Compositions and methods for the treatment of disorders related to dystrophia myotonica protein kinase

Abstract

The disclosure relates to compositions and methods for modulating, e.g., reducing or eliminating, the expression of mutated DMPK via delivery using an adeno-associated viral (AAV) capsid variant. The compositions and methods of the present disclosure are useful in the treatment of subjects diagnosed with, suspected of having, or having myotonic dystrophy type 1 or another DMPK-related disorder.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF DISORDERSRELATED TO DYSTROPHIA MYOTONICA PROTEIN KINASERELATED APPLICATIONS

[0001] This application claims the benefit of and priority to US Provisional Application Serial No. 63 / 613,678, filed December 21, 2023, and US Provisional Application Serial No. 63 / 617,287, filed January 3, 2024, the contents of each which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 14640_0204-00304_SL.xml, was created on December 11, 2024, and is 51,535 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.FIELD

[0003] Disclosed herein arc compositions and methods relating to adcno-associatcd virus (AAV) viral particles for the delivery of polynucleotides, e.g., modulatory polynucleotides, for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA and their use in the treatment of myotonic dystrophy type 1 (DM1) and other disorders associated with or caused bymutated DMPK mRNA. In some embodiments, compositions disclosed herein may be used to treat a subject in need thereof, such as a human subject diagnosed with DM1 or another condition resulting from mutated DMPK mRNA expression.BACKGROUND

[0004] Dystrophia myotonica protein kinase (DMPK) (also referred to as myotonic dystrophy protein kinase) is an enzyme encoded by the DMPK gene (Ensembl Gene ID No. ENSG00000104936), which is found on chromosome 19. The DMPK gene is also known as DM15, DM1PK, DMK, MDPK, and MT- PK.

[0005] The DMPK protein is thought to be involved in cell communication and appears to play important roles in tissues such as heart, muscle, and brain.

[0006] Mutation(s) in DMPK cause myotonic dystrophy type 1 (DM1). DM1 is characterized by progressive muscle wasting and weakness. Muscle weakness is most often observed in distal muscles, and myotonia, cataracts, hypogonadism, frontal balding and electrocardiogram changes are also observed. There are an estimated 500,000 cases of DM1 worldwide.

[0007] DM1 is typically caused by trinucleotide repeat expansions in the DMPK gene. Trinucleotide repeat expansions occur in a repeated CTG segment in the 3‘ UTR of DMPK. Non-diseased humans typically have 5-37 CTG repeats (e.g., SEQ ID NO: 31). The number of CTG repeats determines theform of DM1 and the severity of disease. CTG repeats of the trinucleotide repeat expansion may be uninterrupted or may be interrupted with one or more other nucleotides.

[0008] Patients with 38-49 CTG repeats (e.g., SEQ ID NO: 32) are typically asymptomatic.

[0009] Patients with 50-150 CTG repeats (e.g., SEQ ID NO: 33) may manifest with mild DM1. Mild DM1 symptoms typically begin between the ages of 20 and 70 years. Mild DM1 is characterized by cataracts, myotonia, and mild muscle weakness, and patients have a normal life span.

[0010] Patients with 50 to 1000 CTG repeats (e.g., SEQ ID NO: 34) may manifest with classic DM1. Classic DM1 symptoms typically begin in adulthood. Classic DM1 is characterized by muscle weakness and wasting, myotonia, cataracts, cardiac conduction abnormalities, and a myopathic face. Adults may be physically disabled and have a shortened lifespan.[Oil] Patients with CTG repeat lengths greater than 800 may manifest with childhood DM1. Symptoms of childhood DM1 typically begin around age 10. Childhood DM1 is characterized by learning difficulties and psychosocial problems (e.g., family problems, depression, anxiety), slurred speech, hand muscle myotonia, and heart conduction abnormalities.

[0012] Patients with CTG repeat lengths greater than 1000 may manifest with congenital DM1. Patients with congenital DM1 show symptoms before birth. Symptoms of congenital DM1 visible before birth include decreased fetal movement in the uterus, polyhydramnios, clubfoot, and ventriculomegaly. Congenital DM1 is characterized by hypotonia, and severe weakness at birth. Further symptoms of congenital DM1 include a tented appearance of the upper lip. dysarthria, intellectual disability, hypotonia, respiratory insufficiency, and early death.

[0013] In DM1 patients, the mutated DMPK gene produces an altered (mutated) mRNA. The altered mRNA is thought to trap proteins and forms foci within cells. Said foci interfere with protein production, which prevents proper cell function. Without being bound by theory, the foci may trap proteins that have a function in mRNA splicing, thereby disrupting proper (normal) mRNA splicing patterns. In muscle cells, this results in muscle weakness. DM1 particularly affects cardiac, smooth, and skeletal muscle.

[0014] There are no specific treatments for DM1, and some forms of DM1 such as congenital DM1 and childhood DM1 result in decreased lifespan. Existing treatments are directed to managing symptoms and maximizing patients’ quality of life and independence. Treatments directed to managing symptoms include anti-diabetic drugs, anti -myotonic drugs (e.g., mexiletine), and non-steroidal anti-inflammatory drugs. The present disclosure provides improved pharmaceutical compositions and methods. In some embodiments, the disclosure provides methods of treatment using AAV capsid variants that are capable of delivering modulatory polynucleotides to a target cell or tissue, e.g., a cell or tissue of the muscle, heart, and / or CNS.SUMMARY

[0015] The present disclosure addresses these challenges by providing AAV-based compositions, AAV-based compositions for use in methods for treating DMPK-related disorders in subjects, andmethods for treating DMPK-related disorders in subjects. In some embodiments, the DMPK-related disorder is DM1. In various embodiments, disclosed herein are compositions and methods directed to AAV -based gene delivery of modulatory polynucleotides for reducing or eliminating expression of DMPK (e.g., mutated DMPK mRNA) to treat DM1. The compositions and methods are useful to reduce the effects of mutated DMPK mRNA expression, and to slow, halt or reverse muscular and other symptoms of DM1. In some embodiments, mutated DMPK mRNA expression refers to expression of DMPK mRNA comprising 50 or more CTG trinucleotide repeats (e.g., SEQ ID NO: 35). Unless otherwise specified, DMPK, DM15, DM1PK. DMK, MDPK, and MT-PK are synonymous tenns and are used interchangeably to refer to the DMPK gene. DMPK is used to refer to the transcript encoding the DMPK protein, DMPK protein refers to the protein encoded by the DMPK gene and mRNA. DM1 is used to refer to myotonic dystrophy type 1.

[0016] In some aspects, the present disclosure provides an AAV particle comprising an AAV capsid variant and a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of a dystrophia myotonica protein kinase (DMPK) mRNA. In some embodiments, the modulatory polynucleotide reduces or eliminates mRNA encoding mutant DMPK (e.g., comprising aberrant CTG repeats). In some embodiments, the AAV capsid is an AAV capsid variant. In some embodiments, the AAV capsid variant is an AAV5 capsid variant.

[0017] In some embodiments, the AAV particles described herein comprising the modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA provide high deliver}’ and / or expression of the modulatory polynucleotide in muscle and / or CNS, strong inhibition of DMPK expression or activity in muscle and / or CNS, and / or reduced immunogenicity’. In some embodiments, the muscle comprises a skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis). In some embodiments, the muscle comprises smooth muscle. In some embodiments, the muscle comprises cardiac muscle. In some embodiments, the CNS comprises the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamcn, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region). In some embodiments, the AAV particles described herein provide reduced modulatory polynucleotide deliver}’ to or expression in the liver while retaining high modulator}’ polynucleotide activity in other areas (e.g.. muscle and / or CNS), e.g., relative to AAV5 or AAV9. In some embodiments, the AAV particles described herein can be administered to a subject having a DMPK-related disorder such as DM1. In some embodiments, the DM1 is congenital DM1.

[0018] In some embodiments, administration to a subject of an AAV particle comprising an AAV capsid variant described herein and a modulatory polynucleotide disclosed herein for reducing or eliminating expression of DMPK mRNA results in greater reduction in the expression and / or activity of DMPK (e.g., of mutated DMPK) in muscle cells or tissues and / or CNS cells or tissue of the subject as compared to administration of an AAV particle comprising a wildtype AAV5 capsid and the modulatory polynucleotide sequence. In some embodiments, the muscle comprises skeletal muscle (e.g.. diaphragm,intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis). In some embodiments, the muscle comprises smooth muscle. In some embodiments, the muscle comprises cardiac muscle. In some embodiments, the CNS comprises the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region).

[0019] In some aspects, the present disclosure provides an adeno-associated virus (AAV) particle comprising: (i) a viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and (ii) an AAV capsid variant comprising in loop VIII: (a) an amino acid sequence of SEQ ID NO: 4, (b) an amino acid sequence comprising at least 4 or at least 5 consecutive amino acids from the amino acid sequence of SEQ ID NO: 4; or (c) an amino acid sequence comprising one. two, or three different amino acids relative to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the AAV particle comprises at least 5 consecutive amino acids from the amino acid sequence of SEQ ID NO: 4. In some embodiments, the consecutive amino acids comprise the amino acid sequence of NAA. In some embodiments, the consecutive amino acids comprise the amino acid sequence of NAAQ (SEQ ID NO: 2). In some embodiments, the consecutive amino acids comprise the amino acid sequence of AQAY (SEQ ID NO: 1). In some embodiments, the consecutive amino acids comprise the amino acid sequence of NAAQA (SEQ ID NO: 3). In some embodiments, the amino acid sequence comprises the amino acid sequence of NAAQ AY (SEQ ID NO: 4).

[0020] In some embodiments, the AAV capsid variant is an AAV5 capsid variant.

[0021] In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 571-592 of the amino acid sequence of SEQ ID NO: 8 or 12. In some embodiments, the amino acid sequence replaces amino acids corresponding to positions 578, 579, 580, 581, 582, and 583 (e.g., corresponding to positions T578, A579, P580, A581, T582, and G583) of tire amino acid sequence of SEQ ID NO: 8. In some embodiments, the amino acid sequence is present at amino acids corresponding to positions 578, 579, 580, 581, 582, and / or 583 (e.g., comprises amino acids T578, A579, P580, A581, T582, and / or G583) of the amino acid sequence of SEQ ID NO: 8 or 12. In some embodiments, the amino acid sequence comprises or consists of NAAQAY (SEQ ID NO: 4) and is present at amino acids corresponding to positions 578, 579, 580, 581, 582, and 583 (e g., comprises amino acids T578, A579, P580, A581, T582, and G583) of the amino acid sequence of SEQ ID NO: 12.

[0022] In some aspects, the present disclosure provides an adeno-associated virus (AAV) particle comprising: (i) a viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and (ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to amino acids 193-724 of the amino acid sequence of SEQ ID NO: 12, wherein the AAV capsid variantcomprises: N at an amino acid corresponding to position 578 of the amino acid sequence of SEQ ID NO: 12, A at an amino acid corresponding to position 580 of the amino acid sequence of SEQ ID NO: 12, Q at an amino acid corresponding to position 581 of the ammo acid sequence of SEQ ID NO: 12, A at an amino acid corresponding to position 582 of the amino acid sequence of SEQ ID NO: 12, and Y at an amino acid corresponding to position 583 of the ammo acid sequence of SEQ ID NO: 12.

[0023] In some embodiments, the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to amino acids 137-724 of the amino acid sequence of SEQ ID NO: 12.

[0024] In some aspects, the present disclosure provides an adeno-associated virus (AAV) particle comprising: (i) a viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and N at an amino acid corresponding to position 578 of the amino acid sequence of SEQ ID NO: 12, A at an amino acid corresponding to position 580 of the amino acid sequence of SEQ ID NO: 12. Q at an amino acid corresponding to position 581 of the amino acid sequence of SEQ ID NO: 12, A at an amino acid corresponding to position 582 of the amino acid sequence of SEQ ID NO: 12. and Y at an amino acid corresponding to position 583 of the amino acid sequence of SEQ ID NO: 12.

[0025] In some embodiments, the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12.

[0026] In some aspects, the present disclosure provides an adeno-associated virus (AAV) particle comprising: (i) a viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and (ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, wherein the AAV capsid variant comprises: N at an amino acid corresponding to position 578 of the amino acid sequence of SEQ ID NO: 12, A at an amino acid corresponding to position 580 of the amino acid sequence of SEQ ID NO: 12, Q at an amino acid corresponding to position 581 of the amino acid sequence of SEQ ID NO: 12, A at an amino acid corresponding to position 582 of the amino acid sequence of SEQ ID NO: 12, and Y at an amino acid corresponding to position 583 of the amino acid sequence of SEQ ID NO: 12.

[0027] In some embodiments, the AAV capsid variant comprises A at a position corresponding to position 579 of the amino acid sequence of SEQ ID NO: 12.

[0028] In some embodiments, the AAV capsid variant comprises: (i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12: (ii) an amino acid sequence that is at least 99% identical to positions 193-724 of the amino acid sequence of SEQ ID NO: 12: and / or (iii) an amino acid sequence that is at least 99% identical to positions 137-724 of the amino acid sequence of SEQ ID NO: 12.

[0029] In some embodiments, the AAV capsid variant comprises the amino acid sequence of ATNNQSSTNAAQAYT (SEQ ID NO: 6) at amino acids corresponding to positions 570-584 of the amino acid sequence of SEQ ID NO: 12, optionally wherein the amino acid sequence of ATNNQSSTNAAQAYT (SEQ ID NO: 6) is present in loop VIII, wherein loop VIII comprises amino acids 571-592 of the amino acid sequence of SEQ ID NO: 8 or 12.

[0030] In some embodiments, the AAV capsid variant has: (i) an increased tropism for a muscle cell or tissue, relative to the muscle cell or tissue tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9: and / or (ii) a decreased tropism for a liver cell or tissue, relative to the liver cell or tissue tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the AAV capsid variant: (i) transduces a muscle cell or tissue, optionally wherein the level of transduction is at least 2, at least 5, at least 10, at least 15. at least 20, or at least 25-fold greater as compared to transduction of a muscle cell or tissue by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., an immunohistochemistry assay or a qPCR assay; (ii) delivers an increased level of the modulatory polynucleotide to a muscle cell or tissue, optionally wherein the level of the modulatory polynucleotide is increased by at least 5. at least 10, at least 12. at least 15, at least 20, at least 21. or at least 25 -fold, as compared to the level of modulatory polynucleotide delivered to a muscle cell or tissue by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g.. when measured by an assay; and / or (iii) delivers an increased level of viral genomes to a muscle cell or tissue, optionally wherein the level of viral genomes is increased by at least 2, at least 2.5, at least 5. at least 5.5, at least 6, at least 6.5, at least 7. at least 7.5, at least 8, at least 8.5, at least 9. at least 9.5, at least 10. at least 10.5, at least 11, at least 11.5, at least 12, at least 12.5, at least 13, at least 13.5, at least 13.8, or at least 14-fold, as compared to the level of viral genomes delivered to a muscle cell or tissue by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay. In some embodiments, tire muscle cell or tissue comprises a cardiac muscle, a smooth muscle, or a skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e g., vastus lateralis).

[0031] In some embodiments, the AAV capsid variant has: (i) an increased tropism for a central nervous system (CNS) cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the CNS cell or tissue tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and / or (ii) a decreased tropism for a liver cell or tissue, relative to the liver cell or tissue tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the AAV capsid variant: (i) transduces a brain cell or tissue of the CNS, optionally wherein the level of transduction is at least 3, at least 4, at least 5, at least 6, at least 7. at least 8. at least 9. at least 10, at least 11, at least 12. at least 13, at least 14. at least 15, at least 20, at least 24. at least 25, at least 29, or at least 30-fold greater as compared to transduction of a brain cell or tissue of the CNS by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g..when measured by an assay, e.g., an immunohistochemistry assay or a qPCR assay; (ii) is enriched at least 4, at least 4.6, at least 10, at least 20, at least 25, at least 28, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 81, at least 80, at least 100, at least 101, or at least 110-fold, in the CNS as compared to enrichment in the CNS of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9; (iii) delivers an increased level of the modulatory poly nucleotide to a brain cell or tissue of the CNS, optionally wherein the level of the modulatory polynucleotide is increased by at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12. at least 13, or at least 14-fold, as compared to the level of modulator,' polynucleotide delivered to a brain cell or tissue of the CNS by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay; and / or (iv) delivers an increased level of viral genomes to a brain cell or tissue of the CNS, optionally wherein the level of viral genomes is increased by at least 3, at least 4, at least 5, at least 6. at least 7. at least 8, at least 9, at least 10. at least 11, at least 12, at least 13. at least 14, at least 15. at least 20. at least 24, at least 25. at least 29, or at least 30-fold, as compared to the level of viral genomes delivered to brain cell or tissue of the CNS by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay. In some embodiments, the CNS comprises caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, thalamus, and / or spinal cord (e.g., con ical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region).

[0032] In some embodiments, the AAV capsid variant comprises: (i) the amino acid sequence of SEQ ID NO: 12; (ii) the amino acid sequence according to positions 193-724 of the amino acid sequence of SEQ ID NO: 12; and / or (iii) the amino acid sequence according to positions 137-724 of the amino acid sequence of SEQ ID NO: 12.

[0033] In some embodiments, the modulatory polynucleotide comprises a molecular scaffold, wherein the molecular scaffold comprises: (i) a 5’ flanking region comprising the nucleotide sequence of any one of SEQ ID NOs: 13-16, or a nucleotide sequence that is at least 95% identical thereto; (ii) a loop region comprising the nucleotide sequence of any one of SEQ ID NOs: 17-21, or a nucleotide sequence that is at least 95% identical thereto; and (iii) a 3 ’ flanking region comprising the nucleotide sequence of any one of SEQ ID NOs: 22-27, or a nucleotide sequence that is at least 95% identical thereto.

[0034] In some embodiments, (i) the 5 ‘ flanking region comprises the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 15, or a nucleotide sequence that is at least 95% identical thereto; (ii) the loop region comprises the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 21. or a nucleotide sequence that is at least 95% identical thereto; and (iii) the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. or a nucleotide sequence that is at least 95% identical thereto.

[0035] In some embodiments, the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises thenucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% identical thereto.

[0036] In some embodiments, the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence that is at least 95% identical thereto, the loop region of the comprises the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence that is at least 95% identical thereto.

[0037] In some embodiments, the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence that is at least 95% identical thereto.

[0038] In some embodiments, the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% identical thereto.

[0039] In some embodiments, the modulatory polynucleotide comprises siRNA or shRNA.

[0040] In some embodiments, the modulatory polynucleotide further comprises a passenger strand and a guide strand. In some embodiments, the guide strand binds to and reduces or eliminates expression of one or more DMPK mRNA transcripts (e.g., one or more mutated DMPK mRNA transcripts). In some embodiments, the modulatory polynucleotide comprises, from 5’ to 3’: the 5’ flanking region, the passenger strand, the loop region, the guide strand, and the 3’ flanking region. In some embodiments, the modulatory polynucleotide comprises, from 5’ to 3’: the 5 ‘ flanking region, the guide strand, the loop region, the passenger strand, and the 3 ’ flanking region.

[0041] In some embodiments, the passenger strand is 15-30 nucleotides in length. In some embodiments, the guide strand is 15-30 nucleotides in length. In some embodiments, the guide strand is 21-25 nucleotides in length and / or the passenger strand is 21-25 nucleotides in length. In some embodiments, the passenger strand is at least 70%, at least 80%, at least 90%, or at least 95%, or is 100%, complementary to the guide strand.

[0042] In some embodiments, the one or more DMPK mRNA transcripts comprises the nucleotide sequence of SEQ ID NOs: 28-29 or a trinucleotide repeat expansion thereof.

[0043] In some embodiments, the viral genome comprises a promoter operably linked to the nucleotide sequence encoding the modulatory polynucleotide.

[0044] In some embodiments, the viral genome further comprises an inverted terminal repeat (ITR) sequence. In some embodiments, the viral genome comprises an ITR sequence positioned 5’ relative totlie nucleotide sequence encoding the modulatory polynucleotide. In some embodiments, the viral genome comprises an ITR sequence positioned 3’ relative to the nucleotide sequence encoding the modulatory polynucleotide. In some embodiments, the viral genome comprises an ITR sequence positioned 5’ relative to the nucleotide sequence encoding the modulatory polynucleotide, and an ITR sequence positioned 3‘ relative to the nucleotide sequence encoding the modulatory polynucleotide.

[0045] In some embodiments, the viral genome further comprises a polyadenylation (poly A) sequence.

[0046] In some aspects, the present disclosure provides a cell comprising an AAV particle provided herein, optionally wherein the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an SI9 cell), or a bacterial cell.

[0047] In some aspects, the present disclosure provides a method of making an AAV particle provided herein, wherein the method comprises (i) providing a cell comprising the viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA and a nucleic acid encoding the AAV capsid variant; and (ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant; thereby making the AAV particle.

[0048] In some embodiments of the method of making, the viral genome comprises (i) a 5 ’ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% identical thereto; (ii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence that is at least 95% identical thereto;(iii) a 5‘ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence drat is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and a 3‘ flanking region comprising the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence that is at least 95% thereto; or (iv) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% thereto; and the AAV capsid variant comprises (a) the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%. at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; (b) the amino acid sequence according to positions 137-724 of the amino acid sequence of SEQ ID NO: 12 or an amino acidsequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; and / or (c) the amino acid sequence according to positions 193-724 of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

[0049] In some embodiments of the method of making, the viral genome comprises: (i) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% identical thereto; (ii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence that is at least 95% identical thereto;(iii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising die nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence that is at least 95% thereto; or (iv) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% thereto; and the AAV capsid variant comprises SEQ ID NO: 12, the amino acid sequence according to positions 137-724 of the amino acid sequence of SEQ ID NO: 12, and / or the amino acid sequence according to positions 193-724 of the amino acid sequence of SEQ ID NO: 12.

[0050] In some embodiments of the method of making, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.

[0051] In some embodiments, the method of making an AAV particle further comprises, prior to step (i), introducing a nucleic acid molecule comprising the viral genome into the cell. In some embodiments, the method of making an AAV particle further comprises, prior to step (i), introducing the nucleic acid encoding the AAV capsid variant into the cell.

[0052] In some embodiments of the method of making, the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an S19 cell), or a bacterial cell.

[0053] In some aspects, the present disclosure provides a pharmaceutical composition comprising an AAV particle provided herein and a pharmaceutically acceptable excipient.

[0054] In some aspects, the present disclosure provides a method of delivering an AAV particle encoding a modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA to a cell, comprising administering an effective amount of a pharmaceutical composition provided herein or an AAV particle provided herein. In some embodiments, the cell is in a subject, optionally wherein the subject has, has been diagnosed with having, or is at risk of having a DMPK-related disorder. In some embodiments, the DMPK-related disorder is myotonic dystrophy type 1 (DM1).

[0055] In some aspects, the present disclosure provides a method of treating a DMPK-related disorder in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition provided herein or an AAV particle provided herein. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having the DMPK-related disorder.

[0056] In some embodiments, the subject has one or more mutations in the DMPK gene. In some embodiments, the one or more mutations in the DMPK gene comprises a trinucleotide repeat expansion. In some embodiments, the trinucleotide repeat expansion in the DMPK gene is or comprises 50 or more CTG repeats (e.g., SEQ ID NO: 35).

[0057] In some embodiments, the treating results in prevention of progression of the DMPK-related disorder in the subject. In some embodiments, the treating results in amelioration of at least one symptom of the DMPK-related disorder in the subject. In some embodiments, the at least one symptom comprises cataracts, myotonia, muscle weakness and wasting, cardiac conduction abnormalities, a myopathic face, learning difficulties, psychosocial problems including depression and / or anxiety, slurred speech, decreased fetal movement in the uterus, polyhydramnios, clubfoot, ventriculomegaly, hypotonia, a tented appearance of the upper lip, dysarthria, intellectual disability, hypotonia, respirator}' insufficiency, or a combination thereof. In some embodiments, the DMPK-related disorder is myotonic dystrophy type 1 (DM1).

[0058] In some aspects, the present disclosure provides a method of treating myotonic dystrophy type 1 (DM1) in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition provided herein or an AAV particle provided herein. In some embodiments, die subject has, has been diagnosed with having, or is at risk of having DM1.

[0059] In some embodiments, the subject is a human.

[0060] In some embodiments, the pharmaceutical composition or AAV particle is delivered to a cell, tissue, or region of muscle. In some embodiments, the muscle is one or more of cardiac, smooth, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e g., vastus lateralis).

[0061] In some embodiments, the pharmaceutical composition or AAV particle is delivered to a cell, tissue, or region of the central nervous system, e.g., the brain. In some embodiments, the pharmaceutical composition or AAV particle is delivered to the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region,lumbar spinal cord region, or thoracic spinal cord region). In some embodiments, the subject has, has been diagnosed with having, or is at risk of having congenital myotonic dystrophy type 1.

[0062] In some embodiments, the AAV particle or the pharmaceutical composition is delivered via intravenous administration. In some embodiments, the AAV particle or the pharmaceutical composition is delivered via intracerebroventricular administration.

[0063] In some embodiments, the method of delivery or treating further comprises evaluating, e.g.. measuring, the level of modulatory polynucleotide expression, the level of mutated DMPK mRNA expression, the level of mutated DMPK protein expression, and / or the level of normal mRNA splicing, optionally DMPK mRNA splicing, in the subject, e.g.. in a cell, tissue, or fluid, of the subject. In some embodiments, evaluating the subject’s level of modulatory polynucleotide expression, the subject’s level of mutated DMPK mRNA expression, the subject’s level of mutated DMPK protein expression, and / or the subject’s level of normal mRNA splicing, optionally DMPK mRNA splicing, is performed prior to and / or subsequent to administration of the pharmaceutical composition or AAV particle, optionally wherein the subject’s level of modulatory polynucleotide expression, the subject’s level of mutated DMPK mRNA expression, the subject’s level of mutated DMPK protein expression, and / or the subject’s level of normal mRNA splicing, optionally DMPK mRNA splicing, prior to administration is compared to the subject’s level of modulatory polynucleotide expression, the subject’s level of mutated DMPK mRNA expression, the subject’s level of mutated DMPK protein expression, and / or the subject’s level of normal mRNA splicing, optionally DMPK mRNA splicing, subsequent to administration. In some embodiments of a method of delivery or treating, the cell or tissue of the subject is a muscle cell or tissue (e.g., cardiac, smooth, and / or skeletal muscle (e.g.. diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis)). In some embodiments of a method of deliver}' or treating, the cell or tissue of the subject is a cell or tissue of the CNS (e.g., caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)).

[0064] In some embodiments of a method of delivery or treating, the subject’s level of mutated DMPK mRNA expression subsequent to administration of the pharmaceutical composition or AAV particle is decreased relative to the subject’s level of mutated DMPK mRNA expression prior to administration of the pharmaceutical composition or AAV particle. In some embodiments of a method of delivery or treating, the subject’s level of mutated DMPK protein expression subsequent to administration of the pharmaceutical composition or AAV particle is decreased relative to the subject’s level of mutated DMPK protein expression prior to administration of the pharmacal composition or AAV particle.

[0065] In some embodiments of a method of delivery or treating, administering the pharmacal composition or AAV particle to the subject results in: (i) an increase in the number and / or level of viral genomes (VG) per cell in a muscle cell or tissue (e.g., cardiac, smooth, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g.. vastus lateralis)) of thesubject relative to the number and / or level of VG per cell in a non-muscle cell or tissue of the subject; (ii) a decrease in mutated DMPK mRNA expression in a muscle cell or tissue (e.g., cardiac, smooth, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e g., vastus lateralis)) of the subject relative to baseline and / or relative to mutated DMPK mRNA expression in a muscle cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle; and / or (iii) an increase in normal mRNA splicing, optionally DMPK mRNA splicing, in a muscle cell or tissue (e.g., cardiac, smooth, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis)) of the subject relative to baseline and / or relative to normal mRNA splicing, optionally DMPK mRNA splicing, in a muscle cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle.

[0066] In some embodiments of a method of delivery or treating, administering the pharmaceutical composition or AAV particle to the subject results in: (i) an increase in the number and / or level of viral genomes (VG) per cell in a CNS tissue (e.g., caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g.. cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)) of the subject relative to the number and / or level of VG per cell in a peripheral tissue of the subject; (ii) a decrease in mutated DMPK mRNA expression in a cell or a tissue of the CNS (e.g., caudate, cerebellum (e.g.. molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g.. cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)) of the subject relative to baseline and / or relative to mutated DMPK mRNA expression in a CNS cell or tissue of an individual with a DMPK- related disorder who has not been administered the pharmaceutical composition or AAV particle; and / or (iii) an increase in normal mRNA splicing, optionally DMPK mRNA splicing, in a cell or tissue of the CNS (e.g., caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)) of the subject relative to baseline and / or relative to normal mRNA splicing, optionally DMPK mRNA splicing, in a CNS cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having congenital myotonic dystrophy type 1.

[0067] In some embodiments, a method of delivery or treating provided herein further comprises administering to the subject at least one additional agent and / or therapy. In some embodiments, the at least one additional agent and / or therapy comprises an agent and / or therapy for treating the DMPK- related disorder, optionally wherein the at least one additional agent and / or therapy comprises an antidiabetic drug, an anti-myotonic drug (e.g., mexiletine), a non-steroidal anti-inflammatory drug, or a combination thereof.

[0068] In some embodiments, a method of deliver}' or treating provided herein further comprises administering an immunosuppressant to the subject. In some embodiments, the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and / or dexamethasone), adrenocorticotropic hormone, rapamycin, mycophenolate mofetil, tacrolimus, rituximab, eculizumab hydroxychloroquine, alemtuzumab, hydroxyurea, fludarabine, and / or busulfan.

[0069] In some aspects, the present disclosure provides a pharmaceutical composition provided herein or an AAV particle provided herein for use in a method of treating a disorder provided herein.

[0070] In some aspects, the present disclosure provides a pharmaceutical composition provided herein or an AAV particle provided herein for use in treating a DMPK-related disorder in a subject, optionally wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1). In some embodiments, the subject has, has been diagnosed with having, or is at risk of having the DMPK-related disorder, optionally wherein the DMPK-related disorder is DM1. In some embodiments, the DMPK- related disorder is congenital DM1.

[0071] In some aspects, the present disclosure provides a use of a pharmaceutical composition provided herein or an AAV particle provided herein in the manufacture of a medicament for treating a DMPK-related disorder provided herein, optionally wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1). In some embodiments, the subject has, has been diagnosed with having, or is at risk of having the DMPK-related disorder, optionally wherein the DMPK-related disorder is DM1. In some embodiments, the DMPK-related disorder is congenital DM1.Enumerated Embodiments1. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises:(a) the amino acid sequence of SEQ ID NO: 4;(b) an amino acid sequence comprising at least 3. at least 4. or at least 5 consecutive amino acids from SEQ ID NO: 4;(c) an amino acid sequence comprising one, two. or three but no more than four different amino acids, relative to the amino acid sequence of SEQ ID NO: 4; or(d) an amino sequence comprising one, two, or three but no more than four modifications, relative to the amino acid sequence of SEQ ID NO: 4.2. The AAV particle of embodiment 1, wherein the AAV capsid variant comprises at least 3, at least 4, or at least 5 consecutive amino acids from SEQ ID NO: 4.3. The AAV particle of embodiment 1 or 2, wherein the 3 consecutive amino acids comprise NAA.4. The AAV particle of any one of embodiments 1-3, wherein the 4 consecutive amino acids comprise NAAQ (SEQ ID NO: 2).5. The AAV particle of any one of embodiments 1-4, wherein the 5 consecutive amino acids comprise NAAQA (SEQ ID NO: 3).6. The AAV particle of any one of embodiments 1-5, wherein amino acid sequence comprises NAAQAY (SEQ ID NO: 4).7. The AAV particle of any one of embodiments 1-6. wherein the AAV capsid variant comprises an amino acid sequence comprising one, two. or three but no more than four different amino acids, relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4).8. The AAV particle of any one of embodiments 1-7, wherein the AAV capsid variant comprises an amino acid sequence comprising one or two (e.g., no more than two) different amino acids, relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4).9. The AAV particle of any one of embodiments 1-8, wherein the AAV capsid variant comprises an amino acid sequence comprising one, two, or three but no more than four modifications, relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4).10. The AAV particle of any one of embodiments 1-9, wherein the AAV capsid variant comprises an amino acid sequence comprising one or two (e.g., no more than two) modifications, relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4).11. The AAV particle of any one of embodiments 1-10, wherein the AAV capsid variant comprises an amino acid sequence encoded by:(i) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six. or at least seven modifications, e.g., substitutions, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 5; or(ii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 5.12. The AAV particle of any one of embodiments 1-11, wherein the nucleotide sequence encoding the amino acid sequence comprises:(i) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, e.g., substitutions, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 5; or(ii) a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six. or at least seven, but no more than ten different nucleotides relative to the nucleotide sequence of SEQ ID NO: 5.13. The AAV of any one of embodiments 1-12, wherein the amino acid sequence is present in loop VIII, optionally wherein loop VIII comprises positions 571-592, numbered according to SEQ ID NO: 8 or 12.14. The AAV particle of any one of embodiments 1-13, wherein the amino acid sequence replaces one, two. three, four, five, or all of positions 578. 579, 580, 581. 582, and / or 583 (e g., positions T578, A579, P580, A581. T582. and / or G583). numbered according to SEQ ID NO: 8.15. The AAV particle of any one of embodiments 1-14, wherein the amino acid sequence replaces positions 578, 579. 580, 581, 582, and 583 (e.g., positions T578, A579, P580, A581. T582, and G583), numbered according to SEQ ID NO: 8.16. The AAV particle of any one of embodiments 1-15, wherein the amino acid sequence corresponds to positions 578, 579, 580, 581, 582, and 583 (e.g., positions N578, A579, A580, Q581, A582, and Y583) of SEQ ID NO: 12.17. The AAV particle of any one of embodiments 1-16, wherein the AAV capsid variant comprises tire amino acid sequence of NAAQAY (SEQ ID NO: 4), optionally wherein the amino acid sequence replaces positions 578. 579, 580, 581, 582, and 583 (e.g., positions T578, A579, P580, A581, T582, and G583), numbered according to SEQ ID NO: 8.18. The AAV particle of any one of embodiments 1-17, wherein the AAV capsid variant comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4), optionally wherein the amino acid sequence corresponds to positions 578. 579, 580. 581, 582, and 583 (e.g., positions N578. A579, A580, Q581, A582, and Y583) of SEQ ID NO: 8 or 12.19. The AAV particle of any one of embodiments 1-18, wherein the AAV capsid variant comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4), optionally wherein the amino acid sequence is present at positions 578-583, numbered according to SEQ ID NO: 8 or 12.20. The AAV particle of any one of embodiments 1-16, wherein the AAV capsid variant comprises the amino acid sequence AQAY (SEQ ID NO: 1), optionally wherein the amino acid sequence replaces positions 580-583 (e.g., P580, A581, T582, and G583), numbered according to SEQ ID NO: 8.21. The AAV particle of any one of embodiments 1-16, wherein the AAV capsid variant comprises the amino acid sequence AQAY (SEQ ID NO: 1), optionally wherein the amino acid sequence is present at positions 580-583. numbered according to SEQ ID NO: 8 or 12.22. The AAV particle of embodiment 20 or 21, wherein the AAV capsid variant further comprises an amino acid other than T at position 578, numbered according to SEQ ID NO: 8.23. The AAV particle of any one of embodiments 20-22, wherein the AAV capsid variant further comprises the amino acid N at position 578, numbered according to SEQ ID NO: 8 or 12.24. The AAV particle of any one of embodiments 1-23, wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1). wherein the amino acid sequence replaces positions 580-583 (e.g., P580, A581, T582, and G583), numbered according to SEQ ID NO: 8; and / or(ii) an amino acid other than T at position 578. numbered according to SEQ ID NO: 8.25. The AAV particle of any one of embodiments 1-24, wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1), wherein the amino acid sequence is present at positions 580-583, numbered according to SEQ ID NO: 8 or 12; and / or(ii) an amino acid other than T at position 578, numbered according to SEQ ID NO: 8.26. The AAV particle of any one of embodiments 1-25, wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1), wherein the amino acid sequence replaces positions 580-583 (e.g., P580, A581, T582, and G583), numbered accoring to SEQ ID NO: 8; and / or(ii) the amino acid N at position 578, numbered according to SEQ ID NO: 8 or 12.27. The AAV particle of any one of embodiments 1-26, wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1), wherein the amino acid sequence is present at positions 580-583, numbered according to SEQ ID NO: 8 or 12; and / or(ii) the amino acidN at position 578, numbered according to SEQ ID NO: 8 or 12.28. The AAV particle of any one of embodiments 1-27, wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1), wherein the amino acid sequence corresponds to positions 580-583 of SEQ ID NO: 8 or 12; and / or(ii) the amino acid N at position 578, numbered according to SEQ ID NO: 8 or 12.29. The AAV capsid variant of any one of the preceding embodiments, wherein the AAV capsid variant comprises one, two, three, four, or all of an amino acid other than T at position 578, an amino acid other than P at position 580, an amino acid other than A at position 581, an amino acid other than T at position582, and / or an amino acid other than G at position 583, numbered according to SEQ ID NO: 8.30. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g.. an AAV 5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises one, tw o. three, four, five, or all of an amino acid other than T at position 578, an amino acid other than P at position 580, an amino acid other than A at position 581, an amino acid other than T at position 582, and / or an amino acid other than G at position583, numbered according to SEQ ID NO: 8 or 12.31. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises an amino acid other than T at position 578, an amino acid other than P at position 580, an amino acid other than A at position 581, an amino acid other than T at position 582, and an amino acid other than G at position 583, numbered according to SEQ ID NO: 8 or 12.32. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises an amino acid other than T at position 578, an amino acid other than P at position 580, an amino acid other than A at position 581, an amino acid other than Tat position 582, and an amino acid other than G at position 583, numbered according to SEQ ID NO: 8 or 12.33. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises one, two, three, four, five, or all of the amino acid N at position 578, A at position 579, A at position 580, Q at position 581, A at position 582, and / or Y at position 583. numbered according to SEQ ID NO: 8 or 12.34. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises one, two, three, four, or all of the amino acid N at position 578, A at position 580. Q at position 581, A at position 582, and / or Y at position 583, numbered according to SEQ ID NO: 8 or 12.35. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g.. an AAV 5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises one, two. three, four, five, or all of the amino acid N at position 578, A at position 579, A at position 580, Q at position 581, A at position 582, and / or Y at position 583, numbered according to SEQ ID NO: 8 or 12.36. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises one, two, three, four, or all of the amino acid N at position 578. A at position 580, Q at position 581, A at position 582, and / or Y at position 583, numbered according to SEQ ID NO: 8 or 12.37. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises the amino acid N at position 578, A at position 579, A at position 580, Q at position 581, A at position 582, and Y at position 583. numbered according to SEQ ID NO: 8 or 12.38. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises the amino acid N at position 578, A at position 580, Q at position 581, A at position 582, and Y at position 583, numbered according to SEQ ID NO: 8 or 12.39. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises the amino acid N at position 578. A at position 579. A at position 580, Q at position 581. A at position 582. and Y at position 583, numbered according to SEQ ID NO: 8 or 12.40. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises the amino acid N at position 578, A at position 580, Q at position 581, A at position 582, and Y at position 583, numbered according to SEQ ID NO: 8 or 12.41. The AAV particle of any one of embodiments 1-32, wherein the AAV capsid variant comprises one, two, three, four, or all of the amino acid substitutions T578N, P580A, A581Q, T582A, and / or G583Y, numbered according to SEQ ID NO: 8 or 12.42. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g.. an AAV 5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises one, two. three, four, or all of the amino acid substitutions T578N, P580A, A581Q, T582A, and / or G583Y, numbered according to SEQ ID NO: 8 or 12.43. The AAV particle of any one of embodiments 1-38, wherein the AAV capsid variant comprises the amino acid substitutions T578N, P580A, A581Q, T582A, and G583Y, numbered according to SEQ ID NO: 8 or 12.44. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises the amino acid substitutions T578N, P580A, A581Q, T582A, and G583Y, numbered according to SEQ ID NO: 8 or 12.45. The AAV particle of any one of the preceding embodiments, wherein the amino acid sequence corresponds to positions 578-583 (e.g., N578, A579, A580, Q581, A582, Y583) of SEQ ID NO: 12.46. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1), optionally wherein the amino acid sequence replaces positions 580-583 (e.g., P580, A581, T582. G583), numbered according to SEQ ID NO: 8; and(ii) an amino acid other than T at position 578, numbered according to SEQ ID NO: 8.47. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g.. an AAV 5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1), wherein the amino acid sequence replaces positions 580-583 (e.g., P580, A581, T582, G583), numbered according to SEQ ID NO: 8; and(ii) an amino acid other than T at position 578, numbered according to SEQ ID NO: 8 or 12.48. Aii adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1), optionally wherein the amino acid sequence is present at positions 580-583, numbered according to SEQ ID NO: 8 or 12; and(ii) an amino acid other than T at position 578. numbered according to SEQ ID NO: 8 or 12.49. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory' polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises:(i) the ammo acid sequence AQAY (SEQ ID NO: 1), wherein the amino acid sequence is present at positions 580-583, numbered according to SEQ ID NO: 8 or 12; and(ii) an amino acid other than T at position 578, numbered according to SEQ ID NO: 8 or 12.50. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1). optionally wherein the amino acid sequence replaces positions 580-583 (e.g., P580, A581, T582, G583), numbered according to SEQ ID NO: 8; and(ii) the amino acid other N at position 578, numbered according to SEQ ID NO: 8.51. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome,wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1), optionally wherein the amino acid sequence replaces positions 580-583 (e.g., P580, A581, T582, G583), numbered according to SEQ ID NO: 12; and(ii) the amino acid other N at position 578, numbered according to SEQ ID NO: 12.52. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1). optionally wherein the amino acid sequence is present at positions 580-583, numbered according to SEQ ID NO: 8; and(ii) the amino acid other N at position 578, numbered according to SEQ ID NO: 8.53. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises:(i) the amino acid sequence AQAY (SEQ ID NO: 1), wherein the amino acid sequence is present at positions 580-583, numbered according to SEQ ID NO: 12; and(ii) the amino acid other N at position 578. numbered according to SEQ ID NO: 12.54. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises the amino acid A at position 579, numbered according to SEQ ID NO: 8 or 12.55. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant corresponds to position 579 of SEQ ID NO: 12.56. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant further comprises a modification, e.g., an insertion, substitution (e.g., conservative substitution), and / or deletion, in loop I, II, IV, and / or VI.57. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one. at least two or at least three modifications, but not more than 30, not more than 20, or not more than 10 modifications, relative to the amino acid sequence of SEQ ID NO: 8 or 12.58. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one. at least tw o or at least three, but no more than 30, no more than 20. or no more than 10, different amino acids relative to the amino acid sequence of SEQ ID NO: 8 or 12.59. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 8 or 12, or an amino acid sequence with at least 80% (e.g., at least 80%, at least 85%. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99%) sequence identity thereto.60. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 8 or 12.61. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 10 or 30, or a sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99%) sequence identity thereto.62. The AAV particle of any one of the preceding embodiments, wherein the nucleotide sequence encoding the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 10 or 30, or a sequence with at least 80% (e.g., at least 80%. at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.63. The AAV particle of any one of the preceding embodiments, wherein the AAV capsid variant comprises a VP1 protein, a VP2 protein, a VP3 protein, or a combmation thereof.64. The AAV particle of any one of embodiments 1-63, wherein the AAV capsid variant comprises the amino acid sequence corresponding to positions 137-724, e.g.. a VP2, of SEQ ID NO: 8. or a sequencewith at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.65. The AAV particle of any one of embodiments 1-64, wherein the AAV capsid variant comprises tire amino acid sequence corresponding to positions 193-724, e.g., a VP3, of SEQ ID NO: 8. or a sequence with at least 80% (e.g.. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.66. The AAV particle of any one of embodiments 1-65, wherein the AAV capsid variant comprises the amino acid sequence corresponding to positions 137-724, e.g., a VP2, of SEQ ID NO: 12, or a sequence with at least 80% (e.g.. at least 80%, at least 85%. at least 90%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99%) sequence identity thereto.67. The AAV particle of any one of embodiments 1-66, wherein the AAV capsid variant comprises the amino acid sequence corresponding to positions 193-724. e.g., a VP3. of SEQ ID NO: 12, or a sequence with at least 80% (e.g., at least 80%. at least 85%, at least 90%. at least 95%, at least 96%, at least 97%. at least 98%. or at least 99%) sequence identity thereto.68. The AAV particle of any one of embodiments 1-67, wherein the AAV capsid variant comprises an amino acid sequence comprising at least 3, 4, or 5, or all 6 consecutive amino acids from the amino acid sequence of NAAQAY (SEQ ID NO: 4), wherein:(i) the at least 3 consecutive amino acids comprise NAA;(ii) the at least 4 consecutive amino acids comprise NAAQ (SEQ ID NO: 2);(iii) the at least 5 consecutive amino acids comprise NAAQA (SEQ ID NO: 3);(iv) the all 6 consecutive amino acids comprise NAAQAY (SEQ ID NO: 4); wherein the AAV capsid variant comprises: (a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12; (b) a VP2 protein comprising the amino acid sequence of positions 137-724 of SEQ ID NO: 8 or positions 137-724 of SEQ ID NO: 12; (c) a VP3 protein comprising the amino acid sequence of positions 193-724 of SEQ ID NO: 8 or positions 193-724 of SEQ ID NO: 12; or (d) an amino acid sequence with at least 90% (e g., at least 90%, at least 95%. at least 96%, at least 97%. at least 98%, or at least 99%) sequence identity to any one of the amino acid sequences in (a)-(c).69. The AAV particle of any one of embodiments 1-68, wherein the AAV capsid variant comprises one, two, or three but no more than four different amino acids relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4), wherein the AAV capsid variant comprises:(a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12;(b) a VP2 protein comprising the amino acid sequence of positions 137-724 of SEQ ID NO: 8 or positions 137-724 of SEQ ID NO: 12;(c) a VP3 protein comprising the amino acid sequence of positions 193-724 of SEQ ID NO: 8 or positions 193-724 of SEQ ID NO: 12; or(d) an amino acid sequence with at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to any one of the amino acid sequences in (a)- (c).70. The AAV particle of any one of embodiments 1-69, wherein the AAV capsid variant comprises one or two, but no more than three substitutions, relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4), wherein the AAV capsid variant comprises an amino acid sequence at least 90% (e.g.. at least 90%, at least 95%. at least 96%, at least 97%. at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 8 or 12.71. The AAV particle of any one of embodiments 1-70, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 8 or 12, or an amino acid sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99%) sequence identity thereto.72. The AAV particle of any one of embodiments 1-71, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 8 or 12 or an amino acid sequence with at least 95% identity thereto.73. The AAV particle of any one of embodiments 1-72, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 8 or 12 or an amino acid sequence with at least 98% identity’ thereto.74. The AAV particle of any one of embodiments 1-73, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 8 or 12 or an amino acid sequence with at least 99% identity thereto.75. The AAV particle of any one of embodiments 1-74, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three modifications, e.g., substitutions (e.g.. conservative substitutions), insertions, or deletions, but not more than 30. not more than 20. or not more than 10 modifications relative to the amino acid sequence of SEQ ID NO: 8 or 12.76. The AAV particle of any one of embodiments 1-75, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least tw o. or at least three, but not more than 30, not more than 20, or not more than 10 different amino acids relative to the amino acid sequence of SEQ ID NO: 8 or 12.77. The AAV particle of any one of embodiments 1-76, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 8 or 12.78. The AAV particle of any one of the preceding embodiments 1-77, wherein the nucleotide sequence encoding the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.79. The AAV particle of any one of the preceding embodiments, wherein the nucleotide sequence encoding the AAV capsid variant is codon optimized.80. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory' polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises the amino acid sequence of any one of embodiments 1-28, and further comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 12.81. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA. optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises an amino acid sequence 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%, or at least 99% identical to positions 193-724 of SEQ ID NO: 12. wherein the AAV capsid variant comprises an Nat position 578, A at position 580, Q at position 581, A at position 582, and Y at position 583, numbered according to SEQ ID NO: 8 or 12.82. The AAV particle of embodiment 81, wherein the AAV capsid variant comprises the amino acid sequence of positions 193-724 of SEQ ID NO: 12.83. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome. wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA. optionally wherein the modulator}' polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises an amino acid sequence 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%, or at least 99% identical to positions 137-724 of SEQ ID NO: 12. wherein the AAV capsid variant comprises an N at position 578, A at position 580, Q at position 581, A at position 582, and Y at position 583, numbered according to SEQ ID NO: 8 or 12.84. The AAV particle of any one of embodiments 81-83, wherein the AAV capsid variant comprises the amino acid sequence of positions 137-724 of SEQ ID NO: 12.85. An adeno-associated virus (AAV) particle particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises an amino acid sequence 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%, or at least 99% identical to SEQ ID NO: 12. wherein the AAV capsid variant comprises an N at position 578, A at position 580. Q at position 581, A at position 582, and Y at position 583, numbered according to SEQ ID NO: 8 or 12.86. The AAV particle of any one of embodiments 81-85, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.87. An adeno-associated virus (AAV) particle particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.88. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g., an AAV5 capsid variant), and a viral genome, wherein the viral genome comprises a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulator}' polynucleotide comprises an RNAi agent targeting DMPK mRNA; and wherein the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence at least 95% identical thereto.89. The AAV particle of any one of embodiments 81-88, wherein the nucleotide sequence encoding the AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence at least 95% identical thereto.90. The AAV particle of any one of embodiments 1-89, wherein the AAV capsid variant has an increased tropism for a muscle cell or tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.91. The AAV particle of any one of embodiments 1-90, wherein the AAV capsid variant transduces a muscle region, optionally wherein the level of transduction is at least 2. at least 5, at least 10, at least 15, at least 20, or at least 25-fold greater as compared to a reference sequence of SEQ ID NO: 8 or 9, e.g., when measured by an assay, e.g.. an immunohistochemistry assay or a qPCR assay.92. The AAV particle of any one of embodiments 1-91, wherein the AAV capsid variant delivers an increased level of a pay load to a muscle cell or region, optionally wherein the level of the pay load is increased by at least 5, at least 10, at least 12, at least 15, at least 20, at least 21, or at least 25-fold, as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g.. a qRT-PCR or a qPCR assay.93. The AAV particle of any one of embodiments 1-92, wherein the AAV capsid variant delivers an increased level of viral genomes to a muscle cell or region, optionally wherein the level of viral genomes is increased by at least 2, at least 2.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 10.5, at least 11, at least 11.5, at least 12, at least 12.5, at least 13, at least 13.5, at least 13.8. or at least 14-fold, as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay.94. The AAV particle of any one of embodiments 1-93, wherein the AAV capsid variant has increased tropism for a heart cell or tissue, relative to the tropism of a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9.95. The AAV particle of any one of embodiments 1-94, wherein the AAV capsid variant delivers an increased level of a pay load to a heart cell or region, optionally wherein the level of the payload is increased by at least 2, at least 2.5, at least 3. at least 3.5, at least 4, at least 4.5, or at least 5-fold, as compared to a reference sequence of SEQ ID NO: 8, e.g.. when measured by an assay, e.g.. a qRT-PCR or a qPCR assay.96. The AAV particle of any one of embodiments 1-95, wherein the AAV capsid variant delivers an increased level of viral genomes to a heart cell or region, optionally wherein the level of viral genomes is increased by at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10-fold, as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., a qRT- PCR or a qPCR assay.97. The AAV particle of any one of embodiments 1-96, wherein the AAV capsid variant has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising die ammo acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.98. The AAV particle of any one of embodiments 1-97, wherein the AAV capsid variant transduces a brain cell or region, e.g., (e.g., the caudate, motor cortex, putamen, thalamus, and / or cerebellum (e.g., the molecular and granule layer of the cerebellum)), optionally wherein the level of transduction is at least 3. at least 4, at least 5, at least 6, at least 7. at least 8, at least 9, at least 10. at least 11, at least 12, at least 13, at least 14, at least 15, at least 20. at least 24, at least 25. at least 29, or at least 30-fold greater as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g.. when measured by an assay, e.g., an immunohistochemistry assay or a qPCR assay.99. The AAV particle of any one of embodiments 1-98, wherein the AAV capsid variant delivers an increased level of a payload to a brain cell or region, optionally wherein the level of the payload is increased by at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12. at least 13, at least 14-fold, as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay.100. The AAV particle of any one of embodiments 1-99, wherein the AAV capsid variant delivers an increased level of viral genomes to a brain cell or region, optionally wherein the level of viral genomes is increased by at least 3 at least 4, at least 5, at least 6. at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14. at least 15, at least 20. at least 24, at least 25, at least 29. or at least 30- fold, as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g.. a qRT-PCR or a qPCR assay.101. The AAV particle of any one of embodiments 98-100, wherein the brain region is a caudate, motor cortex, putamen, thalamus, and / or cerebellum (e.g., the molecular and granule layer of the cerebellum).102. The AAV particle of any one of embodiments 1-101, which is enriched at least 4. at least 4.6, at least 10. at least 20, at least 25, at least 28. at least 30, at least 40, at least 50, at least 60, at least 70. at least 80. at least 81, at least 90, at least 100, at least 101, or at least 110-fold, in the brain compared to a reference sequence of SEQ ID NO: 8.103. The AAV particle of any one of embodiments 1-102, which is enriched in the brain of at least two to at least three species, e.g., a non-human primate and rodent (e.g., mouse), e.g., as compared to a reference sequence of SEQ ID NO: 8.104. The AAV particle of any one of embodiments 1-103, which is enriched at least 4, at least 4.6, at least 10, at least 20, at least 25, at least 28, at least 30, at least 40. at least 50, at least 60, at least 70, at least 80, at least 81, at least 90, at least 100, at least 101, or at least 110-fold, in the brain of at least Evo to at least three species, e.g., a non-human primate and rodent (e.g., mouse), compared to a reference sequence of SEQ ID NO: 8.105. The AAV particle of embodiment 103 or 104, wherein the at least two to at least three species are Macaco fascicularis. Chlorocebus sabaeus, Callithrix jacchus. and / or mouse (e.g., BALB / c and / or C57BL6 mice).106. The AAV particle of any one of embodiments 1-105, which shows preferential transduction in a muscle cell or region relative to the transduction in the liver.107. The AAV particle of any one of embodiments 1-106, which shows preferential transduction in a heart cell or region relative to the transduction in the liver.108. The AAV particle of any one of embodiments 1-107, which has an decreased tropism for a liver cell or tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.109. The AAV particle of any one of embodiments 1-108, which shows preferential transduction in a brain cell or region relative to the transduction in the liver.[Embodiments 110-311 are intentionally absent.]312. The AAV particle of any one of embodiments 1-109, wherein the viral genome is single-stranded.313. The AAV particle of any one of embodiments 1-109, wherein the viral genome is self- complementary.314. The AAV particle of any one of embodiments 1-109, wherein the viral genome further comprises a nucleotide sequence encoding a Rep protein, e.g., a non-structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68 protein, a Rep52 protein, and / or a Rep40 protein (e.g., a Rep 78 protein and a Rep52 protein).315. The AAV particle of any one of embodiments 1-109, wherein the AAV particle further comprises a nucleotide sequence encoding a Rep protein, e.g., a non-structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68 protein, a Rep52 protein, and / or a Rep40 protein (e.g., a Rep78 protein and a Rep52 protein).316. The AAV particle of embodiment 314 or 315, wherein the Rep78 protein, the Rep68 protein, the Rep52 protein, and / or the Rep40 protein is encoded by at least one Rep gene.317. The AAV particle of any one of embodiments 1-109 and 312-316. wherein the AAV particle is an isolated AAV particle.318. The AAV particle of any one of embodiments 1-109 and 312-317. wherein the AAV particle is a recombinant AAV particle.319. A cell, e.g., a host cell, comprising the AAV particle of any one of embodiments 1-109 and 312-318.320. The cell of embodiment 319, wherein tire cell is a mammalian cell or an insect cell.321. The cell of embodiment 319 or 320, wherein the cell is a cell of a brain region or a spinal cord region, optionally wherein the cell is a cell of the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)).[Embodiments 322-325 are intentionally absent.]326. A pharmacal composition comprising the AAV particle of any one of embodiments 1-109 and 312-318, and a pharmaceutically acceptable excipient.327. A method of delivering to a cell or tissue (e.g., a CNS cell or CNS tissue) a modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA, comprising administering an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-109 and 312-318, thereby delivering the modulator}' polynucleotide.328. The method of embodiment 327, wherein the cell is a cell (e.g., a neuron such as a sensory neuron and / or a motor neuron) of a brain region or a spinal cord region, optionally a cell of the caudate, cerebellum (e.g.. molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus.329. The method of embodiment 327 or 328, wherein the cell is a cell of a muscle tissue or region (e.g., a cardiac, smooth, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis).330. The method of any one of embodiments 327-329, wherein the cell or tissue is within a subject.331. The method of embodiment 330, wherein the subject has, has been diagnosed with having, or is at risk of having a genetic disorder.332. The method of embodiment 330 or embodiment 331, wherein the subject has, has been diagnosed with having, or is at risk of having a muscular disorder or a neuromuscular disorder, such as a muscular dystrophy.333. The method of embodiment 330 or embodiment 331, wherein the subject has, has been diagnosed with having, or is at risk of having a DMPK-related disorder.334. The method of embodiment 330 or embodiment 331, wherein the subject has, has been diagnosed with having, or is at risk of having myotonic dystrophy type 1 (DM1).335. A method of treating a DMPK-related disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or die AAV particle of any one of embodiments 1-109 and 312-318, optionally wherein the subject has, has been diagnosed with having, or is at risk of having the DMPK-related disorder.336. A method of treating a muscular dystrophy in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-109 and 312-318. optionally wherein the subject has. has been diagnosed with having, or is at risk of having the muscular dystrophy.337. A method of treating a muscular disorder or a neuromuscular disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-109 and 312-318, optionally wherein the subject has. has been diagnosed with having, or is at risk of having the muscular disorder or the neuromuscular disorder.338. The method of any one of embodiments 327-337, wherein the genetic disorder, neurological disorder, ncurodcgcncrativc disorder, muscular disorder, or neuromuscular disorder is DM1.339. The method of embodiment 338, wherein the DM1 is congenital DM1.340. The method of any one of embodiments 335-339, where treating comprises prevention of progression of the disorder in the subject.341. The method of any one of embodiments 330-340. wherein the subject is a human.342. The method of any one of embodiments 330-341. wherein the AAV particle or the pharmaceutical composition is administered to the subject intravenously, via intra-cisterna magna injection (ICM). intracerebrally, intrathecally. intracerebroventricularly, via intraparenchymal administration. intraarterially, or intramuscularly.343. The method of any one of embodiments 330-342, wherein the AAV particle or pharmaceutical composition is administered to the subject via focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.344. The method of any one of embodiments 330-343. wherein the AAV particle or pharmaceutical composition is administered to the subject intravenously or intraarterially.345. The method of any one of embodiments 330-344. wherein the AAV particle or pharmaceutical composition is administered to the subject via intra-cistema magna injection (ICM).346. The method of any one of embodiments 330-345. wherein the AAV particle or pharmaceutical composition is administered intracerebroventricularly.347. The method of any one of embodiments 330-346, wherein the subject has aberrant expression or activity of DMPK, e g., of a DMPK gene, DMPK mRNA, and / or DMPK protein.348. The method of any one of embodiments 330-347, wherein the subject has mutated DMPK mRNA.349. The method of any one of embodiments 342-348, wherein administration of the AAV particle or pharmaceutical composition results in amelioration of at least one symptom of the DMPK-related disorder in the subject.350. The method of any one of embodiments 342-349, wherein administration of the AAV particle or pharmaceutical composition results in a decreased activity of the DMPK gene, mRNA, or protein, or a combination thereof.351. The pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-109 and 312-318, for use in a method of delivering the modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA to a cell or tissue.352. The pharmacal composition of embodiment 326 or the AAV particle of any one of embodiments 1-109 and 312-318, for use in a method of treating a genetic disorder, a muscular dystrophy, a muscular disorder, or a neuromuscular disorder.353. The pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-109 and 312-318, for use in the manufacture of a medicament.354. Use of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-109 and 312-318 in the manufacture of a medicament.355. Use of the pharmacal composition of embodiment 326 or the AAV particle of any one of embodiments 1-109 and 312-318 in the manufacture of a medicament for treating a genetic disorder, a muscular dystrophy, a muscular disorder, or a neuromuscular disorder.356. An adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and(ii) an AAV capsid variant comprising, in loop VIII:(a) an amino acid sequence of SEQ ID NO: 4,(b) an amino acid sequence comprising at least 4 or at least 5 consecutive amino acids from SEQ ID NO: 4; or(c) an amino acid sequence comprising one. two, or three different amino acids, relative to the amino acid sequence of SEQ ID NO: 4.357. The AAV particle of embodiment 356, comprising at least 5 consecutive amino acids from SEQ ID NO: 4.358. The AAV particle of embodiment 356 or embodiment 357, wherein the consecutive amino acids comprise NAA.359. The AAV particle of any one of embodiments 356-358, wherein the consecutive amino acids comprise NAAQ (SEQ ID NO: 2).360. The AAV particle of any one of embodiments 356-359, wherein the consecutive amino acids comprise AQAY (SEQ ID NO: 1).361. The AAV particle of any one of embodiments 356-360, wherein the consecutive amino acids comprise NAAQA (SEQ ID NO: 3).362. The AAV particle of any one of embodiments 356-361, wherein the amino acid sequence comprises NAAQAY (SEQ ID NO: 4).363. The AAV particle of any one of embodiments 356-362, wherein the AAV capsid variant is an AAV5 capsid variant.364. The AAV particle of any one of embodiments 356-363, wherein the loop VIII comprises amino acids 571-592, numbered according to SEQ ID NO: 8 or 12.365. The AAV particle of any one of embodiments 356-364, wherein the amino acid sequence replaces positions 578. 579, 580, 581, 582, and 583 (e.g., positions T578. A579, P580, A581, T582, and G583), numbered according to SEQ ID NO: 8.366. The AAV particle of any one of embodiments 356-365, wherein the amino acid sequence corresponds to positions 578, 579. 580, 581. 582, and / or 583 (e.g., amino acids T578, A579, P580, A581. T582, and / or G583), numbered according to SEQ ID NO: 8 or 12.367. The AAV particle of any one of embodiments 356-366, wherein the amino acid sequence comprises or consists of NAAQAY (SEQ ID NO: 4) and corresponds to amino acids 578, 579, 580, 581, 582. 583 (e.g., amino acids T578, A579, P580, A581, T582, and G583). numbered according to SEQ ID NO: 8 or 12.368. An adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and(ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to amino acids 193-724 of SEQ ID NO: 12, wherein the AAV capsid variant comprises:N at position 578,A at position 580,Q at position 581,A at position 582, andY at position 583; wherein the amino acids are numbered according to SEQ ID NO: 8 or 12.369. An adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and(ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to amino acids 137-724 of SEQ ID NO: 12, wherein the AAV capsid variant comprises:N at position 578,A at position 580,Q at position 581,A at position 582, andY at position 583; wherein the amino acids are numbered according to SEQ ID NO: 8 or 12.370. An adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and(ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 12, wherein the AAV capsid variant comprises:N at position 578,A at position 580,Q at position 581,A at position 582, andY at position 583; wherein the amino acids are numbered according to SEQ ID NO: 8 or 12.371. The AAV particle of any one of embodiments 368-370, wherein the AAV capsid variant comprises A at position 579.372. The AAV particle of any one of embodiments 368-371, wherein the AAV capsid variant comprises:(i) a VP1 protein comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 12:(ii) a VP2 protein comprising an amino acid sequence having at least 99% identity to amino acids(iii) a VP3 protein comprising an amino acid sequence having at least 99% identity to amino acids 137-724 of SEQ ID NO: 12.373. The AAV particle of any one of embodiments 368-372, wherein the AAV capsid variant comprises the amino acid sequence of ATNNQSSTNAAQAYT (SEQ ID NO: 6) at amino acids 570-584 as numbered according to SEQ ID NO: 8 or 12.374. The AAV particle of any one of embodiments 368-373, wherein the amino acid sequence of ATNNQSSTNAAQAYT (SEQ ID NO: 6) is present in loop VIII, wherein loop VIII comprises amino acids 571-592, numbered according to SEQ ID NO: 8 or 12.375. The AAV particle of any one of embodiments 368-374, wherein the AAV capsid variant has at least one of the following properties:(i) an increased tropism for a muscle cell or tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and / or(ii) a decreased tropism for a liver cell or tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.376. The AAV particle of any one of embodiments 368-375, wherein the AAV capsid variant has at least one of the following properties:(i) transduces a muscle region, optionally wherein the level of transduction is at least 2, at least 5. at least 10, at least 15, at least 20, or at least 25-fold greater as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., an immunohistochemistry assay or a qPCR assay;(ii) delivers an increased level of a payload to a muscle cell or region, optionally wherein the level of the payload is increased by at least 5, at least 10, at least 12, at least 15, at least 20, at least 21, or at least 25-fold, as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay; and / or(iii) delivers an increased level of viral genomes to a muscle cell or region, optionally wherein the level of viral genomes is increased by at least 2, at least 2.5, at least 5. at least 5.5, at least 6, at least 6.5. at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 10.5. at least 11, at least 11.5. at least 12, at least 12.5, at least 13. at least 13.5, at least 13.8, or at least 14-fold, as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g.. when measured by an assay, e.g., a qRT-PCR or a qPCR assay.377. The AAV particle of embodiment 375 or embodiment 376, wherein the muscle cell or region comprises a cardiac muscle, a smooth muscle, or a skeletal muscle (e g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis).378. The AAV particle of any one of embodiments 356-377, wherein the AAV capsid variant has:(i) an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and / or(ii) a decreased tropism for a liver cell or tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.379. The AAV particle of any one of embodiments 356-378, wherein the AAV capsid variant has at least one of the following properties:(i) transduces a brain cell or region of the CNS, optionally wherein the level of transduction is at least 3, at least 4. at least 5. at least 6, at least 7, at least 8, at least 9. at least 10, at least 11, at least 12. at least 13. at least 14, at least 15, at least 20. at least 24, at least 25, at least 29, or at least 30-fold greater as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g.. an immunohistochemistry assay or a qPCR assay;(ii) is enriched at least 4, at least 4.6, at least 10, at least 20. at least 25, at least 28, at least 30, at least 40. at least 50, at least 60, at least 70, at least 80, at least 81, at least 80, at least 100, at least 101, or at least 110-fold, in the CNS compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9;(iii) delivers an increased level of a pay load to a brain cell or region of the CNS, optionally wherein the level of the payload is increased by at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14-fold, as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., a qRT- PCR or a qPCR assay; and / or(iv) delivers an increased level of viral genomes to a brain cell or region of the CNS. optionally wherein the level of viral genomes is increased by at least 3, at least 4, at least 5, at least 6. at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20. at least 24, at least 25. at least 29, or at least 30-fold, as compared to a reference sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay.380. The AAV particle of embodiment 378 or embodiment 379. wherein the CNS comprises caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, thalamus, and / or spinal cord (e.g.. cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region).381. The AAV particle of any one of embodiments 356-380, wherein the modulatory polynucleotide comprises a molecular scaffold, wherein the molecular scaffold comprises:(a) a 5’ flanking region, optionally comprising any one of SEQ ID NOs: 13-16;(b) a loop region, optionally comprising any one of SEQ ID NOs: 17-21; and(c) a 3’ flanking region, optionally comprising any one of SEQ ID NOs: 22-27.382. The AAV particle of embodiment 381, wherein:(a) the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 14 or SEQ ID NO: 15;(b) the loop region of the molecular scaffold comprises SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 21; and(c) the 3’ flanking region of the molecular scaffold comprises SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.383. The AAV particle of embodiment 382. wherein the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 14. the loop region of the molecular scaffold comprises SEQ ID NO: 17, and the 3’ flanking region of the molecular scaffold comprises SEQ ID NO: 23.384. The AAV particle of embodiment 382, wherein the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 15. the loop region of the molecular scaffold comprises SEQ ID NO: 21, and the 3’ flanking region of tire molecular scaffold comprises SEQ ID NO: 25.385. The AAV particle of embodiment 382, wherein the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 14, the loop region of the molecular scaffold comprises SEQ ID NO: 17, and the 3’ flanking region of tire molecular scaffold comprises SEQ ID NO: 24.386. The AAV particle of embodiment 382, wherein the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 14, the loop region of the molecular scaffold comprises SEQ ID NO: 18, and the 3’ flanking region of the molecular scaffold comprises SEQ ID NO: 23.387. The AAV particle of any one of embodiments 356-386, wherein the modulatory polynucleotide comprises siRNA.388. The AAV particle of embodiment 387, wherein the modulatory polynucleotide further comprises a passenger strand and a guide strand, wherein the guide strand binds to and reduces or eliminates expression of one or more DMPK mRNA transcripts (e.g.. one or more mutated DMPK mRNA transcripts), and wherein the passenger strand and the guide strand are located, respectively, on a 5’ armand a 3‘ arm of a stem loop structure, wherein the passenger strand is located betw een the 5’ flanking region and the loop region and the guide strand is located between the loop region and the 3 ‘ flanking region.389. The AAV particle of embodiment 387, wherein the modulatory polynucleotide further comprises a passenger strand and a guide strand, wherein the guide strand binds to and reduces or eliminates expression of one or more DMPK mRNA transcripts (e.g., one or more mutated DMPK mRNA transcripts), and wherein the guide strand and the passenger strand are located, respectively, on a 5’ arm and a 3’ arm of a stem loop structure, wherein the guide strand is located between the 5’ flanking region and the loop region and the passenger strand is located between the loop region and the 3’ flanking region.390. The AAV particle of embodiment 388 or embodiment 389. wherein the passenger strand is 15-30 nucleotides in length.391. The AAV particle of any one of embodiments 388-390. wherein the guide strand is 15-30 nucleotides in length.392. The AAV particle of embodiment 391, wherein the guide strand is 21-25 nucleotides in length.393. The AAV particle of any one of embodiments 388-392, wherein the passenger strand is at least 70% complementary to the guide strand.394. The AAV particle of any one of embodiments 388-393, wherein the one or more DMPK mRNA transcripts comprises SEQ ID NO: 28 or SEQ ID NO: 29, or a trinucleotide repeat expansion thereof.395. The AAV particle of any one of embodiments 356-394, wherein the viral genome comprises a promoter operably linked to the sequence encoding the modulatory polynucleotide.396. The AAV particle of any one of embodiments 356-395, wherein the viral genome further comprises an inverted terminal repeat (ITR) sequence.397. The AAV particle of embodiment 396, wherein the viral genome comprises an ITR sequence positioned 5’ relative to the sequence encoding the modulatory polynucleotide.398. The AAV particle of embodiment 396 or embodiment 397. w herein the viral genome comprises an ITR sequence positioned 3’ relative to the sequence encoding the modulatory' polynucleotide.399. The AAV particle of any of embodiments 396-398, wherein the viral genome comprises an ITR sequence positioned 5‘ relative to the sequence encoding the modulatory polynucleotide, and an ITR sequence positioned 3‘ relative to the sequence encoding the modulatory polynucleotide.400. A cell comprising the AAV particle of any one of embodiments 356-399, optionally wherein the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an SI9 cell), or a bacterial cell.401. A method of making the AAV particle of any one of embodiments 356-399, wherein the method comprises:(i) providing a host cell comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA and a nucleic acid encoding an AAV capsid variant; and(ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant; thereby making the AAV particle.402. The method of embodiment 401, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.403. The method of embodiment 401 or embodiment 402. further comprising, prior to step (i), introducing a nucleic acid comprising the viral genome into the cell.404. The method of any one of embodiments 401-403, further comprising, prior to step (i) introducing die nucleic acid encoding the AAV capsid variant into the cell.405. The method of any one of embodiments 401-404, wherein the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e g., an SI9 cell), or a bacterial cell.406. A pharmaceutical composition comprising the AAV particle of any one of embodiments 356-399 and a pharmaceutically acceptable excipient.407. A method of delivering a modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA to a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 406 or the AAV particle of any one of embodiments 356-. thereby delivering the modulatory polynucleotide.408. The method of embodiment 407, wherein the subject has, has been diagnosed with having, or is at risk of having a DMPK-related disorder.409. The method of embodiment 408, wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1).410. A method of treating a DMPK-related disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 406 or the AAV particle of any one of embodiments 356-399, thereby treating the DMPK-related disorder.411. The method of embodiment 410, wherein the subject has, has been diagnosed with having, or is at risk for having the DMPK-related disorder.412. The method of embodiment 410 or embodiment 411, wherein the subj ect has one or more mutations in the DMPK gene.413. The method of embodiment 412, wherein the one or more mutations in the DMPK gene comprises a trinucleotide repeat expansion.414. The method of embodiment 413, wherein the trinucleotide repeat expansion in the DMPK gene is or comprises 50 or more CTG repeats (e.g., SEQ ID NO: 35).415. The method of any one of embodiments 410-414, wherein the treating results in prevention of progression of the DMPK-related disorder in the subject.416. The method of any one of embodiments 410-415, wherein the treating results in amelioration of at least one symptom of the DMPK-related disorder in the subject.417. The method of embodiment 416, wherein the at least one symptom comprises cataracts, myotonia, muscle weakness and wasting, cardiac conduction abnonnalities, a myopathic face, learning difficulties, psychosocial problems including depression and / or anxiety, slurred speech, decreased fetal movement in the uterus, polyhydramnios, clubfoot, ventriculomegaly, hypotonia, a tented appearance of the upper lip, dysarthria, intellectual disability, hypotonia, respiratory insufficiency, or a combination thereof.418. The method of any one of embodiments 410-417, wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1).419. A method of treating myotonic dystrophy type 1 (DM1) in a subject, comprising administering to die subject an effective amount of the pharmaceutical composition of embodiment 51 or the AAV particle of any one of embodiments 356-399, thereby treating DM1.420. The method of embodiment 419, wherein the subject has, has been diagnosed with having, or is at risk of having DM1.421. The method of any one of embodiments 407-420, wherein the subject is a human.422. The method of any one of embodiments 407-421, wherein the pharmaceutical composition or AAV particle is delivered to a cell, tissue, or region of muscle, optionally wherein the AAV particle or the pharmaceutical composition is delivered via intravenous administration.423. The method of embodiment 422, wherein the muscle is one or more of cardiac, smooth, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g.. vastus lateralis), optionally wherein the AAV particle or the pharmaceutical composition is delivered via intravenous administration.424. The method of any one of embodiments 407-421, wherein the pharmaceutical composition or AAV particle is delivered to a cell, tissue, or region of the central nervous system, e.g., the brain, optionally wherein the AAV particle or the pharmaceutical composition is delivered via intravenous administration or intracerebroventricular administration.425. The method of embodiment 424, wherein the pharmaceutical composition or AAV particle is delivered to the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region), optionally wherein the AAV particle or the pharmaceutical composition is delivered via intravenous administration or intracerebroventricular administration.426. The method of embodiment 424 or embodiment 425, wherein the subject has, has been diagnosed with having, or is at risk of having congenital myotonic dystrophy type 1.427. The method of any one of embodiments 407-426, further comprising evaluating, e.g., measuring, the level of modulatory polynucleotide expression, the level of mutated DMPK mRNA expression. and / or the level of normal mRNA splicing, optionally DMPK mRNA splicing, in the subject, e.g., in a cell, tissue, or fluid, of the subject.428. The method of embodiment 427, wherein evaluating the subject’s level of modulatory polynucleotide expression, the subject’s level of mutated DMPK mRNA expression, and / or the subject’s level of normal mRNA splicing, optionally DMPK mRNA splicing, is performed prior to and / or subsequent to administration of the pharmaceutical composition or AAV particle, optionally wherein the subject's level of modulatory polynucleotide expression, the subject’s level of mutated DMPK mRNA expression, and / or the subject’s level of normal mRNA splicing, optionally DMPK mRNA splicing, prior to administration is compared to the subject’s level of modulatory polynucleotide expression, the subject’s level of mutated DMPK mRNA expression, and / or the subject’s level of normal mRNA splicing, optionally DMPK mRNA splicing, subsequent to administration.429. The method of embodiment 427 or embodiment 428, wherein the cell or tissue of the subject is a muscle cell or tissue.430. The method of embodiment 427 or embodiment 428. wherein the cell or tissue of the subject is a cell or tissue of the CNS (e.g., caudate, cerebellum (e g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e g., con ical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)).431. The method of any one of embodiments 407-430, wherein the subject’s level of mutated DMPK mRNA expression subsequent to administration of the pharmaceutical composition or AAV particle is decreased relative to the subject’s level of mutated DMPK mRNA expression prior to administration of the pharmaceutical composition or AAV particle.432. The method of any one of embodiments 407-423 or any one of embodiments 427-429, wherein administering the pharmaceutical composition or AAV particle results in:(i) an increase in the number and / or level of viral genomes (VG) per cell in a muscle cell or tissue of the subject relative to the number and / or level of VG per cell in a non-muscle cell or tissue of the subject;(ii) a decrease in mutated DMPK mRNA expression in a muscle cell or tissue of the subject relative to baseline and / or relative to mutated DMPK mRNA expression in a muscle cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle; and / or(iii) an increase in normal mRNA splicing, optionally DMPK mRNA splicing, in a muscle cell or tissue of the subject relative to baseline and / or relative to normal mRNA splicing, optionally DMPK mRNA splicing, in a muscle cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle.433. The method of any one of embodiments407-411, any one of embodiments424-427, or embodiment 430, wherein administering the pharmaceutical composition or AAV particle results in:(i) an increase in the number and / or level of viral genomes (VG) per cell in a CNS tissue (e.g., caudate, cerebellum (e g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region) of the subject relative to the number and / or level of VG per cell in a peripheral tissue of the subject;(ii) a decrease in mutated DMPK mRNA expression in a cell or a tissue of the CNS (e.g., caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen. and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)) of the subject relative to baseline and / or relative to mutated DMPK mRNA expression in a CNS cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle; and / or(iii) an increase in normal mRNA splicing, optionally DMPK mRNA splicing, in a cell or tissue of the CNS (e.g.. caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen. and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)) of the subject relative to baseline and / or relative to normal mRNA splicing, optionally DMPK mRNA splicing, in a CNS cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle.434. The method of embodiment 433, wherein the subject has, has been diagnosed with having, or is at risk of having congenital myotonic dystrophy ty pe 1.435. The method of any one of embodiments 407-434, further comprising administering to the subject at least one additional therapeutic agent and / or therapy.436. The method of embodiment 435, wherein the at least one additional therapeutic agent and / or therapy comprises an agent and / or therapy for treating the DMPK-related disorder, optionally wherein the at least one additional therapeutic agent and / or therapy comprises an anti-diabetic drug, an anti- myotonic drug (e.g., mexiletine), a non-steroidal anti-inflammatory drug, or a combination thereof.437. The method of any one of embodiments 407-436, further comprising administering an immunosuppressant to the subject.438. The method of embodiment 437, wherein the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and / or dexamethasone).adrenocorticotropic hormone, rapamycin, mycophenolate mofetil, tacrolimus, rituximab, eculizumab hydroxychloroquine, alemtuzumab, hydroxyurea, fludarabine, and / or busulfan.439. The pharmaceutical composition of embodiment 406 or the AAV particle of any one of embodiments 356-399 for use in a method of treating a disorder according to any one of embodiments 410-438.440. The pharmaceutical composition of embodiment 406 or the AAV particle of any one of embodiments 356-399 for use in treating a DMPK-related disorder in a subject, optionally wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1).441. The pharmaceutical composition or AAV particle for use of embodiment 440. wherein the subject has. has been diagnosed with having, or is at risk of having the DMPK-related disorder, optionally wherein the DMPK-related disorder is DM1.442. The pharmaceutical composition or AAV particle for use of embodiment 440 or embodiment 441. wherein the DMPK-related disorder is congenital DM1.443. Use of the pharmaceutical composition of embodiment 406 or the AAV particle of any one of embodiments 356-399 in the manufacture of a medicament for treating a DMPK-related disorder in a subject, optionally wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1).444. The use of embodiment 443, wherein the subject has, has been diagnosed with having, or is at risk of having the DMPK-related disorder, optionally wherein the DMPK-related disorder is DM1.445. The use of embodiment 443 or embodiment 444, wherein the DMPK-related disorder is congenital DM1.

[0072] The details of various aspects or embodiments of the present disclosure are set forth below. Other features, objects, and advantages of the disclosure will be apparent from the description and the claims. In the description, the singular fonns also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of this disclosure. In the case of conflict, the present description will control.DETAILED DESCRIPTIONOverview

[0073] Described herein, inter alia, are compositions comprising an AAV capsid variant comprising a sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA. In some embodiments, the present disclosure provides a method of delivering the modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA to a cell or tissue in a subject by administering the AAV capsid variant. In some embodiments, the present disclosure provides a method for treating myotonic dystrophy type 1 (D 1) or another disorder associated with mutated DMPK mRNA expression (i.e.. another DMPK-related disorder) by administering the AAV capsid variant. In various embodiments, AAV capsid variants disclosed herein have enhanced tropism for a cell or tissue, for example a cell or tissue of the muscle, heart, and / or brain. In some embodiments, the AAV capsid variants described herein have enhanced tropism for a cell or tissue of the heart. In some embodiments, the AAV capsid variants described herein have enhanced tropism for a cell or tissue of muscle. In some embodiments, the AAV capsid variants described herein have enhanced tropism for a cell or tissue of cardiac muscle. In some embodiments, the AAV capsid variants described herein have enhanced tropism for a cell or tissue of skeletal muscle. In some embodiments, the AAV capsid variants described herein have enhanced tropism for a cell or tissue of smooth muscle. In some embodiments, the AAV capsid variants described herein have enhanced tropism for more than one tissue (e.g., heart and brain).

[0074] AAVs have proven to be useful as a biological tool due to their relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile. For example, engineered adeno-associated virus (AAV) capsids with improved brain tropism represent an attractive solution to the limitations of CNS delivery. AAV-derived vectors are promising tools for clinical gene transfer because of their non-pathogenic nature, their low immunogenic profile, low rate of integration into the host genome, and long-tenn transgene expression in non-dividing cells. However, the transduction efficiency of naturally occurring AAVs in certain organs is too low for clinical applications, and capsid neutralization by pre-existing neutralizing antibodies may prevent treatment of a large proportion of patients. For these reasons, considerable efforts have been devoted to obtaining capsid variants with enhanced properties. Of many approaches tested so far, significant advances have resulted from directed evolution of AAV capsids using in vitro or in vivo selection of capsid variants created by capsid sequence randomization using either error -prone PCR. shuffling of various parent serotypes, or insertion of fully randomized short peptides at defined positions.

[0075] The genome of the virus may be modified to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver a modulatory polynucleotide, e.g., a modulatory polynucleotide for reducing or eliminating expression of DMPK mRNA, and the viral particle comprising said genomemay be delivered to a target cell, tissue, or organism. In some embodiments, the target cell or tissue is in die muscle, heart, and / or brain. In some embodiments, the target cell is a muscle cell, i.e., a skeletal muscle, smooth muscle, and / or cardiac muscle cell. In some embodiments, the target tissue is a muscle tissue, i.e., a skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis), smooth muscle, and / or cardiac muscle tissue. In some embodiments, the target tissue is a muscle of the head or neck. In some embodiments, the target tissue is a respiratory muscle. In some embodiments, the target tissue is the diaphragm. In some embodiments, the target tissue is an intercostal muscle. In some embodiments, the target cell or tissue is in the brain (e.g.. the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus).

[0076] Gene therapy presents an alternative approach for treating DM1 and related diseases sharing single-gene etiology. AAVs are commonly used in gene therapy approaches as a result of a number of advantageous features. Without being bound by theory, it is believed in some embodiments, that an AAV particle described herein can be used to administer and / or deliver a modulatory polynucleotide, in order to achieve sustained, high concentrations, allowing for longer lasting efficacy, fewer dose treatments, broad biodistribution, and / or more consistent levels of the modulatory polynucleotide, relative to a non- AAV therapy.

[0077] Provided herein are compositions and methods which may provide for improved features compared to prior AAV-mediated approaches, including (i) increased biodistribution throughout muscle and / or heart tissue, (ii) elevated modulatory polynucleotide expression in muscle and / or heart tissue; (iii) preferential biodistribution in muscle and / or heart tissue over other tissues or cells such as the liver and / or DRG; and (iv) increased biodistribution throughout the CNS (e.g., the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region)), (v) elevated modulatory’ poly nucleotide expression in multiple brain regions (e.g., cortex, thalamus, and brain stem); and (vi) preferential biodistribution in the CNS over the liver and / or DRG, of the subject.

[0078] In some embodiments, the AAV capsid variants with enhanced muscle and / or heart tropism described herein increase modulatory polynucleotide expression in muscle and / or heart cells and / or tissues affected by mutated DMPK mRNA expression. In some embodiments, the AAV capsid variants with enhanced muscle and / or heart tropism reduce or eliminate expression of DMPK mRNA in muscle and / or heart.

[0079] The modulatory' polynucleotides provided herein modulate mutated DMPK by altering levels of mutated protein and / or mRNA in cells and / or tissues. In some embodiments, the modulatory' polynucleotides may reduce or eliminate the expression of the mutated DMPK mRNA by reducing its amount and / or activity. In some embodiments, the modulatory polynucleotides reduce or eliminate expression of mutated DMPK mRNA, e.g., by binding to mutated DMPK mRNA and thereby reducing its level in a subject (e.g., via RISC-mediated degradation). In some embodiments, the modulatory polynucleotides reduce or eliminate one or more effects of mutated DMPK mRNA. In someembodiments, the modulatory polynucleotides reduce or eliminate expression of mutated DMPK by reducing or eliminating translation of the mutated mRNA into protein. In some embodiments, the modulatory polynucleotides reduce or eliminate expression of mutated DMPK by reducing or eliminating effects on DMPK protein. In some embodiments, the modulatory polynucleotide may reduce or eliminate formation of hairpin loops, ribonuclear foci, sequestration of RNA-binding proteins, and / or alterations in splicing, translation, localization, polyadenylation and mRNA stability of RNA-binding proteins. In some embodiments, the modulatory polynucleotide may reduce or eliminate aggregation of mutant protein, disruption of ribosome function, and / or disruption of mRNA metabolism, e.g., including translation, stability, and degradation.

[0080] Also provided herein are AAV capsid variants (e.g., AAV5 capsid variants) with improved properties compared to wildtype AAV5, such as (i) increased penetrance through the blood brain barrier following intravenous administration, (ii) wider distribution throughout the multiple brain regions, e.g.. the caudate, cerebellum (e.g.. molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, (iii) elevated expression of a modulatory polynucleotide in multiple brain regions, (iv) wider distribution of a modulatory polynucleotide in one or more peripheral tissues, e.g.. the heart, kidney, muscle (e.g.. diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis), pancreas, and / or liver, and / or (v) elevated expression of a modulatory polynucleotide in one or more peripheral tissues. In some embodiments, the AAV capsid variants described herein enhance the delivery of a modulator}' polynucleotide to multiple regions of the brain. Exemplar}' regions may comprise the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen. and / or thalamus. In some embodiments, the AAV capsid variants described herein enhance the delivery of a modulatory poly nucleotide to the forebrain. In some embodiments, the AAV capsid variants described herein enhance the delivery of a modulatory polynucleotide to the spinal cord. In some embodiments, die AAV capsid variants with enhanced brain tropism described increase expression of a modulatory polynucleotide in the brain. In some embodiments, the AAV capsid variants increase expression of a modulatory polynucleotide in the forebrain. In some embodiments, the AAV capsid variants are capable of increasing expression of a modulatory polynucleotide in the dentate nucleus. In some embodiments, the AAV capsid variants are capable of increasing expression of a modulatory polynucleotide in the somatosensory cortex. In some embodiments, the modulatory polynucleotide is a modulatory polynucleotide for reducing or eliminating mutated DMPK mRNA. In some embodiments, the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA.

[0081] Thus, the compositions and methods described herein can be used in the treatment of a DMPK-related disorders. In some embodiments, the disclosure provides an AAV particle comprising an AAV capsid variant disclosed herein and an AAV viral genome comprising a nucleotide sequence comprising a promoter and a sequence encoding a modulatory polynucleotide for use in treating a DMPK-related disorder. In some embodiments, the modulatory polynucleotide reduces or eliminates expression of mutated DMPK mRNA. In some embodiments, the modulatory polynucleotide comprisesan RNAi agent targeting DMPK mRNA. In some embodiments, the RNAi agent comprises siRNA. In some embodiments, the DMPK-related disorder is DM1. In some embodiments, tire DM1 is congenital DM1.I. CompositionsAdeno-associated viral (AA V) Particles

[0082] AAVs have a genome of about 5.000 nucleotides in length and contains tw o open reading frames encoding the proteins responsible for replication (Rep) and the structural protein of the capsid (Cap). The open reading frames are flanked by two Inverted Terminal Repeat (ITR) sequences, which serve as the origin of replication of the viral genome. The wild-tj pe AAV viral genome comprises nucleotide sequences for two open reading frames, one for the four non-structural Rep proteins (Rep78. Rep68, Rep52. Rep40, encoded by Rep genes) and one for the three capsid, or structural, proteins (VP1, VP2, VP3, encoded by capsid genes or Cap genes). The Rep proteins are important for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid. Alternative splicing and alternate initiation codons and promoters result in the generation of four different Rep proteins from a single open reading frame and the generation of three capsid proteins from a single open reading frame. Though it varies by AAV seroty pe, as a non-limiting example, for AAV9 / 11U.14 (SEQ ID NO: 123 of US 7,906,111, the contents of which are herein incorporated by reference in their entirety), VP1 refers to amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. In some embodiments, with reference to tire amino acid sequence of SEQ ID NO: 12. VP1 comprises amino acids 1-736, VP2 comprises amino acids 138-736, and VP3 comprises amino acids 203-736. In other words, VP1 is the full-length capsid protein sequence, while VP2 and VP3 are shorter components of the whole. As a result, changes in the sequence in the VP3 region are also changes to VP1 and VP2, however, the percent difference as compared to the parent sequence will be greatest for VP3 since it is the shortest sequence of the three. Though described here in relation to the amino acid sequence, the nucleic acid sequence encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid. Without being bound by theory, the AAV capsid typically comprises a molar ratio of 1 : 1: 10 of VP1:VP2:VP3.

[0083] The AAV particle typically requires a co-helper (e.g.. adenovirus) to undergo productive infection in cells. In the absence of such helper functions, the AAV virions essentially enter host cells but do not integrate into the cells’ genome.

[0084] AAV particles have been investigated for delivery of gene therapeutics because of several unique features. Non-limiting examples of the features include (i) the ability to infect both dividing and non-dividing cells; (ii) a broad host range for infectivity, including human cells; (iii) wild-type AAV has not been associated with any disease and has not been shown to replicate in infected cells; (iv) the lack of cell-mediated immune response against the particle, and (v) the non-integrative nature in a host chromosome thereby reducing potential for long-term genetic alterations. Moreover, infection with AAV particles has minimal influence on changing the pattern of cellular gene expression (Stilwell andSamulski et al., Biotechniques, 2003, 34, 148, the contents of which are herein incorporated by reference in their entirety ).

[0085] Typically, AAV particles for delivery' of a modulatory' polynucleotide may be recombinant viral particles which are replication defective as they lack sequences encoding functional Rep and Cap proteins within the viral genome. In some cases, the replication defective AAV particles may lack most or all coding sequences and essentially only contain one or two AAV ITR sequences and a nucleic acid sequence encoding a modulatory polynucleotide (e.g., a modulatory polynucleotide for reducing or eliminating expression of DMPK (e.g., mutated DMPK mRNA).

[0086] In some embodiments, the AAV particles of the present disclosure may be introduced into mammalian cells.

[0087] AAV particles may be modified to enhance the efficiency of delivery. Such modified AAV particles of the present disclosure can be packaged efficiently and can be used to successfully infect the target cells at high frequency and with minimal toxicity.

[0088] In other embodiments, AAV particles of the present disclosure may be used to deliver a modulatory polynucleotide to the central nervous system (see, e.g., U.S. Pat. No. 6,180,613; the contents of which are herein incorporated by reference in their entirety) or to specific tissues of the CNS.

[0089] It is understood that the compositions described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.

[0090] In some embodiments, an AAV capsid variant comprises one or more modifications in loop VIII of AAV5. e.g., at one or more amino acids corresponding to positions 571-592 of VP1 of AAV5 or a variant thereof (e.g., of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12). In some embodiments, an AAV capsid variant comprises a substitution at one. two, three, four, or five of amino acids corresponding to position(s) 578, 580, 581, 582, and / or 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant does not have a substitution at an amino acid corresponding to position 579 (e.g., corresponding to A579) of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the term loop (e.g., loop VIII) is used interchangeably herein with the term variable region (e.g., variable region VIII), or VR (e.g., VR-VIII). In some embodiments, AAV capsid variants disclosed herein comprise a modification in loop VIII of AAV5 or a variant thereof, e.g., at amino acids corresponding to positions 578-583, e.g.. corresponding to amino acids 578, 580, 581, 582, and 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12.

[0091] The AAV particles and modulatory polynucleotides of the disclosure may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles demonstrate enhanced tropism for a target cell type, tissue, or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively). In some embodiments, an AAV particle may, in addition, or alternatively, have decreased tropism for a cell-type, tissue or organ.

[0092] In some embodiments, AAV particles are used as a biological tool due to a relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile. The genome of the virus may be manipulated to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver a desired pay load.

[0093] In some embodiments, the AAV particle is a recombinant AAV particle. In some embodiments, the wild-type AAV viral genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. In some embodiments, inverted terminal repeats (ITRs) cap the viral genome at both the 5 ’ and the 3 ’ end. providing origins of replication for the viral genome. In some embodiments, an AAV viral genome comprises two ITR sequences. In some embodiments, the ITRs have a characteristic T-shaped hairpin structure defined by a self-complementary region (145nt in wild-type AAV) at the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region. In some embodiments, the double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.

[0094] AAV particles of the present disclosure may be produced recombinantly and may be based on AAV reference sequences. In addition to single-stranded AAV viral genomes (e.g., ssAAVs), the present disclosure also provides for self-complementary AAV (scAAV) viral genomes. scAAV viral genomes contain DNA strands that amreal together to form double-stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the transduced cell. In some embodiments, the AAV particle of the present disclosure is an scAAV. In some embodiments, the AAV particle of the present disclosure is an ssAAV.

[0095] Methods for producing and / or modiy ing AAV particles are disclosed in the art such as pseudotyped AAV particles (PCT Patent Publication Nos. W0200028004; W0200123001;W02004112727; W02005005610; and W02005072364, the contents of each of which are incorporated herein by reference in their entirety).

[0096] As described herein, the AAV particles of the disclosure comprising an AAV capsid variant, and a viral genome, have enhanced tropism for a cell-type or a tissue, e g., a CNS cell-type, region, or tissue or a muscle or heart cell-type, region, or tissue.AAV Capsid Variants

[0097] Disclosed herein are AAV particles comprising an AAV capsid variant comprising one or more modifications (e.g., comprising one or more substitutions relative to a wildtype AAV capsid) for enhanced or improved transduction of a target tissue (e.g., cells, regions, and / or tissues of muscle, such as cardiac muscle, smooth muscle, and / or skeletal muscle; and / or the CNS; and / or PNS). In some embodiments, the peptide (e.g., comprising one or more substitutions relative to a wildtype AAV capsid) is present in VP1, VP2. and / or VP3 proteins of the AAV capsid variant. In some embodiments, themodification (e.g., comprising one or more substitutions relative to a wildty pe AAV capsid) is present in VP1, VP2, and VP3 proteins of the AAV capsid variant.

[0098] In some embodiments, the modification (e.g., comprising the one or more substitutions relative to a wildtype AAV capsid) is in loop VIII. In some embodiments, the AAV capsid variant is an AAV5 capsid variant.

[0099] In some embodiments, the one or more embodiments in the AAV capsid variant may increase distribution of an AAV particle to a cell, tissue, or region of muscle. In some embodiments, the muscle is cardiac muscle. In some embodiments, the muscle is smooth muscle. In some embodiments, the muscle is skeletal muscle. In some embodiments, the one or more embodiments in the AAV capsid variant may increase distribution of an AAV particle to a cell, tissue, or region of one or more of cardiac, smooth, and / or skeletal muscle. In some embodiments, the one or more embodiments in the AAV capsid variant may increase distribution of an AAV particle to a cell, tissue, or region of two or more (e.g.. two or all of) cardiac, smooth, and skeletal muscle.

[0100] In some embodiments, the one or more substitutions in the AAV capsid variant may increase distribution of an AAV particle to a cell, region, or tissue of the CNS. The cell of the CNS may be, but is not limited to, neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic. Purkinje, Betz, etc ), glial cells (e.g., microglia, astrocytes, oligodendrocytes) and / or supporting cells of the brain such as immune cells (e.g., T cells). The tissue of the CNS may be, but is not limited to, the caudate, cerebellum (e.g.. molecular layer and / or granule layer), motor cortex, putamen, thalamus, and / or spinal cord (e.g.. cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region).

[0101] In some embodiments, the one or more substitutions may increase distribution of an AAV particle to a cell, region, or tissue of the CNS. In some embodiments, the one or more substitutions may decrease distribution of an AAV particle to the DRG. In some embodiments, the one or more substitutions may decrease distribution of an AAV particle to the cervical DRG.

[0102] In some embodiments, the one or more substitutions may increase distribution of an AAV particle to the CNS (e.g., the cortex) after intravenous administration or after intracerebroventricular administration. In some embodiments, tire one or more substitutions may increase distribution of an AAV particle to the CNS (e.g., the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region)) following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.

[0103] In some embodiments, the one or more substitutions may increase distribution of an AAV particle to the PNS (e.g.. DRG) after intravenous administration or after intracerebroventricular administration. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to non-DRG cells of the PNS following focused ultrasound (FUS), e.g.. coupled with theintravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration. In some embodiments, the one or more substitutions may decrease distribution of an AAV particle to the DRG following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration, hi some embodiments, the one or more substitutions may increase distribution of an AAV particle to the PNS (e.g., DRG) following focused ultrasound (FUS), e.g.. coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.

[0104] In some embodiments, the one or more modifications, e.g., substitutions, may increase distribution of an AAV particle to a cell, region, or tissue of a heart, e.g., a heart atrium or a heart ventricle. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to a heart cell, region, or tissue after intravenous administration or after intracerebroventricular administration.

[0105] In some embodiments, the one or more modifications, e.g., substitutions, may increase distribution of an AAV particle to a cell, region, or tissue of a muscle. In some embodiments, the muscle is a heart muscle (e.g., a heart atrium or a heart ventricle), a diaphragm, an intercostal muscle, a gastrocnemius muscle, and / or a quadriceps muscle, e.g., the vastus lateralis muscle. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to a muscle cell, region, or tissue after intravenous administration.

[0106] In some embodiments, the one or more modifications, e.g., substitutions, may increase distribution an AAV particle to a cell, region, or tissue of the kidney. In some embodiments, the one or more substitutions may increase distribution an AAV particle to a cell, region, or tissue of the pancreas.

[0107] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII, e.g., in an AAV5 variant) an amino acid sequence as set forth in Table 1 (comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6). In some embodiments, the peptide is isolated. In some embodiments, the peptide is recombinant.Table 1. Exemplary Peptide Sequences

[0108] In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least 3, at least 4, or at least 5 consecutive amino acids from the amino acid sequence of SEQ ID NO: 4. In some embodiments, the at least 3 consecutive amino acids comprise NAA. In some embodiments, the at least 4 consecutive amino acids comprise NAAQ (SEQ ID NO: 2). In some embodiments, the at least 5 consecutive amino acids comprise NAAQA (SEQ ID NO: 3). In some embodiments, the AAV capsid variant comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4).

[0109] In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three, but no more than four different amino acids, relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4). In some embodiments, the amino acid sequence comprises one or two (e.g., no more than two) different amino acids relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4).

[0110] In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three, but no more than four modifications, relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4). In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one or two (e.g., no more than two) modifications, relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4).

[0111] In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of the sequences provided in Table 1. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 6.

[0112] In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence, e.g.. a nucleotide sequence of Table 1. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least two. at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, but no more than ten different nucleotides, relative to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence substantially identical (e.g., having at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity) thereto. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven, but no more than ten different nucleotides, relative to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 7. or a nucleotide sequence substantially identical (e.g., having at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, at least 92%. at least 95%, at least 97%, at least 98%. or at least 99% sequence identity) thereto.

[0113] In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence that comprises a nucleotide sequence, e.g., as described in Table 1. In some embodiments, the nucleotide sequence is codon optimized. In some embodiments, the nucleotide sequence is isolated. In some embodiments, the nucleotide sequence is recombinant.

[0114] In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the amino acid sequence is encoded by a nucleotide sequence that comprises a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six. or at least seven, but no more than ten different nucleotides, relative to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the amino acid sequence is encoded by a nucleotide sequence that comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 7. or a nucleotide sequence substantially identical (e.g., having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%. at least 95%, at least 97%. at least 98%, or at least 99% sequence identity) thereto. In some embodiments, an AAV capsid variant comprises a region comprising an amino acid sequence as set forth in Tables 1 or 10.

[0115] In some embodiments, an AAV capsid variant comprises an amino acid sequence comprising at least 3, at least 4, or at least 5 consecutive amino acids from SEQ ID NO: 4. In some embodiments, the amino acid sequence is present in loop VIII. In some embodiments, the amino acid sequence replaces one. two, three, four, five, or all of amino acids corresponding to position(s) 578, 579, 580, 581, 582, and / or 583 (e.g., corresponding to positions T578, A579. P580. A581, T582, and / or G583) of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant comprises one or more amino acid substitutions at amino acids corresponding to position(s) 578, 579, 580, 581, 582, and / or 583 (e.g., corresponding to positions T578, A579, P580, A581, T582, and / or G583) of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12.

[0116] In some embodiments, the at least 3 consecutive ammo acids comprise NAA. In some embodiments, the at least 4 consecutive amino acids comprise NAAQ (SEQ ID NO: 2). In some embodiments, the at least 5 consecutive amino acids comprise NAAQA (SEQ ID NO: 3). In some embodiments, the AAV capsid variant comprises the amino acid sequence of NAAQ AY (SEQ ID NO: 4).

[0117] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising one, two, or three, but no more than four modifications, relative to the amino acid sequence of any one of the sequences provided in Table 1. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one or two (e.g.. no more than two) modifications, relative to the amino acid sequence of any one of the sequences provided in Table 1. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one. two, or three,but no more than four different amino acids, relative to the amino acid sequence of any one of the sequences provided in Table 1. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one or two (e.g., no more than two) different amino acids relative to the amino acid sequence of any one of the sequences provided in Table 1.

[0118] In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three, but no more than four modifications, relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4). In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one or two (e.g., no more than two) modifications relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4). In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising one, two, or three, but no more than four, different amino acids relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4). In some embodiments, the AAV capsid variant comprises one or two (e.g.. no more than two) different amino acids relative to the amino acid sequence of NAAQAY (SEQ ID NO: 4). In some embodiments, the amino acid sequence is present in loop VIII. In some embodiments, the amino acid sequence replaces one, two, three, four, five, or all of amino acids corresponding to positions 578, 579. 580, 581. 582, and / or 583 (e.g., corresponding to positions T578, A579, P580. A581. T582. and / or G583) of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the amino acid sequence replaces one or more amino acids corresponding to positions 578. 579. 580, 581, 582. and / or 583 (e.g., corresponding to positions T578, A 79, P580, A581, T582, and / or G583) of the amino acid sequence of SEQ ID NO: 8.

[0119] In some embodiments, the AAV capsid variant comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4), wherein the amino acid sequence is present at positions corresponding to positions 578, 579. 580, 581, 582, and 583 (e.g., replacing positions corresponding to T578, A579, P580, A581, T582, and G583) of the amino acid sequence of SEQ ID NO: 8.

[0120] In some embodiments, the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, wherein die AAV capsid variant comprises NAAQAY (SEQ ID NO: 4), and wherein SEQ ID NO: 4 is present at amino acids corresponding to positions 578, 579, 580, 581, 582, and 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.

[0121] In some embodiments, the AAV capsid variant comprises an AAV5 capsid variant comprising an amino acid other than T (e.g., N) at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8, an amino acid other than P (e.g., A) at a position corresponding to position 580 of the amino acid sequence of SEQ ID NO: 8. an amino acid other than A (e.g.. Q) at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 8, an amino acid other than T (e.g.. A) at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 8. and / or an amino acid other than G (e.g.. Y) at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant comprises an N at a positioncorresponding to position 578 of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant comprises an A at a position corresponding to position 580 of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant comprises a Q at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant comprises an A at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant comprises a Y at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the substituted amino acids of the AAV capsid variant correspond to positions 578-583 of the amino acid sequence of SEQ ID NO: 12.

[0122] In some embodiments, the AAV capsid variant comprises an AAV5 capsid variant comprising one, two, three, four, five, or all of the amino acid N at position 578, A at position 579. A at position 580, Q at position 581, A at position 582. and / or Y at position 583. numbered according to SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, Hie AAV capsid variant comprises the amino acid N at position 578, A at position 579, A at position 580, Q at position 581, A at position 582, and Y at position 583. numbered according to SEQ ID NO: 8 or SEQ ID NO: 12.

[0123] In some embodiments, the AAV capsid variant comprises an AAV5 variant comprising one. two. three, four, or all of an amino acid other than T at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, an amino acid other than P at a position corresponding to position 580 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, an amino acid other than A at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. an amino acid other than T at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, and / or an amino acid other than G at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises an amino acid other than T at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, an amino acid other than P at a position corresponding to position 580 of tire amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, an amino acid other than A at a position corresponding to position 581 of the ammo acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, an amino acid other than T at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, and an amino acid other than G at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12.

[0124] In some embodiments, the AAV capsid variant comprises an AAV5 variant comprising one, two, three, four, five, or all of the amino acid N at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. A at a position corresponding to position 579 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. A at a position corresponding to position 580 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, Q at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, A at a positioncorresponding to position 582 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, and / or Y at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid N at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, A at a position corresponding to position 579 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, A at a position corresponding to position 580 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. Q at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. A at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, and / or Y at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12.

[0125] In some embodiments, the AAV capsid variant comprises an AAV5 capsid variant comprising one. two, three, four, or all of the amino acid N at position 578, A at position 580, Q at position 581, A at position 582, and / or Y at position 583, numbered according to SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid N at position 578, A at position 580, Q at position 581. A at position 582. and Y at position 583, numbered according to SEQ ID NO: 8 or SEQ ID NO: 12.

[0126] In some embodiments, the AAV capsid variant comprises an AAV5 variant comprising one. two. three, four, or all of the amino acid N at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, A at a position corresponding to position 580 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, Q at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, A at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, and / or Y at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid N at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, A at a position corresponding to position 580 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, Q at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, A at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, and / or Y at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12.

[0127] In some embodiments, the AAV capsid variant comprises an AAV5 capsid variant comprising one, two, three, four, or all of the amino acid substitutions T578N, P580A, A581Q, T582A, and / or G583Y, numbered according to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid substitutions T578N, P580A, A581Q. T582A. and G583Y. numbered according to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12.

[0128] In some embodiments, the AAV capsid variant comprises (i) the amino acid sequence AQAY (SEQ ID NO: 1), optionally wherein the amino acid sequence replaces amino acids corresponding to positions 580-583 (e.g., corresponding to P580, A581, T582, G583of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12; and (ii) an amino acid other than T at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid N at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12.

[0129] In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence substantially identical (e.g., having at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%. or at least 99% sequence identity) thereto. In some embodiments, the AAV capsid variant described herein comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 5. or a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least tw o. at least three, at least four, at least five, at least six, or at least seven, but no more than ten. different nucleotides relative to the nucleotide sequence of SEQ ID NO: 5.

[0130] In some embodiments, the nucleotide sequence encoding the AAV capsid variant (e.g., an AAV capsid variant described herein), comprises the nucleotide sequence of SEQ ID NO: 5, or a nucleotide sequence substantially identical (e.g., having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%. at least 98%, or at least 99% sequence identity ) thereto. In some embodiments, the nucleic acid sequence encoding the AAV capsid variant comprises a nucleotide sequence comprising at least one, at least tw o. at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequences of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein comprises a nucleotide sequence comprising at least one, at least tw o. at least three, at least four, at least five, at least six, or at least seven, but no more than ten, different nucleotides relative to the nucleotide sequence of SEQ ID NO: 5.

[0131] In some embodiments, the AAV capsid variant comprises the amino acid N at position 578 and the amino acid A at position 580, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant further comprises one, two, or all of an amino acid other than A at position 581, an amino acid other than T at position 582, and / or an amino acid other than G at position 583, numbered according to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant further comprises one, two, or all of the amino acid Q at position 581, A at position 582, and Y at position 583. numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid N atposition 578, A at position 580, Q at position 581, A at position 582, and Y at position 583, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12. or an amino acid sequence at least 95% identical thereto.

[0132] In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto, wherein the AAV capsid variant comprises the amino acid N at position 578 and the amino acid A at position 580, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant further comprises one, tw o. or all of an amino acid other than A at position 581. an amino acid other than T at position 582. and / or an amino acid other than G at position 583. numbered according to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant further comprises one. two, or all of the amino acid Q at position 581, A at position 582, and Y at position 583. numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid N at position 578, A at position 580. Q at position 581, A at position 582, and Y at position 583, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.

[0133] In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto, wherein the AAV capsid variant comprises the amino acid N at position 578 and the amino acid A at position 580, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant further comprises one, tw o, or all of an amino acid other than A at position 581, an amino acid other than T at position 582, and / or an amino acid other than G at position 583, numbered according to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant further comprises one, two, or all of the amino acid Q at position 581, A at position 582, and Y at position 583, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid N at position 578, A at position 580, Q at position 581. A at position 582, and Y at position 583, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.

[0134] In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto, wherein the AAV capsid variant comprises the amino acid N at position 578 and the amino acid A at position 580, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant further comprises one, two, or all of an amino acid other than A at position 581, an amino acid other than T at position 582, and / or an amino acid other than G at position 583, numbered according to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant further comprises one, two, or all of the amino acid Q at position 581, A at position 582, and Y at position 583, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid N at position 578. A at position 580, Q at position 581, A at position 582, and Y at position 583, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.

[0135] In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto, wherein the AAV capsid variant comprises the amino acid N at position 578, A at position 580, Q at position 581. A at position 582, and Y at position 583. numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.

[0136] In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto, wherein the AAV capsid variant comprises the amino acid N at position 578, A at position 580, Q at position 581, A at position 582, and Y at position 583, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12.

[0137] In some embodiments, the AAV capsid variant comprises the amino acid sequence SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto, wherein the AAV capsid variant comprises the amino acid N at position 578, A at position 580, Q at position 581, A at position 582, and Y at position 583, numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.

[0138] In some embodiments, the AAV capsid variant further comprises a modification in loop I, II, IV, and / or VI. In some embodiments, loop I, II, IV, VI, and VIII can be identified as described in Govindasamy et al. Structurally Mapping the Diverse Phenotype of Adeno-Associated Virus Serotype 4. Journal of Virology. 2006 Dec. 80(23): 11556-11570; and Govindasamy et al. Structural Insights into Adeno-Associated Virus Serotype 5. Journal of Virology. 2013 Oct. 87(20): 11187-11199; the contents of each of which are hereby incorporated by reference in their entirety.

[0139] In some embodiments, additional modifications can be introduced into tire AAV capsid variant at positions determined using a structural map of wild-type AAV5, e.g., a structural map described and generated by Govindasamy et al. et al. Structural Insights into Adeno-Associated Virus Seroty pe 5. Journal of Virology. 2013 Oct. 87(20): 11187-11199 (the contents of which are hereby incorporated herein by reference in their entirety) or Walters et al. “Structure of Adeno-Associated Virus Seroty pe 5," Journal of Virology. 2004, 78(7):3361-3371 (the contents of each of which are hereby incorporated by reference in their entirety).

[0140] In some embodiments, an AAV capsid variant comprises a modification as described in Jose et al. “High-Resolution Structural Characterization of a New Adenoassociated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors.” Journal of Virology. 2020, 93(1): e01394-18; Qian et al. “Directed Evolution of AAV Seroty pe 5 for Increased Hepatocyte Transduction and Retained Low Humoral Seroreactivity,” Molecular Therapy: Methods and Clinical Development. 2021, 20: 122-132; Afione et al. “Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region,” Journal of Virology, 2015, 89(3): 1660-1672; and / or Wang et al. “Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism,” Mol Ther Nucleic Acids, 2022, 28:293-306; the contents of each of which are hereby incorporated by reference in their entirety .

[0141] In some embodiments, the AAV capsid variant further comprises an amino acid sequence comprising at least one, at least two, or at least three modifications, but not more than 30, not more than 20, or not more than 10 modifications of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant, further comprises an amino acid sequence comprising at least one. at least two. or at least three, but not more than 30, not more than 20, or not more than 10 different amino acids relative to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence with at least 70% (c.g., 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%, or at least 99%) sequence identity' thereto.

[0142] In some embodiments, the AAV capsid variant comprises an amino acid sequence as disclosed herein, e.g., an amino acid sequence of TTK-001, e.g., as described in Tables 2 and 3. In some embodiments, the AAV capsid variant is or comprises TTK-001. In some embodiments, the AAV capsid variant comprises an AAV5 variant wherein the AAV5 capsid sequence is modified to comprise (e.g., wherein the only modification comprises) the amino acid sequence of SEQ ID NO: 4 in a loop VIII region.

[0143] In some embodiments, the AAV capsid variant comprises a VP1, VP2, and / or VP3 protein comprising amino acid sequences disclosed herein, e.g., VP1, VP2. and / or VP3 amino acid sequences from TTK-001, e.g.. as disclosed in Tables 2 and 3.

[0144] In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence as disclosed herein, e.g., a nucleotide sequence encoding TTK-001, e.g., as disclosed in Tables 2 and 4.

[0145] In some embodiments, a polynucleotide or nucleic acid encoding tire AAV capsid variant comprises a nucleotide sequence described herein, e g., a nucleotide sequence encoding the AAV capsid variant of TTK-001, e.g., encoding a capsid variant disclosed in Tables 2 and 3. In some embodiments, the polynucleotide or nucleic acid comprises the sequence in Table 4.Table 2. Exemplary full length capsid sequencesTable 3. Exemplary full length capsid amino acid sequencesTable 4. Exemplary full length capsid nucleic acid sequences

[0146] In some embodiments, an AAV capsid variant comprises the amino acid sequence of a VP 1, VP2, and / or VP3 from the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence with at least 70% (e.g., 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%, or at least 99%) sequence identity thereto. In some embodiments, an AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with at least 70% (e.g., 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%, or at least 99%) sequence identity thereto. In some embodiments, an AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with at least 90% sequence identity thereto. In some embodiments, an AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with at least 95% sequence identity thereto. In some embodiments, an AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with at least 96% sequence identity thereto. In some embodiments, an AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with at least 97% sequence identity thereto. In some embodiments, an AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence with at least 98% sequence identity thereto. In some embodiments, an AAV capsid variant comprises the amino acid sequence of SEQ IDNO: 12 or an amino acid sequence with at least 99% sequence identity thereto. In some embodiments, an AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three modifications, but not more than 30. not more than 20, or not more than 10 modifications relative to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three, but not more than 30, not more than 20, or not more than 10 different amino acids relative to the amino acid sequence of SEQ ID NO: 12.

[0147] In some embodiments, an AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 30. or a nucleotide sequence with at least 70% (e.g.. 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%, or at least 99%) sequence identity thereto. In some embodiments, an AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least two, or at least three, but not more than 30, not more than 20, or not more than 10 different nucleotides relative to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, an AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one. at least two. or at least three modifications, but not more than 30. not more than 20, or not more than 10 modifications relative to the nucleotide sequence of SEQ ID NO: 30.

[0148] In some embodiments, the nucleotide sequence encoding an AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence with at least 70% (e.g., 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%, or at least 99%) sequence identity thereto. In some embodiments, the nucleotide sequence encoding an AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence with at least 70% (e.g., 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%, or at least 99%) sequence identity thereto. In some embodiments, the nucleotide sequence encoding an AAV capsid variant comprises a nucleotide sequence comprising at least one, at least Evo, or at least three modifications, but not more than 30, not more than 20, or not more than 10 modifications relative to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the nucleotide sequence encoding an AAV capsid variant comprises a nucleotide sequence comprising at least one, at least two, or at least three, but not more than 30, not more than 20, or not more than 10 different nucleotides relative to the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the nucleic acid sequence encoding an AAV capsid variant is codon optimized.

[0149] In some embodiments, an AAV capsid variant comprises a VP1, VP2, or VP3 protein, or a combination thereof. In some embodiments, an AAV capsid variant comprises the amino acid sequence corresponding to positions 137-724, e.g., a VP2. of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence with at least 70% (e.g., 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%, or at least 99%) sequence identity thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequencecorresponding to positions 193-724, e.g., a VP3, of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence with at least 70% (e.g., 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%, or at least 99%) sequence identity thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence corresponding to positions 1-724. e.g., a VP1, of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence with at least 70% (e.g., 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%, or at least 99%) sequence identity thereto.

[0150] In some embodiments, an AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12, or an amino acid sequence 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%. or at least 99% identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of SEQ ID NO: 12, or an amino acid sequence 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%, or at least 99% sequence identity thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at 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%, or at least 99% identical thereto.

[0151] In some embodiments, an AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12, or an amino acid sequence at least 90%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of SEQ ID NO: 12, or an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity’ thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0152] In some embodiments, an AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12, or an amino acid sequence at least 95% identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of SEQ ID NO: 12. or an amino acid sequence at least 95% identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12. or an amino acid sequence at least 95% identical thereto.

[0153] In some embodiments, an AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of SEQ ID NO: 12, or an amino acid sequence at least 98% identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of SEQ ID NO: 12. or an amino acid sequence at least 98% identical thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12. or an amino acid sequence at least 98% identical thereto.

[0154] In some embodiments, an AAV capsid variant has an increased tropism for a muscle cell or tissue (e.g., a cardiac muscle, a smooth muscle, or a skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis), relative to the tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the AAV capsid variant delivers an increased level of a modulatory polynucleotide to a muscle cell or region, optionally wherein the level of the modulatory' polynucleotide is increased by at least 5, at least 10. at least 12, at least 15, at least 20, at least 21, or at least 25 -fold, as compared to an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the AAV capsid variant delivers an increased level of viral genomes to a muscle cell or region, optionally wherein the level of viral genomes is increased by at least 2. at least 2.5, at least 5, at least 5.5. at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5. at least 9, at least 9.5, at least 10, at least 10.5. at least 11, at least 11.5. at least 12, at least 12.5, at least 13. at least 13.5, at least 13.8, or at least 14-fold, as compared to an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the muscle cell or region, is a cardiac, smooth, and / or skeletal muscle. In some embodiments, the skeletal muscle is a quadriceps muscle (e.g., vastus lateralis) and / or gastrocnemius. In some embodiments, the muscle is the diaphragm or an intercostal muscle.

[0155] In some embodiments, an AAV capsid variant has an increased tropism for a heart cell or tissue, relative to the tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the AAV capsid variant delivers an increased level of a modulatory polynucleotide to a heart cell or region, optionally wherein the level of the modulatory' polynucleotide is increased by at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5 or at least 5-fold, as compared to an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant delivers an increased level of viral genomes to a heart cell or region, optionally wherein the level of viral genomes is increased by at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10-fold, as compared to an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

[0156] In some embodiments, an AAV capsid variant has an increased tropism for a CNS cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AAV capsid variant transduces a brain cell or region, optionally wherein the level of transduction is at least 3, at least 4, at least 5, at least 6, at least 7. at least 8, at least 9, at least 10. at least 11, at least 12, at least 13, at least 14, at least 15, at least 20. at least 24, at least 25, at least 29, or at least 30-fold greater as compared to an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the AAV capsid variant delivers an increased level of a modulatory polynucleotide to a brain cell or region, optionally wherein the level of the modulatory polynucleotide is increased by at least 3, at least 4, at least 5, at least 6. at least 7, at least 8, at least 9, at least 10. at least 11, at least 12, at least 13, or at least 14-fold, as compared to an AAV capsid comprising the amino acid sequence of SEQID NO: 8 or SEQ ID NO: 9. In some embodiments, the AAV capsid variant delivers an increased level of viral genomes to a brain cell or region, optionally wherein the level of viral genomes is increased by at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 24, at least 25. at least 29, or at least 30-fold, as compared to an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, the brain region is the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus.

[0157] In some embodiments, an AAV capsid variant is enriched at least 4, at least 4.6, at least 10, at least 20, at least 25, at least 28. at least 30, at least 40, at least 50. at least 60, at least 70, at least 80, at least 81, at least 90, at least 100, at least 101, or at least 110-fold in the brain compared to enrichment in the brain of an AAV capsid comprising the amino acid of SEQ ID NO: 8.

[0158] In some embodiments, an AAV capsid variant is enriched in the brain of at least two to at least three species, e.g., a non-human primate and rodent (e.g.. mouse) species, compared to enrichment in the brain of an AAV capsid comprising the amino acid of SEQ ID NO: 8. In some embodiments, an AAV capsid variant is enriched at least 4, at least 4.6, at least 10, at least 20. at least 25, at least 28. at least 30. at least 40, at least 50, at least 60. at least 70, at least 80, at least 81, at least 90, at least 100, at least 101, or at least 110-fold in the brain of at least two to at least three species, e.g., a non-human primate and rodent (e.g., mouse) species, compared to enrichment in the brain of an AAV capsid comprising the amino acid of SEQ ID NO: 8. In some embodiments, the at least two to at least three species are Macaca fascicularis, Chlorocebus sabaeus, Callithrix jacchus, rat and / or mouse (e.g., BALB / c mice and / or C57BL6 mice).

[0159] In some embodiments an AAV capsid variant has preferential transduction in a muscle region relative to the transduction in the liver.

[0160] In some embodiments an AAV capsid variant has preferential transduction in a heart region relative to the transduction in the liver.

[0161] In some embodiments an AAV capsid variant has preferential transduction in a brain region relative to the transduction in the liver.

[0162] In some embodiments, an AAV capsid variant has decreased tropism for a liver cell or tissue, relative to the tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

[0163] In some embodiments, an AAV capsid variant is capable of transducing neuronal cells.

[0164] In some embodiments, an AAV capsid variant is an isolated capsid variant. In some embodiments, the AAV capsid variant is a recombinant capsid variant. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g.. an AAV capsid variant, is a polynucleotide encoding an isolated and / or a recombinant AAV capsid polypeptide.

[0165] Also provided herein are polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same.Exemplary Properties of AAV Capsids

[0166] In some embodiments, the AAV particle may comprise a capsid protein or variant of any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to. packaging, tropism, transduction and immunogenic profdes. Without being bound by theory, it is believed in some embodiments that the AAV capsid protein, e.g., an AAV capsid variant, can modulate AAV particle tropism in a particular tissue.10167] In some embodiments, the AAV capsid variant allows for blood brain barrier penetration following intravenous administration. In some embodiments, the AAV capsid variant allows for blood brain barrier penetration following intravenous administration, focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration. In some embodiments, the AAV capsid variant allows for increased distribution to a brain region. In some embodiments, the brain region comprises the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen. and / or thalamus. In some embodiments, the AAV capsid variant allows for preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG). In some embodiments, the AAV capsid variant allows for preferential transduction in a brain region relative to the transduction in the liver. In some embodiments, the AAV capsid variant allows for transduction in neuronal cells. In some embodiments, the AAV capsid variant allows for transduction in a non-neuronal cell, e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination thereol). In some embodiments, the AAV capsid variant allows for transduction in both neuronal cells and non-neuronal cell, e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination thereol).

[0168] In some embodiments, an AAV capsid variant allows for increased distribution to a spinal cord region. In some embodiments, the spinal region comprises a cervical spinal cord region, thoracic spinal cord region, and / or lumbar spinal cord region.

[0169] In some embodiments, the AAV capsid variant allows for increased distribution to a heart region.

[0170] In some embodiments, the AAV capsid variant is suitable for intramuscular administration and / or transduction of muscle fibers. In some embodiments, the AAV capsid variant, allows for increased distribution to a muscle region. In some embodiments, the muscle region comprises a heart muscle, quadriceps muscle, a respiratory muscle region, or a combination thereof. In some embodiments, the muscle region comprises a heart muscle region, e.g.. a heart atrium muscle region or a heart ventricle muscle region. In some embodiments, the muscle region comprises smooth muscle. In some embodiments, the muscle region comprises skeletal muscle. In some embodiments, the skeletal muscle is the diaphragm, an intercostal muscle, the gastrocnemius, and / or a quadriceps muscle, e.g., the vastus lateralis.

[0171] In some embodiments, the AAV capsid variant is suitable for increased distribution to a kidney. In some embodiments, the AAV capsid variant is suitable for increased distribution to a pancreas.

[0172] In some embodiments, the initiation codon for translation of the AAV VP1 capsid protein, e.g., a capsid variant, described herein may be CTG, TTG, or GTG as described in US Patent No. US8163543, the contents of which are herein incorporated by reference in its entirety.

[0173] The present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (e.g. capsid) of a viral vector such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Metl), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for a first-methionine (Metl) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases. This “Met / AA-clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g.. alanine, valine, serine, threonine, etc.). Met-clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins.

[0174] Where the Met / AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid may be produced, some of which may include a Metl / AAl amino acid (Met+ / AA+) and some of which may lack a Metl / AAl amino acid as a result of Met / AA- clipping (Met- / AA-). For further discussion regarding Met / AA-clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno- Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017 Oct. 28(5):255- 267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 February 19. 327(5968): 973-977; the contents of which are each incorporated herein by reference in its entirety.

[0175] According to the present disclosure, references to capsid proteins, e.g., AAV capsid variants, is not limited to either clipped (Met- / AA-) or unclipped (Met+ / AA+) and may, in context, refer to independent capsid proteins, viral capsids comprised of a ixture of capsid proteins, and / or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid proteins of the present disclosure. A direct reference to a capsid protein or capsid polypeptide (such as VP1, VP2 or VP2) may also comprise VP capsid proteins which include a Metl / AAl amino acid (Met+ / AA+) as well as corresponding VP capsid proteins which lack the Metl / AAl amino acid as a result of Met / AA-clipping (Met- / AA-).

[0176] Further according to the present disclosure, a reference to a specific SEQ ID NO (whether a protein or nucleic acid) which comprises or encodes, respectively, one or more capsid proteins which include a Metl / AAl amino acid (Met+ / AA+) should be understood to teach the VP capsid proteins which lack the Metl / AAl amino acid as upon review of the sequence, it is readily apparent any sequence which merely lacks the first listed amino acid (whether or not Metl / AAl).

[0177] As a non-limiting example, reference to a VP 1 polypeptide sequence which is 736 amino acids in length and which includes a “Metl” amino acid (Met+) encoded by the AUG / ATG start codonmay also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “Metl” amino acid (Met-) of the 736 amino acid Met+ sequence. As a second non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes an “AA1” amino acid (AA1+) encoded by any NNN initiator codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “AA1" amino acid (AA1-) of the 736 amino acid AA1+ sequence.

[0178] References to viral capsids formed from VP capsid proteins (such as reference to specific AAV capsid serotypes), can incorporate VP capsid proteins which include a Metl / AAl amino acid (Met+ / AA1+), corresponding VP capsid proteins which lack the Metl / AAl amino acid as a result of Met / AAl -clipping (Met- / AA1-), and combinations thereof (Met+ / AA1+ and Met- / AA1-).

[0179] As a non-limiting example, an AAV capsid serotype can include VP1 (Met+ / AA1+). VP1 (Met- / AA1-), or a combination of VP1 (Met+ / AA1+) and VP1 (Met- / AA1-). An AAV capsid serotype can also include VP3 (Met+ / AA1+), VP3 (Met- / AA1-), or a combination of VP3 (Met+ / AA1+) and VP3 (Met- / AA1-); and can also include similar optional combinations of VP2 (Met+ZAAl) and VP2 (Met- / AA1-).Additional AAV Capsid Sequences

[0180] In some embodiments, immediately subsequent to an amino acid corresponding to position 577 of the amino acid sequence of SEQ ID NO: 8, the AAV capsid variant comprises at least 3, at least 4. or at least 5 consecutive amino acids of any amino acid sequence provided in Table 1. In some embodiments, the amino acid sequence replaces amino acids corresponding to positions 578, 579, 580, 581, 582, and / or 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12.

[0181] In some embodiments, the AAV capsid variant, comprises a modification at an amino acid corresponding to position 578, 580, 581, 582, and / or 583 of the amino acid sequence of SEQ ID NO: 8 (e.g., corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO. AAVrh32.33, AAVrh74, PHP.N. PHP.B, or an AAV serotype as provided in Table 6 of WO 2021 / 230987 (the contents of which are hereby incorporated by reference in their entirety))). In some embodiments, the amino acid sequence of NAAQAY (SEQ ID NO: 4) replaces amino acids corresponding to positions 578, 579, 580, 581, 582, and / or 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12 (e.g.. corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2. AAV3, AAV3b. AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8. AAVrhlO, AAVrh32.33. AAVrh74, PHP.N, PHP.B. or an AAV serotype as provided in Table 6 of WO 2021 / 230987 (the contents of which are hereby incorporated by reference in their entirety ))).

[0182] In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 8, wherein the AAV capsid variant comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4) in loop VIII. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence having at least95% identity to the amino acid sequence of SEQ ID NO: 8, wherein the AAV capsid variant comprises die amino acid sequence of NAAQAY (SEQ ID NO: 4) in loop VIII. In some embodiments, an AAV capsid variant described herein an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 8, wherein the AAV capsid variant comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4) in loop VIII. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 8, wherein the AAV capsid variant comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4) in loop VIII. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence having at least 98% identity to die amino acid sequence of SEQ ID NO: 8. wherein the AAV capsid variant comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4) in loop VIII. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 8, wherein the AAV capsid variant comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4) in loop VIII.

[0183] In some embodiments the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three modifications, but no more than 30, not more than 20. or not more than 10 modifications relative to the amino acid sequence of SEQ ID NO: 8. In some embodiments the AAV capsid variant, comprises an amino acid sequence comprising at least one, at least tw o. or at least three, but no more than 30, not more than 20, or not more than 10 different amino acids relative to the amino acid sequence of SEQ ID NO: 8.

[0184] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence or is encoded by the nucleotide sequence of any of SEQ ID NO: 1 or 2 of US7427396; SEQ ID NO: 114 of US20030138772; SEQ ID NO: 199 of US20150315612; or SEQ ID NOs: 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, or 44 of US20160289275A1 (the contents of each are incorporated herein by reference in their entirety), or a sequence substantially identical (e.g., having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%. at least 98%, or at least 99% sequence identity) thereto.

[0185] In some embodiments, the AAV capsid variant described herein comprises a modification, e.g., substitution, at a position corresponding to position 569 (e.g., M569V), 652 (e.g., D652A), 362 (e.g., T362M), 359 (e.g., Q359D), 350 (e.g., E350Q), 533 (e.g., P533S), 585 (e.g., Y585V), 587 (e.g., L587T), 581 (e.g., A581T), 582 (e.g., T582A), 584 (e.g., T584A), or a combination thereof, all numbered relative to SEQ ID NO: 8.

[0186] In some embodiments, an AAV capsid variant described herein comprises an amino acid from a wild-type AAV5 sequence, e.g., die amino acid sequence of SEQ ID NO: 8. at one or more of positions 581 to 589, numbered relative to SEQ ID NO: 8. In some embodiments, the AAV capsid variant comprises 1, 2, 3, 4, 5. 6, 7, 8, or all of: the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 8) at position 581 (e.g., comprises the amino acid A at position 581); the amino acid from a wildtype AAV5 sequence (e.g.. SEQ ID NO: 8) at position 582 (e.g.. comprises the amino acid T at position582); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 8) at position 583 (e.g., comprises the amino acid G at position 583); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 8) at position 584 (e.g.. comprises the amino acid T at position 584); the amino acid from a wildtype AAV5 sequence (e.g., SEQ ID NO: 8) at position 585 (e.g., comprises the amino acid Y at position 585); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 8) at position 586 (e.g., comprises the amino acid N at position 586); the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 8) at position 587 (e.g.. comprises the amino acid L at position 587); the amino acid from a wildtype AAV5 sequence (e.g.. SEQ ID NO: 8) at position 588 (e.g., comprises the amino acid Q at position 588); and / or the amino acid from a wild-type AAV5 sequence (e.g., SEQ ID NO: 8) at position 589 (e.g.. comprises the amino acid E at position 589).

[0187] In certain embodiments, an AAV capsid described herein does not comprise a T at position 581, an A at position 582, an A at position 584, a V at position 585, a T at position 585, a V at position 569, an A at position 652, an M at position 362. a Q at position 359, a Q at position 350. an S at position 533, or a combination thereof, all numbered relative to the amino acid sequence of SEQ ID NO: 8.

[0188] In certain embodiments, an AAV capsid variant described herein does not comprise a T at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 8, an A at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 8. an A at a position corresponding to position 584 of the amino acid sequence of SEQ ID NO: 8, a V at a position corresponding to position 585 of the amino acid sequence of SEQ ID NO: 8, a T at a position corresponding to position 585 of the amino acid sequence of SEQ ID NO: 8, a V at a position corresponding to position 569 of the amino acid sequence of SEQ ID NO: 8, an A at a position corresponding to position 652 of the amino acid sequence of SEQ ID NO: 8, an M at a position corresponding to position 362 of the amino acid sequence of SEQ ID NO: 8, a Q at a position corresponding to position 359 of the amino acid sequence of SEQ ID NO: 8, a Q at a position corresponding to position 350 of the amino acid sequence of SEQ ID NO: 8, an S at a position corresponding to position 533 of the amino acid sequence of SEQ ID NO: 8, or a combination thereof.

[0189] In some embodiments, an AAV capsid described herein does not comprise a modification, e.g., substitution, at amino acids corresponding to positions 581-589 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, wherein the modification has the amino acid sequence of any one of the sequences provided in Table 2, 7. 8, 10. 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36. 39, 41, 43, 45, 47, 49, 51, 53. 55, 57, 59, 61. 63, 65, 67, 69, or 71-86 of WO 2021 / 242909.

[0190] In any of the embodiments described herein, a position or amino acid numbered relative to SEQ ID NO: 8 (which may be interchangeably referred to as a position corresponding to the amino acid sequence of SEQ ID NO: 8) can be identified by providing an alignment of a reference sequence and a query sequence, wherein the reference sequence is SEQ ID NO: 8, and identifying the residues corresponding to the positions in the query sequence that correspond to positions in the reference sequence.Table 5. AAV SequencesAAV Viral Genome

[0191] In some embodiments, the AAV particle of the present disclosure serves as an expression vector comprising a viral genome that encodes a modulatory polynucleotide. In various embodiments, a gene product can be expressed from an expression vector. In some embodiments, an mRNA encoding a protein is transcribed from the expression vector and translated into a protein. In some embodiments, a modulatory polynucleotide is transcribed from an expression vector and serves to reduce or eliminate expression of a separate gene product, such as a mutant DMPK mRNA. In some embodiments, various regulatory elements may be used in an expression vector.

[0192] In some embodiments, an AAV particle, e.g.. an AAV particle for the vectorized delivery of a modulatory polynucleotide described herein, comprises a viral genome, e.g., an AAV viral genome. In some embodiments, the viral genome, e.g., the AAV viral genome, further comprises an inverted terminal repeat (ITR) region, an enhancer, a promoter, an intron region, an exon region, a nucleic acid encoding a modulatory polynucleotide (e.g., a modulatory polynucleotide for reducing or eliminating expression of DMPK described herein), a poly A region, or a combination thereof. In some embodiments, the viral genome, e.g., the AAV viral genome, further comprises a nucleotide sequence encoding at least one miRNA binding site.Viral Genome Component: Inverted Terminal Repeats (ITRs)

[0193] In some embodiments, the viral genome may comprise at least one inverted terminal repeat (ITR) region. The AAV particles of the present disclosure comprise a viral genome with at least one ITR region and a region encoding a modulatory polynucleotide (e.g., a modulatory polynucleotide for reducing or eliminating expression of DMPK, e.g., a mutated DMPK gene, a mutated DMPK mRNA, and / or a mutated DMPK protein). In some embodiments, the viral genome has two ITRs. These two ITRs flank the region comprising the modulatory’ polynucleotide at the 5’ and 3’ ends. In some embodiments, the viral genome comprises a 5’ ITR and a 3’ ITR. In some embodiments, the viral genome comprises an ITR present 5‘ relative to the nucleotide sequence encoding the modulatory polynucleotide and an ITR present 3’ relative to the nucleotide sequence encoding the modulatory polynucleotide. In some embodiments, die ITR functions as an origin of replication comprising a recognition site for replication. In some embodiments, the ITR comprises a sequence region which can be complementary and symmetrically arranged. In some embodiments, the ITR incorporated into a viral genome described herein may be comprised of a naturally occurring polynucleotide sequence or a recombinantly derived polynucleotide sequence.

[0194] In some embodiments, the ITR is of the same serotype as the capsid, selected from any one of the serotypes described herein, or a derivative thereof. In some embodiments, the ITR is of a different serotype than the capsid. In some embodiments, the AAV particle has more than one ITR. In some embodiments, the AAV particle comprises a viral genome comprising two ITRs. In some embodiments,die ITRs are of the same seroty pe as one another. In some embodiments, the ITRs are of different serotypes. Non-limiting examples include zero, one, or both of the ITRs having the same seroty pe as the capsid.Viral Genome Component: Promoters and Enhancers

[0195] hi some embodiments, the viral genome comprises at least one element to enhance the transgene target specificity and expression. See, e.g, Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity' and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in their entirety'. Non-limiting examples of elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) region, upstream enhancers (USEs). CMV enhancers, and introns.

[0196] In some embodiments, expression of the modulatory polynucleotide in a target cell may be driven by a specific promoter, including but not limited to. a promoter that is species specific, inducible, tissue-specific, or cell cycle-specific (Parr et al. , Nat. Med.3 1145-9 ( 1997); the contents of which are herein incorporated by reference in their entirety ).

[0197] In some embodiments, the viral genome comprises a promoter that is sufficient for expression, e g., in a target cell, of a modulatory' polynucleotide for reducing or eliminating expression of DMPK, e.g.. mutated DMPK mRNA. In some embodiments, the promoter is deemed to be efficient when it drives expression of the modulatory polynucleotide encoded in the viral genome of the AAV particle.

[0198] In some embodiments, the promoter is deemed to be efficient when it drives expression of the modulatory polynucleotide in the cell or tissue being targeted.

[0199] Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters. In some embodiments, the promoter may be a human promoter. In some embodiments, the promoter may be truncated.

[0200] In some embodiments, the viral genome comprises a promoter that results in expression of the modulatory polynucleotide in one or more cells and / or tissues. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the promoter is a human elongation factor la-subunit (EFla) promoter, a cytomegalovirus (CMV) immediate-early enhancer and / or promoter, a chicken [1- actin (CBA) promoter, a CAG promoter, a (3 glucuronidase (GUSB) promoter, or a ubiquitin C (UBC) promoter.

[0201] In some embodiments, the viral genome comprises a nervous system specific promoter, i.e.. a promoter that results in expression of a modulatory polynucleotide in a neuron, an astrocyte, and / or an oligodendrocyte. Non-limiting examples of tissue-specific expression elements for neurons include synapsin (Syn) or synapsin 1 (Synl), e.g.. human synapsin or synapsin 1.

[0202] In some embodiments, the promoter may be less than 1 kb.

[0203] In some embodiments, the promoter may be a combination of two or more components of the same or different starting or parental promoters.

[0204] In some embodiments, the viral genome comprises an enhancer.

[0205] In some embodiments, the viral genome comprises an engineered promoter.Viral Genome Component: Intron and Exon Sequences

[0206] In some embodiments, tire AAV viral genome comprises at least one intron or a fragment or derivative thereof. In some embodiments, the AAV viral genome comprises at least one exon or a fragment or derivative thereof.

[0207] In some embodiments, the intron may be 100-600 nucleotides in length.

[0208] In certain embodiments, the intron sequence is not an enhancer sequence. In some embodiments, the intron sequence is not a sub-component of a promoter sequence. In some embodiments, the intron sequence is a sub-component of a promoter sequence.Viral Genome Component: Untranslated Regions (UTRs)

[0209] In some embodiments, a wildtype untranslated region (UTR) of a gene is transcribed but not translated. Generally, the 5’ UTR starts at the transcription start site and ends at the start codon and tire 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.

[0210] Features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production.

[0211] In some embodiments, the viral genome encoding a modulatory polynucleotide described herein (e.g., a modulatory polynucleotide for reducing or eliminating expression of DMPK mRNA) comprises a Kozak sequence. Without being bound by theory, wild-type 5' untranslated regions (UTRs) include features that play roles in translation initiation. Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another 'G1.

[0212] In some embodiments, the 5’UTR in the viral genome includes a Kozak sequence.

[0213] In some embodiments, the 5’UTR in the viral genome does not include a Kozak sequence.

[0214] Without being bound by theory , wild-ty pe 3' UTRs are known to have stretches of adenosines and uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al. 1995. the contents of which are herein incorporated by reference in their entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs, such as, but not limited to. GM-CSF and TNF-a, possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Class III ARES, such as. but not limited to. c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR. have been documented to increase the stability' of mRNA. HuR binds to AREs of all the three classes. Engineeringdie HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0215] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides. When engineering specific polynucleotides, one or more copies of an ARE can be introduced to make polynucleotides less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.

[0216] In some embodiments, the 3' UTR of the viral genome may include an oligo(dT) sequence for templated addition of a polyA tail.

[0217] Any UTR from any gene known in the art may be incorporated into the viral genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected or they may be altered in orientation or location. In some embodiments, the UTR used in the viral genome of the AAV particle may be inverted, shortened, lengthened, or made with one or more other 5' UTRs or 3' UTRs known in the art. As used herein, the term “altered,” as it relates to a UTR. means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.

[0218] In some embodiments, the viral genome of the AAV particle comprises at least one artificial UTR, which is not a variant of a wild type UTR.

[0219] In some embodiments, the viral genome of the AAV particle comprises UTRs which have been selected from a family of transcripts whose proteins share a common function, structure, feature, or property'.Viral Genome Component: Filler (Staffer) Sequence

[0220] As used herein, the terms “stuffcr sequence” and “filler sequence” arc used interchangeably. In some embodiments, the AAV particle viral genome comprises at least one filler sequence.

[0221] In some embodiments, the viral genome comprises one or more filler sequences. The filler sequence may be a wild-type sequence or an engineered sequence. A filler sequence may be a variant of a wild-type sequence.

[0222] In some embodiments, the viral genome comprises one or more filler sequences in order to have the length of the viral genome be the optimal size for packaging. In some embodiments, the viral genome comprises at least one filler sequence in order to have the length of the viral genome be about 2.3 kb. In some embodiments, the viral genome comprises at least one filler sequence in order to have the length of the viral genome be about 4.6 kb.Viral Genome Component: Modulatory polynucleotide-encoding sequence

[0223] In some embodiments, the disclosure provides an AAV particle comprising a viral genome encoding a modulatory' polynucleotide for reducing or eliminating expression of DMPK, e.g., a mutatedDMPK gene, a mutated DMPK mRNA, and / or a mutated DMPK protein. In some embodiments, reduction or elimination of DMPK expression thereby reduces or eliminates DMPK protein (e.g., mutated DMPK protein). In some embodiments, the modulatory polynucleotide comprises or encodes an inhibitory polynucleotide, e.g., an RNA interference (RNAi) agent or an antisense oligonucleotide (ASO). In some embodiments, the modulatory polynucleotide comprises or encodes an RNAi agent. In some embodiments, the RNAi agent comprises an siRNA.

[0224] Exemplary DMPK mRNA sequences are provided in Table 6. In some embodiments, the modulatory polynucleotide of the disclosure reduces or eliminates expression of one or both of SEQ ID NO: 28 or SEQ ID NO: 29, or a trinucleotide repeat expansion of either of the foregoing. The exemplary DMPK sequence information from the National Center for Biotechnology' Infonnation cited in Table 6 is hereby incorporated by reference in its entirety.Table 6. Exemplary DMPK mRNA transcripts

[0225] In some embodiments, the modulatory polynucleotide comprises a guide strand and a passenger strand. In some embodiments, the guide strand targets and thus reduces or eliminates expression of one or both of SEQ ID NO: 28 or SEQ ID NO: 29 or a trinucleotide repeat expansion thereof. In some embodiments, the guide strand targeting SEQ ID NO: 28 or SEQ ID NO: 29 or a trinucleotide repeat expansion thereof is fully complementary to a region of that DMPK mRNA sequence. In some embodiments, the guide strand targeting SEQ ID NO: 28 or SEQ ID NO: 29 or a trinucleotide repeat expansion thereof is partially (e.g., substantially) complementary (e.g., at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%. at least 98% or at least 99% complementary) to a region of that DMPK mRNA sequence. In some embodiments, the guide strand has up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9. or up to 10 mismatches relative to a region of DMPK mRNA, e.g.. to a region of SEQ ID NO: 28 or SEQ ID NO: 29 or a trinucleotide repeat expansion thereof.

[0226] In some embodiments, the passenger strand and guide strand are fully complementary. In some embodiments, the passenger strand and guide strand are partially (e.g.. substantially) complementary (permitting one or more mismatches). In some embodiments, the passenger strand is at least 70% (e.g., at least 70%, at least 75%. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99%, or 100%) complementary to the guide strand. In some embodiments, the passenger strand has up to 1, up to2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 mismatches relative to the guide strand.

[0227] In some embodiments, the modulatory polynucleotide binds to a coding region of DMPK mRNA. In some embodiments, the modulatory polynucleotide binds to a noncoding region of DMPK mRNA.

[0228] In some embodiments, the disclosure herein provides constructs that allow for improved expression of a modulatory polynucleotide delivered by gene therapy vectors.

[0229] In some embodiments, the disclosure provides constructs that allow for improved biodistribution of a modulatory polynucleotide delivered by gene therapy vectors.

[0230] In some embodiments, the disclosure provides constructs that allow for improved sub-cellular distribution or trafficking of a modulatory polynucleotide delivered by gene therapy vectors.

[0231] In some embodiments, the disclosure provides constructs that allow for improved trafficking of a modulatory polynucleotide to lysosomal membranes delivered by gene therapy vectors.

[0232] In some embodiments, the present disclosure relates to a composition containing or comprising a nucleic acid sequence encoding modulatory polynucleotide for reducing or eliminating expression of DMPK mRNA or a functional fragment or variant thereof and methods of administering the composition in vitro or in vivo in a subject, e.g., a human subject and / or an animal model of disease, e.g.. a DMPK-related disorder.

[0233] In some embodiments, the disclosure provides a nucleotide sequence encoding a modulatory polynucleotide for use in an AAV genome. In some embodiments, the nucleotide sequence further comprises one or more, e.g., all of, a 5’ ITR sequence, an enhancer sequence, a promoter sequence, an intron sequence, a poly A sequence, and a 3’ ITR sequence.

[0234] In some embodiments, the AAV genome encodes a payload construct that comprises a combination of coding and non-coding nucleic acid sequences.

[0235] In some embodiments, the viral genome encodes more than one modulatory’ polynucleotide. As a non-limiting example, a viral genome encoding more than one modulatory polynucleotide may be replicated and packaged into a viral particle. A target cell transduced with a viral particle comprising more than one modulatory polynucleotide may express each of the modulatory polynucleotides in a single cell.

[0236] In some embodiments, the viral genome encodes an inhibitory polynucleotide, e.g.. an antisense oligonucleotide (ASO) or RNAi agent (e.g., a dsRNA, siRNA, shRNA, pre-miRNA, pri- miRNA, miRNA, stRNA, IncRNA. piRNA. or snoRNA). In some embodiments, the modulatory agent is an RNAi agent. In some embodiments, the RNAi agent is a siRNA. In some embodiments, the modulatory agent is an ASO. In some embodiments, the ASO or siRNA comprises at least one (e.g., one or more or all) modified nucleotides. In some embodiments, the viral genome encodes an siRNA. In some embodiments, the viral genome encodes an siRNA comprising a sense strand (e.g., a passenger strand) and an antisense strand (e.g., a guide strand).

[0237] A viral genome encoding a modulatory polynucleotide may further comprise or encode a selectable marker. A selectable marker may comprise a gene sequence or a protein or polypeptide encoded by a gene sequence expressed in a host cell that allows for the identification, selection, and / or purification of the host cell from a population of cells that may or may not express the selectable marker. In some embodiments, the selectable marker provides resistance to survive a selection process that would otherwise kill the host cell, such as treatment with an antibiotic. In some embodiments, an antibiotic selectable marker may comprise one or more antibiotic resistance factors, including but not limited to neomycin resistance (e.g., neo), hygromycin resistance, kanamycin resistance, and / or puromycin resistance.

[0238] In some embodiments, a viral genome encoding a modulatory polynucleotide may comprise a selectable marker including, but not limited to, -lactamase, luciferase, (3-galactosidase, or any other reporter gene as that term is understood in the art, including cell-surface markers, such as CD4 or the truncated nerve growth factor (NGFR) (for GFP, see WO 96 / 23810; Heim et al., Current Biology 2: 178- 182 (1996); Heim et al.. Proc. Natl. Acad. Sci. USA (1995); or Heim et al.. Science 373:663-664 (1995); for P-lactamase, see WO 96 / 30540); the contents of each of which are herein incorporated by reference in their entirety.

[0239] In some embodiments, a viral genome encoding a selectable marker may comprise a fluorescent protein. A fluorescent protein as herein described may comprise any fluorescent marker including but not limited to green, yellow, and / or red fluorescent protein (GFP, YFP, and / or RFP). In some embodiments, a payload construct encoding a selectable marker may comprise a human influenza hemagglutinin (HA) tag.

[0240] In certain embodiments, a nucleic acid for expression of a modulatory polynucleotide in a target cell will be incorporated into the viral genome and located betw een two ITR sequences.Piral Genome Component: Molecular Scaffold-encoding sequences

[0241] In some embodiments, a modulatory polynucleotide described herein comprises a molecular scaffold. In some embodiments, the molecular scaffold is or comprises a known or wild type pri- or pre- microRNA. In other embodiments, the molecular scaffold of die modulatory polynucleotide is designed ab initio. In some embodiments, the molecular scaffold along with the payload (e.g., passenger strand and guide strand) form a stem-loop structure.

[0242] In some embodiments, the molecular scaffold comprises a 5’ flanking region. In some embodiments, the molecular scaffold comprises a 3’ flanking region. In some embodiments, the molecular scaffold comprises a loop region, wherein the loop region is present between the passenger and guide strand of the stem loop structure. In some embodiments, the molecular scaffold comprises one or more spacers between one or more modules of the modulatory polynucleotide. A module of the modulatory polynucleotide may include a 5’ flanking region, a 5’ arm, a loop region, a 3’ arm. or a 3’ flanking region. In some embodiments, a spacer is of sufficient length to form approximately one helical turn of the sequence.

[0243] In some embodiments, the molecular scaffold comprises a 5’ flanking region, a loop region, and a 3‘ flanking region. In some embodiments, the modulator}' polynucleotide comprises, in the 5' to 3' direction, a 5' flanking sequence, a 5' ami comprising a passenger strand or guide strand, a loop region, a 3' arm comprising a guide strand or passenger strand, respectively, and a 3' flanking sequence.

[0244] hi some embodiments, a modulatory polynucleotide described herein comprises one, two, or all of a 5 ’ flanking region, a loop region, and / or a 3 ‘ flanking region. In some embodiments, the modulatory polynucleotide comprises a guide strand and a passenger strand and further comprises a 5 ’ flanking region, a loop region, and / or a 3’ flanking region. In some embodiments, the modulatory polynucleotide comprises a guide strand and a passenger strand and further comprises a 5’ flanking region, a loop region, and a 3’ flanking region.

[0245] In some embodiments, an encoded modulatory polynucleotide described herein comprises from 5’ to 3’: a 5’ flanking region, a passenger strand, a loop region, a guide strand, and a 3’ flanking region. In some embodiments, an encoded modulatory polynucleotide described herein comprises from 5’ to 3’: a 5’ flanking region, a guide strand, a loop region, a passenger strand, and a 3’ flanking region.

[0246] Exemplary sequences for the 5' flanking region, the loop region, and the 3' flanking region that may be used in the molecular scaffolds described herein are shown in Tables 7-9.Table 7. Exemplary 5’ Flanking Regions for a Molecular ScaffoldTable 8. Exemplary Loop Regions for a Molecular ScaffoldTable 9. Exemplary 3’ Flanking Regions for a Molecular Scaffold

[0247] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising any one of SEQ ID NOs: 13-16, or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 13-16; a loop region comprising any one of SEQ ID NOs: 17-21, or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 17-21; and a 3’ flanking region comprising any one of SEQ ID NOs: 22-27, or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NOs: 22-27.

[0248] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising any one of SEQ ID NOs: 13-16; a loop region comprising any one of SEQ ID NOs: 17-21; and a 3’ flanking region comprising any one of SEQ ID NOs: 22-27.

[0249] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 14 or SEQ ID NO: 15; a loop region comprising SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 21; and a 3’ flanking region comprising SEQ ID NO: 22. SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 14 or SEQ ID NO: 15; a loop region comprising SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 21; and a 3’ flanking region comprising SEQ ID NO: 23, SEQ ID NO: 24. or SEQ ID NO: 25.

[0250] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 14, or a nucleotide sequence at least 75%, at least 80%. at least 85%, at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto; a loop region comprising SEQ ID NO: 17, or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; and a 3’ flanking region comprising SEQ ID NO: 23 or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0251] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQID NO: 15, or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical thereto; a loop region comprising SEQ ID NO: 21, or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical thereto; and a 3’ flanking region comprising SEQ ID NO: 25 or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0252] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 14, or a nucleotide sequence at least 75%. at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical thereto; a loop region comprising SEQ ID NO: 17. or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto; and a 3’ flanking region comprising SEQ ID NO: 24 or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto.

[0253] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprises SEQ ID NO: 14, or a nucleotide sequence at least 75%, at least 80%. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto; a loop region comprising SEQ ID NO: 18, or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto;and a 3’ flanking region comprising SEQ ID NO: 23 or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0254] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 14, a loop region comprising SEQ ID NO: 17, and a 3' flanking region comprising SEQ ID NO: 23. In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 15. a loop region comprising SEQ ID NO: 21, and a 3’ flanking region comprising SEQ ID NO: 25. In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 14, a loop region comprising SEQ ID NO: 17. and a 3’ flanking region comprising SEQ ID NO: 24. In some embodiments, the molecular scaffold comprises a 5’ flanking region comprises SEQ ID NO: 14, a loop region comprising SEQ ID NO: 18, and a 3’ flanking region comprising SEQ ID NO: 23.Exemplary AAV Capsid Sequences

[0255] In some embodiments, the AAV viral genome further comprises a nucleic acid encoding a capsid protein, e.g., a structural protein. In some embodiments, the capsid protein comprises a VP1 polypeptide, a VP2 polypeptide, and / or a VP3 polypeptide. In some embodiments, the VP1 polypeptide, the VP2 polypeptide, and / or the VP3 polypeptide are encoded by at least one Cap gene. In some embodiments, the AAV viral genome further comprises a nucleic acid encoding a Rep protein, e.g., a non-structural protein. In some embodiments, the Rep protein comprises a Rep78 protein, a Rep68 protein, a Rep52 protein, and / or a Rep40 protein. In some embodiments, the Rep78 protein, the Rep68 protein, the Rep52 protein, and / or the Rep40 protein are encoded by at least one Rep gene.

[0256] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising an amino acid sequence selected from Table 1 or Table 2. In some embodiments, the amino acid sequence of NAA, NAAQ (SEQ ID NO: 2), AQAY (SEQ ID NO: 1), NAAQA (SEQ ID NO: 3), or NAAQAY (SEQ ID NO: 4) is present in loop VIII. wherein loop VIII comprises amino acids 571 - 592 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises: (i) a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of tire amino acid sequence at positions 137-724 of SEQ ID NO: 12 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; and / or (iii) a VP3 protein comprising or consisting of the amino acid sequence at positions 193- 724 of SEQ ID NO: 12 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises N at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, A at a position corresponding to position 580 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, Q at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, A at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12, and Y at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 12.

[0257] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the amino acid N at position 578. A at position 580, Q at position 581, A at position 582, and Y at position 583. numbered according to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%. at least 97%, at least 98%. or at least 99% identical) thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical) thereto. In someembodiments, die AAV capsid variant comprises the amino acid sequence SEQ ID NO: 12, or an amino acid sequence that is at least 95% identical (e.g.. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

[0258] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the amino acid N at a position corresponding to position 578 of the amino acid sequence of SEQ ID NO: 12. A at a position corresponding to position 580 of the amino acid sequence of SEQ ID NO: 12. Q at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 12. A at a position corresponding to position 582 of the amino acid sequence of SEQ ID NO: 12, and Y at a position corresponding to position 583 of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 193-724 of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 95% identical (e.g., at least 95%. at least 96%, at least 97%, at least 98%. or at least 99% identical) thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 137-724 of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%. at least 97%, at least 98%. or at least 99% identical) thereto. In some embodiments, the AAV capsid variant comprises the amino acid sequence SEQ ID NO: 12, or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto.

[0259] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the ammo acid sequence of amino acids 193-724 of the amino acid sequence of SEQ ID NO: 12.

[0260] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the ammo acid sequence of amino acids 137-724 of the amino acid sequence of SEQ ID NO: 12.

[0261] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the amino acid sequence of SEQ ID NO: 12.

[0262] The present disclosure provides in some embodiments, vectors, cells, and / or AAV particles comprising the above identified viral genomes.Self-Complementary and Single Stranded Viral Genomes

[0263] In some embodiments, the AAV viral genome used in the present disclosure is singlestranded (ssAAV).

[0264] In some embodiments, the AAV viral genome is capable of forming double-stranded DNA. In some embodiments, the AAV viral genome is self-complementary. See, e.g., US Patent No. 7,465,583. scAAV particles contain both DNA strands that anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.

[0265] Methods for producing and / or modifying AAV viral genome and particles are disclosed in the art such as pseudotyped AAV particles (International Patent Publication Nos. W0200028004;W0200123001; W02004112727; WO 2005005610 and WO 2005072364, the content of each of which are incorporated herein by reference in their entirety).II. AAV Production

[0266] Viral production disclosed herein describes processes and methods for producing AAV particles (with enhanced, improved and / or increased tropism for a target tissue), e.g., an AAV particle comprising an AAV capsid variant that may be used to contact a target cell to deliver a modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA.

[0267] In some embodiments, disclosed herein is a method of making an AAV particle of the present disclosure, e.g., an AAV particle comprising an AAV capsid variant disclosed herein, wherein the method comprises: (i) providing a cell comprising a viral genome comprising a nucleotide sequence encoding a modulatory7polynucleotide for reducing or eliminating expression of mutated DMPK mRNA and a nucleic acid encoding an AAV capsid variant and (ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant, e.g., an AAV capsid variant described herein, thereby making the AAV particle. In some embodiments, the AAV capsid variant comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV particle comprises a viral genome that is packaged in an AAV capsid variant comprising the amino acid sequence of amino acids 193-724 of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV particle comprises a viral genome that is packaged in an AAV capsid variant comprising the ammo acid sequence of amino acids 137-724 of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12, and the viral genome comprises a molecular polynucleotide comprising: (a) a 5’ flanking region comprising any one of SEQ ID NOs: 13-16, or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 13-16; (b) a loop region comprising any one of SEQ ID NOs: 17-21, or a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical to any one of SEQ ID NOs: 17-21; and a 3’ flanking region comprising any one of SEQ ID NOs: 22-27, or a nucleotide sequence at least 75%. at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%. or at least 99% identical to any one of SEQ ID NOs: 22-27. In some embodiments, the method comprises, prior to step (i), introducing a nucleic acid comprising the viral genome into the cell. In some embodiments, the method comprises, prior to step (i). introducing the nucleic acid encoding die AAV capsid variant into the cell. In some embodiments, the AAV particle described herein is an isolated AAV particle. In some embodiments, the AAV particle described herein is a recombinant AAV particle.

[0268] Any method known in the art may be used for the preparation of AAV particles. In some embodiments, AAV particles are produced in mammalian cells (e.g., HEK293 cells). In some embodiments, AAV particles are produced in insect cells (e g., S® cells).

[0269] Methods of making AAV particles are well known in the art and are described in e.g., U.S. Patent Nos. US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, US5064764, US6194191, US6566118, US8137948; or International Publication Nos. WO1996039530, W01998010088, WO1999014354, WO1999015685, WO1999047691, W02000055342, W02000075353 and W02001023597: Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. E / r.63:3822-8 (1989); Kajigaya et al.. Proc. Nat'l. Acad. Set. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir., 219:37-44 (1996); Zhao et al., 17r.272:382-93 (2000); the contents of each of which are herein incorporated by reference in their entirety. In some embodiments, the AAV particles are made using the methods described in International Patent Publication WO2015191508, the contents of which are herein incorporated by reference in their entirety- ill. Pharmaceutical Compositions

[0270] The present disclosure additionally provides a method for treating a DMPK-related disorder, e.g.. in a human subject, comprising administering to the subject any of the AAV polynucleotides or AAV genomes described herein or administering to the subject a particle comprising said AAV polynucleotide or AAV genome, or administering to the subject any of the described compositions, including pharmaceutical compositions.

[0271] In some embodiments, the pharmaceutical composition comprises an AAV particle comprising a viral genome that is packaged in an AAV capsid variant comprising the amino acid sequence of amino acids 193-724 of the amino acid sequence of SEQ ID NO: 12, or an AAV particle comprising a viral genome that is packaged in an AAV capsid variant comprising the amino acid sequence of amino acids 137-724 of tire ammo acid sequence of SEQ ID NO: 12, or an AAV particle comprising a viral genome that is packaged in an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the present disclosure provides pharmaceutical compositions of an AAV particle comprising a viral genome encoding a modulatory polynucleotide for reducing or eliminating expression of DMPK (e.g., a mutated DMPK gene, a mutated DMPK mRNA, and / or a mutated DMPK protein) and an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 12.10272] Although pharmaceutical compositions provided herein, e.g., comprising AAV particles comprising a modulatory polynucleotide-encoding sequence, are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions may be suitable for administration to any other animal, e.g., non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various non-human annuals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merelyordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0273] hi some embodiments, compositions are administered to humans, e.g., human patients or human subjects.

[0274] In some embodiments, the AAV particle formulations described herein may contain a nucleic acid encoding at least one modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA. In some embodiments, the formulations may contain a nucleic acid encoding 1, 2, 3, 4. or 5 modulatory polynucleotide(s) for reducing or eliminating expression of mutated DMPK mRNA.

[0275] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.IV. Formulations

[0276] Formulations of the AAV pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association w ith an excipient and / or one or more other accessory' ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0277] Relative amounts of the active ingredient, tire pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance ith the disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered.

[0278] For example, the composition may comprise about 0.1% to about 99% (w / w) of the active ingredient. By way of example, the composition may comprise about 0.1% to about 100%, e.g., about 0.5% to about 50%, about 1% to about 30%, about 5% to about 80%. or at least 80% (w / w) active ingredient.|0279| The AAV particles of the disclosure can be formulated using one or more excipients to: (1) increase stability’ ; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encoded protein in vivo-, (6) alter the release profile of encoded protein in vivo and / or (7) allow for regulatable expression of the modulatory polynucleotide.

[0280] Formulations of the present disclosure can include, without limitation, saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics and combinations thereof. Further, the viral vectors of the present disclosure may be formulated using selfassembled nucleic acid nanoparticles.

[0281] In some embodiments, tire viral vectors encoding a modulatory polynucleotide may be formulated to optimize baricity and / or osmolality. In some embodiments, the baricity and / or osmolality of the formulation may be optimized to ensure optimal drug distribution in the central nervous system or a region or component of the central nervous system.

[0282] In some embodiments, a fonnulation comprises an AAV particle comprising a viral genome that is packaged in an AAV capsid variant comprising the amino acid sequence of amino acids 193-724 of the amino acid sequence of SEQ ID NO: 12, or an AAV particle comprising a viral genome that is packaged in an AAV capsid variant comprising the amino acid sequence of amino acids 137-724 of the amino acid sequence of SEQ ID NO: 12, or an AAV particle comprising a viral genome that is packaged in an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the present disclosure provides formulations of an AAV particle comprising a viral genome encoding a modulatory' polynucleotide for reducing or eliminating expression of mutated DMPK (e.g., a mutated DMPK gene, a mutated DMPK mRNA, and / or a mutated DMPK protein) and an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 12.Excipients

[0283] The formulations of the disclosure can include one or more excipients, each in an amount that together increases the stability of the AAV particle, increases cell transfection or transduction by the viral particle, increases the expression of viral particle encoded protein, and / or alters the release profile of AAV particle encoded proteins. In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Phannacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0284] Excipients, which, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition. A. R. Gennaro, Lippincott. Williams & Wilkins. Baltimore, MD. 2006; the contents of which are herein incorporated by reference in their entirety ). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing anyundesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.Inactive Ingredients

[0285] In some embodiments, AAV formulations may comprise at least one excipient which is an inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more agents that do not contribute to the activity of the pharmaceutical composition included in formulations. In some embodiments, all, none, or some of the inactive ingredients which may be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA).

[0286] Formulations of AAV particles may include cations or anions. In one embodiment, the formulations include metal cations such as, but not limited to. Zn2+, Ca2+, Cu2+, Mg+. or combinations thereof. In some embodiments, formulations may include polymers or polynucleotides complexed with a metal cation (See, e.g., U.S. Pat. Nos. 6,265.389 and 6.555.525. the contents of each of which are herein incorporated by reference in their entirety)V. Uses and Applications

[0287] The compositions of the disclosure may be administered to a subject or used in the manufacture of a medicament for administration to a subject having a DMPK-related disorder. The DMPK-related disorder may be a muscular dystrophy or another muscular or neuromuscular disorder. Also specifically contemplated herein is myotonic dystrophy type 1 (DM1) and other disorder(s) that arise from expression of a DMPK gene product with trinucleotide repeat expansions, e.g., DMPK with 50 or more CTG repeats (e.g., SEQ ID NO: 35) (polyQ expansions).

[0288] The present disclosure addresses the need for new technologies by providing DMPK-related treatment deliverable by AAV-based compositions and complexes for the treatment of DMPK-related disorders.

[0289] The compositions of the disclosure may be administered to a subject, e.g., to deliver a modulatory polynucleotide, e.g., to a subject who has, has been diagnosed with having, or is at risk of having a DMPK-related disorder (e.g., DM1). The compositions may similarly be used in the manufacture of a medicament for administration to a subject having a DMPK-related disorder (e.g., DM1).

[0290] In some embodiments, the disclosure provides a method of delivering a modulatory polynucleotide to a subject comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding a modulatory polynucleotide, thereby delivering the modulatory polynucleotide. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having a DMPK-related disorder. In some embodiments, the DMPK-related disorder is DM1. In some embodiments, the DM1 is congenital DM1.

[0291] In some embodiments, the disclosure provides a method for treating a DMPK-related disorder (e.g., DM1). In certain embodiments, a pharmaceutical composition or AAV particle disclosed herein, comprising a viral genome encoding a modulatory’ polynucleotide, may be administered to a subject totreat a DMPK-related disorder (e.g., DM1), thereby treating the disorder (e.g., DM1). In some embodiments, the subject has, has been diagnosed with having, or is at risk of having a DMPK-related disorder, such as DM1. In some embodiments, the treatment results in a reduction in the subject's DMPK mRNA level (e.g., levels of mutated DMPK mRNA) as compared to baseline. In some embodiments, the treatment results in a reduction in the subject's DMPK protein level (e.g., levels of aberrantly expressed, aberrantly active, and / or mutant DMPK protein) as compared to baseline.

[0292] In some embodiments, the disclosure provides an AAV particle or pharmaceutical composition according to any one of the embodiments disclosed herein for treating a DMPK-related disorder, such as DM1. In some embodiments, the present disclosure provides the pharmaceutical composition or the AAV particle of any one the embodiments disclosed herein for use in a method of treating a disorder as disclosed herein, such as DM1.

[0293] In some embodiments, the disclosure provides a method of treating DM1 in a subject. In certain embodiments, a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding a modulatory polynucleotide may be administered to a subject to treat DM1. In some embodiments, the subject has. has been diagnosed with having, or is at risk of having DM1.

[0294] In some embodiments, a pharmaceutical composition or AAV particle may be administered to a subject may have one or more mutations in the DMPK gene. In some embodiments, the one or more mutations in the DMPK gene comprises a trinucleotide repeat expansion. In some embodiments, the trinucleotide repeat expansion in the DMPK gene is or comprises 50 or more CTG repeats (e.g., SEQ ID NO: 35).

[0295] In some embodiments, the treatment may result in prevention of progression of a DMPK- related disorder. For example, the treatment may result in amelioration of at least one symptom of the DMPK-related disorder in the subject. In some embodiments, the treatment results in amelioration of at least one symptom of DM1. In some embodiments, at least one symptom comprises cataracts, myotonia, muscle weakness and wasting, cardiac conduction abnormalities, a myopathic face, learning difficulties, psychosocial problems including depression and / or anxiety, slurred speech, decreased fetal movement in the uterus, polyhydramnios, clubfoot, ventriculomegaly, hypotonia, a tented appearance of the upper lip, dysarthria, intellectual disability, hypotonia, respiratory insufficiency, or a combination thereof.

[0296] hi some embodiments, tire methods or uses disclosed herein further comprise evaluating, e.g., measuring, the level of modulatory polynucleotide expression in the subject, e.g., in a cell, tissue, or fluid of the subject. In some embodiments, the methods or uses disclosed herein further comprise evaluating, e.g., measuring, the level of DMPK expression, e.g.. DMPK gene, DMPK mRNA, and / or DMPK protein expression, in the subject, e.g., in a cell, tissue, or fluid of the subject. In some embodiments, the level of DMPK protein is measured by an ELISA, a Western blot, or an immunohistochemistry assay. In some embodiments, the methods or uses disclosed herein further comprise evaluating, e.g., measuring, the level of normal mRNA splicing in the subject, e.g., in a cell, tissue, or fluid of the subject. In some embodiments, the mRNA splicing is DMPK mRNA splicing.

[0297] In some embodiments, evaluating the subject’s level of modulatory polynucleotide expression, level of DMPK expression, and / or level of normal mRNA splicing (e.g., DMPK mRNA splicing) is performed prior to and / or subsequent to administration of the pharmaceutical composition or AAV particle, optionally wherein the subject’s level of modulatory polynucleotide expression, level of DMPK expression, and / or level of normal mRNA splicing (e.g., DMPK mRNA splicing) prior to administration is compared to the subject’s level of modulatory polynucleotide expression, level of DMPK expression, and / or level of normal mRNA splicing (e.g., DMPK mRNA splicing) subsequent to administration. In some embodiments, the level of modulatory polynucleotide expression, level of DMPK expression, and / or level of normal mRNA splicing (e.g., DMPK mRNA splicing) is evaluated in a muscle, heart, and / or brain cell or tissue. In some embodiments, the level of modulatory polynucleotide expression, level of DMPK expression, and / or level of nonnal mRNA splicing (e.g.. DMPK mRNA splicing) is evaluated in a muscle cell or tissue. In some embodiments, the subject’s level of mutated DMPK mRNA expression subsequent to administration is decreased relative to the subject’s level of mutated DMPK mRNA expression prior to administration. In some embodiments, the subject’s level of mutated DMPK protein expression subsequent to administration is decreased relative to the subject’s level of mutated DMPK protein expression prior to administration.

[0298] In some embodiments, the administration of the effective amount of a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding a modulatory polynucleotide may be a treatment that results in: (i) an increase in the number and / or level of viral genomes (VG) per cell in a muscle cell or tissue of the subject relative to the number and / or level of VG per cell in a non-muscle cell or tissue of the subject; (ii) a decrease in mutated DMPK mRNA expression in a muscle cell or tissue of the subject relative to baseline and / or relative to mutated DMPK mRNA expression in a muscle cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle; and / or (iii) an increase in normal mRNA splicing, optionally DMPK mRNA splicing, in a muscle cell or tissue of the subject relative to baseline and / or relative to normal mRNA splicing, optionally DMPK mRNA splicing, in a muscle cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle. In some embodiments, the muscle cell or tissue is cardiac, smooth, and / or skeletal muscle cell or tissue.

[0299] In some embodiments, the administration of the effective amount of a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding a modulatory polynucleotide may be a treatment that results in an increase in: (i) an increase in the number and / or level of viral genomes (VG) per cell in a CNS tissue (e.g.. the caudate, cerebellum (e g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region), of the subject relative to the number and / or level of VG per cell in a peripheral tissue of the subject; and / or (ii) a decrease in mutated DMPK mRNA expression in a cell or tissue of the CNS (e.g., the caudate, cerebellum (e.g., molecularlayer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region) of the subject relative to baseline and / or relative to mutated DMPK mRNA expression in a CNS cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle: and / or (iii) an increase in normal mRNA splicing, optionally DMPK mRNA splicing, in a cell or tissue of the CNS (e.g., the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region)) of the subject relative to baseline and / or relative to normal mRNA splicing, optionally DMPK mRNA splicing, in a CNS cell or tissue of an individual with a DMPK- related disorder who has not been administered the pharmaceutical composition or AAV particle.

[0300] In some embodiments, at least one additional agent for treating a DMPK-related disorder (e.g., DM1) may be administered together with the effective amount of a pharmaceutical composition or AAV particle disclosed herein.

[0301] In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce, and / or modify their metabolism, and / or modify their distribution within the body.

[0302] In certain embodiments, the pharmaceutical compositions described herein are used as research tools, particularly in in vitro investigations using human cell lines such as HEK293T and in vivo testing in nonhuman primates which will occur prior to human clinical trials.

[0303] The present disclosure provides a method for treating a disease, disorder, and / or condition in a mammalian subject, including a human subject, comprising administering to the subject any of the AAV particles that produces a modulatory polynucleotide described herein or administering to the subject a composition or formulation comprising said AAV particle.

[0304] Delivery of a construct comprising a modulatory’ polynucleotide-encoding sequence may alleviate or reduce symptoms that result from abnormal level and / or function of a gene product (e.g., an absence or defect in a protein) in a subject in need thereof or that otherwise confers a benefit to a CNS disorder in a subject in need thereof.Neurological and neuromuscular diseases

[0305] In some embodiments, AAV particles of the present disclosure, through delivery of a modulatory polynucleotide, can reduce or eliminate DMPK expression in the CNS. In some embodiments. AAV particles of the present disclosure can reduce or eliminate the level of mutant DMPK mRNA and / or mutant DMPK protein in the CNS.

[0306] In some embodiments, AAV particles of the present disclosure may be used to treat neurological and / or neuromuscular disorders. In some embodiments, the neurological or neuromuscular disorder is DMPK-related disorder. In some embodiments, the delivery of the AAV particles may halt or slow the disease progression of DMPK-related disorders as assessed using a known analysis method and comparator group for DMPK-related disorders. As a non-limiting example, the delivery of the AAVparticles may halt or slow progression of DM1.

[0307] In some embodiments, the AAV particle may decrease the amount of DMPK mRNA or protein in a tissue effective to reduce one or more symptoms of a DMPK-related disorder, optionally caused by trinucleotide repeat (and thus polyQ) expansion.

[0308] hi some embodiments, tire AAV particles and AAV viral genomes described herein, upon administration to subject or introduction to a target cell, decrease DMPK activity compared to baseline DMPK activity. In the case of subjects or target cells with aberrant DMPK activity (e.g.. formation of foci that trap proteins involved in mRNA splicing), as in the case of certain subjects having a DMPK- related disorder or cells or tissues harboring one or more mutation in a DMPK gene, the AAV particles and AAV viral genomes described herein deliver a modulatory polynucleotide to inhibit, decrease, or eliminate activity of mutated DMPK (e.g., inhibit, decrease, or eliminate foci), relative to DMPK activity levels in subjects, tissues, and cells not afflicted with a DMPK-related disorder or not harboring a DMPK gene mutation.Therapeutic applications

[0309] The present disclosure additionally provides methods for treating non-infectious diseases and / or disorders in a mammalian subject, including a human subject, comprising administering to the subject any of the AAV particles or pharmaceutical compositions described herein. In some embodiments, non-infectious diseases and / or disorders treated according to the methods described herein include, but are not limited to, myotonic dystrophy type 1 (DM1).

[0310] The present disclosure provides a method for administering to a subject in need thereof, including a human subject, a therapeutically effective amount of the AAV particles of the invention to slow, stop or reverse disease progression. As a non-limiting example, disease progression may be measured by tests or diagnostic tool(s) known to those skilled in the art. As another non-limiting example, disease progression may be measured by change in the pathological features of the brain, CSF, muscle, heart, or other tissues of the subject.VI. Delivery of AAV ParticlesDelivery to Cells

[0311] In some aspects, the present disclosure provides a method of delivering to a cell or tissue any of the above-described AAV particles, comprising contacting the cell or tissue with said AAV particle or contacting the cell or tissue with a formulation comprising said AAV particle, or contacting the cell or tissue with any of the described compositions, including pharmaceutical compositions. The method of delivering the AAV particle to a cell or tissue can be accomplished in vitro, ex vivo, or in vivo.

[0312] In some embodiments, the AAV particles are delivered to a cell, tissue, or region of the CNS. In some embodiments, the AAV particles are delivered to a cell or tissue of the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamcn, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region).Delivery to Subjects

[0313] In some aspects, the present disclosure additionally provides a method of delivering to a subject, including a mammalian subject, any of the above-described AAV particles comprising administering to the subject said AAV particle, or administering to the subject a formulation comprising said AAV particle, or administering to the subject any of the described compositions, including pharmaceutical compositions.|0314| In some embodiments, the AAV particles may be delivered to bypass anatomical blockages (e.g., the blood brain barrier).

[0315] In some embodiments, the AAV particles may be formulated and delivered to a subject by a route which increases the speed of drug effect as compared to oral delivery.

[0316] In some embodiments, the AAV particles may be delivered using intrathecal infusion.

[0317] In some embodiments, a subject may be administered the AAV particles described herein using a bolus infusion.

[0318] In some embodiments, the AAV particles may be delivered in a continuous and / or bolus infusion. Each site of delivery may use a different dosing regimen or the same dosing regimen may be used for each site of delivery’. As a non-limiting example, the sites of delivery may be in the cervical and the lumbar region. As another non-limiting example, the sites of delivery may be in the cervical region. As another non-limiting example, the sites of delivery’ may be in the lumbar region.

[0319] In some embodiments, the AAV particles may be delivered to a subject via a single route of administration.

[0320] In some embodiments, the AAV particles may be delivered to a subject via a multi-site route of administration. For example, a subject may be administered the AAV particles at 2, 3, 4, 5, or more than 5 sites.

[0321] hi some embodiments, a subject may be administered the AAV particles described herein using sustained delivery over a period of minutes, hours, or days. The infusion rate may be changed depending on the subject, distribution, formulation, or another delivery parameter known to those in the art.

[0322] In some embodiments, if continuous delivery (continuous infusion) of the AAV particles is used, the continuous infusion may be for 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours. 7 hours. 8 hours, 9 hours, 10 hours. 11 hours, 12 hours, 13 hours. 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours. 22 hours, 23 hours, 24 hours, or more than 24 hours.

[0323] In some embodiments, the intracranial pressure may be evaluated prior to administration. The route, volume. AAV particle concentration, infusion duration and / or vector titer may be optimized based on the intracranial pressure of a subject.

[0324] In some embodiments, the AAV particles may be delivered by systemic delivery. In some embodiments, the systemic delivery’ may be by intravascular administration. In some embodiments, the systemic delivery may be by intravenous administration.

[0325] In some embodiments, the AAV particles may be delivered by injection into the CSF pathway. Non-limiting examples of delivery to the CSF pathway include intrathecal and intracerebroventricular administration.

[0326] In some embodiments, an AAV particle is administered intravenously. In some embodiments, an AAV particle is administered intracerebroventricularly .

[0327] In some embodiments, the AAV particle may be delivered by direct (intraparenchymal) injection into the substance of an organ, e.g., one or more regions of the brain.

[0328] In some embodiments, the AAV particles may be delivered by subpial injection into the spinal cord. For example, subjects may be placed into a spinal immobilization apparatus. A dorsal laminectomy may be performed to expose the spinal cord. Guiding tubes and XYZ manipulators may be used to assist catheter placement. Subpial catheters may be placed into the subpial space by advancing the catheter from the guiding tube and AAV particles may be injected through the catheter (Miyanohara et al.. Mol Ther Methods Clin Dev. 2016; 3: 16046). In some cases, the AAV particles may be injected into the cervical subpial space. In some cases, the AAV particles may be injected into the thoracic subpial space.

[0329] In some embodiments, the AAV particles may be delivered by direct injection to the CNS of a subject. In some embodiments, direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intra-cistema magna injection, or any combination thereof. In some embodiments, direct injection to the CNS of a subject comprises convection enhanced delivery (CED). In some embodiments, administration comprises peripheral injection. In some embodiments, peripheral injection is intravenous injection.

[0330] In some embodiments, the AAV particles may be delivered to a subject in order to increase a modulatory polynucleotide level in the muscle, heart, and / or brain as compared to a baseline level in the subject.

[0331] In some embodiments, the AAV particles may be delivered to a subject in order to increase a modulatory polynucleotide level in a cell or tissue of the CNS (e.g., the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region) as compared to a baseline level in the subject.

[0332] In some embodiments, the AAV particles may be delivered to a subject in order to decrease the number of DMPK mRNA foci as compared to a baseline level in the subject. In some embodiments, the AAV particles may be delivered to a subject in order to decrease the number of trapped splicing proteins as compared to a baseline level in the subject. In some embodiments, the AAV particles may be delivered to a subject in order to increase a level of normal mRNA splicing in the muscle, heart, and / or brain as compared to a baseline level in the subject. In some embodiments, the mRNA splicing is DMPK mRNA splicing.

[0333] In some embodiments, the AAV particles may be delivered to a subject in order to reduce or eliminate a DMPK mRNA and / or protein level in the muscle, heart, and / or brain as compared to a baseline level in the subject.

[0334] In some embodiments, the AAV particles are delivered by transducing cells in these regions. Transduction may also be referred to as the number of cells that are positive for modulatory polynucleotide.

[0335] In some embodiments, the AAV particles may be delivered to a subject in order to establish desired distribution of modulatory polynucleotides, e.g., throughout muscle tissue. In some embodiments, the increased expression of modulatory polynucleotide may lead to a reduction in at least one symptom of a DMPK-related disorder in a subject (e.g.. DM1), the at least one symptom includes cataracts, myotonia, muscle weakness and wasting, cardiac conduction abnormalities, a myopathic face, learning difficulties, psychosocial problems including depression and / or anxiety, slurred speech, decreased fetal movement in the uterus, polyhydramnios, clubfoot, ventriculomegaly, hypotonia, a tented appearance of the upper lip, dysarthria, intellectual disability, hypotonia, respiratory insufficiency, or a combination thereof.Administration

[0336] In some embodiments, the present disclosure provides methods comprising administering viral vectors in accordance with the disclosure to a subject in need thereof. Viral vector pharmaceutical, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any amount and any route of administration effective for treating, or diagnosing a DMPK-related disorder. In some embodiments, the DMPK-related disorder is DM1. In some embodiments, the DM1 is congenital DM1.

[0337] Compositions in accordance with the disclosure may be formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophy tactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific protein employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[0338] In certain embodiments, the desired dosage may be delivered using multiple administrations e.g., two. three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic composition administered in one dose / at onetime / single route / single point of contact, e.g. , single administration event. In some embodiments, a single unit dose is provided as a discrete dosage form (e.g., a tablet, capsule, patch, loaded syringe, vial, etc.). As used herein, a “total daily dose’’ is an amount given or prescribed in 24-hour period. It may be administered as a single unit dose. The viral particles may be formulated in buffer only or in a formulation described herein.

[0339] In some embodiments, a pharmaceutical composition described herein can be formulated into a topical, intranasal, pulmonary, intratracheal, or injectable dosage form. In some embodiments, a pharmaceutical composition described herein can be formulated in a dosage form suitable for intravenous, intracerebroventricular, intraocular, intravitreal. intramuscular, intracardiac, intraperitoneal, and / or subcutaneous administration.

[0340] In some embodiments, the pharmaceutical composition may be formulated for intravenous administration. In some embodiments, the pharmaceutic composition may be formulated for intracerebroventrical administration.

[0341] In some embodiments, an AAV particle described herein is administered via intravenous administration. In some embodiments, an AAV particle described herein is formulated for intravenous administration. In some embodiments, an AAV particle described herein is administered via intracerebroventrical administration. In some embodiments, an AAV particle described herein is formulated for intracerebroventrical administration.

[0342] In some embodiments, a pharmaceutical composition described herein is administered via intravenous administration. In some embodiments, a pharmaceutical composition described herein is formulated for intravenous administration. In some embodiments, a pharmaceutical composition described herein is administered via intracerebroventrical administration. In some embodiments, a pharmaceutical composition described herein is formulated for intracerebroventrical administration.

[0343] In some embodiments, an AAV particle or pharmaceutical composition described herein is administered via intravenous administration.

[0344] In some embodiments, delivery' of the AAV particles described herein results in minimal serious adverse events (SAEs) as a result of die delivery of the AAV particles.Combinations

[0345] The AAV particles may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. The phrase “in combination with.” is not intended to require that the agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and / or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that mayimprove their bioavailability, reduce and / or modify their metabolism, and / or modify their distribution within the body.

[0346] The therapeutic agents may be approved by the US Food and Drug Administration or may be in clinical trial or at the preclinical research stage. The therapeutic agents may utilize any therapeutic modality known in the art, with non-limiting examples including gene silencing or interference (e.g., miRNA, siRNA, RNAi, shRNA). gene editing (e.g., TALEN, CRISPR / Cas9 systems, zinc finger nucleases), and gene, protein, or enzyme replacement.

[0347] In some embodiments, an AAV particle described herein, or a pharmaceutical composition comprising the AAV particle, may be administered in combination with at least one additional therapeutic agent and / or therapy. In some embodiments, the at least one additional therapeutic agent and / or therapy comprises an agent and / or therapy for treating a DMPK-related disorder, e.g.. DM1.

[0348] In some embodiments, the at least one additional therapeutic agent and / or therapy comprises an anti-diabetic drug, an anti-myotonic drug (e.g.. mexiletine). a non-steroidal anti-inflammatory drug, or a combination thereof.

[0349] In some embodiments, the at least one additional therapeutic agent and / or therapy comprises an immunosuppressant. In some embodiments, the immunosuppressant may be administered to the subject prior to administration of an AAV particle or pharmaceutical composition described herein. In some embodiments, the immunosuppressant may be administered to the subject simultaneously with administration of an AAV particle or pharmaceutical composition described herein. In some embodiments, the immunosuppressant may be administered to the subject after administration of an AAV particle or pharmaceutical composition described herein. In some embodiments, the AAV particle or pharmaceutical composition is administered to a subject who is receiving or has received an immunosuppressant. In some embodiments, the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and / or dexamethasone), rapamycin, mycophcnolatc mofctil, tacrolimus, rituximab, cculizumab hydroxychloroquine, and / or alemtuzumab, hydroxyurea, fludarabine, and / or busulfan. In some embodiments, the corticosteroid comprises prednisone, prednisolone, methylprednisolone, and / or dexamethasone. In some embodiments, the immunosuppressant comprises adrenocorticotropic hormone.Measurement of Expression

[0350] In some embodiments, expression of the modulatory polynucleotide as described herein and / or expression of DMPK mRNA and / or expression of DMPK protein may be determined using various methods known in the art such as. but not limited to hybridization to a probe (e.g.. by detecting a labeled probe that is complementary to the DMPK mRNA), a nuclease protection assay. Northern blot, sequencing the mRNA or a complement thereof, immunochcmistrv (e.g., IHC), enzyme-linked immunosorbent assay (ELISA), affinity ELISA, ELISPOT. flow cytometry, immunocytology, surface plasmon resonance analysis, kinetic exclusion assay, liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography (HPLC). BCA assay, immunoelectrophoresis, Western blot,SDS-PAGE. protein immunoprecipitation, PCR, and / or in situ hybridization (ISH). In some embodiments, modulatory polynucleotides delivered using different AAV capsids may have different expression levels in dorsal root ganglion (DRG).

[0351] In certain embodiments. DMPK protein is detectable by an enzyme-linked immunosorbent assay (ELISA).

[0352] In certain embodiments. DMPK protein is detectable by an immunohistochemistry assay.

[0353] In certain embodiments. DMPK protein is detectable by Western blot.

[0354] In certain embodiments. DMPK mRNA is detected by Northern blot.

[0355] In certain embodiments. DMPK mRNA is detected by a nuclease protection assay.

[0356] In certain embodiments. DMPK mRNA is detected by hybridization of the mRNA to a probe(e.g., in situ hybridization).

[0357] In certain embodiments. DMPK mRNA is detected by RT-PCR.

[0358] In certain embodiments, expression of a modulatory polynucleotide as described herein and / or expression of DMPK mRNA or protein and / or level of normal mRNA splicing (e.g., DMPK mRNA splicing) is measured in a cell or tissue of a subject who is receiving or has received an AAV particle described herein. In certain embodiments, expression of the modulatory7polynucleotide and / or DMPK mRNA or protein and / or level of normal mRNA splicing (e.g., DMPK mRNA splicing) is measured in a brain cell or tissue. In certain embodiments, expression of the modulatory7polynucleotide and / or DMPK mRNA or protein and / or level of normal mRNA splicing (e.g., DMPK mRNA splicing) is measured in a peripheral cell or tissue. In certain embodiments, expression of t...

Claims

CLAIMS1. An adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory’ poly nucleotide comprises an RNAi agent targeting DMPK mRNA, and(ii) an AAV capsid variant comprising in loop VIII:(a) the amino acid sequence of SEQ ID NO: 4,(b) an amino acid sequence comprising at least 4 or at least 5 consecutive ammo acids from the amino acid sequence of SEQ ID NO: 4; or(c) an amino acid sequence comprising one. Ivo. or three different amino acids relative to the amino acid sequence of SEQ ID NO: 4.

2. The AAV particle of claim 1. comprising at least 5 consecutive amino acids from the amino acid sequence of SEQ ID NO: 4.

3. The AAV particle of claim 2. wherein the consecutive amino acids comprise the amino acid sequence of NAA.

4. The AAV particle of any one of claims 1-3, wherein the consecutive amino acids comprise the amino acid sequence of NAAQ (SEQ ID NO: 2).

5. The AAV particle of any one of claims 1-4, wherein the consecutive amino acids comprise the amino acid sequence of AQAY (SEQ ID NO: 1).

6. The AAV particle of any one of claims 1-5, wherein the consecutive amino acids comprise the amino acid sequence of NAAQA (SEQ ID NO: 3).

7. The AAV particle of any one of claims 1-6, wherein die amino acid sequence comprises the amino acid sequence of NAAQAY (SEQ ID NO: 4).

8. The AAV particle of any one of claims 1-7, wherein the AAV capsid variant is an AAV5 capsid variant.

9. The AAV particle of any one of claims 1-8, wherein loop VIII is present at amino acids comprising those corresponding to positions 571-592 of the amino acid sequence of SEQ ID NO: 8 or 12.

10. The AAV particle of any one of claims 1-9, wherein the amino acid sequence replaces amino acids corresponding to positions 578, 579, 580, 581, 582, and 583 (e.g., corresponding to positions T578, A579, P580, A581, T582, and G583) of the amino acid sequence of SEQ ID NO: 8.

11. The AAV particle of any one of claims 1-10, wherein the amino acid sequence is present at amino acids corresponding to positions 578. 579, 580, 581, 582, and / or 583 (e.g., comprises amino acids T578. A579, P580, A581, T582, and / or G583) of the amino acid sequence of SEQ ID NO: 8 or 12.

12. The AAV particle of any one of claims 1-11, wherein the amino acid sequence comprises or consists of NAAQAY (SEQ ID NO: 4) and is present at amino acids corresponding to positions 578. 579, 580, 581, 582. and 583 (e.g., comprises amino acids T578. A579, P580, A581, T582, and G583) of the amino acid sequence of SEQ ID NO: 12.

13. An adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and(ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to amino acids 193-724 of the amino acid sequence of SEQ ID NO: 12, wherein the AAV capsid variant comprises:N at an amino acid corresponding to position 578 of the amino acid sequence of SEQ ID NO: 12,A at an amino acid corresponding to position 580 of the amino acid sequence of SEQ ID NO: 12,Q at an amino acid corresponding to position 581 of the amino acid sequence of SEQ ID NO: 12,A at an amino acid corresponding to position 582 of the amino acid sequence of SEQ ID NO: 12, andY at an amino acid corresponding to position 583 of the amino acid sequence of SEQ ID NO: 12.

14. An adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and(ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to amino acids 137-724 of the amino acid sequence of SEQ ID NO: 12, wherein the AAV capsid variant comprises:N at an amino acid corresponding to position 578 of the amino acid sequence of SEQ ID NO: 12,A at an amino acid corresponding to position 580 of the amino acid sequence of SEQ ID NO: 12.Q at an amino acid corresponding to position 581 of the amino acid sequence of SEQ ID NO: 12.A at an amino acid corresponding to position 582 of the amino acid sequence of SEQ ID NO: 12, andY at an amino acid corresponding to position 583 of the amino acid sequence of SEQ ID NO: 12.

15. An adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated dystrophia myotonica protein kinase (DMPK) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting DMPK mRNA, and(ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, wherein the AAV capsid variant comprises:N at an amino acid corresponding to position 578 of the amino acid sequence of SEQ ID NO: 12,A at an amino acid corresponding to position 580 of the amino acid sequence of SEQ ID NO: 12,Q at an amino acid corresponding to position 581 of the amino acid sequence of SEQ ID NO: 12,A at an amino acid corresponding to position 582 of the amino acid sequence of SEQ ID NO: 12, andY at an amino acid corresponding to position 583 of the amino acid sequence of SEQ ID NO: 12.

16. The AAV particle of any one of claims 13-15, wherein the AAV capsid variant comprises A at a position corresponding to position 579 of the amino acid sequence of SEQ ID NO: 12.

17. The AAV particle of any one of claims 13-16, wherein the AAV capsid variant comprises:(i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12;(ii) an amino acid sequence that is at least 99% identical to positions 193-724 of the amino acid sequence of SEQ ID NO: 12; and / or(iii) an amino acid sequence that is at least 99% identical to positions 137-724 of the amino acid sequence of SEQ ID NO: 12.

18. The AAV particle of any one of claims 13-17. wherein the AAV capsid variant comprises the amino acid sequence of ATNNQSSTNAAQAYT (SEQ ID NO: 6) at amino acids corresponding to positions 570-584 of the amino acid sequence of SEQ ID NO: 12, optionally wherein the amino acid sequence of ATNNQSSTNAAQAYT (SEQ ID NO: 6) is present in loop VIII, wherein loop VIII comprises amino acids 571-592 of the amino acid sequence of SEQ ID NO: 8 or 12.

19. The AAV particle of any one of claims 1-18, wherein the AAV capsid variant has:(i) an increased tropism for a muscle cell or tissue, relative to tire muscle cell or tissue tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and / or(ii) a decreased tropism for a liver cell or tissue, relative to the liver cell or tissue tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

20. The AAV particle of any one of claims 1-19, wherein the AAV capsid variant:(i) transduces a muscle cell or tissue, optionally wherein the level of transduction is at least 2, at least 5, at least 10. at least 15, at least 20, or at least 25-fold greater as compared to transduction of a muscle cell or tissue by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO:

9. e.g., when measured by an assay, e.g., an immunohistochemistry assay or a qPCR assay;(ii) delivers an increased level of the modulatory polynucleotide to a muscle cell or tissue, optionally wherein the level of the modulatory polynucleotide is increased by at least 5, at least 10. at least 12, at least 15, at least 20, at least 21, or at least 25-fold, as compared to the level of modulatory polynucleotide delivered to a muscle cell or tissue by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g.. when measured by an assay; and / or(iii) delivers an increased level of viral genomes to a muscle cell or tissue, optionally wherein the level of viral genomes is increased by at least 2, at least 2.5, at least 5. at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8. at least 8.5, at least 9, at least 9.5, at least 10, at least 10.5, at least 11, at least 11.5, at least 12, at least 12.5, at least 13, at least 13.5, at least 13.8, or at least 14-fold, as compared to the level of viral genomes delivered to a muscle cell or tissue by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay.

21. The AAV particle of claim 19 or claim 20, wherein the muscle cell or tissue comprises a cardiac muscle, a smooth muscle, or a skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis).

22. The AAV particle of any one of claims 1-21, wherein the AAV capsid variant has:(i) an increased tropism for a central nervous system (CNS) cell or tissue, e.g., a brain cell, brain tissue, spinal cord cell, or spinal cord tissue, relative to the CNS cell or tissue tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and / or(ii) a decreased tropism for a liver cell or tissue, relative to the liver cell or tissue tropism of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9.

23. The AAV particle of any one of claims 1-22, wherein the AAV capsid variant:(i) transduces a brain cell or tissue of the CNS, optionally wherein the level of transduction is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 24, at least 25, at least 29, or at least 30-fold greater as compared to transduction of a brain cell or tissue of the CNS by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., an immunohistochemistry assay or a qPCR assay;(ii) is enriched at least 4, at least 4.6, at least 10, at least 20, at least 25, at least 28. at least 30, at least 40, at least 50, at least 60. at least 70, at least 80, at least 81. at least 80, at least 100, at least 101, or at least 110-fold, in the CNS as compared to enrichment in the CNS of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9;(iii) delivers an increased level of the modulatory polynucleotide to a brain cell or tissue of the CNS. optionally wherein the level of the modulatory polynucleotide is increased by at least 3, at least 4, at least 5. at least 6, at least 7, at least 8. at least 9. at least 10, at least 11. at least 12. at least 13, or at least 14-fold, as compared to the level of modulatory polynucleotide delivered to a brain cell or tissue of the CNS by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO:

9. e.g.. when measured by an assay, e.g.. a qRT-PCR or a qPCR assay; and / or(iv) delivers an increased level of viral genomes to a brain cell or tissue of the CNS, optionally wherein the level of viral genomes is increased by at least 3, at least 4, at least 5. at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14. at least 15, at least 20, at least 24, at least 25, at least 29, or at least 30-fold, as compared to the level of viral genomes delivered to brain cell or tissue of the CNS by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay.

24. The AAV particle of claim 22 or claim 23, wherein the CNS comprises caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamcn, thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region).

25. The AAV particle of any one of claims 1-24, wherein the AAV capsid variant comprises:(i) the amino acid sequence of SEQ ID NO: 12;(ii) the amino acid sequence according to positions 193-724 of the amino acid sequence of SEQ ID NO: 12; and / or(iii) the amino acid sequence according to positions 137-724 of the amino acid sequence of SEQ ID NO: 12.

26. The AAV particle of any one of claims 1-25. wherein the modulatory polynucleotide comprises a molecular scaffold, wherein the molecular scaffold comprises:(i) a 5’ flanking region comprising the nucleotide sequence of any one of SEQ ID NOs: 13-16, or a nucleotide sequence that is at least 95% identical thereto;(ii) a loop region comprising the nucleotide sequence of any one of SEQ ID NOs: 17-21, or a nucleotide sequence that is at least 95% identical thereto; and(iii) a 3‘ flanking region comprising the nucleotide sequence of any one of SEQ ID NOs: 22-27, or a nucleotide sequence that is at least 95% identical thereto.

27. The AAV particle of claim 26, wherein:(i) the 5' flanking region comprises the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 15, or a nucleotide sequence that is at least 95% identical thereto;(ii) the loop region comprises the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO:

21. or a nucleotide sequence that is at least 95% identical thereto; and(iii) the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25, or a nucleotide sequence that is at least 95% identical thereto.

28. The AAV particle of claim Tl, wherein the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% identical thereto.

29. The AAV particle of claim Tl, wherein the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence that is at least 95% identical thereto, the loop region of the comprises the nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence that is at least 95% identical thereto.

30. The AAV particle of claim Tl, wherein the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence that is at least 95% identical thereto.

31. The AAV particle of claim 27, wherein the 5 ’ flanking region comprises the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence that is at least 95%identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% identical thereto.

32. The AAV particle of any one of claims 1-31, wherein tire modulatory polynucleotide comprises siRNA or shRNA.

33. The AAV particle of any one of claims 1-32, wherein the modulatory polynucleotide further comprises a passenger strand and a guide strand.

34. The AAV particle of claim 33, wherein the guide strand binds to and reduces or eliminates expression of one or more DMPK mRNA transcripts (e.g.. one or more mutated DMPK mRNA transcripts).

35. The AAV particle of claim 33 or claim 34, wherein the modulatory polynucleotide comprises, from 5’ to 3’: the 5’ flanking region, the passenger strand, the loop region, the guide strand, and the 3’ flanking region.

36. The AAV particle of claim 33 or claim 34. wherein the modulatory polynucleotide comprises, from 5’ to 3’: the 5’ flanking region, the guide strand, the loop region, the passenger strand, and the 3’ flanking region.

37. The AAV particle of any one of claims 33-36, wherein the passenger strand is 15-30 nucleotides in length.

38. The AAV particle of any one of claims 33-37, wherein the guide strand is 15-30 nucleotides in length.

39. The AAV particle of any one of claims 33-38, wherein the guide strand is 21-25 nucleotides in length and / or the passenger strand is 21-25 nucleotides in length.

40. The AAV particle of any one of claims 33-39, wherein the passenger strand is at least 70%, at least 80%, at least 90%, or at least 95%, or is 100%, complementary to the guide strand.

41. The AAV particle of any one of claims 33-40. wherein the one or more DMPK mRNA transcripts comprises the nucleotide sequence of SEQ ID NO: 28 or SEQ ID NO: 29 or a trinucleotide repeat expansion thereof.

42. The AAV particle of any one of claims 1-41, wherein the viral genome comprises a promoter operably linked to the nucleotide sequence encoding the modulatory polynucleotide.

43. The AAV particle of any one of claims 1-42, wherein the viral genome further comprises an inverted terminal repeat (ITR) sequence.

44. The AAV particle of any one of claims 1-43, wherein the viral genome comprises an ITR sequence positioned 5’ relative to the nucleotide sequence encoding the modulatory polynucleotide.

45. The AAV particle of any one of claims 1-44, wherein the viral genome comprises an ITR sequence positioned 3’ relative to the nucleotide sequence encoding the modulatory polynucleotide.

46. The AAV particle of any one of claims 1-45, wherein the viral genome comprises an ITR sequence positioned 5 ’ relative to the nucleotide sequence encoding the modulatory polynucleotide, and an ITR sequence positioned 3 ’ relative to the nucleotide sequence encoding the modulatory polynucleotide.

47. A cell comprising the AAV particle of any one of claims 1 -46, optionally wherein the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g.. an S1 cell), or a bacterial cell.

48. A method of making the AAV particle of any one of claims 1-46, wherein the method comprises:(i) providing a cell comprising the viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA and a nucleic acid encoding the AAV capsid variant; and(ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV capsid variant; thereby making the AAV particle.

49. The method of claim 48, wherein the viral genome comprises:(i) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising die nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% identical thereto;(ii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising die nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking regioncomprising the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence that is at least 95% identical thereto;(iii) a 5‘ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence that is at least 95% thereto; or(iv) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising die nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% thereto; and wherein the AAV capsid variant comprises(a) the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical (e.g., at least 90%. at least 91%, at least 92%, at least 93%. at least 94%, at least 95%. at least 96%, at least 97%, at least 98%. or at least 99% identical) thereto;(b) the amino acid sequence according to positions 137-724 of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; and / or(c) the amino acid sequence according to positions 193-724 of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical (e.g., at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

50. The method of claim 48, wherein the viral genome comprises:(i) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising tire nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% identical thereto;(ii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising die nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence that is at least 95% identical thereto;(iii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence that is at least 95% thereto; or(iv) a 5‘ flanking region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising die nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is at least 95% thereto; and wherein the AAV capsid variant comprises SEQ ID NO: 12, the amino acid sequence according to positions 137-724 of the amino acid sequence of SEQ ID NO: 12, and / or the amino acid sequence according to positions 193-724 of the amino acid sequence of SEQ ID NO: 12.

51. The method of claim 48, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 12.

52. The method of any one of claims 48-51, further comprising, prior to step (i). introducing a nucleic acid molecule comprising the viral genome into the cell.

53. The method of any one of claims 48-52, further comprising, prior to step (i). introducing the nucleic acid encoding the AAV capsid variant into the cell.

54. The method of any one of claims 48-53, wherein the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (c.g., an S19 cell), or a bacterial cell.

55. A pharmaceutical composition comprising the AAV particle of any one of claims 1-46 and a pharmaceutically acceptable excipient.

56. A method of delivering an AAV particle encoding a modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA to a cell, comprising administering an effective amount of the pharmaceutical composition of claim 55 or the AAV particle of any one of claims 1-46.

57. The method of claim 56, wherein the cell is in a subject, optionally wherein the subject has, has been diagnosed with having, or is at risk of having a DMPK-related disorder.

58. The method of claim 57, wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1).

59. A method of treating a DMPK-related disorder in a subject, comprising administering to the subject an effective amount of tire pharmaceutical composition of claim 55 or the AAV particle of any one of claims 1-46.

60. The method of claim 59, wherein the subject has, has been diagnosed with having, or is at risk of having the DMPK-related disorder.

61. The method of claim 59 or claim 60, wherein the subject has one or more mutations in the DMPK gene.

62. The method of claim 61, wherein the one or more mutations in the DMPK gene comprises a trinucleotide repeat expansion.

63. The method of claim 62, wherein the trinucleotide repeat expansion in the DMPK gene is or comprises 50 or more CTG repeats (e.g., SEQ ID NO: 35).

64. The method of any one of claims 59-63, wherein the treating results in prevention of progression of the DMPK-related disorder in the subject.

65. The method of any one of claims 59-64, wherein the treating results in amelioration of at least one symptom of the DMPK-related disorder in the subject.

66. The method of claim 65, wherein the at least one symptom comprises cataracts, myotonia, muscle weakness and wasting, cardiac conduction abnormalities, a myopathic face, learning difficulties, psychosocial problems including depression and / or anxiety, slurred speech, decreased fetal movement in the uterus, polyhydramnios, clubfoot, ventriculomegaly, hypotonia, a tented appearance of the upper lip, dysarthria, intellectual disability, hypotonia, respiratory insufficiency, or a combination thereof.

67. The method of any one of claims 59-66. wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1).

68. A method of treating myotonic dystrophy type 1 (DM1) in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 55 or the AAV particle of any one of claims 1-46.

69. The method of claim 68, wherein the subject has, has been diagnosed with having, or is at risk of having DM1.

70. The method of any one of claims 57-69. wherein the subject is a human.

71. The method of any one of claims 57-70. wherein the pharmaceutical composition or AAV particle is delivered to a cell, tissue, or region of muscle.

72. The method of claim 71, wherein the muscle is one or more of cardiac muscle, smooth muscle, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, (e.g., vastus lateralis)).

73. The method of any one of claims 57-70, wherein the pharmaceutical composition or AAV particle is delivered to a cell, tissue, or region of the central nervous system (CNS), e.g., the brain.

74. The method of claim 73, wherein the pharmaceutical composition or AAV particle is delivered to the caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g.. cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region).

75. The method of claim 73 or claim 74, wherein the subject has, has been diagnosed with having, or is at risk of having congenital myotonic dystrophy type 1.

76. The method of any one of claims 57-75, wherein the AAV particle or the pharmaceutical composition is delivered via intravenous administration.

77. The method of any one of claims 57-70 or any one of claims 73-75. wherein the AAV particle or the pharmaceutical composition is delivered via intracerebroventricular administration.

78. The method of any one of claims 57-77. further comprising evaluating, e.g.. measuring, the level of modulatory polynucleotide expression, the level of mutated DMPK mRNA expression, the level of mutated DMPK protein expression, and / or the level of normal mRNA splicing, optionally DMPK mRNA splicing, in the subject, e.g., in a cell, tissue, or fluid, of the subject.

79. The method of claim 78. wherein evaluating the subject’s level of modulatory polynucleotide expression, the subject’s level of mutated DMPK mRNA expression, the subject’s level of mutatedDMPK protein expression, and / or the subject's level of normal mRNA splicing, optionally DMPK mRNA splicing, is performed prior to and / or subsequent to administration of the pharmaceutical composition or AAV particle, optionally wherein the subject’s level of modulatory polynucleotide expression, the subject’s level of mutated DMPK mRNA expression, the subject’s level of mutated DMPK protein expression, and / or the subject's level of normal mRNA splicing, optionally DMPK mRNA splicing, prior to administration is compared to the subject's level of modulatory polynucleotide expression, the subject’s level of mutated DMPK mRNA expression, the subject’s level of mutated DMPK protein expression, and / or the subject’s level of normal mRNA splicing, optionally DMPK mRNA splicing, subsequent to administration.

80. The method of claim 78 or claim 79, wherein the cell or tissue of the subject is a muscle cell or a muscle tissue (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle (e.g., vastus lateralis))).

81. The method of claim 78 or claim 79. wherein the cell or tissue of the subject is a cell or tissue of the CNS (e.g., caudate, cerebellum (e.g.. molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)).

82. The method of any one of claims 57-81, wherein the subject’s level of mutated DMPK mRNA expression subsequent to administration of the pharmaceutical composition or AAV particle is decreased relative to the subject’s level of mutated DMPK mRNA expression prior to administration of the pharmaceutical composition or AAV particle, and / or wherein the subject’s level of mutated DMPK protein expression subsequent to administration of the pharmaceutical composition or AAV particle is decreased relative to the subject’s level of mutated DMPK protein expression prior to administration of the pharmaceutical composition or AAV particle.

83. The method of any one of claims 57-72. wherein administering the pharmaceutical composition or AAV particle to the subject results in:(i) an increase in the number and / or level of viral genomes (VG) per cell in a muscle cell or a muscle tissue (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis)) of the subject relative to the number and / or level of VG per cell in a non-muscle cell or tissue of the subject;(ii) a decrease in mutated DMPK mRNA expression in a muscle cell or a muscle tissue (e.g.. cardiac muscle, smooth muscle, and / or skeletal muscle (e.g.. diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis)) of the subject relative to baseline and / orrelative to mutated DMPK mRNA expression in a muscle cell or tissue of an individual with a DMPK- related disorder who has not been administered the pharmaceutical composition or AAV particle; and / or(iii) an increase in normal mRNA splicing, optionally DMPK mRNA splicing, in a muscle cell or a muscle tissue (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle (e.g., diaphragm, intercostal muscle, gastrocnemius, and / or quadriceps muscle, e.g., vastus lateralis)) of the subject relative to baseline and / or relative to normal mRNA splicing, optionally DMPK mRNA splicing, in a muscle cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle.

84. The method of any one of claims 57-70 or any one of claims 73-75. wherein administering the pharmaceutical composition or AAV particle to the subject results in:(i) an increase in the number and / or level of viral genomes (VG) per cell in a CNS tissue (e.g., caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen. and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)) of the subject relative to the number and / or level of VG per cell in a peripheral tissue of the subject;(ii) a decrease in mutated DMPK mRNA expression in a cell or a tissue of the CNS (e.g.. caudate, cerebellum (e.g.. molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)) of the subject relative to baseline and / or relative to mutated DMPK mRNA expression in a CNS cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle; and / or(iii) an increase in normal mRNA splicing, optionally DMPK mRNA splicing, in a cell or tissue of the CNS (e.g., caudate, cerebellum (e.g., molecular layer and / or granule layer), motor cortex, putamen, and / or thalamus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)) of the subject relative to baseline and / or relative to normal mRNA splicing, optionally DMPK mRNA splicing, in a CNS cell or tissue of an individual with a DMPK-related disorder who has not been administered the pharmaceutical composition or AAV particle.

85. The method of claim 84, wherein the subject has, has been diagnosed with having, or is at risk of having congenital myotonic dystrophy type 1.

86. The method of any one of claims 57-85. further comprising administering to the subject at least one additional agent and / or therapy.

87. The method of claim 86. wherein the at least one additional agent and / or therapy comprises an agent and / or therapy for treating the DMPK-related disorder, optionally wherein the at least oneadditional agent and / or therapy comprises an anti-diabetic drug, an anti-myotonic drug (e.g., mexiletine), a non-steroidal anti-inflammatory drug, or a combination thereof.

88. The method of any one of claims 57-87. further comprising administering an immunosuppressant to the subject.

89. The method of claim 88, wherein the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and / or dexamethasone), adrenocorticotropic hormone, rapamycin, mycophenolate mofetil, tacrolimus, rituximab, eculizumab hydroxychloroquine, alemtuzumab. hydroxyurea, fludarabine, and / or busulfan.

90. The pharmaceutical composition of claim 55 or the AAV particle of any one of claims 1-46 for use in a method of treating a disorder according to any one of claims 59-89.

91. The pharmaceutical composition of claim 55 or the AAV particle of any one of claims 1-46 for use in treating a DMPK-related disorder in a subject, optionally wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1).

92. The pharmaceutical composition or AAV particle of claim 91, wherein the subject has. has been diagnosed with having, or is at risk of having the DMPK-related disorder, optionally wherein the DMPK- related disorder is DM1.

93. The pharmaceutical composition or AAV particle for use of claim 91 or claim 92, wherein the DMPK-related disorder is congenital DM1.

94. Use of the pharmaceutical composition of claim 55 or the AAV particle of any one of claims 1- 46 in the manufacture of a medicament for treating a DMPK-related disorder in a subject, optionally wherein the DMPK-related disorder is myotonic dystrophy type 1 (DM1).

95. The use of claim 94, wherein the subject has, has been diagnosed with having, or is at risk of having the DMPK-related disorder, optionally wherein the DMPK-related disorder is DM1.

96. The use of claim 94 or claim 95, wherein the DMPK-related disorder is congenital DM1.

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