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-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 63 / 328,241 filed on April 6, 2022 and U.S. Provisional Application Serial No. 63 / 483,075 filed on February 3, 2023, the contents of which are incorporated herein by reference. Submission of sequence listing

[0001] The following content submitted as an XML file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: 737870_SA9-363PC_ST26.xml, Creation Date: April 3, 2023, Size: 82,900 bytes). Technical Field

[0002] The present invention relates to variant RNAi molecules.In some aspects, the present invention relates to variant RNAi molecules for treating muscular dystrophy. Background Art

[0003] In basic research on gene function, RNA interference (RNAi) has been demonstrated to be 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 [Nature] 431: 350-355; Krol et al., (2010) Nat. Rev. Genet. [Nature Review: Genetics] 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 suppression of target genes. Elucidation of the mechanisms involved in miRNA processing has allowed scientists to harness endogenous cellular RNAi machinery and use artificial miRNAs to direct degradation of target gene products (see, e.g., US PG Pub. [Pre-Grant Publication] 2014 / 0163214 and Davidson et al., (2012) Cell 150:873-875).

[0004] Myotonic dystrophy type 1 (DM1) is a monogenic, autosomal dominant progressive disease caused by the amplification of CTG repeats (>50) in the DMPK locus. DMPK with repeats is transcribed into mRNA, which forms a hairpin and binds to RNA binding proteins, thereby preventing these binding proteins from performing their normal functions. This leads to the appearance of nuclear foci, the mis-splicing of mRNA and ultimately myotonia. DM1 mainly affects skeletal muscle, cardiac muscle and smooth muscle, thereby causing significant physical, cognitive and behavioral impairment and disability. There is currently no approved therapy for DM1. Therefore, there is a high unmet medical need for the treatment of DM1.

[0005] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the invention

[0006] 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 duplex; b) the first strand comprises a guide region, wherein the guide region comprises a nucleic acid having a sequence of 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) or a sequence having about 90% identity to the sequence of SEQ ID NO: 1; and c) the second strand comprises a non-guide region. In some embodiments, the non-guide region comprises a nucleic acid having a sequence of 5'ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2) or a sequence having about 90% identity to the sequence of SEQ ID NO: 2. In some embodiments, the first strand comprises a nucleic acid having a sequence of SEQ ID NO: 1, and the non-guide region comprises a nucleic acid having a sequence of SEQ ID NO: 2. In some embodiments, the first strand and the second strand are connected by means of an RNA linker capable of forming a loop structure. In some embodiments, the RNA linker 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 a sequence of SEQ ID NO: 3 or a sequence having about 90% identity to a 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 a sequence of SEQ ID NO: 7 or a sequence having about 90% identity to a sequence of SEQ ID NO: 7. In some embodiments, the RNAi is a small inhibitory RNA (siRNA), a micro RNA (miRNA), or a small hairpin RNA (shRNA).

[0007] In some embodiments of the present invention, the RNAi further comprises a scaffold. In some embodiments, the scaffold comprises all or part of a nucleic acid of SEQ ID No: 11. In some embodiments, the miRNA is embedded in 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, 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 having a sequence of SEQ ID NO: 9 or a sequence having about 90% identity with the sequence of SEQ ID NO: 9 located at the 5' of the RNAi. In some embodiments, the miR-155 scaffold comprises a nucleic acid having a sequence of SEQ ID NO: 10 or a sequence having about 90% identity with the sequence of SEQ ID NO: 10 located at the 3' of the RNAi.

[0008] 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 comprises a mutation associated with DM1. In some embodiments, the gene encoding DMPK comprises 5 or more CTG trinucleotide repeats.

[0009] In some aspects, the present invention provides an expression cassette comprising a nucleic acid encoding any RNAi described herein. In some embodiments, the nucleic acid encoding the RNAi is operably 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 a human desmin gene. In some embodiments, the desmin promoter comprises two enhancer elements and a promoter of a 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 a nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having about 90% identity to a sequence of SEQ ID NO: 21, and / or one or more enhancer elements comprising a nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having about 90% identity to a sequence of SEQ ID NO: 22. In some embodiments, the desmin promoter comprises a nucleotide sequence of SEQ ID NO: 12 or a sequence having about 90% identity to 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 a nucleotide sequence of SEQ ID NO: 13 or a sequence having about 90% identity to 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 a nucleotide sequence of SEQ ID NO: 14 or a sequence having about 90% identity to the sequence of SEQ ID NO: 14. In some embodiments, the 3' arm of the intron comprises a nucleotide sequence of SEQ ID NO: 15 or a sequence having about 90% identity to 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 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 about 90% identity to the sequence of SEQ ID NO: 16.In some embodiments, the expression cassette comprises the nucleotide sequence of SEQ ID NO:17, or a sequence having about 90% identity to the sequence of SEQ ID NO:17.

[0010] In some aspects, the present invention provides an expression cassette, wherein the expression cassette comprises a modified desmin promoter, wherein the modified desmin promoter comprises one or more enhancer elements and a promoter of a human desmin gene. In some embodiments, the modified desmin promoter comprises two enhancer elements and a promoter of a 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 a nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having about 90% identity to a sequence of SEQ ID NO: 21, and / or one or more enhancer elements comprising a nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having about 90% identity to a sequence of SEQ ID NO: 22. In some embodiments, the desmin promoter comprises a nucleotide sequence of SEQ ID NO: 12 or a sequence having about 90% identity to a 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 a nucleotide sequence of SEQ ID NO: 13 or a sequence having about 90% identity to the sequence of SEQ ID NO: 13. In some embodiments, the nucleic acid encoding the 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 a nucleotide sequence of SEQ ID NO: 14 or a sequence having about 90% identity to the sequence of SEQ ID NO: 14. In some embodiments, the 3' arm of the intron comprises a nucleotide sequence of SEQ ID NO: 15 or a sequence having about 90% identity to 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 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 about 90% identity to 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.

[0011] In some aspects, the present invention provides a vector comprising any expression cassette described herein. In some embodiments, the expression cassette is flanked by 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 about 90% identity to 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 about 90% identity to the sequence of SEQ ID NO: 19.

[0012] In some embodiments of the present 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, cattle AAV or mouse AAV serotype ITRs. In some embodiments, these AAV ITRs are AAV2 ITRs. In some embodiments, the rAAV vector comprises a nucleotide sequence of SEQ ID NO: 20 or a sequence having about 90% identity to a sequence of SEQ ID NO: 20. In some embodiments, the vector is a self-complementary rAAV vector.

[0013] In some embodiments, the invention provides a cell comprising any expression cassette described herein, any vector described herein, or any rAAV vector described herein.

[0014] In some aspects, the invention provides a viral particle comprising any of the vectors described herein. In some aspects, the invention provides a recombinant AAV particle comprising any of the rAAV vectors described herein. In some embodiments, the AAV viral particle comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, AAVrh74 N502I, AAVrh74 W505R, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2N708A, AAV V708K, AAV2-HBKO, AAVDJ8, AAVPHP.B, AAVPHP.eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1 / AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAV2 / HBoV1 serotype capsid. In some embodiments, the ITR and the capsid of the rAAV virus particle are derived from the same AAV serotype. In some embodiments, the ITR and the capsid of the rAAV virus particle are derived from different AAV serotypes. In some embodiments, the AAV virus particle comprises an AAVrh74 N502I serotype capsid. In some embodiments, the ITR is an AAV2 ITR and the capsid of the rAAV particle is an AAVrh74 N502I serotype capsid. In some embodiments, the AAV virus particle comprises an AAVrh74 W505R serotype capsid. In some embodiments, the ITR is an AAV2 ITR and the capsid of the rAAV particle is an AAVrh74W505R serotype capsid. In some embodiments, the invention provides 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, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimal bovine growth hormone polyadenylation sequence, nucleic acid encoding stuffer nucleic acid sequence from the human serpinA1 gene, and AAV2 ITRs; and wherein the capsid is an AAVrh74 N502I capsid. In some embodiments, the present invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:18, a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:21, a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:22, a desmin promoter comprising the polynucleotide sequence of SEQ ID NO:23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:40, and a DMPK comprising the polynucleotide sequence of SEQ ID NO:4. 204 miRNA guide sequence, miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, DMPK comprising the polynucleotide sequence of SEQ ID NO: 5 204 miRNA passenger sequence, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is an AAVrh74 N502I capsid. In some embodiments, the AAVrh74 N502I capsid comprises a capsid protein comprising the amino acid sequence of SEQ ID NO:50.

[0015] In some embodiments, the present invention provides 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, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimal bovine growth hormone polyadenylation sequence, nucleic acid encoding stuffer nucleic acid sequence from the human serpinA1 gene, and AAV2 ITRs; and wherein the capsid is an AAVrh74 W505R capsid. In some embodiments, the present invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:18, a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:21, a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:22, a desmin promoter comprising the polynucleotide sequence of SEQ ID NO:23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:40, and a DMPK comprising the polynucleotide sequence of SEQ ID NO:4. 204 miRNA guide sequence, miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, DMPK comprising the polynucleotide sequence of SEQ ID NO: 5 204 miRNA passenger sequence, a 3'miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is an AAVrh74 W505R capsid. In some embodiments, the AAVrh74 W505R capsid comprises a capsid protein comprising the amino acid sequence of SEQ ID NO:52.

[0016] In some aspects, the present invention provides a composition comprising any viral particle or rAAV particle described herein. In some embodiments, the present invention provides a pharmaceutical composition comprising any viral particle or rAAV particle described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0017] In some aspects, the present invention provides a modified desmin promoter (e.g., for expressing a transgene in a muscle cell), wherein the modified desmin promoter comprises one or more enhancer elements and a promoter of a human desmin gene. In some embodiments, the modified desmin promoter comprises two enhancer elements and a promoter of a 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 sequence having about 90% identity to the sequence of SEQ ID NO: 21, and / or one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 22, or a sequence having about 90% identity to the sequence of SEQ ID NO: 22. In some embodiments, the modified desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12, or a sequence having about 90% identity to the sequence of SEQ ID NO: 12.

[0018] In some aspects, the invention provides kits comprising one or more of the RNAi as described herein, the viral particles as described herein, the AAV particles as described herein, or the compositions as described herein. In some embodiments, the kit further comprises instructions for use.

[0019] In some aspects, the invention provides methods for treating myotonic dystrophy type 1 (DM1) in a mammal in need, comprising administering to the mammal an effective amount of any RNAi described herein. In some aspects, the invention provides methods for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal suffering from DM-1 in need, comprising administering to the mammal an effective amount of any RNAi described herein. In some aspects, the invention provides methods for inhibiting the accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need, comprising administering to the mammal an effective amount of any RNAi described herein.

[0020] In some aspects, the present invention provides methods for treating myotonic dystrophy type 1 (DM1) in a mammal in need, 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 a mammal with DM-1 in need, 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 cells of a mammal with DM-1 in need, comprising administering to the mammal an effective amount of any viral particles as described herein (e.g., rAAV particles).

[0021] In some embodiments of the invention, the effective amount of a viral particle (e.g., a rAAV particle) is about 1×10 8 About 2×10 13 In some embodiments of the present invention, the dose is about 5×10 12 In some embodiments of the invention, the dose is about 1×10 13 In some embodiments of the present invention, the dose is about 2×10 13 genome copies / mL.

[0022] In some embodiments of the invention, the effective amount of a viral particle (e.g., a rAAV particle) is about 1×10 8 About 2×10 14 In some embodiments of the invention, the dose is about 5×10 13 In some embodiments of the present invention, the dose is about 1×10 14 In some embodiments of the present invention, the dose is about 2×10 14 genome copies / kg body weight.

[0023] In some aspects, the invention provides methods for treating myotonic dystrophy type 1 (DM1) in a mammal in need, comprising administering an effective amount of any composition as described herein to the mammal. In some aspects, the invention provides methods for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal suffering from DM-1 in need, comprising administering an effective amount of any composition as described herein to the mammal. In some aspects, the invention provides methods for inhibiting the accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need, comprising administering an effective amount of any composition as described herein to the mammal.

[0024] In some embodiments of the invention, the RNAi is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with, and / or after the RNAi is administered. In some embodiments, 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 viral particle or the rAAV particle is administered. In some embodiments, the composition is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with, and / or after the composition is administered. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present application may be understood by referring to the following description in conjunction with the accompanying drawings.

[0026] Figure 1A nDes-miR155-amiR-DMPK is depicted 204 Schematic diagram of the sequence of the gene cassette. The hybrid muscle promoter is located at miR155-amiR-DMPK 204 The cassette is flanked by two AAV2 ITRs, yielding a combined vector genome size of 3739 bp.

[0027] Figure 1B The method for cloning nDes-miR155-amiR-DMPK is described. 204 ITR plasmid for gene cassette. The ITR plasmid contains the A1AT stuffer sequence flanked by the AAV2 5' and 3' ITRs. The A1AT stuffer sequence contains NcoI and SphI restriction sites for cloning.

[0028] Figure 1C Depicted is a small-scale packaging assay in HEK 293 cells to confirm DC969-nDes-miR155-amiR-DMPK 204Results of plasmid packaging. Small-scale production using AAV rep / cap plasmid. The y-axis shows the amount of vector produced per HEK 293 cell compared to a standard EGFP plasmid gene cassette (CD627-CBA-GFP). DRP: DNase resistant particles.

[0029] Figure 2A-2C Depicts the effect of AAV nDes-miR155-amiR-DMPK in DMSXL mice after tail vein injection. 204 Evaluation of the (amiR155-204) expression cassette for DMPK knockdown. Figure 2A The transduction efficiency and biodistribution of AAV evaluated by quantification of transgene copy number in different organs are shown. qPCR results are expressed as the average ratio of AAV copy number / nucleus. Figure 2B The expression of amiR-DMPK in transduced tissues was shown 204 MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to cells treated with BSS (balanced salt solution). Figure 2C Silencing of DMPK in transduced tissues is shown. Total DMPK was determined by qRT-PCR. mRNA input was normalized to tata box binding protein (TBP) and set relative to BSS-treated cells. The dotted line indicates 50% DMPK expression relative to TBP expression. Figure 2A-2C , Data were evaluated using paired Student's T-test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. n=13 (BSS), n=12 (amiR155-204).

[0030] Figure 3A-3B AAV nDes-miR155-amiR-DMPK was depicted 204 Inhibition of human DMPK. DMSXL mice were systemically injected with AAV nDes-miR155-amiR-DMPK in a dose-dependent manner. 204 Mice were euthanized after 8 weeks, organs were harvested, and amiR-DMPK was measured. 204 and DMPK transcript levels. Figure 3A Delineation of amiR-DMPK normalized to U6 in various tissues 204 abundance. Figure 3B The abundance of hDMPK transcripts normalized to mTBP in various tissues is depicted. Data were evaluated using ANOVA, multiple comparison test: *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).

[0031] Figure 4 Depicted is the use of AAV nDes-miR155-amiR-DMPK 204 Correction of splicing abnormalities in DMSXL mice after systemic treatment. After 8 weeks of treatment, splicing of alternative exon 11 in LDB3 was assessed in gastrocnemius muscle using RT-PCR. Data were evaluated using ANOVA, multiple comparison test: *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).

[0032] Figure 5A and Figure 5B AAV nDes-miR155-amiR-DMPK is shown 204 Survival rate and body weight gain in female DMSXL mice treated in a dose-dependent manner. Figure 5A Kaplan-Meier survival curves are shown, demonstrating improved survival after 8 weeks of treatment with the medium dose compared to animals treated with the low dose or BSS. Figure 5B Compared with BSS-treated or low-dose treated animals, the expression of AAV nDes-miR155-amiR-DMPK was significantly higher in the control group (P<0.05). 204 Increased body weight was observed in treated DMSXL animals.

[0033] Figure 6 Depicted is the use of AAV nDes-miR155-amiR-DMPK 204 Reversal of electrophysiological features of DM1 disease after systemic treatment of the DMSXLDM1 mouse model in a dose-dependent manner. Data were evaluated using ANOVA, multiple comparison test: *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).

[0034] Fig. 7A A schematic diagram of the experimental protocol is shown. Blood was collected on day -1 To confirm neutralizing antibodies again. AAV encoding GFP reporter was administered to NHPs at a dose of 1e13 vg / kg on day 0, and necropsies were performed on D21 and multiple tissues were collected to evaluate biodistribution. Figure 7B-Figure 7E Quantification of GFP expression in various tissues from animals injected with AAV9, AAV rh74, and AAV rh74 N502I (rh74M) is shown. Bar graphs represent tibialis anterior muscle (TA; Figure 7B ), biceps femoris( Figure 7C), quadriceps( Fig.7D ),heart( Fig. 7E ) and liver( Figure 7F ) GFP amount measured by ELISA in . Data were evaluated using ANOVA, multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0035] Fig. 8A and Figure 8B AAV rh74 N502InDes-miR155-amiR-DMPK is depicted 204 Inhibition of human DMPK. DMSXL mice were systemically injected with AAV rh74 N502I nDes-miR155-amiR-DMPK in a dose-dependent manner. 204 Mice were euthanized after 8 weeks, organs were harvested, and amiR-DMPK was measured. 204 and DMPK transcript levels. Fig. 8A Delineation of amiR-DMPK normalized to U6 in various tissues 204 abundance. Figure 8B The abundance of hDMPK transcripts normalized to mTBP in various tissues is depicted. Data were evaluated using ANOVA, multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0036] Fig. 9 AAVrh74N502I nDes-miR155-amiR-DMPK is shown 204 Silencing of endogenous DMPK. Total DMPK levels were determined by qRT-PCR on RNA extracted from human DM1 cardiomyocytes differentiated from iPSCs. These cardiomyocytes were transfected with AAVrh74N502InDes-miR155-amiR-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.

[0037] Fig.10 Is to show 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 with 3′UTR seed complementation, FDR < 1% and FDR < 5%.

[0038] Fig.11Multiple sequences are shown in amiRDMPK 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.

[0039] Fig.12 Illustrated is the biodistribution of viral genome copies / cell in various tissues in the indicated treatment groups.

[0040] Fig.13 Shown is the dose-dependent amiR-DMPK expression in different muscle tissues in the indicated treatment groups.

[0041] Fig.14 Shown are the DMPK expression levels after treatment in the different muscles of each indicated treatment group. DETAILED DESCRIPTION

[0042] In some aspects, the invention provides an RNAi comprising a first strand and a second strand, wherein a) the first strand and the second strand form a duplex; b) the first strand comprises a guide region, wherein the guide region comprises a nucleic acid having a sequence of 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) or a sequence having about 90% identity to the sequence of SEQ ID NO: 1; and c) the second strand comprises a non-guide region, wherein the non-guide region comprises a nucleic acid having a sequence of 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2) or a sequence having about 90% identity to the sequence of SEQ ID NO: 2. In some embodiments, the invention provides an expression cassette for expressing a nucleic acid encoding an RNAi; for example, for expressing the RNAi in the muscle of a mammal. In some embodiments, the expression cassette is in a rAAV vector.

[0043] In some aspects, the invention provides a method of treating myotonic dystrophy type 1 (DM-1) in a mammal by administering the RNAi of the invention to the 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 techniques

[0044] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are routinely employed using routine methods, such as the widely used methods described in 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 (FM Ausubel et al., eds., 2003); Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology (Humana Press, 1998); Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., Academic Press, 1998); Introduction to Cell and Tissue Culture (J. P. Heater and P. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A.Doyle, JB Griffiths and DG Newell, eds., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 1993-8); Sons [John Wiley & Sons, 2002); Immunobiology [Immunobiology] (CA Janeway et al., 2004); Antibodies [Antibodies] (P. Finch, 1997); Antibodies: A Practical Approach [Antibodies: A Practical Approach] (D. Catty, ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach [Monoclonal Antibodies: A Practical Approach] (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual [Using Antibodies: A Laboratory Manual] (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies [Antibodies] (M. Zanetti and JD Capra, eds., Harwood Academic Press, 1996); Publishers [Harwood Academic Publishers], 1995); and Cancer: Principles and Practice of Oncology [Cancer: Principles and Practice of Oncology] (VT DeVita et al., eds., JBLippincott Company, 2011). definition

[0045] As used herein, "vector" refers to a recombinant plasmid or virus comprising a nucleic acid to be delivered into a host cell in vitro or in vivo.

[0046] 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 comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of a polynucleotide may include sugar and phosphate groups (as can be found in RNA or DNA generally), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide may include a polymer of synthetic subunits such as phosphoramidates, and therefore may be oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. In addition, double-stranded polynucleotides may be obtained from chemically synthesized single-stranded polynucleotide products in the following manner: by synthesizing complementary chains and annealing the chains under appropriate conditions, or by synthesizing complementary chains de novo using DNA polymerase with appropriate primers.

[0047] The terms "polypeptide" 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 may contain natural or non-natural 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 encompassed in this definition. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of the present invention, "polypeptide" refers to proteins including modifications to the native sequence, such as deletions, additions and substitutions (usually conservative in nature), as long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host producing the protein or due to errors in PCR amplification.

[0048] "Recombinant viral vector" refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one, and in some embodiments, two inverted terminal repeats (ITRs).

[0049] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences from non-AAV sources), which are flanked by at least one, and in some embodiments, two AAV inverted terminal repeats (ITRs). When present in a host cell that has been infected with a suitable helper virus (or is expressing a suitable auxiliary function) and is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), such rAAV vectors can be replicated and packaged into infectious viral particles. When the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or another vector such as a plasmid for cloning or transfection), the rAAV vector can be referred to as a "front vector", which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable auxiliary functions. The rAAV vector can be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated in viral particles, particularly AAV particles. rAAV vectors can be packaged into AAV viral capsids to produce "recombinant adeno-associated virus particles (rAAV particles)".

[0050] "Heterologous" means derived from a genotypically different entity from the rest of the entity to which it is being compared or into which it is introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (and when expressed, may encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0051] The term "transgenic" refers to a polynucleotide that is introduced into a cell and can be transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In some aspects, it imparts the desired properties to the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic result. In another aspect, it can be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA or shRNA.

[0052] The terms "genomic particles (gp)", "genomic equivalents" or "genomic copies (gc)" as used in relation to viral titer refer to the number of virions containing the recombinant AAV DNA genome, without regard to infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured by procedures such as those described in the Examples herein or, for example, in Clark et al. (1999) Hum. Gene Ther. [Human Gene Therapy], 10: 1031-1039; Veldwijk et al. (2002) Mol. Ther. [Molecular Therapy], 6: 272-278.

[0053] The term "vector genome (vg)" as used herein may 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 encapsidated in a viral particle. Depending on the specific viral vector, the vector genome may comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA or double-stranded RNA. The vector genome may include endogenous sequences associated with a specific viral vector and / or any heterologous sequences inserted into a specific viral vector by recombinant techniques. For example, a recombinant AAV vector genome may include at least one ITR sequence flanking a promoter, a stuffer sequence, a target sequence (e.g., RNAi), and a polyadenylation sequence. A complete vector genome may include a set of polynucleotide sequences of a vector. In certain embodiments, the nucleic acid titer of a viral vector may be measured according to vg / mL. Methods suitable for measuring this titer are known in the art (e.g., quantitative PCR).

[0054] As used herein, the term "inhibit" may refer to blocking, reducing, eliminating or otherwise antagonizing the presence or activity of a specific target. Inhibition may refer to partial inhibition or complete inhibition. For example, inhibiting the expression of a gene may refer to any effect of blocking, reducing, eliminating or any other antagonism that may result in the expression of the gene, including a reduction in mRNA abundance (e.g., silencing mRNA transcription), mRNA degradation, inhibition of mRNA translation, etc. In certain embodiments, inhibiting the expression of DMPK may refer to the retardation, reduction, elimination or any other antagonism of DMPK expression, including a reduction in DMPK mRNA abundance (e.g., silencing DMPK mRNA transcription), DMPK mRNA degradation, inhibition of DMPK mRNA translation, etc. For example, inhibiting the accumulation of protein in a cell may refer to any effect of blocking, reducing, eliminating or other antagonism that may result in the expression of the protein, including a reduction in mRNA abundance (e.g., silencing mRNA transcription), mRNA degradation, inhibition of mRNA translation, degradation of protein, etc. In some embodiments, inhibiting the accumulation of DMPK protein in cells refers to the blocking, reduction, elimination or other antagonism of DMPK protein expression in cells, including reduction of DMPK mRNA abundance (e.g., silencing DMPK mRNA transcription), degradation of DMPK mRNA, inhibition of DMPK mRNA translation, degradation of DMPK protein, etc.

[0055] The terms "infectious unit (iu)", "infectious particles" or "replication units" as used in reference to viral titers refer to the number of infectious and replication-competent recombinant AAV vector particles measured by the infectious center assay (also called the replication center assay), as described, for example, in McLaughlin et al. (1988) J. Virol., 62: 1963-1973.

[0056] The term "transducing unit (tu)" as used in reference to viral titer refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured in a functional assay such as in the Examples herein or as described, for example, in Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or Fisher et al. (1996) J. Virol., 70: 520-532.

[0057] "Inverted terminal repeat" or "ITR" sequence is a term well known in the art and refers to relatively short sequences in opposite orientation found at the ends of a viral genome.

[0058] The "AAV inverted terminal repeat (ITR)" sequence is a term well known in the art and is a sequence of approximately 145 nucleotides present at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can exist in either of two alternative 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 (designated as region A, region A', region B, region B', region C, region C', and region D) that allow intrastrand base pairing to occur within this portion of the ITR.

[0059] "Terminal resolution sequence" or "trs" is a sequence in the D region of the AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. Mutant terminal resolution sequences are resistant to cleavage by the AAV rep protein.

[0060] "AAV helper function" refers to a function that allows AAV to be replicated and packaged by a host cell. AAV helper function can be provided in any of a variety of forms, including but not limited to a helper virus or helper virus gene that assists in AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0061] The "helper virus" of AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by host cells. The helper virus provides a "helper function" that allows AAV to replicate. Many such helper viruses have been identified, including adenovirus, herpes virus and poxvirus such as vaccinia and baculovirus. Adenovirus covers many different subgroups, but adenovirus type 5 (Ad5) of subgroup C is the most commonly used. Many adenoviruses of human, non-human mammalian and avian origin are known and can be obtained from depository institutions such as ATCC. Viruses of the herpes family that can also be obtained from depository institutions such as ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and pseudorabies virus (PRV). Examples of adenovirus helper functions for AAV replication include E1A function, E1B function, E2A function, VA function and E4orf6 function. Baculoviruses available from depository institutions include Autographacalifornica nuclear polyhedrosis virus.

[0062] A rAAV preparation 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 preparation also does not contain an equivalent amount of helper virus protein (i.e., protein present due to such levels of helper virus if the above-mentioned 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-stained bands (e.g., the appearance of bands other than those corresponding to the AAV capsid proteins VP1, VP2, and VP3).

[0063] "Percent (%) sequence identity" with respect to 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 the amino acid residues or nucleotides in a reference polypeptide or nucleic acid sequence, after the sequences have been aligned and spaces have been introduced (if necessary) to achieve the maximum percentage of sequence identity, and without regard to any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining amino acid or nucleic acid sequence percent identity can be accomplished in various ways within the skill of the art, e.g., using publicly available computer software programs, e.g., those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supplement 30, Section 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 alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, and or for a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, and or for 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 scored as identical matches by the sequence alignment program in the program's alignment of A and B, 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 to B will not be equal to the % amino acid sequence identity of B to A. For the purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, and or to a given nucleic acid sequence D (which may alternatively be expressed as a given nucleic acid sequence C having or comprising a certain % nucleic acid sequence identity to, and or to a given nucleic acid sequence D) is calculated as follows: 100 multiplied by a fraction W / Z, where W is the number of nucleotides scored as identical matches by a sequence alignment program in the program's alignment of C and D, and where 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 % nucleic acid sequence identity of C to D will not be equal to the % nucleic acid sequence identity of D to C.

[0064] An "isolated" molecule (eg, nucleic acid or protein) or cell means that it has been identified and separated and / or recovered from a component of its natural environment.

[0065] An "effective amount" is an amount sufficient to achieve beneficial or desired results, including clinical results (e.g., improvement of symptoms, achievement of clinical endpoints, etc.). An effective amount can be administered in one or more administrations. With respect to a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or delay the progression of the disease.

[0066] An "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 certain embodiments, the individual or subject is a human.

[0067] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of disease state (e.g., no deterioration), prevention of disease transmission (e.g., metastasis), delay or slowing of disease progression, improvement or alleviation of disease state, and detectable or undetectable relief (partial or complete). "Treatment" can also mean extending survival rate compared to the expected survival rate without treatment.

[0068] As used herein, the term "prophylactic treatment" refers to treatment in which an individual is known or suspected to have a disorder or is at risk for a disorder, but has not yet exhibited symptoms of the disorder or has exhibited minimal symptoms of the disorder. An individual undergoing prophylactic treatment may be treated prior to the onset of symptoms.

[0069] As used herein, the term "myotonic dystrophy type 1" or "DM1" refers to a multisystem disorder that affects skeletal and smooth muscle 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]. In: Adam MP, Ardinger HH, Pagon RA et al., eds. [Internet]. Seattle (WA): University of Washington, Seattle [Seattle Washington University]; 1993-2022). Mild DM1 is characterized by cataracts and mild myotonia, and classic DM1 is characterized by muscle weakness and emaciation, myotonia, cataracts, and often cardiac conduction abnormalities. Congenital DM1 is characterized by hypotonia and severe generalized weakness at birth, often with respiratory insufficiency and early death; intellectual disability is common.

[0070] As used herein, the term "dystrophic myotonic protein kinase", "DMPK", "myotonic protein kinase", "MT-PK", "myotonic dystrophy protein kinase" or "MDPK" may refer to the gene or its polypeptide product associated with the majority of DM1 cases. The 3' untranslated region of the DMPK gene contains 5-37 copies of the CTG trinucleotide repeat sequence. Expansion of this unstable motif to 50-1,000 copies results in myotonic dystrophy type I, the severity of which increases with increasing copy number of the repeat element.

[0071] As used herein, "RNAi" may refer to any RNA molecule that induces RNA interference in a cell. Examples of RNAi include, but are not limited to, small inhibitory RNA (siRNA), microRNA (miRNA), and small hairpin RNA (shRNA).

[0072] "miRNA" can refer to a polynucleotide containing the following: (i) a double-stranded sequence that targets a target gene to be knocked down by RNAi and (ii) an additional sequence that forms a stem-loop structure similar to an endogenous miRNA. In some embodiments, miRNA includes nucleic acids flanking the stem-loop structure. These flanking sequences are called "miRNA scaffolds". The sequence of the target gene of RNAi targeting (e.g., a short sequence of ~20-nt) can be connected to the sequence that produces the miRNA-like stem-loop and the sequence that base pairs with the target sequence to form a duplex when the polynucleotide is assembled into a miRNA-like secondary structure. As described herein, the duplex can be incompletely hybridized, for example, it can contain one or more unpaired or mismatched bases. When the polynucleotide is cut by Dicer, the duplex containing the sequence of the target gene can be unfolded and 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. [Contemporary Biology] 12: 735-9) and the mirGE scaffold (WO 2014016817A2). Commercially available kits for cloning sequences into miRNA scaffolds are known in the art (e.g., from Life Technologies, Invitrogen, Thermo Fisher Scientific, and others). TM BLOCK-iT TM Pol II miR RNAi Expression Vector Kit; Waltham, MA).

[0073] As used herein, the term "sense" nucleic acid is a nucleic acid comprising a sequence encoding all or a portion of a transgene. In some examples, the mRNA of a transgene is a sense nucleic acid.

[0074] As used herein, an "antisense" nucleic acid is a nucleic acid sequence that is complementary to a "sense" nucleic acid. For example, an antisense nucleic acid can be complementary to an mRNA encoding a transgene.

[0075] As used herein, the "guidance region" of RNAi is an RNAi strand that typically binds to a target mRNA based on complementarity. The complementary region can encompass all or a portion of the guide region. Typically, the complementary region includes at least the seed region. In many cases, the antisense region of RNAi is the guide region.

[0076] As used herein, the "passenger region" or "non-guide region" of an RNAi, which are used interchangeably herein, is a region of the RNAi that is complementary to the guide region. In many cases, the sense region of an RNAi is the passenger region.

[0077] As used herein, the "seed region" of RNAi (eg, miRNA) is a region of microRNA that is about 1-8 nucleotides in length. In some examples, the seed region and the 3'-UTR of its target mRNA can be key determinants in RNAi recognition.

[0078] As used herein, "off-target gene silencing" refers to the pairing of the seed region of RNAi with sequences in the 3'-UTR of unintended mRNAs and directing translational repression and destabilization of those transcripts (eg, reducing expression of the unintended mRNA).

[0079] Reference herein to "about" a value or parameter includes (and describes) embodiments involving the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0080] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0081] It should be understood that the aspects and embodiments of the present invention described herein include "comprising the aspects and embodiments," "consisting of," and / or "consisting essentially of," the aspects and embodiments. RNAi

[0082] In some aspects, the invention provides improved RNAi for the targeted DMPK RNA for the treatment of myotonic dystrophy type 1 (DM1). In certain embodiments, the RNAi is a small inhibitory RNA (siRNA), microRNA (miRNA) or a small hairpin RNA (shRNA). Small inhibitory or interfering RNA (siRNA) known in the art is a double-stranded RNA molecule with a length of about 19-25 (e.g., 19-23) base pairs, which induces RNAi in cells. MiRNA is typically smaller than siRNA, can have multiple targets, and plays a role in suppressing translation, degrading mRNA and in some cases endonucleolytic cleavage of mRNA. Small hairpin RNA (shRNA) is known in the art as an RNA molecule comprising about 19-25 (e.g., 19-23) base pairs of double-stranded RNA connected by a short loop (e.g., about 4-11 nucleotides), which induces RNAi in cells. In some embodiments, the RNAi comprises a first strand and a second strand, wherein a) the first strand and the second strand form a duplex; b) the first strand comprises a guide region, wherein the guide region comprises the nucleic acid sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1); and c) the second strand comprises a non-guide region. In some embodiments, the guide region comprises the nucleic acid sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1), and the non-guide region comprises the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2).

[0083] In some embodiments, the first strand comprises a guide region, wherein the guide region comprises a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1). In some embodiments, the first strand comprises a guide region, wherein the guide region comprises a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1), but retains at least one CpG motif. In some embodiments, the second strand comprises a non-guide region, wherein the non-guide region comprises a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2). In some embodiments, the second strand comprises a non-guide region, wherein the non-guide region comprises a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2), but retains at least one CpG motif.

[0084] In some embodiments, the RNAi comprises a nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the RNAi comprises a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 7. In some embodiments, the RNAi comprises a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 7, but maintaining at least one sequence (e.g., in a seed sequence).

[0085] In some embodiments, the present invention provides a nucleic acid encoding an RNAi comprising a first strand and a second strand, wherein a) the first strand and the second strand form a duplex; b) the first strand comprises a guide region, and c) the second strand comprises a non-guide region. In some embodiments, the nucleic acid encoding the RNAi comprises a nucleic acid sequence of SEQ ID NO:4 and / or a nucleic acid of SEQ ID NO:5. In some embodiments, the nucleic acid encoding the RNAi comprises a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:4 and / or a nucleic acid sequence having greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:5. In some embodiments, the RNAi is encoded by a nucleic acid sequence of SEQ ID NO:8. In some embodiments, the RNAi is encoded by a nucleic acid sequence that is greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:8.

[0086] MicroRNA (miRNA) is known in the art as an RNA molecule that induces RNAi in cells, which comprises a short (e.g., 19-25 base pairs) sequence of double-stranded RNA connected by a loop and contains one or more additional sequences of double-stranded RNA containing one or more protrusions (e.g., mispaired or unpaired base pairs). As used herein, the term "miRNA" encompasses endogenous miRNAs as well as 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 in the following manner to produce pre-miRNA: excision of one or more sequences leaves a pre-miRNA with a 5' flanking region, a guide strand, a loop region, a non-guide strand, and a 3' flanking region; or a 5' flanking region, a non-guide strand, a loop region, a guide strand, and a 3' flanking region. The pre-miRNA is then exported to the cytoplasm and processed by Dicer to produce siRNA with a guide strand and a non-guide (or passenger) strand. The RISC complex then uses the guide strand to catalyze gene silencing, for example by identifying a target RNA sequence complementary to the guide strand. Further descriptions of miRNA can be found, for example, in WO 2008 / 150897. The recognition of the target sequence by 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 Acids Research] 41: e9).

[0087] In the pri / pre-miRNA structure, the guide strand: non-guide strand interface in the duplex is formed in part by complementary base pairing (e.g., Watson-Crick base pairing). However, in some embodiments, the complementary base pairing does not extend through the entire duplex. In some embodiments, the protrusion in the interface may be present at one or more nucleotide positions. As used herein, the term "protrusion" may refer to a nucleic acid region that is non-complementary to the nucleic acid opposite to it in the duplex. In some embodiments, a protrusion is formed when the regions of complementary nucleic acids are bound to each other and the regions of the central non-complementary regions are not bound. In some embodiments, a protrusion is formed when the two nucleic acid chains located between the two complementary regions have different lengths. As described below, a protrusion may include 1 or more nucleotides.

[0088] During miRNA processing, the miRNA is cleaved at a cleavage site adjacent to the guide strand: non-guide strand interface, thereby releasing the siRNA duplex of the guide strand and the non-guide strand. In some embodiments, the miRNA comprises a bulge in the sense strand or the antisense strand adjacent to the cleavage site. In other words, in some embodiments, the miRNA comprises a bulge in the guide strand or the non-guide strand adjacent to the seed sequence.

[0089] In some embodiments, the miRNA comprises a bulge in the guide strand opposite the 5' cleavage site of the mature non-guide strand. In some embodiments, the miRNA comprises a bulge opposite the 5' nucleotide of the non-guide strand. In some embodiments, the miRNA comprises a bulge in the sense strand opposite the 3' cleavage site of the mature guide strand. In some embodiments, the miRNA comprises a bulge opposite the 3' nucleotide of the guide strand.

[0090] The safety of RNAi-based therapies can be hampered by the ability of small inhibitory RNAs (siRNAs) to bind to and reduce the expression of unintended mRNAs, an effect known as off-target gene silencing. Off-targeting occurs primarily when the seed region (nucleotides 2-8 of the small RNA) pairs with sequences in the 3′-UTR of unintended mRNAs and directs translational repression and destabilization of those transcripts. Reduced off-target RNAi can be designed by replacing bases within the guide sequence and non-guide sequence; for example, by generating CpG motifs. Potential substitutions that can produce significantly lower off-target scores can be evaluated using the SiSPOTR algorithm, which is a specifically 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 January; 41(1)e9). Reduced SiSPOTR scores predict sequences with a lower number of potential human off-targets compared to the parent RNAi molecule. In some embodiments of the present invention, RNAi is improved to reduce off-target gene silencing.

[0091] In some embodiments, the first strand and the second strand are connected by means of an RNA (e.g., an RNA joint) capable of forming a ring structure. As is generally known in the art, when an RNA molecule comprises two RNA sequences, an RNA ring structure (e.g., a stem-loop or a hairpin) is formed, and the two RNA sequences are base-paired together and separated by RNA sequences that are not base-paired together. For example, if sequence A and sequence C are complementary or partially complementary so that they are base-paired together, but the bases in sequence B are not base-paired together, a ring structure can be formed in RNA molecule ABC.

[0092] In some embodiments, the RNA capable of forming a ring structure comprises 4 to 50 nucleotides. In certain embodiments, the RNA capable of forming a ring structure comprises 13 nucleotides. In some embodiments, the number of nucleotides in the RNA capable of forming a ring is 4 to 50 nucleotides or any integer therebetween. In some embodiments, 0-50% of the ring can be complementary to another part of the ring. As used herein, the term "ring structure" is a sequence connecting two complementary chains of nucleic acid. In some embodiments, 1-3 nucleotides of the ring structure are adjacent to the complementary chain of nucleic acid and can be complementary to 1-3 nucleotides of the distal part of the ring structure. For example, the three nucleotides at the 5' end of the ring structure can be complementary to the three nucleotides at the 3' end of the ring structure.

[0093] In some embodiments, the nucleic acid encoding the RNAi disclosed herein comprises a heterologous miRNA scaffold. In some embodiments, a heterologous miRNA scaffold is used to regulate miRNA expression; for example, to increase miRNA expression or to reduce miRNA expression. Any miRNA scaffold known in the art can be used. In some embodiments, the miRNA scaffold is derived from a miR-155 scaffold (see, e.g., Lagos-Quintana, M. et al. (2002) Curr. Biol. [Contemporary Biology] 12: 735-9 and Invitrogen from Life Technologies, Thermo Fisher Scientific TM BLOCK-iT TM Pol II miR RNAi Expression Vector Kit; Waltham, MA) or mirGE scaffold (WO 2014 / 016817). Methods of treating myotonic dystrophy type 1 (DM-1)

[0094] Myotonic dystrophy type 1 (DM1) is a monogenic, autosomal dominant, progressive disease caused by an expansion of the CTG repeat sequence (>50) in the DMPK locus (dystrophic myotonic protein kinase). The DMPK with the repeat sequence is transcribed into mRNA, which forms a hairpin and binds to RNA binding proteins, isolating them from their normal function. This leads to the appearance of nuclear foci, mis-splicing and ultimately myotonia. DM1 primarily affects skeletal muscle, cardiac muscle and smooth muscle, causing significant physical, cognitive and behavioral impairment and disability.

[0095] In some aspects, the present invention provides methods and compositions for treating myotonic dystrophy type 1 (DM1) in mammals, comprising administering to the mammal a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising rAAV particles of the present disclosure). In some aspects, the present invention provides methods and compositions for inhibiting the expression of DMPK in mammals suffering from DM-1, comprising administering to the mammal a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising rAAV particles of the present disclosure). In some aspects, the present invention provides methods and compositions for inhibiting the accumulation of DMPK in cells of mammals suffering from DM1, comprising administering to the mammal a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising rAAV particles of the present disclosure). In some aspects, the present invention provides methods and compositions for improving DM1 symptoms, comprising administering to the muscle brain of a mammal an effective amount of rAAV particles comprising a vector encoding RNAi of the present disclosure.

[0096] In some aspects, the 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 comprising a sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1), and the second strand comprising a second nucleic acid comprising a sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2). The RNAi described herein (e.g., as part of an rAAV vector) is particularly useful for treating DM1.

[0097] In some embodiments, the RNAi is a small inhibitory RNA (siRNA), microRNA (miRNA) or small hairpin RNA (shRNA). Small inhibitory or interfering RNA (siRNA) known in the art is a double-stranded RNA molecule of about 19-25 (e.g., 19-23) base pairs in length, which induces RNAi in cells. MiRNA is typically smaller than siRNA, can have multiple targets, and plays a role in suppressing translation, degrading mRNA and in some cases endonucleolytic cleavage of mRNA. Small hairpin RNA (shRNA) is known in the art as an RNA molecule of about 19-25 (e.g., 19-23) base pairs of double-stranded RNA connected by a short loop (e.g., about 4-11 nucleotides), which induces RNAi in cells.

[0098] In some embodiments, the miRNA comprises a guide sequence that is about 90% identical to SEQ ID NO: 1. In some embodiments, the miRNA comprises a guide sequence that is any of about 90% identical, 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical to SEQ ID NO: 1.

[0099] In some embodiments, the miRNA comprises a non-guide sequence (passenger strand) that is about 90% identical to SEQ ID NO: 2. In some embodiments, the miRNA comprises a non-guide sequence that is about 90% identical, 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical to SEQ ID NO: 2.

[0100] In some embodiments, the first strand and the second strand are connected by means of an RNA capable of forming a loop structure. As is generally known in the art, an RNA loop structure (e.g., a stem-loop or hairpin) is formed when an RNA molecule comprises two RNA sequences that are base paired together and separated by RNA sequences that are not base paired together. For example, if sequence A and sequence C are complementary or partially complementary so that they are base paired together, but the bases in sequence B are not base paired together, a loop structure can be formed in RNA molecule ABC.

[0101] In some embodiments, the RNA capable of forming a loop structure comprises 4 to 50 nucleotides. In certain embodiments, the RNA capable of forming a loop structure comprises 13 nucleotides. In certain 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.

[0102] In some aspects, the present invention provides a method comprising administering to a mammal (e.g., a mammal with DM1) a first strand comprising a first nucleic acid comprising the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) and a second strand comprising a second nucleic acid comprising 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 encapsidating a rAAV vector, wherein the rAAV vector encodes RNAi.

[0103] In some embodiments, the delivery of rAAV particles is by systemic injection of rAAV particles into a mammal. In some embodiments, systemic injection is intravenous injection, intraarterial injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, intra-CSF and intrathecal administration (IT).

[0104] In some aspects, the invention provides methods for treating DM1 in mammals, including administering the pharmaceutical composition of the present disclosure to mammals. In some aspects, the invention provides methods for suppressing the accumulation of DMPK in cells of mammals suffering from DM1, including administering the pharmaceutical composition of the present disclosure to mammals. In some aspects, the invention provides methods for suppressing the expression of DMPK in mammals suffering from DM1, including administering the pharmaceutical composition of the present disclosure to mammals. In certain embodiments, DMPK is a mutant DMPK (e.g., a DMPK comprising greater than 37 or greater than 50 CTG repeats).

[0105] In some embodiments, the present invention provides methods of treating a human suffering from DM1 by administering an effective amount of a pharmaceutical composition comprising a rAAV vector encoding the RNAi disclosed herein to suppress the activity of a mutant DMPK. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0106] In some embodiments, the methods include administering an effective amount of a pharmaceutical composition comprising a rAAV vector encoding an RNAi disclosed herein to suppress the activity of a mutant DMPK. In some embodiments, the viral titer of the rAAV particles is at least about 5×10 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 In some embodiments, the viral titer of the rAAV particles is about 5×10 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 In some embodiments, the viral titer of the rAAV particles is about 5×10 12 Up to 10×10 12 , 10×10 12 Up to 25×10 12 or 25×10 12 Up to 50×10 12 In some embodiments, the viral titer of the rAAV particles is at least about 5×10 9, 6×10 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 In some embodiments, the viral titer of the rAAV particles is about 5×10 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 In some embodiments, the viral titer of the rAAV particles is about 5×10 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 In some embodiments, the viral titer of the rAAV particles is at least about 5×10 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 In some embodiments, the viral titer of the rAAV particles is at least about 5×10 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 In some embodiments, the viral titer of the rAAV particles is at least about 5×10 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 infectious units / mL.

[0107] In some embodiments, the dose concentration of rAAV particles administered to an individual is about 1×10 8 About 2×10 13In some embodiments, the dose concentration of the rAAV particles administered to an individual is about 1×10 8 About 5×10 8 , about 5×10 8 to about 10×10 8 , about 10×10 8 About 20×10 8 , about 20×10 8 About 30×10 8 , about 30×10 8 About 40×10 8 , about 40×10 8 About 50×10 8 or about 50×10 8 About 100×10 8 In some embodiments, the dose concentration of the rAAV particles administered to an individual is about 1×10 9 About 5×10 9 , about 5×10 9 to about 10×10 9 , about 10×10 9 About 20×10 9 , about 20×10 9 About 30×10 9 , about 30×10 9 About 40×10 9 , about 40×10 9 About 50×10 9 or about 50×10 9 About 100×10 9 In some embodiments, the dose concentration of the rAAV particles administered to an individual is about 1×10 10 About 5×10 10 , about 5×10 10 to about 10×10 10 , about 10×10 10 About 20×10 10 , about 20×10 10 About 30×10 10 , about 30×10 10 About 40×10 10 , about 40×10 10 About 50×10 10 or about 50×10 10 About 100×10 10 In some embodiments, the dose concentration of the rAAV particles administered to an individual is about 1×1011 About 5×10 11 , about 5×10 11 to about 10×10 11 , about 10×10 11 About 20×10 11 , about 20×10 11 About 30×10 11 , about 30×10 11 About 40×10 11 , about 40×10 11 About 50×10 11 or about 50×10 11 About 100×10 11 In some embodiments, the dose concentration of the rAAV particles administered to an individual is about 1×10 12 About 5×10 12 , about 5×10 12 to about 10×10 12 , about 10×10 12 About 20×10 12 , about 20×10 12 About 30×10 12 , about 30×10 12 About 40×10 12 , about 40×10 12 About 50×10 12 or about 50×10 12 About 100×10 12 In some embodiments, the dose concentration of the rAAV particles administered to an individual is about 1×10 13 About 2×10 13 In some embodiments, the dose concentration of the rAAV particles administered to an individual is about 1×10 8 , about 5×10 8 , about 1×10 9 , about 5×10 9 , about 1×10 10 , about 5×10 10 , about 1×10 11 , about 5×10 11 , about 1×10 12 , about 5×10 12 , about 1×10 13 or about 2×10 13 In some embodiments, the dose concentration of rAAV particles administered to an individual is about 5×10 12In some embodiments, the dose concentration of the rAAV particles administered to an individual is about 1×10 13 In some embodiments, the dose concentration of the rAAV particles administered to an individual is about 2×10 13 genome copies / mL.

[0108] In some embodiments, the dose of rAAV particles administered to a subject is at least about 1×10 8 About 2×10 14 In some embodiments, the dose of rAAV particles administered to an individual is between about 1×10 8 About 2×10 14 In some embodiments, the dose of rAAV particles administered to a subject is between about 1×10 8 About 1×10 14 , 5×10 8 About 1×10 14 , 1×10 9 About 1×10 14 , 5×10 9 About 1×10 14 , 1×10 10 About 1×10 14 , 5×10 10 About 1×10 14 , 1×10 11 About 1×10 14 , 5×10 11 About 1×10 14 , 1×10 12 About 1×10 14 , 5×10 12 About 1×10 14 , 1×10 13 About 1×10 14 , 5×10 13 About 1×10 14 , 1×10 8 About 5×10 13 , 5×10 8 About 5×10 13 , 1×10 9 About 5×10 13 , 5×10 9 About 5×10 13 , 1×10 10 About 5×10 13 , 5×10 10 About 5×10 13 , 1×1011 About 5×10 13 , 5×10 11 About 5×10 13 , 1×10 12 About 5×10 13 , 5×10 12 About 5×10 13 , 1×10 13 About 5×10 13 , 1×10 8 About 1×10 13 , 5×10 8 About 1×10 13 , 1×10 9 About 1×10 13 , 5×10 9 About 1×10 13 , 1×10 10 About 1×10 13 , 5×10 10 About 1×10 13 , 1×10 11 About 1×10 13 , 5×10 11 About 1×10 13 , 1×10 12 About 1×10 13 , 5×10 12 About 1×10 13 , 1×10 8 About 5×10 12 , 5×10 8 About 5×10 12 , 1×10 9 About 5×10 12 , 5×10 9 About 5×10 12 , 1×10 10 About 5×10 12 , 5×10 10 About 5×10 12 , 1×10 11 About 5×10 12 , 5×10 11 About 5×10 12 , 1×10 12 About 5×10 12 , 1×10 8 About 1×10 12 , 5×10 8 About 1×10 12 , 1×10 9 About 1×10 12 , 5×109 About 1×10 12 , 1×10 10 About 1×10 12 , 5×10 10 About 1×10 12 , 1×10 11 About 1×10 12 , 5×10 11 About 1×10 12 , 1×10 8 About 5×10 11 , 5×10 8 About 5×10 11 , 1×10 9 About 5×10 11 , 5×10 9 About 5×10 11 , 1×10 10 About 5×10 11 , 5×10 10 About 5×10 11 , 1×10 11 About 5×10 11 , 1×10 8 About 1×10 11 , 5×10 8 About 1×10 11 , 1×10 9 About 1×10 11 , 5×10 9 About 1×10 11 , 1×10 10 About 1×10 11 , 5×10 10 About 1×10 11 , 1×10 8 About 5×10 10 , 5×10 8 About 5×10 10 , 1×10 9 About 5×10 10 , 5×10 9 About 5×10 10 , 1×10 10 About 5×10 10 , 1×10 8 About 1×10 10 , 5×10 8 About 1×10 10 , 1×10 9 About 1×10 10 , 5×10 9 About 1×10 10 , 1×108 About 5×10 9 , 5×10 8 About 5×10 9 , 1×10 9 About 5×10 9 , 1×10 8 About 1×10 9 , 5×10 8 About 1×10 9 or 1×10 8 About 5×10 8 In some embodiments, the dose of rAAV particles administered to an individual is about 1×10 9 , about 5×10 9 , about 1×10 10 , about 5×10 10 , about 1×10 11 , about 5×10 11 , about 1×10 12 , about 5×10 12 , about 1×10 13 , about 5×10 13 , about 1×10 14 or about 2×10 14 In some embodiments, the dose of rAAV particles administered to an individual is about 5×10 13 In some embodiments, the dose of rAAV particles administered to an individual is about 1×10 14 In some embodiments, the dose of rAAV particles administered to an individual is about 2×10 14 genome copies / kg body weight.

[0109] In some embodiments, the total amount of rAAV particles administered to a subject is at least about 1×10 9 About 2×10 14 In some embodiments, the total amount of rAAV particles administered to an individual is about 1×10 9 About 2×10 14 In some embodiments of the invention, the volume of the composition injected into the striatum is greater than 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, or any amount therebetween.

[0110] The compositions of the invention (e.g., rAAV particles comprising vectors encoding RNAi disclosed herein) can be used alone or in combination with one or more additional therapeutic agents for treating DM1. The interval between sequential administrations can be at least (or alternatively, less than) a few minutes, hours, or days.

[0111] In some embodiments, the RNAi for treating DM1 is administered in combination with an immunosuppressant; for example, to suppress the immune response to the RNAi. In some embodiments, the immunosuppressant is administered before the RNAi is administered. In some embodiments, the immunosuppressant is administered while the RNAi is administered. In some embodiments, the immunosuppressant is administered after the RNAi is administered. In some embodiments, the immunosuppressant is administered in any combination before, during, or after the RNAi is administered.

[0112] In some embodiments, the rAAV particles used to treat DM1 are administered in combination with an immunosuppressant; for example, to suppress an immune response to the rAAV particles and / or the transgenic products of the rAAV particles. In some embodiments, the immunosuppressant is administered before the rAAV particles are administered. In some embodiments, the immunosuppressant is administered while the rAAV particles are administered. In some embodiments, the immunosuppressant is administered after the rAAV particles are administered. In some embodiments, the immunosuppressant is administered in any combination before, during, or after the administration of the rAAV particles.

[0113] In some embodiments, the invention provides an effective amount of any RNAi described herein for use in the manufacture of a medicament for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides an effective amount of any RNAi described herein for use in the manufacture of a medicament for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with DM-1 in need thereof. In some embodiments, the invention provides an effective amount of any RNAi described herein for use in the manufacture of a medicament for inhibiting the accumulation of DMPK RNA in cells of a mammal with DM-1 in need thereof.

[0114] In some embodiments, the present invention provides the use of an effective amount of any RNAi described herein for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the present invention provides the use of an effective amount of any RNAi described herein for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal suffering from DM-1 in need thereof. In some embodiments, the present invention provides the use of an effective amount of any RNAi described herein for inhibiting the accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof.

[0115] In some embodiments, the invention provides RNAi described herein for use in treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides any RNAi described herein for use in inhibiting expression of dystrophic myotonic protein kinase (DMPK) in a mammal with DM-1 in need thereof. In some embodiments, the invention provides any RNAi described herein for use in inhibiting accumulation of DMPK RNA in cells of a mammal with DM-1 in need thereof.

[0116] In some embodiments, the present invention provides an effective amount of any of the viral particles described herein (e.g., AAV particles) for use in the manufacture of a medicament for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the present invention provides an effective amount of any of the viral particles described herein (e.g., AAV particles) for use in the manufacture of a medicament for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with DM-1 in need thereof. In some embodiments, the present invention provides an effective amount of any of the viral particles described herein (e.g., AAV particles) for use in the manufacture of a medicament for inhibiting the accumulation of DMPK RNA in cells of a mammal with DM-1 in need thereof.

[0117] In some embodiments, the present invention provides an effective amount of any of the viral particles described herein (e.g., AAV particles) for use in treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the present invention provides an effective amount of any of the viral particles described herein (e.g., AAV particles) for use in inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal suffering from DM-1 in need thereof. In some embodiments, the present invention provides an effective amount of any of the viral particles described herein (e.g., AAV particles) for use in inhibiting the accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof.

[0118] In some embodiments, the invention provides a viral particle (e.g., AAV particle) described herein for use in treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides any viral particle (e.g., AAV particle) described herein for use in inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with DM-1 in need thereof. In some embodiments, the invention provides any viral particle (e.g., AAV particle) described herein for use in inhibiting the accumulation of DMPK RNA in cells of a mammal with DM-1 in need thereof.

[0119] 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 a mammal in need thereof. 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 a mammal with DM-1 in need thereof. 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 cells of a mammal with DM-1 in need thereof.

[0120] In some embodiments, the present invention provides an effective amount of any of the compositions described herein for use in treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the present invention provides an effective amount of any of the compositions described herein for use in inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal suffering from DM-1 in need thereof. In some embodiments, the present invention provides an effective amount of any of the compositions described herein for use in inhibiting the accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof.

[0121] In some embodiments, the present invention provides compositions described herein for use in treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the present invention provides any compositions described herein for use in inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal suffering from DM-1 in need thereof. In some embodiments, the present invention provides any compositions described herein for use in inhibiting the accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof. RNAi expression constructs and vectors

[0122] The present invention provides expression constructs, vectors and rAAV particles for expressing the RNAi described herein.

[0123] In some embodiments, the nucleic acid encoding the RNAi disclosed herein comprises a heterologous miRNA scaffold. In some embodiments, a heterologous miRNA scaffold is used to regulate miRNA expression; for example, to increase miRNA expression or to reduce miRNA expression. Any miRNA scaffold known in the art can be used. In some embodiments, the miRNA scaffold is derived from a miR-155 scaffold (see, e.g., Lagos-Quintana, M. et al. (2002) Curr. Biol. [Contemporary Biology] 12: 735-9 and Invitrogen from Life Technologies, Thermo Fisher Scientific TMBLOCK-iT TM Pol II miR RNAi expression vector kit; Waltham, MA) or mirGE scaffold (WO 2014 / 016817). In some embodiments, the nucleic acid encoding the RNAi of the present disclosure 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 to the nucleic acid sequence of SEQ ID NO: 11.

[0124] In some embodiments, RNAi targets RNA encoding a polypeptide associated with DM1 (e.g., mutant DMPK). Without wishing to be bound by theory, it is believed that RNAi can be used to reduce or eliminate the expression and / or activity of a polypeptide (e.g., mutant DMPK) whose function has been associated with DM1.

[0125] In some embodiments, the transgene (e.g., encoding the RNAi disclosed herein) is operably linked to a promoter. Exemplary promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, RSV LTR, 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., Gene Ther., 1996, 3(9):802-10). In some embodiments, the promoter comprises a human β-glucuronidase promoter or a cytomegalovirus enhancer linked to a chicken β-actin (CBA) promoter.The promoter may be a constitutive promoter, an inducible promoter, or a repressible promoter.

[0126] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors (such as temperature), or the presence of a specific physiological state, 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 [Proceedings of the National Academy of Sciences of the United States of America], 93:3346-3351 (1996)), the tetracycline repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 89:5547-5551 (1992)), the tetracycline inducible system (Gosse ...). n et al., Science [Science], 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol. [Current Opinion in Chemical Biology], 2:512-518 (1998)), the RU486 inducible system (Wang et al., Nat. Biotech. [Nature Biotechnology], 15:239-243 (1997) and Wang et al., Gene Ther. [Gene Ther.], 4:432-441 (1997)) and the rapamycin inducible system (Magari et al., J. Clin. Invest. [Journal of Clinical Research], 100:2865-2872 (1997)). Other types of inducible promoters useful in this context are promoters that are regulated by specific physiological states, such as temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells.

[0127] In some embodiments, the regulatory sequence confers tissue-specific gene expression capability. 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 a 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 comprises desmin promoter elements (Li and Paulin et al. 1991. Journal of Biol Chem. [Journal of Biological Chemistry]) that are important for high-level expression in muscle cells. In some embodiments, the desmin promoter comprises at least one copy of the Byrne desmin enhancer (e.g., SEQ ID NO: 21). In some embodiments, the desmin promoter comprises 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).

[0128] In some aspects, the invention provides expression cassettes (e.g., and expression cassettes for expressing a transgene (e.g., a therapeutic transgene) in a muscle cell), wherein the expression cassette comprises a modified desmin promoter, wherein the desmin promoter comprises one or more enhancer elements, and a promoter of a human desmin gene. In some embodiments, the desmin promoter comprises two enhancer elements, and a promoter of a 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 to 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 to 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 to 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 to the nucleotide sequence of SEQ ID NO: 22.

[0129] In some embodiments, the expression cassette comprising a modified desmin promoter further comprises an intron. In some embodiments, the intron is a rabbit β-globin intron. In some embodiments, the intron comprises a nucleotide sequence of SEQ ID NO: 13. In some embodiments, the intron comprises a nucleotide sequence having at least about 80%, 85%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, a nucleic acid encoding a transgenic (e.g., a therapeutic transgenic) 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 transgenic and the 3' arm is located at the 3' of the nucleic acid encoding the transgenic. In some embodiments, the 5' arm of the intron comprises a nucleic acid having a sequence of SEQ ID NO: 14. In some embodiments, the 5' arm of the intron comprises a nucleic acid having a sequence of at least about 80%, 85%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the 3' arm of the intron comprises a nucleic acid having a 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 to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the 5' arm of the intron comprises a nucleic acid having a sequence of SEQ ID NO: 14, and the 3' arm of the intron comprises a nucleic acid having a 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 to 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 to the nucleotide sequence of SEQ ID NO: 15.

[0130] In some embodiments, the expression cassette comprising a modified desmin promoter further comprises 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 a 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 to the nucleotide sequence of SEQ ID NO: 16.

[0131] In some embodiments, the invention provides expression cassettes comprising a modified desmin promoter for expressing a transgene (e.g., a therapeutic transgene) in a muscle cell. 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.

[0132] In some aspects, the present invention provides a modified desmin promoter, wherein the desmin promoter comprises one or more enhancer elements and a promoter of a human desmin gene. In some embodiments, the desmin promoter comprises two enhancer elements and a promoter of a 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 a nucleotide sequence of SEQ ID NO: 21. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence of SEQ ID NO: 22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence of SEQ ID NO: 21 and one or more enhancer elements comprising a nucleotide sequence of SEQ ID NO: 22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence of SEQ ID NO: 21 and one or more enhancer elements comprising a 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 to 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 to 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 to 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 to the nucleotide sequence of SEQ ID NO: 22.

[0133] In some aspects, the present invention provides rAAV particles comprising a recombinant self-complementary genome (e.g., a self-complementary rAAV vector). AAV virus particles with self-complementary vector genomes and methods using self-complementary AAV genomes are described in U.S. Patents 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 [gene therapy] 10: 2105-2111, each reference is incorporated herein by reference in its entirety. rAAV comprising a self-complementary genome will rapidly form a double-stranded DNA molecule by relying on its partially complementary sequence (e.g., complementary coding strands and non-coding strands of heterologous nucleic acids). In some embodiments, the vector comprises a first nucleic acid sequence encoding a heterologous nucleic acid and a second nucleic acid sequence encoding the complement of the nucleic acid, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length.

[0134] In some embodiments, the first heterologous nucleic acid sequence encoding RNAi and the second heterologous nucleic acid sequence encoding the complementary sequence of RNAi are connected by a mutated ITR (e.g., the right ITR). In some embodiments, the ITR comprises a polynucleotide sequence 5'-CCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACC AAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG AGCGAGCGCGCAGAGAGGGA-3 (SEQ ID NO: 27). The mutated ITR comprises a deletion of the D region containing the terminal resolution sequence. Therefore, when the AAV viral genome is replicated, the rep protein will not cut the viral genome at the mutated ITR, and therefore, the recombinant viral genome comprising the following items in 5' to 3' order will be packaged in the viral capsid: AAV ITR, a first heterologous polynucleotide sequence comprising a regulatory sequence, a mutated AAV ITR, a second heterologous polynucleotide in the opposite orientation to the first heterologous polynucleotide, and a third AAV ITR. rAAV particles and methods of producing rAAV particles

[0135] The present invention provides rAAV particles comprising RNAi as disclosed herein. In some embodiments, the present invention provides methods for delivering RNAi to treat DM1 using recombinant viral particles. In some embodiments, rAAV particles comprise sequences encoding RNAi disclosed herein, the flanks of which are one or two ITRs. The nucleic acid is encapsidated in AAV particles. AAV particles also comprise capsid proteins. In some embodiments, the nucleic acid comprises a target coding sequence (e.g., a nucleic acid encoding RNAi disclosed herein) of a component operably connected in the transcription direction, a control sequence (including a transcription start sequence and a termination sequence), thereby forming an expression cassette. The expression cassette is flanked by at least one functional AAV ITR sequence at the 5' end and the 3' end. "Functional AAV ITR sequence" means that the function of the ITR sequence is as expected for the rescue, replication and packaging of AAV virions. See Davidson et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 2000, 97(7):3428-32; Passini et al., J. Virol. [Journal of Virology], 2003, 77(12):7034-40; and Pechan et al., Gene Ther. [Gene therapy], 2009, 16:10-16, all of which are incorporated herein by reference in their entirety. To practice some aspects of the present invention, the recombinant vector comprises at least all AAV sequences necessary for encapsidation and a physical structure for rAAV infection. The AAV ITRs used in the vectors of the present invention need not have wild-type nucleotide sequences (e.g., as described in Kotin, Hum. Gene Ther. [Human Gene Ther.], 1994, 5:793-801), and may be altered by insertion, deletion or substitution of nucleotides, or the AAV ITRs may be derived from any of several AAV serotypes. Currently, more than 40 serotypes of AAV are known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 2002, 99(18):11854-6; Gao et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 2003, 100(10):6081-6; and Bossis et al., J. Virol. [Journal of Virology], 2003, 77(12):6799-810. The use of any AAV serotype is considered within the scope of the present invention. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including but not limited to AAV ITRs that are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVrh74, AAV DJ, goat AAV, bovine AAV or mouse AAV capsid serotype ITRs, etc.In some embodiments, the nucleic acid in AAV comprises ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, AAV DJ, goat AAV, bovine AAV, or mouse AAV capsid serotype ITRs, etc. In some embodiments, the nucleic acid in AAV further encodes an RNAi as described herein. For example, the nucleic acid in AAV can comprise at least one ITR of any AAV serotype contemplated herein, and can further encode an RNAi comprising one strand comprising a guide region and another strand comprising a non-guide region. In one embodiment, the nucleic acid in the AAV can comprise at least one ITR of any AAV serotype and can further encode an RNAi comprising a first strand comprising a first nucleic acid comprising the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1), or a sequence having 80%, 85%, 90% or 95% identity to SEQ ID NO: 1, and a second strand comprising a second nucleic acid comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2), or a sequence having 80%, 85%, 90% or 95% identity to SEQ ID NO: 2.

[0136] In some embodiments, the nucleic acid in AAV comprises from 5' to 3' a nucleic acid encoding an ITR (eg, AAV2 ITR), a promoter, a nucleic acid encoding an RNAi as disclosed herein, a polyadenylation signal, and an AAV ITR (eg, AAV2 ITR). In some embodiments, the nucleic acid in the AAV comprises, from 5' to 3', a nucleic acid encoding an ITR (e.g., AAV2 ITR), a promoter, a nucleic acid encoding an RNAi, a polyadenylation signal, and an AAV ITR (e.g., AAV2 ITR), wherein the RNAi comprises a first strand and a second strand, wherein the first strand comprises a first nucleic acid comprising the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1), or a sequence with 80%, 85%, 90%, or 95% identity to SEQ ID NO: 1, and the second strand comprises a second nucleic acid comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2), or a sequence with 80%, 85%, 90%, or 95% identity to SEQ ID NO: 2. In some embodiments, the nucleic acid in the AAV comprises, from 5' to 3', nucleic acid encoding an ITR (e.g., AAV2 ITR), a desmin promoter, a nucleic acid encoding an RNAi as disclosed herein, a polyadenylation signal (e.g., bovine growth hormone polyA), and an AAV ITR (e.g., AAV2 ITR). In some embodiments, the nucleic acid in the AAV comprises, from 5' to 3', a nucleic acid encoding the following: all or a functional portion of an ITR (e.g., an AAV2 ITR), a desmin promoter, an intron (e.g., a chimeric intron), a nucleic acid encoding an RNAi, a polyadenylation signal (e.g., bovine growth hormone polyA), 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 comprising the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) or a sequence with 80%, 85%, 90% or 95% identity to SEQ ID NO: 1, the second strand comprising a second nucleic acid comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2) or a sequence with 80%, 85%, 90% or 95% identity to SEQ ID NO: 2. In some embodiments, the first strand and the second strand form a duplex. In some embodiments, the first strand is connected to the second strand by a linker. In some embodiments, the linker comprises the nucleic acid sequence of SEQ ID NO:3, or a sequence that is 80%, 85%, 90%, or 95% identical to SEQ ID NO:3.

[0137] In some embodiments, the nucleic acid in AAV comprises, from 5' to 3', a nucleic acid encoding the following: all or a functional portion of an ITR (e.g., AAV2 ITR), a stuffer sequence (e.g., all or a portion of a human α1-antitrypsin (AAT) stuffer sequence), a desmin promoter, the 5' arm of an intron (e.g., a rabbit β-globin intron), a nucleic acid encoding an RNAi, the 3' arm of an intron (e.g., a rabbit β-globin intron), a polyadenylation signal (e.g., bovine growth hormone polyA), a stuffer sequence (e.g., all or a portion of a human α1-antitrypsin (AAT) stuffer sequence), and an AAV ITR (e.g., AAV2 ITR), the RNAi comprising a first strand and a second strand, the first strand comprising a sequence comprising 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) or a sequence corresponding to SEQ ID NO: 2 ID NO:1 has a first nucleic acid with a sequence that is 80%, 85%, 90% or 95% identical, and the second strand comprises a second nucleic acid comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2) or a sequence that is 80%, 85%, 90% or 95% identical to SEQ ID NO:2.

[0138] In some embodiments, the nucleic acid in the AAV comprises, from 5' to 3', a nucleic acid encoding an ITR (e.g., AAV2 ITR), a desmin promoter, a nucleic acid encoding an RNAi, a polyadenylation signal (e.g., bovine growth hormone polyA), and an AAVITR (e.g., AAV2 ITR), the RNAi comprising a first strand and a second strand, the first strand comprising a first nucleic acid comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2) or a sequence with 80%, 85%, 90% or 95% identity to SEQ ID NO: 2, and the second strand comprising a second nucleic acid comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 1) or a sequence with 80%, 85%, 90% or 95% identity to SEQ ID NO: 1. In some embodiments, the first strand and the second strand form a duplex. In some embodiments, the first strand is connected to the second strand by a linker. In some embodiments, the linker comprises the nucleic acid sequence of SEQ ID NO: 6.

[0139] In some embodiments, the nucleic acid in AAV comprises, from 5' to 3', a nucleic acid encoding the following: all or a functional portion of an ITR (e.g., AAV2 ITR), a stuffer sequence (e.g., all or a portion of a human α1-antitrypsin (AAT) stuffer sequence), a desmin promoter, the 5' arm of an intron (e.g., a rabbit β-globin intron), a nucleic acid encoding an RNAi, the 3' arm of an intron (e.g., a rabbit β-globin intron), a polyadenylation signal (e.g., bovine growth hormone polyA), a stuffer sequence (e.g., all or a portion of a human α1-antitrypsin (AAT) stuffer 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 comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO:2), the second strand comprising a second nucleic acid comprising the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1).

[0140] In some embodiments, the vector may include (one or more) filler nucleic acids. In some embodiments, the filler nucleic acid may include a sequence encoding a reporter polypeptide. As will be appreciated by those skilled in the art, 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 in a single position) or multiple sequences (e.g., more than one filler nucleic acid in more than one position (e.g., 2 positions, 3 positions, etc.)). In some embodiments, the filler nucleic acid may be located downstream of the RNAi sequence. In an embodiment, the filler nucleic acid may be located upstream of the RNAi sequence (e.g., between a promoter and a nucleic acid encoding RNAi). Those skilled in the art will also appreciate that a variety of nucleic acids may be used as filler nucleic acids. In some embodiments, the filler nucleic acid includes all or part 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 includes all or part of a gene. For example, the filler sequence includes a portion of a human AAT sequence. Those skilled in the art will recognize that different portions of a gene (e.g., a human AAT sequence) may be used as a filler fragment. For example, the stuffing fragment can be from the 5' end of the gene, the 3' end of the gene, the middle of the gene, the non-coding portion of the gene (e.g., intron), the coding region of the gene (e.g., exon), or a mixture of the non-coding portion and the coding portion of the gene. Those skilled in the art will also recognize that all or part of the stuffing sequence can be used as a stuffing sequence. In some embodiments, the vector comprises a 5' stuffing sequence containing a nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having greater than about 80%, 85%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the vector comprises a 3' stuffing sequence containing a nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having greater than about 80%, 85%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the vector comprises a 5' stuffer sequence comprising a nucleotide sequence of SEQ ID NO:18, or a nucleotide sequence having greater than about 80%, 85%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:18 and a 3' stuffer sequence comprising a nucleotide sequence of SEQ ID NO:19, or a nucleotide sequence having greater than about 80%, 85%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO:19.

[0141] In other embodiments, the rAAV particles comprise AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh8R, AAVrh.10, AAV11, AAV12, AAVrh74, AAVrh74 N502I, AAVrh74 W505R capsid proteins or mutants of these capsid proteins. In some embodiments, the mutant capsid protein retains the ability to form AAV capsids. In some embodiments, the rAAV particles comprise AAV5 tyrosine mutant capsids (Zhong L. et al., (2008) Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States] 105 (22): 7827-7832). In further embodiments, the rAAV particles comprise capsid proteins of AAV serotypes from clade AF (Gao, et al., J. Virol. 2004, 78(12):6381).

[0142] 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). 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 AAV1 capsid proteins and at least one AAV2 ITR, or it may contain AAV2 capsid proteins and at least one AAV1 ITR. Any combination of AAV serotypes for producing rAAV particles is provided herein, as if each combination is explicitly stated herein. In some embodiments, the present invention provides rAAV particles comprising an AAV1 capsid and an rAAV vector disclosed herein (e.g., an expression cassette comprising a nucleic acid encoding an RNAi disclosed herein), flanked by at least one AAV2 ITR. In some embodiments, the present invention provides rAAV particles comprising an AAV2 capsid.

[0143] In some aspects, the present invention provides virus particles comprising a recombinant self-complementary genome. rAAV particles with self-complementary genomes and methods using self-complementary AAV genomes are described in U.S. Patents 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 [Gene Therapy] 10: 2105-2111, each reference is incorporated herein by reference in its entirety. rAAV comprising a self-complementary genome will rapidly form double-stranded DNA molecules by relying on its partially complementary sequences (e.g., complementary coding strands and non-coding strands of transgenics). In some embodiments, the present invention provides rAAV particles comprising an AAV genome, wherein the rAAV genome comprises a first heterologous polynucleotide sequence (e.g., RNAi disclosed herein) and a second heterologous polynucleotide sequence (e.g., the antisense strand of RNAi disclosed herein), wherein the first heterologous polynucleotide sequence can form an intrachain base pair with the second polynucleotide sequence along most or all of its length. In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are connected by a sequence that promotes intrachain base pairing (e.g., a hairpin DNA structure). Hairpin structures are known in the art, such as in miRNA or siRNA molecules. In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are connected by a mutated ITR (e.g., right ITR). In some embodiments, the ITR comprises a polynucleotide sequence 5'-CCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACC AAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA-3 (SEQ ID NO: 27). The mutated ITR comprises a deletion of the D region containing the terminal resolution sequence. Thus, when the AAV viral genome is replicated, the rep protein will not cleave the viral genome at the mutated ITR, and therefore, a recombinant viral genome comprising the following items in 5' to 3' order will be packaged in a viral capsid: AAV ITR, a first heterologous polynucleotide sequence comprising a regulatory sequence, the mutated AAV ITR, a second heterologous polynucleotide in the opposite orientation to the first heterologous polynucleotide, and a third AAV ITR.In some embodiments, the present invention provides an AAV virus particle comprising a recombinant viral genome, wherein the recombinant viral genome comprises: 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 terminal resolution sequence, a second polynucleotide sequence comprising a complementary sequence of the first polynucleotide sequence encoding the RNAi disclosed herein, and a functional AAV2 ITR.

[0144] rAAV particles can be produced using methods known in the art. See, for example, U.S. Pat. Nos. 6,566,118; 6,989,264; and 6,995,006. In practicing the present invention, host cells for producing rAAV particles include mammalian cells, insect cells, plant cells, microorganisms, and yeast. The host cell may also be a packaging cell in which the AAV rep and cap genes are stably maintained in the host cell or a production cell in which the AAV vector genome is stably maintained. Exemplary packaging cells and production cells are derived from 293 cells, A549 cells, or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.

[0145] 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: 1) in the case of a baculovirus production system, a suitable host cell, including, for example, a cell line of human origin such as HeLa cells, A549 cells or 293 cells, or a cell line of insect origin such as SF-9; 2) suitable helper virus functions provided by wild-type or mutant adenovirus (such as a temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) nucleic acids flanked by at least one AAV ITR sequence (such as a therapeutic nucleic acid); and 5) suitable culture media and culture media components that support rAAV production. In some embodiments, AAV rep and cap gene products can be from any AAV serotype. Generally speaking, but not necessarily, the AAV rep gene product has the same serotype as the ITR of the rAAV vector genome, as long as the rep gene product can play the role of replicating and packaging the rAAV genome. Suitable culture media known in the art can be used to produce rAAV vectors. These culture media include, but are not limited to, culture media produced by Hyclone Laboratories and JRH, including modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), custom preparations such as those described in U.S. Patent No. 6,566,118, and Sf-900 II SFM culture media 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 media preparations for producing recombinant AAV vectors. In some embodiments, AAV helper functions are provided by adenovirus or HSV. In some embodiments, the AAV helper functions are provided by a baculovirus and the host cell is an insect cell (eg, a Spodoptera frugiperda (Sf9) cell).

[0146] In some embodiments, rAAV particles can be produced by a triple transfection method, such as the exemplary triple transfection method provided below. In short, a plasmid containing a rep gene and a capsid gene can be transfected (e.g., using a calcium phosphate method) into a cell line (e.g., HEK-293 cells) together with an auxiliary adenovirus plasmid, and the virus can be collected and optionally purified. Therefore, in some embodiments, rAAV particles are produced by triple transfection of a nucleic acid encoding a rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding an AAV helper virus function into a host cell, wherein the nucleic acid is transfected into a host cell to produce a host cell capable of producing rAAV particles.

[0147] In some embodiments, rAAV particles can be produced by a production cell line method, such as the exemplary production cell line method provided below (see also references in Martin et al., (2013) Human Gene Therapy Methods [Human Gene Therapy Methods] 24: 253-269). Briefly, a cell line (e.g., a HeLa cell line) can be stably transfected with a plasmid containing a heterologous nucleic acid sequence of a rep gene, a capsid gene, and a promoter. The cell line can be screened to select a leading clone for rAAV production, which can then be amplified into a production bioreactor and infected with an adenovirus (e.g., a wild-type adenovirus) as a helper 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. Therefore, in some embodiments, rAAV particles are produced by a production cell line comprising one or more of the following: nucleic acid encoding a rAAV vector, nucleic acid encoding AAV rep and cap, and nucleic acid encoding AAV helper virus function.

[0148] In some aspects, a method for producing any rAAV particles as disclosed herein is provided, the method comprising (a) culturing a host cell under conditions that produce rAAV particles, wherein the host cell comprises (i) one or more AAV packaging genes, wherein each of the AAV packaging genes encodes an AAV replication and / or encapsidation protein; (ii) a rAAV provector comprising a nucleic acid encoding an RNAi of the present disclosure as described herein, the flank of the nucleic acid being at least one AAV ITR, and (iii) AAV helper functions; and (b) recovering the rAAV particles produced 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 encapsidation protein is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., a wild-type AAV6 capsid or a variant AAV6 capsid such as ShH10, as described in U.S. Pre-Grant Publication No. 2012 / 0164106), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., a wild-type AAV9 capsid or a modified AAV9 capsid as described in U.S. Pre-Grant Publication No. 2013 / 0323226), AAV10, AAVrh10, AAV11, AAV12, AAVrh74 (e.g., a wild-type AAVrh74 capsid or a variant AAVrh74 capsid, such as AAVrh74 N502I or AAVrh74 as described in WO 2019178412 In some embodiments, the mutant capsid protein retains the ability to form an AAV capsid. In some embodiments, the encapsidation protein is an AAV5 tyrosine mutant capsid protein. In other embodiments, the rAAV particles comprise capsid proteins of AAV serotypes from clade AF. In some embodiments, the rAAV particles comprise an AAVrh74 N502I capsid and a recombinant genome comprising an AAV2 ITR and a nucleic acid encoding the RNAi disclosed herein. In some embodiments, the rAAV particles comprise an AAVrh74 W505R capsid and a recombinant genome comprising an AAV2 ITR and a nucleic acid encoding the RNAi disclosed herein. In another embodiment, the rAAV particles are purified. As used herein, the term "purification" includes preparations of rAAV particles that do not contain at least some other components, which may also be present in places where the rAAV particles are naturally present or initially prepared. Thus, for example, isolated rAAV particles can be prepared by enriching them from a source mixture such as a culture lysate or a production culture supernatant using purification techniques.Enrichment can be measured in a variety of ways, such as by the proportion of DNase-resistant particles (DRP) or genome copies (gc) present in 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, media components, etc.

[0149] Also provided herein are pharmaceutical compositions comprising rAAV particles and a pharmaceutically acceptable carrier, the rAAV particles comprising a transgene encoding the RNAi disclosed herein. These pharmaceutical compositions may be suitable for any mode of administration described herein. Pharmaceutical compositions comprising rAAV particles encoding the nucleic acid of the RNAi disclosed herein may be systemically introduced. For example, recombinant viral particles comprising nucleic acid encoding the RNAi disclosed herein may be administered intravenously, intra-arterially, subcutaneously or intraperitoneally.

[0150] In some embodiments, a pharmaceutical composition comprising a recombinant viral particle comprising a transgene encoding the RNAi disclosed herein as described herein and a pharmaceutically acceptable carrier is suitable for administration to a human. Such carriers are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 15th edition, pages 1035-1038 and 1570-1580). In some embodiments, a pharmaceutical composition comprising a rAAV as described herein and a pharmaceutically acceptable carrier is suitable for systemic injection into a mammal.

[0151] Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Saline solutions and dextrose, polyethylene glycol (PEG) and glycerol aqueous solutions can also be used as liquid carriers, particularly for injections. The pharmaceutical composition may further comprise additional ingredients, such as preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting agents or clarifying agents, tackifiers, etc. The pharmaceutical compositions described herein can be packaged in single unit doses or in multiple doses. These compositions are typically formulated as sterile and substantially isotonic solutions. Products and kits

[0152] Also provided are kits or articles used in the methods described herein. In various aspects, the kit comprises a composition described herein (e.g., rAAV particles of the present disclosure comprising nucleic acids encoding RNAi disclosed herein) in a suitable package. Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These articles can be further sterilized and / or sealed.

[0153] The present invention also provides a kit comprising the composition described herein, and may further include instructions for methods of using the composition, such as the uses described herein. The kit described herein may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for performing any of the methods described herein. For example, in some embodiments, the kit comprises: a composition comprising a transgene encoding the RNAi disclosed herein to deliver an effective amount of rAAV particles to a mammal, a pharmaceutically acceptable carrier suitable for injection into a mammal, and one or more of the following: a buffer, a diluent, a filter, a needle, a syringe, and a package insert containing instructions for injection into a mammal. In some embodiments, the kit includes instructions for treating DM-1 with 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

[0154] The present invention includes the exemplary embodiments listed below.

[0155] 1. An RNAi comprising a first strand and a second strand, wherein a) the first strand and the second strand form a duplex; b) the first strand comprises a guide region, wherein the guide region comprises a nucleic acid having a sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) or a sequence having about 90% identity to a sequence of SEQ ID NO: 1; and c) the second strand comprises a non-guide region.

[0156] 2. The RNAi of embodiment 1, wherein the non-guide region comprises a nucleic acid having the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2) or a sequence having about 90% identity to the sequence of SEQ ID NO: 2.

[0157] 3. The RNAi of embodiment 1 or 2, wherein the first strand comprises a nucleic acid having a sequence of SEQ ID NO: 1, and the non-guide region comprises a nucleic acid having a sequence of SEQ ID NO: 2.

[0158] 4. The RNAi of any one of embodiments 1-3, wherein the first strand and the second strand are connected by means of an RNA linker capable of forming a loop structure.

[0159] 5. The RNAi of embodiment 4, wherein the RNA linker comprises about 4 to about 50 nucleotides.

[0160] 6. The RNAi of embodiment 4 or 5, wherein the loop structure comprises about 4 to about 20 nucleotides.

[0161] 7. The RNAi of any one of embodiments 4-6, wherein the loop structure comprises a nucleic acid sequence having a sequence of SEQ ID NO: 3 or a sequence having about 90% identity with a sequence of SEQ ID NO: 3.

[0162] 8. The RNAi of any one of embodiments 4-7, wherein the RNAi comprises from 5' to 3' the second strand, the RNA linker and the first strand.

[0163] 9. The RNAi of any one of embodiments 4-7, wherein the RNAi comprises from 5' to 3' the first strand, the RNA linker, and the second strand.

[0164] 10. The RNAi of any one of embodiments 1-8, wherein the RNAi comprises a nucleic acid having a sequence of SEQ ID NO:7 or a sequence having about 90% identity to a sequence of SEQ ID NO:7.

[0165] 11. The RNAi of any one of embodiments 1-10, wherein the RNAi is a small inhibitory RNA (siRNA), a micro RNA (miRNA), or a small hairpin RNA (shRNA).

[0166] 12. The RNAi of any one of embodiments 1-11, wherein the RNAi further comprises a scaffold.

[0167] 13. The RNAi of embodiment 12, wherein the scaffold comprises all or part of the nucleic acid of SEQ ID No: 11.

[0168] 14. The RNAi of embodiment 13, wherein the miRNA is embedded in the scaffold.

[0169] 15. The RNAi of embodiment 14, wherein the scaffold has a 5' arm and a 3' arm, wherein the 5' arm is located 5' of the nucleic acid encoding the RNAi, and wherein the 3' arm is located 3' of the nucleic acid encoding the RNAi.

[0170] 16. The RNAi of any one of embodiments 12-15, wherein the scaffold is a miR-155 scaffold.

[0171] 17. The RNAi of any one of embodiments 12-16, wherein the miR-155 scaffold comprises a nucleic acid having a sequence of SEQ ID NO: 9 or a sequence having about 90% identity to a sequence of SEQ ID NO: 9 located 5′ of the RNAi.

[0172] 18. The RNAi of any one of embodiments 12-17, wherein the miR-155 scaffold comprises a nucleic acid having a sequence of SEQ ID NO: 10 or a sequence having about 90% identity to a sequence of SEQ ID NO: 10 located 3' of the RNAi.

[0173] 19. The RNAi of any one of embodiments 1-18, wherein the RNAi targets RNA encoding a polypeptide associated with myotonic dystrophy type 1 (DM1).

[0174] 20. The RNAi of embodiment 19, wherein the polypeptide is dystrophic myotonia protein kinase (DMPK).

[0175] 21. The RNAi of embodiment 20, wherein the DMPK comprises a mutation associated with DM 1.

[0176] 22. The RNAi of embodiment 20 or 21, wherein the gene encoding DMPK comprises 5 or more CTG trinucleotide repeat sequences.

[0177] 23. An expression cassette comprising a nucleic acid encoding the RNAi of any one of embodiments 1-22.

[0178] 24. The expression cassette of embodiment 23, wherein the nucleic acid encoding the RNAi is operably linked to a promoter.

[0179] 25. The expression cassette of embodiment 24, wherein the promoter is a muscle-specific promoter.

[0180] 26. The expression cassette of embodiment 24 or 25, wherein the promoter is a desmin promoter or a variant thereof.

[0181] 27. The expression cassette of embodiment 26, wherein the desmin promoter comprises one or more enhancer elements, and a promoter of a human desmin gene.

[0182] 28. The expression cassette of embodiment 26 or 27, wherein the desmin promoter comprises two enhancer elements, and the promoter of the human desmin gene.

[0183] 29. The expression cassette of any one of embodiments 26-28, wherein the desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.

[0184] 30. An expression cassette as described in any of embodiments 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.

[0185] 31. The expression cassette of any one of embodiments 26-30, wherein the desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence having about 90% identity to the nucleotide sequence of SEQ ID NO: 12.

[0186] 32. The expression cassette of any one of embodiments 23-31, wherein the expression cassette further comprises an intron.

[0187] 33. The expression cassette of embodiment 32, wherein the intron is a rabbit β-globin intron.

[0188] 34. The expression cassette of embodiment 32 or 33, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 13 or a sequence having about 90% identity to the sequence of SEQ ID NO: 13.

[0189] 35. The expression cassette of any one of embodiments 32-34, wherein the nucleic acid encoding the RNAi is embedded in the intron.

[0190] 36. The expression cassette of embodiment 35, wherein the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located 5' of the nucleic acid encoding the RNAi and the 3' arm is located 3' of the nucleic acid encoding the RNAi.

[0191] 37. The expression cassette of embodiment 36, wherein the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 14 or a sequence having about 90% identity to the sequence of SEQ ID NO: 14.

[0192] 38. The expression cassette of embodiment 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 to the sequence of SEQ ID NO: 15.

[0193] 39. The expression cassette of any one of embodiments 23-38, wherein the expression cassette further comprises a polyadenylation signal.

[0194] 40. The expression cassette of embodiment 39, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal, a SV40 polyadenylation signal, or a HSV TK pA.

[0195] 41. The expression cassette of embodiment 40, wherein the polyadenylation signal is a minimal bovine growth hormone polyadenylation signal.

[0196] 42. The expression cassette of any one of embodiments 39-41, wherein the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO: 16 or a sequence with about 90% identity to the sequence of SEQ ID NO: 16.

[0197] 43. The expression cassette of any one of embodiments 23-42, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO: 17 or a sequence having about 90% identity to the sequence of SEQ ID NO: 17.

[0198] 44. An expression cassette, wherein the expression cassette comprises a modified desmin promoter, wherein the modified desmin promoter comprises one or more enhancer elements, and a promoter of a human desmin gene.

[0199] 45. The expression cassette of embodiment 44, wherein the modified desmin promoter comprises two enhancer elements, and the promoter of the human desmin gene.

[0200] 46. ​​The expression cassette of embodiment 44 or 45, wherein the modified desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.

[0201] 47. An expression cassette as described in any of embodiments 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.

[0202] 48. The expression cassette of any one of embodiments 44-47, wherein the desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence having about 90% identity to the nucleotide sequence of SEQ ID NO: 12.

[0203] 49. The expression cassette of any one of embodiments 44-48, wherein the expression cassette further comprises an intron.

[0204] 50. The expression cassette of embodiment 49, wherein the intron is a rabbit β-globin intron.

[0205] 51. The expression cassette of embodiment 49 or 50, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 13 or a sequence having about 90% identity to the sequence of SEQ ID NO: 13.

[0206] 52. The expression cassette of any one of embodiments 44-51, wherein the nucleic acid encoding the transgene is embedded in the intron.

[0207] 53. The expression cassette of embodiment 52, wherein the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located 5' to the nucleic acid encoding the transgene and the 3' arm is located 3' to the nucleic acid encoding the transgene.

[0208] 54. The expression cassette of embodiment 53, wherein the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 14 or a sequence having about 90% identity to the sequence of SEQ ID NO: 14.

[0209] 55. The expression cassette of embodiment 53 or 54, wherein the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 15 or a sequence having about 90% identity to the sequence of SEQ ID NO: 15.

[0210] 56. The expression cassette of any one of embodiments 44-55, wherein the expression cassette further comprises a polyadenylation signal.

[0211] 57. The expression cassette of embodiment 56, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or HSV TK pA.

[0212] 58. The expression cassette of embodiment 57, wherein the polyadenylation signal is a minimal bovine growth hormone polyadenylation signal.

[0213] 59. The expression cassette of any one of embodiments 56-58, wherein the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO: 16 or a sequence with about 90% identity to the sequence of SEQ ID NO: 16.

[0214] 60. The expression cassette of any one of embodiments 44-59, wherein the transgene encodes a polypeptide or a nucleic acid.

[0215] 61. The expression cassette of any one of embodiments 44-60, wherein the transgene encodes RNAi.

[0216] 62. A vector comprising the expression cassette of any one of embodiments 23-61.

[0217] 63. The vector of embodiment 62, wherein the expression cassette is flanked by one or more filler nucleic acid sequences.

[0218] 64. The vector of embodiment 63, wherein the one or more stuffer nucleic acid sequences are derived from human SerpinA1 gene.

[0219] 65. The vector of embodiment 63 or 64, wherein the stuffer nucleic acid sequence located 5' of the expression cassette is derived from the human SerpinA1 gene.

[0220] 66. The vector of any one of embodiments 63-65, wherein the stuffer sequence located 5' of the expression cassette comprises the nucleotide sequence of SEQ ID NO: 18 or a sequence having about 90% identity to the sequence of SEQ ID NO: 18.

[0221] 67. The vector of any one of embodiments 63-66, wherein the stuffer nucleic acid sequence located 3' to the expression cassette is derived from the human SerpinA1 gene.

[0222] 68. The vector of any one of embodiments 63-67, wherein the stuffer sequence located 3' to the expression cassette comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence having about 90% identity to the sequence of SEQ ID NO: 19.

[0223] 69. The vector of any one of embodiments 62-68, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

[0224] 70. The rAAV vector of Example 69, wherein the expression cassette is flanked by one or more AAV inverted terminal repeat (ITR) sequences.

[0225] 71. The rAAV vector of Example 70, wherein the expression cassette is flanked by two AAV ITRs.

[0226] 72. The rAAV vector of embodiment 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.

[0227] 73. The rAAV vector of any of embodiments 70-72, wherein the AAV ITRs are AAV2 ITRs.

[0228] 74. The rAAV vector of any one of embodiments 69-73, wherein the rAAV vector comprises the nucleotide sequence of SEQ ID NO: 20 or a sequence having about 90% identity to the sequence of SEQ ID NO: 20.

[0229] 75. The rAAV vector of any one of embodiments 69-74, wherein the vector is a self-complementary rAAV vector.

[0230] 76. A cell comprising the expression cassette of any one of embodiments 23-61, the vector of any one of embodiments 62-68, or the rAAV vector of any one of embodiments 69-75.

[0231] 77. A viral particle comprising the vector of any one of embodiments 62-68.

[0232] 78. A recombinant AAV particle comprising the rAAV vector of any one of Examples 69-75.

[0233] 79. The rAAV particle of embodiment 78, wherein the AAV virus particle comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, AAVrh74N502I, AAVrh74 W505R, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2N587A, AAV2 E548A, AAV2 N708A, AAV V708K, AAV2-HBKO, AAVDJ8, AAVPHP.B, AAVPHP.eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1 / AAV2 chimera, bovine AAV, or mouse AAV capsid rAAV2 / HBoV1 serotype capsid.

[0234] 80. The rAAV particle of embodiment 78 or 79, wherein the ITRs and the capsid of the rAAV viral particle are derived from the same AAV serotype.

[0235] 81. The rAAV particle of embodiment 78 or 79, wherein the ITR and the capsid of the rAAV viral particle are derived from different AAV serotypes.

[0236] 82. The rAAV particle of embodiment 78, 79 or 81, wherein the AAV viral particle comprises an AAVrh74 N502I serotype capsid.

[0237] 83. The rAAV particle of embodiment 82, wherein the ITR is an AAV2 ITR and the capsid of the rAAV particle is an AAVrh74 N502I serotype capsid.

[0238] 84. The rAAV particle of embodiment 78, 79 or 81, wherein the AAV virus particle comprises an AAVrh74 W505R serotype capsid.

[0239] 85. The rAAV particle of embodiment 84, wherein the ITR is an AAV2 ITR and the capsid of the rAAV particle is an AAVrh74 W505R serotype capsid.

[0240] 86. 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, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimal bovine growth hormone polyadenylation sequence, nucleic acid encoding stuffer nucleic acid sequence from the human serpinA1 gene, and AAV2 ITRs; and wherein the capsid is an AAVrh74 N502I capsid. 87. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:18, a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:21, a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:22, a desmin promoter comprising the polynucleotide sequence of SEQ ID NO:23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:40, a DMPK comprising the polynucleotide sequence of SEQ ID NO:4 204 miRNA guide sequence, miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, DMPK comprising the polynucleotide sequence of SEQ ID NO: 5 204 miRNA passenger sequence, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is an AAVrh74 N502I capsid.

[0241] 88. The rAAV particle of embodiment 86 or 87, wherein the AAVrh74N502I capsid comprises a capsid protein comprising an amino acid sequence of SEQ IDNO:50.

[0242] 89. 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, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimal bovine growth hormone polyadenylation sequence, nucleic acid encoding stuffer nucleic acid sequence from the human serpinA1 gene, and AAV2 ITRs; and wherein the capsid is an AAVrh74 W505R capsid.

[0243] 90. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:18, a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:21, a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:22, a desmin promoter comprising the polynucleotide sequence of SEQ ID NO:23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:40, a DMPK comprising the polynucleotide sequence of SEQ ID NO:4 204 miRNA guide sequence, miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, DMPK comprising the polynucleotide sequence of SEQ ID NO: 5 204miRNA passenger sequence, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is an AAVrh74 W505R capsid.

[0244] 91. The rAAV particle of embodiment 89 or 90, wherein the AAVrh74W505R capsid comprises a capsid protein comprising the amino acid sequence of SEQ IDNO:52.

[0245] 92. A composition comprising the viral particles of Example 77 or the rAAV particles of any one of Examples 78-91.

[0246] 93. A pharmaceutical composition comprising the viral particles of Example 77 or the rAAV particles of any one of Examples 78-91.

[0247] 94. The composition of embodiment 92 or 93, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0248] 95. A modified desmin promoter, wherein the modified desmin promoter comprises one or more enhancer elements and a promoter of a human desmin gene.

[0249] 96. The modified desmin promoter of embodiment 95, wherein the modified desmin promoter comprises two enhancer elements and a promoter of a human desmin gene.

[0250] 97. The modified desmin promoter of embodiment 95 or 96, wherein the modified desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.

[0251] 98. A modified desmin promoter as described in any one of embodiments 95-97, wherein the modified desmin promoter comprises: one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21 or a 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 sequence having about 90% identity with the sequence of SEQ ID NO:22.

[0252] 99. The modified desmin promoter of any one of embodiments 95-98, wherein the modified desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence having about 90% identity to the sequence of SEQ ID NO: 12.

[0253] 100. A kit comprising the RNAi of any one of embodiments 1-22.

[0254] 101. A kit comprising the viral particles of Example 77 or the AAV particles of any one of Examples 78-91.

[0255] 102. A kit comprising the composition of any one of embodiments 92-94.

[0256] 103. The kit of any one of embodiments 100-102, further comprising instructions for use.

[0257] 104. A method for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof, the method comprising administering to the mammal an effective amount of the RNAi of any one of embodiments 1-22.

[0258] 105. A method for inhibiting expression of dystrophic myotonia protein kinase (DMPK) in a mammal with DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the RNAi of any one of embodiments 1-22.

[0259] 106. A method for inhibiting accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the RNAi of any one of embodiments 1-22.

[0260] 107. A method for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof, the method comprising administering to the mammal an effective amount of the viral particle of Example 77 or the rAAV particle of any one of Examples 78-91.

[0261] 108. A method for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the viral particles of Example 77 or an effective amount of the rAAV particles of any one of Examples 78-91.

[0262] 109. A method for inhibiting accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the viral particle of Example 77 or an effective amount of the rAAV particle of any one of Examples 78-91.

[0263] 110. The method of any one of embodiments 102-104, wherein the effective amount of the viral particle or rAAV particle is about 1×10 8 About 2×10 13 A dose of genomic copies / mL.

[0264] 111. The method of embodiment 110, wherein the dose is about 5×10 12 genome copies / mL.

[0265] 112. The method of embodiment 110, wherein the dose is about 1×10 13 genome copies / mL.

[0266] 113. The method of embodiment 110, wherein the dose is about 2×10 13 genome copies / mL.

[0267] 114. The method of any one of embodiments 107-109, wherein the effective amount of the viral particle or rAAV particle is about 1×10 8 About 2×10 14 The dose is 1 genome copy / kg body weight.

[0268] 115. The method of embodiment 114, wherein the dose is about 5×10 13 genome copies / kg body weight.

[0269] 116. The method of embodiment 114, wherein the dose is about 1×10 14 genome copies / kg body weight.

[0270] 117. The method of embodiment 114, wherein the dose is about 2×10 14 genome copies / kg body weight.

[0271] 118. A method for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof, the method comprising administering to the mammal an effective amount of the composition of any one of embodiments 92-94.

[0272] 119. A method for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal suffering from DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the composition of any one of embodiments 92-94.

[0273] 120. A method for inhibiting accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the composition of any one of embodiments 92-94.

[0274] 121. The method of any one of embodiments 104-106, wherein the RNAi is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with, and / or after administration of the RNAi.

[0275] 122. The method of any one of embodiments 107-109, 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 administration of the viral particle or the rAAV particle.

[0276] 123. The method of any one of embodiments 118-120, wherein the composition is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with, and / or after administration of the composition. Examples

[0277] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the invention and not by way of limitation. Example 1: amiR-DMPK 204 Generation of expression cassettes

[0278] We generated a microRNA (amiR-DMPK) designed to target the DMPK gene. 204 ) of single-stranded AAV viral vector ( Figure 1A ). The construct was designed such that amiR-DMPK 204 The microRNAs were embedded in the optimized microRNA backbone miR155 (BLOCK-iT; Thermo Fisher catalog numbers K4935-00, K4936-00, K4937-00, K4938-00) and are referred to hereinafter as “miR155 amiR-DMPK 204 " or "amiR155-DMPK204 miR155 amiR-DMPK 204 is flanked by rabbit β-globin intronic sequences and placed under the control of a hybrid muscle promoter (nDes).

[0279] 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 by conventional oligonucleotide synthesis (Genscript, USA).

[0280] Placing the bovine growth hormone polyadenylation sequence into the amiR-DMPK flanking introns 204 3' of the microRNA (minBGHpA). A filler sequence ("stuffer sequence") is included. 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 AAV capsids. The sequences were engineered into an ITR plasmid, which was used to generate vectors for in vivo efficacy studies. 5' and 3' ITR sequences

[0281] The ITR sequence is the 145 bp AAV2 wild-type sequence. The 3'ITR (downstream of the expression cassette) is in the Flip orientation (GenBank: LQ493091.1). The 5'ITR (upstream of the expression cassette) is in the Flop 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 promoter

[0282] The nDes promoter was constructed using desmin promoter elements described in the literature (Li and Paulin et al., 1991, J Biol Chem. 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). 5' and 3' arms of the rabbit β-globin intron

[0283] This intron is used to flank amiR-DMPK 204 cassette, as intronic expression of miRNAs is known to enhance target knockdown. amiR-DMPK with miR155 scaffold 204 (miR155-amiRDMPK 204 )

[0284] Endogenous miRNAs are hairpin-like secondary structures found in many primary RNA transcripts (pri-miRNAs). In the nucleus, the microprocessor Drosha / DGCR8 complex binds and cuts the base 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 cut by Dicer / TRBP to form a mature RNA duplex. The guide strand, also known as the targeting strand, is separated from the passenger strand and loaded onto the argonaute protein in the RNA-induced silencing complex (RISC), which then targets the complementary mRNA transcript for degradation or translation repression.

[0285] amiR-DMPK 204 Sequences identified as targets for DM1 treatment. amiR-DMPK 204 The target sequence of amiR-DMPK is located upstream of the "CUG" repeat sequence within the 3'UTR of the DMPK nucleotide sequence. 204 Both wild-type and mutant DMPK transcripts can be repressed.

[0286] In addition, amiR-DMPK 204 The target region is conserved in non-human primates (NHP-cynomolgus monkeys) and humans, allowing preclinical evaluation of DMPK knockdown in NHPs ( Fig.11 Evaluation of amiR-DMPK 204 microRNAs, and showed that the miR155 scaffold has efficient guide strand processing and minimal passenger strand processing, reducing the possibility of off-target effects (see Example 2 below).

[0287] The final construct selected for development was amiR-DMPK with the miR155 scaffold 204 (amiR155-DMPK 204 ), wherein when processed, 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 internal loop resulted in a higher knockdown rate than the 5-nucleotide / 3-nucleotide internal loop found in the native miR-155 molecule (Source: BLOCK-iT TMPol II miR RNAi expression vector, Invitrogen). The vector nDes-miR155-amiR-DMPK was confirmed 204 , in the context of AAV capsids, has potent in vivo activity in the DMSXL mouse model of DM1 (see Example 3 below). minBGH polyA

[0288] Insertion of a 186 bp minimal BGH polyA site into amiR-DMPK 204 sequences downstream to allow transcription termination and polyadenylation of the mRNA. Filler sequence from the A1AT intron

[0289] A stuffer sequence from intron 4 of the alpha-1 antitrypsin gene was used to bring the gene cassette within the packaging limit of the rAAV vector. nDes-miR155-amiR-DMPK 204 ITR plasmid containing cassette (nDes-miR155-amiR-DMPK 204 )

[0290] Design the 1938 bp ITR-ITR sequence of the expression construct nDes-miR155-amiR-DMPK204-minBGHpolyA cassette. 204 -minBGHpolyA cassette was cloned into the ITR plasmid. The synthesized nDes-miR155-amiR-DMPK204-minBGHpolyA included a 5'Nco1 site and a 3'Sph1 site for cloning into the ITR plasmid ( Figure 1B ). Briefly, the synthetic nDes-miR155-amiR-DMPK2 04 -minBGHpolyA sequence, and gel purified 1.9 kB fragment. The ITR plasmid was digested with NcoI and SphI, dephosphorylated using calf intestinal alkaline phosphatase (New England Biolabs; catalog number M0290) and gel purified 8.2 kB vector backbone fragment. The digested 1.9 kB fragment containing the expression cassette and the digested ITR plasmid were ligated to generate the plasmid ITR-nDes-miR155-amiR-DMPK 204 . Small-scale rAAV vector production

[0291] Small-scale packaging assay was performed in HEK 293 cells to confirm the expression of ITR-nDes-miR155-amiR-DMPK 204 Plasmid packaging. Use AAV rep / cap plasmids for small-scale production. Figure 1C Shown is the amount of vector produced per HEK 293 cell compared to a standard EGFP plasmid gene cassette.

[0292] To determine whether amiR-DMPK 204 The potential to correct the DM1 phenotype was tested in skeletal muscle myoblast cultures from DM1 patients to silence human DMPK transcripts and correct splicing defects. Due to the CTG expansion in the 3'UTR of the DMPK gene, the DMPK mRNA hairpin structure aggregates into insoluble ribonuclear foci and sequesters several RNA binding proteins. The resulting redistribution of essential splicing factors such as myoblind-like protein 1 (MBNL1) leads to mis-splicing of downstream effectors responsible for muscle tissue differentiation. 204 Treatment of DM1 patient cells resulted in more than 50% silencing of DMPK mRNA and splicing correction as measured by MBNL1 exon 7 inclusion. Example 2: miR155 amiR-DMPK 204 Processing

[0293] amiR-DMPK 204 The cells were packaged into AAV capsids and analyzed for DMPK knockdown efficacy, passenger chain activity, and processing patterns in vivo. 204 The construct was packaged into AAV. The vector was injected intravenously into the DMSXL adult humanized DM1 mouse model expressing human DMPK with >1,000 CTG repeats. After eight weeks, the animals were euthanized, multiple tissues were collected to measure DMPK knockdown efficacy, and cardiac tissue was selected to measure passenger chain activity and processing patterns.

[0294] RT-PCR analysis showed that amiR-DMPK 204 Robust expression in multiple muscle tissues, with higher expression in the heart ( Figure 2B ). Accompanying amiR-DMPK 204 Expression, RT PCR analysis confirmed robust DMPK inhibition in the heart, with an average of >70% and approximately 30% DMPK inhibition in different skeletal muscles ( Figure 2C ). In cardiac tissue, DMPK expression was less than 50% relative to TBP (TATA binding protein) expression. It is noteworthy that low levels of DMPK expression relative to TBP (about 50%, Figure 2C DSMXL mice (indicated by the dashed line in the figure) had no obvious DM1 phenotype. Interestingly, even though higher transduction was observed in the liver ( Figure 2A ), but the nDesmin promoter showed strong activity in cardiac tissue and similar levels in skeletal muscle ( Figure 2B ). This suggests that expression is primarily restricted to cardiac and skeletal muscles affected by DM1 pathology.

[0295] To evaluate amiR-DMPK 204 Processing and analysis of mature amiR-DMPK in cardiac tissue by NGS for small transcriptome analysis 204 Length and sequence composition of guide and passenger chains. amiR-DMPK 204 Processing of amiR-DMPK does not generate the passenger chain. 204 Processing is exclusive to the guide strand (>99%), but often produces a longer strand than predicted from the miRBase database (Table 1). Processing of miR155 most often produces a mature length of 22 to 26 nucleotides long, but is precisely processed at the 5' end (Table 1). Mapping to miR155 amiR-DMPK 204 The sequence distribution of different guide strand lengths (nt) was calculated as percentage (% reads). 204 The guide strand is underlined and the seed sequence is in bold. Asterisks indicate the corresponding amiR-DMPK 204 Reads of predicted length of the guide strand. Table 1. AmiR-DMPKs mapped to miR155 204 The sequence distribution of different guide strand lengths (nt) was calculated as percentage (% reads). 204 The guide strand is underlined. Asterisks indicate the corresponding amiR-DMPK 204 Reads of predicted length of the guide strand. miR-155 read sequences length %sequence SEQ ID NO <![CDATA[ AGTCGAAGACAGTTCTAGGGT GT]]> 23 52 33 <![CDATA[ AGTCGAAGACAGTTCTAGGGT GTT]]> 24 24 34 <![CDATA[ AGTCGAAGACAGTTCTAGGGT GTTT]]> 25 11 35 <![CDATA[ AGTCGAAGACAGTTCTAGGGT *]]> 22 6 36 <![CDATA[ AGTCGAAGACAGTTCTAGGGT GTTTT]]> 26 3 37 <![CDATA[ AGTCGAAGACAGTTCTAGGGT G]]> 22 4 38

[0296] In conclusion, using amiR-DMPK with miR155 204 No passenger strand was detected (% guide strand > 99%). Therefore, miR155 was selected as a lead for preclinical studies because the miR155 miRNA scaffold is well validated for RNAi. Example 3: Systemic injection of transgenic mice encoding miR155-amiRDMPK 204 Dose-dependent inhibition of human DMPK by AAV

[0297] To determine the most effective dose, three separate doses of the myotropic AAV (WO / 2019 / 207132) capsid were studied. 204 The expression cassette (amiR155-DMPK 204) AAV. 8-week-old DMSXL mice were intravenously injected with 5.0×10 11 vector genomes (vg) / kg, 5×10 12 vg / kg and 1.0×10 13 vg / kg (corresponding to low, medium and high doses). Clinical symptoms of mice, such as body weight, survival rate, myotonia and cardiac function, were analyzed 8 weeks after AAV infusion. Mice were euthanized 8 weeks after gene transfer and DMPK inhibition and splicing correction were measured. miR155-amiRDMPK was measured by small RNA TaqMan 204 Expression levels were calculated and mRNA input levels were normalized to u6 small nuclear RNA.

[0298] The effects of amiR155-DMPK on 204 The expression ( Figure 3A ) and caused a dose-dependent decrease in total DMPK expression in multiple tissues ( Figure 3B In total, approximately 10 amiR155-DMPK 204 Copies / U6 are sufficient to reduce DMPK in the heart and diaphragm by ≥50%, an amount sufficient to treat DM1 patients.

[0299] Next, we investigated the consequences of DMPK inhibition on characteristic DM1 phenotypes such as splicing abnormalities. Aberrant splicing of LIM domain binding protein 3 (Ldb3) containing exon 11 in Ldb3 transcripts has been shown to be a DM1-specific phenotype caused by a CUG repeat RNA sequestering RNA splicing mechanism (Yamashita et al. 2014. Neurobiol Dis. [Disease Neurobiology] 69: 200-5). 204 A significant reduction in Ldb3 transcripts including exon 11 was observed in gastrocnemius muscle of mice treated with amiR155-DMPK 204 The splicing defect was effectively corrected in treated muscles ( Figure 4 ).

[0300] In addition to these molecular corrections, AAV nDes-miR155-amiR-DMPK was then measured in terms of physiological and functional manifestations of the disease 204 efficacy.

[0301] To determine whether treatment could improve survival and reduce weight loss, three different doses of AAV nDes-miR155-amiR-DMPK were used. 204DMSXL mice were treated and survival and body weight were monitored. Improvements in body weight and survival were observed after 8 weeks of treatment with the medium and high doses. On the other hand, no improvements were observed with the low dose or balanced salt solution (BSS) control ( Figure 5A and Figure 5B ).

[0302] Next, AAV nDes-miR155-amiR-DMPK was measured in terms of functional manifestations of the disease, such as prevention of myotonia and cardiac abnormalities. 204 Efficacy of AAV nDes-miR155-amiR-DMPK 204 The myotonia of the treated mice was significantly reduced (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. AAV nDes-miR155-amiR-DMPK 204 Number of DMSXL DM1 mice with myotonia after treatment. 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; and 3, myotonic discharges in almost every insertion.

[0303] Cardiac function of DMSXL mice was also monitored using surface echocardiography along with skeletal muscle function 8 weeks after treatment. 204 Improved cardiac output. After 8 weeks of treatment, a significant improvement in cardiac output was noted in the medium dose group (5e12 vg / kg) ( Figure 6 ). Example 4: AAVrh74N502I capsid improves muscle transduction and reduces liver transduction

[0304] Experiments were performed to test the transduction efficiency of AAV capsids containing AAVrh74N502I VP1 capsid protein (WO 2019178412; SEQ ID NO: 50) in various tissues of non-human primates. Fig. 7A The outline of the experiment is shown in FIG. 13Nonhuman primates were treated intravenously with AAV9, AAVrh74 or AAVrh74N502I capsids containing eGFP expression cassettes at 50 vg / kg. Twenty-one days after treatment, animals were sacrificed and eGFP expression levels were measured in tibialis anterior (TA), biceps femoris, quadriceps, heart and liver.

[0305] Capsids containing the AAVrh74N502I capsid protein have improved muscle transduction in nonhuman primates compared with comparator capsids ( Figure 7B-Figure 7E ) and decreased liver transduction ( Figure 7F ). (Table 3). Table 3. eGFP levels in tissues of non-human primates treated with AAVrh74N502I capsid are increased relative 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 5: AAVrh74N502InDes-miR155-amiR-DMPK in the DMSXL mouse model 204 Evaluation of target binding

[0306] To determine the most effective dose, delivery of two separate doses of AAVrh74N502I capsid was studied. 204 The expression cassette (miR155-amiR-DMPK 204 ) AAV. 8-week-old DMSXL mice were intravenously injected with 9×10 13 vector genomes (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 by small RNA TaqMan 204 Expression levels were calculated and mRNA input levels were normalized to u6 small nuclear RNA.

[0307] The amiR-DMPK 204 The expression ( Fig. 8A ) and caused a dose-dependent decrease in total DMPK expression in multiple tissues ( Figure 8B In total, ~10 amiR-DMPK 204 Copies / U6 are sufficient to reduce DMPK in the heart and diaphragm by ≥50%, an amount sufficient to treat DM1 patients.

[0308] Finally, it was confirmed that the leading vector nDes-miR155-amiR-DMPK204 , in the context of the myotropic capsid AAVrh74N502I (SEQ ID NO: 50), had potent in vitro activity in cardiomyocyte-derived DM1 iPSCs ( Fig. 9 ). Example 6: AAVrh74N502I nDes-miR155-amiR-DMPK on the transcriptome 204 Reviews

[0309] To determine whether amiR-DMPK 204 To determine whether the treatment had any significant effect on the transcriptome, whole-genome RNA sequencing (RNA-seq) was performed by CBA miR155-amiR-DMPK 204 Plasmids were transfected into HEK293 cell line to compare amiR-DMPK 204 To evaluate whether the observed changes in expression of non-DMPK genes were due to amiR-DMPK 204 The enrichment of seed complementarity in significantly downregulated targets was evaluated due to off-target effects. Using a 5% false discovery rate (FDR) significance threshold, there were four differentially expressed genes, 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 complementarity sequences (Table 4). For 1% FDR, only OPN4 (12.5-fold) and DMPK (1.7-fold) showed differential expression ( Fig.10 As expected, DMPK was one of the most significantly affected mRNA levels (44% silencing). 204 Minimal off-target effects were observed after overexpression in HEK293 cells. Table 4 Example 7: Exploration of AAVrh74N502I nDes-miR155-amiR-DMPK 204 Dose-range finding study of biodistribution and activity in nonhuman primates To determine the AAVrh74MN502I nDes-miR155-amiR-DMPK 204 To investigate the biodistribution and activity of ANTIBASE, a single intravenous infusion (IV) dose was administered to cynomolgus monkeys. The duration of the study was 12 weeks after the single injection. A total of 16 cynomolgus monkeys (8 males, 8 females; 24 to 48 months old) were dosed once via saphenous vein IV on day 1 of the study. The doses are shown in Table 5 below. The animals were grouped (2 males and 2 females per group) into four different categories based on the following dose levels (vg / kg): formulation buffer (Group 1); 5×10 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 sacrificed and tissues were harvested for analysis. Table 5 Harvested tissues were mechanically homogenized for RNA and DNA extraction, and samples were analyzed by digital PCR (dPCR). Dose-dependent biodistribution and activity of AAVrh74N502I nDes-miR155-amiR-DMPK204 were demonstrated in several muscle and non-muscle tissues. Several skeletal muscles (tibialis anterior (TA); gastrocnemius; quadriceps; biceps; soleus; extensor digitorum longus (EDL); diaphragm) as well as myocardial and liver tissues were analyzed. Viral genome copies were found in all tissues tested, and the number of copies / cell in each tissue was dose-dependent ( Fig.12 amiR-DMPK expression ( Fig.13 ) and DMPK downregulation ( Fig.14 ) was also in a dose-dependent manner in various muscle tissues, heart tissues, and liver tissues. A dose-dependent reduction in DMPK expression was found to be up to 90% compared to the control group. All doses tested in animals were found to be safe and well tolerated by the animals. sequence Unless otherwise indicated, all polypeptide sequences are presented from N-terminus to C-terminus. Unless otherwise indicated, all nucleic acid sequences are presented from 5' to 3'. Byrne desmin enhancer sequence CACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTC CCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTG CTGCCAGGGAGATGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGT GTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCAATTGGAAACTG AAAGCTTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCCCACCCTCGA(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 (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 AGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCAATTGGAAACTGAA (SEQ ID NO: 22) Source: Li and Paulin et al. 1991. "Highlevel 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 (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) One base pair difference from the published sequence (C instead of A, shown in bold and underlined). 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 (NCBI Reference Sequence: NG_046330.1) The Paulin promoter sequence corresponds to 18535-18844 in the reference sequence Complete nDes promoter sequence CACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTC 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 intron 5' arm of rabbit β-globin intron Source: Oryctolagus cuninculus Hemoglobin, beta (HBB2) Gene ID 100009084 The sequence of the present invention has an additional CATG (shown in bold and underlined) which is not present in Gene ID 100009084. 3' arm of rabbit β-globin intron Source: Oryctolagus haemoglobin, beta (HBB2) Gene ID 100009084 The sequence of the present invention has two T residues (shown in bold and underlined) instead of the two C residues in Gene ID 100009084. miR155-DMPK 204 sequence: 5' miR155 flanking sequence RNA-seq CUGGAGGCUUGCUGAAGGCUGUAUGCU(SEQ ID NO:9) DNA sequence CTGGAGGCTTGCTGAAGGCTGTATGCT(SEQ ID NO:40) Source: BLOCK-iT TMPol II miR RNAi Expression Vector Kit Catalog # K493500 The engineered pre-miRNA sequence structure is based on the murine 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 loop-DNA GTTTTGGCCACTGACTGAC(SEQ ID NO:6) Source: BLOCK-iT TM Pol II miR RNAi Expression Vector Kit Catalog # K493500 The engineered pre-miRNA sequence structure is based on the murine 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 Guide-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 flanking sequence RNA-seq GACACAAGGCCUGUUACUAGCACUCACAUGGAACAAAUGGCC (SEQ ID NO: 10) DNA sequence GACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC (SEQ ID NO: 41) Source: BLOCK-iT TM Pol II miR RNAi Expression Vector Kit Catalog #K493500 The engineered pre-miRNA sequence structure was based on the murine 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) All-miR155-DMPK 204 duplex sequence-RNA CUGGAGGCUUGCUGAAGGCUGUAUGCUGAGUCGAAGACAGUUCUAGGGUGUUUUGGCCACUGACUGACACCCUAGAUGUCUUCGAUUCAGGACACAAGGCCUGUUACUAGCACUCACAUGGAACAAAUGGCC (SEQ ID NO: 24) All-miR155-DMPK 204 Duplex sequence-DNA CTGGAGGCTTGCTGAAGGCTGTATGCTGAGTCGAAGACAGTTCTAGGGTGTTTTGGCCACTGACTGACACCCTAGATGTCTTCGATTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC (SEQ ID NO: 25) Minimal BGHpA sequence TCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC(SEQ ID NO:16) Source: Bos taurus growth hormone 1 (GH1) mRNA NCBI Reference Sequence NM_180996.1 1138 bp A1AT intron stuffer sequence (upstream of expression cassette) TACGTACAATTGGGATCCTTCGAACTTGAGAGAAAACATCCCAGGGATTTACAGATCAC ATGCAGGCAGGGACCAGCTCAACCCTTCTTTAATGTCATCCAGGGAGGGGGCCAGGGAT GGAGGGGAGGGGTTGAGGAGCGAGAGGCAGTTATTTTTGGGTGGGATTCACCACTTTTC CCATGAAGAGGGGAGACTTGGTATTTTGTTCAATCATTAAGAAGACAAAGGGTTTGTTG AACTTGACCTCGGGGGGGATAGACATGGGTATGGCCTCTAAAAACATGGCCCCAGCAGC TTCAGTCCCTTTCTCGTCGATGGTCAGCACAGCCTTATGCACGGCCTGGAGGGGAGAGA AGCAGAGACACGTTGTAAGGCTGATCCCAGGCCTCGAGCAAGGCTCACGTGGACACCTC CCAGGAAGCGCTCACTCCCCCTGGACGGCCCTGGCCCTGCACATCCTCTCCCTCCCTGT CACATAGGCCTTGCTCCTCCTCAAGGCTTTGGCTGATGGGGCTGGCTCCCCTCTGTCCA TCTTCCTGACAAGCGCCTCTCCCCCTGCTCAGGTGCACCCACAACTCAGAACAGGGAAG 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 stuffer 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-stuffer sequence-ITR (3739 bp) Table 5. ITR-nDes-miR155-amiR-DMPK from 5' ITR to 3' ITR 204 Sequence annotation (3739bp) Sequence of the synthetic 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 TTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCGCATGCGTCGACT(SEQ ID NO:26) nDes-miR155-204 promoter to polyA (SEQ ID NO:17) Amino acid sequences of AAVrh74 variants (WO 2019178412) Amino acid sequence of AAVrh74N502I MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSIFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL(SEQ ID NO:50) Nucleotide sequence encoding AAVrh74 N502I capsid Amino acid sequence of AAVrh74W505R (SEQ ID NO:52) Nucleotide sequence encoding AAVrh74W505R

Claims

1. An RNAi comprising a first strand and a second strand, wherein a) the first strand and the second strand form a duplex; b) the first strand comprises a guide region, wherein the guide region comprises a nucleic acid having the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1), or a sequence having about 90% identity to the sequence of SEQ ID NO: 1; and c) the second strand comprises a non-guiding region.

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

2.

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

2.

4. The RNAi according to any one of claims 1 to 3, wherein the first strand and the second strand are connected via an RNA linker capable of forming a loop structure.

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

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

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

3.

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

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

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

7.

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

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

13. The RNAi of claim 12, wherein the scaffold comprises all or part of the nucleic acid of SEQ ID No:

11. The RNAi of claim 13 , wherein the miRNA is embedded in the scaffold.

15. The RNAi of claim 14, wherein the scaffold has a 5' arm and a 3' arm, wherein the 5' arm is located 5' to the nucleic acid encoding the RNAi, and wherein the 3' arm is located 3' to the nucleic acid encoding the RNAi.

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

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

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

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

20. The RNAi of claim 19, wherein the polypeptide is dystrophic myotonia protein kinase (DMPK).

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

22. The RNAi of claim 20 or 21, wherein the gene encoding DMPK comprises 5 or more CTG trinucleotide repeat sequences.

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

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

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

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

27. The expression cassette of claim 26, wherein the desmin promoter comprises one or more enhancer elements, and the 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 to the sequence of SEQ ID NO:21, and / or one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:22, or a nucleotide sequence having about 90% identity to the sequence of SEQ ID NO:

22.

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

12.

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

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

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

13.

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

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

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

14.

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

15.

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

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

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

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

16.

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

17.

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

22.

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

12.

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

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

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

13.

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

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

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

14.

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

15.

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

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

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

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

16.

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

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

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

63. The vector of claim 62, wherein the expression cassette is flanked by one or more stuffer nucleic acid sequences. The vector of claim 63 , wherein the one or more stuffer nucleic acid sequences are derived from human SerpinA1 gene.

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

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

18.

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

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

19.

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

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

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

72. The rAAV vector of claim 70 or 71, wherein the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITRs.

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

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

20.

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

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

77. A viral particle comprising the vector of any one of claims 62-68.

78. A recombinant AAV particle comprising the rAAV vector of any one of claims 69-75.

79. The rAAV particle of claim 78, wherein the AAV virus particle comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAVrh74, AAVrh74N502I, AAVrh74 W505R, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2N587A, AAV2 E548A, AAV2 N708A, AAV V708K, AAV2-HBKO, AAVDJ8, AAVPHP.B, AAVPHP.eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1 / AAV2 chimera, bovine AAV, or mouse AAV capsid rAAV2 / HBoV1 serotype capsid.

80. The rAAV particle of claim 78 or 79, wherein the ITRs and the capsid of the rAAV viral particle are derived from the same AAV serotype.

81. The rAAV particle of claim 78 or 79, wherein the ITRs and the capsid of the rAAV viral particle are derived from different AAV serotypes.

82. The rAAV particle of claim 78, 79 or 81, wherein the AAV viral particle comprises an AAVrh74 N502I serotype capsid.

83. The rAAV particle of claim 82, wherein the ITR is an AAV2 ITR and the capsid of the rAAV particle is an AAVrh74 N502I serotype capsid.

84. The rAAV particle of claim 78, 79 or 81, wherein the AAV viral particle comprises an AAVrh74 W505R serotype capsid.

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

86. An rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises the following nucleic acid from 5' to 3': AAV2 ITR, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, Byrne desmin enhancer element, Paulin desmin enhancer element, Desmin promoter, 5' arm of rabbit β-globin intron, 5' miR155 scaffold sequence, DMPK 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 miRNA passenger sequences, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, Minimal bovine growth hormone polyadenylation sequence, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, and AAV2 ITR; and wherein the capsid is the AAVrh74 N502I capsid.

87. An rAAV particle comprising a rAAV vector, wherein the rAAV vector comprises the following nucleic acid from 5' to 3': An AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 18, A Byrne's knot protein 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, The 5' arm of the 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, DMPK comprising the polynucleotide sequence of SEQ ID NO: 4 204 miRNA guide sequence, A miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, DMPK comprising the polynucleotide sequence of SEQ ID NO: 5 204 miRNA passenger sequences, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, The 3' arm of the rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO: 15, A minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO: 16, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 19, and An AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is the AAVrh74 N502I capsid.

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

50.

89. 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, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK 204 miRNA guide sequence, miR155 terminal loop sequence, DMPK 204 miRNA passenger sequence, 3' miR155 scaffold sequence, 3' arm of rabbit β-globin intron, minimal bovine growth hormone polyadenylation sequence, nucleic acid encoding stuffer nucleic acid sequence from the human serpinA1 gene, and AAV2 ITRs; and wherein the capsid is an AAVrh74 W505R capsid.

90. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:43, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:18, a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:21, a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO:22, a desmin promoter comprising the polynucleotide sequence of SEQ ID NO:23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:40, a DMPK comprising the polynucleotide sequence of SEQ ID NO:4 204 miRNA guide sequence, miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, DMPK comprising the polynucleotide sequence of SEQ ID NO: 5 204 miRNA passenger sequence, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO:41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO:15, a minimal bovine growth hormone polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO:16, a nucleic acid encoding a stuffer nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO:19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO:49; and wherein the capsid is an AAVrh74 W505R capsid.

91. The rAAV particle of claim 86a or 86b, wherein the AAVrh74 W505R capsid comprises a capsid protein comprising the amino acid sequence of SEQ ID NO:

52.

92. A composition comprising the viral particle of claim 77 or the rAAV particle of any one of claims 78-86.

93. A pharmaceutical composition comprising the viral particle of claim 77 or the rAAV particle of any one of claims 78-86.

94. The composition of claim 87 or 88, wherein the composition further comprises a pharmaceutically acceptable carrier.

95. A modified desmin promoter, wherein the modified desmin promoter comprises one or more enhancer elements and a promoter of a human desmin gene.

96. The modified desmin promoter of claim 90, wherein the modified desmin promoter comprises two enhancer elements and the promoter of the human desmin gene.

97. The modified desmin promoter of claim 90 or 91, wherein the modified desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.

98. A modified desmin promoter as described in any one of claims 90-92, wherein the modified desmin promoter comprises: one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:21 or a sequence with about 90% identity to the sequence of SEQ ID NO:21, and / or one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO:22 or a sequence with about 90% identity to the sequence of SEQ ID NO:

22.

99. The modified desmin promoter of any one of claims 90-93, wherein the modified desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence having about 90% identity to the sequence of SEQ ID NO:

12.

100. A kit comprising the RNAi of any one of claims 1-22.

101. A kit comprising the viral particles of claim 77 or the AAV particles of any one of claims 78-86.

102. A kit comprising the composition of any one of claims 87-89.

103. The kit of any one of claims 95-97, further comprising instructions for use.

104. A method for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof, the method comprising administering to the mammal an effective amount of the RNAi of any one of claims 1-22.

105. A method for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal suffering from DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the RNAi of any one of claims 1-22.

106. A method for inhibiting accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the RNAi of any one of claims 1-22.

107. A method for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof, the method comprising administering to the mammal an effective amount of the viral particle of claim 77 or the rAAV particle of any one of claims 78-86.

108. A method for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the viral particles of claim 77 or an effective amount of the rAAV particles of any one of claims 78-86.

109. A method for inhibiting accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the viral particle of claim 77 or an effective amount of the rAAV particle of any one of claims 78-86.

110. The method of any one of claims 102-104, wherein the effective amount of the viral particle or rAAV particle is about 1×10 8 About 2×10 13 A dose of genomic copies / mL.

111. The method of claim 105, wherein the dose is about 5×10 12 genome copies / mL.

112. The method of claim 105, wherein the dose is about 1×10 13 genome copies / mL.

113. The method of claim 105, wherein the dose is about 2×10 13 genome copies / mL.

114. The method of any one of claims 102-104, wherein the effective amount of the viral particle or rAAV particle is about 1×10 8 About 2×10 14 The dose is 1 genome copy / kg body weight.

115. The method of claim 109, wherein the dose is about 5×10 13 genome copies / kg body weight.

116. The method of claim 109, wherein the dose is about 1×10 14 genome copies / kg body weight.

117. The method of claim 109, wherein the dose is about 2×10 14 genome copies / kg body weight.

118. A method for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof, the method comprising administering to the mammal an effective amount of the composition of any one of claims 87-89.

119. A method for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal suffering from DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the composition of any one of claims 87-89.

120. A method for inhibiting accumulation of DMPK RNA in cells of a mammal suffering from DM-1 in need thereof, the method comprising administering to the mammal an effective amount of the composition of any one of claims 87-89.

121. The method of any one of claims 99-101, wherein the RNAi is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with, and / or after administration of the RNAi.

122. The method of any one of claims 102-104, 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 administration of the viral particle or the rAAV particle.

123. The method of any one of claims 113-115, wherein the composition is administered in combination with an immunosuppressant, wherein the immunosuppressant is administered before, simultaneously with, and / or after administration of the composition.

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