in combination with an immunosuppressant; for example, to suppress the immune response to RNAi. In some embodiments, the immunosuppressant is administered before RNAi administration. In some embodiments, the immunosuppressant is administered concurrently with RNAi administration. In some embodiments, the immunosuppressant is administered after RNAi administration. In some embodiments, the immunosuppressant is administered in any combination before, during, or after RNAi administration.

[0128] In some embodiments, rAAV particles for treating DM1 are administered in combination with an immunosuppressant; for example, to suppress an immune response to the rAAV particles and / or the transgenic product of the rAAV particles. In some embodiments, the immunosuppressant is administered before administration of the rAAV particles. In some embodiments, the immunosuppressant is administered concurrently with administration of the rAAV particles. In some embodiments, the immunosuppressant is administered after administration of the rAAV particles. In some embodiments, the immunosuppressant is administered in any combination before, during, or after administration of the rAAV particles.

[0129] In some embodiments, the present invention provides the use of an effective amount of any of the RNAi described herein in the manufacture of a medicament for treating myotonic dystrophy type 1 (DM1) in mammals in need. In some embodiments, the present invention provides the use of an effective amount of any of the RNAi described herein in the manufacture of a medicament for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1. In some embodiments, the present invention provides the use of an effective amount of any of the RNAi described herein in the manufacture of a medicament for inhibiting the accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1.

[0130] In some embodiments, the present invention provides the use of an effective amount of any of the RNAi described herein for treating myotonic dystrophy type 1 (DM1) in mammals in need. In some embodiments, the present invention provides the use of an effective amount of any of the RNAi described herein for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1. In some embodiments, the present invention provides the use of an effective amount of any of the RNAi described herein for inhibiting the accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1.

[0131] In some embodiments, the present invention provides the RNAi described herein for treating myotonic dystrophy type 1 (DM1) in mammals in need. In some embodiments, the present invention provides any of the RNAi described herein for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1. In some embodiments, the present invention provides any of the RNAi described herein for inhibiting the accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1.

[0132] In some embodiments, the present invention provides the use of an effective amount of any viral particles described herein (e.g., AAV particles) in the manufacture of a medicament for treating myotonic dystrophy type 1 (DM1) in mammals in need. In some embodiments, the present invention provides the use of an effective amount of any viral particles described herein (e.g., AAV particles) in the manufacture of a medicament for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1. In some embodiments, the present invention provides the use of an effective amount of any viral particles described herein (e.g., AAV particles) in the manufacture of a medicament for inhibiting the accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1.

[0133] In some embodiments, the present invention provides the use of an effective amount of any viral particle described herein (e.g., AAV particles) for the treatment of myotonic dystrophy type 1 (DM1) in mammals in need. In some embodiments, the present invention provides the use of an effective amount of any viral particle described herein (e.g., AAV particles) for the inhibition of expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1. In some embodiments, the present invention provides the use of an effective amount of any viral particle described herein (e.g., AAV particles) for the inhibition of accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1.

[0134] In some embodiments, the present invention provides viral particles (e.g., AAV particles) as described herein for treating myotonic dystrophy type 1 (DM1) in mammals in need. In some embodiments, the present invention provides any viral particles (e.g., AAV particles) as described herein for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1. In some embodiments, the present invention provides any viral particles (e.g., AAV particles) as described herein for inhibiting the accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1.

[0135] In some embodiments, the present invention provides the use of an effective amount of any of the compositions described herein in the manufacture of a medicament for treating myotonic dystrophy type 1 (DM1) in mammals of need. In some embodiments, the present invention provides the use of an effective amount of any of the compositions described herein in the manufacture of a medicament for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals of need suffering from DM-1. In some embodiments, the present invention provides the use of an effective amount of any of the compositions described herein in the manufacture of a medicament for inhibiting the accumulation of DMPK RNA in the cells of mammals of need suffering from DM-1.

[0136] In some embodiments, the present invention provides the use of an effective amount of any of the compositions described herein for the treatment of myotonic dystrophy type 1 (DM1) in mammals in need. In some embodiments, the present invention provides the use of an effective amount of any of the compositions described herein for the inhibition of expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1. In some embodiments, the present invention provides the use of an effective amount of any of the compositions described herein for the inhibition of accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1.

[0137] In some embodiments, the present invention provides the compositions described herein for treating myotonic dystrophy type 1 (DM1) in mammals in need. In some embodiments, the present invention provides any of the compositions described herein for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in mammals in need suffering from DM-1. In some embodiments, the present invention provides any of the compositions described herein for inhibiting the accumulation of DMPK RNA in the cells of mammals in need suffering from DM-1. RNAi expression constructs and vectors

[0138] This invention provides expression constructs, vectors, and rAAV particles for expressing the RNAi described herein.

[0139] In some embodiments, the nucleic acid encoding the RNAi disclosed herein comprises a heterologous miRNA scaffold. In some embodiments, the heterologous miRNA scaffold is used to regulate miRNA expression; for example, to increase or decrease miRNA expression. Any miRNA scaffold known in the art can be used. In some embodiments, the miRNA scaffold is derived from the miR-155 scaffold (see, for example, Lagos-Quintana, M. et al. (2002) Curr. Biol. [Current Biology] 12:735-9) and Invitrogen from Thermo Fisher Scientific, a life sciences company. TMBLOCK-iT TM Pol II miR RNAi Expression Vector Kit; Waltham, Massachusetts) or mirGE scaffold (WO 2014 / 016817). In some embodiments, the nucleic acid encoding the RNAi disclosed herein comprises a miRNA scaffold. In some embodiments, the miRNA scaffold is provided by SEQ ID NO:11. In some embodiments, the miRNA scaffold comprises a nucleic acid having greater than 80%, 85%, 90%, 95%, or 99% identity with the nucleic acid sequence of SEQ ID NO:11.

[0140] In some embodiments, RNAi targets RNA encoding a DM1-associated peptide (e.g., mutant DMPK). It is not intended to be theoretically rigorous, but rather to suggest that RNAi can be used to reduce or eliminate the expression and / or activity of peptides already associated with DM1 (e.g., mutant DMPK).

[0141] In some embodiments, the transgene (e.g., encoding the RNAi disclosed herein) is operatively linked to a promoter. Exemplary promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, RSV LTR, and MoMLV. LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter and CK6 promoter, transthyretin promoter (TTR), TK promoter, tetracycline-responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; cytomegalovirus enhancer / chicken β-actin / rabbit β-globin promoter (CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9) and elongation factor 1-α promoter (EFl-α) promoter (Kim et al., Gene, 1990, 91(2):217-23 and Guo et al., GeneTher, 1996, 3(9):802-10). In some embodiments, the promoter includes a human β-glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken β-actin (CBA) promoter. The promoter can be a constitutive promoter, an inducible promoter, or a repressive promoter.

[0142] Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors (such as temperature), or specific physiological states, such as the acute phase, a specific differentiation state of the cell, or only in replicating cells. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline repressor system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), and the tetracycline inducible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)). See also N et al., Science, 268:1766-1769 (1995), Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Other types of inducible promoters available in this context are promoters regulated by specific physiological states, such as temperature, acute phase, specific differentiation state of the cell, or only in replicating cells.

[0143] In some embodiments, the regulatory sequence confers tissue-specific gene expression. In some cases, the tissue-specific regulatory sequence binds to a tissue-specific transcription factor that induces transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the promoter is a desmin promoter. In some embodiments, the promoter is a human desmin promoter (e.g., -228 to +75 of the human desmin gene; e.g., SEQ ID NO:23). In some embodiments, the promoter is a modified desmin promoter. In some embodiments, the desmin promoter includes a desmin promoter element that is important for high-level expression in muscle cells (Li and Paulin et al. 1991. Journal of BiolChem.). In some embodiments, the desmin promoter includes at least one copy of the Byrne desmin enhancer (e.g., SEQ ID NO:21). In some embodiments, the desmin promoter includes at least one copy of the Paulin desmin enhancer (-973 to -693) (e.g., SEQ ID NO:22). In some embodiments, the desmin promoter comprises one copy of the Byrne desmin enhancer and one copy of the Paulin desmin enhancer (-973 to -693). In some embodiments, the desmin promoter comprises one copy of the Byrne desmin enhancer and one copy of the Paulin desmin enhancer (-973 to -693) and the promoter of the human desmin gene (-228 to +75).

[0144] In some aspects, the present invention provides expression cassettes (e.g., and expression cassettes for expressing transgenes (e.g., therapeutic transgenes) in muscle cells), wherein the expression cassette comprises a modified desmin promoter, wherein the desmin promoter comprises one or more enhancer elements, and a promoter of the human desmin gene. In some embodiments, the desmin promoter comprises two enhancer elements, and a promoter of the human desmin gene. In some embodiments, the desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21 and one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:21. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:21, and one or more enhancer elements comprising a nucleotide sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:22.

[0145] In some embodiments, the expression cassette containing the modified desmin promoter further includes an intron. In some embodiments, the intron is a rabbit β-globin intron. In some embodiments, the intron contains the nucleotide sequence of SEQ ID NO:13. In some embodiments, the intron contains a nucleotide sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:13. In some embodiments, a nucleic acid encoding a transgene (e.g., a therapeutic transgene) is embedded in the intron. In some embodiments, the intron contains a 5' arm and a 3' arm, wherein the 5' arm is located at the 5' of the nucleic acid encoding the transgene and the 3' arm is located at the 3' of the nucleic acid encoding the transgene. In some embodiments, the 5' arm of the intron contains a nucleic acid having the sequence of SEQ ID NO:14. In some embodiments, the 5' arm of the intron contains a nucleic acid having a sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:14. In some embodiments, the 3' arm of the intron comprises a nucleic acid having the sequence of SEQ ID NO:15. In some embodiments, the 3' arm of the intron comprises a nucleic acid having a sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:15. In some embodiments, the 5' arm of the intron comprises a nucleic acid having the sequence of SEQ ID NO:14, and the 3' arm of the intron comprises a nucleic acid having the sequence of SEQ ID NO:15. In some embodiments, the 5' arm of the intron comprises a nucleic acid having a sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:14, and the 3' arm of the intron comprises a nucleic acid having a sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:15.

[0146] In some embodiments, the expression cassette containing the modified desmin promoter further includes a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or an HSV TK pA. In some embodiments, the polyadenylation signal is a minimal bovine growth hormone polyadenylation signal. In some embodiments, the bovine growth hormone polyadenylation signal comprises a nucleic acid having the sequence of SEQ ID NO:16. In some embodiments, the bovine growth hormone polyadenylation signal comprises a nucleic acid having a sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:16.

[0147] In some embodiments, the present invention provides an expression cassette comprising a modified desmin promoter for expressing a transgene (e.g., a therapeutic transgene) in muscle cells. In some embodiments, the transgene encodes a polypeptide (e.g., a therapeutic polypeptide). In some embodiments, the transgene encodes a nucleic acid (e.g., a therapeutic nucleic acid). In some embodiments, the transgene encodes RNAi. In some embodiments, the transgene encodes siRNA, shRNA, or miRNA.

[0148] In some aspects, the present invention provides a modified desmin promoter comprising one or more enhancer elements and a promoter of the human desmin gene. In some embodiments, the desmin promoter comprises two enhancer elements and a promoter of the human desmin gene. In some embodiments, the desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21 and one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:21. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:21, and one or more enhancer elements comprising a nucleotide sequence having at least about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:22.

[0149] In some aspects, the present invention provides rAAV particles comprising a recombinant self-complementary genome (e.g., a self-complementary rAAV vector). AAV viral particles having a self-complementary vector genome and methods using a self-complementary AAV genome are described in U.S. Patents 6,596,535, 7,125,717, 7,465,583, 7,785,888, 7,790,154, 7,846,729, 8,093,054, and 8,361,457; and Wang Z. et al., (2003) GeneTher [Gene Therapy] 10:2105-2111, each reference incorporated herein by reference in its entirety. rAAV containing a self-complementary genome rapidly forms a double-stranded DNA molecule based on its partially complementary sequences (e.g., complementary coding and non-coding strands of a heterologous nucleic acid). In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding a complementary sequence of the nucleic acid, wherein the first nucleic acid sequence may form intra-strand base pairs with the second nucleic acid sequence along most or all of its length.

[0150] In some embodiments, a first heteronucleotide sequence encoding RNAi and a second heteronucleotide sequence encoding a complementary sequence of RNAi are linked by a mutated ITR (e.g., a right-hand ITR). In some embodiments, the ITR contains the polynucleotide sequence 5'-CCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAA AGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAGGGA-3 (SEQ ID NO:27). The mutated ITR contains a deletion of the D region containing the terminal resolution sequence. Therefore, during the replication of the AAV viral genome, the rep protein will not cleave the viral genome at the mutated ITR, and thus, a recombinant viral genome containing the following in 5' to 3' order will be packaged in the viral capsid: AAV ITR, a first heteronucleotide sequence containing the regulatory sequence, the mutated AAV ITR, a second heteronucleotide in the opposite orientation to the first heteronucleotide, and a third AAV ITR. rAAV particles and methods for generating rAAV particles

[0151] This invention provides rAAV particles comprising RNAi as disclosed herein. In some embodiments, this invention provides a method for delivering RNAi to treat DM1 using recombinant viral particles. In some embodiments, the rAAV particle comprises a sequence encoding the RNAi disclosed herein, flanked by one or two ITRs. Nucleic acid is capsidated in the AAV particle. The AAV particle also comprises a capsid protein. In some embodiments, the nucleic acid comprises a target coding sequence (e.g., a nucleic acid encoding the RNAi disclosed herein) and control sequences (including transcription start and stop sequences) of components operatively linked in the transcriptional direction, thereby forming an expression cassette. The expression cassette is flanked by at least one functional AAVITR sequence at the 5' and 3' ends. “Functional AAV ITR sequence” means that the ITR sequence functions as intended for the rescue, replication, and packaging of AAV virions. See Davidson et al., PNAS, 2000, 97(7): 3428-32; Passini et al., J. Virol., 2003, 77(12): 7034-40; and Pechan et al., Gene Ther., 2009, 16: 10-16, all of which are incorporated herein by reference in their entirety. In order to practice some aspects of the present invention, the recombinant vector contains at least all the AAV sequences necessary for capsidation and the physical structure for rAAV infection. The AAV ITR used in the vectors of the present invention does not need to have a wild-type nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5: 793-801) and can be altered by nucleotide insertion, deletion, or substitution, or the AAV ITR can be derived from any of several AAV serotypes. More than 40 serotypes of AAV are currently known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810. The use of any AAV serotype is considered to be within the scope of this invention. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including but not limited to AAV ITRs such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVrh74, AAVDJ, goat AAV, bovine AAV, or mouse AAV capsid serotype ITRs.In some embodiments, the nucleic acid in the AAV comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, AAVDJ, ITRs of goat AAV, bovine AAV, or mouse AAV capsid serotypes, etc. In some embodiments, the nucleic acid in the AAV further encodes RNAi as described herein. For example, the nucleic acid in the AAV may contain at least one ITR of any AAV serotype considered herein, and may further encode RNAi comprising one strand containing a guide region and another strand containing a non-guide region. In one embodiment, the nucleic acid in the AAV may contain at least one ITR of any AAV serotype and may further encode an RNAi comprising a first strand and a second strand, the first strand comprising a first nucleic acid containing the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1) or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:1, and the second strand comprising a second nucleic acid containing the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2) or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:2.

[0152] In some embodiments, the nucleic acid in the AAV from 5' to 3' includes a nucleic acid encoding the following: an ITR (e.g., AAV2ITR), a promoter, a nucleic acid encoding RNAi as disclosed herein, a polyadenylation signal, and an AAV ITR (e.g., AAV2ITR). In some embodiments, the nucleic acid in the AAV from 5' to 3' comprises a nucleic acid encoding the following: an ITR (e.g., AAV2 ITR), a promoter, a nucleic acid encoding RNAi, a polyadenylation signal, and an AAV ITR (e.g., AAV2 ITR), the RNAi comprising a first strand and a second strand, the first strand comprising a first nucleic acid containing the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1) or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:1, and the second strand comprising a second nucleic acid containing the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2) or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:2. In some embodiments, the nucleic acids in the AAV from 5' to 3' include nucleic acids encoding the following: ITR (e.g., AAV2 ITR), desmin promoter, nucleic acid encoding RNAi as disclosed herein, polyadenylation signal (e.g., bovine growth hormone polyA), and AAV ITR (e.g., AAV2 ITR). In some embodiments, the nucleic acid in the AAV from 5' to 3' comprises nucleic acids encoding all or a functional portion of an ITR (e.g., AAV2 ITR), a desmin promoter, an intron (e.g., a chimeric intron), a nucleic acid encoding RNAi, a polyadenylation signal (e.g., bovine growth hormone polyA), and an AAV ITR (e.g., AAV2 ITR). The RNAi comprises a first strand and a second strand. The first strand comprises a first nucleic acid containing the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1) or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:1. The second strand comprises a second nucleic acid containing the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2) or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:2. In some embodiments, the first and second strands form a double helix. In some embodiments, the first strand is connected to the second strand via a connector. In some embodiments, the adapter comprises the nucleic acid sequence of SEQ ID NO:3 or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:3.

[0153] In some embodiments, the nucleic acid in the AAV from 5' to 3' comprises nucleic acid encoding the following: all or a functional portion of an ITR (e.g., AAV2ITR), a filler sequence (e.g., all or a portion of a human α1-antitrypsin (AAT) filler sequence), a desmin promoter, the 5' arm of an intron (e.g., a rabbit β-globin intron), nucleic acid encoding RNAi, the 3' arm of an intron (e.g., a rabbit β-globin intron), a polyadenylation signal (e.g., bovine growth hormone polyA), a filler sequence (e.g., all or a portion of a human α1-antitrypsin (AAT) filler sequence), and an AAV ITR (e.g., AAV2 ITR), the RNAi comprising a first strand and a second strand, the first strand comprising the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1) or the sequence SEQ ID NO:1. NO:1 is a first nucleic acid with a sequence having 80%, 85%, 90%, or 95% identity, and the second strand contains a second nucleic acid containing the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2) or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:2.

[0154] In some embodiments, the nucleic acid in the AAV from 5' to 3' comprises nucleic acids encoding the following: ITR (e.g., AAV2ITR), desmin promoter, nucleic acid encoding RNAi, polyadenylation signal (e.g., bovine growth hormone polyA), and AAVITR (e.g., AAV2 ITR). The RNAi comprises a first strand and a second strand. The first strand comprises a first nucleic acid containing the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2) or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:2. The second strand comprises a second nucleic acid containing the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:1) or a sequence having 80%, 85%, 90%, or 95% identity with SEQ ID NO:1. In some embodiments, the first and second strands form a double helix. In some embodiments, the first strand is connected to the second strand via a adapter. In some embodiments, the adapter comprises the nucleic acid sequence of SEQ ID NO:6.

[0155] In some embodiments, the nucleic acid in the AAV from 5' to 3' comprises nucleic acid encoding the following: all or a functional portion of an ITR (e.g., AAV2ITR), a filler sequence (e.g., all or a portion of a human α1-antitrypsin (AAT) filler sequence), a desmin promoter, the 5' arm of an intron (e.g., a rabbit β-globin intron), a nucleic acid encoding RNAi, the 3' arm of an intron (e.g., a rabbit β-globin intron), a polyadenylation signal (e.g., bovine growth hormone polyA), a filler sequence (e.g., all or a portion of a human α1-antitrypsin (AAT) filler sequence), and an AAV ITR (e.g., AAV2 ITR), the RNAi comprising a first strand and a second strand, the first strand comprising a first nucleic acid containing the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2), and the second strand comprising a second nucleic acid containing the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1).

[0156] In some embodiments, the vector may include one or more filler nucleic acids. In some embodiments, the filler nucleic acid may contain a sequence encoding a reporter polypeptide. As those skilled in the art will understand, the filler nucleic acid may be located in multiple regions within the vector and may consist of a continuous sequence within the vector (e.g., a single filler nucleic acid at a single location) or multiple sequences (e.g., more than one filler nucleic acid at more than one location (e.g., two locations, three locations, etc.)). In some embodiments, the filler nucleic acid may be located downstream of the RNAi sequence. In embodiments, the filler nucleic acid may be located upstream of the RNAi sequence (e.g., between the promoter and the nucleic acid encoding RNAi). Those skilled in the art will also understand that a variety of nucleic acids can be used as filler nucleic acids. In some embodiments, the filler nucleic acid comprises all or a portion of a human α1-antitrypsin (AAT) filler sequence or a C16 P1 chromosome 16P1 clone (human C16) filler sequence. In some embodiments, the filler sequence comprises all or a portion of a gene. For example, the filler sequence comprises a portion of a human AAT sequence. Those skilled in the art will recognize that different parts of a gene (e.g., the human AAT sequence) can be used as filler fragments. For example, the filler fragment may originate from the 5' end of a gene, the 3' end of a gene, the middle of a gene, a non-coding portion of a gene (e.g., an intron), a coding region of a gene (e.g., an exon), or a mixture of non-coding and coding portions of a gene. Those skilled in the art will also recognize that all or part of the filler sequence can be used as the filler sequence. In some embodiments, the vector comprises a 5' filler sequence containing the nucleotide sequence of SEQ ID NO:18 or a nucleotide sequence having greater than about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:18. In some embodiments, the vector comprises a 3' filler sequence containing the nucleotide sequence of SEQ ID NO:19 or a nucleotide sequence having greater than about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:19. In some embodiments, the vector comprises a 5' filler sequence containing the nucleotide sequence of SEQ ID NO:18 or a nucleotide sequence having greater than about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:18, and a 3' filler sequence containing the nucleotide sequence of SEQ ID NO:19 or a nucleotide sequence having greater than about 80%, 85%, 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID NO:19.

[0157] In a further embodiment, the rAAV particle comprises a capsid protein comprising AAVrh74 or a variant thereof. In some embodiments, the AAVrh74 capsid is a variant capsid. In some embodiments, the AAVrh74 variant capsid protein retains the ability to form an AAV capsid. In some embodiments, the AAVrh74 variant capsid is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the wild-type AAVrh74 capsid protein. In some embodiments, the AAVrh74 variant capsid contains a substitution at amino acid position N502. In some embodiments, the substitution at amino acid position N502 is isoleucine (I). In some embodiments, the AAVrh74 variant capsid protein contains a substitution at amino acid position W505. In some embodiments, the substitution at amino acid position W505 is arginine (R).

[0158] Different AAV serotypes are used to optimize the transduction of specific target cells or to target specific cell types within specific target tissues (e.g., diseased tissues, such as muscle tissue). rAAV particles may contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes. For example, in some embodiments, rAAV particles may contain AAVrh74 capsid protein or a variant thereof and at least one ITR of a different AAV serotype. In some embodiments, rAAV particles may contain AAVrh74 capsid protein or a variant thereof and at least one AAV2 ITR. In some embodiments, rAAV particles contain AAVrh74N502I capsid protein and at least one AAV2 ITR. In some embodiments, rAAV particles contain AAVrhW505R capsid protein and at least one AAV2 ITR.

[0159] In some aspects, the present invention provides viral particles comprising a recombinant self-complementary genome. rAAV particles having a self-complementary genome and methods for using a self-complementary AAV genome are described in U.S. Patent Nos. 6,596,535, 7,125,717, 7,465,583, 7,785,888, 7,790,154, 7,846,729, 8,093,054, and 8,361,457; and Wang Z. et al., (2003) Gene Ther 10:2105-2111, each reference incorporated herein by reference in its entirety. rAAVs comprising a self-complementary genome rapidly form double-stranded DNA molecules based on their partially complementary sequences (e.g., complementary coding and non-coding strands of the transgene). In some embodiments, the present invention provides rAAV particles comprising an AAV genome, wherein the rAAV genome comprises a first heteropolynucleotide sequence (e.g., the RNAi disclosed herein) and a second heteropolynucleotide sequence (e.g., the antisense strand of the RNAi disclosed herein), wherein the first heteropolynucleotide sequence may form intrastrand base pairs with the second polynucleotide sequence along most or all of its length. In some embodiments, the first and second heteropolynucleotide sequences are linked by a sequence that promotes intrastrand base pairing (e.g., a hairpin DNA structure). Hairpin structures are known in the art, for example, in miRNA or siRNA molecules. In some embodiments, the first and second heteropolynucleotide sequences are linked by a mutated ITR (e.g., a right-hand ITR). In some embodiments, the ITR comprises the polynucleotide sequence 5'-CCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAA AGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCGCAGAGAGGGA-3 (SEQ ID NO:27). The mutated ITR contains a deletion of the D region containing the terminal resolution sequence. Therefore, during the replication of the AAV viral genome, the rep protein will not cleave the viral genome at the mutated ITR, and thus, a recombinant viral genome containing the following in 5' to 3' order will be packaged in the viral capsid: AAV ITR, a first heteropolynucleotide sequence containing the regulatory sequence, the mutated AAVITR, a second heteropolynucleotide in the opposite orientation to the first heteropolynucleotide, and a third AAVITR.In some embodiments, the present invention provides an AAV viral particle comprising a recombinant viral genome comprising: a functional AAV2 ITR, a first polynucleotide sequence encoding the RNAi disclosed herein, a mutant AAV2 ITR comprising a D region deletion and lacking a functional end resolution sequence, a second polynucleotide sequence comprising a complementary sequence of the first polynucleotide sequence encoding the RNAi disclosed herein, and the functional AAV2 ITR.

[0160] rAAV particles can be generated using methods known in the art. See, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006. In practicing this invention, host cells used for generating rAAV particles include mammalian cells, insect cells, plant cells, microorganisms, and yeast. The host cell can also be a packaging cell in which the AAV rep and cap genes are stably maintained, or a production cell in which the AAV vector genome is stably maintained. Exemplary packaging and production cells are derived from 293 cells, A549 cells, or HeLa cells. The AAV vector is purified and formulated using standard techniques known in the art.

[0161] Methods known in the art for producing rAAV vectors include, but are not limited to, transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids. The rAAV production culture for producing rAAV viral particles requires all of the following: 1) in the case of a baculovirus production system, suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect-derived cell lines such as SF-9; 2) suitable helper viral function provided by wild-type or mutant adenovirus (such as temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper function; 3) AAV rep and cap genes and gene products; 4) nucleic acids flanking at least one AAV ITR sequence (such as therapeutic nucleic acids); and 5) suitable culture media and culture medium components supporting rAAV production. In some embodiments, the AAV rep and cap gene products can be derived from any AAV serotype. Generally, but not necessarily, the AAV rep gene product has the same serotype as the ITR of the rAAV vector genome, provided that the rep gene product can function to replicate and package the rAAV genome. Suitable culture media known in the art can be used to generate rAAV vectors. These media include, but are not limited to, those produced by Hyclone Laboratories and JRH, including modified Eagle Medium (MEM), Durbeco Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Patent No. 6,566,118, and Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom culture medium formulations for generating recombinant AAV vectors. In some embodiments, AAV helper functions are provided by adenovirus or HSV. In some embodiments, the AAV assistive function is provided by a baculovirus, and the host cell is an insect cell (e.g., a fall armyworm (Spodoptera frugiperda) (Sf9) cell).

[0162] In some embodiments, rAAV particles can be generated via a triple transfection method, such as the exemplary triple transfection method provided below. In short, a plasmid containing the rep gene and capsid gene can be transfected (e.g., using the calcium phosphate method) together with a helper adenovirus plasmid into a cell line (e.g., HEK-293 cells), and the virus can be collected and optionally purified. Therefore, in some embodiments, rAAV particles are generated by triple transfecting a host cell with nucleic acid encoding the rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus function, wherein transfecting the nucleic acid into the host cell produces host cells capable of producing rAAV particles.

[0163] In some embodiments, rAAV particles can be produced by production cell line methods, such as the exemplary production cell line methods provided below (see also references in Martin et al., (2013) Human Gene Therapy Methods 24:253-269). Briefly, a cell line (e.g., HeLa cell line) can be stably transfected with a plasmid containing a rep gene, a capsid gene, and a promoter heterologous nucleic acid sequence. Cell lines can be screened to select a leader clone for rAAV production, which can then be amplified in a production bioreactor and infected with an adenovirus (e.g., wild-type adenovirus) as an assistant to initiate rAAV production. The virus can then be harvested, the adenovirus can be inactivated (e.g., by heating) and / or removed, and the rAAV particles can be purified. Thus, in some embodiments, rAAV particles are produced by production cell lines comprising one or more of the following: nucleic acids encoding an rAAV vector, nucleic acids encoding AAV rep and cap, and nucleic acids encoding AAV helper viral functions.

[0164] In some aspects, methods for generating any rAAV particles as disclosed herein are provided, the method comprising (a) culturing a host cell under conditions for generating rAAV particles, wherein the host cell contains (i) one or more AAV packaging genes, wherein each of the AAV packaging genes encodes an AAV replication and / or capsidation protein; (ii) an rAAV protovector containing a nucleic acid encoding an RNAi as described herein, flanked by at least one AAV ITR; and (iii) an AAV helper function; and (b) recovering the rAAV particles generated by the host cell. In some embodiments, the RNAi comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the at least one AAV ITR is selected from the group consisting of the following AAV ITRs: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, AAVrh74 N502I, AAVrh74 W505R, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV capsid serotype ITR, etc. In some embodiments, the capsid protein is selected from AAVrh74 or its variants, such as the group consisting of AAVrh74 N502I or AAVrh74 W505R. In some embodiments, the capsid protein is the AAVrh74 protein. In some embodiments, the capsid protein is a variant of the AAVrh74 protein. In some embodiments, the capsid protein is the AAVrh74N502I protein. In some embodiments, the capsid protein is the AAVrh74W505R protein. In some embodiments, the variant AAVrh74 capsid protein retains the ability to form an AAV capsid. In some embodiments, the rAAV particle comprises an AAVrh74N502I capsid and a recombinant genome comprising an AAV2 ITR and a nucleic acid encoding the RNAi disclosed herein. In some embodiments, the rAAV particle comprises an AAVrh74W505R capsid and a recombinant genome comprising an AAV2 ITR and a nucleic acid encoding the RNAi disclosed herein. In another embodiment, the rAAV particle is purified. As used herein, the term "purified" includes a formulation of rAAV particles that does not contain at least some other components, which may also be present where the rAAV particle is naturally present or was originally prepared. Thus, for example, isolated rAAV particles can be prepared by enriching them from source mixtures such as culture lysates or production culture supernatants using purification techniques.Enrichment can be measured in a variety of ways, such as by the proportion of DNAase-resistant particles (DRP) or genome copies (gc) present in the solution, or by infectivity, or it can be measured relative to a second potential interfering substance present in the source mixture, such as a contaminant, including production culture contaminants or process contaminants, including helper viruses, culture medium components, etc.

[0165] This document also provides pharmaceutical compositions comprising rAAV particles and a pharmaceutically acceptable carrier, wherein the rAAV particles contain a transgene encoding the RNAi disclosed herein. These pharmaceutical compositions are suitable for any of the administration methods described herein. Pharmaceutical compositions comprising rAAV particles encoding nucleic acids encoding the RNAi disclosed herein can be administered systemically. For example, recombinant viral particles containing nucleic acids encoding the RNAi disclosed herein can be administered intravenously, intra-arterially, subcutaneously, or intraperitoneally.

[0166] In some embodiments, a pharmaceutical composition comprising a recombinant viral particle and a pharmaceutically acceptable carrier is suitable for administration to humans, the recombinant viral particle comprising a transgene encoding the RNAi disclosed herein. Such carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). In some embodiments, a pharmaceutical composition comprising the rAAV described herein and a pharmaceutically acceptable carrier is suitable for systemic injection into mammals.

[0167] Pharmaceutically acceptable carriers of this type can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, and mineral oil. Saline solutions and aqueous solutions of dextran, polyethylene glycol (PEG), and glycerol can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions may further contain additional components such as preservatives, buffers, tonics, antioxidants and stabilizers, nonionic wetting or clarifying agents, thickeners, etc. The pharmaceutical compositions described herein can be packaged in single-unit doses or in multiple-dose formulations. These compositions are typically formulated as sterile and substantially isotonic solutions. Products and reagent kits

[0168] Kits or articles of manufacture used in the methods described herein are also provided. In various respects, the kits contain the compositions described herein (e.g., rAAV particles of this disclosure containing nucleic acids encoding the RNAi disclosed herein) in suitable packaging. Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These articles of manufacture may be further sterilized and / or sealed.

[0169] The present invention also provides kits comprising the compositions described herein, and may further include instructions on methods of using the compositions, such as those described herein. The kits described herein may further include other materials deemed commercially and user-appropriate, including additional buffers, diluents, filters, needles, syringes, and a packaging insert containing instructions for performing any of the methods described herein. For example, in some embodiments, the kit comprises: a recombinant viral particle containing a transgene encoding the RNAi disclosed herein for delivering an effective amount of rAAV particles to a mammal, a composition of a pharmaceutically acceptable carrier suitable for injection into a mammal, and one or more of the following: buffers, diluents, filters, needles, syringes, and a packaging insert containing instructions for injection into a mammal. In some embodiments, the kit includes instructions for treating DM-1 with the rAAV particles described herein. In some embodiments, the kit includes instructions for using the rAAV particles described herein according to any of the methods described herein. Exemplary embodiments

[0170] The present invention includes the exemplary embodiments listed below. 1. A recombinant adeno-associated virus (rAAV) particle, the rAAV particle comprising: a) RNAi containing both first and second strands, wherein i) The first chain and the second chain form a double chain; ii) The first strand contains a guide region, wherein the guide region contains a nucleic acid having the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1) or having a sequence having approximately 90% identity with the sequence of SEQ ID NO:1; and iii) The second chain contains a non-guided region; and b) An AAV capsid containing an amino acid sequence that is approximately 90% identical to that of the wild-type AAVrh74 capsid.

[0171] 2. The rAAV particle as described in Example 1, wherein the unguided region comprises a nucleic acid having the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2) or having a sequence having approximately 90% identity with the sequence of SEQ ID NO:2.

[0172] 3. The rAAV particle as described in Example 1 or 2, wherein the first chain comprises a nucleic acid having the sequence of SEQ ID NO:1, and the non-guided region comprises a nucleic acid having the sequence of SEQ ID NO:2.

[0173] 4. The rAAV particle as described in any one of Examples 1-3, wherein the first strand and the second strand are connected by an RNA adapter capable of forming a loop structure.

[0174] 5. The rAAV particle as described in Example 4, wherein the RNA adapter comprises about 4 to about 50 nucleotides.

[0175] 6. The rAAV particle as described in Example 4 or 5, wherein the ring structure comprises about 4 to about 20 nucleotides.

[0176] 7. The rAAV particle as described in any one of Examples 4-6, wherein the loop structure comprises a nucleic acid sequence having the sequence of SEQ ID NO:3 or having a sequence having approximately 90% identity with the sequence of SEQ ID NO:3.

[0177] 8. The rAAV particle as described in any one of Examples 4-7, wherein the RNAi comprises the second strand, the RNA adapter, and the first strand from 5' to 3'.

[0178] 9. The rAAV particle as described in any one of Examples 4-7, wherein the RNAi comprises the first strand, the RNA adapter, and the second strand from 5' to 3'.

[0179] 10. The rAAV particle as described in any one of Examples 1-8, wherein the RNAi comprises a nucleic acid having a sequence having SEQ ID NO:7 or having a sequence having approximately 90% identity with the sequence having SEQ ID NO:7.

[0180] 11. The rAAV particle as described in any one of Examples 1-10, wherein the RNAi is a small repressive RNA (siRNA), microRNA (miRNA), or small hairpin RNA (shRNA).

[0181] 12. The rAAV particle as described in any one of Examples 1-11, wherein the RNAi further comprises a scaffold.

[0182] 13. The rAAV particles as described in Example 12, wherein the scaffold contains all or part of the nucleic acid of SEQ ID No: 11.

[0183] 14. The rAAV particle as described in Example 13, wherein the miRNA is embedded within the scaffold.

[0184] 15. The rAAV particle as described in Example 14, wherein the scaffold has a 5' arm and a 3' arm, wherein the 5' arm is located at the 5' of the nucleic acid encoding the RNAi, and wherein the 3' arm is located at the 3' of the nucleic acid encoding the RNAi.

[0185] 16. The rAAV particles as described in any one of Examples 12-15, wherein the scaffold is a miR-155 scaffold.

[0186] 17. The rAAV particle as described in any one of Examples 12-16, wherein the miR-155 scaffold comprises a nucleic acid located at the 5' of the RNAi having a sequence having SEQ ID NO:9 or a sequence having approximately 90% identity with the sequence having SEQ ID NO:9.

[0187] 18. The rAAV particle as described in any one of Examples 12-17, wherein the miR-155 scaffold comprises a nucleic acid located at the 3' of the RNAi having the sequence of SEQ ID NO:10 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:10.

[0188] 19. The rAAV particle as described in any one of Examples 1-18, wherein the RNAi targets RNA encoding a polypeptide associated with myotonic dystrophy type 1 (DM1).

[0189] 20. The rAAV particles as described in Example 19, wherein the polypeptide is dystrophic myotonic kinase (DMPK).

[0190] 21. The rAAV particle as described in Example 20, wherein the DMPK contains a mutation associated with DM 1.

[0191] 22. The rAAV particle as described in Example 20 or 21, wherein the gene encoding DMPK contains five or more CTG trinucleotide repeat sequences.

[0192] 23. An expression cassette comprising a nucleic acid sequence encoding an RNAi as described in any one of Examples 1-22.

[0193] 24. The expression cassette as described in Example 23, wherein the nucleic acid encoding the RNAi is operatively linked to a promoter.

[0194] 25. The expression cassette as described in Example 24, wherein the promoter is a muscle-specific promoter.

[0195] 26. The expression cassette as described in Example 24 or 25, wherein the promoter is a desmin promoter or a variant thereof.

[0196] 27. The expression cassette as described in Example 26, wherein the desmin promoter comprises one or more enhancer elements and a promoter for the human desmin gene.

[0197] 28. The expression cassette as described in Example 26 or 27, wherein the desmin promoter comprises two enhancer elements and the promoter of the human desmin gene.

[0198] 29. The expression cassette as described in any one of Examples 26-28, wherein the desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.

[0199] 30. The expression cassette of any one of Examples 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 having about 90% identity with 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 having about 90% identity with the sequence of SEQ ID NO:22.

[0200] 31. The expression cassette as described in any one of Examples 26-30, wherein the desmin promoter comprises the nucleotide sequence of SEQ ID NO:12 or a sequence having approximately 90% identity with the nucleotide sequence of SEQ ID NO:12.

[0201] 32. The expression box as described in any one of Examples 23-31, wherein the expression box further comprises introns.

[0202] 33. The expression cassette as described in Example 32, wherein the intron is a rabbit β-globin intron.

[0203] 34. The expression cassette as described in Example 32 or 33, wherein the intron comprises the nucleotide sequence of SEQ ID NO:13 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:13.

[0204] 35. The expression cassette as described in any one of Examples 32-34, wherein the nucleic acid encoding the RNAi is embedded in the intron.

[0205] 36. The expression cassette as described in Example 35, wherein the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at the 5' of the nucleic acid encoding the RNAi and the 3' arm is located at the 3' of the nucleic acid encoding the RNAi.

[0206] 37. The expression cassette as described in Example 36, wherein the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO:14 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:14.

[0207] 38. The expression cassette as described in Example 36 or 37, wherein the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO:15 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:15.

[0208] 39. The expression cassette as described in any one of Examples 23-38, wherein the expression cassette further comprises a polyadenylation signal.

[0209] 40. The expression cassette as described in Example 39, wherein the polyadenylation signal is bovine growth hormone polyadenylation signal, SV40 polyadenylation signal, or HSV TK pA.

[0210] 41. The expression cassette as described in Example 40, wherein the polyadenylation signal is the minimum bovine growth hormone polyadenylation signal.

[0211] 42. The expression cassette as described in any one of Examples 39-41, wherein the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO:16 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:16.

[0212] 43. The expression cassette as described in any one of Examples 23-42, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO:17 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:17.

[0213] 44. An expression cassette comprising a modified desmin promoter, wherein the modified desmin promoter comprises one or more enhancer elements and a promoter of a human desmin gene.

[0214] 45. The expression cassette as described in Example 44, wherein the modified desmin promoter comprises two enhancer elements and a promoter for the human desmin gene.

[0215] 46. ​​The expression cassette as described in Example 44 or 45, wherein the modified desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.

[0216] 47. The expression cassette of any one of Examples 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 having about 90% identity with 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 having about 90% identity with the sequence of SEQ ID NO:22.

[0217] 48. The expression cassette as described in any one of Examples 44-47, wherein the desmin promoter comprises the nucleotide sequence of SEQ ID NO:12 or a sequence having approximately 90% identity with the nucleotide sequence of SEQ ID NO:12.

[0218] 49. The expression box as described in any one of Examples 44-48, wherein the expression box further comprises introns.

[0219] 50. The expression cassette as described in Example 49, wherein the intron is a rabbit β-globin intron.

[0220] 51. The expression cassette as described in Example 49 or 50, wherein the intron comprises the nucleotide sequence of SEQ ID NO:13 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:13.

[0221] 52. The expression cassette as described in any one of Examples 44-51, wherein the nucleic acid encoding the transgene is embedded in the intron.

[0222] 53. The expression cassette as described in Example 52, wherein the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at the 5' of the nucleic acid encoding the transgene and the 3' arm is located at the 3' of the nucleic acid encoding the transgene.

[0223] 54. The expression cassette as described in Example 53, wherein the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO:14 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:14.

[0224] 55. The expression cassette as described in Example 53 or 54, wherein the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO:15 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:15.

[0225] 56. The expression cassette as described in any one of Examples 44-55, wherein the expression cassette further comprises a polyadenylation signal.

[0226] 57. The expression cassette as described in Example 56, wherein the polyadenylation signal is bovine growth hormone polyadenylation signal, SV40 polyadenylation signal, or HSV TK pA.

[0227] 58. The expression cassette as described in Example 57, wherein the polyadenylation signal is the minimum bovine growth hormone polyadenylation signal.

[0228] 59. The expression cassette as described in any one of Examples 56-58, wherein the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO:16 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:16.

[0229] 60. The expression cassette as described in any one of Examples 44-59, wherein the transgene encodes a polypeptide or nucleic acid.

[0230] 61. The expression cassette as described in any one of Examples 44-60, wherein the transgene encodes RNAi.

[0231] 62. A carrier comprising an expression cassette as described in any one of Examples 23-61.

[0232] 63. The vector as described in Example 62, wherein the flanks of the expression cassette are one or more filler nucleic acid sequences.

[0233] 64. The vector as described in Example 63, wherein the one or more filler nucleic acid sequences are derived from the human SerpinA1 gene.

[0234] 65. The vector as described in Example 63 or 64, wherein the filler nucleic acid sequence located at the expression cassette 5' is derived from the human SerpinA1 gene.

[0235] 66. The vector as described in any one of Examples 63-65, wherein the filling sequence located in the expression cassette 5' comprises the nucleotide sequence of SEQ ID NO:18 or a sequence having about 90% identity with the sequence of SEQ ID NO:18.

[0236] 67. The vector as described in any one of Examples 63-66, wherein the filler nucleic acid sequence located at the expression cassette 3' is derived from the human SerpinA1 gene.

[0237] 68. The vector as described in any one of Examples 63-67, wherein the filling sequence located in the expression cassette 3' comprises the nucleotide sequence of SEQ ID NO:19 or a sequence having about 90% identity with the sequence of SEQ ID NO:19.

[0238] 69. The vector as described in any one of Examples 62-68, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

[0239] 70. The rAAV vector as described in Example 69, wherein the flanking sides of the expression cassette are one or more AAV inverted terminal repeat (ITR) sequences.

[0240] 71. The rAAV vector as described in Example 70, wherein the expression cassette has two AAV ITRs on its flanks.

[0241] 72. The rAAV vector as described in Example 70 or 71, wherein these AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITRs.

[0242] 73. The rAAV vector as described in any one of Examples 70-72, wherein these AAV ITRs are AAV2 ITRs.

[0243] 74. The rAAV vector as described in any one of Examples 69-73, wherein the rAAV vector comprises the nucleotide sequence of SEQ ID NO:20 or a sequence having approximately 90% identity with the sequence of SEQ ID NO:20.

[0244] 75. The rAAV vector as described in any one of Examples 69-74, wherein the vector is a self-complementary rAAV vector.

[0245] 76. A cell comprising an expression cassette as described in any one of Examples 23-61, a vector as described in any one of Examples 62-68, or an rAAV vector as described in any one of Examples 69-75.

[0246] 77. The rAAV particle as described in any one of Examples 1-22, wherein the AAV capsid contains an amino acid sequence with an amino acid substitution at position 502.

[0247] 78. The rAAV particles as described in Example 77, wherein the amino acid substitution at position 502 is isoleucine (I).

[0248] 79. The rAAV particles as described in Examples 77 or 78, wherein the rAAV particles comprise an AAVrh74 N502I serum-type capsid.

[0249] 80. The rAAV particle as described in Example 77 or 78, wherein the ITR is AAV2ITR and the capsid of the rAAV particle is an AAVrh74 N502I serum-type capsid.

[0250] 81. The rAAV particle as described in any one of Examples 1-22, wherein the AAV capsid contains an amino acid sequence with an amino acid substitution at position 505.

[0251] 82. The rAAV particle as described in Example 81, wherein the amino acid substitution at position 505 is arginine (R).

[0252] 83. The rAAV particle as described in any one of Examples 1-22, wherein the rAAV particle comprises an AAVrh74 W505R serological capsid.

[0253] 84. The rAAV particle as described in Example 83, wherein the ITR is AAV2 ITR and the capsid of the rAAV particle is an AAVrh74 W505R serological capsid.

[0254] 85. An rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: AAV2 ITR, nucleic acid encoding a filler sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, the 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, and DMPK. 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 The miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimum bovine growth hormone polyadenylated sequence, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, and AAV2 ITR; and wherein the capsid is AAVrh74 N502I capsid.

[0255] 86. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43; a nucleic acid encoding a filler 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; and a DMPK comprising the polynucleotide sequence of SEQ ID NO:4. 204 miRNA guide sequence, miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6, and DMPK containing the polynucleotide sequence of SEQ ID NO:5. 204 The miRNA passenger sequence comprises a 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; wherein the capsid is an AAVrh74 N502I capsid.

[0256] 87. The rAAV particle as described in Example 85 or 86, wherein the AAVrh74N502I capsid comprises a capsid protein containing the amino acid sequence of SEQ ID NO: 50.

[0257] 88. An rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: AAV2 ITR, nucleic acid encoding a filler 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, and DMPK. 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204The miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimum bovine growth hormone polyadenylated sequence, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, and AAV2 ITR; and wherein the capsid is AAVrh74 W505R capsid.

[0258] 89. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43; a nucleic acid encoding a filler 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; and a DMPK comprising the polynucleotide sequence of SEQ ID NO:4. 204 miRNA guide sequence, miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6, and DMPK containing the polynucleotide sequence of SEQ ID NO:5. 204 The miRNA passenger sequence comprises a 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; wherein the capsid is an AAVrh74 W505R capsid.

[0259] 90. The rAAV particle as described in Example 88 or 89, wherein the AAVrh74W505R capsid comprises a capsid protein containing the amino acid sequence of SEQ ID NO: 52.

[0260] 91. A composition comprising rAAV particles as described in any one of claims 1-22 and 77-90.

[0261] 92. A pharmaceutical composition comprising rAAV particles as described in any one of claims 1-22 and 77-90.

[0262] 93. The composition as described in Example 91 or 92, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0263] 94. A kit comprising rAAV particles as described in any one of Examples 1-22 and 77-90.

[0264] 95. A kit comprising rAAV particles as described in any one of claims 1-22 and 77-90.

[0265] 96. A kit comprising the composition as described in any one of Examples 91-93.

[0266] 97. The kit as described in any one of Examples 94-96, further comprising instructions for use.

[0267] 98. A method for treating myotonic dystrophy type 1 (DM1) in a mammal in need, the method comprising administering to the mammal an effective amount of rAAV particles as described in any one of Examples 1-22 and 77-90.

[0268] 99. A method for inhibiting the expression of dystrophic myotonic kinase (DMPK) in a mammal with DM-1, the method comprising administering to the mammal an effective amount of rAAV particles as described in any one of Examples 1-22 and 77-90.

[0269] 100. A method for inhibiting the accumulation of DMPK RNA in the cells of a mammal with DM-1, the method comprising administering to the mammal an effective amount of RNAi as described in any one of Examples 1-22 and 77-90.

[0270] 101. A method for treating myotonic dystrophy type 1 (DM1) in a mammal in need, the method comprising administering to the mammal an effective amount of rAAV particles as described in any one of claims 1-22 and 77-90.

[0271] 102. A method for inhibiting the expression of dystrophic myotonic kinase (DMPK) in a mammal with DM-1, the method comprising administering to the mammal an effective amount of rAAV particles as described in any one of claims 1-22 and 77-90.

[0272] 103. A method for inhibiting the accumulation of DMPK RNA in the cells of a mammal with DM-1, the method comprising administering to the mammal an effective amount of rAAV particles as described in any one of claims 1-22 and 77-90.

[0273] 104. The method as described in any one of Examples 100-104, wherein the effective amount of the virus particle or rAAV particle is about 1 × 10⁻⁶. 8 Approximately 2×10 13 A dose of one genome copy / mL.

[0274] 105. The method as described in Example 104, wherein the dose is approximately 5 × 10⁻⁶. 12 One genome copy / mL.

[0275] 106. The method as described in Example 104, wherein the dose is approximately 1 × 10⁻⁶. 13 One genome copy / mL.

[0276] 107. The method as described in Example 104, wherein the dose is approximately 2 × 10⁻⁶. 13 One genome copy / mL.

[0277] 108. The method as described in any one of Examples 101-103, wherein the effective amount of the virus particle or rAAV particle is about 1 × 10⁻⁶. 8 Approximately 2×10 14 A dose of one genome copy per kg of body weight.

[0278] 109. The method as described in Example 108, wherein the dose is approximately 5 × 10⁻⁶. 13 One genome copy per kg of body weight.

[0279] 110. The method as described in Example 108, wherein the dose is approximately 1 × 10⁻⁶. 14 One genome copy per kg of body weight.

[0280] 111. The method as described in Example 108, wherein the dose is approximately 2 × 10⁻⁶. 14 One genome copy per kg of body weight.

[0281] 112. A method for treating myotonic dystrophy type 1 (DM1) in a mammal in need, the method comprising administering to the mammal an effective amount of the composition as described in any one of Examples 91-93.

[0282] 113. A method for inhibiting the expression of dystrophic myotonic kinase (DMPK) in a mammal with DM-1, the method comprising administering to the mammal an effective amount of the composition as described in any one of Examples 91-93.

[0283] 114. A method for inhibiting the accumulation of DMPK RNA in the cells of a mammal with DM-1, the method comprising administering to the mammal an effective amount of the composition as described in any one of Examples 91-93.

[0284] 115. The method as described in any one of Examples 98-100, wherein the RNAi is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with and / or after the RNAi administration.

[0285] 116. The method of any one of Examples 101-103, wherein the viral particle or the rAAV particle is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with and / or after the administration of the viral particle or the rAAV particle.

[0286] 117. The method of any one of Examples 112-114, wherein the composition is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with and / or after the composition is administered. Example

[0287] The subject matter of this disclosure will be better understood by referring to the following examples, which are provided as examples of the invention and not as limiting. Example 1: amir-DMPK 204 The generation of expression boxes

[0288] A series of nine transcription promoters (S1-S9) were rationally designed to identify candidates with high activity in muscle and low activity in liver. A set of promoters was screened in primary human myoblasts and Huh7 hepatocellular carcinoma cells to represent muscle and liver, respectively. The newly designed promoters were compared with the ubiquitous CAG promoter and a set of previously disclosed promoter sequences with high activity in myocytes: a synthetic MH promoter containing four muscle-specific transcription factor binding sites (TFBS) and a core promoter fragment of the chicken skeletal muscle α-actin gene; a chimeric MHCK7 promoter designed to target and combine myocardium; an α-MCK enhancer and an MCK promoter plus an enhancer from the mouse α-myosin heavy chain; and sk-CRM4 / Des, a synthetic promoter combining a skeletal muscle-specific cis-regulatory module, a desmin promoter, and an MVM intron. For screening, cells were transfected with a plasmid containing a luciferase gene controlled by the target promoter. After 48 hours, myoblasts were measured (…). Figure 16A ) and Huh7 cells ( Figure 16B The relative luciferase activity in the AAV expression construct was measured. Compared to all other newly designed promoters, the S6 promoter drove the highest expression in muscle cells, except for the CAG promoter which drives high systemic expression. S6 promoter expression was relatively low in the liver. Therefore, the S6 promoter was chosen for the AAV expression construct.

[0289] This resulted in the development of a microRNA (amiR-DMPK) designed to target the DMPK gene. 204 Single-stranded AAV viral vector ( Figure 1A The constructor is designed to enable amir-DMPK. 204 MicroRNAs are embedded in the optimized microRNA backbone miR155 (BLOCK-iT; Thermo Fisher catalog numbers K4935-00, K4936-00, K4937-00, K4938-00) and hereinafter referred to as "miR155amiR-DMPK". 204 "or "amiR155-DMPK 204 miR155 amiR-DMPK 204 Its flank is a rabbit β-globin intron sequence and is regulated by a hybrid muscle promoter (nDes; “S6” promoter).

[0290] The nDesmin promoter, containing the Byrne desmin enhancer, one copy of the Paulin desmin enhancer (-973 to -693), and the promoter of the human desmin gene (-228 to +75), was synthesized using conventional oligonucleotide synthesis (Genscript, USA).

[0291] The bovine growth hormone polyadenylated sequence was placed in amiR-DMPK with a lateral intron. 204 The 3' end of the microRNA (minBGHpA) includes a filler sequence (“filler sequence”). The entire gene cassette is flanked by wild-type AAV serotype 2 inverted terminal repeat (ITR) sequences for DNA rescue and replication, as well as packaging into the AAV capsid. The sequence is engineered into an ITR plasmid, which is then used to generate a vector for in vivo efficacy studies. 5' and 3' ITR sequences

[0292] The ITR sequence is a 145 bp wild-type AAV2 sequence. The 3' ITR (downstream of the expression cassette) is in a flip orientation (GenBank: LQ493091.1). The 5' ITR (upstream of the expression cassette) is in a flip orientation (145 bp) (Miller et al., 2004, Nature Genetics 36.7(2004):767-773). The accuracy of the sequence was confirmed by Sanger sequencing. nDes starter

[0293] The nDes promoter was constructed using desmin promoter elements shown in the literature (Li and Paulin et al., 1991, J Biol Chem. [Journal of Biochemistry] 266.10:6562-6570). The nDes promoter contains one copy of the Byrne desmin enhancer, one copy of the Paulin desmin enhancer (-973 to -693), and the promoter of the human desmin gene (-228 to +75). The 5' and 3' arms of the rabbit β-globin intron

[0294] This intron is used for side-connection of amir-DMPK. 204 The box, because it is known that intron expression of miRNAs enhances target knockdown. amiR-DMPK with miR155 stand 204 (miR155-amiRDMPK 204 )

[0295] Endogenous miRNAs are hairpin-like secondary structures found in many primary RNA transcripts (pri-miRNAs). In the nucleus, the microprocessor Drosha / DGCR8 complex binds to and cleaves the basal stem of the pri-miRNA to release the stem-loop precursor miRNA (pre-miRNA). The pre-miRNA is then exported from the nucleus, where the loop is cleaved by Dicer / TRBP to form the mature RNA duplex. The guide strand, also known as the target strand, separates from the passenger strand and is loaded onto the argonaute protein in the RNA-induced silencing complex (RISC), which then targets the complementary mRNA transcript for degradation or translational repression.

[0296] amiR-DMPK 204 The sequence was identified as a target for DM1 therapy. amiR-DMPK 204 The target sequence is located upstream of the "CUG" repeat sequence within the 3' UTR of the DMPK nucleotide sequence. Therefore, amiR-DMPK 204 It can inhibit both wild-type and mutant DMPK transcripts.

[0297] Additionally, amiR-DMPK 204The target region is conserved in non-human primates (NHP - cynomolgus monkeys) and humans, thus allowing for preclinical evaluation of DMPK knockdown in NHP. Figure 11 Evaluation of amiR-DMPK 204 MicroRNAs were analyzed, and the miR155 scaffold was shown to have efficient guide strand processing and minimal passenger strand processing, reducing the likelihood of off-target effects (see Example 2 below).

[0298] The final build chosen for development is the amiR-DMPK with a miR155 support. 204 (amiR155-DMPK 204 During processing, the guide strand targets DMPK mRNA for degradation. The engineered pre-miRNA sequence structure is based on the mouse miR-155 sequence (Lagos-Quintana et al., 2002, Current Biology, 12:9, 735-739). The 5' and 3' flanking regions derived from the miR-155 transcript were inserted into the vector to preserve the miR-155 structure as much as possible. The stem-loop structure was optimized, and the 2-nucleotide inner loop resulted in a higher knockdown rate than the 5-nucleotide / 3-nucleotide inner loop found in the native miR-155 molecule (Source: BLOCK-iT). TM Pol II miR RNAi expression vector (Invitrogen). The validated vector nDes-miR155-amiR-DMPK. 204 It exhibits potent in vivo activity in the DMSXL mouse model of DM1 against the background of the AAV capsid (see Example 4 below). minBGH polyA

[0299] The smallest BGH polyA site of 186 bp was inserted into amiR-DMPK. 204 The downstream of the sequence allows for transcriptional termination and polyadenylation of the mRNA. Filling sequences from A1AT introns

[0300] The gene cassette was made to reach the packaging limit of the rAAV vector by using a filler sequence from intron sequence 4 of the α-1 antitrypsin gene. With nDes-miR155-amiR-DMPK 204 The ITR plasmid of the box (nDes-miR155-amiR-DMPK) 204 cloning

[0301] Design the expression construct nDes-miR155-amiR-DMPK204-minBGHpolyA box with a 1938bp ITR-ITR sequence. Then, use nDes-miR155-amiR-DMPK... 204The -minBGHpolyA box was cloned into the ITR plasmid. The synthesized nDes-miR155-amiR-DMPK204-minBGHpolyA box includes the 5'Nco1 site and 3'Sph1 site for cloning into the ITR plasmid. Figure 1B In short, digestion of synthetic nDes-miR155-amiR-DMPK2 with NcoI and SphI. 04 The plasmid containing the -minBGHpolyA sequence was extracted and the 1.9 kB fragment was purified by gel electrophoresis. The ITR plasmid was digested with NcoI and SphI, dephosphorylated using bovine intestinal alkaline phosphatase (New England Biolabs; catalog number M0290), and the 8.2 kB vector backbone fragment was purified by gel electrophoresis. The digested 1.9 kB fragment containing the expression cassette was ligated to the digested ITR plasmid to generate plasmid ITR-nDes-miR155-amiR-DMPK. 204 . Small-scale production of rAAV carriers

[0302] A small-scale packaging assay was performed in HEK 293 cells to confirm the ITR-nDes-miR155-amiR-DMPK assay. 204 Plasmid packaging. Small-scale production was carried out using AAV rep / cap plasmids. Figure 1C The amount of vector produced per HEK 293 cell is shown compared to the standard EGFP plasmid cassette.

[0303] To determine amir-DMPK 204 The potential to correct the DM1 phenotype was tested by silencing human DMPK transcripts and correcting splicing defects in skeletal muscle myoblast cultures from DM1 patients. Due to CTG amplification in the 3'UTR of the DMPK gene, DMPK mRNA hairpin structures aggregate into insoluble ribonucleofoci and isolate several RNA-binding proteins. The resulting redistribution of essential splicing factors such as blind muscle-like protein 1 (MBNL1) leads to missplicing of downstream effectors responsible for muscle tissue differentiation. This was investigated using amiR-DMPK. 204 Treatment of DM1 patient cells resulted in over 50% silencing of DMPK mRNA and splicing correction, as measured via MBNL1 exon 7. Example 2: miR155-amiRDMPK 204 Expression of DM1 phenotype in vitro

[0304] Testing miR155-amiRDMPK driven by the nDes promoter in two in vitro DM1 models 204Expression of the construct. DM1 immortalized human muscle tubes were transduced using an AAV2 vector containing an artificial miRNA targeting DMPK under the control of the nDes promoter. DMPK amiRNA levels were measured by PCR. Figure 17A ) and DMPK mRNA ( Figure 17B As expected, DMPK amiRNA was detectable only in treated cells, where DMPK levels were reduced by 65% ​​compared to untreated cells (untreated = 1 ± 0.04; treated = 0.34 ± 0.018; mean ± SD; p < 0.0001, two-tailed unpaired t-test).

[0305] One of the hallmarks of DM1 is the presence of nuclear RNA foci. Amplification of the CUG repeat sequence in the 3'-UTR of DMPK mRNA results in the mRNA remaining in the nucleus, where it binds to and insulates important RNA-binding proteins, including blind muscle-like protein 1 (MBNL1), thereby regulating pre-mRNA splicing and mRNA localization. Furthermore, the presence of mutant DMPK transcripts is thought to lead to the hyperphosphorylation and stabilization of CELF1 (a CUG RNA-binding protein and embryonic lethal visual RNA-binding protein 3-like factor 1), which also plays a role in the regulation of alternative splicing and RNA stability. Fluorescence in situ hybridization (FISH) was used to quantify the percentage of cells presenting nuclear foci and the number of foci / nuclei in treated and untreated DM1 human myotubes. Figures 17C-17D Using nDes-miR155-amiR-DMPK 204 Treatment reduced the percentage of focal cells by 60% (untreated = 74.36% ± 9.3; treated = 15.36% ± 4.2, mean ± SEM; p = 0.0044, two-tailed unpaired t-test) Figure 17E Furthermore, compared with untreated cells, treated cells had significantly fewer foci per cell (untreated = 1.55 ± 0.08; treated = 0.15 ± 0.03, mean ± SEM; p < 0.0001, two-tailed unpaired t-test). Figure 17F ). Example 3: miR155 amiR-DMPK 204 Processing

[0306] amiR-DMPK 204 The product was packaged into an AAV capsid and its knockdown efficacy, passenger chain activity, and processing mode were analyzed in vivo. The product was packaged with nDes-miR155-amiR-DMPK. 204The construct was packaged into an AAV. The vector was intravenously injected into a DMSXL adult humanized DM1 mouse model expressing human DMPK with >1,000 CTG repeat sequences. Eight weeks later, the animals were euthanized, and multiple tissues were collected to measure DMPK knockdown efficacy, and cardiac tissue was selected to measure passenger chain activity and processing patterns.

[0307] RT-PCR analysis showed that amiR-DMPK 204 It is robustly expressed in various muscle tissues and exhibits high expression in the heart. Figure 2B Accompanied by amir-DMPK 204 Expression, RT-PCR analysis confirmed robust DMPK inhibition in the heart, with an average DMPK inhibition of >70% and approximately 30% in different skeletal muscles ( Figure 2C In cardiac tissue, DMPK expression is less than 50% relative to TBP (TATA-binding protein) expression. Notably, DMPK expression is low relative to TBP expression (approximately 50%, derived from…). Figure 2C DSMXL mice (indicated by the dashed line in the diagram) did not exhibit a significant DM1 phenotype. Interestingly, even with elevated transduction observed in the liver (…),… Figure 2A However, the nDesmin promoter shows strong activity in cardiac tissue and similar levels in skeletal muscle. Figure 2B This indicates that expression is primarily limited to myocardial and skeletal muscle affected by DM1 pathology.

[0308] To evaluate amir-DMPK 204 The processing of mature amiR-DMPK in cardiac tissue was analyzed by NGS for small transcriptome analysis. 204 The length and sequence composition of the guide chain and passenger chain.

[0309] amiR-DMPK 204 The processing does not produce a passenger chain. In mouse cardiomyocytes, amiR-DMPK... 204 Specialized processing yields a guide strand (>99%), but often produces longer strands than predicted from the miRBase database (Table 1). miR155 processing most commonly produces mature strands of 22 to 26 nucleotides, but with precise processing at the 5' end (Table 1). Mapping to miR155 amiR-DMPK 204 The sequence distribution of different guide chain lengths (nt) is calculated as a percentage (% reads). Expected amiR-DMPK 204 The guide chain is underlined, and the seed sequence is in bold. An asterisk indicates the correspondence to amiR-DMPK. 204 The read segment indicating the predicted length of the guide chain. Table 1. Mapping to miR155 amir-DMPK204 The sequence distribution of different guide chain lengths (nt) is calculated as a percentage (% reads). Expected amiR-DMPK 204 The guide chain is underlined. An asterisk indicates the correspondence between amiR-DMPK. 204 The read segment indicating the predicted length of the guide chain. miR-155 read sequence length %sequence SEQ ID NO AGTCGAAGACAGTTCTAGGGTGT 23 52 33 AGTCGAAGACAGTTCTAGGGTGTT 24 24 34 AGTCGAAGACAGTTCTAGGGTGTTT 25 11 35 AGTCGAAGACAGTTCTAGGGT* 22 6 36 AGTCGAAGACAGTTCTAGGGTGTTTT 26 3 37 AGTCGAAGACAGTTCTAGGGTG 22 4 38

[0310] In summary, using an amiR-DMPK with miR155 204 No passenger chain was detected (% guide chain > 99%). Therefore, miR155 was chosen as the lead compound for preclinical studies because the miR155 miRNA scaffold is well-validated for RNAi. Example 4: Systemic injection of transgenic mice encoding miR155-amiRDMPK 204 AAV's dose-dependent inhibition of human DMPK

[0311] To determine the most effective dose, delivery of three individual doses of the skin-friendly AAV (WO / 2019 / 207132) capsid was investigated. The amiR-DMPK encoded with a miR155 scaffold was evaluated in a dose-escalation study. 204 Expression box (amiR155-DMPK) 204 AAV of ) was administered intravenously to 8-week-old DMSXL mice carrying human DMPK transgenes with CTG amplification containing more than 1,000 repeat sequences. 11 One vector genome (vg) / kg, 5×10 12 vg / kg and 1.0×10 13 vg / kg (corresponding to low, medium, and high doses). Clinical signs in mice, such as body weight, survival rate, muscle rigidity, and cardiac function, were analyzed 8 weeks after AAV infusion. Mice were euthanized 8 weeks after gene transfer, and DMPK repression and splicing correction were measured. miR155-amiRDMPK was measured using the small RNA TaqMan. 204 Expression levels were determined, and mRNA input levels were normalized for u6 small nuclear RNA.

[0312] AmiR155-DMPK was observed in a dose-dependent manner. 204 The expression ( Figure 3A ), and caused a dose-dependent decrease in total DMPK expression in multiple tissues ( Figure 3B In summary, approximately 10 amiR155-DMPKs were observed. 204 A single copy of U6 is sufficient to reduce DMPK in the heart and diaphragm by ≥50%, a dose sufficient to treat patients with DM1.

[0313] The DMSXL mouse cohort was treated with a single dose of miR155-DMPK. 204 With 9×10 13 Treatment with a dose of vg / kg resulted in significant silencing of artificial miRNA expression in skeletal and cardiac muscle, as well as DMPK mRNA in the heart, TA, diaphragm, and gastrocnemius muscles, but not in the liver, thus confirming the muscle specificity of this platform at higher doses. Figure 18 ).

[0314] Nuclear RNA foci in the hearts of treated mice were measured by in situ hybridization. Foci often exhibited large formations, therefore the linear size of each formation in positive cells was measured. miR155-amiRDMPK was used. 204 Treatment significantly reduced the size of the foci (untreated = 7 ± 0.32 μm; treated = 2.7 ± 0.08 μm; mean ± SEM; p < 0.0001, one-way ANOVA); Figure 19 ).

[0315] Next, the consequences of DMPK repression on characteristic DM1 phenotypes such as splicing abnormalities were investigated. To determine whether treatment could improve the splicing phenotype in mice, cardiac and tibialis anterior (TA) muscle samples were collected and RNA-Seq analyses were performed in wild-type, buffer-negative control, and treated mice (Shen et al. (2014) Proc Natl Acad Sci [Proceedings of the National Academy of Sciences] 111(51):E5593-E5601; Park et al. (2013) Methods Mol Biol [Methods in Molecular Biology] 1038:171-9; Shen et al. (2012) Nucleic Acids Res [Nucleic Acid Research] 40(8):e61). Overall alterations in alternative splicing events were found in the heart and TA, indicating molecular changes in the disease with treatment. The altered genes in the heart of DMSXL mice were Ldb3, Mbnl2, and Spag9 ( Figure 20 Aberrant splicing of Ldb3, which contains the LIM domain of exon 11 in the Ldb3 transcript, has been shown to be caused by the CUG repetitive RNA-isolated RNA splicing mechanism, resulting in a DM1-specific phenotype (Yamashita et al. 2014. Neurobiol Dis. [Neurobiology of Disease] 69:200-5). This was achieved with medium or high doses of AAV nDes-miR155-amiR-DMPK. 204 A significant reduction in Ldb3 transcripts containing exon 11 was observed in the gastrocnemius muscle of treated mice, confirming the effect of amiR155-DMPK. 204 The splicing defects in the treated muscle were effectively corrected. Figure 4Previously, it has been confirmed that the persistence of fetal isotypes of these genes contributes to DM1 pathology. In TA, a distinct but overlapping set of alternative splicing events was altered when comparing wild-type and DMSXL mice. Among them, the following three genes showed significant splicing rescue in the treatment group: DNase I, Tnnt3, and Zbtb49. Figure 20 For these transcripts, there were no significant differences between wild-type and treated DMSXL mice in both the heart and TA, indicating normal splicing restoration after treatment.

[0316] In addition to these molecular corrections, AAV nDes-miR155-amiR-DMPK was then measured based on the physiological and functional manifestations of the disease. 204 Its effects.

[0317] To determine whether treatment could improve survival and reduce weight loss, three different doses of AAV nDes-miR155-amiR-DMPK were used. 204 DMSXL mice were treated, and survival and body weight were monitored. Improvements in body weight and survival were observed after 8 weeks of treatment with medium and high doses. On the other hand, no improvement was observed with low doses or balanced salt solution (BSS) as a control. Figure 5A and Figure 5B ).

[0318] Next, AAV nDes-miR155-amiR-DMPK was measured based on the functional manifestations of the disease, such as prevention of myotonia and cardiac abnormalities. 204 The efficacy of AAV nDes-miR155-amiR-DMPK was revealed by electromyography measurements. 204 Myotonia was significantly reduced in the treated mice (Table 2). Specifically, only 7.6% of the animals treated with the medium dose had myotonia after treatment. In contrast, >50% of the mice in the control (treated with BSS) or low-dose groups had persistent myotonia (score 1). Table 2. Using AAV nDes-miR155-amiR-DMPK 204 The number of DMSXL DM1 mice with myotonia after treatment.

[0319] Myotonic discharges were graded on a 4-point scale: 0, no myotonic discharges; 1, occasional myotonic discharges in less than 50% of needle insertions; 2, myotonic discharges in more than 50% of needle insertions; 3, myotonic discharges in almost every insertion.

[0320] In the treatment groups (wild-type mice, vector control, and amiR155-DMPK in DMSXL mice)204 Compare the number of grade 0 events with the number of grade 1-3 events in a single intravenous dose. Figure 21 As expected, all wild-type mice scored 0 in all three muscle groups analyzed. In DMSXL mice treated with the mediator, the number of grade 0 events recorded in all three muscle groups analyzed was significantly reduced (gastrocnemius, grade 0: WT = 30; DMSXL mediator = 1; grades 1–3: WT = 0, DMSXL mediator = 9; Fisher exact test, Bonferroni-Holm adjusted p < 0.0001; quadriceps, grade 0: WT = 30; DMSXL mediator = 2; grades 1–3: WT = 0, DMSXL mediator = 8; Fisher exact test, Bonferroni-Holm adjusted p < 0.0001; TA, grade 0: WT = 30; DMSXL mediator = 7; grades 1–3: WT = 0; DMSXL mediator = 3; Fisher exact test, Bonferroni-Holm adjusted p = 0.036). However, compared with the mediator group, both doses significantly improved the tetanic phenotype in the gastrocnemius muscle and showed a positive tendency to improve in the other two muscles (Gastrocnemius: DMSXL 9 x 10¹³, grade 0 = 9; grades 1-3 = 3; Fisher exact test, Bonferroni-Holm adjusted p = 0.0041; DMSXL 1.8 x 10¹⁴, grade 0 = 15; grades 1-3 = 6; Fisher exact test, Bonferroni-Holm adjusted p = 0.0041; Quadriceps: DMSXL 9 x 10¹³, grade 0 = 9; grades 1-3 = 3; Fisher exact test, Bonferroni-Holm adjusted p = 0.059; DMSXL 1.8 x 10¹⁴, grade 0 = 13; grades 1-3 = 8; Fisher exact test, Bonferroni-Holm adjusted p = 0.059; TA: DMSXL 9 x 10¹³, grade 0 = 12; grades 1-3 = 0; Fisher exact test, Bonferroni-Holm adjusted p-value = 0.155; DMSXL 1.8 x 10¹⁴, grade 0 = 19; grades 1-3 = 2; Fisher exact test, Bonferroni-Holm adjusted p-value = 0.295). To determine the use of miR155-DMPK 204 Whether AAV capsid treatment of the construct can correct for this defect was investigated by measuring the grade of myotonic events in the gastrocnemius, quadriceps, and TA muscles via EMG. While wild-type mice did not exhibit myotonic events in any of the muscles analyzed, most untreated DMSXL mice showed events at grades between 1 and 2, and most treated mice showed myotonic events at grades between 0 and 1. Figure 21 ).

[0321] Another clinical hallmark of this disease is cardiac dysfunction. Conduction defects are found in up to 75% of adult DM1 patients, and arrhythmias are a leading cause of death. Cardiac function in DMSXL mice was also monitored using surface echocardiography along with skeletal muscle function 8 weeks after treatment. Compared with the BSS-treated control, AAV nDes-miR155-amiR-DMPK 204 Improved cardiac output. A significant improvement in cardiac output was observed in the medium-dose group (5e12 vg / kg) after 8 weeks of treatment. Figure 6 ).

[0322] Echocardiographic analysis of the hearts of DSMXL mice showed that, compared with their wild-type littermates, the mice exhibited aortic stenosis (Fig. 22, p < 0.001, univariate ANOVA). However, using 9 × 10 13 vg / kg amiR-DMPK 204 Treatment significantly increased aortic diameter (p<0.05). There was no significant difference between treated DMSXL mice and wild-type mice. Figure 22A Furthermore, it was found that DMSXL mice had reduced ascending aortic blood flow velocity (inadequate cardiac output) compared to wild-type mice; Figure 22B (p<0.0001), and this defect was also corrected by treatment (p<001). Example 5: The AAVrh74N502I capsid improved muscle transduction and reduced liver transduction.

[0323] Experiments were conducted to test the transduction efficiency of AAV capsids containing AAVrh74N502I VP1 capsid protein (SEQ ID NO:50) in various tissues of non-human primates. Figure 7A The experiment is summarized in the diagram. Using 1×10⁻⁶... 13 Non-human primates were treated intravenously with AAV9, AAVrh74, or AAVrh74N502I capsid containing eGFP expression cassettes at a dose of vg / kg. Twenty-one days post-treatment, animals were sacrificed, and eGFP expression levels in the tibialis anterior (TA), biceps femoris, quadriceps femoris, heart, and liver were measured.

[0324] Compared to the control capsid, capsids containing the AAVrh74N502I capsid protein exhibited improved muscle transduction in non-human primates. Figures 7B-7E ) and reduced liver transduction ( Figure 7F (Table 3). Table 3. The level of eGFP in tissues of non-human primates treated with AAVrh74N502I capsids increased compared to non-human primates treated with AAV9 and AAVrh74 capsids. organize AAV9 AAVrh74 Tibialis anterior muscle +13x +178x biceps femoris +303x +56x quadriceps NS* +32x heart NS +13x liver -15x -2x *NS = Not significant Example 6: AAVrh74N502I nDes-miR155-amiR-DMPK in the DMSXL mouse model 204 Evaluation of target bonding

[0325] To determine the most effective dose, delivery of two separate doses of the AAVrh74N502I capsid was investigated. The amiR-DMPK encoded with a miR155 scaffold was evaluated in a dose escalation study. 204 Expression box (miR155-amiR-DMPK) 204 AAV of 9×10⁸ was administered intravenously to 8-week-old DMSXL mice. 13 One vector genome (vg) / kg and 1.8×10 14 vg / kg (corresponding to low-high and high doses). Mice were euthanized 8 weeks after gene transfer, and DMPK repression and amiR-DMPK were measured using small RNA TaqMan. 204 Expression levels were determined, and mRNA input levels were normalized for u6 small nuclear RNA.

[0326] amiR-DMPK was observed in a dose-dependent manner. 204 The expression ( Figure 8A ), and caused a dose-dependent decrease in total DMPK expression in multiple tissues ( Figure 8B In summary, ~10 amiR-DMPK were observed. 204 A single copy of U6 is sufficient to reduce DMPK in the heart and diaphragm by ≥50%, a dose sufficient to treat patients with DM1.

[0327] Finally, the lead vector nDes-miR155-amiR-DMPK was confirmed. 204 In the context of the skin-friendly capsid AAVrh74N502I (SEQ ID NO:50), it exhibits potent in vitro activity in cardiomyocyte-derived DM1iPSCs. Figure 9 ). Example 7: AAVrh74N502InDes-miR155-amiR-DMPK on the transcriptome 204 Evaluation

[0328] To determine amir-DMPK 204 To determine whether the treatment had any significant impact on the transcriptome, whole-genome RNA sequencing (RNA-seq) was performed using CBA miR155-amiR-DMPK. 204Plasmid transfection into HEK293 cell line to compare amiR-DMPK 204 Treatment with CTL3 (scrambled miRNA). To evaluate whether the observed changes in non-DMPK gene expression were due to amiR-DMPK. 204 The off-target effect was evaluated by assessing the enrichment of seed complementarity in significantly downregulated targets. Using a 5% false discovery rate (FDR) significance threshold, four differentially expressed genes were identified: OPN4 (12.5-fold), DMPK (1.7-fold), KRTAP21-2 (1.7-fold), and C8ORF44-SGK3 (1.7-fold), whose 3′ UTRs contained TTCGAC seed complement sequences (Table 4). For a 1% FDR, only OPN4 (12.5-fold) and DMPK (1.7-fold) showed differential expression. Figure 10 As expected, DMPK was one of the most significantly affected mRNA levels (44% silencing). In summary, amiR-DMPK... 204 Minimal off-target effects were observed after overexpression in HEK293 cells. Table 4 Example 8: Exploring AAVrh74N502InDes-miR155-amiR-DMPK 204 Biodistribution and dose range of activity in non-human primates: a study

[0329] To determine AAVrh74MN502I nDes-miR155-amiR-DMPK 204 The biodistribution and activity of the drug were determined by administering a single intravenous (IV) dose to cynomolgus monkeys. The study lasted for 12 weeks following the single injection.

[0330] A total of 16 cynomolgus macaques (8 males and 8 females; aged 24 to 48 months) were administered the drug once via the saphenous vein IV on day 1 of the study. Dosages are shown in Table 5 below. Animals were grouped (2 males and 2 females per group) into four distinct categories based on the following dose levels (vg / kg): Buffer preparation (Group 1); 5 × 10⁻⁶ 13 vg / kg (Group 2); 1×10 14 vg / kg (Group 3); and 2×10 14 vg / kg (Group 4). After 12 weeks, the animals were euthanized and tissues were harvested for analysis. Table 5

[0331] Mechanically homogenized harvested tissues were subjected to RNA and DNA extraction, and the samples were analyzed using digital PCR (dPCR).

[0332] AAVrh74N502I nDes-miR155-amiR-DMPK 204 The dose-dependent biodistribution and activity of the virus were demonstrated in several muscle and non-muscle tissues. Several skeletal muscle groups (tibialis anterior (TA); gastrocnemius; quadriceps; biceps; soleus; extensor digitorum longus (EDL); diaphragm) as well as cardiac and liver tissues were analyzed. Viral genome copies were found in all tissues tested, and the copy number / cell ratio in each tissue was dose-dependent. Figure 12 ). amiR-DMPK expression ( Figure 13 ) and DMP reduction ( Figure 14 It also occurs in a dose-dependent manner in various muscle, heart, and liver tissues. A dose-dependent reduction in DMPK expression was found to be up to 90% lower than in the control group. All doses tested in animals were found to be safe and well-tolerated. Example 9: Using amir-DMPK 204b Salvage of shearing defects after treatment

[0333] DMPK-mediated splicing defects are a molecular marker in DM1 tissues. To determine amiR-DMPK... 204 To determine whether the treatment affected these splicing defects, targeted splicing analysis was performed using RNA sequencing (RNA-seq) and the Nanostring platform, focusing on 36 genes (Tanner et al. 2021. Nucleic Acids Res. [Nucleic Acid Research] 49(4):2240-2254. doi:10.1093 / nar / gkab022).

[0334] The data between the two platforms is comparable. (Using AAV2-nDesmin-nDES-miR155-DMPK) 204 Immortalized human DM1 myotubes were transduced and compared with untreated DM1 myotubes and healthy control myotubes. 25 out of 36 genes showed statistically significant changes in their splicing before treatment. Compared with untreated cells, 6 out of 25 genes (MBNL1, SOS1, PKM, zTTN, GOLGA4, and CLASP1) showed significant restoration in treated cells (Figure 15). In summary, the data demonstrate the effectiveness of amiR-DMPK... 204 Treating human myotubes can at least partially salvage splicing defects caused by DMPK mutations. Example 10: Up to 2×10 14 vg / kg of AAVrh74N502I nDes-miR155-amiR-DMPK 204 It is well tolerated in non-human primates.

[0335] The review is based on AAVrh74N502I nDes-miR155-amiR-DMPK. 204 Tissue sections from treated animals were examined to assess tolerance to the AAV construct. All animals in the study were monitored in vivo, including detailed cage-side observations, body weight, food consumption, and vital sign measurements.

[0336] No macroscopic observations related to the test sample were noted. After application of 1×10 14 A male and an animal given 2×10 vg / kg 14 Minimal individual hepatocyte necrosis associated with the test substance was observed in the liver of one female at a dose of vg / kg (Table 6). This was observed after administration of a single dose ≥1×10⁻⁶. 14 The study showed a reduction in minor clinical chemical effects associated with the test substance in animals, characterized by minimal to mild increases in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glutamate dehydrogenase (GDH) activities (Table 7). Enzyme increases were observed only at early time points, completely reversing to baseline values ​​by day 43, and no associated microscopic liver findings were found in the affected animals, except in cases of administration of 2 × 10⁻⁶. 14 Minimal individual hepatocyte necrosis was found in one female at a dose of vg / kg. After administration of 5×10 13 No evidence of liver effects was identified in animals at vg / kg. No test-sample-related effects on hematological or coagulation parameters were identified at any dose level. These changes are considered non-adverse based on the generally small magnitude of variation, minimal severity and incidence, and / or lack of dose-relatedness. Table 6. When applying amiR-DMPK 204 The incidence and severity of test-sample-related microscopic findings in animals. Table 7. Application of amiR-DMPK 204 Changes in liver enzymes related to the test sample in selected animals. sequence Unless otherwise indicated, all polypeptide sequences are presented from the N-terminus to the C-terminus. Unless otherwise indicated, all nucleic acid sequences are presented from 5' to 3'. Byrne desmin enhancer sequence CACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGTCCTTCCTGTGCCTTGTTTC CCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTG CTGCCAGGGAGATGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGT GTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCAATTGGAAACTG AAAGCTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCCCACC CTCGA(SEQID NO:21) Source: Li and Paulin et al., 1991. "High level desmin expression depends on amuscle-specific enhancer." Journal of Biol Chem. 266.10:6562-6570. Homo sapiens desmin locus control region (DES-LCR) on chromosome 2 [Homo sapiens desmin locus control region (DES-LCR) on chromosome 2] (NCBI reference sequence: NG_046330.1) The Byrne enhancer sequence corresponds to 17767-18125 in the reference sequence. Paulin desmin enhancer sequence CCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTCT ATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGG TTGACATGCGGCTCCTGACAAAACACAAACCCTGGGTGTGTGGGCGTGGGTGGTGTG AGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGT CTGTGCGGGGGTGGGAGCGCACATAGCAATTGGAAACTGAA(SEQ ID NO:22) Source: Li and Paulin et al., 1991. "High level desmin expression depends on amuscle-specific enhancer." Journal of Biol Chem. 266.10:6562-6570. Homo sapiens desmin locus control region (DES-LCR) on chromosome 2 [Homo sapiens desmin locus control region (DES-LCR) on chromosome 2] (NCBI reference sequence: NG_046330.1) The Paulin enhancer sequence corresponds to 17787-18063 in the reference sequence. Paulin desmin promoter sequence (-228 to +75) There is one base pair difference from the published sequence (C instead of A, shown in bold and underline). Source: Li and Paulin et al., 1991. "High level desmin expression depends on a muscle-specific enhancer." Journal of Biol Chem. 266.10:6562-6570. Homo sapiens desmin locus control region (DES-LCR) on chromosome 2 [Homo sapiens desmin locus control region (DES-LCR) on chromosome 2] (NCBI reference sequence: NG_046330.1) The Paulin promoter sequence corresponds to 18535-18844 in the reference sequence. Complete nDes starter sequence CACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGTCCTTCCTGTGCCTTGTTTC CCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTG CTGCCAGGGAGATGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGT GTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGG GGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCAATTGGAAACTG AAAGCTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCCCACCCTCGAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTCCCAGCCATGCG TTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAG ATGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTG GGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCC ACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCAATTGGAAACTGAAAGCTTCTGC AGACCTGCTTGCTGCCTGCCCTGGCGAAGGATTGGCAGGCTTGCCCGTCACAGGACCCC CGCTGGCTGACTCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACG GCCACGGGCCGCCCTTTCCTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGGAGGGGA GGCGGGGGCTGATGTCAGGAGGGATACAAATAGTGCCGACGGCTGGGGGCCCTGTCTCC CCTCGCCGCATCCACTCTCCGGCCGGCCGCCTGCCCGCCGCCTCCTCCGTGCGCCCGCCAGCCTCGCCCG (SEQ ID NO: 12) Source: Li and Paulin et al., 1991. "High level desmin expression depends on a muscle-specific enhancer." Journal of Biol Chem. 266.10:6562-6570. Rabbit β-globin introns The 5' arm of the rabbit β-globin intron Source: Rabbit (Oryctolagus cuninculus) hemoglobin, β(HBB2) gene ID 100009084 The sequence of this invention has an additional CATG (shown in bold and underline) that is not present in gene ID 100009084. The 3' arm of the rabbit β-globin intron Source: Rabbit hemoglobin, β(HBB2) gene ID 100009084 The sequence of this invention has two T residues (shown in bold and underline) instead of the two C residues in gene ID 100009084. miR155-DMPK 204 sequence: 5'miR155 wing sequence RNA sequence CUGGAGGCUUGCUGAAGGCUGUAUGCU(SEQ ID NO:9) DNA sequence CTGGAGGCTTGCTGAAGGCTGTATGCT(SEQ ID NO:40) Source: BLOCK-iT TM Pol II miR RNAi Expression Vector Kit Catalog Number #K493500 The engineered pre-miRNA sequence structure is based on the mouse miR-155 sequence (Lagos-Quintana et al., 2002, Current Biology, 12:9, 735-739). amiR-DMPK 204 Guidance - DNA AGTCGAAGACAGTTCTAGGGT(SEQ ID NO:4) miR155 terminal circular DNA GTTTTGGCCACTGACTGAC(SEQ ID NO:6) Source: BLOCK-iT TM Pol II miR RNAi Expression Vector Kit Catalog Number #K493500 The engineered pre-miRNA sequence structure is based on the mouse miR-155 sequence (Lagos-Quintana et al., 2002, Current Biology, 12:9, 735-739). amiR-DMPK 204 Passenger-DNA ACCCTAGATGTCTTCGATT(SEQ ID NO:5) amiR-DMPK 204 Guided RNA-miR155 terminal loop-amiR-DMPK 204 Passenger-RNA AGUCGAAGACAGUUCUAGGGUUGUUUUGGCCACUGACUGACACCCUAGAUGUCUUCGAUU(SEQ IDNO:7) amiR-DMPK 204 Guide-DNA-miR155 terminal loop-amiR-DMPK 204 Passenger-DNA AGTCGAAGACAGTTCTAGGGTTGTTTTGGCCACTGACTGACACCCTAGATGTCTTCGATT(SEQ IDNO:8) 3'miR155 wing sequence RNA sequence GACACAAGGCCUGUUACUAGCACUCACAUGGAACAAAUGGCC(SEQ ID NO:10) DNA sequence GACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC(SEQ ID NO:41) Source: BLOCK-iT TM Pol II miR RNAi Expression Vector Kit Catalog Number #K493500 The engineered pre-miRNA sequence structure is based on the mouse miR-155 sequence (Lagos-Quintana et al., 2002, Current Biology, 12:9, 735-739). miR155 RNA CUGGAGGCUUGCUGAAGGCUGUAUGCUGACACAAGGCCUGUUACUAGCACUCACAUGGAACAAAUGGCC(SEQ ID NO:11) DNA CTGGAGGCTTGCTGAAGGCTGTATGCTGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC(SEQ ID NO:42) full miR155-DMPK 204 Double-stranded sequence-RNA CUGGAGGCUUGCUGAAGGCUGUAUGCUGAGUCGAAGACAGUUCUAGGGUGUUUUGGCCACUGACUGACACCCUAGAUGUCUUCGAUUCAGGACACAAGGCCUGUUACUAGCACUCACAUGGAACAAAUGGCC (SEQ ID NO: 24) full miR155-DMPK 204 Double-stranded sequence - DNA CTGGAGGCTTGCTGAAGGCTGTATGCTGAGTCGAAGACAGTTCTAGGGTGTTTTGGCCACTGACTGACACCCTAGATGTCTTCGATTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC (SEQ ID NO: 25) Minimal BGHpA sequence TCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGG TGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTA GGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAA GACAATAGC(SEQ ID NO:16) Source: Bovine (Bos taurus) growth hormone 1 (GH1) mRNA NCBI Reference Sequence NM_180996.1 1138bp A1AT intron-filling sequence (upstream of the expression cassette) TACGTACAATTGGGATCCTTCGAACTTGAGAGAAAACATCCCAGGGATTTACAGATCAC ATGCAGGCAGGGACCAGCTCAACCCTTCTTTAATGTCATCCAGGGAGGGGGCCAGGGAT GGAGGGGAGGGGTTGAGGAGCGAGAGGCAGTTATTTTTGGGTGGGATTCACCACTTTTC CCATGAAGAGGGGAGACTTGGTATTTTGTTCAATCATTAAGAAGACAAAGGGTTTGTTG AACTTGACCTCGGGGGGGATAGACATGGGTATGGCCTCTAAAAACATGGCCCCAGCAGC TTCAGTCCCTTTCTCGTCGATGGTCAGCACAGCCTTATGCACGGCCTGGAGGGGAGAGA ​​​​​​​​AGCATCGTCACTCCACTAGTCTGCCTCCAGGGCTCTCTCCTTTCTAGTACACGGCTTGA AGCTCCTTGAGGACACGGACCCTGGCAGTGACCTTCACAGTGCCCAGACCCCAAGATAA TGCAGCCATTCATGGAACTGCAGGTTGTTCATTGGTCGCCTTTAGTTTTCCAAAATAAG TGTCACTTTAGCTGAAATCATTCATTAATTCAGACACCAAATCTCACAGATCGAAGGAG TCAGAAATTCCTTTGAAACAACTTAGCCCAAACCTTTCTGTGTCAGTATGGATAAATCA AGGCCCAATGTCTAGAAGGTCTTGGGCAAAGTTGAAATTCAGGGTCAGTGACACAACCT CAAGGGAGGCCCCGAAAGTGCCAGCTGCACAGCAGCCCCTGCCTGGCTTTGCTGTTTGC CCACCGTCCCGTGTCAGTGAATCACGGGCATCTTCAGGAGCTCAGCCTGGGTCTTCATT TGTTTCCCTCGGCCCCTTCCTCAGCCTCAGGACAGTGCTAGCAGCCCCCACACATTCTTCCCTACAGATACCATGG(SEQ ID NO:18) Source: Plasmid DC 969 (SerpinA1 = A1AT) Chromosome 14 NG_008290.1 398bp A1AT intron-filling sequence (downstream of the expression cassette) GCATGCAGAGTGGACAGGGGCCTCAGGGACCCCTGATCCCAGCTTTCTCATTGGACAGA AGGAGGAGACTGGGGCTGGAGAGGGACCTGGGCCCCCACTAAGGCCACAGCAGAGCCAG GACTTTAGCTGTGCTGACTGCAGCCTGGCTTGCCTCCACTGCCCTCCTTTGCCTCAAGA GCAAGGGAGCCTCAGAGTGGAGGAAGCAGCCCCTGGCCTTGCCTCCCACCTCCCCTCCC CTATGCTGTTTTCCTGGGACAGTGGGAGCTGGCTTAGAATGCCCTGGGGCCCCCAGGAC CCTGGCATTTTAACCCCTCAGGGGCAGGAAGGCAGCCTGAGATACAGAAGAGTCCATCA CCTGCTGTATGCCACACACCATCCCCACAGTCGACATTTAAATT(SEQ ID NO:19) Source: Plasmid DC 969 (SerpinA1 = A1AT) Chromosome 14 NG_008290.1 ITR-nDes-miR155-amiR-DMPK 204 -BGHpA-filler sequence-ITR (3739bp) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTACGTACAATTGGGATCCTTCGAACTTGAGAGAAAACATCCCAGGGATTTACAGATCACATGCAGGCA GGGACCAGCTCAACCCTTCTTTAATGTCATCCAGGGAGGGGGCCAGGGATGGAGGGGAGGGGTTGAGGAGC GAGAGGCAGTTATTTTTGGGTGGGATTCACCACTTTTCCCATGAAGAGGGGAGACTTGGTATTTTGTTCAA TCATTAAGAAGACAAAGGGTTTGTTGAACTTGACCTCGGGGGGGATAGACATGGGTATGGCCTCTAAAAAC ATGGCCCCAGCAGCTTCAGTCCCTTTCTCGTCGATGGTCAGCACAGCCTTATGCACGGCCTGGAGGGGAGA GAAGCAGAGACACGTTGTAAGGCTGATCCCAGGCCTCGAGCAAGGCTCACGTGGACACCTCCCAGGAAGCG CTCACTCCCCCTGGACGGCCCTGGCCCTGCACATCCTCTCCCTCCCTGTCACATAGGCCTTGCTCCTCCTC AAGGCTTTGGCTGATGGGGCTGGCTCCCCTCTGTCCATCTTCCTGACAAGCGCCTCTCCCCCTGCTCAGGT GCACCCACAACTCAGAACAGGGAAGAGCATCGTCACTCCACTAGTCTGCCTCCAGGGCTCTCTCCTTTCTA GTACACGGCTTGAAGCTCCTTGAGGACACGGACCCTGGCAGTGACCTTCACAGTGCCCAGACCCCAAGATA ATGCAGCCATTCATGGAACTGCAGGTTGTTCATTGGTCGCCTTTAGTTTTCCAAAATAAGTGTCACTTTAG CTGAAATCATTCATTAATTCAGACACCAAATCTCACAGATCGAAGGAGTCAGAAATTCCTTTGAAACAACT TAGCCCAAACCTTTCTGTGTCAGTATGGATAAATCAAGGCCCAATGTCTAGAAGGTCTTGGGCAAAGTTGA AATTCAGGGTCAGTGACACAACCTCAAGGGAGGCCCCGAAAGTGCCAGCTGCACAGCAGCCCCTGCCTGGC TTTGCTGTTTGCCCACCGTCCCGTGTCAGTGAATCACGGGCATCTTCAGGAGCTCAGCCTGGGTCTTCATT TGTTTCCCTCGGCCCCTTCCTCAGCCTCAGGACAGTGCTAGCAGCCCCCACACATTCTTCCCTACAGATAC CATGGCACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTCCCAGCCA TGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGG GTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTGGTGTGAGTAGGGGGAT GAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACA TAGCAATTGGAAACTGAAAGCTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCC CACCCTCGAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTC TATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGGTTGACATGCGG CTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGG GGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCAATTGGAA ACTGAAAGCTTCTGCAGACCTGCTTGCTGCCTGCCCTGGCGAAGGATTGGCAGGCTTGCCCGTCACAGGAC CCCCGCTGGCTGACTCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACGGCCACGGGC CGCCCTTTCCTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGGAGGGGAGGCGGGGGCTGATGTCAGGAG GGATACAAATAGTGCCGACGGCTGGGGGCCCTGTCTCCCCTCGCCGCATCCACTCTCCGGCCGGCCGCCTG CCCGCCGCCTCCTCCGTGCGCCCGCCAGCCTCGCCCGGAGCTCTGAGTAGACGAAGCTAAGGCGCGCCTGA GAACTTCAGGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATATG GAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGCATGGACCCTC ATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTT CTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCA GATTGTAAGATCCCATCGATTCGGATCCCTGGAGGCTTGCTGAAGGCTGTATGCTGAGTCGAAGACAGTTC TAGGGTGTTTTGGCCACTGACTGACACCCTAGATGTCTTCGATTCAGGACACAAGGCCTGTTACTAGCACT CACATGGAACAAATGGCCCTCGAGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACAA TTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATT GGTAGAAACAACTACATCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGA GATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCC TACAGCTCCTGGGCAACGTGCTGACCGGTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCGCATGCAGAGTGGACAGGGGCCTCAGGGACCCCTGATCCCAGCTTTCTCATTGGACAGAAGGAGGAGACTGGGGCTGGAGAGGGACCTGGGCCCCCACTAAGGCCACAGCAGAGCCAGGACTTTAGCTGTGCTGACTGCAG CCTGGCTTGCCTCCACTGCCCTCCTTTGCCTCAAGAGCAAGGGAGCCTCAGAGTGGAGGAAGCAGCCCCTG GCCTTGCCTCCCACCTCCCCTCCCCTATGCTGTTTTCCTGGGACAGTGGGAGCTGGCTTAGAATGCCCTGG GGCCCCCAGGACCCTGGCATTTTAACCCCTCAGGGGCAGGAAGGCAGCCTGAGATACAGAAGAGTCCATCA CCTGCTGTATGCCACACACCATCCCCACAGTCGACATTTAAATTAGGAACCCCTAGTGATGGAGTTGGCCA CTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGT CGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO:20) Table 5. ITR-nDes-miR155-amiR-DMPK from 5' ITR to 3' ITR[[ID=!17]] 204 Sequence annotation (3739bp) The sequence of the synthesized nDes-miR155-204 fragment: AGATCTCCATGGCACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTC CCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGA TGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTGGTGTGAGTA GGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGA GCGCACATAGCAATTGGAAACTGAAAGCTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTT GAGGCCCCACCCTCGAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTCCCAGCCATGCGT TCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGGTTGA CATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAATC AGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCA ATTGGAAACTGAAAGCTTCTGCAGACCTGCTTGCTGCCTGCCCTGGCGAAGGATTGGCAGGCTTGCCCGTC ACAGGACCCCCGCTGGCTGACTCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACGGC CACGGGCCGCCCTTTCCTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGGAGGGGAGGCGGGGGCTGATG TCAGGAGGGATACAAATAGTGCCGACGGCTGGGGGCCCTGTCTCCCCTCGCCGCATCCACTCTCCGGCCGG CCGCCTGCCCGCCGCCTCCTCCGTGCGCCCGCCAGCCTCGCCCGGAGCTCTGAGTAGACGAAGCTAAGGCG CGCCTGAGAACTTCAGGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATG TTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGCATG GACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTT TCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTA TTTGTCAGATTGTAAGATCCCATCGATTCGGATCCCTGGAGGCTTGCTGAAGGCTGTATGCTGAGTCGAAG ACAGTTCTAGGGTGTTTTGGCCACTGACTGACACCCTAGATGTCTTCGATTCAGGACACAAGGCCTGTTAC TAGCACTCACATGGAACAAATGGCCCTCGAGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAG AGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATAT TCTTATTGGTAGAAACAACTACATCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACAC TGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTC TTTTTCCTACAGCTCCTGGGCAACGTGCTGACCGGTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCC GTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA TTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG ACAATAGCGCATGCGTCGACT(SEQ ID NO:26) nDes-miR155-204 promoter to polyA CACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTC CCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGA TGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTGGTGTGAGTA GGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGA GCGCACATAGCAATTGGAAACTGAAAGCTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTT GAGGCCCCACCCTCGAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTCCCAGCCATGCGT TCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGGTTGA CATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAATC AGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCA ATTGGAAACTGAAAGCTTCTGCAGACCTGCTTGCTGCCTGCCCTGGCGAAGGATTGGCAGGCTTGCCCGTC ACAGGACCCCCGCTGGCTGACTCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACGGC CACGGGCCGCCCTTTCCTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGGAGGGGAGGCGGGGGCTGATG TCAGGAGGGATACAAATAGTGCCGACGGCTGGGGGCCCTGTCTCCCCTCGCCGCATCCACTCTCCGGCCGG CCGCCTGCCCGCCGCCTCCTCCGTGCGCCCGCCAGCCTCGCCCGGAGCTCTGAGTAGACGAAGCTAAGGCG CGCCTGAGAACTTCAGGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATG TTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGCATG GACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTT TCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTA TTTGTCAGATTGTAAGATCCCATCGATTCGGATCCCTGGAGGCTTGCTGAAGGCTGTATGCTGAGTCGAAG ACAGTTCTAGGGTGTTTTGGCCACTGACTGACACCCTAGATGTCTTCGATTCAGGACACAAGGCCTGTTAC TAGCACTCACATGGAACAAATGGCCCTCGAGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAG AGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATAT TCTTATTGGTAGAAACAACTACATCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACAC TGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTC TTTTTCCTACAGCTCCTGGGCAACGTGCTGACCGGTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCC GTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA TTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC (SEQ ID NO:17) The amino acid sequence of AAVrh74N502I MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSIFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL(SEQ ID NO:50) Nucleotide sequence encoding the AAVrh74 N502I capsid The amino acid sequence of AAVrh74W505R (SEQ ID NO:52) Nucleotide sequence encoding AAVrh74W505R

Claims

1. A recombinant adeno-associated virus (rAAV) particle, the rAAV particle comprising: a) RNAi containing both a first strand and a second strand, wherein: i) The first chain and the second chain form a double chain; ii) The first strand contains a guide region, wherein the guide region contains a nucleic acid having the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO:1) or having a sequence having approximately 90% identity with the sequence of SEQ ID NO:1; and iii) The second chain contains a non-guided region; and b) An AAV capsid containing an amino acid sequence that is approximately 90% identical to that of the wild-type AAVrh74 capsid.

2. The rAAV particle of claim 1, wherein the unguided region comprises a nucleic acid having the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2) or having a sequence having approximately 90% identity with the sequence of SEQ ID NO:

2.

3. The rAAV particle of claim 1 or 2, wherein the first chain comprises a nucleic acid having the sequence of SEQ ID NO:1, and the unguided region comprises a nucleic acid having the sequence of SEQ ID NO:

2.

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

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

6. The rAAV particle as claimed in claim 4 or 5, wherein the ring structure comprises about 4 to about 20 nucleotides.

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

3.

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

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

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

7.

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

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

13. The rAAV particle of claim 12, wherein the scaffold comprises all or part 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 claim 14, wherein the scaffold has a 5' arm and a 3' arm, wherein the 5' arm is located at the 5' of the nucleic acid encoding the RNAi, and wherein the 3' arm is located at the 3' of the nucleic acid encoding the RNAi.

16. The rAAV particles as claimed in any one of claims 12-15, wherein the scaffold is a miR-155 scaffold.

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

9.

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

10.

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

20. The rAAV particle of claim 19, wherein the polypeptide is dystrophic myotonic kinase (DMPK).

21. The rAAV particle of claim 20, wherein the DMPK contains a DM-1-related mutation.

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

23. An expression cassette comprising a nucleic acid sequence encoding an RNAi as described in any one of claims 1-22.

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

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

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

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

28. The expression cassette of claim 26 or 27, wherein the desmin promoter comprises two enhancer elements and the promoter of 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 having about 90% identity with 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 having about 90% identity with 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 having about 90% identity with the nucleotide sequence of SEQ ID NO:

12.

32. The expression box of any one of claims 23-31, wherein the expression box further comprises introns.

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 having about 90% identity with the sequence of SEQ ID NO:

13.

35. The expression cassette according to 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 at the 5' of the nucleic acid encoding the RNAi and the 3' arm is located at the 3' of 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 having approximately 90% identity with 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 having about 90% identity with 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 bovine growth hormone polyadenylation signal, SV40 polyadenylation signal, or HSV TK pA.

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

42. The expression cassette according to 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 having about 90% identity with the sequence of SEQ ID NO:

16.

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

17.

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

45. The expression cassette of claim 44, wherein the modified desmin promoter comprises two enhancer elements and the promoter of 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 having about 90% identity with 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 having about 90% identity with 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 having about 90% identity with the nucleotide sequence of SEQ ID NO:

12.

49. The expression box of any one of claims 44-48, wherein the expression box further comprises introns.

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 having about 90% identity with the sequence of SEQ ID NO:

13.

52. The expression cassette according to 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 at the 5' of the nucleic acid encoding the transgene and the 3' arm is located at the 3' of 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 having approximately 90% identity with 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 having approximately 90% identity with 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 bovine growth hormone polyadenylation signal, SV40 polyadenylation signal, or HSV TK pA.

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

59. The expression cassette according to 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 having about 90% identity with the sequence of SEQ ID NO:

16.

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

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

62. A carrier comprising an expression cassette as claimed in any one of claims 23-61.

63. The vector of claim 62, wherein the flanks of the expression cassette are one or more filler nucleic acid sequences.

64. The vector of claim 63, wherein the one or more filler nucleic acid sequences are derived from the human SerpinA1 gene.

65. The vector of claim 63 or 64, wherein the filler nucleic acid sequence located at the expression cassette 5' is derived from the human SerpinA1 gene.

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

18.

67. The vector according to any one of claims 63-66, wherein the filler nucleic acid sequence located at the expression cassette 3' is derived from the human SerpinA1 gene.

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

19.

69. The vector according to 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 flanking sides of the expression cassette are one or more AAV inverted terminal repeat (ITR) sequences.

71. The rAAV carrier of claim 70, wherein the flanks of the expression cassette are 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, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITRs.

73. The rAAV carrier as claimed in any one of claims 70-72, wherein these AAV ITRs are AAV2 ITRs.

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

20.

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

76. A cell comprising an expression cassette as claimed in any one of claims 23-61, a vector as claimed in any one of claims 62-68, or an rAAV vector as claimed in any one of claims 69-75.

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

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

79. The rAAV particle of claim 77 or 78, wherein the rAAV particle comprises an AAVrh74 N502I serum-type capsid.

80. The rAAV particle of claim 1, wherein the ITR is AAV2 ITR and the capsid of the rAAV particle is AAVrh74 N502I serum-type capsid.

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

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

83. The rAAV particle according to any one of claims 1-22, wherein the rAAV particle comprises an AAVrh74 W505R serological capsid.

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

85. An rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': AAV2 ITR, Nucleic acid encoding a filler nucleic acid sequence derived from the human serpinA1 gene. Byrne desmin enhancer element, Paulin desmin enhancer element Desmin promoter, The 5' arm of the rabbit β-globin intron, 5'miR155 stent sequence, DMPK 204 miRNA guide sequence, miR155 terminal loop sequence DMPK 204 miRNA passenger sequence 3'miR155 stent sequence, The 3' arm of the rabbit β-globin intron, The smallest bovine growth hormone polyadenylated sequence, Nucleic acid encoding the filler nucleic acid sequence from the serpinA1 gene of this individual, and AAV2 ITR; Furthermore, the outer shell is an AAVrh74 N502I outer shell.

86. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:43, Nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:

18. Byrne desmin enhancer element containing the polynucleotide sequence of SEQ ID NO:21 Paulin desmin enhancer element containing the polynucleotide sequence of SEQ ID NO:22 The desmin promoter containing the polynucleotide sequence of SEQ ID NO:23 The 5' arm of the rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:14 The 5' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:40 DMPK containing the polynucleotide sequence of SEQ ID NO:4 204 miRNA guide sequence, The miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6 DMPK containing the polynucleotide sequence of SEQ ID NO:5 204 miRNA passenger sequence The 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41 The 3' arm of the rabbit β-globin intron contains the polynucleotide sequence of SEQ ID NO:

15. The smallest bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:

16. Nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; Furthermore, the outer shell is an AAVrh74 N502I outer shell.

87. The rAAV particle of claim 85 or 86, wherein the AAVrh74 N502I capsid comprises a capsid protein containing 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, from 5' to 3', the following nucleic acids: AAV2 ITR, nucleic acid encoding a filler 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, and DMPK. 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 The miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimum bovine growth hormone polyadenylated sequence, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, and AAV2 ITR; and wherein the capsid is AAVrh74 W505R capsid.

89. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises, from 5' to 3', the following nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43; a nucleic acid encoding a filler 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; and a DMPK comprising the polynucleotide sequence of SEQ ID NO:

4. 204 miRNA guide sequence, miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6, and DMPK containing the polynucleotide sequence of SEQ ID NO:

5. 204 The miRNA passenger sequence comprises a 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; wherein the capsid is an AAVrh74 W505R capsid.

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

52.

91. A composition comprising rAAV particles as described in any one of claims 1-22 and 77-90.

92. A pharmaceutical composition comprising rAAV particles as described in 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 rAAV particles as described in any one of claims 1-22 and 77-90.

95. A kit comprising rAAV particles as described in any one of claims 1-22 and 77-90.

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

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

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

99. A method for inhibiting the expression of dystrophic myotonic kinase (DMPK) in a mammal with DM-1, the method comprising administering to the mammal an effective amount of rAAV particles as described in any one of claims 1-22 and 77-90.

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

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

102. A method for inhibiting the expression of dystrophic myotonic kinase (DMPK) in a mammal with DM-1, the method comprising administering to the mammal an effective amount of rAAV particles as described in any one of claims 1-22 and 77-90.

103. A method for inhibiting the accumulation of DMPK RNA in the cells of a mammal with DM-1, the method comprising administering to the mammal an effective amount of rAAV particles as described in any one of claims 1-22 and 77-90.

104. The method according to any one of claims 100-103, wherein the effective amount of the rAAV particles is about 1 × 10⁻⁶. 8 Approximately 2×10 13 A dose of one genome copy / mL.

105. The method of claim 104, wherein the dose is about 5 × 10⁻⁶. 12 One genome copy / mL.

106. The method of claim 104, wherein the dose is about 1 × 10⁻⁶. 13 One genome copy / mL.

107. The method of claim 104, wherein the dose is about 2 × 10⁻⁶. 13 One genome copy / mL.

108. The method of any one of claims 101-103, wherein the effective amount of the rAAV particles is about 1 × 10⁻⁶. 8 Approximately 2×10 14 A dose of one genome copy per kg of body weight.

109. The method of claim 108, wherein the dose is about 5 × 10⁻⁶. 13 One genome copy per kg of body weight.

110. The method of claim 108, wherein the dose is about 1 × 10⁻⁶. 14 One genome copy per kg of body weight.

111. The method of claim 108, wherein the dose is about 2 × 10⁻⁶. 14 One genome copy per kg of body weight.

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

113. A method for inhibiting the expression of dystrophic myotonic kinase (DMPK) in a mammal with DM-1, the method comprising administering to the mammal an effective amount of the composition as described in any one of claims 91-93.

114. A method for inhibiting the accumulation of DMPK RNA in the cells of a mammal with DM-1, the method comprising administering to the mammal an effective amount of the composition as described in 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 immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with and / or after the RNAi administration.

116. The method of any one of claims 101-103, wherein the viral particle or the rAAV particle is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with and / or after the 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 immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with and / or after the composition is administered.

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

119. The method of claim 118, wherein the splicing of these gene transcripts is a measure of therapeutic efficacy.

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

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

122. An rAAV particle comprising a recombinant adeno-associated virus (rAAV) vector and a capsid, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': AAV2 ITR, Nucleic acid encoding a filler nucleic acid sequence derived from the human serpinA1 gene. Byrne desmin enhancer element, Paulin desmin enhancer element Desmin promoter, The 5' arm of the rabbit β-globin intron, 5'miR155 stent sequence, DMPK 204 miRNA guide sequence, miR155 terminal loop sequence DMPK 204 miRNA passenger sequence 3'miR155 stent sequence, The 3' arm of the rabbit β-globin intron, The smallest bovine growth hormone polyadenylated sequence, Nucleic acid encoding the filler nucleic acid sequence from the serpinA1 gene of this individual, and AAV2 ITR; Furthermore, the capsid contains a capsid protein containing the amino acid sequence of SEQ ID NO:

50.

123. An rAAV particle comprising a recombinant adeno-associated virus (rAAV) vector and a capsid, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': AAV2 ITR, Nucleic acid encoding a filler nucleic acid sequence derived from the human serpinA1 gene. Byrne desmin enhancer element, Paulin desmin enhancer element Desmin promoter, The 5' arm of the rabbit β-globin intron, 5'miR155 stent sequence, DMPK 204 miRNA guide sequence, miR155 terminal loop sequence DMPK 204 miRNA passenger sequence 3'miR155 stent sequence, The 3' arm of the rabbit β-globin intron, The smallest bovine growth hormone polyadenylated sequence, Nucleic acid encoding the filler nucleic acid sequence from the serpinA1 gene of this individual, and AAV2 ITR; Furthermore, the capsid contains a capsid protein containing the amino acid sequence SEQ ID NO:

52.

124. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:43, Nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:

18. Byrne desmin enhancer element containing the polynucleotide sequence of SEQ ID NO:21 Paulin desmin enhancer element containing the polynucleotide sequence of SEQ ID NO:22 The desmin promoter containing the polynucleotide sequence of SEQ ID NO:23 The 5' arm of the rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:14 The 5' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:40 DMPK containing the polynucleotide sequence of SEQ ID NO:4 204 miRNA guide sequence, The miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6 DMPK containing the polynucleotide sequence of SEQ ID NO:5 204 miRNA passenger sequence The 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41 The 3' arm of the rabbit β-globin intron contains the polynucleotide sequence of SEQ ID NO:

15. The smallest bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:

16. Nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; Furthermore, the capsid contains a capsid protein containing 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 from 5' to 3': AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:43, Nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:

18. Byrne desmin enhancer element containing the polynucleotide sequence of SEQ ID NO:21 Paulin desmin enhancer element containing the polynucleotide sequence of SEQ ID NO:22 The desmin promoter containing the polynucleotide sequence of SEQ ID NO:23 The 5' arm of the rabbit β-globin intron containing the polynucleotide sequence of SEQ ID NO:14 The 5' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:40 DMPK containing the polynucleotide sequence of SEQ ID NO:4 204 miRNA guide sequence, The miR155 terminal loop sequence containing the polynucleotide sequence of SEQ ID NO:6 DMPK containing the polynucleotide sequence of SEQ ID NO:5 204 miRNA passenger sequence The 3' miR155 scaffold sequence containing the polynucleotide sequence of SEQ ID NO:41 The 3' arm of the rabbit β-globin intron contains the polynucleotide sequence of SEQ ID NO:

15. The smallest bovine growth hormone polyadenylated sequence containing the polynucleotide sequence of SEQ ID NO:

16. Nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene containing the polynucleotide sequence of SEQ ID NO:19, and AAV2 ITR containing the polynucleotide sequence of SEQ ID NO:49; Furthermore, the capsid contains a capsid protein containing the amino acid sequence SEQ ID NO:52.

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