Targeted gene therapy for type 1 myotonic dystrophy

Description

Targeted gene therapy for type 1 myotonic dystrophy CROSS-REFERENCE TO RELATED ORDERS

[001] This application claims priority to Provisional Patent Applications under Serial Nos. US 63 / 494,453, filed April 5, 2023; and US 63 / 589,417, filed October 11, 2023; the content of which is incorporated herein by reference in its entirety. PRESENTATION OF THE SEQUENCE LISTING

[002] The content of the following presentation in XML file is incorporated into this document by reference in its entirety: a machine-readable form (CRF) of the Sequence Listing (file name: 752749-SA9-363BPC.xml, creation date: April 3, 2024, size: 84754 bytes). FIELD

[003] The present invention relates to variant RNAi molecules. In some respects, the invention relates to variant RNAi molecules for treating muscular dystrophy. BACKGROUND

[004] RNA interference (RNAi) has proven to be a useful tool for gene silencing in basic research on genetic function and shows great promise as a therapeutic agent for suppressing genes associated with the development of a range of diseases. In nature, gene regulation by RNAi occurs through small RNAs known as microRNAs (miRNAs) (Ambros, (2004) Nature 431:350-355; Krol et al., (2010) Nat. Rev. Genet. 11:597-610). MicroRNAs have emerged as powerful regulators of various cellular processes and, when administered by viral vectors, artificial miRNAs are continuously expressed, resulting in robust and sustained suppression of target genes. The elucidation of the mechanisms Petition 870250090423, dated 03 / 10 / 2025, page 9 / 218 2 / 144 The processes involved in miRNA processing have allowed scientists to co-opt the endogenous cellular RNAi machinery and direct the degradation of a target genetic product using artificial miRNAs (see, for example, PG Publication No. US 2014 / 0163214 and Davidson et al., (2012) Cell 150:873-875).

[005] Myotonic dystrophy type 1 (DM1) is a progressive monogenic, autosomal dominant disease caused by the expansion of CTG repeats (>50) at the DMPK locus. DMPK with repeats is transcribed into mRNA, which forms hairpin shapes and binds RNA-binding proteins, hijacking them from their normal function. This leads to the appearance of nuclear foci, incorrect splicing of mRNAs, and ultimately, myotonia. DM1 primarily affects skeletal, cardiac, and smooth muscle, resulting in significant physical, cognitive, and behavioral impairments and disability. Currently, there is no approved therapy for DM1. Therefore, there is a high unmet medical need for therapies to treat DM1.

[006] All references cited in this document, including patent applications and publications, are incorporated by reference in their entirety. BRIEF SUMMARY

[007] In some respects, the invention provides a recombinant adeno-associated virus (rAAV) particle comprising an RNAi comprising a first strand and a second strand, wherein the first strand and the second strand form a duplex, the first strand comprising a guide region, wherein the guide region comprises a nucleic acid with the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) or with a sequence with about 90% identity to the sequence of SEQ ID NO: 1, and the second strand comprises a non-guide region; and an AAV capsid comprising an amino acid sequence with about 90% identity to a capsid. Petition 870250090423, dated 03 / 10 / 2025, p. 10 / 218 3 / 144 wild-type AAVrh74. In some embodiments, the first strand comprises nucleic acid with the sequence SEQ ID NO: 1, and the non-guide region comprises nucleic acid with the sequence SEQ ID NO: 2. In some embodiments, the first strand and the second strand are linked by 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 with SEQ ID NO: 3 or with a sequence with about 90% identity to the sequence of SEQ ID NO: 3. In some embodiments, the RNAi comprises 5' to 3' of the second strand, the RNA linker, and the first strand. In some embodiments, RNAi comprises 5' to 3' of the first strand, the RNA linker, and the second strand.In some embodiments, RNAi comprises nucleic acid with the sequence SEQ ID NO: 7 or with a sequence approximately 90% identical to the sequence SEQ ID NO: 7. In some embodiments, RNAi is a small inhibitory RNA (siRNA), a microRNA (miRNA), or a small hairpin RNA (shRNA).

[008] In some embodiments of the invention, the RNAi further comprises a scaffold. In some embodiments, the scaffold comprises all or a portion of the nucleic acid of SEQ ID NO: 11. In some embodiments, the miRNA is incorporated into the scaffold. In some embodiments, the scaffold has a 5' arm, wherein the 5' arm is located at 5' of the nucleic acid encoding the RNAi, and a 3' arm, wherein the 3' arm is located at 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 the nucleic acid of SEQ ID NO: 9 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 9 located Petition 870250090423, dated 03 / 10 / 2025, p. 11 / 218 4 / 144 in 5' of RNAi. In some embodiments, the miR-155 scaffold comprises the nucleic acid of SEQ ID NO: 10 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 10 located in 3' of RNAi.

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

[0010] In some aspects, the invention provides an expression cassette comprising nucleic acid encoding any of the RNAi described herein. In some embodiments, the nucleic acid encoding the RNAi is operationally linked to a promoter. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the promoter is a desmin promoter or variant thereof. In some embodiments, the desmin promoter comprises one or more enhancer elements and the promoter for the human desmin gene. In some embodiments, the desmin promoter comprises two enhancer elements and the promoter for the human desmin gene. In some embodiments, the desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence with approximately 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 of nucleotides with approximately 90% identity to the sequence of SEQ ID NO: 21. Petition 870250090423, dated 03 / 10 / 2025, p. 12 / 218 5 / 144 nucleotide sequence with approximately 90% identity to the sequence of SEQ ID NO: 22. In some embodiments, the desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence with approximately 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 the nucleotide sequence of SEQ ID NO: 13 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 13. In some embodiments, the nucleic acid encoding RNAi is incorporated into the intron. In some embodiments, the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at 5' of the nucleic acid encoding RNAi and the 3' arm is located at 3' of the nucleic acid encoding RNAi.In some embodiments, the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 14 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 14. In some embodiments, the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 15 or a sequence with approximately 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 SEQ ID NO: 16 or a sequence with approximately 90% identity to the sequence SEQ ID NO: 16.In some forms, the expression cassette comprises the nucleotide sequence of... Petition 870250090423, dated 03 / 10 / 2025, p. 13 / 218 6 / 144 SEQ ID NO: 17 or a sequence with approximately 90% identity to the sequence with SEQ ID NO: 17.

[0011] In some aspects, the invention provides an expression cassette, wherein the expression cassette comprising a modified desmin promoter, wherein the modified desmin promoter comprises one or more enhancer elements and the promoter for the human desmin gene. In some embodiments, the modified desmin promoter comprises two enhancer elements and the promoter for the human desmin gene. In some embodiments, the modified desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.In some embodiments, the modified desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence with approximately 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 approximately 90% identity to the sequence of SEQ ID NO: 22. In some embodiments, the desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence with approximately 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 the nucleotide sequence of SEQ ID NO: 13 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 13.In some embodiments, the nucleic acid encoding the transgene is incorporated into the intron. In some embodiments, the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at 5' of the nucleic acid encoding the transgene and the 3' arm is at 3'. Petition 870250090423, dated 03 / 10 / 2025, p. 14 / 218 7 / 144 is located 3' from the nucleic acid encoding the transgene. In some embodiments, the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 14 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 14. In some embodiments, the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 15 or a sequence with approximately 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 with approximately 90% identity to the sequence of SEQ ID NO: 16. In some embodiments, the transgene encodes a polypeptide or a nucleic acid. In some embodiments, the transgene encodes an RNAi.

[0012] In some aspects, the invention provides a vector comprising any of the expression cassettes described in this document. In some embodiments, the expression cassette is flanked by one or more filler nucleic acid sequences. In some embodiments, one or more filler nucleic acid sequences are derived from the human SerpinA1 gene. In some embodiments, a filler nucleic acid sequence located 5' from the expression cassette is derived from the human SerpinA1 gene. In some embodiments, a filler sequence located 5' from the expression cassette comprises the nucleotide sequence of SEQ ID NO: 18 or a sequence with approximately 90% identity to the sequence of SEQ Petition 870250090423, dated 03 / 10 / 2025, p. 15 / 218 8 / 144 ID NO: 18. In some embodiments, a 3' filler nucleic acid sequence in the expression cassette is derived from the human SerpinA1 gene. In some embodiments, a 3' filler sequence for the expression cassette comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 19.

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

[0014] In some embodiments, the invention provides a cell comprising any of the expression cassettes described herein, any of the vectors described herein, or any of the rAAV vectors described herein.

[0015] In some respects, the invention provides a viral particle comprising any of the vectors described herein. In some respects, the invention provides a recombinant AAV particle comprising any of the rAAV vectors. Petition 870250090423, dated 03 / 10 / 2025, page 16 / 218 9 / 144 described in this document. In some embodiments, the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 502. In some embodiments, the amino acid substitution at position 502 is an isoleucine (I). In some embodiments, the rAAV viral particle comprises a capsid of the AAVrh74 N502I serotype. In some embodiments, the ITR is an AAV2 ITR and the rAAV particle capsid is a capsid of the AAVrh74 N502I serotype. In some embodiments, the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 505. In some embodiments, the amino acid substitution at position 505 is an arginine (R). In some embodiments, the rAAV particle comprises a capsid of the AAVrh74 W505R serotype. In some forms, the ITR is an AAV2 ITR and the rAAV particle capsid is an AAVrh74 W505R serotype capsid.

[0016] In some aspects, 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': an AAV2 ITR, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204 miRNA passenger sequence, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a minimal hormone polyadenylation sequence. of bovine growth, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene and an ITR of AAV2; and wherein the capsid is a Petition 870250090423, dated 03 / 10 / 2025, page 17 / 218 10 / 144 AAVrh74 N502I capsid.

[0017] In some respects, the invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR comprising the polynucleotide sequence SEQ ID NO: 43, a nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence SEQ ID NO: 18, a Byrne desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 21, a Paulin desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 22, a desmin promoter comprising the polynucleotide sequence SEQ ID NO: 23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 14, a sequence of framework 5' miR155 comprising the polynucleotide sequence of SEQ ID NO: 40,a DMPK204 miRNA guide sequence comprising the polynucleotide sequence of SEQ ID NO: 4, a miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, a DMPK204 miRNA passenger sequence comprising the polynucleotide sequence of SEQ ID NO: 5, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO: 41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO: 15, a bovine growth hormone minimal polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO: 16, nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence of SEQ ID NO: 19, and an AAV2 ITR comprising the sequence polynucleotide of SEQ ID NO: 49; and in which the capsid is an AAVrh74 N502I capsid. In al, Petition 870250090423, dated 03 / 10 / 2025, page 18 / 218 11 / 144 in some embodiments, the AAVrh74 N502I capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 50.

[0018] 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': an AAV2 ITR, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204 miRNA passenger sequence, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a minimal polyadenylation sequence of bovine growth hormone, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene and an ITR of AAV2;and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 50.;

[0019] In some respects, the invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR comprising the polynucleotide sequence SEQ ID NO: 43, a nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence SEQ ID NO: 18, a Byrne desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 21, a Paulin desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 22, a pro Petition 870250090423, dated 03 / 10 / 2025, p. 19 / 218 12 / 144 desmin motor comprising the polynucleotide sequence SEQ ID NO: 23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence SEQ ID NO: 40, a DMPK204 miRNA guide sequence comprising the polynucleotide sequence SEQ ID NO: 4, a miR155 terminal loop sequence comprising the polynucleotide sequence SEQ ID NO: 6, a DMPK204 miRNA passenger sequence comprising the polynucleotide sequence SEQ ID NO: 5, a 3' miR155 scaffold sequence comprising the polynucleotide sequence SEQ ID NO: 41, a 3' arm of a β-globin intron rabbit growth hormone comprising the polynucleotide sequence SEQ ID NO: 15, a minimal polyadenylation sequence of bovine growth hormone comprising the polynucleotide sequence SEQ ID NO: 16,nucleic acid encoding a filler nucleic acid sequence of the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 19 and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO: 49; and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 50.

[0020] In some aspects, the invention provides an rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, a miR155 terminal loop sequence, and a miRNA passenger sequence. Petition 870250090423, dated 03 / 10 / 2025, p. 20 / 218 13 / 144 DMPK204, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a minimal polyadenylation sequence of bovine growth hormone, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene and an ITR from AAV2; and wherein the capsid is an AAVrh74 W505R capsid.

[0021] In some embodiments, the invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR comprising the polynucleotide sequence SEQ ID NO: 43, a nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence SEQ ID NO: 18, a Byrne desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 21, a Paulin desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 22, a desmin promoter comprising the polynucleotide sequence SEQ ID NO: 23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 14, a 5' miR155 framework sequence comprising the polynucleotide sequence of SEQ ID NO: 40,a DMPK204 miRNA guide sequence comprising the polynucleotide sequence SEQ ID NO: 4, a miR155 terminal loop sequence comprising the polynucleotide sequence SEQ ID NO: 6, a DMPK204 miRNA passenger sequence comprising the polynucleotide sequence SEQ ID NO: 5, a 3' miR155 scaffold sequence comprising the polynucleotide sequence SEQ ID NO: 41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 15, a bovine growth hormone minimal polyadenylation sequence comprising the polynucleotide sequence, Petition 870250090423, dated 03 / 10 / 2025, page 21 / 218 14 / 144 typicity of SEQ ID NO: 16, nucleic acid encoding a filler nucleic acid sequence of the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 19 and an ITR of AAV2 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 capsid proteins comprising the amino acid sequence of SEQ ID NO: 52.

[0022] 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': an AAV2 ITR, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204 miRNA passenger sequence, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a minimal polyadenylation sequence of bovine growth hormone, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene and an ITR of AAV2;and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 52.;

[0023] In some embodiments, the invention provides an rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following 5' to 3' nucleic acids, an AAV2 ITR comprising the polynucleotide sequence SEQ ID NO: 43, nucleic acid encoding a nucleic acid sequence of Petition 870250090423, dated 03 / 10 / 2025, p. 22 / 218 15 / 144 filling of the human serpinAI gene comprising the polynucleotide sequence SEQ ID NO: 18, a Byrne desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 21, a Paulin desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 22, a desmin promoter comprising the polynucleotide sequence SEQ ID NO: 23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence SEQ ID NO: 40, a DMPK204 miRNA guide sequence comprising the polynucleotide sequence SEQ ID NO: 4, a miR155 terminal loop sequence comprising the polynucleotide sequence SEQ ID NO: 6, a DMPK204 miRNA passenger sequence comprising the polynucleotide sequence of SEQ ID NO: 5,a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO: 41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO: 15, a bovine growth hormone minimal polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO: 16, nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence of SEQ ID NO: 19 and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO: 49; and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 52.

[0024] In some aspects, the invention provides a composition comprising any of the viral particles or rAAV particles described herein. In some embodiments, the invention provides a pharmaceutical composition comprising any of the viral particles or rAAV particles described in the Petition 870250090423, dated 03 / 10 / 2025, page 23 / 218 16 / 144 of this document. In some embodiments, the composition also includes a pharmaceutically acceptable carrier.

[0025] In some aspects, the invention provides kits comprising one or more of an RNAi, as described herein, a viral particle, as described herein, an AAV particle, as described herein, or a composition, as described herein. In some embodiments, the kit also includes instructions for use.

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

[0027] In some aspects, the invention provides methods for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof, comprising administering to the mammal an effective amount of any of the viral particles (e.g., rAAV particles) as described herein. In some aspects, the invention provides methods for inhibiting the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 in need thereof, comprising administering to the mammal an effective amount of any of the viral particles (e.g., rAAV particles). Petition 870250090423, dated 03 / 10 / 2025, page 24 / 218 17 / 144 of rAAV) as described in this document. In some respects, the invention provides methods for inhibiting the accumulation of DMPK RNA in a mammalian cell with DM-1 that requires the same, comprising administering to the mammal an effective amount of any of the viral particles (e.g., rAAV particles) as described in this document.

[0028] In some embodiments of the invention, the effective quantity of viral particles (e.g., rAAV particles) is a dose of about 1 x 10⁸ to about 2 x 10¹³ genome copies / mL. In some embodiments of the invention, the dose is about 5 x 10¹² genome copies / mL. In some embodiments of the invention, the dose is about 1 x 10¹³ genome copies / mL. In some embodiments of the invention, the dose is about 2 x 10¹³ genome copies / mL.

[0029] In some embodiments of the invention, the effective quantity of viral particles (e.g., rAAV particles) is a dose of about 1 x 10⁸ to about 2 x 10¹⁴ genome copies / kg of body weight. In some embodiments of the invention, the dose is about 5 x 10¹³ genome copies / kg of body weight. In some embodiments of the invention, the dose is about 1 x 10¹⁴ genome copies / kg of body weight. In some embodiments of the invention, the dose is about 2 x 10¹⁴ genome copies / kg of body weight.

[0030] In some aspects, the invention provides methods for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof, comprising administering to the mammal an effective amount of any of the compositions described herein. In some aspects, the invention provides methods for inhibiting the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 in need thereof, comprising administering to the mammal an effective amount of the composition of any of the compositions as described herein. In some Petition 870250090423, dated 03 / 10 / 2025, page 25 / 218 18 / 144 aspects, the invention provides methods for inhibiting the accumulation of DMPK RNA in a mammalian cell with DM-1 that requires the same, comprising administering to the mammal an effective amount of any of the compositions as described herein.

[0031] In some embodiments of the invention, RNAi is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time as, and / or after administration of the RNAi. In some embodiments, the viral particle or rAAV particle is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time as, and / or after administration of the viral particle or rAAV particle. In some embodiments, the composition is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time as, and / or after administration of the composition. Description of the Drawings

[0032] The present application can be understood by reference to the following description taken in conjunction with the attached Figures.

[0033] Figure 1A represents a sequence diagram of the nDes-miR155-amiR-DMPK204 gene cassette. A hybrid muscle promoter is located upstream of the miR155-amiRDMPK204 sequence. Downstream of the miRNA is a bovine growth hormone (minBGHpA) polyadenylation sequence. A filler sequence from the A1AT intron flanks both sides of the cassette. All of these are flanked by two AAV2 ITRs, generating a combined vector genome size of 3739 bp.

[0034] Figure 1B represents the ITR plasmid used for cloning the nDes-miR155-amiR-DMPK204 gene cassette. The ITR plasmid contains flanked A1AT filler sequence. Petition 870250090423, dated 03 / 10 / 2025, page 26 / 218 19 / 144 by the 5' and 3' AAV2 ITRs. The A1AT fill sequence comprises NcoI and SphI restriction sites for cloning.

[0035] Figure 1C represents the results of a small-scale packaging assay that was performed in HEK 293 cells to confirm the packaging of the DC969-nDesmiR155-amiR-DMPK204 plasmid. Small-scale production was performed using the AAV rep / cap plasmid. The geometric y-axis shows the amount of vector produced per HEK 293 cell compared to a standard EGFP plasmid gene cassette (CD627-CBAGFP). DRP: DNase resistance particle.

[0036] Figures 2A to 2C represent an evaluation of DMPK knockdown by the nDes-miR155amiR-DMPK204 (amiR155-204) AAV expression cassette in DMSXL mice after caudal vein injection. Figure 2A shows AAV transduction and biodistribution efficiency as assessed by quantifying transgene copy numbers in different organs. qPCR results are expressed as the average copy number / nucleus ratio of AAV. Figure 2B shows amiR-DMPK204 levels in transduced tissues. MicroRNA entry levels were normalized to U6 small nuclear RNA and adjusted for cells treated with BSS (balanced saline solution). Figure 2C shows DMPK silencing in transduced tissues. Total DMPK was determined by qRT-PCR. mRNA entry was normalized with tata-box binding protein (TBP) and defined relative to BSS-treated cells. The dashed line indicates 50% DMPK expression relative to TBP expression.For Figures 2A to 2C, the data were evaluated using the 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).

[0037] Figures 3A to 3B represent the suppression of human DMPK by AAV nDes-miR155-amiR-DMPK204. Mice Petition 870250090423, dated 03 / 10 / 2025, p. 27 / 218 20 / 144 DMSXL mice received a dose-dependent systemic injection of AV nDes-miR155amiR-DMPK204. Mice were euthanized after 8 weeks, organs were collected, and amiR-DMPK204 and DMPK transcript levels were measured. Figure 3A represents the abundance of U6-normalized amiR-DMPK204 in various tissues. Figure 3B represents the abundance of mTBP-normalized hDMPK transcripts in various tissues. 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, intermediate dose 13 and high dose 7).

[0038] Figure 4 represents the correction of splicing abnormalities in DMSXL mice after systemic treatment with AAV nDes-miR155-amiR-DMPK204. Splicing of alternative exon 11 in LDB3 was evaluated using RT-PCR in the gastrocnemius muscle after 8 weeks of treatment. 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, intermediate dose 13 and high dose 7).

[0039] Figures 5A and 5B show increased survival rate and body weight in female DMSXL mice treated with the dose-dependent AAV nDes-miR155-amiR-DMPK204. Figure 5A shows Kaplan-Meier survival curves showing improved survival rate with the medium dose after 8 weeks of treatment compared to animals treated with a low dose or BSS. Figure 5B shows improved body weight observed in DMSXL animals treated with the AAV nDes-miR155-amiR-DMPK204 compared to animals treated with BSS or low dose.

[0040] Figure 6 represents the reversal of the electrophysiological characteristics of DM1 disease after systemic treatment of the model. Petition 870250090423, dated 03 / 10 / 2025, page 28 / 218 21 / 144 DM1 DMSXL mice with AAV nDes-miR155-amiR-DMPK204 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, intermediate dose 13 and high dose 7).

[0041] Figure 7A shows a schematic representation of the experimental protocol. Blood (β) was collected on day 1 to reconfirm neutralizing antibodies. NHPS were administered with AAV encoding the GFP reporter at a dose of 1e13gv / kg on day 0, and necropsy was performed on D21 and multiple tissues were collected to assess biodistribution. Figures 7B to 7E show quantification of GFP expression from various tissues of animals injected with AAV9, AAVrh74, and AAVrh74 N502I (rh74M). The bar graphs represent the amounts of GFP measured by ELISA in the tibialis anterior muscle (TA; Figure 7B), biceps femoris (Figure 7C), quadriceps (Figure 7D), heart (Figure 7E), and liver (Figure 7F). The data were evaluated using ANOVA, multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0042] Figures 8A and 8B show suppression of human DMPK by AAV rh74 N502I nDes-miR155-amiR-DMPK204. DMSXL mice were systemically injected with AAV rh74 N502I nDesmiR155-amiR-DMPK204 in a dose-dependent manner. Mice were euthanized after 8 weeks, organs were collected, and amiR-DMPK204 and DMPK transcript levels were measured. Figure 8A represents the abundance of U6-normalized amiR-DMPK204 in various tissues. Figure 8B represents the abundance of mTBP-normalized hDMPK transcripts in various tissues. The data were evaluated using ANOVA, multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0043] Figure 9 shows silencing of endogenous DMPK by Petition 870250090423, dated 03 / 10 / 2025, page 29 / 218 22 / 144 AAVrh74N502I nDes-miR155-amiR-DMPK204. Total DMPK levels were determined by qRT-PCR in RNA extracted from human DM1 cardiomyocytes differentiated from iPSCs. These cardiomyocytes were transduced with AAVrh74N502I nDes-miR155-amiR-DMPK204. Input mRNA levels were normalized to TBP mRNA. miRCTL3 served as a negative control and was adjusted by 1. Paired Student's t-test: **p < 0.01.

[0044] Figure 10 is a volcano plot showing changes in gene expression across the genome in HEK293 cells treated with miR-DMPK204 (Benjamini Hochberg FDR<1%). The table shows the four major differentially expressed (DE) genes with a complementary 3' UTR initial particle with FDR < 1% and FDR<5%.

[0045] Figure 11 shows the conservation of the miRDMPK204 target sequence among multiple sequences. The human target is SEQ ID NO: 28, the monkey target is SEQ ID NO: 29, the mouse target is SEQ ID NO: 30, the rat target is SEQ ID NO: 31, and the pup target is SEQ ID NO: 32.

[0046] Figure 12 illustrates the biodistribution of viral genome copies / cell in multiple tissues in the indicated treatment groups.

[0047] Figure 13 shows the dose-dependent expression of amiR-DMPK in various muscle tissues in the indicated treatment groups.

[0048] Figure 14 shows the DMPK expression levels after treatment in various muscles for each indicated treatment group.

[0049] Figures 15A to 15F illustrate changes in the splicing of 6 genes (MBNL1, SOS1, PKM, zTTN, GOLGA4 and CLASP1), comparing healthy myotubes, untreated DM1 myotubes and DM1 myotubes treated with Amir-DMPK204. Petition 870250090423, dated 03 / 10 / 2025, page 30 / 218 23 / 144

[0050] Figures 16A and 16B illustrate an in vitro screening to identify promoters with high activity in skeletal muscles (human myotubes; Figure 16A) and low activity in liver cells (Huh7 cells; Figure 16B). The S1-S9 promoters were compared to published muscle promoters, as well as to the ubiquitous CAG promoter.

[0051] Figures 17A to 17F illustrate data from immortalized human DM1 myotubes transduced with AAV2 carrying a miRNA that targets DMPK expressed under the desmin promoter. PCR was used to quantify miRNA (Figure 17A) and DMPK mRNA levels (Figure 17B). Nuclear CTG RNA foci were imaged using fluorescence in situ hybridization (FISH) and quantified in untreated (Figure 17C) and treated (Figure 17D) cells. Foci quantification can be seen in Figures 17E and 17F.

[0052] Figure 18 illustrates DMPK levels in DMSXL mice dosed with 9 x 1013 gv / kg of AAV.amiR155-DMPK204. Eight weeks after dosing, DMPK expression was measured by PCR. There was a significant reduction in DMPK in treated versus untreated mice in several muscles, while no significant reduction in DMPK mRNA was detected in the liver.

[0053] Figure 19 illustrates a significant reduction in the size of RNA foci in mouse hearts after treatment (DMSXL+) compared with a control (buffer; DMSXL-).

[0054] Figure 20 illustrates the splicing of several genes (Ldb3, MBNL2, Spag9, DNAse1, Tnnt3 and Zbtb49) in wild-type mice, buffer-treated DMSXL mice (DMSXL-) and treatment-treated DMSXL mice (DMSXL+).

[0055] Figure 21 represents the functional benefits in DMSXL mice treated with AAV.amiR155-DMPK204. Myotonia was measured in three different muscle groups - gastrocnemius, qua Petition 870250090423, dated 03 / 10 / 2025, page 31 / 218 24 / 144 triceps and tibialis anterior (TA) muscles in wild-type mice, untreated DMSXL mice (DMSXL-), and treated DMSXL mice (DMSXL+) using electromyography (EMG). The number of grade 0 events (no myotonia) versus grade 1-3 events was compared between the groups.

[0056] Figures 22A and 22B represent cardiac changes in DMSXL mice with or without treatment with AAV.amiR155DMPK204 compared to WT mice. Figure 22A is a graph describing changes in aortic diameter (mm). Figure 22B describes changes in cardiac output (μl / s). DETAILED DESCRIPTION

[0057] 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 nucleic acid with the sequence 5'-AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) or with a sequence with about 90% identity to the sequence of SEQ ID NO: 1; and c) the second strand comprises a non-guide region, wherein the non-guide region comprises nucleic acid with the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2) or with a sequence with about 90% identity to the sequence of SEQ ID NO: 2. In some embodiments, the invention provides expression cassettes for expressing nucleic acid encoding RNAi; for example, for expressing RNAi in mammalian muscles. In some modalities, the expression cassette is in an rAAV vector.

[0058] In some respects, the invention provides methods for 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 the dystrophy protein kinase. Petition 870250090423, dated 03 / 10 / 2025, page 32 / 218 25 / 144 myotonic (DMPK) in mammals; thus improving type 1 diabetes in mammals. General techniques

[0059] The techniques and procedures described or mentioned in this document are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely used methodologies 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); the 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); Animal Cell Culture: A Handbook of Basic Technique and Specialized Applications (RI Freshney, 6th ed., J. Wiley and Sons, 2010); Synthesis of oligonucleotides (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., Academic Press, 1998); Introduction to Cell and Tissue Culture (J.P. Mather e P.E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, e D.G. Newell, eds., J. Wiley e Sons, 1993-8); Handbook of Experimental Immunology (D.M. Weir e C.C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (J.M. Miller e M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley e Sons, 2002); Immunobiology (C.A. Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd e C. Dean, eds., Oxford University Press, 2000);. Petição 870250090423, de 03 / 10 / 2025, pág. 33 / 218 26 / 144 Using Antibodies: A Laboratory Handbook (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 2011). Definitions

[0060] A vector, as used in this document, refers to a plasmid or recombinant virus comprising a nucleic acid to be delivered into a host cell, in vitro or in vivo.

[0061] The term polynucleotide or nucleic acid, as used herein, refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The polynucleotide backbone may comprise sugars and phosphate groups (as is typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the polynucleotide backbone may comprise a polymer of synthetic subunits, such as phosphoramidates, and thus may be a phosphoramidate oligodeoxynucleoside (PNH2) or a phosphoramidate-phosphodiester oligomer blend.Furthermore, a double-stranded polynucleotide can be obtained from the product of a single-stranded polynucleotide by chemical synthesis, either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer. Petition 870250090423, dated 03 / 10 / 2025, page 34 / 218 27 / 144

[0062] The terms polypeptide and protein are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of the present invention, a polypeptide refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally of a conservative nature), to the native sequence, provided that the protein maintains the desired activity.These modifications can be deliberate, such as through site-directed mutagenesis, or they can be accidental, such as through host mutations that produce the proteins or errors due to PCR amplification.

[0063] A recombinant viral vector refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences not of viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid is flanked by at least one, and in some embodiments, two, inverted terminal repeat (ITR) sequences.

[0064] A recombinant AAV vector (rAAV vector) refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) that are flanked by at least one, and in some embodiments, two, inverted terminal repeat (ITR) sequences of AAV. Such rAAV vectors can be replicated and packaged. Petition 870250090423, dated 03 / 10 / 2025, p. 35 / 218 28 / 144 in infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger polynucleotide (e.g., into a chromosome or another vector, such as a plasmid used for cloning or transfection), then the rAAV vector can be called a provector that can be rescued by replication and encapsulation in the presence of suitable AAV packaging functions and helper functions. An rAAV vector can be in any of several forms, including, but not limited to, plasmids, linear artificial chromosomes, lipid-complexed, encapsulated in liposomes, and encapsulated in a viral particle, particularly an AAV particle.An rAAV vector can be packaged into an AAV virus capsid to generate a recombinant adeno-associated viral particle (rAAV particle).

[0065] Heterologous means derived from an entity genotypically distinct from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, may encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0066] The term transgene refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and, optionally, translated and / or expressed under appropriate conditions. In some respects, it confers a desired property to a cell in which it has been introduced. Petition 870250090423, dated 03 / 10 / 2025, p. 36 / 218 29 / 144 introduced, or otherwise leads to a desired therapeutic or diagnostic result. In another aspect, it can be transcribed into a molecule that mediates interfering RNA, such as miRNA, siRNA, or shRNA.

[0067] The terms genomic particles (pg), genomic equivalents or genome copies (cg), as used in reference to a viral titer, refer to the number of virions containing the recombinant DNA genome of AAV, regardless of infectivity or functionality. The amount of genomic particles in a particular vector preparation can be measured by procedures as described in the Examples in this document or, for example, in et al. (1999) Hum. Gene Ther., 10: 1031 to 1039; Veldwijk et al. (2002) Mol. Ther., 6:272 to 278.

[0068] The term vector genome (vg), as used in this document, may refer to one or more polynucleotides comprising a set of polynucleotide sequences of a vector, for example, a viral vector. A vector genome may be encapsulated in a viral particle. Depending on the particular viral vector, a vector genome may comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA or double-stranded RNA. A vector genome may include endogenous sequences associated with a particular viral vector and / or any heterologous sequences inserted into a particular viral vector via recombinant techniques. For example, a recombinant AAV vector genome may include at least one ITR sequence flanking a promoter, a filler, a sequence of interest (e.g., an RNAi), and a polyadenylation sequence.A complete vector genome may include a full set of polynucleotide sequences of a vector. In some embodiments, the nucleic acid titer of a viral vector can be measured in terms of gv / mL. Methods. Petition 870250090423, dated 03 / 10 / 2025, page 37 / 218 30 / 144 suitable methods for measuring this titer are known in the art (e.g., quantitative PCR).

[0069] As used in this document, the term inhibition may refer to the act of 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 gene expression may refer to any act that leads to a blocking, reduction, elimination, or any other antagonism of gene expression, including reducing mRNA abundance (e.g., silencing mRNA transcription), degrading mRNA, inhibiting mRNA translation, and so forth. In some embodiments, inhibiting DMPK expression may refer to a blocking, reduction, elimination, or any other antagonism of DMPK expression, including reducing DMPK mRNA abundance (e.g., silencing DMPK mRNA transcription), degrading DMPK mRNA, inhibiting DMPK mRNA translation, and so forth.As another example, inhibiting the accumulation of a protein in a cell can refer to any act that leads to a blockage, reduction, elimination, or other antagonism of protein expression, including reducing mRNA abundance (e.g., silencing mRNA transcription), degrading mRNA, inhibiting mRNA translation, degrading the protein, and so on. In some embodiments, inhibiting the accumulation of DMPK protein in a cell refers to a blockage, reduction, elimination, or other antagonism of DMPK protein expression in a cell, including reducing DMPK mRNA abundance (e.g., silencing DMPK mRNA transcription), degrading DMPK mRNA, inhibiting DMPK mRNA translation, degrading the DMPK protein, and so on.

[0070] The terms infection unit (IU), infectious particle Petition 870250090423, dated 03 / 10 / 2025, page 38 / 218 31 / 144 or replication unit, as used in reference to a viral titrator, refers to the number of infectious and replicating recombinant AAV vector particles, as measured by the infectious center assay, also known as the replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0071] The term transduction unit (TU), as used in reference to a viral titer, refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgenic product, as measured in functional assays, as described in Examples in this document, or for example, in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or in Fisher et al. (1996) J. Virol., 70:520-532.

[0072] An inverted terminal repeat sequence or ITR is a well-understood term in the art and refers to relatively short sequences found at the ends of viral genomes that are in opposite orientation.

[0073] An AAV inverted terminal repeat (ITR) sequence, a term well understood in the art, is a sequence of approximately 145 nucleotides that is present at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR may be present in either of two alternative orientations, leading to 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 A, A', B, B', C, C', and D regions), allowing intrastrand base pairing to occur within this portion of the ITR.

[0074] A terminal resolution sequence or trs is a sequence in the D region of the AAV ITR that is cleaved by rep proteins. Petition 870250090423, dated 03 / 10 / 2025, page 39 / 218 32 / 144 AAV during viral DNA replication. A mutant terminal resolution sequence is refractory to cleavage by AAV rep proteins.

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

[0076] A helper virus for AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by a host cell. A helper virus provides auxiliary functions that enable AAV replication. Several such helper viruses have been identified, including adenoviruses, herpesviruses, and poxviruses such as vaccinia and baculovirus. Adenoviruses encompass several different subgroups, although adenovirus type 5 subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non-human, and avian origin are known and available from repositories such as ATCC. Viruses of the herpes family, which are also available from repositories 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 functions, E1B functions, E2A functions, VA functions, and E4orf6 functions. Baculoviruses available from repositories include the nuclear polyhedrosis virus of Autographa californica.

[0077] A rAAV preparation is said to be substantially free of helper viruses if the ratio of infectious AAV particles to infectious helper virus particles is at least about 102:l; at least about 104:l; at least about 106:l; or at least Petition 870250090423, dated 03 / 10 / 2025, p. 40 / 218 33 / 144 approximately 108:l or more. In some embodiments, the preparations are also free of equivalent amounts of helper virus proteins (i.e., proteins that would be present as a result of such a level of helper virus if the helper virus particle impurities observed above were present in the disrupted form). Contamination with viral and / or cellular protein can generally be observed as the presence of Coomassie staining bands in SDS gels (e.g., the appearance of bands other than those corresponding to the capsid proteins of AAV VPL, VP2, and VP3).

[0078] Percent sequence identity (%) with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid or nucleotide residues in a candidate sequence that are identical to the amino acid or nucleotide residues in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in several ways that are within the skill of the art, for example, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Sup. 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 alignment. Petition 870250090423, dated 03 / 10 / 2025, page 41 / 218 34 / 144 to the maximum along the total length of the sequences being compared. For the purposes described herein, the % amino acid sequence identity of a given amino acid sequence A with or in relation to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity with or in relation to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program in the alignment of A and B, and where Y is the total number of amino acid residues in B.It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % identity of the amino acid sequence from A to B will not be equal to the % identity of the amino acid sequence from B to A. For the purposes described in this document, the % identity of the nucleic acid sequence of a given nucleic acid sequence C to, with or against, a given nucleic acid sequence D (which may alternatively be expressed as a given nucleic acid sequence C that has or comprises a given % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W / Z, where W is the number of nucleotides scored as identical matches by the sequence alignment program in the program alignment of C and D, and where Z is the total number of nucleotides in D.It will be understood that when the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the percentage identity of nucleic acid sequence C with D will not be present. Petition 870250090423, dated 03 / 10 / 2025, p. 42 / 218 35 / 144 equals the % identity of the nucleic acid sequence of D with C.

[0079] An isolated molecule (e.g., nucleic acid or protein) or cell means that it has been identified and separated and / or recovered from a component of its natural environment.

[0080] An effective amount is an amount sufficient to effect beneficial or desired results, including clinical results (e.g., improvement of symptoms, achievement of clinical endpoints, and the like). An effective amount may be administered in one or more administrations. In terms of a disease state, an effective amount is an amount sufficient to improve, stabilize, or slow the progression of a disease.

[0081] An individual or subject is a mammal. Mammals include, but are not limited to, domestic 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 being.

[0082] As used in this document, treatment is an approach to achieving beneficial or desired clinical outcomes. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, symptom attenuation, reduction in disease extent, stabilized disease status (e.g., without worsening), prevention of disease spread (e.g., metastasis), delay or retardation of disease progression, attenuation or palliation of disease status, and detectable or undetectable remission (partial or total). Treatment may also mean prolonged survival compared to the expected survival if no treatment is received.

[0083] As used in this document, the term prophylactic treatment refers to treatment where it is known or suspected to be true or likely to be true. Petition 870250090423, dated 03 / 10 / 2025, page 43 / 218 36 / 144 if there is a risk of an individual having a disorder, but they have not shown symptoms or have shown minimal symptoms of the disorder. An individual undergoing prophylactic treatment may be treated before the onset of symptoms.

[0084] As used in this document, the term myotonic dystrophy type 1 or DM1 refers to the multisystem disorder affecting 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. September 17, 1999 [Updated March 25, 2021]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2022). Mild DM1 is characterized by cataracts and mild myotonia, classic DM1 is characterized by muscle weakness and wasting, myotonia, cataracts, and often cardiac conduction abnormalities. Congenital type 1 diabetes mellitus (DM1) is characterized by hypotonia and severe generalized weakness at birth, often with respiratory failure and early death; intellectual disability is common.

[0085] As used in this document, the term myotonic dystrophy protein kinase, DMPK myotonin-protein kinase, MT-PK, myotonic dystrophy protein kinase, or MDPK may refer to the gene or a polypeptide product thereof associated with most cases of type 1 DM. The 3' untranslated region of the DMPK gene contains 5–37 copies of a CTG trinucleotide repeat. Expansion of this unstable motif to 50–1000 copies causes type I myotonic dystrophy, which increases in severity with increasing copy numbers of the repeat element.

[0086] As used in this document, an RNAi can refer to any RNA molecule that induces interfering RNA in a cell. Examples of RNAs include, without limitation, inhibitory RNAs. Petition 870250090423, dated 03 / 10 / 2025, page 44 / 218 37 / 144 small thoriatic RNAs (siRNAs), microRNAs (miRNAs), and small hairpin RNAs (shRNAs).

[0087] A miRNA can refer to a polynucleotide containing (i) a double-stranded sequence that directs a gene of interest for knockdown by RNAi and (ii) additional sequences that form a stem-loop structure similar to that of endogenous miRNAs. In some embodiments, the miRNA includes nucleic acid that flanks the stem-loop structure. These flanking sequences are known as the miRNA scaffold. A sequence that directs a gene of interest for RNAi (e.g., a short sequence, ~20 nt) can be linked to sequences that create a miRNA-like stem-loop structure and a base-pair sequence with the sequence of interest to form a duplex when the polynucleotide is assembled into the miRNA-like secondary structure. As described in this document, this duplex can hybridize imperfectly, for example, it can contain one or more unpaired or mismatched bases.After cleavage of this polynucleotide by Dicer, this duplex containing the sequence targeting a gene of interest can be unwound and incorporated into the RISC complex. A miRNA scaffold can refer to the miRNA itself or to a DNA polynucleotide encoding the miRNA. Examples of a miRNA scaffold include the miR-155 sequence (Lagos-Quintana, M. et al. (2002) Curr. Biol. 12:735-9) and the mirGE scaffold (WO2014016817A2). Commercially available kits for cloning a sequence into a miRNA scaffold are known in the art (e.g., the Pol II miR RNAi nvitrogen™ BLOCK-iT™ expression vector kit from Life Technologies, Thermo Fisher Scientific; Waltham, MA).

[0088] As used in this document, the term DMPK-mediated splicing defects refers to a dysregulation of Petition 870250090423, dated 03 / 10 / 2025, p. 45 / 218 38 / 144 Alternative splicing occurs in tissues affected by type 1 diabetes mellitus (DM1). These splicing defects contribute to the core symptoms of the disease, such as insulin resistance, myotonia, muscle weakness, and cardiac arrhythmia. CUG repeat expansion in DMPK transcripts accumulates in the cell nucleus, impairing the physiological function of proteins involved in transcription, splicing, or RNA export. These aggregations lead to dysregulation of alternative splicing of different transcripts due to alteration of the splicing machinery. In some modalities, genetic transcripts known to have dysregulated splicing in DM1 can be used to measure the effect of treatment with the constructs as described in this document. In some modalities, splicing rescue can be measured.In some modalities, splicing salvage can be approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 100% compared to comparable healthy tissue. In some modalities, the measured genetic transcripts may be any one or more of MBNL1, SOS1, PKM, zTTN, GOGL4, and CLASP1. Splicing can be measured by any one or more techniques known in the art, for example, RNA sequencing (RNA-seq) or platform technology, such as the Nanostring platform.

[0089] As used in this document, the term sense nucleic acid is a nucleic acid comprising a sequence that encodes all or part of a transgene. In some examples, the mRNA for a transgene is a sense nucleic acid.

[0090] As used in this document, an antisense nucleic acid is a nucleic acid sequence that is complementary to a sense nucleic acid. For example, an antisense nucleic acid may be complementary to an mRNA that encodes a transgene. Petition 870250090423, dated 03 / 10 / 2025, page 46 / 218 39 / 144

[0091] As used in this document, the guide region of an RNAi is the RNAi strand that binds the target mRNA, typically based on complementarity. The complementarity region may encompass all or a portion of the guide region. Typically, the complementarity region includes at least the initial region. In many cases, the antisense region of an RNAi is the guide region.

[0092] As used herein, the transient region, or non-guide region, used interchangeably herein, of an RNAi is the region of the RNAi that is complementary to the guide region. In many cases, the sense region of an RNAi is the transient region.

[0093] As used in this document, the start region of an RNAi (e.g., miRNA) is a region of approximately 1-8 nucleotides in length of a microRNA. In some instances, the start region and the 3' UTR of its target mRNA may be a key determinant in RNAi recognition.

[0094] As used in this document, off-target gene silencing refers to the pairing of an early region of an RNAi with sequences in UTRs 3μ of unintended mRNAs and directing translational repression and destabilization of those transcripts (e.g., reducing the expression of the unintended mRNAs).

[0095] The reference to about a value or parameter here includes (and describes) modalities that are directed to that value or parameter per se. For example, the description referring to about X includes the description of X.

[0096] As used in this document, the singular form of the articles a, an, the and includes plural references, unless otherwise indicated.

[0097] It is understood that the aspects and embodiments of the invention described in this document include that it comprises, that Petition 870250090423, dated 03 / 10 / 2025, p. 47 / 218 40 / 144 consists of and / or essentially consists of aspects and modalities. RNAi

[0098] In some respects, the invention provides enhanced RNAi that targets DMPK RNA for the treatment of myotonic dystrophy type 1 (DM1). In some embodiments, the RNAi is a small inhibitory RNA (siRNA), a microRNA (miRNA), or a small hairpin RNA (shRNA). A small inhibitory or interfering RNA (siRNA) is known in the art as a double-stranded RNA molecule of approximately 19-25 (e.g., 19-23) base pairs in length that induces RNAi in a cell. miRNAs are typically smaller than siRNAs, can have multiple targets, and function to repress translation, degrade mRNA, and in some cases, cleave mRNA endonucleolytically. A small hook RNA (shRNA) is known in the art as an RNA molecule comprising approximately 19-25 (e.g., 19-23) double-stranded RNA base pairs linked by a short loop (e.g., about 4-11 nucleotides) that induces RNAi in a cell.In some embodiments, 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 nucleic acid of 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).

[0099] In some forms, the first tape comprises a guide region, wherein the guide region comprises a sequence of acids Petition 870250090423, dated 03 / 10 / 2025, page 48 / 218 41 / 144 of the nucleic acids have more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to 5'-AGUCGAAGACAGUUCUAGGGU3' (SEQ ID NO: 1). In some embodiments, the first strand comprises a guide region, wherein the guide region comprises a nucleic acid sequence with more 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 with more 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 with more 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.

[00100] In some embodiments, the RNAi comprises the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the RNAi comprises a nucleic acid sequence that has more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 7. In some embodiments, the RNAi comprises a nucleic acid sequence that has more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 7, but retains at least one sequence (e.g., in an initial sequence).

[00101] In some embodiments, the 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 guide region. Petition 870250090423, dated 03 / 10 / 2025, p. 49 / 218 42 / 144 gunda fita comprehends a non-guide region. In some embodiments, the nucleic acid encoding RNAi comprises the nucleic acid sequence of SEQ ID NO: 4 and / or the nucleic acid of SEQ ID NO: 5. In some embodiments, the nucleic acid encoding RNAi comprises a nucleic acid sequence that has more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4 and / or a nucleic acid sequence that has more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 5. In some embodiments, RNAi is encoded by the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, RNAi is encoded by a nucleic acid sequence that has more than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% match with SEQ ID NO: 8.

[00102] A microRNA (miRNA) is known in the art as an RNA molecule that induces RNAi in a cell comprising a short sequence (e.g., 19–25 base pairs) of double-stranded RNA linked by a loop and containing one or more additional double-stranded RNA sequences comprising one or more overhangs (e.g., mismatched or unpaired base pairs). As used in this document, the term miRNA encompasses endogenous miRNAs as well as exogenous or heterologous miRNAs. In some embodiments, miRNA may refer to a pri-miRNA or a pre-miRNA. During miRNA processing, a pri-miRNA transcript is produced.The pri-miRNA is processed by Drosha-DGCR8 to produce a pre-miRNA by excising one or more sequences to leave 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 region, and a 3' flanking region. The pre-miRNA is then exported to the cytoplasm and processed. Petition 870250090423, dated 03 / 10 / 2025, page 50 / 218 43 / 144 used by Dicer to yield a siRNA with a guide strand and a non-guide (or passenger) strand. The guide strand is then used by the RISC complex to catalyze gene silencing, for example, by recognizing a target RNA sequence complementary to the guide strand. A further description of miRNAs can be found, for example, in document no. WO 2008 / 150897. Recognition of a target sequence by a miRNA is primarily determined by pairing between the target and the miRNA starter sequence, for example, nucleotides 1-8 (5' to 3') of the guide strand (see, for example, Boudreau, RL et al. (2013) Nucleic Acids Res. 41: e9).

[00103] In the pri / pre-miRNA structure, the guide strand:non-guide strand interface in a duplex is formed in part through complementary base pairing (e.g., Watson-Crick base pairing). However, in some embodiments, this complementary base pairing does not extend throughout the duplex. In some embodiments, a bulge at the interface may exist at one or more nucleotide positions. As used herein, the term bulge may refer to a region of nucleic acid that is not complementary to the nucleic acid opposite it in a duplex. In some embodiments, the bulge is formed when complementary nucleic acid regions bind to each other, while non-complementary core regions do not bind. In some embodiments, the bulge is formed when the two nucleic acid strands positioned between the two complementary regions are of different lengths.As described below, a protrusion can comprise one or more nucleotides.

[00104] During miRNA processing, the miRNA is cleaved at a cleavage site adjacent to the guide:non-guide strand interface, thus releasing the siRNA duplex from the guide and non-guide strands. Petition 870250090423, dated 03 / 10 / 2025, p. 51 / 218 44 / 144 guide. In some embodiments, the miRNA comprises a protrusion on the sense or antisense strand adjacent to the cleavage site. To put it another way, in some embodiments, the miRNA comprises a protrusion on the guide or non-guide strand adjacent to the initial sequence.

[00105] In some embodiments, the miRNA comprises a protrusion on the guide strand opposite the 5' cleavage site of the mature non-guide strand. In some embodiments, the miRNA comprises a protrusion opposite the 5' nucleotide of the non-guide strand. In some embodiments, the miRNA comprises a protrusion on the sense strand opposite the 3' cleavage site of the mature guide strand. In some embodiments, the miRNA comprises a protrusion opposite the 3' nucleotide of the guide strand.

[00106] The safety of RNAi-based therapies can be hampered by the ability of small inhibitory RNAs (siRNAs) to bind to unintended mRNAs and reduce their expression, an effect known as off-target gene silencing. Off-target targeting occurs primarily when the initial region (nucleotides 2-8 of the small RNA) pairs with sequences in 3' UTRs of unintended mRNAs and directs the repression and translational destabilization of these transcripts. Off-target targeting of reduced RNAi can be engineered by replacing bases within the guide and non-guide sequences; for example, by creating CPG motifs.Potential substitutions that could result in a significantly lower off-target score can be evaluated using the SiSPOTR algorithm, a specificity-focused siRNA design algorithm that identifies candidate sequences with off-target targeting potential and potent silencing capabilities (Boudreau et al, NucleicosRes. January 2013; 41(1) e9). A reduced SiSPOTR score predicts sequences that have a me number. Petition 870250090423, dated 03 / 10 / 2025, page 52 / 218 45 / 144 nor of potential off-target human RNAi compared with progenitor RNAi molecules. In some embodiments of the invention, RNAi is enhanced to reduce off-target gene silencing.

[00107] In some embodiments, the first strand and second strand are linked by means of an RNA linker (e.g., an RNA ligand) capable of forming a loop structure. As is commonly known in the art, an RNA loop structure (e.g., a stem-loop or hairpin shape) is formed when an RNA molecule comprises two base-pairing RNA sequences separated by a non-base-pairing RNA sequence. For example, a loop structure may form in the ABC RNA molecule if sequences A and C are complementary or partially complementary, such that they base-pair, but the bases in sequence B do not base-pair.

[00108] In some embodiments, the RNA capable of forming a loop structure comprises from 4 to 50 nucleotides. In certain embodiments, the RNA capable of forming a loop structure comprises 13 nucleotides. In some embodiments, the number of nucleotides in the RNA capable of forming a loop is from 4 to 50 nucleotides or any integer in between. In some embodiments, 0 to 50% of the loop may be complementary to another portion of the loop. As used herein, the term loop structure is a sequence that joins two complementary strands of nucleic acid. In some embodiments, 1-3 nucleotides of the loop structure are contiguous to the complementary strands of nucleic acid and may be complementary to 1-3 nucleotides of the distal portion of the loop structure. For example, the three nucleotides at the 5' end of the loop structure may be complementary to the three nucleotides at the 3' end of the loop structure.

[00109] In some forms, the nucleic acid that codes for a Petition 870250090423, dated 03 / 10 / 2025, p. 53 / 218 46 / 144 The RNAi of this disclosure comprises a heterologous miRNA scaffold. In some embodiments, the use of a heterologous miRNA scaffold is used to modulate miRNA expression; for example, to increase miRNA expression or to decrease miRNA expression. Any miRNA scaffold known in the art may be used. In some embodiments, the miRNA scaffold is derived from a miR-155 scaffold (see, for example, Lagos-Quintana, M. et al. (2002) Curr. Biol. 12:735-9 and the Pol II miR RNAi Invitrogen™ BLOCK-iT™ expression vector kit from Life Technologies, Thermo Fisher Scientific; Waltham, MA) or a mirGE scaffold (WO 2014 / 016817). Methods for treating myotonic dystrophy type 1 (DM-1)

[00110] Myotonic dystrophy type 1 (DM1) is a progressive monogenic, autosomal dominant disease caused by the expansion of CTG repeats (>50) at the DMPK locus (myotonic dystrophy protein kinase). DMPK with repeats is transcribed into mRNA, which forms hairpin shapes and binds RNA-binding proteins, hijacking them from their normal function. This leads to the appearance of nuclear foci, missplicing, and ultimately, myotonia. DM1 primarily affects skeletal, cardiac, and smooth muscle, resulting in significant physical, cognitive, and behavioral impairments and disability.

[00111] In some aspects, the invention provides methods and compositions for treating type 1 myotonic dystrophy (T1D) in a mammal, comprising administering to the mammal a pharmaceutical composition of the present disclosure (for example, a pharmaceutical composition comprising an rAAV particle of the present disclosure). In some aspects, the invention provides methods and compositions for inhibiting DMPK expression in a mammal with T1D, comprising administering to the mammal a pharmaceutical composition of the present disclosure (for example, a pharmaceutical composition that Petition 870250090423, dated 03 / 10 / 2025, page 54 / 218 47 / 144 comprises an rAAV particle of the present disclosure). In some aspects, the invention provides methods and compositions for inhibiting the accumulation of DMPK in a mammalian cell with T1DM, comprising administering to the mammal a pharmaceutical composition of the present disclosure (for example, a pharmaceutical composition comprising an rAAV particle of the present disclosure). In some aspects, the invention provides methods and compositions for attenuating a symptom of T1DM, comprising administering an effective amount of rAAV particles comprising a vector encoding an RNAi of the present disclosure to the brain muscle of a mammal.

[00112] In some aspects, the invention provides an RNAi to target DMPK mRNA in a mammal with T1DM. In some embodiments, the RNAi comprises a first strand comprising a first nucleic acid comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 1) and a second strand comprising a second nucleic acid comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2). An RNAi described herein (e.g., as part of an rAAV vector) may find use, among others, in the treatment of T1DM.

[00113] In some embodiments, RNAi is a small inhibitory RNA (siRNA), a microRNA (miRNA), or a small hairpin RNA (shRNA). A small inhibitory or interfering RNA (siRNA) is known in the art as a double-stranded RNA molecule of approximately 19–25 (e.g., 19–23) base pairs in length that induces RNAi in a cell. miRNAs are typically smaller than siRNAs, can have multiple targets, and function to repress translation, degrade mRNA, and in some cases, cleave mRNA endonucleolytically. A hairpin RNA Petition 870250090423, dated 03 / 10 / 2025, p. 55 / 218 Small RNA (shRNA) is known in the art as an RNA molecule comprising approximately 19-25 (e.g., 19-23) double-stranded RNA base pairs linked by a short loop (e.g., about 4-11 nucleotides) that induces RNAi in a cell.

[00114] In some embodiments, the miRNA comprises a guide sequence that is approximately 90% identical to SEQ ID NO: 1. In some embodiments, the miRNA comprises a guide sequence that is approximately any of 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.

[00115] In some embodiments, the miRNA comprises a non-guide sequence (passing ribbon) that is about 90% identical to SEQ ID NO: 2. In some embodiments, the miRNA comprises a non-guide sequence that is about any one of 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.

[00116] In some embodiments, the first strand and the second strand are linked by RNA capable of forming a loop structure. As is commonly known in the art, an RNA loop structure (e.g., a stem-loop or hairpin shape) is formed when an RNA molecule comprises two base-pairing RNA sequences separated by a non-base-pairing RNA sequence. For example, a loop structure may form in the RNA molecule ABC if sequences A and C are complementary or partially complementary, such that they base-pair, but the bases in sequence B do not base-pair.

[00117] In some embodiments, the RNA capable of forming a loop structure comprises 4 to 50 nucleotides. In certain embodiments Petition 870250090423, dated 03 / 10 / 2025, p. 56 / 218 49 / 144 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 approximately any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 3.

[00118] In some aspects, the invention provides methods comprising administering to a mammal (e.g., a mammal with DM1) an RNAi comprising 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, a recombinant viral particle comprises the RNAi. In some embodiments, the recombinant viral particle is an AAV particle that encapsulates an rAAV vector, wherein the rAAV vector encodes the RNAi.

[00119] In some embodiments, the delivery of rAAV particles is done by systemic injection of rAAV particles into the mammal. In some embodiments, systemic injection is intravenous injection, intra-arterial injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, intra-CSF and intrathecal (TI) administrations.

[00120] In some aspects, the invention provides methods for treating type 1 diabetes in a mammal comprising administering to the mammal the pharmaceutical composition of the present disclosure. In some aspects, the invention provides methods for inhibiting the accumulation of DMPK in a mammalian cell with type 1 diabetes comprising administering to Petition 870250090423, dated 03 / 10 / 2025, page 57 / 218 50 / 144 mammal the pharmaceutical composition of the present disclosure. In some aspects, the invention provides methods for inhibiting DMPK expression in a mammal with DM1 comprising administering to the mammal the pharmaceutical composition of the present disclosure. In some embodiments, the DMPK is a mutant DMPK (e.g., a DMPK comprising more than 37 or more than 50 CTG repeats).

[00121] In some embodiments, the invention provides a method for treating a human with T1DM by administering an effective amount of a pharmaceutical composition comprising an rAAV vector encoding an RNAi of the present disclosure to suppress the activity of a mutant DMPK. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[00122] In some embodiments, the methods comprise administering an effective amount of a pharmaceutical composition comprising an rAAV vector encoding an RNAi of the present disclosure to suppress the activity of a mutant DMPK. In some embodiments, the viral titer of the rAAV particles is at least about any one of 5 χ 1012, 6 χ 1012, 7 χ 1012, 8 χ 1012, 9 χ 1012, 10 χ 1012, 11 χ 1012, 15 χ 1012, 20 χ 1012, 25 χ 1012, 30 χ 1012 or 50 χ 1012 genome copies / mL. In some embodiments, the viral titer of the rAAV particles is about any one of 5 χ 1012a 6 χ 1012. 6 χ 1012a 7 χ 1012. 1012a 10 χ 1012, 10 χ 1012a 11 χ 1012, 11 χ 1012a 15 χ 1012, 15 χ 1012a 20 χ 1012, 20 χ 1012a 25 χ 1012, 25 χ 1012a 30 χ 1012, 30 χ 1012a 50 χ 1012or 50 χ 1012a 100 χ 1012genome copies / mL.In some embodiments, the viral titer of rAAV particles is approximately any one of 5 χ¹⁰¹² to 10 χ¹⁰¹², 10 χ¹⁰¹² to 25 χ¹⁰¹², or 25 χ¹⁰¹² to 50 χ¹⁰¹² genome copies / mL. In some embodiments, the... Petition 870250090423, dated 03 / 10 / 2025, page 58 / 218 51 / 144 viral titer of rAAV particles is at least about any one of 5 χ 109, 6 χ 109, 7 χ 109, 8 χ 109, 9 χ 109, 10 χ 109, 11 χ 109, 15 χ 109, 20 χ 109, 25 χ 109, 30 χ 109 or 50 χ 109 transduction units / mL. In some embodiments, the viral titer of the rAAV particles is about 5 χ 109a 6 χ 109.6 χ 109a 7 χ 109.7 χ 109a 8 χ 109.8 χ 109a 9 χ 109.9 χ 109a 10 χ 109.10 χ 109a 11 χ 109.11 χ 109a 15 χ 109. χ 109a 50 χ 109 or 50 χ 109 to 100 χ 109 transduction units / mL. In some forms, the viral titer of rAAV particles is approximately any of 5 χ 109 to 10 χ 109, 10 χ 109 to 15 χ 109, 15 χ 109 to 25 χ 109, or 25 χ 109 to 50 χ 109 transduction units / mL.In some embodiments, the viral titer of the rAAV particles is at least somewhere around 5 x 1010, 6 x 1010, 7 x 1010, 8 x 1010, 9 x 1010, 10 x 1010, 11 x 1010, 15 x 1010, 20 x 1010, 25 x 1010, 30 x 1010, 40 x 1010 or 50 x 1010 unidades infecciosas / mL. In some embodiments, the viral titer of the rAAV particles is at least any one of about 5 x 1010a 6 x 1010, 6 x 1010a 7 x 1010, 7 x 1010a 8 x 1010, 8 x 1010a 9 x 1010, 9 x 1010a 10 x 1010, 10 x 1010a 11 x 1010, 11 x 1010a 15 x 1010, 15 x 1010a 20 x 1010, 20 x 1010a 25 x 1010, 25 x 1010a 30 x 1010, 30 x 1010a 40 x 1010, 40 x 1010a 50 x 1010ou 50 x 1010a 100 x 1010unidades infecciosas / mL. In some embodiments, the viral titer of the rAAV particles is at least somewhere between 5 x 1010a 10 x 1010, 10 x 1010a 15 x 1010, 15 x 1010a 25 x 1010 or 25 x 1010a 50 x 1010 unidades infecciosas / mL.

[00123] In some embodiments, the dose concentration of rAAV particles administered to the individual is anywhere from about 1 x 10⁸ to about 2 x 10¹³ genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to the individual is anywhere from about 1 x 10⁸ to about 5 x 10⁸, about 5 x 10⁸ to about 10 x 10⁸, about 10 x 10⁸ to about Petition 870250090423, dated 03 / 10 / 2025, p. 59 / 218 52 / 144 from 20 x 108, about 20 x 108 to about 30 x 108, about 30 x 108 to about 40 x 108, about 40 x 108 to about 50 x 108, or about 50 x 108 to about 100 x 108 genome copies / mL. In some modalities, the dose concentration of rAAV particles administered to the individual is any one of approximately 1 x 10⁹ to approximately 5 x 10⁹, approximately 5 x 10⁹ to approximately 10 x 10⁹, approximately 10 x 10⁹ to approximately 20 x 10⁹, approximately 20 x 10⁹ to approximately 30 x 10⁹, approximately 30 x 10⁹ to approximately 40 x 10⁹, approximately 40 x 10⁹ to approximately 50 x 10⁹, or approximately 50 x 10⁹ to approximately 100 x 10⁹ genome copies / mL.In some modalities, the dose concentration of rAAV particles administered to the individual is anywhere from about 1 x 1010 to about 5 x 1010, about 5 x 1010 to about 10 x 1010, about 10 x 1010 to about 20 x 1010, about 20 x 1010 to about 30 x 1010, about 30 x 1010 to about 40 x 1010, about 40 x 1010 to about 50 x 1010, or about 50 x 1010 to about 100 x 1010 genome copies / mL. In some modalities, the concentration of the dose of rAAV particles administered to the individual is approximately 1 x 1011 to approximately 5 x 1011, approximately 5 x 1011 to approximately 10 x 1011, approximately 10 x 1011 to approximately 20 x 1011, approximately 20 x 1011 to approximately 30 x 1011, approximately 30 x 1011 to approximately 40 x 1011, approximately 40 x 1011 to approximately 50 x 1011, or approximately 50 x 1011 to approximately 100 x 1011 genome copies / mL.In some modalities, the dose concentration of rAAV particles administered to the individual is any one of approximately 1 x 1012 to approximately 5 x 1012, approximately 5 x 1012 to approximately 10 x 1012, approximately 10 x 1012 to approximately 20 x 1012, approximately 20 x 1012 to approximately 30 x 1012, approximately 30 x 1012 to approximately 40 x 1012, approximately 40 x 1012 to approximately 50 x 1012, or approximately 50 x 1012 to approximately 100 x 1012 genome copies / mL. In some modalities, the dose concentration of rAAV particles administered to the individual is anywhere from about 1 x 10¹³ to about 2 x 10¹³ genome copies / mL. In some modalities, a. Petition 870250090423, dated 03 / 10 / 2025, p. 60 / 218 53 / 144 The dose concentration of rAAV particles administered to the individual is approximately 1 x 10⁸, approximately 5 x 10⁸, approximately 1 x 10⁹, approximately 5 x 10⁹, approximately 1 x 10¹⁰, approximately 5 x 10¹⁰, approximately 1 x 10¹¹, approximately 5 x 10¹¹, approximately 1 x 10¹², approximately 5 x 10¹², approximately 1 x 10¹³, or approximately 2 x 10¹³ genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to the individual is approximately 5 x 10¹² genome copies / mL. In some embodiments, the dose concentration of rAAV particles administered to the individual is approximately 1 x 10¹³ genome copies / mL. In some modalities, the dose concentration of rAAV particles administered to the individual is approximately 2 x 1013 genome copies / mL.

[00124] In some embodiments, the dose of rAAV particles administered to the individual is at least about any one of 1 x 10⁸ to about 2 x 10¹⁴ genome copies / kg of body weight. In some embodiments, the dose of rAAV particles administered to the individual is between about any one of 1 x 10⁸ to about 2 x 10¹⁴ genome copies / kg of body weight. In some embodiments, the dose of rAAV particles administered to the subject is between about 1 χ 108 to about 1 χ 1014.5 χ 108 to about 1 χ 1014. 1 χ 109 to about 1 χ 1014. χ 1010a about 1 χ 1014, 5 χ 1010a about 1 χ 1014, 1 χ 1011a about 1 χ 1014, 5 χ 1011a about 1 χ 1014, 1 χ 1012a about 1 χ 1014, 5 χ 1012a about 1 χ 1014, 1 χ 1013a about 1 χ 1014, 5 χ 1013a about 1χ 1014. 1 χ 108a about 5 χ 1013. 5 χ 108a about 5 χ 1013. 1013, 5 x 1010a about 5 x 1013, 1 x 1011a about 5 x 1013, 5 x 1011a about 5 x 1013, 1 x 1012a about 5 x 1013, 5 x 1012a about 5 x 1013, 1 x 1013a about 5 x 1013, 1 x 108a about 1 x 1013, 5 x 108a about 1 x 1013, 1 x 109a about 1 x 1013, 1 x 1010a about 1 x 1013, 5 x 1010a about 1 x 1013, Petition 870250090423, de 03 / 10 / 2025, pág. 61 / 218 54 / 144 x 1011a about 1 x 1013, 5 x 1011a about 1 x 1013, 1 x 1012a about 1 x 1013, 5 x 1012a about 1 x 1013, 1 x 108a about 5 x 1012, 5 x 108a about 5 x 1012, 1 x 109a about 5 x 1012, 5 x 109a about 5 x 1012, 1 x 1010a about 5 x 1012, 5 x 1010a about 5 x 1012, 1 x 1011a about 5 x 1012, 5 x 1011a about 5 x 1012, 1 x 1012a about 5 x 1012, 1 x 108a about 1 x 1012, 5 x 108a about 1 x 1012, 1 x 109a about 1 x 1012, 5 x 109a about 1 x 1012, 1 x 1010a about 1 x 1012, 5 x 1010a about 1 x 1012, 1 x 1011a about 1 x 1012, 5 x 1011a about 1 x 1012, 1 x 108a about 5 x 1011, 1 x 109a about 5 x 1011, 5 x 109a about 5 x 1011, 1 x 1010a about 5 x 1011, 5 x 1010a about 5 x 1011, 1 x 1011a about 5 x 1011, 1 x 108a about 1 x 1011, 5 x 108a about 1 x 1011, 1 x 109a about 1 x 1011, 1 x 1010a about 1 x 1011, 5 x 1010a about 1 x 1011,1 χ 108a about 5 χ 1010, 5 χ 108a about 5 χ 1010, 1 χ 109a about 5 χ 1010, 5 χ 109a about 5 χ 1010, 1 χ 1010a about 5 χ 1010, 1 χ 108a about 1 χ 1010, 5 χ 108a about 1 χ 1010, 1 χ 109a about 1 χ 1010, 5 χ 109a about 1 χ 1010, 1 χ 108a about 5 χ 109, 5 χ 108a about 5 χ 109, 1 χ 109a about 5 χ 109, 1 χ 108a about 1 χ 109, 5 χ 108a about 1 χ 109, ou 1 χ 108a about 5 χ 108, gc / kg body weight. In some embodiments, the dose of rAAV particles administered to the individual is about 1 x 109, about 5 x 109, about 1 x 1010, about 5 x 1010, about 1 x 1011, about 5 x 1011, about 1 x 1013, about 5 x 1013, about 1 x 1014or about 2 x 1014 genome copies / kg body weight. In some embodiments, the dose of rAAV particles administered to the individual is about 5 x 1013 genome copies / kg body weight. In some ways,The dose of rAAV particles administered to an individual is approximately 1 x 10¹⁴ genome copies / kg of body weight. In some modalities, the dose of... Petition 870250090423, dated 03 / 10 / 2025, page 62 / 218 55 / 144 particles of rAAV administered to an individual is approximately 2 x 1014 genome copies / kg of body weight.

[00125] In some embodiments, the total amount of rAAV particles administered to the individual is at least about any one of 1 x 10⁹ to about 2 x 10¹⁴ genome copies / kg of body weight. In some embodiments, the total amount of rAAV particles administered to the individual is about 1 x 10⁹ to about 2 x 10¹⁴ genome copies / kg of body weight. In some embodiments of the invention, the volume of the composition injected into the groove is more than about any one of 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 between them.

[00126] The compositions of the invention (for example, rAAV particles comprising a vector encoding an RNAi of the present disclosure) can be used alone or in combination with one or more additional therapeutic agents for the treatment of DM1. The interval between sequential administration can be in terms of at least (or, alternatively, less than) minutes, hours or days.

[00127] In some embodiments, RNAi for treating type 1 diabetes is administered in combination with an immunosuppressive agent; for example, to suppress an immune response to RNAi. In some embodiments, the immunosuppressive agent is administered before the administration of RNAi. In some embodiments, the immunosuppressive agent is administered at the same time as the administration of RNAi. In some embodiments, the immunosuppressive agent is administered after the administration of RNAi. In some embodiments, the immunosuppressive agent is administered in any combination before, during, or after the administration of RNAi.

[00128] In some modalities, rAAV particles are used to treat Petition 870250090423, dated 03 / 10 / 2025, page 63 / 218 56 / 144 DM1s are administered in combination with an immunosuppressive agent; for example, to suppress an immune response to the rAAV particle and / or the transgenic product of the rAAV particle. In some embodiments, the immunosuppressive agent is administered before the administration of the rAAV particle. In some embodiments, the immunosuppressive agent is administered at the same time as the administration of the rAAV particle. In some embodiments, the immunosuppressive agent is administered after the administration of the rAAV particle. In some embodiments, the immunosuppressive agent is administered in any combination before, during, or after the administration of the rAAV particle.

[00129] In some embodiments, the invention provides the use of an effective amount of any of the RNAi described herein in the manufacture of a medicament to treat myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides the use of an effective amount of any of the RNAi described herein in the manufacture of a medicament to inhibit the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 that needs the same. In some embodiments, the invention provides the use of an effective amount of any of the RNAi described herein in the manufacture of a medicament to inhibit the accumulation of DMPK RNA in a cell of a mammal with DM-1 that needs the same.

[00130] In some embodiments, the invention provides the use of an effective amount of any of the RNAi described herein to treat myotonic dystrophy 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides the use of an effective amount of any of the RNAi described herein to inhibit the expression of protein kinase of Petition 870250090423, dated 03 / 10 / 2025, page 64 / 218 57 / 144 myotonic dystrophy (DMPK) in a mammal with type 1 diabetes mellitus (DM-1) that needs it. In some embodiments, the invention provides the use of an effective amount of any of the RNAi described herein to inhibit the accumulation of DMPK RNA in a cell of a mammal with DM-1 that needs it.

[00131] In some embodiments, the invention provides an RNAi described herein to treat myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides any of the RNAi described herein to inhibit the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 that needs it. In some embodiments, the invention provides any of the RNAi described herein to inhibit the accumulation of DMPK RNA in a cell of a mammal with DM-1 that needs it.

[00132] In some embodiments, the invention provides the use of an effective amount of any of the viral particles (e.g., AAV particles) described herein in the manufacture of a medicament to treat myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides the use of an effective amount of any of the viral particles (e.g., AAV particles) described herein in the manufacture of a medicament to inhibit the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 that needs thereof. In some embodiments, the invention provides the use of an effective amount of any of the viral particles (e.g., AAV particles) described herein in the manufacture of a medicament to inhibit the accumulation of DMPK RNA in a cell of a mammal with DM-1 that needs thereof.

[00133] In some embodiments, the invention provides the use of Petition 870250090423, dated 03 / 10 / 2025, page 65 / 218 58 / 144 an effective amount of any of the viral particles (e.g., AAV particles) described herein to treat myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides the use of an effective amount of any of the viral particles (e.g., AAV particles) described herein to inhibit the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 in need thereof. In some embodiments, the invention provides the use of an effective amount of any of the viral particles (e.g., AAV particles) described herein to inhibit the accumulation of DMPK RNA in a cell of a mammal with DM-1 in need thereof.

[00134] In some embodiments, the invention provides viral particles (e.g., AAV particles) described herein to treat myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides any of the viral particles (e.g., AAV particles) described herein to inhibit the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 that needs the same. In some embodiments, the invention provides any of the viral particles (e.g., AAV particles) described herein to inhibit the accumulation of DMPK RNA in a cell of a mammal with DM-1 that needs the same.

[00135] In some embodiments, the invention provides the use of an effective amount of any of the compositions described herein in the manufacture of a medicament for treating type 1 myotonic dystrophy (T1DM) in a mammal in need thereof. In some embodiments, the invention provides the use of an effective amount of any of the compositions described herein. Petition 870250090423, dated 03 / 10 / 2025, page 66 / 218 59 / 144 ment in the manufacture of a medicament to inhibit the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with type 1 diabetes mellitus (DM-1) that requires it. In some embodiments, the invention provides the use of an effective amount of any of the compositions described herein in the manufacture of a medicament to inhibit the accumulation of DMPK RNA in a cell of a mammal with DM-1 that requires it.

[00136] In some embodiments, the invention provides the use of an effective amount of any of the compositions described herein to treat myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides the use of an effective amount of any of the compositions described herein to inhibit the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 that needs thereof. In some embodiments, the invention provides the use of an effective amount of any of the compositions described herein to inhibit the accumulation of DMPK RNA in a cell of a mammal with DM-1 that needs thereof.

[00137] In some embodiments, the invention provides compositions described herein for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof. In some embodiments, the invention provides any of the compositions described herein for inhibiting the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 that needs it. In some embodiments, the invention provides any of the compositions described herein for inhibiting the accumulation of DMPK RNA in a cell of a mammal with DM-1 that needs it. RNAi Expression Constructs and Vectors Petition 870250090423, dated 03 / 10 / 2025, page 67 / 218 60 / 144

[00138] The invention provides expression constructs, vectors and rAAV particles for RNAi expression described in this document.

[00139] In some embodiments, the nucleic acid encoding an RNAi of the present disclosure comprises a heterologous miRNA scaffold. In some embodiments, the use of a heterologous miRNA scaffold is used to modulate miRNA expression; for example, to increase miRNA expression or to decrease miRNA expression. Any miRNA scaffold known in the art may be used. In some embodiments, the miRNA scaffold is derived from a miR-155 scaffold (see, for example, Lagos-Quintana, M. et al. (2002) Curr. Biol. 12:735-9 and the Pol II miR RNAi Invitrogen™ BLOCK-iT™ expression vector kit from Life Technologies, Thermo Fisher Scientific; Waltham, MA) or the mirGE scaffold (WO 2014 / 016817). In some embodiments, the nucleic acid encoding an RNAi of this 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 with more than 80%, 85%, 90%, 95%, or 99% identity to the nucleic acid sequence of SEQ ID NO: 11.

[00140] In some embodiments, RNAi targets RNA encoding a polypeptide associated with DM1 (e.g., mutant DMPK). Without wishing to be overly theoretical, it is believed that RNAi can be used to reduce or eliminate the expression and / or activity of a polypeptide whose gain of function has been associated with DM1 (e.g., mutant DMPK).

[00141] In some embodiments, the transgene (for example, encoding an RNAi of the present disclosure) is operationally linked to a promoter. Exemplary promoters include, however, Petition 870250090423, dated 03 / 10 / 2025, p. 68 / 218 61 / 144 without limitation, the immediate early promoter of cytomegalovirus (CMV), the RSV LTR, the MoMLV LTR, the phosphoglycerate kinase 1 (PGK) promoter, a simian virus 40 (SV40) promoter and a CK6 promoter, a transthyretin (TTR) promoter, a TK promoter, a tetracycline-responsive (TRE) promoter, an HBV promoter, an hAAT promoter, an LSP promoter, liver-specific chimeric promoters (LSPs), the E2F promoter, the telomerase promoter (hTERT); The cytomegalovirus / chicken beta-actin / rabbit β-globin enhancer promoter (CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9) and the elongation factor 1-alpha (EF1-alpha) 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, inducible, or repressible promoter.

[00142] Inducible promoters allow the 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, for example, acute phase, a particular cell differentiation state, or only in replicating cells. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein promoter (MT), the dexamethasone (Dex)-inducible mouse mammary tumor virus promoter (MMTV), the T7 polymerase promoter system (WO 98 / 10088); the insect ecdysone promoter (No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al, Science, 268:1766-1769 (1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518 (1998)), the system induces Petition 870250090423, of 03 / 10 / 2025, page 69 / 218. 62 / 144 vel by RU486 (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are those that are regulated by a specific physiological state, for example, temperature, acute phase, a particular state of cell differentiation or only in replicating cells.

[00143] In some embodiments, regulatory sequences confer tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory sequences link tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the promoter is a desmin promoter. In some embodiments, the promoter is a human desmin promoter (e.g., -228 to +75 of the human desmin gene; e.g., SEQ ID NO: 23). In some embodiments, the promoter is a modified desmin promoter. In some embodiments, the desmin promoter comprises desmin promoter elements important for high-level expression in muscle cells (Li and Paulin, et al. 1991).(Journal of Biol Chem.). 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. Petition 870250090423, dated 03 / 10 / 2025, p. 70 / 218 63 / 144 In some embodiments, the desmin promoter comprises one copy of Byrne's desmin enhancer and one copy of Palin's desmin enhancer (-973 to -693) and the human desmin gene promoter (-228 to +75).

[00144] In some aspects, the invention provides an expression cassette (e.g., and an expression cassette for the expression of 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 the promoter for the human desmin gene. In some embodiments, the desmin promoter comprises two enhancer elements and the promoter for the human desmin gene. In some embodiments, the desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 21.In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 21 and one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence with 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 nu sequence. Petition 870250090423, dated 03 / 10 / 2025, p. 71 / 218 64 / 144 nucleotides with 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 with at least about any one of 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 with at least about any one of 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of SEQ ID NO: 22.

[00145] In some embodiments, the expression cassette comprising the modified desmin promoter further comprises an intron. In some embodiments, the intron is a rabbit β-globin intron. In some embodiments, the intron comprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the intron comprises a nucleotide sequence with at least about 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleic acid encoding the transgene (e.g., a therapeutic transgene) is incorporated into the intron. In some embodiments, the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at 5' of the nucleic acid encoding the transgene and the 3' arm is located at 3' of the nucleic acid encoding the transgene. In some embodiments, the 5' arm of the intron comprises nucleic acid with the sequence SEQ ID NO: 14.In some embodiments, the 5' arm of the intron comprises nucleic acid with a sequence having 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 nucleic acid with the sequence of SEQ ID NO: 15. In some embodiments, the 3' arm of the intron... Petition 870250090423, dated 03 / 10 / 2025, p. 72 / 218 65 / 144 comprises nucleic acid with a sequence that has 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 nucleic acid with the sequence of SEQ ID NO: 14 and the 3' arm of the intron comprises nucleic acid with the sequence of SEQ ID NO: 15. In some embodiments, the 5' arm of the intron comprises nucleic acid with a sequence that has at least about any one of 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of SEQ ID NO: 14, and the 3' arm of the intron comprises nucleic acid with a sequence that has at least about any one of 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of SEQ ID NO: 14. identity to the nucleotide sequence of SEQ ID NO: 15.

[00146] In some embodiments, the expression cassette comprising the 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 nucleic acid with the sequence of SEQ ID NO: 16. In some embodiments, the bovine growth hormone polyadenylation signal comprises nucleic acid with a sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 16.

[00147] In some embodiments, the invention provides an expression cassette comprising a modified desmin promoter for expression of a transgene (e.g., a therapeutic transgene) in a muscle cell. In some embodiments, the transgene encodes a polypeptide (e.g., a therapeutic polypeptide). Petition 870250090423, dated 03 / 10 / 2025, p. 73 / 218 66 / 144 In some embodiments, the transgene encodes a nucleic acid (for example, a therapeutic nucleic acid). In some embodiments, the transgene encodes an RNAi. In some embodiments, the transgene encodes an siRNA, a shRNA, or a miRNA.

[00148] In some aspects, the invention provides a modified desmin promoter, wherein the desmin promoter comprises one or more enhancer elements and the promoter for the human desmin gene. In some embodiments, the desmin promoter comprises two enhancer elements and the promoter for the human desmin gene. In some embodiments, the desmin promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 22.In some embodiments, the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 21 and one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 22. In some embodiments, the desmin promoter comprises one or more enhancer elements comprising a nucleotide sequence with 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 with 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. Petition 870250090423, dated 03 / 10 / 2025, p. 74 / 218 67 / 144 enhancer elements comprising a nucleotide sequence that has 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 that has at least about 80%, 85%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO: 22.

[00149] In some respects, the invention provides rAAV particles comprising a recombinant self-complementary genome (e.g., a self-complementary rAAV vector). AAV viral particles with self-complementary vector genomes and methods of 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 10:2105 to 2111, each of which is incorporated herein by reference in its entirety. A rAAV comprising a self-complementary genome will rapidly form a double-stranded DNA molecule by virtue of its partially complementary sequences (e.g., complementing coding and non-coding strands of a heterologous nucleic acid).In some embodiments, the vector comprises a first nucleic acid sequence encoding the heterologous nucleic acid and a second nucleic acid sequence encoding a nucleic acid complement, wherein the first nucleic acid sequence may form intrastrand base pairs with the second nucleic acid sequence along most or all of its length.

[00150] In some embodiments, the first heterologous nucleic acid sequence encoding an RNAi and a second heterologous nucleic acid sequence encoding the complement of the RNAi are linked by a mutated ITR (e.g., the right ITR). Petition 870250090423, dated 03 / 10 / 2025, p. 75 / 218 68 / 144 In some embodiments, the ITR comprises the polynucleotide sequence 5'CCACTCCCTCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA-3 (SEQ ID NO: 27). The mutated ITR comprises a deletion of the D region comprising the terminal resolution sequence. As a result, upon replication of an AAV viral genome, the rep proteins will not cleave the viral genome at the mutated ITR, and thus a recombinant viral genome comprising the following in order from 5' to 3' will be packaged into a viral capsid: an AAV ITR, the first heterologous polynucleotide sequence including regulatory sequences, a mutated AAV ITR, the second heterologous polynucleotide in reverse orientation to the first heterologous polynucleotide, and a third AAV ITR. rAAV Particles and Methods for Producing rAAV Particles

[00151] The invention provides rAAV particles comprising RNAi as disclosed herein. In some embodiments, the invention provides methods for using recombinant viral particles to deliver RNAi to treat DM1. In some embodiments, the rAAV particle comprises a sequence encoding the RNAi of the present disclosure flanked by one or two ITRs. The nucleic acid is encapsulated in the AAV particle. The AAV particle also comprises capsid proteins. In some embodiments, the nucleic acid comprises the coding sequence(s) of interest (e.g., nucleic acid encoding the RNAi of the present disclosure), components operationally linked in the direction of transcription, control sequences including transcription initiation and termination sequences, thus forming an expression cassette. The expression cassette is Petition 870250090423, dated 03 / 10 / 2025, p. 76 / 218 69 / 144 flanked at the 5' and 3' ends by at least one functional AAV ITR sequence. By functional AAV ITR sequences it is understood that the ITR sequences function as intended for the rescue, replication and packaging of the AAV virion. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated herein in their entirety by reference. For the practice of some aspects of the invention, the recombinant vectors comprise at least all the AAV sequences essential for encapsulation and the physical structures for rAAV infection. AAV ITRs for use in the vectors of the invention need not have a wild-type nucleotide sequence (e.g., as described in Kotin, Hum. Gene Ther.( , 1994, 5:793-801) and can be altered by the insertion, deletion, or substitution of nucleotides or ITRs of AAVs can be derived from any of several AAV serotypes. More than 40 AAV serotypes are currently known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18): 11,854 to 11,856; Gao et al., PNAS, 2003, 100(10):6,081 to 6,086; and Bossis et al., J. Virol., 2003, 77(12):6,799 to 6,810. The use of any AAV serotype is considered within the scope of the present invention. In some embodiments, an rAAV vector is a vector derived from an AAV serotype, including, without limitation, AAV ITRs that are capsid serotype ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVrh74, AAV DJ, a goat AAV, bovine AAV, or mouse AAV, or similar.In some embodiments, the nucleic acid in AAV comprises an ITR among capsid ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AA. Petition 870250090423, dated 03 / 10 / 2025, page 77 / 218 70 / 144 Vrh10, AAV11, AAV12, AAVrh74, AAV DJ, a goat AAV, bovine AAV, or mouse AAV, or similar. In some embodiments, the nucleic acid in the AAV additionally encodes an RNAi as described in this document. For example, the nucleic acid in the AAV may comprise at least one ITR of any AAV serotype contemplated in this document and may additionally 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 AAV may comprise at least one ITR of any AAV serotype and may additionally 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 with 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 with 80%, 85%, 90% or 95% identity to SEQ ID NO: 2.

[00152] In some embodiments, the nucleic acid in the AAV comprises 5' to 3' nucleic acid encoding the following: an ITR (e.g., an AAV2 ITR), a promoter, a nucleic acid encoding an RNAi as disclosed in this document, a polyadenylation signal, and an AAV ITR (e.g., an AAV2 ITR). In some embodiments, the nucleic acid in the AAV comprises 5' to 3' nucleic acid encoding the following: an ITR (e.g., an AAV2 ITR), a promoter, a nucleic acid encoding an RNAi comprising a 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 with SEQ ID NO: 1 and a se Petition 870250090423, dated 03 / 10 / 2025, p. 78 / 218 71 / 144 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, a polyadenylation signal and an AAV ITR (e.g., an AAV2 ITR). In some embodiments, the nucleic acid in the AAV comprises 5' to 3' nucleic acid encoding the following: an ITR (e.g., an AAV2 ITR), a desmin promoter, a nucleic acid encoding an RNAi as disclosed herein, a polyadenylation signal (e.g., a bovine growth hormone polyA) and an AAV ITR (e.g., an AAV2 ITR).In some embodiments, the nucleic acid in AAV comprises 5' to 3' 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 comprising a 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, and a 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, a polyadenylation signal (e.g., a bovine growth hormone polyA) and an AAV ITR (e.g., an AAV2 ITR). In some embodiments, the first strand and the second strand form a duplex. In some embodiments, the first strand is linked to the second strand by a linker.In some embodiments, the ligand comprises the nucleic acid sequence of SEQ ID NO: 3 or a sequence with 80%, 85%, 90%, or 95% identity with SEQ ID NO: 3.

[00153] In some modalities, the nucleic acid in the AAV with Petition 870250090423, dated 03 / 10 / 2025, p. 79 / 218 72 / 144 comprises 5' to 3' nucleic acid encoding the following: all or a functional portion of an ITR (e.g., an AAV2 ITR), a filler sequence (e.g., all or a portion of a human alpha-1-antitrypsin (AAT) filler sequence), a desmin promoter, a 5' arm of an intron (e.g., a rabbit β-globin intron), a nucleic acid encoding an RNAi comprising a 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 with 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 with 80%, 85%, 90%, or 95% identity with SEQ ID NO: 2, a 3' arm of an intron (e.g., a rabbit β-globin intron), a polyadenylation signal (e.g., a bovine growth hormone polyA),a filler sequence (e.g., all or a portion of a human alpha-1-antitrypsin (AAT) filler sequence) and an AAV ITR (e.g., an AAV2 ITR).

[00154] In some embodiments, the nucleic acid in AAV comprises 5' to 3' nucleic acid encoding the following: an ITR (e.g., an AAV2 ITR), a desmin promoter, a nucleic acid encoding an RNAi comprising a 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 with SEQ ID NO: 2 and a 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 with SEQ ID NO: 1, a polyadenylation signal (e.g., a Petition 870250090423, dated 03 / 10 / 2025, p. 80 / 218 73 / 144 bovine growth hormone polyA) and an AAV ITR (e.g., an AAV2 ITR). In some embodiments, the first strand and the second strand form a duplex. In some embodiments, the first strand is linked to the second strand by a linker. In some embodiments, the linker comprises the nucleic acid sequence SEQ ID NO: 6.

[00155] In some embodiments, the nucleic acid in AAV comprises 5' to 3' nucleic acid encoding the following: all or a functional portion of an ITR (e.g., an AAV2 ITR), a filler sequence (e.g., all or a portion of a human alpha-1-antitrypsin (AAT) filler sequence), a desmin promoter, a 5' arm of an intron (e.g., a rabbit β-globin intron), a nucleic acid encoding an RNAi comprising a first strand comprising a first nucleic acid comprising the sequence 5'-ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2), and a second strand comprising a second nucleic acid comprising the sequence 5'AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1), a 3' arm of an intron (e.g., a rabbit β-globin intron), a polyadenylation signal (e.g., a bovine growth hormone polyA), a filling sequence (e.g.,all or a portion of a human alpha-1-antitrypsin (AAT) filling sequence and an AAV ITR (e.g., an AAV2 ITR).

[00156] In some embodiments, a vector may include one (or more) filler nucleic acids. In some embodiments, the filler nucleic acid may comprise a sequence encoding a reporter polypeptide. As will be understood by those skilled in the art, the filler nucleic acid may be located in a variety of regions within the vector, and may be composed of a continuous sequence (e.g., a single Petition 870250090423, dated 03 / 10 / 2025, page 81 / 218 74 / 144 filler nucleic acid at a single site) or multiple sequences (e.g., more than one filler nucleic acid at more than one site (e.g., 2 sites, 3 sites, etc.) within the vector. In some embodiments, the filler nucleic acid may be located downstream of the RNAi sequence. In other embodiments, the filler nucleic acid may be located upstream of the RNAi sequence (e.g., between the promoter and the nucleic acid encoding RNAi). As will also be understood by those skilled in the art, a variety of nucleic acids may be used as a filler nucleic acid. In some embodiments, the filler nucleic acid comprises all or a portion of a human alpha-1-antitrypsin (AAT) filler sequence or a chromosome 16 clone P1 (human C16) C16 P1 filler sequence.For example, the filler sequence comprises a portion of the human AAT sequence. One skilled in the art would recognize that different portions of a gene (e.g., the human AAT sequence) can be used as a filler fragment. For example, the filler fragment can be from the 5' end of the gene, the 3' end of the gene, the middle of a gene, a non-coding portion of the gene (e.g., an intron), a coding region of the gene (e.g., an exon), or a mixture of coding and non-coding portions of a gene. One skilled in the art would also recognize that all or a portion of the filler sequence can be used as a filler sequence. In some embodiments, the vector comprises a 5' filler sequence comprising the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence with more than about 80%, 85%, 90%, 95%, or 99% of it. Petition 870250090423, dated 03 / 10 / 2025, p. 82 / 218 75 / 144 identity to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the vector comprises a 3' filler sequence comprising the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence with more 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' filler sequence comprising the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence with more than about 80%, 85%, 90%, 95% or 99% identity to the nucleotide sequence of SEQ ID NO: 18 and comprises a 3' filler sequence comprising the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence. with more than approximately 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of SEQ ID NO: 19.

[00157] In other embodiments, the rAAV particle comprises a capsid protein comprising AAVrh74 capsid proteins or variants thereof. In some embodiments, the AAVrh74 capsid is a variant capsid. In some embodiments, the AAVrh74 variant capsid protein retains the ability to form an AAV capsid. In some embodiments, the AAVrh74 variant capsid is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% identical to a wild-type AAVrh74 capsid protein. In some embodiments, the AAVrh74 variant capsid comprises a substitution at the N502 amino acid position. In some embodiments, the substitution at the N502 amino acid position is an isoleucine (I). In some embodiments, the AAVrh74 variant capsid protein comprises a substitution at the W505 amino acid position.In some sports, the substitution in the position of... Petition 870250090423, dated 03 / 10 / 2025, p. 83 / 218 76 / 144 amino acid W505 is an arginine (R).

[00158] Different AAV serotypes are used to optimize transduction of specific target cells or to target specific cell types within a specific target tissue (e.g., a diseased tissue, e.g., muscle tissue). An rAAV particle may comprise viral proteins and viral nucleic acids of the same serotype or a mixed serotype. For example, in some embodiments, an rAAV particle may comprise AAVrh74 capsid proteins or variants thereof and at least one ITR of a different AAV serotype. In some embodiments, the rAAV particle may comprise AAVrh74 capsid proteins or variants thereof and at least one AAV2 ITR. In some embodiments, the rAAV particle comprises AAVrh74N502I capsid proteins and at least one AAV2 ITR. In some embodiments, the rAAV particle comprises AAVrhW505R capsid proteins and at least one AAV2 ITR.

[00159] In some respects, the invention provides viral particles comprising a recombinant self-complementary genome. rAAV particles with self-complementary genomes and methods of 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 10:2105 to 2111, each of which is incorporated herein by reference in its entirety. An rAAV comprising a self-complementary genome will rapidly form a double-stranded DNA molecule by virtue of its partially complementary sequences (e.g., complementing coding and non-coding strands of a transgene). In some embodiments, the invention provides an rAAV particle comprising an AAV genome, wherein the rAAV genome comprises a primary... Petition 870250090423, dated 03 / 10 / 2025, page 84 / 218 77 / 144 a heterologous polynucleotide sequence (e.g., an RNAi of the present disclosure) and a second heterologous polynucleotide sequence (e.g., an antisense strand of an RNAi of the present disclosure) wherein the first heterologous polynucleotide sequence can form intrastrand base pairs with the second polynucleotide sequence along most or all of its length. In some embodiments, the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a sequence that facilitates intrastrand base pairing; for example, a hairpin DNA structure. Hairpin structures are known in the art, for example, in miRNA or siRNA molecules. In some embodiments, the first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a silenced ITR (e.g., the correct ITR).In some embodiments, the ITR comprises the polynucleotide sequence 5'CCACTCCCTCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA-3 (SEQ ID NO: 27). The mutated ITR comprises a deletion of the D region comprising the terminal resolution sequence. As a result, upon replication of an AAV viral genome, the rep proteins will not cleave the viral genome at the mutated ITR, and thus a recombinant viral genome comprising the following in order from 5' to 3' will be packaged into a viral capsid: an AAV ITR, the first heterologous polynucleotide sequence including regulatory sequences, a mutated AAV ITR, the second heterologous polynucleotide in reverse orientation to the first heterologous polynucleotide, and a third AAV ITR. In some embodiments, the invention provides AAV viral particles comprising a re-viral genome. Petition 870250090423, dated 03 / 10 / 2025, p. 85 / 218 78 / 144 combinator comprising a functional AAV2 ITR, a first polynucleotide sequence encoding an RNAi of the present disclosure, a mutated AAV2 ITR comprising a D-region deletion and lacking a functional terminal resolution sequence, a second polynucleotide sequence comprising the sequence complementary to the RNAi-encoding sequence of the present disclosure, the first polynucleotide sequence, and a functional AAV2 ITR.

[00160] rAAV particles can be produced using methods known in the art. See, for example, US Patent Nos. 6,566,118; 6,989,264; and 6,995,006. In the practice of the invention, host cells for the production of rAAV particles include mammalian cells, insect cells, plant cells, microorganisms, and yeasts. Host cells can also be packaging cells in which the AAV rep and cap genes are stably maintained in the host cell or producer cells in which the AAV vector genome is stably maintained. Exemplary packaging and producer cells are derived from 293, A549, or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.

[00161] Known methods in the art for producing rAAV vectors include, but are not limited to, transfection, stable cell line production, and infectious hybrid virus production systems that include adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789), and baculovirus-AAV hybrids. rAAV production cultures for the production of rAAV virus particles require: 1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect-derived cell lines such as SF-9, in the case of production systems. Petition 870250090423, dated 03 / 10 / 2025, page 86 / 218 79 / 144 baculovirus infection; 2) suitable helper virus function, provided by wild-type or mutant adenovirus (such as temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct that provides helper functions; 3) AAV rep and cap genes and gene products; 4) a nucleic acid (such as a therapeutic nucleic acid) flanked by at least one AAV ITR sequence; and 5) suitable media and media components to support rAAV production. In some embodiments, the AAV rep and cap gene products may be of any AAV serotype. Generally, but not necessarily, the AAV rep gene product is of the same serotype as the ITRs of the rAAV vector genome, provided that the rep gene products can function to replicate and package the rAAV genome. Suitable media known in the art may be used for the production of rAAV vectors.These media include, without limitation, media produced by HyClone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco Modified Eagle Medium (DMEM), customized formulations as described in U.S. Patent No. 6,566,118 and SFM Sf-900 II medium as described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to customized media formulations for use in the production of recombinant AAV vectors. In some embodiments, the AAV auxiliary functions are provided by adenovirus or HSV. In some embodiments, the AAV auxiliary functions are provided by baculovirus, and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cells).

[00162] In some embodiments, rAAV particles can be produced by a triple transfection method, such as the exemplary triple transfection method provided below. Briefly, a plasmid containing a rep gene and a capsid gene, jun Petition 870250090423, dated 03 / 10 / 2025, page 87 / 218 80 / 144 directly with a helper adenoviral plasmid, it can be transfected (e.g., using the calcium phosphate method) into a cell line (e.g., HEK-293 cells) and the virus can be collected and optionally purified. Thus, in some embodiments, the rAAV particle was produced by triple transfection of a nucleic acid encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding the functions of AAV helper viruses into a host cell, wherein transfection of the nucleic acids into the host cells generates a host cell capable of producing rAAV particles.

[00163] In some embodiments, rAAV particles can be produced by a producer cell line method, such as the exemplary producer cell line method provided below (see also (mentioned in Martin et al., (2013) 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 rep gene, a capsid gene, and a heterologous promoter nucleic acid sequence. Cell lines can be screened to select a master clone for rAAV production, which can then be expanded into a production bioreactor and infected with an adenovirus (e.g., a wild-type adenovirus) as an auxiliary to initiate rAAV production. The virus can subsequently be collected, the adenovirus can be inactivated (e.g., by heat) and / or removed, and the rAAV particles can be purified.Thus, in some embodiments, the rAAV particle was produced by a producer cell line comprising one or more nucleic acids encoding the rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding AAV helper virus functions. Petition 870250090423, dated 03 / 10 / 2025, page 88 / 218 81 / 144

[00164] In some aspects, a method is provided for producing any rAAV particle as disclosed herein comprising (a) culturing a host cell under a condition in which rAAV particles are produced, wherein the host cell comprises (i) one or more AAV packaging genes, wherein each said AAV packaging gene encodes an AAV replication and / or encapsulation protein; (ii) an rAAV provector comprising a nucleic acid encoding an RNAi of the present disclosure as described herein flanked by at least one AAV ITR and (iii) an AAV helper function; 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, at least one AAV ITR is selected from the group consisting of 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, an ITR of a capsid serotype of goat AAV, bovine AAV, or mouse AAV, or similar. In some embodiments, the capsid protein is selected from the group consisting of AAVrh74, or a variant thereof, such as AAVrh74 N502I or AAVrh74 W505R. In some embodiments, the capsid protein is an AAVrh74 protein. In some embodiments, the capsid protein is a variant of an AAVrh74 protein. In some embodiments, the capsid protein is an AAVrh74N502I protein. In some embodiments, the capsid protein is an AAVrh74W505R protein. In some embodiments, the variant AAVrh74 capsid protein retains the ability to form an AAV capsid.In some embodiments, rAAV particles comprise an AAVrh74 N502I capsid and a recombinant genome that comprises... Petition 870250090423, dated 03 / 10 / 2025, page 89 / 218 82 / 144 ende AAV2 ITRs and nucleic acid encoding an RNAi of the present disclosure. In some embodiments, the rAAV particles comprise an AAVrh74 W505R capsid and a recombinant genome comprising AAV2 ITRs and nucleic acid encoding an RNAi of the present disclosure. In a further embodiment, the rAAV particles are purified. The term purified, as used herein, includes a preparation of rAAV particles devoid of at least some of the other components that may also be present where rAAV particles occur naturally or are initially prepared. Thus, for example, isolated rAAV particles may be prepared using a purification technique to enrich them from a source mixture, such as a culture lysate or production culture supernatant.Enrichment can be measured in a variety of ways, such as, for example, by the proportion of DNase-resistant particles (DRPs) or genome copies (cg) present in a solution, or by infectivity, or it can be measured in relation to a second potentially interfering substance present in the source mixture, such as contaminants, including auxiliary culture contaminants or in-process contaminants, including helper viruses, media components and the like.

[00165] Pharmaceutical compositions comprising a rAAV particle containing a transgene encoding an RNAi of the present disclosure and a pharmaceutically acceptable carrier are also provided in this document. The pharmaceutical compositions may be suitable for any route of administration described in this document. A pharmaceutical composition of a rAAV particle comprising a nucleic acid encoding an RNAi of the present disclosure may be introduced systemically. For example, a recombinant viral particle that purchases Petition 870250090423, dated 03 / 10 / 2025, p. 90 / 218 83 / 144 where a nucleic acid encoding an RNAi of the present disclosure can be administered intravenously, intra-arterially, subcutaneously, or periperitoneally.

[00166] In some embodiments, pharmaceutical compositions comprising a recombinant viral particle comprising a transgene encoding an RNAi of the present disclosure described herein and a pharmaceutically acceptable carrier are suitable for administration to humans. Such carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pages 1035-1038 and 1570-1580). In some embodiments, pharmaceutical compositions comprising a recombinant AAV described herein and a pharmaceutically acceptable carrier are suitable for systemic injection into a mammal.

[00167] Such pharmaceutically acceptable carriers may be sterile liquids, such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil and the like. Saline solutions and aqueous solutions of dextrose, polyethylene glycol (PEG) and glycerol may also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical composition may also include additional ingredients, for example, preservatives, buffers, tonicity agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, viscosity-increasing agents and the like. The pharmaceutical compositions described herein may be packaged in unit doses or in multi-dose forms. The compositions are generally formulated as a sterile and substantially isotonic solution. Manufacturing Supplies and Kits

[00168] Manufacturing kits or articles are also provided for Petition 870250090423, dated 03 / 10 / 2025, page 91 / 218 84 / 144 use in the methods described herein. In aspects, the kits comprise the compositions described herein (for example, an rAAV particle of the present disclosure comprising nucleic acid encoding an RNAi of the present disclosure) in suitable packaging. Suitable packaging for the compositions described herein are known in the art and include, for example, vials (such as sealed vials), vessels, ampoules, bottles, jars, flexible packaging (for example, sealed Mylar or plastic bags) and the like. These articles of manufacture may also be sterilized and / or sealed.

[00169] The present invention also provides kits comprising compositions described herein and may further include instruction(s) on methods of use of the composition, such as the uses described herein. The kits described herein may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein. For example, in some embodiments, the kit comprises a recombinant viral particle composition comprising a transgene encoding an RNAi of the present disclosure for delivering an effective quantity of rAAV particles to a mammal, a pharmaceutically acceptable carrier suitable for injection into the mammal, and one or more of: a buffer, a diluent, a filter, a needle, a syringe, and a package insert with instructions for performing injections into the mammal.In some embodiments, the kit includes instructions for treating type 1 diabetes with the rAAV particles described in this document. In some embodiments, the kit includes instructions for using the rAAV particles described in this document according to any of the methods described in this document. Petition 870250090423, dated 03 / 10 / 2025, p. 92 / 218 85 / 144 ment. EXEMPLARY MODALITIES

[00170] The invention includes the following enumerated exemplary embodiments.

[00171] 1. A recombinant adeno-associated virus (rAAV) particle comprising: a) an RNAi comprising a first strand and a second strand, wherein: i) the first tape and the second tape form a duplex; ii) the first strand comprises a guide region, wherein the guide region comprises nucleic acid with the sequence 5'AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) or with a sequence approximately 90% identical to the sequence of SEQ ID NO: 1; and iii) the second strand comprises a non-guide region; and b) an AAV capsid comprising an amino acid sequence with approximately 90% identity to a wild-type AAVrh74 capsid.

[00172] 2. The rAAV particle of embodiment 1, in which the non-guide region comprises nucleic acid with the sequence 5' ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2) or with a sequence with approximately 90% identity to the sequence of SEQ ID NO: 2.

[00173] 3. The rAAV particle of embodiment 1 or 2, wherein the first strand comprises nucleic acid with the sequence SEQ ID NO: 1, and the non-guide region comprises nucleic acid with the sequence SEQ ID NO: 2.

[00174] 4. The rAAV particle of any of the embodiments 1 to 3, in which the first strand and the second strand are linked by means of an RNA linker capable of forming a loop structure.

[00175] 5. The rAAV particle of embodiment 4, in which the ligand Petition 870250090423, dated 03 / 10 / 2025, p. 93 / 218 RNA (86 / 144) comprises approximately 4 to approximately 50 nucleotides.

[00176] 6. The rAAV particle of embodiment 4 or 5, in which the loop structure comprises about 4 to about 20 nucleotides.

[00177] 7. The rAAV particle of any of the embodiments 4 to 6, wherein the loop structure comprises the nucleic sequence with SEQ ID NO: 3 or with a sequence with approximately 90% identity to the sequence with SEQ ID NO: 3.

[00178] 8. The rAAV particle of any of the claims to 7, wherein the RNAi comprises 5' to 3' the second strand, the RNA linker and the first strand.

[00179] 9. The rAAV particle of any of the embodiments 4 to 7, in which the RNAi comprises 5' to 3' of the first strand, the RNA linker and the second strand.

[00180] 10. The rAAV particle of any of the embodiments in 8, wherein the RNAi comprises nucleic acid with the sequence SEQ ID NO: 7 or with a sequence approximately 90% identical to the sequence SEQ ID NO: 7.

[00181] 11. The rAAV particle of any one of claims 1 to 10, wherein the RNAi is a small inhibitory RNA (siRNA), a microRNA (miRNA) or a small hairpin RNA (shRNA).

[00182] 12. The rAAV particle of any of the embodiments in 11, in which RNAi also comprises a framework.

[00183] 13. The rAAV particle of embodiment 12, wherein the framework comprises all or a portion of the nucleic acid of SEQ ID NO: 11.

[00184] 14. The rAAV particle of embodiment 13, in which the miRNA is incorporated into the scaffold.

[00185] 15. The rAAV particle of embodiment 14, in which the framework has an arm 5', in which the arm 5' is located at 5' of Petition 870250090423, dated 03 / 10 / 2025, p. 94 / 218 87 / 144 nucleic acid that encodes RNAi, and a 3' arm, in which the 3' arm is located at 3' of the nucleic acid that encodes RNAi.

[00186] 16. The rAAV particle of any of claims 12 to 15, wherein the framework is a miR-155 framework.

[00187] 17. The rAAV particle of any of claims 12 to 16, wherein the miR-155 scaffold comprises the nucleic acid of SEQ ID NO: 9 or a sequence with about 90% identity to the sequence of SEQ ID NO: 9 located at 5' of RNAi.

[00188] 18. The rAAV particle of any one of claims 12 to 17, wherein the miR-155 scaffold comprises the nucleic acid of SEQ ID NO: 10 or a sequence with about 90% identity to the sequence of SEQ ID NO: 10 located at 3' of RNAi.

[00189] 19. The rAAV particle of any of the modalities in 18, in which RNAi directs RNA encoding a polypeptide associated with myotonic dystrophy 1 (DM1).

[00190] 20. The rAAV particle of modality 19, in which the polypeptide is dystrophic myotonic protein kinase (DMPK).

[00191] 21. The rAAV particle of modality 20, in which the DMPK comprises a mutation associated with DM-1.

[00192] 22. The rAAV particle of the 20 or 21 modality, in which the gene encoding DMPK comprises five or more CTG trinucleotide repeats.

[00193] 23. An expression cassette comprising nucleic acid encoding RNAi of any of the embodiments 1 to 22.

[00194] 24. The modality 23 expression cassette, in which the nucleic acid encoding RNAi is operationally linked to a promoter.

[00195] 25. The modality 24 expression cassette, in which the promoter is a muscle-specific promoter.

[00196] 26. The expression cassette of modality 24 or 25, in Petition 870250090423, dated 03 / 10 / 2025, page 95 / 218 88 / 144 that the promoter is a demining promoter or a variant thereof.

[00197] 27. The modality 26 expression cassette, in which the desmin promoter comprises one or more enhancer elements and the human desmin gene promoter.

[00198] 28. The expression cassette of modality 26 or 27, in which the desmin promoter comprises two enhancer elements and the human desmin gene promoter.

[00199] 29. The expression cassette of any of claims 26 to 28, in which the demining promoter comprises one or more Byrne intensifying elements and / or one or more Paulin intensifying elements.

[00200] 30. The expression cassette of embodiments 26 to 29, wherein the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence with approximately 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 approximately 90% identity to the sequence of SEQ ID NO: 22.

[00201] 31. The expression cassette of any of the embodiments 26 to 30, wherein the desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence with approximately 90% identity to the nucleotide sequence of SEQ ID NO: 12.

[00202] 32. The expression cassette of any of the embodiments 23 to 31, wherein the expression cassette further comprises an intron.

[00203] 33. The modality 32 expression cassette, where the intron is a rabbit β-globin intron.

[00204] 34. The expression cassette of modality 32 or 33, where the intron comprises the nucleotide sequence of SEQ ID NO: Petition 870250090423, dated 03 / 10 / 2025, p. 96 / 218 89 / 144 or a sequence with approximately 90% identity to the sequence SEQ ID NO: 13.

[00205] 35. The expression cassette of any of the modalities 32-34, in which the nucleic acid encoding RNAi is incorporated into the intron.

[00206] 36. The expression cassette of modality 35, wherein the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at 5' of the nucleic acid encoding RNAi and the 3' arm is located at 3' of the nucleic acid encoding RNAi.

[00207] 37. The expression cassette of modality 36, wherein the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 14 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 14.

[00208] 38. The expression cassette of modality 36 or 37, in which the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 15 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 15.

[00209] 39. The expression cassette of any of the modalities 23 to 38, in which the expression cassette also includes a polyadenylation signal.

[00210] 40. The modality 39 expression cassette, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or an HSV TK pA.

[00211] 41. The expression cassette of modality 40, in which the polyadenylation signal is a minimal polyadenylation signal of bovine growth hormone.

[00212] 42. The expression cassette of any of the modalities 39 to 41, in which the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID Petition 870250090423, dated 03 / 10 / 2025, p. 97 / 218 90 / 144 NO: 16 or a sequence with approximately 90% identity to the sequence with SEQ ID NO: 16.

[00213] 43. The expression cassette of any one of claims 23 to 42, wherein the expression cassette comprises the nucleotide sequence of SEQ ID NO: 17 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 17.

[00214] 44. Expression cassette, comprising a modified desmin promoter, with the modified desmin promoter comprising one or more enhancer elements and the human desmin gene promoter.

[00215] 45. The modality 44 expression cassette, in which the modified desmin promoter comprises two enhancer elements and the human desmin gene promoter.

[00216] 46. The expression cassette of modality 44 or 45, in which the modified demining promoter comprises one or more Byrne enhancer elements and / or one or more Paulin enhancer elements.

[00217] 47. The expression cassette of any of claims 44 to 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.

[00218] 48. The expression cassette of any of the embodiments 44 to 47, in which the desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence with approximately 90% identity to the nucleotide sequence of SEQ ID NO: 12. Petition 870250090423, dated 03 / 10 / 2025, pp. 98 / 218 91 / 144

[00219] 49. The expression cassette of any of the modalities 44 to 48, in which the expression cassette also comprises an intron.

[00220] 50. The expression cassette of modality 49, where the intron is a rabbit β-globin intron.

[00221] 51. The expression cassette of modality 49 or 50, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 13 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 13.

[00222] 52. The expression cassette of any of the modalities 44 to 51, in which the nucleic acid encoding the transgene is incorporated into the intron.

[00223] 53. The expression cassette of modality 52, wherein the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at 5' of the nucleic acid encoding the transgene and the 3' arm is located at 3' of the nucleic acid encoding the transgene.

[00224] 54. The expression cassette of modality 53, wherein the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 14 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 14.

[00225] 55. The expression cassette of modality 53 or 54, in which the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 15 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 15.

[00226] 56. The expression cassette of any of the modalities 44-55, in which the expression cassette also includes a polyadenylation signal.

[00227] 57. The expression cassette of modality 56, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or an HSV signal. Petition 870250090423, dated 03 / 10 / 2025, page 99 / 218 92 / 144 TK pA.

[00228] 58. The expression cassette of modality 57, in which the polyadenylation signal is a minimal polyadenylation signal of bovine growth hormone.

[00229] 59. The expression cassette of any of the embodiments 56 to 58, in which the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO: 16 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 16.

[00230] 60. The expression cassette of any of the modalities 44 to 59, in which the transgene encodes a polypeptide or a nucleic acid.

[00231] 61. The expression cassette of any of the modalities 44 to 60, in which the transgene encodes an RNAi.

[00232] 62. A vector comprising the expression cassette of any of the modalities 23 to 61.

[00233] 63. The modality 62 vector, in which the expression cassette is flanked by one or more filler nucleic acid sequences.

[00234] 64. The vector of modality 63, in which one or more filler nucleic acid sequences are derived from the human SerpinA1 gene.

[00235] 65. The vector of modality 63 or 64, in which a filler nucleic acid sequence located at 5' of the expression cassette is derived from the human SerpinA1 gene.

[00236] 66. The vector of any of the embodiments 63 to 65, in which the fill sequence located at 5' of the expression cassette comprises the nucleotide sequence of SEQ ID NO: 18 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 18. Petition 870250090423, dated 03 / 10 / 2025, p. 100 / 218 93 / 144

[00237] 67. The vector of any of the embodiments 63 to 66, in which a filler nucleic acid sequence located at 3' of the expression cassette is derived from the human SerpinA1 gene.

[00238] 68. The vector of any of the embodiments 63 to 67, in which the filler sequence located at 3' of the expression cassette comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 19.

[00239] 69. The vector of any of embodiments 62 to 68, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

[00240] 70. The rAAV vector of embodiment 69, in which the expression cassette is flanked by one or more AAV inverted terminal repeat (ITR) sequences.

[00241] 71. The rAAV vector of modality 70, in which the expression cassette is flanked by two AAV ITRs.

[00242] 72. The rAAV vector of modality 70 or 71, in which the AAV ITRs are ITRs of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV.

[00243] 73. The rAAV vector of any of the modalities 70 to 72, where the AAV ITRs are AAV2 ITRs.

[00244] 74. The rAAV vector of any of claims 69 to 73, wherein the rAAV vector comprises the nucleotide sequence of SEQ ID NO: 20 or a sequence with approximately 90% identity to the sequence of SEQ ID NO: 20.

[00245] 75. The rAAV vector of any of claims 69 to 74, wherein the vector is a self-complementary rAAV vector.

[00246] 76. A cell comprising the expression cassette of any of the modalities 23 to 61, the vector of any of the Petition 870250090423, dated 03 / 10 / 2025, pp. 101 / 218 94 / 144 modalities 62 to 68, or the rAAV vector of any of the modalities 69 to 75.

[00247] 77. The rAAV particle of any of the embodiments to 22, wherein the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 502.

[00248] 78. The rAAV particle of embodiment 77, in which the amino acid substitution at position 502 is an isoleucine (I).

[00249] 79. The rAAV particle of embodiment 77 or 78, wherein the rAAV particle comprises a capsid of serotype AAVrh74 N502I.

[00250] 80. The rAAV particle of modality 77 or 78, in which the ITR is an AAV2 ITR, and the capsid of the rAAV particle is a capsid of the AAVrh74 N502I serotype.

[00251] 81. The rAAV particle of any of the embodiments in 22, wherein the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 505.

[00252] 82. The rAAV particle of embodiment 81, in which the amino acid substitution at position 505 is an arginine (R).

[00253] 83. The rAAV particle of any of the embodiments in 22, wherein the rAAV particle comprises a capsid of serotype AAVrh74 W505R.

[00254] 84. The rAAV particle of embodiment 83, wherein the ITR is an AAV2 ITR and the rAAV particle capsid is a capsid of the AAVrh74 W505R serotype.

[00255] 85. An rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises the following nucleic acids from 5' to 3', an ITR of AAV2, a nucleic acid encoding a sequence of filler nucleic acids from Petition 870250090423, dated 03 / 10 / 2025, page 102 / 218 95 / 144 of the human serpinAI 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, a miR155 terminal loop sequence, a DMPK204 miRNA passenger sequence, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a bovine growth hormone polyadenylation minimal sequence, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene and an AAV2 ITR; and wherein the capsid is an AAVrh74 N502I capsid.

[00256] 86. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR comprising the polynucleotide sequence SEQ ID NO: 43, a nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence SEQ ID NO: 18, a Byrne desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 21, a Paulin desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 22, a desmin promoter comprising the polynucleotide sequence SEQ ID NO: 23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 14, a sequence of 5' miR155 framework comprising the polynucleotide sequence SEQ ID NO: 40, a miRNA guide sequence DMPK204 comprising the polynucleotide sequence SEQ ID NO: 4,a terminal loop sequence of miR155 comprising the polynucleotide sequence of SEQ ID NO: 6, a miRNA DMPK204 passenger sequence comprising the polynucleotide sequence of SEQ ID NO: 5, a 3' scaffold sequence, Petition 870250090423, dated 03 / 10 / 2025, page 103 / 218 96 / 144 miR155 comprising the polynucleotide sequence SEQ ID NO: 41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 15, a bovine growth hormone minimal polyadenylation sequence comprising the polynucleotide sequence SEQ ID NO: 16, nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence SEQ ID NO: 19 and an AAV2 ITR comprising the polynucleotide sequence SEQ ID NO: 49; and wherein the capsid is an AAVrh74 N502I capsid.

[00257] 87. The rAAV particle of embodiment 85 or 86, wherein the AAVrh74 N502I capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 50.

[00258] 88. An rAAV particle comprising an rAAV vector and a capsid, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': an AAV2 ITR, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204 miRNA passenger sequence, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a growth hormone polyadenylation minimal sequence bovine nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene and an ITR of AAV2; and where the capsid is an AAVrh74 W505R capsid.

[00259] 89. An rAAV particle comprising an rAAV vector, wherein the rAAV vector comprises the following nu acids Petition 870250090423, dated 03 / 10 / 2025, page 104 / 218 97 / 144 cleicos from 5' to 3', an AAV2 ITR comprising the polynucleotide sequence SEQ ID NO: 43, nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence SEQ ID NO: 18, a Byrne desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 21, a Paulin desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 22, a desmin promoter comprising the polynucleotide sequence SEQ ID NO: 23, a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence SEQ ID NO: 40, a sequence of 5' miR155 framework comprising the miRNA guide sequence polynucleotide sequence DMPK204 comprising the polynucleotide sequence of SEQ ID NO: 4, a miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6,a DMPK204 miRNA passenger sequence comprising the polynucleotide sequence SEQ ID NO: 5, a 3' miR155 scaffold sequence comprising the polynucleotide sequence SEQ ID NO: 41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 15, a bovine growth hormone minimal polyadenylation sequence comprising the polynucleotide sequence SEQ ID NO: 16, nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence SEQ ID NO: 19 and an AAV2 ITR comprising the polynucleotide sequence SEQ ID NO: 49; and wherein the capsid is an AAVrh74 W505R capsid.

[00260] 90. The rAAV particle of embodiment 88 or 89, wherein the AAVrh74 W505R capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 52. Petition 870250090423, dated 03 / 10 / 2025, page 105 / 218 98 / 144

[00261] 91. A composition comprising the rAAV particle of any one of claims 1 to 22 and 77 to 90.

[00262] 92. A pharmaceutical composition comprising rAAV particles of any of the embodiments 1 to 22 and 77 to 90.

[00263] 93. The composition of embodiment 91 or 92, wherein the composition further comprises a pharmaceutically acceptable carrier.

[00264] 94. A kit comprising the rAAV particle of any of embodiments 1 to 22 and 77 to 90.

[00265] 95. A kit comprising the rAAV particle of any one of claims 1 to 22 and 77 to 90.

[00266] 96. A kit comprising the composition of any of the modalities 91 to 93.

[00267] 97. The kit for any of the modalities 94 to 96 which also includes instructions for use.

[00268] 98. A method for treating myotonic dystrophy-1 (DM1) in a mammal in need thereof, comprising administering to the mammal an effective amount of rAAV particles of any of the modalities 1 to 22 and 77 to 90.

[00269] 99. A method for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with DM-1 in need thereof, comprising administering to the mammal an effective amount of rAAV particle of any of the modalities 1 to 22 and 77 to 90.

[00270] 100. A method for inhibiting the accumulation of DMPK RNA in a mammalian cell with DM-1 in need thereof, comprising administering to the mammal an effective amount of RNAi of any of the modes 1 to 22 and 77 to 90.

[00271] 101. A method for treating myotonic dystrophy-1 (DM1) in a mammal in need thereof, comprising administering to the mammal an effective amount of rAAV particles of any Petition 870250090423, dated 03 / 10 / 2025, pp. 106 / 218 99 / 144 one of the modalities 1 to 22 and 77 to 90.

[00272] 102. A method for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with DM-1 in need thereof, comprising administering to the mammal an effective amount of rAAV particle of either of the 1 to 22 and 77 to 90 modalities.

[00273] 103. A method for inhibiting the accumulation of DMPK RNA in a mammalian cell with DM-1 in need thereof, comprising administering to the mammal an effective amount of rAAV particle of any of the embodiments 1 to 22 and 77 to 90.

[00274] 104. The method of any of the modalities 100 to 104, where the effective amount of viral particle or rAAV particle is a dose of approximately 1 x 108 to approximately 2 x 1013 genome copies / mL.

[00275] 105. The method of modality 104, where the dose is approximately 5 x 1012 copies of the genome / mL.

[00276] 106. The method of modality 104, where the dose is approximately 1 x 1013 copies of the genome / mL.

[00277] 107. The modality 104 method, where the dose is approximately 2 x 1013 genome copies / mL.

[00278] 108. The method of any of the modalities 101 to 103, where the effective amount of viral particle or rAAV particle is a dose of approximately 1 x 108 to approximately 2 x 1014 genome copies / kg of body weight.

[00279] 109. The method of modality 108, in which the dose is approximately 5 x 1013 copies of the genome / kg of body weight.

[00280] 110. The modality 108 method, in which the dose is approximately 1 x 1014 genome copies / kg of body weight.

[00281] 111. The modality 108 method, in which the dose is approximately 2 x 1014 genome copies / kg of body weight. Petition 870250090423, dated 03 / 10 / 2025, p. 107 / 218 100 / 144

[00282] 112. A method for treating myotonic dystrophy 1 (DM1) in a mammal which requires the same, comprising administering to the mammal an effective amount of the composition of any of the modalities 91 to 93.

[00283] 113. A method for inhibiting the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 that requires the same, comprising administering to the mammal an effective amount of the composition of any of the modalities 91 to 93.

[00284] 114. A method for inhibiting the accumulation of DMPK RNA in a mammalian cell with DM-1 that requires the same, comprising administering to the mammal an effective amount of the composition of any of the modalities 91-93.

[00285] 115. The method of any of the modalities 98 to 100, in which RNAi is administered in combination with an immunosuppressive agent, in which the immunosuppressive agent is administered before, at the same time and / or after administration of RNAi.

[00286] 116. The method of any of the modalities 101 to 103, in which the viral particle or rAAV particle is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time as, and / or after administration of the viral particle or rAAV particle.

[00287] 117. The method of any of the claims 112 to 114, wherein the composition is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time and / or after administration of the composition. EXAMPLES

[00288] The material presented in this document will be better understood by reference to the following Examples, which are provided. Petition 870250090423, dated 03 / 10 / 2025, pages 108 / 218 101 / 144 as examples of the invention, and not as a limitation. Example 1: Generation of amiR-DMPK204

[00289] expression cassettes. A series of nine transcriptional promoters (S1-S9) was rationally designed with the aim of identifying candidates with high activity in muscle and lower activity in liver. A panel of promoters was screened in primary human myoblast and Huh7 hepatoma cells to represent muscle and liver, respectively.The newly designed promoters were compared to the ubiquitous CAG promoter and a panel of previously published promoter sequences with high activity in muscle cells: the synthetic MH promoter comprising four muscle-specific transcription factor binding sites (TFBSs) and a core promoter fragment of the chicken skeletal muscle α-actin gene; the chimeric MHCK7 promoter, which was designed to target cardiac muscle and combines the α-MCK enhancer and the MCK promoter with the addition of a mouse α-myosin heavy chain enhancer; and sk-CRM4 / Des, a synthetic promoter combining a skeletal muscle-specific cisregulatory module, the desmin promoter, and the MVM intron. For screening, cells were transfected with a plasmid containing a luciferase gene under the control of the promoter of interest. After 48 hours, the relative activity of luciferase was measured in myoblasts (Figure 16A) and in Huh7 cells (Figure 16B).In addition to the CAG promoter, which drives high expression throughout the body, the S6 promoter led to the highest expression in muscle cells compared to all other newly designed promoters. S6 promoter expression in the liver was relatively low. Thus, the S6 promoter was selected for the AAV expression construct.

[00290] A single-stranded AAV viral vector encoding a microRNA (amiR-DMPK204) designed to target the gene was generated. Petition 870250090423, dated 03 / 10 / 2025, pp. 109 / 218 102 / 144 DMPK (Figure 1A). The construct was designed so that the microRNA amiR-DMPK204 was incorporated into an optimized microRNA structure, miR155 (BLOCK-iT; Thermofisher Catalogue Nos. K4935-00, K4936-00, K4937-00, K4938-00), and is hereinafter referred to as miR155 amiRDMPK204 or amiR155-DMPK204. miR155 amiR-DMPK204 is flanked by rabbit β-globin intron sequences and has been placed under the regulation of a hybrid muscle promoter (nDes; the S6 promoter).

[00291] The nDesmin promoter comprising the Byrne desmin enhancer, a copy of the Paulin desmin enhancer (-973 to -693) and the human Desmin gene promoter (-228 to +75) were synthesized by conventional oligonucleotide synthesis (Genscript, USA).

[00292] A bovine growth hormone polyadenylation sequence was placed in the 3' of the intron-flanked amiR-DMPK204 microRNA (minBGHpA). A filler sequence was included. The entire genetic cassette is flanked by wild-type AAV serotype 2 inverted terminal repeat (ITR) sequences for DNA rescue and replication, as well as packaging into an AAV capsid. The sequences were genetically modified in the ITR plasmid that was used to generate the vector for in vivo efficacy studies. ITR sequences 5' and 3'

[00293] The ITR sequences were the 145 bp wild-type AAV2 sequence. The 3' ITR (downstream of the expression cassette) was in the Flip orientation (GenBank: LQ493091.1). The 5' ITR (upstream of the expression cassette) was in the Flop orientation (145 bp) (Miller et al., 2004, Nature Genetics 36.7 (2004): 767-773). Sequence accuracy was confirmed by Sanger sequencing. Prosecutor nDes Petition 870250090423, dated 03 / 10 / 2025, pages 110 / 218 103 / 144

[00294] The nDes promoter was constructed using desmin promoter elements shown in the literature (Li and Paulin, et al. 1991, J Biol Chem. 266.10: 6562-6570). The nDes promoter comprises one copy of the Byrne desmin enhancer, one copy of the Palin desmin enhancer (-973 to -693), and the human desmin gene promoter (-228 to +75). Arms 5' and 3' of the rabbit β-globin intron

[00295] This intron was used to flank the amiRDMPK204 cassettes due to the fact that intronic expression of miRNAs is known to improve target knockdown. amiR-DMPK204 with miR155 scaffold (miR155-amiRDMPK204)

[00296] Endogenous miRNAs are hairpin-like secondary structures found in many primary RNA transcripts (pri-miRNAs). In the nucleus, the microprocessor, the Drosha / DGCR8 complex, ligates and cleaves the basal stem of pri-miRNAs to release the stem-loop precursor miRNA (pre-miRNA). The pre-miRNAs are then exported from the nucleus where the loop is cleaved 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 an argonaut protein in the RNA-induced silencing complex (RISC), which then directs complementary mRNA transcripts for degradation or translational repression.

[00297] The amiR-DMPK204 sequence has been identified as a target for DM1 therapy. The amiR-DMPK204 target sequence is located upstream of the CUG repeat sequences within the 3' UTR of the DMPK nucleotide sequence. Therefore, amiR-DMPK204 can suppress both wild-type and mutant DMPK transcripts.

[00298] Additionally, the amiR-DMPK204 target region is conserved in non-human primates (NHPS-cynomolgus monkey) and humans, allowing for preclinical evaluation of DMPK knockdown. Petition 870250090423, dated 03 / 10 / 2025, p. 111 / 218 104 / 144 in NHPS (Figure 11). The amiR-DMPK204 microRNA was evaluated and the miR155 scaffold was shown to have efficient guide processing with minimal passenger strand processing, reducing the likelihood of off-target effects (see Example 2 below).

[00299] The final construct that was selected for development was amiR-DMPK204 with the miR155 scaffold (amiR155-DMPK204), in which the guide strand, when processed, targeted DMPK mRNA for degradation. The designed pre-miRNA sequence structure is based on the murine 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 as much of the miR-155 structure as possible. The stem-loop structure was optimized, and a 2-nucleotide inner loop resulted in a higher knockdown rate than the 5-nucleotide / 3-nucleotide inner loop found in the native miR-155 molecule (source: Pol II miR RNAi BLOCK-iT™ Expression Vector (Invitrogen)). The vector, nDes-miR155-amiR-DMPK204, in the context of an AAV capsid, showed potent in vivo activity in the DMSXL mouse model of DM1 (see Example 4 below). MinBGH poliA

[00300] A minimal BGH polyA site of 186 bp was inserted downstream of the amiR-DMPK204 sequence to allow transcription termination and mRNA polyadenylation. Fill sequence starting from intron A1AT

[00301] A filler sequence from the intron 4 sequence of the alpha-1 antitrypsin gene was used to bring the gene cassette to the packaging boundaries for rAAV vectors. Cloning of the ITR plasmid with the nDes-miR155-amiRDMPK204 cassette (nDes-miR155-amiR-DMPK204)

[00302] A 1938 bp ITR-ITR sequence was projected from the cons Petition 870250090423, dated 03 / 10 / 2025, pp. 112 / 218 105 / 144 expression structure, nDes-miR155-amiR-DMPK204-minBGHpolyA cassette. Cloning of the nDes-miR155amiR-DMPK204-minBGHpolyA cassette into the ITR plasmid was performed. The synthesized nDes-miR155amiR-DMPK204-minBGHpolyA included a 5' Nco1 site and a 3' Sph1 site for cloning into the ITR plasmid (Figure 1B). Briefly, the plasmid containing the synthesized nDes-miR155amiR-DMPK204-minBGHpolyA sequence was digested with Nco1 and Sph1, and the 1.9 kB fragment was purified on a gel. The ITR plasmid was digested with NcoI and SphI, dephosphorylated using calf intestinal alkaline phosphatase (New England Biolabs; catalog number M0290), and the 8.2 kB vector main chain fragment was purified on a gel. The 1.9 kB digested fragment containing the expression cassette and the digested ITR plasmid were ligated to yield the ITR-nDes-miR155-amiR-DMPK204 plasmid. Small-scale rAAV vector production

[00303] A small-scale packaging assay was performed in HEK 293 cells to confirm the packaging of the ITR-nDes-miR155-amiR-DMPK204 plasmid. Small-scale production was performed using the AAV rep / cap plasmid. Figure 1C shows the amount of vector produced per HEK 293 cell compared to a standard EGFP plasmid gene cassette.

[00304] To determine the potential of amiR-DMPK204 to correct the DM1 phenotype, its ability to silence human DMPK transcription and correct splicing defects in skeletal myoblast cultures from DM1 patients was tested. Due to CTG expansion in the 3' UTR of the DMPK gene, hairpin-shaped mRNA structures of DMPK aggregate as insoluble ribonuclear foci and sequester several RNA-binding proteins. The resulting distribution of essential splicing factors, such as muscleblind-like 1 (MBNL1), causes incorrect splicing of the downstream effectors responsible. Petition 870250090423, dated 03 / 10 / 2025, pp. 113 / 218 106 / 144 by muscle tissue differentiation. Treatment of DM1 patient cells with amiR-DMPK204 caused more than 50% DMPK mRNA silencing and splicing correction, as measured by MBNL1 exon 7 inclusion. Example 2: The expression of miR155-amiRDMPK204 corrects DM1 phenotypes in vitro.

[00305] The expression of the miR155-amiRDMPK204 construct driven by the NDES promoter was tested in two in vitro models of DM1. Immortalized human DM1 myotubes were transduced with an AAV2 vector containing an artificial miRNA that directs DMPK under the control of the NDES promoter. DMPK amiRNA (Figure 17A) and DMPK mRNA (Figure 17B) were measured by PCR. As expected, DMPK amiRNA was only detectable in treated cells, where DMPK levels were reduced by 65% ​​compared to untreated cells (untreated by 1±0.04; treated by 0.34±0.018; mean ±SD; p > 0.0001, two-tailed unpaired t-test).

[00306] One of the characteristics of DM1 is the presence of nuclear RNA foci. The expansion of the CUG repeat in the 3' UTR of DMPK mRNA leads to mRNA retention in the nucleus, where it binds to and sequesters important RNA-binding proteins, including muscle-like protein 1 (MBNL1), which regulates premRNA splicing at an mRNA location. Furthermore, the presence of the mutant DMPK transcript is believed to lead to hyperphosphorylation and stabilization of CELF1 (CUG RNA-binding protein and embryonically lethal vision-like RNA-binding factor 1), which also functions in the regulation of alternative splicing and RNA stability. Using fluorescence in situ hybridization (FISH), the percentage of cells exhibiting nuclear foci, as well as the number of foci / nuclei in treated and untreated human DM1 myotubes, was quantified (Figure 17C-17D). The tra Petition 870250090423, dated 03 / 10 / 2025, pp. 114 / 218 Treatment with NDES-miR155-Amir-DMPK204 reduced the percentage of cells containing foci by 60% (untreated: 74.36%±9.3; treated: 15.36%±4.2, MEAN±SEM; p: 0.0044, two-tailed unpaired t-test) (Figure 17E). Furthermore, when compared with untreated cells, treated cells had significantly fewer foci per cell (untreated: 1.55±0.08; treated: 0.15±0.03, MEAN±SEM; p±0.0001, two-tailed unpaired t-test) (Figure 17F). Example 3: Processing miR155 amiR-DMPK204

[00307] amiR-DMPK204 was packaged into an AAV capsid, and DMPK knockdown efficacy, passenger tape activity, and processing patterns were analyzed in vivo. Constructs harboring nDes-miR155-amiR-DMPK204 were packaged into AAVs. Vectors were injected intravenously into the DMSXL humanized adult DM1 mouse model, which expresses human DMPK with >1000 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 tape activity and processing patterns.

[00308] RT-PCR analysis showed robust expression of amiRDMPK204 in multiple muscle tissues with the highest expression in the heart (Figure 2B). Concomitant with amiR-DMPK204 expression, RT-PCR analysis confirmed robust DMPK suppression in the heart with an average of >70% and ~30% DMPK suppression in different skeletal muscles (Figure 2C). In cardiac tissue, DMPK expression is less than 50% relative to TBP (TATA-binding protein) expression. Notably, DSMXL mice expressing low levels of DMPK relative to TBP (~50%, indicated by the dashed line in Figure 2C) do not have a phenomenon Petition 870250090423, dated 03 / 10 / 2025, pages 115 / 218 108 / 144 obvious type of DM1. Interestingly, the nDesmin promoter showed strong activity in cardiac tissue and similar levels in skeletal muscle (Figure 2B), although higher transduction was observed in the liver (Figure 2A). This suggests that expression was primarily restricted to cardiac and skeletal muscle, affected by DM1 pathology.

[00309] To evaluate amiR-DMPK204 processing, cardiac tissues were analyzed for mature amiRDMPK204 lengths and guide and passenger strand sequence composition by NGS for small transcriptome analysis.

[00310] Processing of amiR-DMPK204 did not produce transient ribbons. amiR-DMPK204 was processed exclusively on guide ribbons (>99%) in mouse cardiomyocytes, but frequently produced ribbons longer than predicted from the miRBase database (Tables 1). Processing of miR155 most frequently generated mature lengths between 22 and 26 nt in length, but processed accurately at the 5' end (Table 1). The sequence distributions of the different guide ribbon (NT) lengths mapping miR155 amiR-DMPK204 were calculated as percentages (% of reads). The expected amiR-DMPK204 guide ribbon is underlined and the initial sequence is in bold. The asterisk indicates reads corresponding to the predicted length of the amiR-DMPK204 guide ribbon. Table 1. The sequence distributions of the different guide tape (NT) lengths mapping miR155 amiR-DMPK204 were calculated as percentages (% of reads). The expected amiR-DMPK204 guide tape is underlined. The asterisk indicates readings corresponding to the predicted length of the amiR-DMPK204 guide tape. miR-155 Read Sequence Length % of Seqs SEQ ID NO AGTCGAAGACAGTTCTAGGGTGT 23 52 33 Petition 870250090423, dated 03 / 10 / 2025, pp. 116 / 218 109 / 144 Read Sequence miR-155 Length % of Seqs SEQ ID NO AGTCGAAGACAGTTCTAGGGTGTT 24 24 34 AGTCGAAGACAGTTCTAGGGTG 1 1 1 25 11 35 AGTCGAAGACAGTTCTAGGGT* 22 6 36 AGTCGAAGACAGTTCTAGGGTG- 26 3 37 llll AGTCGAAGACAGTTCTAGGGTG 22 4 38

[00311] Overall, no transient strands were detected by amiR-DMPK204 with miR155 (the guide % is > 99%). Therefore, miR155 was selected as a lead for preclinical studies, since the miR155 miRNA framework is well validated for RNAi. Example 4: Dose-dependent suppression of human DMPK by systemic injection of AAV encoding miR155-amiRDMPK204 in transgenic mice.

[00312] To determine the most effective dose, the administration of three separate doses of myotropic AAV capsid was investigated (WO / 2019 / 207132). AAV encoding the expression cassette for amiR-DMPK204 with the miR155 scaffold (amiR155-DMPK204) was evaluated in a dose-escalation study. Eight-week-old DMSXL mice, carrying a human DMPK transgene containing a CTG expansion of more than 1000 repeats, were intravenously injected with 5.0 χ 1011 vector genomes (vg) / kg, 5 χ 1012 gv / kg, and 1.0 χ 1013 gv / kg, corresponding to low, intermediate, and high doses. Mice were analyzed for clinical symptoms such as body weight, survival, myotonia, and cardiac function 8 weeks after AAV infusion. Mice were euthanized 8 weeks after gene transfer, and DMPK suppression and splicing correction were measured.The expression levels of miR155-amiRDMPK204 were measured by TaqMan small RNA, and mRNA input levels were normalized. Petition 870250090423, dated 03 / 10 / 2025, pp. 117 / 218 110 / 144 for small nuclear RNA u6.

[00313] The expression of amiR155-DMPK204 was observed in a dose-dependent manner (Figure 3A) and resulted in a dose-dependent reduction in total DMPK expression (Figure 3B) in multiple tissues. In general, it was observed that approximately ~10 copies of amiR155DMPK204 / U6 were sufficient to reduce DMPK by > 50% in the heart and diaphragm, an amount that may be sufficient to treat patients with type 1 diabetes.

[00314] A cohort of DMSXL mice was treated with a single dose of miR155-DMPK204 at a dose of 9 x 1013 gv / kg, which resulted in the expression of the artificial miRNA in skeletal and cardiac muscles and significant silencing of DMPK mRNA in the heart, TA, diaphragm and gastrocnemius, but not in the liver, confirming the muscle specificity of the platform at a higher dose (Figure 18).

[00315] Nuclear RNA foci in the hearts of treated mice were measured by in situ hybridization. The foci were frequently present as large formations, and therefore the linear size of each formation in positive cells was measured. Treatment with miR155-amiRDMPK204 significantly reduced the size of the foci (untreated: 7±0.32 μm; treated: 2.7±0.08 μm; Mean ± SEM; p<0.0001, one-way ANOVA; Figure 19).

[00316] Next, the consequences of DMPK suppression on characteristic DM1 phenotypes, such as splicing abnormalities, were investigated. To determine whether treatment could improve the splicing phenotype in mice, hearts and anterior tibial (TA) muscles were collected and RNA-seq analysis was performed on wild-type, buffer-negative control, and treated mice (Shen et al. (2014) Proc Natl Acad Sci 111(51):E5593E5601; Park et al. (2013) Methods Mol Biol 1038:171-9; Shen et al. Petition 870250090423, dated 03 / 10 / 2025, pages 118 / 218 111 / 144 (2012) Nucleic Acids Res 40(8):e61). Global alterations in alternative splicing events were found in the heart and TA, indicating molecular alterations in the disease and with treatment. Among the genes that were altered in the hearts of DMSXL mice were Ldb3, MBNL2, and SPAG9 (Figure 20). Aberrant splicing of the LIM 3 domain (Ldb3), resulting in the inclusion of exon 11 in Ldb3 transcripts, has been demonstrated as a specific DM1 phenotype resulting from the hijacking of RNA splicing machinery by CUG repeat RNA (Yamashita et al. 2014. Neurobiol Dis. 69:200-5). A significant reduction in the Ldb3 transcript was observed with the inclusion of exon 11 in the gastrocnemius muscle in mice that were treated with the medium or high dose of AAV nDesmiR155-amiR-DMPK204, confirming that splicing defects were efficiently corrected in the muscle treated with amiR155DMPK204 (Figure 4).It has also been previously shown that the persistence of fetal isoforms of these genes contributes to DM1 pathology. In TA, a different, yet overlapping, set of alternative splicing events was altered when comparing wild-type and DMSXL mice. Among others, three genes demonstrated significant splicing rescue in the treated group – DNAsel, Tnnt3, and Zbtb49 (Figure 20). For these transcripts, in both the heart and TA, there were no significant differences between wild-type and treated DMSXL mice, suggesting restoration of normal splicing after treatment.

[00317] In addition to these molecular corrections, the efficacy of AAV nDes-miR155-amiR-DMPK204 was then measured in terms of physiological and functional manifestations of the disease.

[00318] To determine whether treatment can improve survival and attenuate body weight loss, DMSXL mice were treated with AAV nDes-miR155-amiR-DMPK204 at three different doses. Petition 870250090423, dated 03 / 10 / 2025, pages 119 / 218 112 / 144 patients, and survival and body weight were monitored. Improved body weight and survival rate were observed after eight weeks of treatment with medium and high doses. On the other hand, no improvement was observed with low-dose control or balanced saline solution (BSS) (Figures 5A and 5B).

[00319] Next, the efficacy of AAV nDes-miR155-amiRDMPK204 was measured in terms of functional disease manifestations, such as prevention of myotonia and cardiac abnormalities. Electromyography measurements revealed a significant decrease in myotonia in mice treated with AAV nDes-miR155-amiRDMPK204 (Table 2). In particular, after treatment, only 7.6% of animals treated with the medium dose had myotonia. In contrast, >50% of mice in the control (treated with BSS) or low-dose groups exhibited persistent myotonia (score 1). Table 2. Number of DM1 DMSXL mice with myotonia after treatment with AAV nDes-miR155-amiR-DMPK204. Group Number of animals with myotonia (gastrocnemius) WT 0 / 10 DMSXL-Ctrl (untreated) 4 / 7 J DMSXL-5E11 GV / kg (low dose) 3 / 5 DMSXL-5E12 GV / kg (intermediate dose) 1 / 13 P<0 DMSXL-5E13 GV / kg (high dose) 1 / 7

[00320] Myotonic discharges were assessed on a 4-point scale: 0, no myotonia; 1, occasional myotonic discharge in less than 50% of needle insertions; 2, myotonic discharge in more than 50% of needle insertions; 3: myotonic discharge with almost every insertion.

[00321] The number of degree 0 events versus degree 1-3 events Petition 870250090423, dated 03 / 10 / 2025, pages 120 / 218 113 / 144 was compared between treatment groups (wild-type, vehicle control in DMSXL mice, and a single intravenous dose of amiR155-DMPK204 in DMSXL mice) (Figure 21). As expected, all wild-type mice scored 0 in all three muscles analyzed. In vehicle-treated DMSXL mice, there was a significant reduction in grade 0 events recorded in all three muscles analyzed (Gastroc, Grade 0: WT = 30; DMSXL vehicle = 1; Grade 1-3: WT: 0, DMSXL vehicle: 9; Fisher's exact test, Bonferroni-Holm adjusted p-value <0.0001; Quad, Grade 0: WT=30; DMSXL Vehicle=2; Grade 1-3: WT=0, DMSXL Vehicle=8; Fisher's exact test, Bonferroni-Holm adjusted p-value <0.0001; TA, Grade 0: WT=30; DMSXL Vehicle=7; Grade 1-3: WT=0; DMSXL Vehicle=3; Fisher's exact test, Bonferroni-Holm adjusted p-value=0.036).However, treatment with both doses significantly improved the myotonia phenotype in the gastrocnemius and showed a positive trend toward improvement in the other two muscles, compared to the Vehicle group (Gastrocnemius: DMSXL 9 X1013, Grade 0 = 9; Grade 1-3 = 3; Fisher's exact test, Bonferroni-Holm adjusted p-value = 0.0041; DMSXL 1.8 X1014, Grade 0 = 15; Grade 1-3 = 6; Fisher's exact test, Bonferroni-Holm adjusted p-value = 0.0041; Quadrilateral: DMSXL 9 X1013, Grade 0 = 9; Grade 1-3 = 3; Fisher's exact test, Bonferroni-Holm adjusted p-value = 0.059; DMSXL 1.8 X1014, Grade 0 = 13; Grade 1-3 = 8; Fisher's exact test, Bonferroni-Holm adjusted p-value = 0.059). Bonferroni-Holm = 0.059; TA: DMSXL 9 X1013 Grade 0 = 12; Grade 1-3 = 0; Fisher's exact test, Bonferroni-Holm adjusted p-value = 0.155; DMSXL 1.8 X1014, Grade 0 = 19; Grade 1-3 = 2: Fisher's exact test, Bonferroni-Holm adjusted p-value = 0.295).To determine whether treatment with the AAV capsid containing the miR155-DMPK204 construct could correct this defect, the degree of myotonic events in the gastrocnemius, quadriceps, and TA, via EMG, was measured. Petition 870250090423, dated 03 / 10 / 2025, pages 121 / 218 114 / 144 measured. Although wild-type mice did not exhibit myotonic events in any of the muscles analyzed, most untreated mice with DMSXL showed events with grades between 1 and 2, and most treated mice showed myotonia grades between 0 and 1 (Figure 21).

[00322] Another clinical feature of the disease is defects in cardiac function. Conduction defects are found in up to 75% of adult DM1 patients, and cardiac arrhythmias are a major cause of death. Cardiac function in DMSXL mice was also monitored using surface echocardiography 8 weeks after treatment, along with skeletal muscle function. AAV nDes-miR155-amiR-DMPK204 improved cardiac output compared to BSS-treated controls. Significant improvement in cardiac output was observed in the medium dose group (5e12 gv / kg) after 8 weeks of treatment (Figure 6).

[00323] Echocardiographic analysis of DSMXL hearts showed that the mice exhibited a narrowed aorta compared to their wild-type littermates (Figure 22, p < 0.001, one-way ANOVA). Treatment with 9 x 1013 gv / kg Amir-DMPK204, however, significantly increased the aortic diameter (p < 0.05). Treated DMSXL mice were not significantly different from the wild type (Figure 22A). Furthermore, DMSXL mice were found to have decreased ascending aortic arterial velocity (cardiac output deficiency; Figure 22B) compared to the wild type (p < 0.0001), and this defect was also corrected with treatment (p < 0.001). Example 5: The AAVrh74N502I capsid improved muscle transduction and reduced liver transduction.

[00324] An experiment was conducted to test the efficiency of Petition 870250090423, dated 03 / 10 / 2025, pages 122 / 218 115 / 144 Transduction of AAV capsids containing the AAVrh74N502I VP1 capsid protein (SEQ ID NO: 50) into various tissues of non-human primates. An outline of the experiment is shown in Figure 7A. Non-human primates were intravenously treated with 1 x 1013 gv / kg of either AAV9, AAVrh74, or AAVrh74N502I capsids, each containing an eGFP expression cassette. Twenty-one days after treatment, the animals were sacrificed and eGFP expression levels in the tibialis anterior (TA) muscle, biceps femoris, quadriceps, heart, and liver were measured.

[00325] Capsids containing the AAVrh74N502I capsid protein had improved muscle transduction (Figures 7B-7E) and reduced liver transduction (Figure 7F) in non-human primates compared to comparator capsids. (Table 3). Table 3. Increase in eGFP levels in tissues of non-human primates treated with AAVrh74N502I capsids compared to those treated with AAV9 and AAVrh74 capsids. Tissue AAV9 AAVrh74 Tibialis anterior + 13x + 178x Biceps femoris +303x +56x Quadriceps NS* +32x Heart NS + 13x Liver -15x -2x *NS = not significant Example 6: Evaluation of target engagement of AAVrh74N502I nDesmiR155-amiR-DMPK204 in a DMSXL mouse model

[00326] To determine the most effective dose, the administration of two separate doses of AAVrh74N502I capsid was investigated. AAV encoding the expression cassette for amiR-DMPK204 with the miR155 framework (miR155-amiR-DMPK204) was evaluated in a dose-escalation study. Eight-week-old DMSXL mice were intravenously injected with 9 x 1013 vector genomes Petition 870250090423, dated 03 / 10 / 2025, pages 123 / 218 116 / 144 rials (gv) / kg and 1.8 x 1014 gv / kg, corresponding to low and high doses. Mice were euthanized 8 weeks after gene transfer, and DMPK suppression and amiRDMPK204 expression levels were measured by TaqMan small RNA, and mRNA entry levels were normalized to u6 small nuclear RNA.

[00327] amiR-DMPK204 expression was observed in a dose-dependent manner (Figure 8A) and resulted in a dose-dependent reduction in total DMPK expression (Figure 8B) in multiple tissues. Overall, it was observed that approximately ~10 copies of amiRDMPK204 / U6 were sufficient to reduce DMPK by > 50% in the heart and diaphragm, an amount that may be sufficient to treat patients with type 1 diabetes.

[00328] Finally, the main vector, nDes-miR155-amiR-DMPK204, in the context of a myotropic capsid, AAVrh74N502I (SEQ ID NO: 50), demonstrated potent in vitro activity in cardiomyocyte-derived DM1 iPSCs (Figure 9). Example 7: Evaluation of AAVrh74N502I nDes-miR155- amiRDMPK204 or transcriptome

[00329] To determine whether treatment with amiR-DMPK204 had any major effect on the transcriptome, genome-wide RNA sequencing (RNA-seq) was performed, comparing amiR-DMPK204-treated cells with CTL3 (miRNA shuffling) transfecting CBA miR155-amiR-DMPK204 plasmids in HEK293 cell lines. To assess whether the observed changes in non-DMPK gene expression were due to off-target effects of amiR-DMPK204, enrichment for initial complementarity in significantly downregulated targets was evaluated. Four differentially expressed genes were found: OPN4 (12.5-fold), DMPK (1.7-fold), KRTAP21-2 (1.7-fold), C8ORF44-SGK3 (1.7-fold), whose 3' UTRs contain the initial complement TTCGAC, using a threshold of Petition 870250090423, dated 03 / 10 / 2025, pp. 124 / 218 117 / 144 significance of false discovery rate (FDR) of 5% (Table 4). With an FDR of 1%, only OPN4 (12.5 times), DMPK (1.7 times), showed differential expression (Figure 10). As expected, DMPK was one of the mRNA levels most significantly impacted (44% silencing). Overall, minimal off-target effects were observed after amiR-DMPK204 expression in HEK293 cells. Table 4 Gene Name log2 times 204 vs CTL3 times 204 vs CTL3 P-Value FDR_BH <0.05 FDR_BH <0.01 OPN4 -3.64 12.5 2.7746E-17 5E-13 yes DMPK -0.77 1.7 3.4308E-10 2E-06 yes KRTAP21-2 -0.76 1.7 0.0001 0.0103 C8orf44SGK3 -0.79 1.7 0.0003 0.0191 Example 8: Dose range identification study to explore the biodistribution and activity of AAVrh74N502I nDes-miR155-amiRDMPK204 in non-human primates.

[00330] To determine the biodistribution and activity of AAVrh74MN502I nDes-miR155-amiR-DMPK204, a single intravenous (IV) infusion dose was administered to cynomolgus monkeys. The study duration was 12 weeks after the single injection.

[00331] Sixteen cynomolgus monkeys in total (8 males, 8 females; 24 to 48 months of age) received a dose intravenously via the saphenous vein once on day 1 of the study. Doses are indicated in Table 5 below. The animals were grouped (2 males and 2 females per group) into four different categories based on dose level (gv / kg): Formulation buffer (Group 1); 5 x 10¹³ gv / kg (Group 2); 1 x 10¹⁴ gv / kg (Group 3); and 2 x 10¹⁴ gv / kg (Group 4). After 12 weeks, the animals were sacrificed, and tissue was collected for analysis. Petition 870250090423, dated 03 / 10 / 2025, pages 125 / 218 118 / 144 Table 5 Group Test Article or Control Article Dose level (gv / kg) Dose concentration (gv / mL) Number of animals Males Female patients 1 Formulation Buffer 0 0 2 2 2 AAVrh74N502I nDes-miR155amiR-DMPK204 5 x 1013 5 x 1012 gv / mL 2 2 3 AAVrh74N502I nDes-miR155amiR-DMPK204 1 x 1014 1 x 1013 gv / mL 2 2 4 AAVrh74N502I nDes-miR155amiR-DMPK204 2 x 1014 2 x 1013 gv / mL 2 2

[00332] The collected tissue was mechanically homogenized for RNA extraction and DNA was used, and the samples were analyzed using PCR. digital rectifier (dPCR).

[00333] Dose-dependent biodistribution and activity of AAVrh74N502I nDes-miR155-amiR-DMPK204 has been demonstrated in various muscle and non-muscle tissues. Several skeletal muscles were analyzed (tibialis anterior (TA) muscle; gastrocnemius; quadriceps; biceps; soleus; extensor digitorum longus (EDL); diaphragm), as well as cardiac muscle and liver tissue. Viral genome copies were found in all tissues tested, and the number of copies / cell in each tissue was dose-dependent (Figure 12). The expression of amiR-DMPK (Figure 13) and the downregulation of DMPK (Figure 14) were also dose-dependent in various muscle tissues, cardiac tissue, and liver tissue. The dose-dependent reduction in DMPK expression was found to be up to 90% reduced compared to a control group. All doses tested on animals were considered safe and well tolerated. Petition 870250090423, dated 03 / 10 / 2025, pages 126 / 218 119 / 144 for the animals. Example 9: Rescue of splicing defects after treatment with AmirDMPK2Q4b

[00334] DMPK-mediated splicing defects are a molecular hallmark in DM1 tissue. To determine whether treatment with Amir-DMPK204 had an effect on these splicing defects, a targeted splicing analysis was performed using RNA sequencing (RNA-seq) and the Nanostring platform, focusing on 36 genes (Tanner et al. 2021. Nucleic Acids Res. 49(4):2240-2254. doi: 10.1093 / nar / gkab022).

[00335] The data between the two platforms were comparable. Immortalized human DM1 myotubes were transduced with AAV2nDesmin-NDES-miR155-DMPK2Q4 and compared to untreated DM1 myotubes and healthy control myotubes. 25 of the 36 genes showed a statistically significant modification of their splicing before treatment. Six of the 25 genes (MBNL1, SOS1, PKM, zTTN, GOLGA4, and CLASP1) demonstrated significant recovery in treated cells compared to untreated cells (Figure 15). Overall, the data demonstrate that treatment of human myotubes with Amir-DMPK2Q4 was able to rescue, at least partially, the splicing defects caused by DMPK mutations. Example 10: AAVrh74N502I nDes-miR155-amiR-DMPK2Q4 up to 2 x 1014 gv / kg is well tolerated in non-human primates.

[00336] Tissue sections from animals treated with AAVrh74N502I NDES-miR155- Amir-DMPK204 were evaluated for tolerability to the AAV construct. All animals in the study were monitored during the life phase, including detailed cage-side observations, body weight, food consumption, and vital sign measurements.

[00337] No macroscopic observations were observed. Petition 870250090423, dated 03 / 10 / 2025, pages 127 / 218 120 / 144 associated with the test article. Minimal individual hepatocyte necrosis related to the test article was observed in the liver of a male that received 1 x 1014 gv / kg and a female that received 2 x 1014 gv / kg (Table 6). Minor clinical chemical effects related to the test article were identified in animals that received a single dose >1 x 1014, particularly aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glutamate dehydrogenase (GDH) activities (Table 7). Enzyme increases were observed only at early time points, fully reverting to baseline values ​​by Day 43, and had no correlative microscopic liver findings in affected animals, except for the finding of minimal individual hepatocyte necrosis in a female administered 2 x 1014 gv / kg. No evidence of a hepatic effect was identified in animals that received 5 x 1013 g / kg.No effects related to the test article were identified on hematology or coagulation parameters at any dose level. These changes were considered non-adverse based on a small overall magnitude of change, minimal severity and incidence, and / or lack of dose-relatedness. Table 6. Incidence and severity of microscopic findings related to the test article in animals administered with AmirDMPK204. Sex rAAVmiR Males Female patients Dose level (gv / kg) 0 5 x 1013 1 x 1014 2 x 1014 0 5 x 1013 1 x 1014 2 x 1014 Liver Number examined 2 2 2 2 2 2 2 2 Petition 870250090423, dated 03 / 10 / 2025, pages 128 / 218 121 / 144 rAAVmiR Sex: Males; Female patients Dose level 0 5 χ 1 χ 2 χ 0 5 χ 1 χ 2 χ (gv / kg) 1013 1014 1014 1013 1014 1014 Necrosis, he- 0 0 1 0 0 0 0 1 individual pathogen Minimum Table 7. Liver enzyme changes related to the test article in selected animals administered with AmirDMPK204. Absolute Enzyme Activity, U / L (Change in times compared to pre-dose)A Pre-phase dosage dose Day -41 4 8 15 29 30 31 43 85 Animal P0202, Male administered with 1 χ 1014 gv / kg AST 33 133 38 35 35 37 40 (4.0X) ALT 32 74 126 52 34 31 35 (2.3X) (3.9X) GDH 22 89 92 35 ? 28 26 28 (4.0X) (4.2X) Animal P0601, female administered with 1 x 1014 gv / kg AST 33 102 52 40 32 29 32 (3.1X) ALT 84 180 171 113 59 60 59 (2.1X) (2.0X) GDH 24 59 68 36 16 18 19 (2.5X) (2.8X) Animal P0401, male administered with 2 x 1014 gv / kg AST 48 91 198 96 60 52 40 (4.1X) (2.0X) ALT 34 63 197 80 50 35 26 (5.8X) (2.4X) Petition 870250090423, dated 03 / 10 / 2025, pages 129 / 218 122 / 144 Absolute Enzyme Activity, U / L (Change in times compared to pre-dose) Pre-dose Dosage Phase GDH 35 45 87 45 ? ? 40 34 32 (2.5X) Animal P0302, male administered with 2 x 1014 gv / kg AST 48 688 58 80 - - 57 52 50 (14.3X) ALT 83 686 306 99 - - 66 64 61 (8.3X) (3.7X) GDH 45 92 75 37 32 31 30 (>2.0X) Animal P0701, female administered with 2 x 1014 gv / kg AST 43 136 82 110 - - 51 54 61 (3.2X) (2.6X) ALT 40 119 109 109 - - 41 59 62 (3.0X) (2.7X) (2.7X) GDH 27 44 54 61 - - 23 36 46 (2.0X) (2.3X) Animal P0702, female administered with 2 x 1014 gv / kg AST 35 58 65 51 - - 34 43 35 ALT 45 69 99 60 - - 34 61 42 (2.2X) GDH 23 32 53 26 - - 16 28 22 (2.3X) SEQUENCES

[00338] All polypeptide sequences are presented as N-terminal to C-terminal unless otherwise indicated. All nucleic acid sequences are presented as 5' to 3' unless otherwise indicated. Intensifying sequence of Byrne's desmina Petition 870250090423, dated 03 / 10 / 2025, pp. 130 / 218 123 / 144 CACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGCTCCTCTC CTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTCTATAAATACCCGC TCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTG GGTTGACATGCGGCTCCTGACAAAACAAACCCCTGGTGTGTGT GGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGC GGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGG TGGGAGCGCACATAGCAATTGGAAACTGAAAGCTTATCAGACCCT TTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCCCACCCTC GA (SEQ ID NO: 21) Source: Li and Paulin, et al. 1991. High-level muscle expression depends on a specific muscle enhancer. Journal of Biol Chem. 266.10: 65626570. Desmin locus control region of Homo sapiens (DES-LCR) on chromosome 2 (NCBI reference sequence: NG_046330.1)

[00339] The Byrne intensifier sequence corresponds to 17767-18125 in Seq Ref. Paulin's desmin-intensifying sequence CCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTCCCAGCC ATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCA GCTGTTGCTGCCAGGGAGATGGTTGGGTTGACATGCGGCTCCTG ACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTGGTGTGAG TAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAG GAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCAAT TGGAAACTGAA (SEQ ID NO: 22) Petition 870250090423, dated 03 / 10 / 2025, pp. 131 / 218 124 / 144 Source: Li and Paulin, et al. 1991. High-level muscle expression depends on a specific muscle enhancer. Journal of Biol Chem. 266.10: 65626570. Desmin locus control region of Homo sapiens (DES-LCR) on chromosome 2 (NCBI reference sequence: NG_046330.1)

[00340] The Paulin intensifier sequence corresponds to 17787-18063 in Seq Ref. Paulin desmin-promoting sequence (-228 to +75) CTGCAGACCTGCTTGCTGCCTGCCCTGGCGAAGGATTGGCAGGC TTGCCCGTCACAGGACCCCCGCTGGCTGACTCAGGGGCGCAGGC CTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACGGCCACGGGC CGCCCTTTCCTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGG AGGGGAGGCGGGGGCTGATGTCAGGAGGGATACAAATAGTGCC GACGGCTGGGGGCCCTGTCTCCCCTCGCCGCATCCACTCTCCGG CCGGCCGCCTGCCCGCCGCCTCCTCCGTGCGCCCGCCAGCCTC GCCCG (SEQ ID NO: 23)

[00341] There is a difference of one base pair from the published sequence (C instead of an A, shown in bold and underlined). Source: Li and Paulin, et al. 1991. High-level muscle expression depends on a specific muscle enhancer. Journal of Biol Chem. 266.10: 65626570. Homo sapiens desmin locus control region (DES-LCR) Petition 870250090423, dated 03 / 10 / 2025, pp. 132 / 218 125 / 144 on chromosome 2 (NCBI reference sequence: NG_046330.1)

[00342] The Paulin promoter sequence corresponds to 1853518844 in Seq Ref. Complete nDes promoter sequenceCACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGCTCCTCTC CTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTCTATAAATACCCGC TCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTG GGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGT GGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGC GGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGG TGGGAGCGCACATAGCAATTGGAAACTGAAAGCTTATCAGACCCT TTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCCCACCCTC GAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTC CCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGG GTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGGTTGACATGCG GCTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTG GTGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAG GGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCAC ATAGCAATTGGAAACTGAAAGCTTCTGCAGACCTGCTTGCTGCCT GCCCTGGCGAAGGATTGGCAGGCTTGCCCGTCACAGGACCCCCG CTGGCTGACTCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCC TCCCCGCCCCCACGGCCACGGGCCGCCCTTTCCTGGCAGGACAG CGGGATCTTGCAGCTGTCAGGGGAGGGGAGGCGGGGGCTGATG TCAGGAGGGATACAAATAGTGCCGACGGCTGGGGGCCCTGTCTC CCCTCGCCGCATCCACTCTCCGGCCGGCCGCCTGCCCGCCGCCT CCTCCGTGCGCCCGCCAGCCTCGCCCG (SEQ ID NO: 12) Petition 870250090423, of 03 / 10 / 2025, p. 133 / 218 126 / 144 Source: Li and Paulin, et. al. 1991. High level desmin expression depends on a muscle-specific enhancer. Journal of Biol Chem. 266.10: 65626570. Rabbit β-globin intron GTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAA AATTCATGTTATATGGAGGGGCAAAGTTTTCAGGGTGTTGTTTAG AATGGGAAGATGTCCCTTGTATCACCATGCATGGACCCTCATGAT AATTTTGTTTCTTTCACTTTCTGTTGATTGTTCCTTGTCCCAT CTTATTTTCTTTTCATTTTCTGTAACTTTTCCGTTAAACTTTAGCTTG CATTTGTAACGAATTTTAATTCACTTTTGTTTATTTGTCAGATTGT AAGATCCCATCGATTCCAATCAGGGTATATTTATTGTACTTCAGC ACAGTTTTAGAGAACAATTGTTATATTAAATGATAAGGTAGAATAT TTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAG AAACAACTACATCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTA CAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCA AACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTGCTCATT: 10:00:00 5′ of rabbit β-globin intron GTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAA AATTCATGTTATATGGAGGGGCAAAGTTTTCAGGGTGTTGTTTAG aatgggaagatgtcccttgtatcaccatgcatggaccctcatgat AATTTTGTTTCTTTCACTTCTACTCTGTTGACCATTTGTCTCCT CTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTG CATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGT AAGATCCCATCGATT C (SEQ ID NO: 14) Petition 870250090423, of 03 / 10 / 2025, p. 134 / 218 127 / 144 Source: hemoglobin of Oryctolagus cuninculus, beta (HBB2) Gene ID 100009084

[00343] The sequence of the present invention has an additional CATG (shown in bold and underlined) that is not present in Gene ID 100009084. a rabbit β-globin intron 3′ arm CAATCAGGGTATATTATATTGTACTTCAGCACAGTTTAGAGACA ATTGTTATAATTAATGATAAGGTAGAATTTCTGCATATAATTC TGGCTGGCGTGGAAATATTCTTATTGGTAGAAAACTACATCCTG GTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATACACTGTT TGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGC TAACCATGTTCATGCCTTCTTCTTTTTCCTACAG (SEQ ID NO: 15) Source: Oryctolagus cuninculus hemoglobin, beta (HBB2) Gene ID 100009084

[00344] The sequence of the present invention has two T residues (shown in bold and underlined above) instead of two C residues in Gene ID 100009084. miR155-DMPK204 sequences: Flanking sequence 5' miR155 Petition 870250090423, dated 03 / 10 / 2025, pp. 135 / 218 128 / 144 RNA sequence CUGGAGGCUUGCUGAAGGCUGUAUGCU (SEQ ID NO: 9) DNA sequence CTGGAGGCTTGCTGAAGGCTGTATGCT (SEQ ID NO: 40) Source: Catalog of miR RNAi BLOCKiT™ Pol II Expression Vector Kits No. K493500

[00345] The projected pre-miRNA sequence structure is based on the murine miR-155 sequence (Lagos-Quintana et al., 2002, Current Biology, 12:9, 735-739). DNA guide amiR-DMPK204 AGTCGAAGACAGTTCTAGGGT (SEQ ID NO: 4) terminal loop of miR155-DNA GTTTTGGCCACTGACTGAC (SEQ ID NO: 6) Source: Catalog of miR RNAi BLOCKiT™ Pol II Expression Vector Kits No. K493500

[00346] The designed pre-miRNA sequence structure is based on the murine miR-155 sequence (Lagos-Quintana et al., 2002, Current Biology, 12:9, 735-739). Passenger DNA amiR-DMPK204 ACCCTAGATGTTCTTCGATT (SEQ ID NO: 5) Petition 870250090423, dated 03 / 10 / 2025, pp. 136 / 218 129 / 144 Guide RNA amiR-DMPK204 - terminal loop miR155 - passenger RNA amiR-DMPK204 AGUCGAAGACAGUUCUAGGGUUGUUUUGGCCACUGACUGACAC CCUAGAUGUCUUCGAUU (SEQ ID NO: 7) DNA-guide amiR-DMPK204 - terminal loop miR155 - DNA passenger amiR-DMPK204 AGTCGAAGACAGTTCTAGGGTTGTTTTGGCCACTGACTGACACCC TAGATGTCTTCGATT (SEQ ID NO: 8) Flanking sequence 3' miR155 RNA sequence GACACAAGGCCUGUUACUAGCACUCACAUGGAACAAAUGGCC (SEQ ID NO: 10) DNA sequence GACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC (SEQ ID NO: 41) Source: Catalog of miR RNAi BLOCKiT™ Pol II Expression Vector Kits No. K493500

[00347] The projected pre-miRNA sequence structure is based on the murine miR-155 sequence (Lagos-Quintana et al., 2002, Current Biology, 12:9, 735-739). miR155 RNA Petition 870250090423, dated 03 / 10 / 2025, pp. 137 / 218 130 / 144 CUGGAGGCUUGCUGAAGGCUGUAUGCUGACACAAGGCCUGUUA CUAGCACUCACAUGGAACAAAUGGCC (SEQ ID NO: 11) DNA CTGGAGGCTTGCTGAAGGCTGTATGCTGACACAAGGCCTGTTACT AGCACTCACATGGAACAAATGGCC (SEQ ID NO: 42) Complete duplex sequence miR155-DMPK204-RNA CUGGAGGCUUGCUGAAGGCUGUAUGCUGAGUCGAAGACAGUUC UAGGGUGUUUUGGCCACUGACUGACACCCUAGAUGUCUUCGAU UCAGGACACAAGGCCUGUUACUAGCACUCACAUGGAACAAAUGG CC (SEQ ID NO: 24) Complete duplex sequence miR155-DMPK204- DNA CTGGAGGCTTGCTGAAGGCTGTATGCTGAGTCGAAGACAGTTCTA GGGTGTTTTGGCCACTGACTGACACCCTAGATGTCTTCGATTCAG GACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC (SEQ ID NO: 25) BGHpA minimum sequence TCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCC TTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATG AGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGG GGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAC AATAGC (SEQ ID NO: 16) Source: Growth hormone 1 (GH1) mRNA from Bos taurus Petition 870250090423, dated 03 / 10 / 2025, pp. 138 / 218 131 / 144 NCBI Ref SEQ NM_180996.1 1138 bp A1AT intron fill sequence (upstream of the expression cassette) TACGTACAATTGGGATCCTTCGAACTTGAGAGAAAACATCCCAGG GATTTACAGATCACATGCAGGCAGGGACCAGCTCAACCCTTCTTT AATGTCATCCAGGGAGGGGGCCAGGGATGGAGGGGAGGGGTTG AGGAGCGAGAGGCAGTTATTTTTGGGTGGGATTCACCACTTTTCC CATGAAGAGGGGAGACTTGGTATTTTGTTCAATCATTAAGAAGACA AAGGGTTTGTTGAACTTGACCTCGGGGGGGATAGACATGGGTATG GCCTCTAAAAACATGGCCCCAGCAGCTTCAGTCCCTTTCTCGTCG ATGGTCAGCACAGCCTTATGCACGGCCTGGAGGGGAGAGAAGCA GAGACACGTTGTAAGGCTGATCCCAGGCCTCGAGCAAGGCTCAC GTGGACACCTCCCAGGAAGCGCTCACTCCCCCTGGACGGCCCTG GCCCTGCACATCCTCTCCCTCCCTGTCACATAGGCCTTGCTCCTC CTCAAGGCTTTGGCTGATGGGGCTGGCTCCCCTCTGTCCATCTTC CTGACAAGCGCCTCTCCCCCTGCTCAGGTGCACCCACAACTCAGA ACAGGGAAGAGCATCGTCACTCCACTAGTCTGCCTCCAGGGCTCT CTCCTTTCTAGTACACGGCTTGAAGCTCCTTGAGGACACGGACCC TGGCAGTGACCTTCACAGTGCCCAGACCCCAAGATAATGCAGCCA TTCATGGAACTGCAGGTTGTTCATTGGTCGCCTTTAGTTTTCCAAA ATAAGTGTCACTTTAGCTGAAATCATTCATTAATTCAGACACCAAAT CTCACAGATCGAAGGAGTCAGAAATTCCTTTGAAACAACTTAGCC CAAACCTTTCTGTGTCAGTATGGATAAATCAAGGCCCAATGTCTAG AAGGTCTTGGGCAAAGTTGAAATTCAGGGTCAGTGACACAACCTCAAGGGAGGCCCCGAAAGTGCCAGCTGCACAGCAGCCCCTGCCTG GCTTTGCTGTTTGCCCACCGTCCCGTGTCAGTGAATCACGGGCAT CTTCAGGAGCTCAGCCTGGGTCTTCATTTGTTTCCCTCGGCCCCT TCCTCAGCCTCAGGACAGTGCTAGCAGCCCCCACACATCTCTCCC Petition 870250090423, of 03 / 10 / 2025, p. 139 / 218 132 / 144 TACAGATACCATGG (SEQ ID NO: 18) Source: Plasmid DC 969 (SerpinA1 = A1AT)chromosome 14 NG_008290.1 A1AT intron filler sequence of 398 bp (upstream of the expression cassette) GCATGCAGAGTGGACAGGGGCCTCAGGGACCCCTGATCCCAGCT TTCTCATTGGACAGAAGGAGGAGACTGGGGCTGGAGAGGGACCT GGGCCCCCACTAAGGCCACAGCAGAGCCAGGACTTTAGCTGTGC TGACTGCAGCCTGGCTTGCCTCCACTGCCTCCATTGACT GCAAGGGAGCCTCAGAGTGGAGGAAGCAGCCCCTGGCCTTGCCT CCCACCTCCCCTCCCCTATGCTGTTTTCCTGGGACAGTGGGAGCT GGCTTAGAATGCCCTGGGGCCCCCAGGACCCTGGCATTTTAACC CCTCAGGGGCAGGAAGGCAGCCTGAGTACAGAGAGGCAGTCCATCC CCTGCTGTATGCCACACACCATCCCCACAGTCGACATTTAAATT (SEQ ID NO: 19) Source: Plasmid DC 969 (SerpinA1= A1AT)chromosome 14 NG_008290.1 ITR-NDES-miR155- Amir-DMPK 204 -BGHpA-filler-ITR (3739 bp) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGC CCGGGCAAAGCCCGGGCGTCGGGCGACCTTTTGGTCGCCCGGCC TCAGTGAGCGAGCGAGCGCAGAGAGGGAGTGGCCAACTCCA TCACTAGGGGTTCCTTACGTACAATTGGGATCCTGAACTTGAACTTGGCC AGAAAACATCCCAGGGATTTACAGATCACATGCAGGCAGGGACCA Petition 870250090423, of 03 / 10 / 2025, p. 140 / 218 133 / 144 GCTCAACCCTTCTTTAATGTCATCCAGGGAGGGGGCCAGGGATG GAGGGGAGGGGTTGAGGAGCGAGAGGCAGTTATTTTTGGGTGGG ATTCACCACTTTTCCCATGAAGAGGGGAGACTTGGTATTTTGTTCA ATCATTAAGAAGACAAAGGGTTTGTTGAACTTGACCTCGGGGGGG ATAGACATGGGTATGGCCTCTAAAAACATGGCCCCAGCAGCTTCA GTCCCTTTCTCGTCGATGGTCAGCACAGCCTTATGCACGGCCTGG AGGGGAGAGAAGCAGAGACACGTTGTAAGGCTGATCCCAGGCCT CGAGCAAGGCTCACGTGGACACCTCCCAGGAAGCGCTCACTCCC CCTGGACGGCCCTGGCCCTGCACATCCTCTCCCTCCCTGTCACAT AGGCCTTGCTCCTCCTCAAGGCTTTGGCTGATGGGGCTGGCTCC CCTCTGTCCATCTTCCTGACAAGCGCCTCTCCCCCTGCTCAGGTG CACCCACAACTCAGAACAGGGAAGAGCATCGTCACTCCACTAGTC TGCCTCCAGGGCTCTCTCCTTTCTAGTACACGGCTTGAAGCTCCT TGAGGACACGGACCCTGGCAGTGACCTTCACAGTGCCCAGACCC CAAGATAATGCAGCCATTCATGGAACTGCAGGTTGTTCATTGGTC GCCTTTAGTTTTCCAAAATAAGTGTCACTTTAGCTGAAATCATTCAT TAATTCAGACACCAAATCTCACAGATCGAAGGAGTCAGAAATTCCT TTGAAACAACTTAGCCCAAACCTTTCTGTGTCAGTATGGATAAATC AAGGCCCAATGTCTAGAAGGTCTTGGGCAAAGTTGAAATTCAGGG TCAGTGACACAACCTCAAGGGAGGCCCCGAAAGTGCCAGCTGCA CAGCAGCCCCTGCCTGGCTTTGCTGTTTGCCCACCGTCCCGTGTCAGTGAATCACGGGCATCTTCAGGAGCTCAGCCTGGGTCTTCATTT GTTTCCCTCGGCCCCTTCCTCAGCCTCAGGACAGTGCTAGCAGCC CCCACACATTCTTCCCTACAGATACCATGGCACCCATGCCTCCTC AGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTCC CAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGGG TTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGGTTGACATGCGG CTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTGG TGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAGG GGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCACA Petição 870250090423, de 03 / 10 / 2025, pág. 141 / 218 134 / 144 TAGCAATTGGAAACTGAAAGCTTATCAGACCCTTTCTGGAAATCAG CCCACTGTTTATAAACTTGAGGCCCCACCCTCGAGGTACCCCCTG CCCCCCACAGCTCCTCTCCTGTGCCTTGTTTCCCAGCCATGCGTT CTCCTCTATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTT GCTGCCAGGGAGATGGTTGGGTTGACATGCGGCTCCTGACAAAA CACAAACCCCTGGTGTGTGTGGGCGTGGGTGGTGTGAGTAGGGG GATGAATCAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCA CACAAAGTCTGTGCGGGGGTGGGAGCGCACATAGCAATTGGAAA CTGAAAGCTTCTGCAGACCTGCTTGCTGCCTGCCCTGGCGAAGG ATTGGCAGGCTTGCCCGTCACAGGACCCCCGCTGGCTGACTCAG GGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCAC GGCCACGGGCCGCCCTTTCCTGGCAGGACAGCGGGATCTTGCAG CTGTCAGGGGAGGGGAGGCGGGGGCTGATGTCAGGAGGGATAC AAATAGTGCCGACGGCTGGGGGCCCTGTCTCCCCTCGCCGCATC CACTCTCCGGCCGGCCGCCTGCCCGCCGCCTCCTCCGTGCGCC CGCCAGCCTCGCCCG GAGCTCTGAGTAGACGAAGCTAAGGCGCG CCTGAGAACTTCAGGGTGAGTTTGGGGACCCTTGATTGTTCTTTC TTTTTCGCTATTGTAAAATTCATGTTATATGGAGGGGGCAAAGTTT TCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATG CATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTG ACAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTC GTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTG TTTATTTGTCAGATTGTAAGATCCCATCGATTCGGATCCCTGGAGG CTTGCTGAAGGCTGTATGCTGAGTCGAAGACAGTTCTAGGGTGTT TTGGCCACTGACTGACACCCTAGATGTCTTCGATTCAGGACACAA GGCCTGTTACTAGCACTCACATGGAACAAATGGCCCTCGAGCAAT CAGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACAATTG TTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGC TGGCGTGGAAATATTCTTATTGGTAGAAACAACTACATCCTGGTCA TCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAG Petição 870250090423, de 03 / 10 / 2025, pág. 142 / 218 135 / 144 ATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAAC CATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGC TG ACCGG TCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGT GCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTA ATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCT ATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATT GGGAAGACAATAGCGCATGCAGAGTGGACAGGGGCCTCAGGGAC CCCTGATCCCAGCTTTCTCATTGGACAGAAGGAGGAGACTGGGG CTGGAGAGGGACCTGGGCCCCCACTAAGGCCACAGCAGAGCCA GGACTTTAGCTGTGCTGACTGCAGCCTGGCTTGCCTCCACTGCCC TCCTTTGCCTCAAGAGCAAGGGAGCCTCAGAGTGGAGGAAGCAG CCCCTGGCCTTGCCTCCCACCTCCCCTCCCCTATGCTGTTTTCCT GGGACAGTGGGAGCTGGCTTAGAATGCCCTGGGGCCCCCAGGA CCCTGGCATTTTAACCCCTCAGGGGCAGGAAGGCAGCCTGAGAT ACAGAAGAGTCCATCACCTGCTGTATGCCACACACCATCCCCACA GTCGACATTTAAATTAGGAACCCCTAGTGATGGAGTTGGCCACTC CCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAA GCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGC GAGCGAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO: 20) Tabela 5. Anotação da sequência ITR-nDes-miR155- amiR-DMPK204de 5'ITR a 3'ITR (3739 pb) Sequence Numbering SEQ ID NO Annotation 1 - 145 43 ITR 5' 146 - 1283 44 A1AT padding 1284 - 2238 12 nDes Promoter (details below) 1284 - 1642 21 Byrne Enhancer 1643 - 1648 n / a Kpn1 restriction site Petition 870250090423, dated 03 / 10 / 2025, pp. 143 / 218 136 / 144 Sequence Numbering SEQ ID NO Annotation 1647 - 1923 22 Paulin Enhancer 1923 - 1928 n / a HindIII Restriction Site 1929 - 1934 n / a PstI Restriction Site 1929 - 2238 23 Paulin Promoter 2268 - 2578 45 Rabbit Globin Intron 5' Arm 2579 - 2584 n / a BamH1 Restriction Site 2585 - 2611 46 miR155 5' Flanking Sequence 2612 n / a Invitrogen Block-IT Kit Additional Base 2613 - 2633 4 miRNA Guide DMPK204 2634 - 2652 6 miR155 Terminal Loop 2653 - 2671 5 miRNA passenger DMPK204 2672 - 2674 Additional base of the Invitrogen Block-IT kit 2675 - 2716 41 3'miR155 flanking sequence 2717 - 2722 n / a Xho1 restriction site 2723 - 3005 47 3' arm of rabbit Bglobin intron 3011 - 3196 16 MinBGH polyA 3197 - 3594 48 A1AT filling 3595 - 3739 49 3'ITR Sequence of the synthesized nDes-miR155-204 fragment: AGATCTCCATGGCACCCATGCCTCCTCAGGTACCCCCTGCCCCCC ACAGCTCCTCTCCTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTCT ATAAATACCCGCTCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCA GGGAGATGGTTGGGTTGACATGCGGCTCCTGACAAAACACAAAC CCCTGGTGTGTGTGGGCGTGGGTGGTGTGAGTAGGGGGATGAAT CAGGGAGGGGGCGGGGGACCCAGGGGGCAGGAGCCACACAAA GTCTGTGCGGGGGTGGGAGCGCACATAGCAATTGGAAACTGAAA GCTTATCAGACCCTTTCTGGAAATCAGCCCACTGTTTATAAACTTG AGGCCCCACCCTCGAGGTACCCCCTGCCCCCCACAGCTCCTCTC Petição 870250090423, de 03 / 10 / 2025, pág. 144 / 218 137 / 144 CTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTCTATAAATACCCGC TCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTG GGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGT GGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGC GGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGG TGGGAGCGCACATAGCAATTGGAAACTGAAAGCTTCTGCAGACCT GCTTGCTGCCTGCCCTGGCGAAGGATTGGCAGGCTTGCCCGTCA CAGGACCCCCGCTGGCTGACTCAGGGGCGCAGGCCTCTTGCGG GGGAGCTGGCCTCCCCGCCCCCACGGCCACGGGCCGCCCTTTC CTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGGAGGGGAGG CGGGGGCTGATGTCAGGAGGGATACAAATAGTGCCGACGGCTGG GGGCCCTGTCTCCCCTCGCCGCATCCACTCTCCGGCCGGCCGCC TGCCCGCCGCCTCCTCCGTGCGCCCGCCAGCCTCGCCCGGAGC TCTGAGTAGACGAAGCTAAGGCGCGCCTGAGAACTTCAGGGTGA GTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTC ATGTTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATG GGAAGATGTCCCTTGTATCACCATGCATGGACCCTCATGATAATTT TGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTAT TTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTT GTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGA TCCCATCGATTCGGATCCCTGGAGGCTTGCTGAAGGCTGTATGCT GAGTCGAAGACAGTTCTAGGGTGTTTTGGCCACTGACTGACACCC TAGATGTCTTCGATTCAGGACACAAGGCCTGTTACTAGCACTCAC ATGGAACAAATGGCCCTCGAGCAATCAGGGTATATTATATTGTACT TCAGCACAGTTTTAGAGAACAATTGTTATAATTAAATGATAAGGTA GAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATT GGTAGAAACAACTACATCCTGGTCATCATCCTGCCTTTCTCTTTAT GGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTT TTCCTACAGCTCCTGGGCAACGTGCTGACCGGTCTAGTTGCCAGC Petition 870250090423, of 03 / 10 / 2025, p. 145 / 218 138 / 144 CATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAG GTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATC GCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG GGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCGCATGC GTCGACT (SEQ ID NO: 26) nDes-miR155-204 promoter for polyA CACCCATGCCTCCTCAGGTACCCCCTGCCCCCCACAGCTCCTCTC CTGTGCCTTGTTTCCCAGCCATGCGTTCTCCTCTATAAATACCCGC TCTGGTATTTGGGGTTGGCAGCTGTTGCTGCCAGGGAGATGGTTG GGTTGACATGCGGCTCCTGACAAAACACAAACCCCTGGTGTGTGT GGGCGTGGGTGGTGTGAGTAGGGGGATGAATCAGGGAGGGGGC GGGGGACCCAGGGGGCAGGAGCCACACAAAGTCTGTGCGGGGG TGGGAGCGCACATAGCAATTGGAAACTGAAAGCTTATCAGACCCT TTCTGGAAATCAGCCCACTGTTTATAAACTTGAGGCCCCACCCTC GAGGTACCCCCTGCCCCCCACAGCTCCTCTCCTGTGCCTTGTTTC CCAGCCATGCGTTCTCCTCTATAAATACCCGCTCTGGTATTTGGG GTTGGCAGCTGTTGCTGCCAGGGAGATGGTTGGGTTGACATGCG GCTCCTGACAAAACACAAACCCCTGGTGTGTGTGGGCGTGGGTG GTGTGAGTAGGGGGATGAATCAGGGAGGGGGCGGGGGACCCAG GGGGCAGGAGCCACACAAAGTCTGTGCGGGGGTGGGAGCGCAC ATAGCAATTGGAAACTGAAAGCTTCTGCAGACCTGCTTGCTGCCT GCCCTGGCGAAGGATTGGCAGGCTTGCCCGTCACAGGACCCCCG CTGGCTGACTCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCC TCCCCGCCCCCACGGCCACGGGCCGCCCTTTCCTGGCAGGACAG CGGGATCTTGCAGCTGTCAGGGGAGGGGAGGCGGGGGCTGATG TCAGGAGGGATACAAATAGTGCCGACGGCTGGGGGCCCTGTCTC CCCTCGCCGCATCCACTCTCCGGCCGGCCGCCTGCCCGCCGCCT CCTCCGTGCGCCCGCCAGCCTCGCCCGGAGCTCTGAGTAGACGAAGCTAAGGCGCGCCTGAGAACTTCAGGGTGAGTTTGGGGACCCT Petition 870250090423, dated 03 / 10 / 2025, pp. 146 / 218 139 / 144 TGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATATGGAG GGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCT TGTATCACCATGCATGGACCCTCATGATAATTTTGTTTCTTTCACTT TCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTT CTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTT AAATTCACTTTTGTTTATTTGTCAGATTGTAAGATCCCATCGATTCG GATCCCTGGAGGCTTGCTGAAGGCTGTATGCTGAGTCGAAGACA GTTCTAGGGTGTTTTGGCCACTGACTGACACCCTAGATGTCTTCG ATTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATG GCCCTCGAGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTT TAGAGAACAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCA TATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAAC TACATCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATA TACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGG CCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTC CTGGGCAACGTGCTGACCGGTCTAGTTGCCAGCCATCTGTTGTTT GCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCC ACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGA GTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGC AAGGGGGAGGATTGGGAAGACAATAGC (SEQ ID NO: 17) Sequência de aminoácidos de AAVrh74N502I MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGL VLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYL RYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVK TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKRLNFGQTGDSESV PDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNT YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFN Petition 870250090423, of 03 / 10 / 2025, p. 147 / 218 140 / 144 IQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPF PADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEF SYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQ QLLFSQAGPNNMSAQAKNWLPGPCYRQRVSTTLSQNNNSIFAWT GATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAG KDNVDYSSVMLTSEEEIKTTNPVATEQYGVADNLQQQNAAPIVGAV NSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLK HPPPQILIKNTPVPADPPTTFNQLASFITQYSTGQVSVEIEWELQKE NSKRWNPEIQYTSNYKSTNVDFAVNTEGTYSEPRPIGTRYLT NORNL (SEQ ID:50) Nucleotide sequence encoding capsid AAVrh74 N502I ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTC TCTGAGGGCATTCGCGAGTGGTGGGACCTGAAACCTGGAGCCCC GAAACCCAAAGCCAACCAGCAAAAGCAGGACAACGGCCGGGGTC TGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCG ACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGA GCACGACAAGGCCTACGACCAGCAGCTCCAAGCGGGTGACAATC CGTACCTGCGGTATAATCACGCCGACGCCGAGTTTCAGGAGCGT CTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGCGCAGT CTTCCAGGCCAAAAAGCGGGTTCTCGAACCTCTGGGCCTGGTTGA ATCGCCGGTTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGC CATCACCCCAGCGCTCTCCAGACTCCTCTACGGGCATCGGCAAG AAAGGCCAGCAGCCCGCAAAAAAGAGACTCAATTTTGGGCAGACT GGCGACTCAGAGTCAGTCCCCGACCCTCAACCAATCGGAGAACC ACCAGCAGGCCCCTCTGGTCTGGGATCTGGTACAATGGCTGCAG GCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGA GTGGGTAGTTCCTCAGGAAATTGGCATTGCGATTCCACATGGCTG GGCGACAGAGTCATCACCACCAGCACCCGCACCTGGGCCCTGCC CACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACCTC GGGAGGAAGCACCAACGACAACACCTACTTCGGCTACAGCACCC Petition 870250090423, dated 03 / 10 / 2025, pages 148 / 218 141 / 144 CCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACC ACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGC CCAAGAGGCTCAACTTCAAGCTCTTCAACATCCAAGTCAAGGAGG TCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTTACCA GCACGATTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGTACG TGCTCGGCTCGGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCG GACGTCTTCATGATTCCTCAGTACGGGTACCTGACTCTGAACAAT GGCAGTCAGGCTGTGGGCCGGTCGTCCTTCTACTGCCTGGAGTA CTTTCCTTCTCAAATGCTGAGAACGGGCAACAACTTTGAATTCAGC TACAACTTCGAGGACGTGCCCTTCCACAGCAGCTACGCGCACAG CCAGAGCCTGGACCGGCTGATGAACCCTCTCATCGACCAGTACTT GTACTACCTGTCCCGGACTCAAAGCACGGGCGGTACTGCAGGAA CTCAGCAGTTGCTATTTTCTCAGGCCGGGCCTAACAACATGTCGG CTCAGGCCAAGAACTGGCTACCCGGTCCCTGCTACCGGCAGCAA CGCGTCTCCACGACACTGTCGCAGAACAACAACAGCATCTTTGCC TGGACGGGTGCCACCAAGTATCATCTGAATGGCAGAGACTCTCTG GTGAATCCTGGCGTTGCCATGGCTACCCACAAGGACGACGAAGA GCGATTTTTTCCATCCAGCGGAGTCTTAATGTTTGGGAAACAGGG AGCTGGAAAAGACAACGTGGACTATAGCAGCGTGATGCTAACCAG CGAGGAAGAAATAAAGACCACCAACCCAGTGGCCACAGAACAGT ACGGCGTGGTGGCCGATAACCTGCAACAGCAAAACGCCGCTCCTATTGTAGGGGCCGTCAATAGTCAAGGAGCCTTACCTGGCATGGTG TGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCCAA GATTCCTCATACGGACGGCAACTTTCATCCCTCGCCGCTGATGGG AGGCTTTGGACTGAAGCATCCGCCTCCAGATCTAAATCTT CACACCTGTTCCCGCCGGATCCTCCGACCACCTTCAATCAGGCCAA GCTGGCTTCTTTCATCACGCAGTACAGTACCGGCCAGGTCAGCGT GGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAACGCTGGA ACCCAGAGATTCAGTACACTTCCAACTACTACAAATCTACAAAT GGACTTTGCTGTCAATACTGAGGGTACTTATTCCGAGCCTCGCCC Petition 870250090423, of 03 / 10 / 2025, p. 149 / 218 142 / 144 CATTGGCACCCGTTACCTCACCCGTAATCTGTAA (SEQ ID NO:51) Amino acid sequence of AAVrh74W505R MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGL VLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYL RYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVK TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKRLNFGQTGDSESV PDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGN WHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNT YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFN IQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPF PADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEF SYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQ QLLFSQAGPNNMSAQAKNWLPGPCYRQRVSTTLSQNNNSNFART GATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAG KDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAV NSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLK HPPPQILIKNTPVPADPPTTFNQKLASFITQYSTGQVSVEIEWELQKE NSKRWNPEIQYTSNYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL (SEQ ID NO:52) Nucleotide sequence encoding AAVrh74W505R ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTC TCTGAGGGCATTCGCGAGTGGTGGGACCTGAAACCTGGAGCCCC GAAACCCAAAGCCAACCAGCAAAAGCAGGACAACGGCCGGGGTC TGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCG ACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGA GCACGACAAGGCCTACGACCAGCAGCTCCAAGCGGGTGACAATC CGTACCTGCGGTATAATCACGCCGACGCCGAGTTTCAGGAGCGT Petition 870250090423, dated 03 / 10 / 2025, pages 150 / 218 143 / 144 CTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGCGCAGT CTTCCAGGCCAAAAAGCGGGTTCTCGAACCTCTGGGCCTGGTTGA ATCGCCGGTTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGC CATCACCCCAGCGCTCTCCAGACTCCTCTACGGGCATCGGCAAG AAAGGCCAGCAGCCCGCAAAAAAGAGACTCAATTTTGGGCAGACT GGCGACTCAGAGTCAGTCCCCGACCCTCAACCAATCGGAGAACC ACCAGCAGGCCCCTCTGGTCTGGGATCTGGTACAATGGCTGCAG GCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGA GTGGGTAGTTCCTCAGGAAATTGGCATTGCGATTCCACATGGCTG GGCGACAGAGTCATCACCACCAGCACCCGCACCTGGGCCCTGCC CACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACCTC GGGAGGAAGCACCAACGACAACACCTACTTCGGCTACAGCACCC CCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACC ACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGC CCAAGAGGCTCAACTTCAAGCTCTTCAACATCCAAGTCAAGGAGG TCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTTACCA GCACGATTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGTACG TGCTCGGCTCGGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCG GACGTCTTCATGATTCCTCAGTACGGGTACCTGACTCTGAACAAT GGCAGTCAGGCTGTGGGCCGGTCGTCCTTCTACTGCCTGGAGTA CTTTCCTTCTCAAATGCTGAGAACGGGCAACAACTTTGAATTCAGCTACAACTTCGAGGACGTGCCCTTCCACAGCAGCTACGCGCACAG CCAGAGCCTGGACCGGCTGATGAACCCTCTCATCGACCAGTACTT GTACTACCTGTCCCGGACTCAAAGCACGGGCGGTACTGCAGGAA CTCAGCAGTTGCTATTTTCTCAGGCCGGGCCTAACAACATGTCGG CTCAGGCCAAGAACTGGCTACCCGGTCCCTGCTACCGGCAGCAA CGCGTCTCCACGACACTGTCGCAGAACAACAACAGCAACTTTGCC AGGACGGGTGCCACCAAGTATCATCTGAATGGCAGAGACTCTCTG GTGAATCCTGGCGTTGCCATGGCTACCCACAAGGACGACGAAGA GCGATTTTTTCCATCCAGCGGAGTCTTAATGTTTGGGAAACAGGG Petição 870250090423, de 03 / 10 / 2025, pág. 151 / 218 144 / 144 AGCTGGAAAAGACAACGTGGACTATAGCAGCGTGATGCTAACCAG CGAGGAAGAAATAAAGACCACCAACCCAGTGGCCACAGAACAGT ACGGCGTGGTGGCCGATAACCTGCAACAGCAAAACGCCGCTCCT ATTGTAGGGGCCGTCAATAGTCAAGGAGCCTTACCTGGCATGGTG TGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCCAA GATTCCTCATACGGACGGCAACTTTCATCCCTCGCCGCTGATGGG AGGCTTTGGACTGAAGCATCCGCCTCCTCAGATCCTGATTAAAAA CACACCTGTTCCCGCGGATCCTCCGACCACCTTCAATCAGGCCAA GCTGGCTTCTTTCATCACGCAGTACAGTACCGGCCAGGTCAGCGT GGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAACGCTGGA ACCCAGAGATTCAGTACACTTCCAACTACTACAAATCTACAAATGT GGACTTTGCTGTCAATACTGAGGGTACTTATTCCGAGCCTCGCCC CATTGGCACCCGTTACCTCACCCGTAATCTGTAA (SEQ ID NO:53)

Claims

1. Recombinant adeno-associated virus particle (rAAV), characterized in that it comprises: a) an RNAi comprising a first strand and a second strand, wherein: i) the first strand and the second strand form a duplex; ii) the first strand comprises a guide region, wherein the guide region comprises nucleic acid with the sequence 5'AGUCGAAGACAGUUCUAGGGU-3' (SEQ ID NO: 1) or with a sequence with approximately 90% identity to the sequence of SEQ ID NO: 1; and iii) the second strand comprises a non-guide region; and b) an AAV capsid comprising an amino acid sequence with approximately 90% identity to a wild-type AAVrh74 capsid.

2. rAAV particle, according to claim 1, characterized in that the non-guide region comprises nucleic acid with the sequence 5'- ACCCUAGAUGUCUUCGAUU-3' (SEQ ID NO: 2) or with a sequence with approximately 90% identity to the sequence of SEQ ID NO:

2.

3. rAAV particle, according to any one of claims 1 to 2, characterized in that the first strand comprises nucleic acid with the sequence SEQ ID NO: 1, and the non-guide region comprises nucleic acid with the sequence SEQ ID NO:

2.

4. rAAV particle, according to any one of claims 1 to 3, characterized in that the first strand and the second strand are linked by means of an RNA linker capable of forming a loop structure.

5. rAAV particle, according to claim 4, ca Petition 870250090423, dated 03 / 10 / 2025, p. 153 / 218 2 / 24 characterized by the fact that the RNA linker comprises about 4 to about 50 nucleotides.

6. rAAV particle, according to any one of claims 4 to 5, characterized in that the loop structure comprises from about 4 to about 20 nucleotides.

7. rAAV particle, according to any one of claims 4 to 6, characterized in that the loop structure comprises the nucleic sequence with SEQ ID NO: 3 or with a sequence with approximately 90% identity to the sequence of SEQ ID NO:

3.

8. rAAV particle, according to any one of claims 4 to 7, characterized in that the RNAi comprises 5' to 3' of the second strand, the RNA linker and the first strand.

9. rAAV particle, according to any one of claims 4 to 7, characterized in that the RNAi comprises 5' to 3' of the first strand, the RNA linker and the second strand.

10. rAAV particle, according to any one of claims 1 to 8, characterized in that the RNAi comprises nucleic acid with the sequence of SEQ ID NO: 7 or with a sequence with approximately 90% identity to the sequence of SEQ ID NO:

7.

11. rAAV particle, according to any one of claims 1 to 10, characterized in that the RNAi is a small inhibitory RNA (siRNA), a microRNA (miRNA) or a small hairpin RNA (shRNA).

12. rAAV particle, according to any one of claims 1 to 11, characterized in that the RNAi further comprises a scaffold.

13. rAAV particle, according to claim 12, characterized in that the scaffold comprises all or a portion of the nucleic acid of SEQ ID NO:

11.

14. rAAV particle, according to claim 13, characterized in that the mRNA is embedded in the scaffold.

15. rAAV particle, according to claim 14, characterized in that the scaffold has a 5' arm, wherein the 5' arm is located at 5' of the RNAi-encoding nucleic acid, and a 3' arm, wherein the 3' arm is located at 3' of the RNAi-encoding nucleic acid.

16. rAAV particle, according to any one of claims 12 to 15, characterized in that the framework is a miR-155 framework.

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

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

19. rAAV particle, according to any one of claims 1 to 18, characterized in that the RNAi targets RNA encoding a polypeptide associated with myotonic dystrophy type 1 (DM1).

20. rAAV particle, according to claim 19, characterized in that the polypeptide is dystrophic myotonic protein kinase (DMPK).

21. rAAV particle, according to claim 20, Petition 870250090423, dated 10 / 03 / 2025, page 155 / 218 4 / 24, characterized in that the DMPK comprises a mutation associated with DM-1.

22. rAAV particle, according to any one of claims 20 to 21, characterized in that the gene encoding DMPK comprises five or more CTG trinucleotide repeats.

23. Expression cassette characterized in that it comprises nucleic acid encoding RNAi, as defined in any one of claims 1 to 22.

24. Expression cassette, according to claim 23, characterized in that the nucleic acid encoding RNAi is operationally linked to a promoter.

25. Expression cassette, according to claim 24, characterized in that the promoter is a muscle-specific promoter.

26. Expression cassette, according to any one of claims 24 to 25, characterized in that the promoter is a demining promoter or variant thereof.

27. Expression cassette, according to claim 26, characterized in that the desmin promoter comprises one or more enhancer elements and the human desmin gene promoter.

28. Expression cassette, according to any one of claims 26 to 27, characterized in that the desmin promoter comprises two enhancer elements and the human desmin gene promoter.

29. Expression cassette, according to any one of claims 26 to 28, characterized in that the demining promoter comprises one or more Byrne intensifying elements and / or one or more Paulin intensifying elements. Petition 870250090423, dated 10 / 03 / 2025, pp. 156 / 218 5 / 24 30. Expression cassette, according to any one of claims 26 to 29, characterized in that the desmin promoter comprises one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence with about 90% identity to the sequence of SEQ ID NO: 21 and / or one or more enhancer elements comprising the nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence with about 90% identity to the sequence of SEQ ID NO:

22.

31. Expression cassette, according to any one of claims 26 to 30, characterized in that the desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence with approximately 90% identity to the nucleotide sequence of SEQ ID NO:

12.

32. Expression cassette, according to any one of claims 23 to 31, characterized in that it further comprises an intron.

33. Expression cassette, according to claim 32, characterized in that the intron is a rabbit β-globin intron.

34. Expression cassette, according to any one of claims 32 to 33, characterized in that the intron comprises the nucleotide sequence of SEQ ID NO: 13 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

13.

35. Expression cassette, according to any one of claims 32 to 34, characterized in that the nucleic acid encoding RNAi is incorporated into the intron.

36. Expression cassette, according to claim 35, characterized in that the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at 5' of the nucleic acid encoding RNAi and the 3' arm is located at 3' of the nucleic acid encoding RNAi.

37. Expression cassette, according to claim 36, characterized in that the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 14 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

14.

38. Expression cassette, according to any one of claims 36 to 37, characterized in that the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 15 or a sequence with about 90% identity to the sequence of SEQ ID NO:

15.

39. Expression cassette, according to any one of claims 23 to 38, characterized in that it further comprises a polyadenylation signal.

40. Expression cassette, according to claim 39, characterized in that the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or an HSV TK pA.

41. Expression cassette, according to claim 40, characterized in that the polyadenylation signal is a minimal polyadenylation signal of bovine growth hormone.

42. Expression cassette, according to any one of claims 39 to 41, characterized in that the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO: 16 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

16.

43. Expression cassette, according to any one of claims 23 to 42, characterized in that the expression cassette comprises the nucleotide sequence of SEQ ID NO: 17 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

17.

44. Expression cassette, characterized in that it comprises a modified desmin promoter, wherein the modified desmin promoter comprises one or more enhancer elements and the human desmin gene promoter.

45. Expression cassette, according to claim 44, characterized in that the modified desmin promoter comprises two enhancer elements and the human desmin gene promoter.

46. ​​Expression cassette, according to any one of claims 44 to 45, characterized in that the modified demine promoter comprises one or more Byrne intensifier elements and / or one or more Paulin intensifier elements.

47. Expression cassette, according to any one of claims 44 to 46, characterized in that 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. Expression cassette, according to any one of claims 44 to 47, characterized in that the desmin promoter comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence with approximately 90% identity to the nucleotide sequence of SEQ ID NO:

12.

49. Expression cassette, according to any of the claims 44 to 48, as described in Petition 870250090423, dated 03 / 10 / 2025, page 159 / 218 8 / 24, characterized by the fact that it also includes an intron.

50. Expression cassette, according to claim 49, characterized in that the intron is a rabbit β-globin intron.

51. Expression cassette, according to any one of claims 49 to 50, characterized in that the intron comprises the nucleotide sequence of SEQ ID NO: 13 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

13.

52. Expression cassette, according to any one of claims 44 to 51, characterized in that the nucleic acid encoding the transgene is incorporated into the intron.

53. Expression cassette, according to claim 52, characterized in that the intron comprises a 5' arm and a 3' arm, wherein the 5' arm is located at 5' of the nucleic acid encoding the transgene and the 3' arm is located at 3' of the nucleic acid encoding the transgene.

54. Expression cassette, according to claim 53, characterized in that the 5' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 14 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

14.

55. Expression cassette, according to any one of claims 53 to 54, characterized in that the 3' arm of the intron comprises the nucleotide sequence of SEQ ID NO: 15 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

15.

56. Expression cassette, according to any one of claims 44 to 55, characterized in that it further comprises a polyadenylation signal. Petition 870250090423, dated 03 / 10 / 2025, p. 160 / 218 9 / 24 57. Expression cassette, according to claim 56, characterized in that the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or an HSV TK pA.

58. Expression cassette, according to claim 57, characterized in that the polyadenylation signal is a minimal polyadenylation signal of bovine growth hormone.

59. Expression cassette, according to any one of claims 56 to 58, characterized in that the bovine growth hormone polyadenylation signal comprises the nucleotide sequence of SEQ ID NO: 16 or a sequence with about 90% identity to the sequence of SEQ ID NO:

16.

60. Expression cassette, according to any one of claims 44 to 59, characterized in that the transgene encodes a polypeptide or a nucleic acid.

61. Expression cassette, according to any one of claims 44 to 60, characterized in that the transgene encodes an RNAi.

62. Vector, characterized in that it comprises the expression cassette, as defined in any one of claims 23 to 61.

63. Vector according to claim 62, characterized in that the expression cassette is flanked by one or more filler nucleic acid sequences.

64. Vector, according to claim 63, characterized in that one or more filler nucleic acid sequences are derived from the human SerpinA1 gene.

65. Vector, according to any one of claims 63 to 64, characterized in that a filler nucleic acid sequence located at 5' of the expression cassette is derived from the human SerpinAI gene.

66. Vector, according to any one of claims 63 to 65, characterized in that the padding sequence located at 5' of the expression cassette comprises the nucleotide sequence of SEQ ID NO: 18 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

18.

67. Vector, according to any one of claims 63 to 66, characterized in that a filler nucleic acid sequence located 3' from the expression cassette is derived from the human SerpinA1 gene.

68. Vector, according to any one of claims 63 to 67, characterized in that the padding sequence located at 3' of the expression cassette comprises the nucleotide sequence of SEQ ID NO: 19 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

19.

69. Vector, according to any one of claims 62 to 68, characterized in that it is a recombinant adeno-associated virus (rAAV) vector.

70. Vector of rAAV, as defined in claim 69, characterized in that the expression cassette is flanked by one or more inverted terminal repeat (ITR) sequences of AAV.

71. Vector of rAAV, according to claim 70, characterized in that the expression cassette is flanked by two AAV ITRs.

72. Vector of rAAV, according to any one of claims 70 to 71, characterized in that the AAV ITRs are serotype ITRs AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV or mouse AAV. Petition 870250090423, dated 10 / 03 / 2025, p. 162 / 218 11 / 24 73. Vector of rAAV, according to any one of claims 70 to 72, characterized in that the ITRs of AAV are ITRs of AAV2.

74. rAAV vector, according to any one of claims 69 to 73, characterized in that it comprises the nucleotide sequence of SEQ ID NO: 20 or a sequence with approximately 90% identity to the sequence of SEQ ID NO:

20.

75. Vector rAAV, according to any one of claims 69 to 74, characterized in that it is a self-complementary vector rAAV.

76. Cell, characterized in that it comprises the expression cassette, as defined in any one of claims 23 to 61, the vector, as defined in any one of claims 62 to 68, or the rAAV vector, as defined in any one of claims 69 to 75.

77. rAAV particle, according to any one of claims 1 to 22, characterized in that the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 502.

78. rAAV particle, according to claim 77, characterized in that the amino acid substitution at position 502 is an isoleucine (I).

79. rAAV particle, according to claim 77 or 78, characterized in that the AAV viral particle comprises a capsid of serotype AAVrh74 N502I.

80. rAAV particle, according to claim 77 or 78, characterized in that the ITR is an AAV2 ITR and the capsid of the rAAV particle is a capsid of serotype AAVrh74 N502I.

81. rAAV particle, according to any one of claims 1 to 22, characterized in that the AAV capsid comprises an amino acid sequence comprising an amino acid substitution at position 505.

82. rAAV particle, according to claim 81, characterized in that the amino acid substitution at position 505 is an arginine (R).

83. rAAV particle, according to any one of claims 1 to 22, characterized in that the rAAV particle comprises a capsid of the AAVrh74 W505R serotype.

84. rAAV particle, according to claim 83, characterized in that the ITR is an AAV2 ITR and the capsid of the rAAV particle is a capsid of serotype AAVrh74 W505R.

85. rAAV particle, characterized in that it comprises an rAAV vector and a capsid, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204 miRNA passenger sequence, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a bovine growth hormone polyadenylation minimal sequence, nucleic acid that encodes a sequence of nucleic acids. Petition 870250090423, dated 03 / 10 / 2025, page.164 / 218 13 / 24 filler cells from the human serpinAI gene, and an ITR of AAV2; and wherein the capsid is an AAVrh74 N502I capsid.

86. rAAV particle, characterized in that it comprises an rAAV vector, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO: 43; a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 18; a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO: 21; a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO: 22; a desmin promoter comprising the polynucleotide sequence of SEQ ID NO: 23; a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO: 14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO: 40,a DMPK204 miRNA guide sequence comprising the polynucleotide sequence of SEQ ID NO: 4, a miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, a DMPK204 miRNA passenger sequence comprising the polynucleotide sequence of SEQ ID NO: 5, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO: 41, a 3' arm of a rabbit β-globin intron that competes with Competition 870250090423, dated 03 / 10 / 2025, p. 165 / 218 14 / 24 includes the polynucleotide sequence of SEQ ID NO: 15, a minimal polyadenylation sequence of bovine growth hormone comprising the polynucleotide sequence of SEQ ID NO: 16, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 19,and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO: 49; and wherein the capsid is an AAVrh74 N502I capsid.

87. rAAV particle, according to any one of claims 85 to 86, characterized in that the capsid of AAVrh74 N502I comprises capsid proteins comprising the amino acid sequence of SEQ ID NO:

50.

88. rAAV particle, characterized in that it comprises an rAAV vector and a capsid, wherein the rAAV vector comprises the following nucleic acids from 5' to 3': an AAV2 ITR, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204 miRNA passenger sequence, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a bovine growth hormone polyadenylation minimal sequence, acid A nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene and an ITR from AAV2; and where the capsid is an AAVrh74 W505R capsid.Petition 870250090423, dated 03 / 10 / 2025, pp. 166 / 218 15 / 24.

89. rAAV particle, characterized in that it comprises an rAAV vector, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR comprising the polynucleotide sequence SEQ ID NO: 43; a nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence SEQ ID NO: 18; a Byrne desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 21; a Paulin desmin enhancer element comprising the polynucleotide sequence SEQ ID NO: 22; a desmin promoter comprising the polynucleotide sequence SEQ ID NO: 23; a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence SEQ ID NO: 14; a scaffold sequence. 5' miR155 comprising the polynucleotide sequence of SEQ ID NO: 40,a DMPK204 miRNA guide sequence comprising the polynucleotide sequence of SEQ ID NO: 4, a miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, a DMPK204 miRNA passenger sequence comprising the polynucleotide sequence of SEQ ID NO: 5, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO: 41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO: 15, a bovine growth hormone minimal polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO: 16, nucleic acid encoding a human serpinA1 gene filler nucleic acid sequence comprising the polynucleotide sequence of SEQ ID NO: 19, and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO: 49; and in which the capsid is an AAVrh74 W505R capsid.

90. rAAV particle, according to any of the claims 88 to 89 in Petition 870250090423, dated 10 / 03 / 2025, page 167 / 218 16 / 24, characterized in that the capsid of AAVrh74 W505R comprises capsid proteins comprising the amino acid sequence of SEQ ID NO:

52.

91. Composition, characterized in that it comprises the rAAV particle, as defined in any one of claims 1 to 22 and 77 to 90.

92. Pharmaceutical composition, characterized in that it comprises the rAAV particle, as defined in any one of claims 1 to 22 and 77 to 90.

93. Composition, according to any one of claims 91 to 92, characterized in that it further comprises a pharmaceutically acceptable carrier.

94. Kit, characterized in that it comprises the rAAV particle, as defined in any one of claims 1 to 22 and 77 to 90.

95. Kit, characterized in that it comprises the rAAV particle, as defined in any one of claims 1 to 22 and 77 to 90.

96. Kit, characterized in that it comprises the composition as defined in any one of claims 91 to 93.

97. Kit, according to any one of claims 94 to 96, characterized in that it also includes instructions for use.

98. Method for treating myotonic dystrophy-1 (DM1) in a mammal in need thereof, characterized in that it comprises administering to the mammal an effective amount of the rAAV particle, as defined in any one of claims 1 to 22 and 77 to 90.

99. Method for inhibiting the expression of myotonic dystrophy protein kinase (DMPK) in a mammal with DM-1 in need of Petition 870250090423, dated 03 / 10 / 2025, page 168 / 218 17 / 24, characterized in that it comprises administering to the mammal an effective amount of the rAAV particle, as defined in any one of claims 1 to 22 and 77 to 90.

100. Method for inhibiting the accumulation of DMPK RNA in a mammalian cell with DM-1 in need thereof, characterized in that it comprises administering to the mammal an effective amount of RNAi, as defined in any one of claims 1 to 22 and 77 to 90.

101. Method for treating myotonic dystrophy-1 (DM1) in a mammal in need thereof, characterized in that it comprises administering to the mammal an effective amount of the rAAV particle, as defined in any one of claims 1 to 22 and 77 to 90.

102. Method for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with type 1 diabetes mellitus in need thereof, characterized in that it comprises administering to the mammal an effective amount of the rAAV particle, as defined in any one of claims 1 to 22 and 77 to 90.

103. Method for inhibiting the accumulation of DMPK RNA in a mammalian cell with DM-1 in need thereof, characterized in that it comprises administering to the mammal an effective amount of the rAAV particle as defined in any one of claims 1 to 22 and 77 to 90.

104. Method, according to any one of claims 100 to 103, characterized in that the effective amount of rAAV particle is a dose of about 1 x 108 to about 2 x 1013 genome copies / mL.

105. Method according to claim 104, characterized in that the dose is approximately 5 x 10¹² genome copies / mL. Petition 870250090423, dated 03 / 10 / 2025, pp. 169 / 218 18 / 24 106. Method according to claim 104, characterized in that the dose is approximately 1 x 1013 genome copies / mL.

107. Method according to claim 104, characterized in that the dose is about 2 x 1013 genome copies / mL.

108. Method, according to any one of claims 101 to 103, characterized in that the effective amount of rAAV particle is a dose of about 1 x 108 to about 2 x 1014 genome copies / kg of body weight.

109. Method according to claim 108, characterized in that the dose is approximately 5 x 1013 genome copies / kg of body weight.

110. Method according to claim 108, characterized in that the dose is approximately 1 x 1014 genome copies / kg of body weight.

111. Method according to claim 108, characterized in that the dose is approximately 2 x 1014 genome copies / kg of body weight.

112. Method for treating myotonic dystrophy type 1 (DM1) in a mammal in need thereof, characterized in that it comprises administering to the mammal an effective amount of the composition, as defined in any one of claims 91 to 93.

113. Method for inhibiting the expression of dystrophic myotonic protein kinase (DMPK) in a mammal with type 1 diabetes mellitus (DM-1) that requires it, characterized in that it comprises administering to the mammal an effective amount of the composition, as defined in any one of claims 91 to 93.

114. Method for inhibiting the accumulation of DMPK RNA in a mammalian cell with DM-1 that needs it, characterized by the fact that it comprises administering to the mammal an effective amount of the composition, as defined in any one of claims 91 to 93.

115. Method, according to any one of claims 98 to 100, characterized in that RNAi is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time and / or after administration of RNAi.

116. Method, according to any one of claims 101 to 103, characterized in that the viral particle or rAAV particle is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time and / or after administration of the viral particle or rAAV particle.

117. Method, according to any one of claims 112 to 114, characterized in that the composition is administered in combination with an immunosuppressive agent, wherein the immunosuppressive agent is administered before, at the same time and / or after administration of the composition.

118. Method, according to any one of claims 98 to 117, characterized in that the administration of the rAAV particle, as defined in any one of claims 1 to 22 and 77 to 90, of the expression cassette, as defined in any one of claims 23 to 61, of the vector, as defined in claim 75, of the composition, as defined in any one of claims 91 to 93, or of the kit, as defined in any one of claims 94 to 97, wherein the splicing of genetic transcripts is measured after administration.

119. Method, according to claim 118, characterized in that the splicing of genetic transcripts is a measure of treatment efficacy. Petition 870250090423, dated 03 / 10 / 2025, page 171 / 218 20 / 24 120. Method, according to any one of claims 118 to 119, characterized in that the splicing of gene transcripts is about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% compared to comparable healthy tissue.

121. Method, according to any one of claims 118 to 119, characterized in that the measured gene transcripts comprise one or more genes selected from MBNL1, SOS1, PKM, zTTN, GOGLA4, CLASP1, LDB3, MBNL2, SPAG9, DNAseI, TNNT3 and ZBTB49.

122. Recombinant adeno-associated virus particle (rAAV), characterized in that it comprises an rAAV vector and a capsid, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204 miRNA passenger sequence, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a minimal sequence of Petition 870250090423, dated 03 / 10 / 2025, page 10.172 / 218 21 / 24 bovine growth, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, and an ITR of AAV2; and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO:

50.

123. Recombinant adeno-associated virus particle (rAAV), characterized in that it comprises an rAAV vector and a capsid, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR, a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, a Byrne desmin enhancer element, a Paulin desmin enhancer element, a desmin promoter, a 5' arm of a rabbit β-globin intron, a 5' miR155 scaffold sequence, a DMPK204 miRNA guide sequence, a miR155 terminal loop sequence, a DMPK204 miRNA passenger sequence, a 3' miR155 scaffold sequence, a 3' arm of a rabbit β-globin intron, a minimal sequence of Polyadenylation of bovine growth hormone, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene, and an ITR of AAV2;and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO:

52. Petition 870250090423, dated 03 / 10 / 2025, pp. 173 / 218 22 / 24; 124. rAAV particle, characterized in that it comprises an rAAV vector, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO: 43; a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 18; a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO: 21; a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO: 22; a desmin promoter comprising the polynucleotide sequence of SEQ ID NO: 23; a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO: 14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO: 40,a guide miRNA DMPK204 sequence comprising the polynucleotide sequence of SEQ ID NO: 4, a terminal loop miR155 sequence comprising the polynucleotide sequence of SEQ ID NO: 6, a passenger miRNA DMPK204 sequence comprising the polynucleotide sequence of SEQ ID NO: 5, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO: 41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO: 15, a bovine growth hormone polyadenylation minimal sequence comprising the polynucleotide sequence Petition 870250090423, dated 03 / 10 / 2025, p. 174 / 218 23 / 24 of SEQ ID NO: 16, nucleic acid encoding a filler nucleic acid sequence of the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 19,and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO: 49; and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO:

50.

125. rAAV particle, characterized in that it comprises an rAAV vector, wherein the rAAV vector comprises the following 5' to 3' nucleic acids: an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO: 43; a nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 18; a Byrne desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO: 21; a Paulin desmin enhancer element comprising the polynucleotide sequence of SEQ ID NO: 22; a desmin promoter comprising the polynucleotide sequence of SEQ ID NO: 23; a 5' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO: 14, a 5' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO: 40,a DMPK204 miRNA guide sequence comprising the polynucleotide sequence of SEQ ID NO: 4, a miR155 terminal loop sequence comprising the polynucleotide sequence of SEQ ID NO: 6, Petition 870250090423, dated 03 / 10 / 2025, p. 175 / 218 24 / 24 a DMPK204 miRNA passenger sequence comprising the polynucleotide sequence of SEQ ID NO: 5, a 3' miR155 scaffold sequence comprising the polynucleotide sequence of SEQ ID NO: 41, a 3' arm of a rabbit β-globin intron comprising the polynucleotide sequence of SEQ ID NO: 15, a bovine growth hormone minimal polyadenylation sequence comprising the polynucleotide sequence of SEQ ID NO: 16, nucleic acid encoding a filler nucleic acid sequence from the human serpinA1 gene comprising the polynucleotide sequence of SEQ ID NO: 19,and an AAV2 ITR comprising the polynucleotide sequence of SEQ ID NO: 49; and wherein the capsid comprises capsid proteins comprising the amino acid sequence of SEQ ID NO: 52.

Images