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 or at least one symptom thereof.

Description

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

[0001] This application claims the benefit of and priority to US Provisional Application Serial No. 63 / 606,204, filed December 5, 2023, the contents of 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_0104-00304_SL.xml, was created on September 18, 2024, and is 2,001.802 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.FIELD

[0003] Disclosed herein are compositions and methods relating to adeno-associated 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 by mutated 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 (SEQ ID NO: 6430). The number of CTG repeats determines the formof 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 (SEQ ID NO: 6431) are typically asymptomatic.

[0009] Patients with 50-150 CTG repeats (SEQ ID NO: 6432) 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 (SEQ ID NO: 6433) 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.

[0011] Patients with CTG repeat lengths greater than 800 (SEQ ID NO: 6434) 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 (SEQ ID NO: 6435) 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 or at least onesymptom thereof in subjects, and methods for treating DMPK-related disorders or at least one symptom thereof 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 (SEQ ID NO: 6436). Unless otherwise specified, DMPK, DM15, DM1PK, DMK, MDPK. and MT-PK are synonymous terms 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 AAV9 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 skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius). In some embodiments, the muscle comprises smooth muscle. In some embodiments, tire muscle comprises cardiac muscle. In some embodiments, the CNS comprises amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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 delivery to or expression in the liver while retaining high modulatory polynucleotide activity in other areas (e.g., muscle and / or CNS), e.g.. relative to 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 ofDMPK (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 w ildly pc AAV9 capsid and the modulatory polynucleotide sequence. In some embodiments, the muscle comprises skeletal muscle. In some embodiments, the muscle comprises smooth muscle. In some embodiments, the muscle comprises cardiac muscle. In some embodiments, the CNS comprises amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus. oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region).

[0019] In some embodiments, the present disclosure provides an AAV particle comprising an AAV capsid variant and a viral genome, wherein the viral genome comprises 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 wherein the AAV capsid variant comprises an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4681) in loop VIII; wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); and wherein: (i) [Nl] comprises amino acids Xi, X2. X3, and X4, wherein X4is Q, K. E, S, P, R, N, or H; and / or (ii) [N3] comprises amino acids X5. X6, and X7, wherein X5is I, V, T, M, S. N, L, or F. In some embodiments, the AAV capsid variant is an AAV9 capsid variant. In some embodiments, the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to positions 203-736 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 981.

[0020] In some embodiments, (i) Xi is T, S, R, A, I, C, N, K, L, or Q; (ii) X2is N, T, G, V, S, Y, K, I,H, D, or F; and (iii) X7, is T, N, K, D, I, S, P, A, Y, E, V, L, M, R, H, Q, or C. In some embodiments, (i)X6is A, Y, P, N. S, T, G, E, V, W, F, or Q; and (ii) X7is Q, G, N, K, H, R, E, L, P, or M.

[0021] In some embodiments, [Nl] comprises the amino acid sequence of TNTQ (SEQ ID NO:4688). In some embodiments, [N3] comprises the amino acid sequence of IAQ. In some embodiments, [N1]-[N2]-[N3] comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343).

[0022] In some embodiments, (i) [Nl] is present at amino acids corresponding to positions 582-585 of the amino acid sequence of SEQ ID NO: 981; (ii) [N2] is present at amino acids corresponding to positions 586-589 of the amino acid sequence of SEQ ID NO: 981; and (iii) [N3] is present at amino acids corresponding to positions 590-592 of the amino acid sequence of SEQ ID NO: 981.

[0023] In some embodiments, the AAV capsid variant further comprises [N4] comprising amino acids X8, X9, Xio, and Xn, wherein: (i) X8is T, S, N, P, A. or I; (ii) X9is G. N, D. R, V, A. S, or Q; (iii) Xiois W, S. C, R, L. or G; and / or (iv) Xu is V, A. S. I. C, G. D, F, L. or T. In some embodiments, [N4]comprises the amino acid sequence of TGWV (SEQ ID NO: 5066). In some embodiments, [N4] is present at amino acids corresponding to positions 593-596 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, [N1]-[N2]-[N3]-[N4] comprises the amino acid sequence of TNTQDWHRIAQTGWV (SEQ ID NO: 201).

[0024] hi some embodiments, tire present disclosure provides an 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 positions 203- 736 of the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981; D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981; W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981; H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981; R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to positions 138- 736 of the amino acid sequence of SEQ ID NO: 981.

[0025] In some embodiments, the present disclosure provides an 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 positions 138- 736 of the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981; D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981; W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981; H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981; R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981. 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: 981.

[0026] In some embodiments, the present disclosure provides an 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 acidsequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises: T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981; D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981; W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981; H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981; R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981.

[0027] 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: 981; (ii) an amino acid sequence that is at least 99% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981; and / or (iii) an amino acid sequence that is at least 99% identical to positions 203-736 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises at least 8. at least 9, or at least 10 consecutive amino acids from the amino acid sequence of TNTQDWHRI AQ (SEQ ID NO: 343). In some embodiments, the AAV capsid variant comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343) present at amino acids corresponding to positions 582-592 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or TNTQDWHRIAQ (SEQ ID NO: 343) is present in loop VIII, wherein loop VIII comprises amino acids 580-599 of the amino acid sequence of SEQ ID NO: 981.

[0028] In some embodiments, the AAV capsid variant comprises: (i) the amino acid sequence of SEQ ID NO: 981; (ii) the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981; and / or (iii) the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981.

[0029] 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: 6413-6416, 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: 6417-6421, or a nucleotide sequence that is at least 95% identical thereto; and (iii) a 3’ flanking region comprising tire nucleotide sequence of any one of SEQ ID NOs: 6422-6427, or a nucleotide sequence that is at least 95% identical thereto.

[0030] In some embodiments, (i) the 5 ’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6414 or SEQ ID NO: 6415. or a nucleotide sequence that is at least 95% identical thereto; (ii) the loop region comprises the nucleotide sequence of SEQ ID NO: 6417. SEQ ID NO: 6418, or SEQ ID NO: 6421, 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: 6423, SEQ ID NO: 6424, or SEQ ID NO: 6425, or a nucleotide sequence that is at least 95% identical thereto.

[0031] In some embodiments, the 5’ flanking region comprises tire nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and the 3‘ flanking region comprises the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% identical thereto.

[0032] In some embodiments, the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6415 or a nucleotide sequence that is at least 95% identical thereto, the loop region of the comprises the nucleotide sequence of SEQ ID NO: 6421 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6425 or a nucleotide sequence that is at least 95% identical thereto.

[0033] In some embodiments, the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6424 or a nucleotide sequence that is at least 95% identical thereto.

[0034] In some embodiments, the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 6418 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% identical thereto.

[0035] In some embodiments, the modulatory polynucleotide comprises siRNA or shRNA.

[0036] 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.

[0037] hi 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.

[0038] 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. In some embodiments, the one or more DMPK mRNA transcripts comprises the nucleotide sequence of SEQ ID NOs: 6428-6434 or a trinucleotide repeat expansion thereof.

[0039] In some embodiments, the viral genome comprises a promoter operably linked to the nucleotide sequence encoding the modulatory polynucleotide.

[0040] In some embodiments, the viral genome further comprises an inverted terminal repeat (ITR) sequence.

[0041] In some embodiments, the viral genome comprises an ITR sequence positioned 5’ relative to the 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.

[0042] In some embodiments, the present disclosure provides a cell comprising an AAV particle of the disclosure. In some embodiments, the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an S19 cell), or a bacterial cell.

[0043] In some embodiments, the present disclosure provides a method of making an AAV particle of the disclosure, 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.

[0044] In some embodiments, the viral genome comprises: (i) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% identical thereto; (ii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6415 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6421 or a nucleotide sequence that is at least 95% identical thereto, and a 3 ’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6425 or a nucleotide sequence that is at least 95% identical thereto; (iii) a 5‘ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and a 3 ' flanking region comprising the nucleotide sequence of SEQ ID NO: 6424 or a nucleotide sequence that is at least 95% thereto; or (iv) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6418 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6423 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: 981 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) theamino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981 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 203-736 of the amino acid sequence of SEQ ID NO: 981 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.

[0045] In some embodiments, the viral genome comprises: (i) a 5 ’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% identical thereto; (ii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6415 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6421 or a nucleotide sequence that is at least 95% identical thereto, and a 3 ’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6425 or a nucleotide sequence that is at least 95% identical thereto; (iii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and a 3 ’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6424 or a nucleotide sequence that is at least 95% thereto; or (iv) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6418 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% thereto; and wherein the AAV capsid variant comprises SEQ ID NO: 981, the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981, and / or the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981.

[0046] hi some embodiments, the method further comprises, prior to step (i), introducing a nucleic acid molecule comprising the viral genome into the cell. In some embodiments, the method further comprises, prior to step (i) introducing the nucleic acid encoding the AAV capsid variant into the cell.

[0047] In some embodiments, the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.

[0048] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an AAV particle of the disclosure and a pharmaceutically acceptable excipient.

[0049] In some embodiments, the present disclosure provides a method of delivering an AAV particle encoding a modulatory polynucleotide for reducing or eliminating expression of mutated DMPKmRNA to a cell, comprising administering an effective amount of a pharmaceutical composition or AAV particle of the disclosure.

[0050] In some embodiments, the delivery is to a cell in a subject. 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 myotonic dystrophy type 1 (DM1).

[0051] In some embodiments, 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 or AAV particle of the disclosure. In some embodiments, the subject has. has been diagnosed with having, or is at risk of having the DMPK-related disorder. 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.

[0052] 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, respiratory’ insufficiency, or a combination thereof. In some embodiments, the DMPK-related disorder is myotonic dystrophy type 1 (DM1).

[0053] In some embodiments, 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 or AAV particle of the disclosure. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having DM1.

[0054] In some embodiments, the subject is a human.

[0055] 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., quadriceps, vastus lateralis, and / or gastrocnemius).

[0056] 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.

[0057] In some embodiments, the pharmaceutical composition or AAV particle is delivered to the amygdala, brainstem, caudate, central grey, cerebellum (e.g.. Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen. substantia nigra.thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region).

[0058] In some embodiments, the subject has, has been diagnosed with having, or is at risk of having congenital myotonic dystrophy type 1.

[0059] hi some embodiments, the AAV particle or the pharmaceutical composition is delivered via intravenous administration.

[0060] In some embodiments, the method of treating or delivering 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.

[0061] In some embodiments, the cell or tissue of the subject is a muscle cell or tissue (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius)).

[0062] In some embodiments, the cell or tissue of the subject is a cell or tissue of the central nervous system (CNS) (e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary' complex, nucleus ambiguus. oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g.. cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)).

[0063] In some embodiments, 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, 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.

[0064] In some embodiments, 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 tissue (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius)) 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 (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius)) 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 muscle, smooth muscle, and / or skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius)) 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.

[0065] In some embodiments, 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., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus. gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus. oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., centical 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., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / orspinal 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.

[0066] 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, the method of treating or delivering 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 suitable for treating a DMPK-related disorder. In some embodiments, the at least one additional 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.

[0068] In some embodiments, the method of treating or delivering 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 embodiments, the present disclosure provides a pharmaceutical composition or AAV particle of the disclosure for use in a method of treating a disorder as disclosed herein.

[0070] In some embodiments, the present disclosure provides a pharmaceutical composition or AAV particle of the disclosure for use in treating a DMPK-related disorder in a subject, optionally wherein tire 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 embodiments, the present disclosure provides use of a pharmaceutical composition or AAV particle of the disclosure in the manufacture of a medicament for treating a DMPK-rclatcd 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.Enumerated Embodiments1. An adeno-associated virus (AAV) particle comprising an AAV capsid variant (e.g.. an AAV9 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; andwherein the AAV capsid variant comprises an amino acid sequence having the following formula: [N1]-[N2]-[N3] (SEQ ID NO: 4681), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein:(i) [Nl] comprises amino acids Xi, X2, X3, and X4. wherein X4 is Q, K, E, S, P, R, N, H; and / or(ii) [N3] comprises amino acids X5, Xe, and X?, wherein X, is I, V, T. M, S, N, L, F.2. The AAV particle of embodiment 1, wherein the AAV capsid variant comprises the amino acid Q at position 585, as numbered according to SEQ ID NO: 138 or 981.3. The AAV particle of embodiment 1, wherein the AAV capsid variant comprises an amino acid other than Q at position 585. as numbered according to SEQ ID NO: 138 or 981.4. The AAV particle of embodiment 1 or 3. wherein the AAV capsid variant comprises the amino acid K at position 585, as numbered according to SEQ ID NO: 138 or 981.5. The AAV particle of any one of embodiments 1-4. wherein the AAV capsid variant comprises one. two. three, or all of:(i) an amino acid other than T at position 582 (e.g.. S, R, A. I. C, N, K, L, or Q);(ii) an amino acid other than N at position 583 (e.g., T, G, V, S. Y, K, I, H, D, or F);(iii) an amino acid other than H at position 584 (e.g., T, N, K. D, I, S, P. A, Y, E, V, L, M, R. Q, or C); and / or(iv) an amino acid other than Q at position 585 (e.g., K, E, S, P. R, N, or H); wherein the amino acids are numbered according to SEQ ID NO: 138.6. The AAV particle of any one of embodiments 1-5, wherein [Nl] comprises amino acids Xi, X2, X3, and X4, wherein X4 is Q, K, E, S, P, R, N, or H.7. The AAV particle of embodiment 6, wherein X4 is Q or K.8. The AAV particle of any one of embodiment 7, wherein X4 is Q.9. The AAV particle of embodiment 7, wherein X4 is K.10. The AAV particle of any one of embodiments 1-9. wherein the AAV capsid variant comprises an amino acid other than H at position 584 (e.g., T), as numbered according to SEQ ID NO: 138.11. The AAV particle of any one of embodiments 1-10, wherein the AAV capsid variant comprises the amino acid T at position 584, as numbered according to SEQ ID NO: 138 or 981.12. The AAV particle of any one of embodiments 1-11, wherein:(i) Xi is T, S, R, A, I, C, N, K, L, or Q;(ii) X2is N, T, G, V, S. Y, K, I, H, D, or F; and / or(iii) X3is T, N, K, D, I. S, P, A. Y, E, V, L, M, R, H. Q, or C.13. The AAV particle of any one of embodiments 1-12, wherein [Nl] comprises TN, NT, NK, SN. TT. RN, TG, TV, ST, TS, TY, AN. TK. TI, IN, TH, TD. CN, NN, KN, LN. SG. TF. RT, SY, SS, QN, ND, NP. GK, TA, VK, NY. TE. SK, NI. YN, GT, TL, TM. YT. TR, NS, IT. NA, KT, GN, HT. DT. NE, NH, YI, HN. NQ, FS, NM, NL. SM, NC, VT, KQ. TQ. DQ, IQ, SQ, PS, KE, AQ, YQ. TP. EQ, VQ, LQ, MQ, KS. IE. RQ. IK. AK. PK, NR, HQ. QQ. or CQ.14. The AAV particle of any one of embodiments 1-13, wherein [Nl] comprises TNT, TNK, TNN. SNN, SNK, SNT, TTN, TND, TTI, RNT, TTK, TTS, TTD. TNP. TTT, TGK, TTA, TVK, TNY, STK, TTE, TSK, TNI. TYN. STI, TTV, TGT, TTL. TTM. ANN, SNI, TKN, TYT, TTR, TNS, TST. TIT, INT. TNA. TKT, STN, ANT, RNN, TGN, TSN, THT, TDT. TNE. CNT. INN, NNN, KNN. LNN. TIN, TNH, STT, SNS, STS. TYI, SGT, THN, TNQ, RNI, TFS, RNS, TNM, RTT, KNT, TNL, TSM, SYT, TNC, SST. TVT, QNT, NTK, NNQ, NKQ, NNE. NTQ. NDQ, TIQ, TKQ, TSQ, TDQ, NPS, NKE, TTQ, GKQ, TAQ, VKQ, NYQ. NTP, TEQ, SKQ, NIQ, YNQ, TVQ, GTQ, NTR, TLQ, TMQ, KNQ, YTQ, NKS, NTE, NIE, TRQ, NSQ, YTK, NIK, NNK, NSK, ITK, NAK, KTK, GNQ, SNQ, HTK, DTK, NEQ, NPK, YTE, NNR, INQ, NHQ, YIQ, HNQ, ITQ. STQ, NSN, NQQ, NNP, ITE, NTN, FSQ, NNH, NMQ, NTS, NLQ, SMQ, NCQ. or VTQ15. The AAV particle of any one of embodiments 1-14, wherein [Nl] is or comprises TNTQ (SEQ ID NO: 4688), TNTK (SEQ ID NO: 4689), TNNQ (SEQ ID NO: 4690), SNNQ (SEQ ID NO: 4691), TNKQ (SEQ ID NO: 4692). TNNE (SEQ ID NO: 4693), SNKQ (SEQ ID NO: 4694), SNTQ (SEQ ID NO: 4695), TTNQ (SEQ ID NO: 4696), TNDQ (SEQ ID NO: 4697), TTIQ (SEQ ID NO: 4698), RNTQ (SEQ ID NO: 4699), TTKQ (SEQ ID NO: 4700), TTSQ (SEQ ID NO: 4701), TTDQ (SEQ ID NO: 4702), TNPS (SEQ ID NO: 4703), TNKE (SEQ ID NO: 4704), TTTQ (SEQ ID NO: 4705), TGKQ (SEQ ID NO: 4706), TTAQ (SEQ ID NO: 4707), TVKQ (SEQ ID NO: 4708). TNYQ (SEQ ID NO: 4709), TNTP (SEQ ID NO: 4710). STKQ (SEQ ID NO: 4711), TTEQ (SEQ ID NO: 4712). TSKQ (SEQ ID NO: 4713), TNIQ (SEQ ID NO: 4714), TYNQ (SEQ ID NO: 4715), STIQ (SEQ ID NO: 4716), TTVQ (SEQ ID NO: 4717), TGTQ (SEQ ID NO: 4718), TNTR (SEQ ID NO: 4719), TTLQ (SEQ ID NO: 4720), TTMQ (SEQ ID NO: 4721), ANNQ (SEQ ID NO: 4722), SNIQ (SEQ ID NO: 4723). TKNQ (SEQ ID NO: 4724), TYTQ (SEQ ID NO: 4725), TNKS (SEQ ID NO: 4726). SNTE (SEQ ID NO: 4727). TNTE(SEQ ID NO: 4728), TNIE (SEQ ID NO: 4729), TTRQ (SEQ ID NO: 4730), TNSQ (SEQ ID NO: 4731), TYTK (SEQ ID NO: 4732), TTTK (SEQ ID NO: 4733), TNIK (SEQ ID NO: 4734), SNTK (SEQ ID NO: 4735), TNNK (SEQ ID NO: 4736), TNSK (SEQ ID NO: 4737), TSTK (SEQ ID NO: 4738), TITK (SEQ ID NO: 4739). INTK (SEQ ID NO: 4740), TNAK (SEQ ID NO: 4741). TKTK (SEQ ID NO: 4742), STNQ (SEQ ID NO: 4743), ANTK (SEQ ID NO: 4744), RNNQ (SEQ ID NO: 4745), TGNQ (SEQ ID NO: 4746). TSNQ (SEQ ID NO: 4747), THTK (SEQ ID NO: 4748), TDTK (SEQ ID NO: 4749), TNEQ (SEQ ID NO: 4750), CNTQ (SEQ ID NO: 4751), TNPK (SEQ ID NO: 4752). INNQ (SEQ ID NO: 4753), TYTE (SEQ ID NO: 4754), NNNQ (SEQ ID NO: 4755), KNNQ (SEQ ID NO: 4756), TNNR (SEQ ID NO: 4757), LNNQ (SEQ ID NO: 4758), TINQ (SEQ ID NO: 4759), TNHQ (SEQ ID NO: 4760), STTQ (SEQ ID NO: 4761). SNSQ (SEQ ID NO: 4762), STSQ (SEQ ID NO: 4763), TYIQ (SEQ ID NO: 4764). SGTQ (SEQ ID NO: 4765), THNQ (SEQ ID NO: 4766), TITQ (SEQ ID NO: 4767), TSTQ (SEQ ID NO: 4768), TNSN (SEQ ID NO: 4769), TNQQ (SEQ ID NO: 4770). RNIQ (SEQ ID NO: 4771), TNNP (SEQ ID NO: 4772), TITE (SEQ ID NO: 4773). TNTN (SEQ ID NO: 4774), TFSQ (SEQ ID NO: 4775), RNSQ (SEQ ID NO: 4776), INTQ (SEQ ID NO: 4777), RNTE (SEQ ID NO: 4778), TNNH (SEQ ID NO: 4779). TNMQ (SEQ ID NO: 4780). RTTQ (SEQ ID NO: 4781), SNIE (SEQ ID NO: 4782), TNTS (SEQ ID NO: 4783). KNTQ (SEQ ID NO: 4784). TNLQ (SEQ ID NO: 4785). TSMQ (SEQ ID NO: 4786), SYTQ (SEQ ID NO: 4787), TNCQ (SEQ ID NO: 4788). SSTQ (SEQ ID NO: 4789), TVTQ (SEQ ID NO: 4790), or QNTQ (SEQ ID NO: 4791).16. The AAV particle of embodiment 15, wherein [Nl] is or comprises TNTQ (SEQ ID NO: 4688).17. The AAV particle of embodiment 15, wherein [Nl] is or comprises TNTK (SEQ ID NO: 4689).18. The AAV particle of any one of embodiments 1-17, wherein [N1]-[N2] comprises:(i) TQDWHR (SEQ ID NO: 4686), TKDWHR (SEQ ID NO: 4792), NQDWHR (SEQ ID NO: 4793), KQDWHR (SEQ ID NO: 4794), NEDWHR (SEQ ID NO: 4795), DQDWHR (SEQ ID NO: 4796), IQDWHR (SEQ ID NO: 4797), SQDWHR (SEQ ID NO: 4798), PSDWHR (SEQ ID NO: 4799), KEDWHR (SEQ ID NO: 4800), AQDWHR (SEQ ID NO: 4801), YQDWHR (SEQ ID NO: 4802). TPDWHR (SEQ ID NO: 4803), EQDWHR (SEQ ID NO: 4804), VQDWHR (SEQ ID NO: 4805), TRDWHR (SEQ ID NO: 4806), LQDWHR (SEQ ID NO: 4807), MQDWHR (SEQ ID NO: 4808). KSDWHR (SEQ ID NO: 4809), TEDWHR (SEQ ID NO: 4810). IEDWHR (SEQ ID NO: 4811), RQDWHR (SEQ ID NO: 4812), IKDWHR (SEQ ID NO: 4813). NKDWHR (SEQ ID NO: 4814), SKDWHR (SEQ ID NO: 4815), AKDWHR (SEQ ID NO: 4816), PKDWHR (SEQ ID NO: 4817), NRDWHR (SEQ ID NO: 4818), HQDWHR (SEQ ID NO: 4819), SNDWHR (SEQ ID NO: 4820). QQDWHR (SEQ ID NO: 4821), NPDWHR (SEQ ID NO: 4822), TNDWHR (SEQ ID NO: 4823). NHDWHR (SEQ ID NO: 4824), TSDWHR (SEQ ID NO: 4825), or CQDWHR (SEQ ID NO: 4826);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).19. The AAV particle of any one of embodiments 1-18, wherein [N1]-[N2] comprises:(i) NTQDWHR (SEQ ID NO: 4827), NTKDWHR (SEQ ID NO: 4828), NNQDWHR (SEQ ID NO: 4829), NKQDWHR (SEQ ID NO: 4830), NNEDWHR (SEQ ID NO: 4831), TNQDWHR (SEQ ID NO: 4832), NDQDWHR (SEQ ID NO: 4833), TIQDWHR (SEQ ID NO: 4834), TKQDWHR (SEQ ID NO: 4835), TSQDWHR (SEQ ID NO: 4836). TDQDWHR (SEQ ID NO: 4837), NPSDWHR (SEQ ID NO: 4838), NKEDWHR (SEQ ID NO: 4839). TTQDWHR (SEQ ID NO: 4840), GKQDWHR (SEQ ID NO: 4841), TAQDWHR (SEQ ID NO: 4842), VKQDWHR (SEQ ID NO: 4843), NYQDWHR (SEQ ID NO: 4844), NTPDWHR (SEQ ID NO: 4845), TEQDWHR (SEQ ID NO: 4846). SKQDWHR (SEQ ID NO: 4847), NIQDWHR (SEQ ID NO: 4848), YNQDWHR (SEQ ID NO: 4849). TVQDWHR (SEQ ID NO: 4850), GTQDWHR (SEQ ID NO: 4851), NTRDWHR (SEQ ID NO: 4852), TLQDWHR (SEQ ID NO: 4853), TMQDWHR (SEQ ID NO: 4854). KNQDWHR (SEQ ID NO: 4855), YTQDWHR (SEQ ID NO: 4856), NKSDWHR (SEQ ID NO: 4857), NTEDWHR (SEQ ID NO: 4858), NIEDWHR (SEQ ID NO: 4859), TRQDWHR (SEQ ID NO: 4860), NSQDWHR (SEQ ID NO: 4861), YTKDWHR (SEQ ID NO: 4862), TTKDWHR (SEQ ID NO: 4863), NIKDWHR (SEQ ID NO: 4864), NNKDWHR (SEQ ID NO: 4865), NSKDWHR (SEQ ID NO: 4866), STKDWHR (SEQ ID NO: 4867), ITKDWHR (SEQ ID NO: 4868), NAKDWHR (SEQ ID NO: 4869), KTKDWHR (SEQ ID NO: 4870), GNQDWHR (SEQ ID NO: 4871), SNQDWHR (SEQ ID NO: 4872), HTKDWHR (SEQ ID NO: 4873), DTKDWHR (SEQ ID NO: 4874), NEQDWHR (SEQ ID NO: 4875), NPKDWHR (SEQ ID NO: 4876), YTEDWHR (SEQ ID NO: 4877), NNRDWHR (SEQ ID NO: 4878), INQDWHR (SEQ ID NO: 4879), NHQDWHR (SEQ ID NO: 4880), YIQDWHR (SEQ ID NO: 4881), HNQDWHR (SEQ ID NO: 4882), ITQDWHR (SEQ ID NO: 4883), STQDWHR (SEQ ID NO: 4884), NSNDWHR (SEQ ID NO: 4885), NQQDWHR (SEQ ID NO: 4886), NNPDWHR (SEQ ID NO: 4887), ITEDWHR (SEQ ID NO: 4888), NTNDWHR (SEQ ID NO: 4889), FSQDWHR (SEQ ID NO: 4890), NNHDWHR (SEQ ID NO: 4891), NMQDWHR (SEQ ID NO: 4892), NTSDWHR (SEQ ID NO: 4893), NLQDWHR (SEQ ID NO: 4894), SMQDWHR (SEQ ID NO: 4895), NCQDWHR (SEQ ID NO: 4896), or VTQDWHR (SEQ ID NO: 4897);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, 5, or 6 amino acids, e.g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).20. The AAV particle of any one of embodiments 1-19, wherein [N1]-[N2] is or comprises:(i) TNTQDWHR (SEQ ID NO: 4898), TNTKDWHR (SEQ ID NO: 4899), TNNQDWHR (SEQ ID NO: 4900), SNNQDWHR (SEQ ID NO: 4901), TNKQDWHR (SEQ ID NO: 4902), TNNEDWHR (SEQ ID NO: 4903). SNKQDWHR (SEQ ID NO: 4904), SNTQDWHR (SEQ ID NO: 4905), TTNQDWHR (SEQ ID NO: 4906), TNDQDWHR (SEQ ID NO: 4907), TTIQDWHR (SEQ ID NO: 4908), RNTQDWHR (SEQ ID NO: 4909), TTKQDWHR (SEQ ID NO: 4910), TTSQDWHR (SEQ ID NO: 4911), TTDQDWHR (SEQ ID NO: 4912), TNPSDWHR (SEQ ID NO: 4913), TNKEDWHR (SEQ ID NO: 4914), TTTQDWHR (SEQ ID NO: 4915), TGKQDWHR (SEQ ID NO: 4916), TTAQDWHR (SEQ ID NO: 4917). TVK.QDWHR (SEQ ID NO: 4918), TNYQDWHR (SEQ ID NO: 4919), TNTPDWHR (SEQ ID NO: 4920). STKQDWHR (SEQ ID NO: 4921). TTEQDWHR (SEQ ID NO: 4922), TSKQDWHR (SEQ ID NO: 4923), TNIQDWHR (SEQ ID NO: 4924), TYNQDWHR (SEQ ID NO: 4925), STIQDWHR (SEQ ID NO: 4926). TTVQDWHR (SEQ ID NO: 4927). TGTQDWHR (SEQ ID NO: 4928), TNTRDWHR (SEQ ID NO: 4929). TTLQDWHR (SEQ ID NO: 4930). TTMQDWHR (SEQ ID NO: 4931), ANNQDWHR (SEQ ID NO: 4932), SNIQDWHR (SEQ ID NO: 4933). TKNQDWHR (SEQ ID NO: 4934). TYTQDWHR (SEQ ID NO: 4935), TNKSDWHR (SEQ ID NO: 4936). SNTEDWHR (SEQ ID NO: 4937). TNTEDWHR (SEQ ID NO: 4938), TNIEDWHR (SEQ ID NO: 4939), TTRQDWHR (SEQ ID NO: 4940), TNSQDWHR (SEQ ID NO: 4941), TYTKDWHR (SEQ ID NO: 4942), TTTKDWHR (SEQ ID NO: 4943). TNIKDWHR (SEQ ID NO: 4944), SNTKDWHR (SEQ ID NO: 4945), TNNKDWHR (SEQ ID NO: 4946), TNSKDWHR (SEQ ID NO: 4947), TSTKDWHR (SEQ ID NO: 4948), TITKDWHR (SEQ ID NO: 4949), INTKDWHR (SEQ ID NO: 4950), TNAKDWHR (SEQ ID NO: 4951), TKTKDWHR (SEQ ID NO: 4952), STNQDWHR (SEQ ID NO: 4953), ANTKDWHR (SEQ ID NO: 4954), RNNQDWHR (SEQ ID NO: 4955), TGNQDWHR (SEQ ID NO: 4956), TSNQDWHR (SEQ ID NO: 4957), THTKDWHR (SEQ ID NO: 4958), TDTKDWHR (SEQ ID NO: 4959), TNEQDWHR (SEQ ID NO: 4960), CNTQDWHR (SEQ ID NO: 4961), TNPKDWHR (SEQ ID NO: 4962), INNQDWHR (SEQ ID NO: 4963), TYTEDWHR (SEQ ID NO: 4964), NNNQDWHR (SEQ ID NO: 4965). KNNQDWHR (SEQ ID NO: 4966), TNNRDWHR (SEQ ID NO: 4967). LNNQDWHR (SEQ ID NO: 4968), TINQDWHR (SEQ ID NO: 4969), TNHQDWHR (SEQ ID NO: 4970), STTQDWHR (SEQ ID NO: 4971), SNSQDWHR (SEQ ID NO: 4972), STSQDWHR (SEQ ID NO: 4973), TYIQDWHR (SEQ ID NO: 4974). SGTQDWHR (SEQ ID NO: 4975), THNQDWHR (SEQ ID NO: 4976). TITQDWHR (SEQ ID NO: 4977), TSTQDWHR (SEQ ID NO: 4978), TNSNDWHR (SEQ ID NO: 4979), TNQQDWHR (SEQ ID NO: 4980), RNIQDWHR (SEQ ID NO: 4981). TNNPDWHR (SEQ ID NO: 4982), TITEDWHR (SEQ ID NO: 4983). TNTNDWHR (SEQ ID NO: 4984), TFSQDWHR (SEQ ID NO: 4985). RNSQDWHR (SEQ ID NO: 4986), INTQDWHR (SEQ ID NO: 4987), RNTEDWHR (SEQ ID NO: 4988), TNNHDWHR (SEQ IDNO: 4989), TNMQDWHR (SEQ ID NO: 4990), RTTQDWHR (SEQ ID NO: 4991), SNIEDWHR (SEQ ID NO: 4992), TNTSDWHR (SEQ ID NO: 4993), KNTQDWHR (SEQ ID NO: 4994), TNLQDWHR (SEQ ID NO: 4995), TSMQDWHR (SEQ ID NO: 4996), SYTQDWHR (SEQ ID NO: 4997), TNCQDWHR (SEQ ID NO: 4998), SSTQDWHR (SEQ ID NO: 4999), TVTQDWHR (SEQ ID NO: 5000), or QNTQDWHR (SEQ ID NO: 5001);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, 5, 6, or 7 amino acids, e.g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions, relative to any one of the amino acid sequences in (i).21. The AAV particle of embodiment 20. wherein [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898).22. The AAV particle of embodiment 20, wherein [N1]-[N2] is or comprises TNTKDWHR (SEQ ID NO: 4899).23. The AAV particle of any one of embodiments 1-22, wherein the AAV capsid variant comprises one, two. or all of: an amino acid other than Q at position 590 (e.g., I, V. T, M. S, N, L. or F). an amino acid other than A at position 591 (e.g.. Y, P, N, S, T, G, E, V, W, F, Q), and / or an amino acid other than Q at position 592 (e.g., G. N, K, H, R, E, L, P, or M). as numbered according to SEQ ID NO: 138 or 981.24. The AAV particle of any one of embodiments 1-23, wherein the AAV capsid variant comprises an amino acid other than Q at position 590 (e.g., I, V, T, M, S, N, L, or F), as numbered according to SEQ ID NO: 138 or 981.25. The AAV particle of any one of embodiments 1-24, wherein the AAV capsid variant comprises tire amino acid I at position 590, as numbered according to SEQ ID NO: 138 or 981.26. The AAV particle of any one of embodiments 1-24, wherein the AAV capsid variant comprises the amino acid V at position 590, as numbered according to SEQ ID NO: 138 or 981.27. The AAV particle of any one of embodiments 1-26, wherein the AAV capsid variant comprises the amino acid A at position 591 and / or the amino acid Q at position 592, as numbered according to SEQ ID NO: 138 or 981.28. The AAV particle of any one of embodiments 1-27, wherein [N3] comprises amino acids X5, X6, andX?, wherein X5 is I, V, T, M, S, N, L, or F.29. The AAV particle of any one of embodiments 1-28, wherein X5 is I or V.30. The AAV particle of embodiment 29, wherein X5 is I.31. The AAV particle of any one of embodiments 1-30, wherein:(i) X6is A, Y, P. N, S, T. G, E, V, W, F, or Q; and / or(ii) X- is Q, G. N, K. H, R, E. L, P, or M.32. The AAV particle of any one of embodiments 1-31, wherein [N3] comprises IA. IY. VP. IN. VN, VY, VA, IS, IT. TA. MA, SA, IG, IE, IV. NA, LA, IP, FA, VS, VT, IW, IF, IQ. VQ, AQ. AG. YQ, PQ, AN, NQ, SG, SQ, TQ, GQ, EQ, AK, AH, AR, AE, AL, AP, TM, SM, WQ, FQ, QQ, FM, AM, or SN.33. The AAV particle of any one of embodiments 1-32, wherein [N3] is or comprises IAQ. IAG, IYQ, VPQ, IAN, INQ. VNQ. VYQ, VAN, ISG. ISQ, VAQ, ITQ, TAQ, MAQ, SAQ, IGQ, IEQ, IVQ, NAQ, LAQ, IAK, IAH. IPQ, IAR, IAE, IAL, IAP, FAQ, VSQ, VTM. ISM, IWQ, IFQ. IQQ, VQQ. IFM, IAM, or ISN.34. The AAV particle of any one of embodiments 1-33, wherein [N3] is or comprises IAQ.35. The AAV particle of any one of embodiments 1-34, wherein [N2]-[N3] comprises: (i)DWHRIA (SEQ ID NO: 5002), DWHRIY (SEQ ID NO: 5003), DWHRVP (SEQ ID NO: 5004), DWHRIN (SEQ ID NO: 5005), DWHRVN (SEQ ID NO: 5006), DWHRVY (SEQ ID NO: 5007), DWHRVA (SEQ ID NO: 5008), DWHRIS (SEQ ID NO: 5009), DWHRIT (SEQ ID NO: 5010), DWHRTA (SEQ ID NO: 5011), DWHRMA (SEQ ID NO: 5012), DWHRSA (SEQ ID NO: 5013). DWHRIG (SEQ ID NO: 5014), DWHRIE (SEQ ID NO: 5015), DWHRIV (SEQ ID NO: 5016), DWHRNA (SEQ ID NO: 5017), DWHRLA (SEQ ID NO: 5018), DWHRIP (SEQ ID NO: 5019), DWHRFA (SEQ ID NO: 5020), DWHRVS (SEQ ID NO: 5021). DWHRVT (SEQ ID NO: 5022), DWHRIW (SEQ ID NO: 5023), DWHRIF (SEQ ID NO: 5024), DWHRIQ (SEQ ID NO: 5025), or DWHRVQ (SEQ ID NO: 5026);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, or 5 amino acids, e.g.. consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).36. The AAV particle of any one of embodiments 1-35, wherein [N2]-[N3] is or comprises:(i) DWHRIAQ (SEQ ID NO: 5027), DWHRIAG (SEQ ID NO: 5028), DWHRIYQ (SEQ ID NO: 5029), DWHRVPQ (SEQ ID NO: 5030). DWHRIAN (SEQ ID NO: 5031), DWHRINQ (SEQ ID NO: 5032), DWHRVNQ (SEQ ID NO: 5033), DWHRVYQ (SEQ ID NO: 5034), DWHRVAN (SEQ ID NO: 5035), DWHRISG (SEQ ID NO: 5036), DWHRISQ (SEQ ID NO: 5037), DWHRVAQ (SEQ ID NO: 5038), DWHRITQ (SEQ ID NO: 5039), DWHRTAQ (SEQ ID NO: 5040), DWHRMAQ (SEQ ID NO: 5041), DWHRSAQ (SEQ ID NO: 5042). DWHRIGQ (SEQ ID NO: 5043), DWHRIEQ (SEQ ID NO: 5044), DWHRIVQ (SEQ ID NO: 5045), DWHRNAQ (SEQ ID NO: 5046), DWHRLAQ (SEQ ID NO: 5047), DWHR1AK (SEQ ID NO: 5048), DWHR1AH (SEQ ID NO: 5049). DWHRIPQ (SEQ ID NO: 5050), DWHRIAR (SEQ ID NO: 5051). DWHRIAE (SEQ ID NO: 5052). DWHRIAL (SEQ ID NO: 5053), DWHRIAP (SEQ ID NO: 5054), DWHRFAQ (SEQ ID NO: 5055), DWHRVSQ (SEQ ID NO: 5056), DWHRVTM (SEQ ID NO: 5057). DWHRISM (SEQ ID NO: 5058). DWHRIWQ (SEQ ID NO: 5059), DWHRIFQ (SEQ ID NO: 5060), DWHRIQQ (SEQ ID NO: 5061), DWHRVQQ (SEQ ID NO: 5062), DWHRIFM (SEQ ID NO: 5063), DWHRIAM (SEQ ID NO: 5064), or DWHRISN (SEQ ID NO: 5065);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i). e.g., any 2, 3, 4. 5, or 6 amino acids, e.g.. consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).37. The AAV particle of any one of embodiments 1-36, wherein [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027).38. The AAV particle of any one of embodiments 1-37, wherein [N1]-[N2]-[N3] is or comprises:(i) the amino acid sequence of any one of SEQ ID NOs: 343-538:(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, 5, 6, 7, 8. 9, or 10 amino acids, e.g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).39. The AAV particle of embodiment 38, wherein [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343).40. The AAV particle of embodiment 38, wherein [N1]-[N2]-[N3] is or comprises TNTKDWHRIAQ (SEQ ID NO: 344).41. The AAV particle of any one of embodiments 1-40, wherein the AAV capsid variant comprises one, two, three, or all of: an amino acid other than T at position 593 (e.g., S, N, P, A. or I), an amino acid other than G at position 594 (e.g., N, D. R, V, A, S, or Q), an amino acid other than W at position 595 (e.g., S. C, R, L. or G). and / or an amino acid other than V at position 596 (e.g., A. S, I, C, G. D, F, L. or T), as numbered according to SEQ ID NO: 138 or 981.42. The AAV particle of any one of embodiments 1-40, wherein the AAV capsid variant comprises the amino acid T at position 593. the amino acid G at position 594, the amino acid W at position 595, and the amino acid V at position 596. as numbered according to SEQ ID NO: 138 or 981.43. The AAV particle of any one of embodiments 1-42, wherein the AAV capsid variant further comprises [N4], wherein [N4] comprises amino acids X8, X9. Xio, and Xu, wherein:(i) X8is T, S, N, P, A, or I;(ii) X9is G, N, D, R, V, A, S, or Q;(iii) Xiois W, S, C, R, L, or G; and / or(iv) Xu is V, A, S, I, C, G, D. F, L, or T.44. The AAV particle of embodiment 43, wherein [N4] comprises TG, TN, SN, NN, SG, PG, TD, AG, IG, NG, TR, TV, TA, TS, SV, TQ, WV, WA, WS, WI, WC, WG, CV, RV, LV, GV, WD, WF, WL, WT, GW, NW, GS, DW, GO, GR, GL, GG, RW, VW, AW, SW, or QW.45. The AAV particle of embodiment 43 or 44, wherein [N4] comprises TGW, TNW, SNW. NNW, SGW, PGW, TGS, TDW, TGC, TGR, TGL, TGG, AGW, IGW, NGW. TRW, TVW, TAW, TSW, SVW, TQW, GWV, GW A. NWS, NWV, NWI, GWS, GWI, GWC, GWG, GSV, DWV, GCV. GRV, GLV, GGV. GWD, GWF, RWV. VWV, GWL, AWV, SWV, GWT, or QWV.46. The AAV particle of any one of embodiments 43-45. wherein [N4] is or comprises TGWV (SEQ ID NO: 5066), TGWA (SEQ ID NO: 5067), TNWS (SEQ ID NO: 5068), SNWV (SEQ ID NO: 5069), TNWV (SEQ ID NO: 5070). TNWI (SEQ ID NO: 5071), NNWV (SEQ ID NO: 5072), TGWS (SEQ ID NO: 5073), TGWI (SEQ ID NO: 5074), TGWC (SEQ ID NO: 5075). TGWG (SEQ ID NO: 5076). SGWV (SEQ ID NO: 5077). PGWV (SEQ ID NO: 5078). TGSV (SEQ ID NO: 5079), TDWV (SEQ IDNO: 5080), TGCV (SEQ ID NO: 5081), TGRV (SEQ ID NO: 5082), TGLV (SEQ ID NO: 5083), TGGV (SEQ ID NO: 5084), AGWV (SEQ ID NO: 5085), IGWV (SEQ ID NO: 5086), TGWD (SEQ ID NO: 5087), NGWV (SEQ ID NO: 5088), TGWF (SEQ ID NO: 5089), TRWV (SEQ ID NO: 5090), TVWV (SEQ ID NO: 5091). TGWL (SEQ ID NO: 5092), TAWV (SEQ ID NO: 5093), TSWV (SEQ ID NO: 5094), TGWT (SEQ ID NO: 5095). SVWV (SEQ ID NO: 5096). TQWV (SEQ ID NO: 5097). or PGWG (SEQ ID NO: 5098).47. The AAV particle of any one of embodiments 43-46. wherein [N4] is or comprises TGWV (SEQ ID NO: 5066).48. The AAV particle of any one of embodiments 43-47, wherein [N 1 ]-[N2]-[N3]-[N4] is or comprises:(i) the amino acid sequence of any one of SEQ ID NOs: 201-245. 247-250, 253-255, 257-265, 268-274, 276-286. 288, 290-297. 299-303, 305-309. 311, 313-319. 323-328, 330-337. 339-342, 539-542. 544, 546, 547, 549-557, 559-589, 592, 593, 595, 596, 598, 599, 601-608, 610-614, 616-622, 625, 628, 630. 631, 633, 636. 638, 639-646. 649, 651-657. 667. 669, 670. 672, 673, 679-683, 685-690, 692. 693, 695. 697, 699-701. 703-705, 708-710. 712-717, 719-723. 728-731, 733-738. 740, and 742:(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i). e.g., any 2, 3, 4. 5, 6, 7, 8, 9. 10, 11, 12, or 13 amino acids, e g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, tw o. or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).49. The AAV particle of embodiment 48, wherein [N1]-[N2]-[N3]-[N4] is or comprises TNTQDWHRIAQTGWV (SEQ ID NO: 201).50. The AAV particle of any one of embodiment 48, wherein [N1]-[N2]-[N3]-[N4] is or comprises TNTKDWHRIAQTGWV (SEQ ID NO: 202).51. An AAV particle comprising: 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 an AAV capsid variant (e.g., an AAV9 capsid variant) comprising an amino acid sequence having the following formula: [N1]-[N2]-[N3] (SEQ ID NO: 4683), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein:(i) [Nl] comprises amino acids Xi, X2, X3, and X4, wherein X4is Q, P, or a conservative substitution drereof; and / or(ii) [N3] comprises amino acids X5, Xs, and X-, wherein X, is I, V, or a conservative substitution thereof.52. The AAV particle of embodiment 51, wherein the AAV capsid variant comprises the amino acid Q at position 585, as numbered according to SEQ ID NO: 138 or 981.53. The AAV particle of embodiment 51 or 52, wherein the AAV capsid variant comprises one, two, three, or all of:(i) an amino acid other than T at position 582 (e.g., S);(ii) an amino acid other than N at position 583 (e.g., T. G, S, I, or V);(iii) an amino acid other than H at position 584 (e.g.. N, I, S. A, V, or L); and / or(iv) an amino acid other than Q at position 585 (e.g., P), wherein the amino acids are as numbered according to SEQ ID NO: 138 or 981.54. The AAV particle of any one of embodiments 51-53, wherein [Nl] comprises amino acids Xi, X2, X3. and X4, wherein X4is Q or P.55. The AAV particle of any one of embodiments 51-54, wherein X4is Q.56. The AAV particle of any one of embodiments 51-55, wherein the AAV capsid variant comprises an amino acid other than H at position 584 (e.g., T), as numbered according to SEQ ID NO: 138 or 981.57. The AAV particle of any one of embodiments 51-56, wherein the AAV capsid variant comprises the amino acid T at position 584, as numbered according to SEQ ID NO: 138 or 981.58. The AAV particle of any one of embodiments 51-57, wherein:(i) Xi is T or S;(ii) X2is N, T, G, S, I, or V; and / or(iii) X3is T, N, I, S, A, V, or L.59. The AAV particle of any one of embodiments 51-58, wherein [Nl] comprises TN. TT. TG, ST, TS, TI, TV, TQ. NQ, IQ. SQ, AQ, VQ, TP, LQ. NT, TA, Nl. GT, IT. NN, TL, NS, or VT.60. The AAV particle of any one of embodiments 51-59, wherein [Nl] comprises TNT, TTN, TTI, TTS, TTT, TTA, TNI, TTV, TGT, STT, TST, TIT, TNN, TTL, TNS, TVT, NTQ, TNQ, TIQ, TSQ, TTQ, TAQ, NIQ, TVQ, GTQ, STQ, ITQ, NTP, NNQ, TLQ, NSQ, or VTQ.61. The AAV particle of any one of embodiments 51-60. wherein [Nl] is or comprises TNTQ (SEQ ID NO: 4688), TTNQ (SEQ ID NO: 4696), TTIQ (SEQ ID NO: 4698), TTSQ (SEQ ID NO: 4701), TTTQ (SEQ ID NO: 4705). TTAQ (SEQ ID NO: 4707), TNIQ (SEQ ID NO: 4714), TTVQ (SEQ ID NO: 4717), TGTQ (SEQ ID NO: 4718), STTQ (SEQ ID NO: 4761), TSTQ (SEQ ID NO: 4768), TITQ (SEQ ID NO: 4767), TNTP (SEQ ID NO: 4710), TNNQ (SEQ ID NO: 4690), TTLQ (SEQ ID NO: 4720), TNSQ (SEQ ID NO: 4731), or TVTQ (SEQ ID NO: 4790).62. The AAV particle of embodiment 61. wherein [Nl] is or comprises TNTQ (SEQ ID NO: 4688).63. The AAV particle of any one of embodiments 51-62, wherein [N1]-[N2] comprises:(i) TQDWHR (SEQ ID NO: 4686), NQDWHR (SEQ ID NO: 4793), IQDWHR (SEQ ID NO: 4797). SQDWHR (SEQ ID NO: 4798), AQDWHR (SEQ ID NO: 4801), VQDWHR (SEQ ID NO: 4805). TPDWHR (SEQ ID NO: 4803), or LQDWHR (SEQ ID NO: 4807);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i). e.g., any 2, 3, 4. or 5 amino acids, e.g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, tw o. or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).64. The AAV particle of any one of embodiments 51-63, w herein [N1]-[N2] comprises:(i) NTQDWHR (SEQ ID NO: 4827), TNQDWHR (SEQ ID NO: 4832), TIQDWHR (SEQ ID NO: 4834), TSQDWHR (SEQ ID NO: 4836), TTQDWHR (SEQ ID NO: 4840), TAQDWHR (SEQ ID NO: 4842), NIQDWHR (SEQ ID NO: 4848), TVQDWHR (SEQ ID NO: 4850), GTQDWHR (SEQ ID NO: 4851), STQDWHR (SEQ ID NO: 4884), ITQDWHR (SEQ ID NO: 4883), NTPDWHR (SEQ ID NO: 4845), NNQDWHR (SEQ ID NO: 4829), TLQDWHR (SEQ ID NO: 4853), NSQDWHR (SEQ ID NO: 4861), VTQDWHR (SEQ ID NO: 4897);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, 5 or 6 amino acids, e.g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).65. The AAV particle of any one of embodiments 51 -64, wherein [N 1 ] - [N2] is or comprises :(i) TNTQDWHR (SEQ ID NO: 4898), TTNQDWHR (SEQ ID NO: 4906), TTIQDWHR (SEQ ID NO: 4908), TTSQDWHR (SEQ ID NO: 4911), TTTQDWHR (SEQ ID NO: 4915), TTAQDWHR (SEQ ID NO: 4917). TNIQDWHR (SEQ ID NO: 4924), TTVQDWHR (SEQ ID NO: 4927), TGTQDWHR (SEQ ID NO: 4928), STTQDWHR (SEQ ID NO: 4971), TSTQDWHR (SEQ ID NO: 4978), TITQDWHR (SEQ ID NO: 4977). TNTPDWHR (SEQ ID NO: 4920). TNNQDWHR (SEQ ID NO: 4900), TTLQDWHR (SEQ ID NO: 4930), TNSQDWHR (SEQ ID NO: 4941), TVTQDWHR (SEQ ID NO: 5000);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, 5 6, or 7 amino acids, e.g.. consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).66. The AAV particle of embodiment 65, wherein [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898).67. The AAV particle of any one of embodiments 51-66, wherein the AAV capsid variant comprises one, two, or all of: an amino acid other than Q at position 590 (e.g., I or V), an amino acid other than A at position 591 (e.g., P, S, Y, or N), and / or an amino acid other than Q at position 592 (e.g., G or N), as numbered according to SEQ ID NO: 138 or 981.68. The AAV particle of any one of embodiments 51-67, wherein the AAV capsid variant comprises an amino acid other than Q at position 590 (e.g., I or V), as numbered according to SEQ ID NO: 138 or 981.69. The AAV particle of any one of embodiments 51-68, wherein the AAV capsid variant comprises the amino acid I at position 590, as numbered according to SEQ ID NO: 138 or 981.70. The AAV particle of any one of embodiments 51-68, wherein the AAV capsid variant comprises the amino acid V at position 590, as numbered according to SEQ ID NO: 138 or 981.71. The AAV particle of any one of embodiments 51-70, wherein the AAV capsid variant comprises the amino acid A at position 591 and / or the amino acid Q at position 592, as numbered according to SEQ ID NO: 138 or 981.72. The AAV particle of any one of embodiments 51-71, wherein [N3] comprises amino acids X5, X6, and X?, wherein X5 is I or V.73. The AAV particle of embodiment 72, wherein X5 is I.74. The AAV particle of any one of embodiments 51-73, wherein:(i) X6is A, P, S, Y, or N; and / or(ii) X7is Q, G. or N.75. The AAV particle of any one of embodiments 51-74, wherein [N3] comprises IA, VP, VA. VS, IY. IN, IS, AQ, AG. PQ. SQ, AN, YQ, or NQ.76. The AAV particle of any one of embodiments 51-75, wherein [N3] is or comprises IAQ. IAG, VPQ, VAQ. VSQ, IAN, IYQ, INQ, or ISQ.77. The AAV particle of any one of embodiments 51-76, wherein [N3] is or comprises IAQ.78. The AAV particle of any one of embodiments 51-77, wherein [N2]-[N3] comprises:(i) DWHRIA (SEQ ID NO: 5002), DWHRVP (SEQ ID NO: 5004), DWHRVA (SEQ ID NO: 5008). DWHRVS (SEQ ID NO: 5021), DWHRIY (SEQ ID NO: 5003). DWHRIN (SEQ ID NO: 5005), or DWHRIS (SEQ ID NO: 5009);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i). e.g., any 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two, or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).79. The AAV particle of any one of embodiments 51-78, wherein [N2]-[N3] is or comprises:(i) DWHRIAQ (SEQ ID NO: 5027), DWHRIAG (SEQ ID NO: 5028), DWHRVPQ (SEQ ID NO: 5030), DWHRVAQ (SEQ ID NO: 5038), DWHRVSQ (SEQ ID NO: 5056), DWHRIAN (SEQ ID NO: 5031), DWHRIYQ (SEQ ID NO: 5029), DWHRINQ (SEQ ID NO: 5032). or DWHRISQ (SEQ ID NO: 5037);(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, 5, or 6 amino acids, e.g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two. or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).80. The AAV particle of any one of embodiments 51-79, wherein [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027).81. The AAV particle of any one of embodiments 51-80, wherein [N1]-[N2]-[N3] is or comprises:(i) the amino acid sequence of any one of SEQ ID NOs: 343, 350, 352, 355. 359, 361, 364. 367, 370, 371, 373. 374, 376, 377. 378, 381, 395, 420, 454. 457, 460, 464. 481, 482, 488, 493, 494, 516, 525, 536;(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, 5, 6, 7. 8. 9, or 10 amino acids, e.g., consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two. or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one. two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).82. The AAV particle of any one of embodiments 51-81, wherein [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343).83. The AAV particle of any one of embodiments 51-82, wherein the AAV capsid variant comprises the amino acid W at position 595, as numbered according to SEQ ID NO: 138 or 981.84. The AAV particle of any one of embodiments 51-83, wherein the AAV capsid variant comprises one, two or all of: an amino acid other than T (e.g., S or N) at position 593, an amino acid other than G (e.g., N) a position 594, and / or an amino acid other than V (e.g., A, I, or S) at position 596, as numbered according to SEQ ID NO: 138 or 981.85. The AAV particle of any one of embodiments 51-84, wherein the AAV capsid variant comprises:(i) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981:(ii) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid A at position 596, as numbered according to SEQ ID NO: 138 or 981:(iii) the amino acid S at position 593, the amino acid N at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981:(iv) the amino acid N at position 593. the amino acid N at position 594. the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981:(v) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid I at position 596, as numbered according to SEQ ID NO: 138 or 981; or(vi) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and tire amino acid S at position 596, as numbered according to SEQ ID NO: 138 or 981.86. The AAV particle of any one of embodiments 51-85, wherein the AAV capsid variant comprises the amino acid T at position 593. the amino acid G at position 594. the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981.87. The AAV particle of any one of embodiments 51-86, wherein the AAV capsid variant further comprises [N4] . wherein [N4] comprises amino acids X8, X9, Xio, and Xu, wherein Xiois W.88. The AAV particle of embodiment 87. wherein:(i) X8is T, S. or N;(ii) X9is G or N; and / or(iv) Xu is V. A, I, or S.89. The AAV particle of embodiment 87 or 88, wherein [N4] comprises TG, SN, NN, WV, WA, WI, WS. GW, or NW.90. The AAV particle of any one of embodiments 87-89, wherein [N4] comprises TGW, SNW, NNW, GWV, GW A, NWV, GWI, or GWS.91. The AAV particle of any one of embodiments 87-90, wherein [N4] is or comprises TGWV (SEQ ID NO: 5066), TGWA (SEQ ID NO: 5067), SNWV (SEQ ID NO: 5069), NNWV (SEQ ID NO: 5072), TGWI (SEQ ID NO: 5074), or TGWS (SEQ ID NO: 5073).92. The AAV particle of any one of embodiments 87-91. wherein [N4] is or comprises TGWV (SEQ ID NO: 5066).93. The AAV particle of any one of embodiments 87-92, wherein [N1]-[N2]-[N3]-[N4] is or comprises:(i) the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214. 216, 219, 220.230, 232, 237. 238, 255, 262-265, 274, 283. 286, 290, 291, 293, 301, 306, 307, 308. 309, 314, and 336;(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, 5, 6, 7. 8. 9, 10. 11, 12, or 13 amino acids, e.g.. consecutive amino acids, thereof;(iii) an amino acid sequence comprising one, two. or three but no more than four modifications relative to any one of the amino acid sequences in (i); or(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).94. The AAV particle of any one of embodiments 87-93, wherein [N1]-[N2]-[N3]-[N4] is or comprises TNTQDWHRIAQTGWV (SEQ ID NO: 201).95. The AAV particle of any one of embodiments 1-94, wherein [N1]-[N2]-[N3] is present in loop VIII, optionally wherein loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 138 or 981.96. The AAV particle of any one of embodiments 43-50 and 87-95, wherein [N4] is present in loop VIII. optionally wherein loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 138 or 981.97. The AAV particle of any one of embodiments 1-96, wherein [Nl] replaces amino acids 582-585 (e.g.. T582, N583. H584. and Q585) of SEQ ID NO: 138.98. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39. 42-49, 51-69, 71-83. and 85-97. wherein [Nl] corresponds to amino acids 582-585 (e.g., T582, N583, T584, and Q585) of SEQ ID NO: 981.99. The AAV particle of any one of embodiments 1-98, wherein [Nl] is present at amino acids 582-585, as numbered according to SEQ ID NO: 138 or 981.100. The AAV particle of any one of embodiments 1-99, wherein [Nl] corresponds to positions 582-585 (e.g., T582, N583, T584, and Q585) of SEQ ID NO: 981.101. The AAV particle of any one of embodiments 1-100, wherein [N2] replaces amino acids 586-589 (e.g., S586, A587, Q588. and A589) of SEQ ID NO: 138.102. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101, wherein [N2] corresponds to amino acids 586-589 (e.g., D586, W587, H588, and R589) of SEQ ID NO: 981.103. The AAV particle of any one of embodiments 1-102, wherein [N2] is present at amino acids 586- 589, as numbered according to SEQ ID NO: 138 or 981.104. The AAV particle of any one of embodiments 1-103, wherein [N1]-[N2] replaces amino acids 582-589 (e.g., T582, N583, H584, Q585, S586, A587, Q588, and A589) of SEQ ID NO: 138.105. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-104, wherein [N1]-[N2] corresponds to amino acids 582-589 (e.g., T582, N583, T584, Q585, D586, W587, H588. and R589) of SEQ ID NO: 981.106. The AAV particle of any one of embodiments 1-105, wherein [N1]-[N2] is present at amino acids 582-589, as numbered according to SEQ ID NO: 138 or 981.107. The AAV particle of any one of embodiments 1-106, wherein [N3] replaces amino acids 590-592 (e.g., Q590. A591. and Q592) of SEQ ID NO: 138.108. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99. and 101-107, wherein [N3] corresponds to amino acids 590-592 (e.g., 1590. A591, and Q592) of SEQ ID NO: 981.109. The AAV particle of any one of embodiments 1-108, wherein [N3] is present at amino acids 590- 592. as numbered according to SEQ ID NO: 138 or 981.110. The AAV particle of any one of embodiments 1-109, wherein [N2]-[N3] replaces amino acids 586- 592 (e.g., S586, A587, Q588. A589, Q590, A591, and Q592) of SEQ ID NO: 138.111. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-110, wherein [N2]-[N3] corresponds to amino acids 586-592 (e.g., D586, W587, H588, R589, 1590, A591, and Q592) of SEQ ID NO: 981.112. The AAV particle of any one of embodiments 1-111, wherein [N2]-[N3] is present at amino acids 586-592, as numbered according to SEQ ID NO: 138 or 981.113. The AAV particle of any one of embodiments 1-112, wherein [N1]-[N2]-[N3] replaces amino acids 582-592 (e.g., T582, N583, H584, Q585, S586, A587, Q588. A589, Q590, A591, and / or Q592) of SEQ ID NO: 138.114. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-113. wherein [N1]-[N2]-[N3] corresponds to amino acids 582-592 (e.g.. T582. N583, T584. Q585, D586, W587. H588, R589, 1590. A591, Q592) of SEQ ID NO: 981.115. The AAV particle of any one of embodiments 1-114, wherein [N1]-[N2]-[N3] is present at amino acids 582-592, as numbered according to SEQ ID NO: 138 or 981.116. The AAV particle of any one of embodiments 43-50 and 87-115, wherein [N4] replaces amino acids 593-596 (e.g., T593, G594, W595, and V596) of SEQ ID NO: 138.117. The AAV particle of any one of embodiments 43-49 and 87-115, wherein [N4] corresponds to amino acids 593-596 (e.g., T593. G594, W595, and V596) of SEQ ID NO: 138 or 981.118. The AAV particle of any one of embodiments 43-50 and 87-117, wherein [N4] is present at amino acids 593-596, as numbered according to SEQ ID NO: 138 or 981.119. The AAV particle of any one of embodiments 43-50 and 87-118, wherein [N2]-[N3]-[N4] replaces amino acids 586-596 (e.g., S586, A587, Q588. A589. Q590, A591, Q592. T593. G594. W595, and V596) of SEQ ID NO: 138.120. The AAV particle of any one of embodiments 43-49 and 87-119. wherein [N2]-[N3]-[N4] corresponds to amino acids 586-596 (e.g., D586. W587, H588, R589, 1590, A591, Q592. T593, G594, W595, and V596) of SEQ ID NO: 981.121. The AAV particle of any one of embodiments 43-50 and 87-120, wherein [N2]-[N3]-[N4] is present at amino acids 586-596, as numbered according to SEQ ID NO: 138 or 981.122. The AAV particle of any one of embodiments 43-50 and 87-121, wherein [N1]-[N2]-[N3]-[N4] replaces amino acids 582-596 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, Q592, T593, G594, W595, and V596), as numbered according to SEQ ID NO: 138.123. The AAV particle of any one of embodiments 43-49 and 87-122, wherein [N1]-[N2]-[N3]-[N4] corresponds to amino acids 582-596 (e.g.. T582, N583, T584, Q585, D586, W587, H588, R589, 1590, A591, Q592, T593, G594, W595, and V596) of SEQ ID NO: 981.124. The AAV particle of any one of embodiments 43-50 and 87-123, wherein [N1]-[N2]-[N3]-[N4] is present at amino acids 582-596, as numbered according to SEQ ID NO: 138 or 981.125. The AAV particle of any one of embodiments 1-124, wherein [N2] is present immediately subsequent to [Nl],126. The AAV particle of any one of embodiments 1-125, wherein [N3] is present immediately subsequent to [N2] .127. The AAV particle of any one of embodiments 43-50 and 87-126, wherein [N4] is present immediately subsequent to [N3],128. The AAV particle of any one of embodiments 1-127, wherein the AAV capsid variant comprises, from N-terminus to C-terminus, [N1]-[N2]-[N3],129. The AAV particle of any one of embodiments 43-50 and 87-128, wherein the AAV capsid variant comprises, from N-terminus to C-terminus, |N1J N2J N3J-|N4J.130. An 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 (e.g., an AAV9 capsid variant) comprising:(a) the amino acid sequence of any one of the sequences provided in Table 1, 2A, 2B, 11, 26, 27, or 28;(b) an amino acid sequence comprising 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 consecutive amino acids from any one of the sequences provided in Table 1, 2A, 2B, 11, 26, 27, or 28; or(c) an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions relative to any one of the sequences provided in Table 1, 2A, 2B, 11, 26, 27, or 28; or(d) an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of any one of the sequences provided in Table 1. 2A, 2B. 11, 26, 27, or 28.131. An 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 (e.g., an AAV9 capsid variant) comprising:(a) the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336;(b) an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least8. at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 consecutive amino acids from any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238. 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336;(c) an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions, relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238. 255, 262-265. 274, 283, 286. 290, 291, 293, 301, 306, 307, 308, 309, 314. and 336; or(d) an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of any one of SEQ ID NOs: 201. 205-209, 211-214, 216. 219, 220, 230. 232, 237, 238. 255, 262-265. 274, 283, 286. 290, 291. 293. 301, 306. 307, 308, 309, 314, and 336.132. The AAV particle of embodiment 130 or 131. wherein the AAV capsid variant does not comprise at least 3, at least 4. at least 5. at least 6, at least 7, at least 8, or at least 9 consecutive amino acids from TNHQSAQAQ (SEQ ID NO: 5100), optionally wherein the TNHQSAQAQ (SEQ ID NO: 5100) corresponds to amino acids 582-592 of SEQ ID NO: 138.133. The AAV particle of any one of embodiments 130-132, wherein the AAV capsid variant does not comprise: TNH, TNHQ (SEQ ID NO: 4760), TNHQS (SEQ ID NO: 5101), TNHQSA (SEQ ID NO: 5102), TNHQSAQ (SEQ ID NO: 5103), TNHQSAQA (SEQ ID NO: 5104), TNHQSAQAQ (SEQ ID NO: 5100), NHQ, NHQS (SEQ ID NO: 5105), NHQSA (SEQ ID NO: 5106), NHQSAQ (SEQ ID NO: 5107), NHQSAQA (SEQ ID NO: 5108), NHQSAQAQ (SEQ ID NO: 5109), HQS, HQSA (SEQ ID NO: 5110), HQSAQ (SEQ ID NO: 5111), HQSAQA (SEQ ID NO: 5112), HQSAQAQ (SEQ ID NO: 5113), QSA, QSAQ (SEQ ID NO: 5114), QSAQA (SEQ ID NO: 5115), QSAQAQ (SEQ ID NO: 5116), SAQA (SEQ ID NO: 5117). or SAQAQ (SEQ ID NO: 5118).134. The AAV particle of any one of embodiments 130-124, wherein the AAV capsid variant comprises an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, at least 7. at least 8, at least9. at least 10, at least 11, at least 12, at least 13, or at least 14 consecutive amino acids from any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219. 220, 230, 232. 237, 238, 255, 262-265, 274. 283, 286, 290, 291, 293. 301, 306, 307. 308, 309, 314, and 336.135. The AAV particle of any one of embodiments 130-134. wherein the at least 3 consecutive amino acids comprise TQD.136. The AAV particle of any one of embodiments 130-135, wherein the at least 4 consecutive amino acids comprise TQDW (SEQ ID NO: 4684).137. The AAV particle of any one of embodiments 130-136, wherein the at least 5 consecutive amino acids comprise TQDWH (SEQ ID NO: 4685).138. The AAV particle of any one of embodiments 130-137, wherein the at least 6 consecutive amino acids comprise TQDWHR (SEQ ID NO: 4686).139. The AAV particle of any one of embodiments 130-138. wherein the at least 7 consecutive amino acids comprise TQDWHRI (SEQ ID NO: 941).140. The AAV particle of any one of embodiments 130-134, wherein the at least 3 consecutive amino acids comprise TNT.141. The AAV particle of any one of embodiments 130-134 and 140, wherein the at least 4 consecutive amino acids comprise TNTQ (SEQ ID NO: 4688).142. The AAV particle of any one of embodiments 130-134, 140. and 141, wherein the at least 5 consecutive amino acids comprise TNTQD (SEQ ID NO: 5119).143. The AAV particle of any one of embodiments 130-134 and 140-142, wherein the at least 6 consecutive amino acids comprise TNTQDW (SEQ ID NO: 5120).144. The AAV particle of any one of embodiments 130-134 and 140-143, wherein the at least 7 consecutive amino acids comprise TNTQDWH (SEQ ID NO: 5121).145. The AAV particle of any one of embodiments 130-134 and 140-144, wherein the at least 8 consecutive amino acids comprise TNTQD WHR (SEQ ID NO: 4898).146. The AAV particle of any one of embodiments 130-134 and 140-145, wherein the at least 9 consecutive amino acids comprise TNTQDWHRI (SEQ ID NO: 746).147. The AAV particle of any one of embodiments 130-146. wherein the AAV capsid variant comprises an amino acid sequence comprising at least one. at least two. or at least three but no more than four modifications relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209. 211-214,216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336.148. The AAV particle of any one of embodiments 130-147, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941).149. The AAV particle of any one of embodiments 130-147, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one. at least two. or at least three but no more than four modifications relative to the amino acid sequence of TNTQDWHRI (SEQ ID NO: 746).150. The AAV particle of any one of embodiments 130-149. wherein the AAV capsid variant comprises an amino acid sequence comprising at least one. at least two. or at least three but no more than four modifications relative to the amino acid sequence of ATNTQDWHRIAQT (SEQ ID NO: 744).151. The AAV particle of any one of embodiments 130-150, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two. or at least three but no more than four substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216. 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291. 293, 301, 306. 307, 308, 309, 314. and 336.152. The AAV particle of any one of embodiments 130-149 and 151, 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 four substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941).153. The AAV particle of any one of embodiments 130-149, 151, and 152, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions relative to the amino acid sequence of TNTQDWHRI (SEQ ID NO: 746).154. The AAV particle of any one of embodiments 130-153, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one. at least two. or at least three but no more than four substitutions relative to the amino acid sequence of ATNTQDWHRIAQT (SEQ ID NO: 744).155. The AAV particle of any one of embodiments 1-154, wherein the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NOs: 201. 205-209, 211-214. 216, 219, 220. 230, 232, 237, 238, 255, 262-265, 274, 283. 286, 290, 291. 293, 301, 306. 307, 308. 309, 314, and 336.156. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-155, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941), optionally wherein the amino acid sequence replaces amino acids 584-590 of SEQ ID NO: 138.157. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-156, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941), optionally wherein the amino acid sequence corresponds to amino acids 584-590 of SEQ ID NO: 981.158. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, 101-149. 151-153, and 155-157. wherein the AAV capsid variant comprises an amino acid sequence encoded by:(i) the nucleotide sequence of SEQ ID NO: 942;(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 modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 942; or(iii) 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: 942.159. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-158, wherein the AAV capsid variant comprises an amino acid sequence encoded by:(i) the nucleotide sequence of SEQ ID NO: 747;(ii) 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 sequence of SEQ ID NO: 747; or(iii) 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: 747.160. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-149, 151-153, and 155-158, wherein the nucleotide sequence encoding the AAV capsid variant comprises:(i) the nucleotide sequence of SEQ ID NO: 942;(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 modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 942; or(iii) 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: 942.161. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-160, wherein the nucleotide sequence encoding the AAV capsid variant comprises:(i) the nucleotide sequence of SEQ ID NO: 747;(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 modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 747; or(iii) 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: 747.162. The AAV particle of any one of embodiments 130-157, w herein the amino acid sequence provided in Table 1. 2 A, 2B. 11, 26, 1 , or 28 is present in loop VIII, optionally wherein loop VIII comprises amino acids 580-599, as numbered according to SEQ ID NO: 138 or 981.163. The AAV particle of any one of embodiments 130-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 11, 26, 27, or 28 replaces ammo acids 584, 585, 586, 587, 588, 589, and / or 590 (e.g., H584. Q585, S586, A587, Q588, A589, and / or Q590), as numbered according to the amino acid sequence of SEQ ID NO: 138.164. The AAV particle of any one of embodiments 130-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 11. 26, 27, or 28 is present at amino acids 584, 585, 586, 587, 588, 589, and / or 590, as numbered according to tire amino acid sequence of SEQ ID NO: 981.165. The AAV particle of any one of embodiments 130-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 11. 26, 27, or 28 corresponds to amino acids 584-590 (e.g., T584, Q585, D586. W587, H588, R589, and / or 1590), numbered according to the amino acid sequence of SEQ ID NO: 981.166. The AAV particle of any one of embodiments 121-157. wherein the amino acid sequence provided in Table 1, 2A, 2B, 11. 26. 27, or 28 replaces amino acids 582. 583, 584, 585. 586, 587. 588. 589, and / or 590 (e.g., T582. N583. H584, Q585, S586, A587, Q588. A589, and / or Q590) of SEQ ID NO: 138.167. The AAV particle of any one of embodiments 121-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 11. 26, 27, or 28 is present at amino acids 582, 583, 584, 585, 586, 587, 588, 589, and / or 590, as numbered according to the amino acid sequence of SEQ ID NO: 981.168. The AAV particle of any one of embodiments 121-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 11. 26, 27, or 28 replaces amino acids 582, 583, 584, 585. 586, 587, 588. 589, 590, 591, 592, 593. 594, 595, and / or 596 (e.g., T582, N583. H584, Q585, S586, A587, Q588. A589, Q590, A591, Q592, T593, G594, W595, and / or V5965) of SEQ ID NO: 138.169. The AAV particle of any one of embodiments 121-157. wherein the amino acid sequence provided in Table 1, 2A, 2B, 11. 26. 27, or 28 is present at amino acids 582, 583. 584, 585, 586, 587, 588. 589, 590, 591, 592. 593, 594. 595, and / or 596. as numbered according to the amino acid sequence of SEQ ID NO: 981.170. The AAV particle of any one of embodiments 1-169, wherein the AAV capsid variant comprises one. two, three, four, five, or all of:(i) an amino acid other than H at position 584 (e g.. T);(ii) an amino acid other than S at position 586 (e.g., D);(iii) an amino acid other than A at position 587 (e.g., W);(iv) an amino acid other than Q at position 588 (e.g., H);(v) an amino acid other than A at position 589 (e.g., R); and / or(vi) an amino acid other than Q at position 590 (e.g., I); wherein the amino acids in (i)-(vi) are numbered according to SEQ ID NO: 138.171. An AAV particle comprising:(a) 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(b) an AAV capsid variant (e g., an AAV9 capsid variant) comprising one, two, three, four, five, or all of:(i) an amino acid other than H at position 584 (e g., T);(ii) an amino acid other than S at position 586 (e.g., D);(iii) an amino acid other than A at position 587 (e.g.. W);(iv) an amino acid other than Q at position 588 (e.g.. H);(v) an amino acid other than A at position 589 (e.g., R); and / or(vi) an amino acid other than Q at position 590 (e.g., I); wherein the amino acids in (i)-(vi) are numbered according to SEQ ID NO: 138.172. The AAV particle of any one of embodiments 1-171, wherein the AAV capsid variant comprises one, two, three, four, five, or all of: T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and / or I at position 590, as numbered according to SEQ ID NO: 981.173. An 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 (e.g., an AAV9 capsid variant) comprising one, two. three, four, five, or all of: T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and / or I at position 590, as numbered according to SEQ ID NO: 981.174. The AAV particle of any one of embodiments 1-173, wherein the AAV capsid variant comprises one. two, three, four, five, or all of the substitutions H584T, S586D, A587W, Q588H, A589R, and / or Q590I, as numbered according to SEQ ID NO: 138.175. An 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’ poly nucleotide comprises an RNAi agent targeting DMPK mRNA; and(ii) an AAV capsid variant (e.g., an AAV9 capsid variant) comprising one, two, three, four, five or all of the substitutions H584T, S586D, A587W. Q588H, A589R, and / or Q590I, as numbered according to SEQ ID NO: 138.176. The AAV particle of any one of embodiments 1-175, wherein the AAV capsid variant comprises an amino acid other than H at position 584 (e.g., T), an amino acid other than S at position 586 (e g., D), an amino acid other than A at position 587 (e.g., W), an amino acid other than Q at position 588 (e.g., H), an amino acid other than A at position 589 (e.g., R), and an amino acid other than Q at position 590 (e.g., I), as numbered according to SEQ ID NO: 138.177. The AAV particle of any one of embodiments 1-176, wherein the AAV capsid variant comprises T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and I at position 590, as numbered according to SEQ ID NO: 981.178. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49. 51-69, 71-83, 85-98, and 100-164, wherein the AAV capsid variant comprises an amino acid at each of positions 584, 586, 587, 588. 589, and 590 that is different from the amino acid at the corresponding positions in SEQ ID NO: 138.179. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-178. wherein the AAV capsid variant comprises an amino acid at each of positions 582, 583, 584, 586. 587, 588, and 589 that is different from the amino acid at the corresponding positions in SEQ ID NO: 138.180. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21. 23-39, 42-49, 51-69. 71-83, 85-99. and 101-179, wherein the AAV capsid variant comprises an amino acid at each of positions 582. 583. 584, 586, 587. 588, 589, 590, 591, 592. 593, 594, 595. and 596 that is different from the amino acid at the corresponding positions in SEQ ID NO: 138.181. The AAV particle of any one of embodiments 1-181, wherein the AAV capsid variant comprises the substitutions H584T, S586D, A587W, Q588H, A589R, and Q590I, as numbered according to SEQ ID NO: 138.182. An 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 (e.g., an AAV9 capsid variant) comprising T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and I at position 590, as numbered according to SEQ ID NO: 981.183. An 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 (e.g., an AAV9 capsid variant) comprising the substitutions H584T, S586D, A587W, Q588H, A589R, and Q590I, as numbered according to SEQ ID NO: 138.184. The AAV particle of any one of embodiments 1-183, wherein the AAV capsid variant further comprises an amino acid other than A position 581, as numbered according to SEQ ID NO: 138 or 981.185. The AAV particle of any one of embodiments 1-184, wherein the AAV capsid variant further comprises a T at position 581 or a V at position 581. as numbered according to SEQ ID NO: 138 or 981.186. The AAV particle of any one of embodiments 1-185, wherein the AAV capsid variant comprises the substitutions A581T or A581V, as numbered according to SEQ ID NO: 138 or 981.187. The AAV particle of any one of embodiments 1, 3-7. 9-15, 17-20. 22-38, 40-48, 50, 130-134. 147, 151, 155, 162, 163, 166-177, and 181-186, wherein the AAV capsid variant comprises an amino acid other than Q at position 585, as numbered according to SEQ ID NO: 138.188. The AAV particle of any one of embodiments 1, 3-7, 9-15, 17-20, 22-38. 40-48, 50. 130-134, 147.151. 155, 162, 163. 166-177, and 181-187, wherein the AAV capsid variant comprises the amino acid K at position 585, as numbered according to SEQ ID NO: 138.189. The AAV particle of any one of embodiments 1-189, wherein the AAV capsid variant further comprises:(i) a modification in loop I, II, IV. and / or VI; and / or(ii) a substitution at position K449, e.g., a K449R substitution, as numbered according to SEQ ID NO: 138 or 981.190. The AAV particle of any one of embodiments 1-189, 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: 138.191. The AAV particle of any one of embodiments 1-190, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three, but no more than not more than 30. not more than 20, or not more than 10 substitutions relative to the amino acid sequence of SEQ ID NO: 138.192. The AAV particle of any one of embodiments 1-191, wherein die AAV capsid variant an amino acid sequence with at least 90% (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%) sequence identity to SEQ ID NO: 138.193. The AAV particle of embodiment 192, wherein the AAV capsid variant an amino acid sequence with at least 95% (e.g.. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 138.194. The AAV particle of any one of embodiments 1-193, wherein the AAV capsid variant comprises an amino acid sequence encoded by a sequence with at least 80% (e.g., 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%) sequence identity to SEQ ID NO: 137.195. The AAV particle of embodiment 194, wherein the AAV capsid variant comprises an amino acid sequence encoded by a sequence with at least 90% (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%) sequence identity to SEQ ID NO: 137.196. The AAV particle of any one of embodiments 1-195, wherein the AAV capsid variant comprises a VP1 protein, a VP2 protein, and a VP3 protein in an about 1:1 :10 ratio.197. The AAV particle of any one of embodiments 1-196, wherein the AAV capsid variant comprises amino acids 138-736, e.g., a VP2, of SEQ ID NO: 981, or a sequence with at least 80% (e.g., 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%) sequence identity thereto.198. The AAV particle of any one of embodiments 1-197, wherein the AAV capsid variant comprises amino acids 203-736, e.g., a VP3, of SEQ ID NO: 981, or a sequence with at least 80% (e.g., 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%) sequence identity thereto.199. The AAV particle of any one of embodiments 1-198, wherein the AAV capsid variant comprises an amino acid sequence that is at least 80% (e.g.. 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 amino acids 138-736. e.g., a VP2, of SEQ ID NO: 138.200. The AAV particle of any one of embodiments 1-199, wherein the AAV capsid variant comprises an amino acid sequence that is at least 80% (e.g., 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 amino acids 203-736, e.g., a VP3, of SEQ ID NO: 138.201. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, 101-186, and 189-200, wherein the AAV capsid variant comprises an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, or all 7 consecutive amino acids from the amino acid sequence of TQDWHRI (SEQ ID NO: 941), wherein:(i) the at least 3 consecutive amino acids comprise TQD:(ii) the at least 4 consecutive amino acids comprise TQDW (SEQ ID NO: 4684);(iii) the at least 5 consecutive amino acids comprise TQDWH (SEQ ID NO: 4685);(iv) the at least 6 consecutive amino acids comprise TQDWHR (SEQ ID NO: 4686); or(v) the 7 consecutive amino acids comprise TQDWHRI (SEQ ID NO: 941); wherein the AAV capsid variant comprises: (a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 981; (b) a VP2 protein comprising amino acids 138-736 of SEQ ID NO: 981 ; (c) a VP3 protein comprising amino acids 203-736 of SEQ ID NO: 981; or (d) an amino acid sequence with at least 80% (e.g., 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%) sequence identity to any one of the amino acid sequences in (a)-(c).202. The AAV particle of embodiment 201, wherein the AAV capsid variant comprises an amino acid sequence with at least 90% (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%) sequence identity to any one of the amino acid sequences in (a)-(c).203. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, 101-186, and 189-202, wherein the AAV capsid variant comprises 1, 2, or 3 substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941), wherein the AAV capsid variant comprises an amino acid sequence at least 90% (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 to the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981.204. The AAV particle of any one of embodiments 1-203, wherein the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NO: 981, or an amino acid sequence with at least 90% (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%) sequence identity thereto.205. The AAV particle of any one of embodiments 1-204, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least tw o. 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: 981.206. The AAV particle of any one of embodiments, 1-205. wherein 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 substitutions, relative to the amino acid sequence of SEQ ID NO: 981.207. The AAV particle of any one of embodiments 1-206, wherein the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983, or a nucleotide sequence with at least 80% (e.g.. 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%) sequence identity thereto.208. The AAV particle of embodiment 207, wherein the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983.209. The AAV particle of embodiment 207, wherein the AAV capsid variant is encoded by a codon- optimized nucleotide sequence.210. An AAV particle comprising an AAV capsid variant (e.g., an AAV9 capsid variant) comprising the amino acid sequence of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85- 99, 101-186, and 189, and further comprising an amino acid sequence at least 95% (e.g.. at least 95%, at least 96%, at least 97%, at least 98%. or at least 99%) identical to SEQ ID NO: 981.211. An 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 (e.g.. an AAV9 capsid variant) comprising the amino acid sequence of SEQ ID NO: 981.212. An 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 (e.g., an AAV9 capsid variant) comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983, or a nucleotide sequence at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical) thereto.213. An 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 (e.g., an AAV9 capsid variant) comprising amino acids 203-736 of SEQ ID NO: 981, or an amino acid sequence at least 80% (e.g.. 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, wherein the AAV capsid variant comprises T at position 584, D at position 586, W at position 587, H at position 588. R at position 589, and I at position 590, as numbered according to SEQ ID NO: 981.214. The AAV particle of embodiment 213, wherein the AAV capsid variant comprises amino acids 203- 736 of SEQ ID NO: 981.215. An 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 (e.g., an AAV9 capsid variant) comprising amino acids 203-736 of SEQ ID NO: 981.216. An 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 (e.g., an AAV9 capsid variant) comprising amino acids 138-736 of SEQ ID NO: 981, or an amino acid sequence at least 80% (e.g., 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, wherein the AAV capsid variant comprises T at position 584, D at position 586, W at position 587, H at position 588, R at position 589. and I at position 590, as numbered according to SEQ ID NO: 981.217. The AAV particle any one of embodiments 213-216, wherein the AAV capsid variant comprises amino acids 138-736 of SEQ ID NO: 981.218. An 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 (e.g.. an AAV9 capsid variant) comprising amino acids 138-736 of SEQ ID NO: 981.219. An 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 (e.g., an AAV9 capsid variant) comprising the amino acid sequence of SEQ ID NO: 981, or an ammo acid sequence at least 80% (e.g., 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, wherein the AAV capsid variant comprises T at position 584, D at position 586, W at position 587. H at position 588, R at position 589, and I at position 590, as numbered according to SEQ ID NO: 981.220. The AAV particle any one of embodiments 213-219, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981.221. The AAV particle of any one of embodiments 1-220, wherein the AAV capsid variant has 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 particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.222. The AAV particle of any one of embodiments 1-221, wherein the AAV capsid variant transduces a brain region, e.g., sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and / or cerebellum; optionally wherein the level of transduction is at least 39, at least 50, at least 100, at least 120, at least 132, at least 146, at least 150, at least 161, at least 174, at least 175, at least 200, at least 225, at least 250, at least 275, at least 283, at least 300, at least 350, at least 400, at least 450, at least 500, at least 525, at least 528, or at least 550-fold greater as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g.. when measured by an assay, e.g., an immunohistochemistry assay or a qPCR or ddPCR assay, e.g.. as described in Example 2.223. The AAV particle of any one of embodiments 1-222, which is enriched at least 10, at least 14, at least 20, at least 24, at least 50. at least 100. at least 150. at least 200. at least 250. at least 300. at least 350, at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1 or 3.224. The AAV particle of any one of embodiments 1-223, which is enriched at least 200. at least 300. at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1.225. The AAV particle of any one of embodiments 1-224, which is enriched in the brain of at least two or at least three species, e.g., a non-human primate and rodent (e.g., mouse), e.g., as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.226. The AAV particle of any one of embodiments 1-225, which is enriched at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65. at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 190, at least 200, at least 205, or at least 210-fold in the brain of at least two or at least three species, e.g., a non-human primate and rodent (e.g., mouse), compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1 or 4.L1. The AAV particle of embodiment 225 or 226, wherein the at least tw o or at least three species comprise Macaca fascicularis, CMorocebus sabaeus, Callithrix jacchus, and / or mouse (e.g., outbred mice).228. The AAV particle of any one of embodiments 1-227, which is enriched at least 2, at least 3, at least 4. at least 5, at least 10, at least 15, at least 17. at least 20, at least 50, at least 75, at least 100, at least 103, at least 107, at least 125, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 750, at least 1000, at least 1200-fold in the brain compared to an AAV particle comprising an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 981, e.g., when measured by an assay as described in Example 3.229. The AAV particle of any one of embodiments 1-228, which delivers an increased level of the modulatory polynucleotide to a brain region, optionally wherein the level of the modulatory polynucleotide is increased by at least 39, at least 50, at least 100, at least 120, at least 132, at least 146, at least 150, at least 161, at least 174, at least 175, at least 200, at least 225, at least 250, at least 275, at least 283, at least 300, at least 350, at least 400, at least 450, at least 500, at least 525, at least 528, or at least 550-fold as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e g., when measured by an assay, e g., a qRT-PCR, a ddPCR, or a qPCR assay (e.g., as described in Example 2).230. The AAV particle of any one of embodiments 1-229, which delivers an increased level of viral genomes to a brain region, optionally wherein the level of viral genomes is increased by at least 2, at least 5, at least 7, at least 10, at least 15, at least 19, at least 20, at least 22, or at least 25-fold as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay (e.g., as described in Example 2).231. The AAV particle of embodiment 229 or 230, wherein the brain region is a sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and / or cerebellum.232. The AAV particle of any one of embodiments 1-231, which is enriched at least 5, at least 10, at least 50, at least 100, at least 115, at least 120, at least 150, at least 175, at least 200, at least 207, at least 225, at least 250, or at least 275-fold in the spinal cord compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g.. when measured by an assay as described in Example 1 or 2.233. The AAV particle of any one of embodiments 1-232, wherein the AAV capsid variant has increased tropism for a muscle cell, tissue, or region, relative to the tropism of an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.234. The AAV particle of any one of embodiments 1-233, wherein the AAV capsid variant transduces a muscle cell, tissue, or region, e.g., cardiac, smooth, and / or skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius).235. The AAV particle of embodiment 234. wherein the level of transduction in the muscle cell, tissue, or region is at least 1.5, at least 2. 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 16, at least 17. at least 18, at least 19, at least 20, at least 21. at least 22, at least 23, at least 24. or at least 25-fold greater as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. e.g., when measured by an assay, e.g., a qPCR or ddPCR assay, e.g., as described in Example 2.236. The AAV particle of any one of embodiments 1-235, which is enriched at least 1.5, at least 2, at least 3, at least 4. at least 5. at least 6, at least 7, at least 10, at least 11, at least 12, at least 13. at least 14, at least 15. at least 16, at least 17, at least 18, at least 19, at least 20. at least 21, at least 22, at least 23, at least 24. at least 25, at least 26, at least 27. at least 28, at least 29, or at least 30-fold in the muscle cell, tissue, or region, as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1 or 3.237. The AAV particle of any one of embodiments 1-236, which delivers an increased level of die modulatory’ polynucleotide to a muscle, optionally wherein the level of the modulatory' polynucleotide is increased by at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30-fold as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay, e.g., a qRT-PCR. a ddPCR, or a qPCR assay (e.g., as described in Example 2).238. The AAV particle of any one of embodiments 1-237, which delivers an increased level of viral genomes to a muscle cell, tissue, or region, optionally wherein the level of viral genomes is increased by at least 1.5, at least 2, at least 5, at least 7. at least 10, at least 15, at least 19, at least 20, at least 23. at least 25, at least 28, at least 30. at least 33, at least 35, at least 38. at least 40, at least 43. or at least 45- fold, as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence ofSEQ ID NO: 138, e.g., when measured by an assay, e.g., a qRT-PCR or a qPCR assay (e.g., as described in Example 2).239. The AAV particle of any one of embodiments 1-238, wherein the AAV capsid variant has increased tropism for a heart cell or heart tissue (e.g., a heart ventricle or heart atrium), relative to the tropism of an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.240. The AAV particle of any one of embodiments 1-239, wherein the AAV capsid variant has an increased level of transduction in the heart cell or heart tissue as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g.. when measured by an assay, e.g.. a qPCR or ddPCR assay, e.g., as described in Example 2.241. The AAV particle of any one of embodiments 1-240. which is enriched at least 1.5. at least 2. at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8-fold in the heart cell or heart tissue compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1 or 3.242. The AAV particle of any one of embodiments 1-241, which delivers an increased level of the modulatory polynucleotide to a heart cell or heart tissue (e.g.. a heart ventricle or heart atrium), optionally wherein the level of the modulatory polynucleotide is increased by at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8-fold, as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay, e.g., a qRT-PCR, a ddPCR, or a qPCR assay (e.g., as described in Example 2).243. The AAV particle of any one of embodiments 1-242, which delivers an increased level of viral genomes to a heart cell or heart tissue (e.g., a heart ventricle or heart atrium), optionally wherein the level of viral genomes is increased by at least 1.5, 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 particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.[Embodiments 244-311 are intentionally absent.]312. The AAV particle of any one of embodiments 1-243, wherein the viral genome is single-stranded.313. The AAV particle of any one of embodiments 1-243, wherein the viral genome is self- complementary.314. The AAV particle of any one of embodiments 1-243, embodiment 312, or embodiment 313, wherein die 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 Rep78 protein and a Rep52 protein).315. The AAV particle of any one of embodiments 1-243, embodiment 312, or embodiment 313, 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 1-243 and 312-316, wherein the AAV particle is an isolated AAV particle.318. The AAV particle of any one of embodiments 1-243 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 1-243 and 312- 318.320. The cell of embodiment 319, wherein the 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 sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, or cerebellum.[Embodiments 322-325 are intentionally absent]326. A pharmacal composition comprising the AAV particle of any one of embodiments 1-243 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 AAVparticle of any one of embodiments 1-243 and 312-318, thereby delivering the modulatory 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 frontal cortex, sensory cortex, motor cortex, caudate, cerebellar cortex, cerebral cortex, brain stem, hippocampus, 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., quadriceps, vastus lateralis, and / or gastrocnemius)).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 1-243 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 1-243 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 1-243 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, neurodegenerative 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.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 to the subject intraarterially.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 pharmacal composition of embodiment 326 or the AAV particle of any one of embodiments 1 -243 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 pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-243 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 1-243 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 1-243 and 312-318 in the manufacture of a medicament.355. Use of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-243 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 an AAV 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 the AAV capsid variant comprises an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4681) in loop VIII: wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); and wherein:(i) [Nl] comprises amino acids Xi, X2, X3, and X4, wherein X4is Q, K, E, S, P, R, N, or H; and / or(ii) [N3] comprises amino acids X5, Xs, and X-, wherein X, is I, V, T, M, S, N, L, or F.357. The AAV particle of embodiment 356, wherein the AAV capsid variant is an AAV9 capsid variant.358. The AAV particle of embodiment 356 or embodiment 357, wherein the AAV capsid variant comprises an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 203-736 of SEQ ID NO: 981.359. The AAV particle of any one of embodiments 356-358. wherein loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 981.360. The AAV particle of any one of embodiments 356-359, wherein:(i) Xi is T, S. R, A. I. C, N, K. L, or Q;(ii) X2is N, T, G. V, S, Y. K, I, H. D, or F; and(iii) X3is T, N. K, D, I, S. P, A, Y, E, V. L, M. R, H, Q, or C.361. The AAV particle of any one of embodiments 356-360, wherein:(i) X6is A. Y, P, N, S, T, G, E, V. W, F, or Q; and(ii) X- is Q, G, N, K, H, R, E, L, P, or M.362. The AAV particle of any one of embodiments 356-361, wherein [Nl] comprises the amino acid sequence of TNTQ (SEQ ID NO: 4688).363. The AAV particle of any one of embodiments 356-362, wherein [N3] comprises the amino acid sequence of IAQ.364. The AAV particle of any one of embodiments 356-363, wherein [N1]-N2]-[N3] comprises the amino acid sequence of TNTQD WHRI AQ (SEQ ID NO: 343).365. The AAV particle of any one of embodiments 356-364, wherein:(i) [Nl] is present at amino acids 582-585;(ii) [N2] is present at amino acids 586-589; and(iii) [N3] is present at amino acids 590-592; wherein the amino acids are numbered according to SEQ ID NO: 981.366. The AAV particle of any one of embodiments 356-365, further comprising [N4] comprising amino acids Xg, X$>, X10, and Xu, wherein:(i) X8is T, S, N, P, A, or I;(ii) X9is G, N, D, R, V, A, S, or Q;(iii) Xiois W, S, C. R, L, or G; and / or(iv) Xu is V, A, S, I, C, G, D, F, L, or T.367. The AAV particle of embodiment 366. wherein [N4] comprises the amino acid sequence of TGWV (SEQ ID NO: 5066).368. The AAV particle of embodiment 366 or embodiment 367, wherein [N4] is present at amino acids 593-596 as numbered according to SEQ ID NO: 981.369. The AAV particle of any one of embodiments 356-368, wherein [N1]-[N2]-[N3]-[N4] comprises the amino acid sequence of TNTQDWHRI AQTGW V (SEQ ID NO: 201).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 at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 203-736 of SEQ ID NO: 981, wherein the AAV capsid variant comprises:T at position 584,D at position 586,W position 587,H at position 588,R at position 589, andI at position 590; wherein the amino acids are numbered according to SEQ ID NO: 981.371. The AAV particle of any one of embodiments 356-370, wherein the AAV capsid variant comprises an amino acid sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%. at least 98%, or at least 99%) identity to amino acids 138-736 of SEQ ID NO: 981.372. 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 at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 138-736 of SEQ ID NO: 981, wherein the AAV capsid variant comprises:T at position 584,D at position 586,W position 587,H at position 588,R at position 589, andI at position 590; wherein the amino acids are numbered according to SEQ ID NO: 981.373. The AAV particle of any one of embodiments 356-372, wherein the AAV capsid variant comprises an amino acid sequence having at least 95% (e g., at least 95%, at least 96%, at least 97%, at least 98%. or at least 99%) identity to SEQ ID NO: 981.374. 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% (c.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 981, wherein the AAV capsid variant comprises:T at position 584,D at position 586,W position 587,H at position 588,R at position 589, andI at position 590; wherein the amino acids are numbered according to SEQ ID NO: 981.375. The AAV particle of embodiment 374. wherein the AAV capsid variant comprises:(i) a VP1 protein comprising an amino acid sequence having at least 99% identity to SEQ IDNO: 981;(ii) a VP2 protein comprising an amino acid sequence having at least 99% identity to amino acids 138-736 of SEQ ID NO: 981; and / or(iii) a VP3 protein comprising an amino acid sequence having at least 99% identity to amino acids 203-736 of SEQ ID NO: 981.376. The AAV particle of any one of embodiments 370-375, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941).377. The AAV particle of any one of embodiments 370-376. wherein the AAV capsid variant comprises at least 8, at least 9, or at least 10 consecutive amino acids from the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343).378. The AAV particle of any one of embodiments 370-377, wherein the AAV capsid variant comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343) at amino acids 582-592 as numbered according to SEQ ID NO: 981.379. The AAV particle of any one of embodiments 376-378, wherein the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or TNTQDWHRIAQ (SEQ ID NO: 343) is present in loop VIII, wherein loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 981.380. The AAV particle of any one of embodiments 356-379, wherein the AAV capsid variant comprises:(i) a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981;(ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981; and / or(iii) a VP3 protein comprising or consisting of die amino acid sequence according to positions 203-736 of SEQ ID NO: 981.381. The AAV particle of any one of embodiments 1-243, 312-318, and 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: 6413-6416, or a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical thereto;(b) a loop region, optionally comprising any one of SEQ ID NOs: 6417-6421. or a nucleotide sequence at least 85%. at least 90%, at least 95%, at least 96%. at least 97%, at least 98%. or at least 99% identical thereto; and(c) a 3’ flanking region, optionally comprising any one of SEQ ID NOs: 6422-6427, or a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.382. The AAV particle of embodiment 381, wherein:(a) the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 6414 or SEQ ID NO: 6415, or a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto;(b) the loop region of the molecular scaffold comprises SEQ ID NO: 6417, SEQ ID NO: 6418, or SEQ ID NO: 6421, or a nucleotide sequence at least 85%, at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; and(c) the 3’ flanking region of the molecular scaffold comprises SEQ ID NO: 6423, SEQ ID NO: 6424. or SEQ ID NO: 6425, or a nucleotide sequence at least 85%. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.383. The AAV particle of embodiment 382, wherein the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 6414. or a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; the loop region of the molecular scaffold comprises SEQ ID NO: 6417, or a nucleotide sequence at least 85%, at least 90%. at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical thereto; and the 3’ flanking region of the molecular scaffold comprises SEQ ID NO: 6423, or a nucleotide sequence at least 85%, at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.384. The AAV particle of embodiment 382, wherein the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 6415, or a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; the loop region of die molecular scaffold comprises SEQ ID NO: 6421, or a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; and the 3’ flanking region of die molecular scaffold comprises SEQ ID NO: 6425, or a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.385. The AAV particle of embodiment 382, wherein the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 6414, or a nucleotide sequence at least 85%, at least 90%. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto: the loop region of the molecular scaffold comprises SEQ ID NO: 6417, or a nucleotide sequence at least 85%. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical thereto; and the 3’ flanking region of themolecular scaffold comprises SEQ ID NO: 6424, or a nucleotide sequence at least 85%, at least 90%, at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.386. The AAV particle of embodiment 382, wherein the 5’ flanking region of the molecular scaffold comprises SEQ ID NO: 6414, or a nucleotide sequence at least 85%, at least 90%. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto: the loop region of the molecular scaffold comprises SEQ ID NO: 6418, or a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical thereto; and the 3’ flanking region of the molecular scaffold comprises SEQ ID NO: 6423, or a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto.387. The AAV particle of any one of embodiments 1-243, 312-318, and 356-386, wherein the modulatory polynucleotide comprises a siRNA.388. The AAV particle of embodiment 387. wherein the modulatory' polynucleotide 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’ arm and a 3’ arm of a stem loop structure, wherein the passenger strand is located between the 5’ flanking region and the loop region and the guide strand is located betw een the loop region and the 3 ’ flanking region.389. The AAV particle of embodiment 387, wherein the modulatory polynucleotide 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 (c.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 betw een 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 and / or wherein the passenger 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: 6428 or SEQ ID NO: 6429, or a trinucleotide repeat expansion thereof.395. The AAV particle of any one of embodiments 1-243, 312-318, and 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 1-243, 312-318, and 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 or an ITR sequence positioned 3’ relative to the sequence encoding the modulator}’ polynucleotide.398. The AAV particle of embodiment 396 or embodiment 397, 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.399. The AAV particle of any one of embodiments 1-243, 312-318, and 356-399, wherein the viral genome further comprises a polyadenylation (poly A) sequence.400. A cell comprising the AAV particle of any one of embodiments 1-243, 312-318. and 356-399, optionally wherein the cell is a mammalian cell (e g., an HEK293 cell), an msect cell (e.g., an S19 cell), or a bacterial cell.401. A method of making the AAV particle of any one of embodiments 1-243. 312-318, and 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: 981.403. 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 the 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 S19 cell), or a bacterial cell.406. A pharmaceutical composition comprising the AAV particle of any one of embodiments 1-243. 312-318, and 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 1-243, 312-318, and 356-399, 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 1-243, 312-318. and 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 subject 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 (SEQ ID NO: 6436).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 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.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 406 or the AAV particle of any one of embodiments 1-243, 312-318, and 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 subject.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.423. The method of embodiment 422, wherein the muscle is one or more of cardiac, smooth, and / or skeletal muscle.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.425. The method of embodiment 424, wherein the pharmaceutical composition or AAV particle is delivered to the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen. substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region).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, tire 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, e.g., cardiac, smooth, and / or skeletal muscle (e.g.. quadriceps, vastus lateralis, and / or gastrocnemius).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., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus. oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g.. cervical 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 embodiments 407-421, any one of embodiments 424-427, or embodiment 430, wherein administering tire 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., amygdala, brainstem, caudate, central grey, cerebellum (e.g.. Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen. substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus. gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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 type 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 1-243, 312-318, and 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 1-243. 312-318, and 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 an effective amount of the pharmaceutical composition of embodiment 406 or the AAV particle of any one of embodiments 1-243, 312-318, and 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-rclatcd 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 AAV capsid variant comprising the sequence encoding the modulatory polynucleotide for reducing or eliminating expression of mutated DMPK mRNA to a cell or tissue in a subject. In some embodiments, the present disclosure provides a method for treating myotonic dystrophy type 1 (DM1) or another disorder associated with mutated DMPK mRNA expression (i.e., another DMPK-related disorder). 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 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-term 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 seroty pes, 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 genome may be delivered to a target cell, tissue, or organism. In some embodiments, the target cell or tissue is indie 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., quadriceps, vastus lateralis, and / or gastrocnemius), smooth muscle, and / or cardiac muscle tissue. In some embodiments, the target cell or tissue is in the brain (e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, and / or vestibular nucleus).

[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 amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g.. frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary’ complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and / or thoracic spinal cord region)), (v) elevated modulatory polynucleotide 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 itsamount 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 some embodiments, the modulator} 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., AAV9 capsid variants) with improved properties compared to wildtype AAV9. such as (i) increased penetrance through the blood brain barrier following intravenous administration, (ii) wider distribution throughout the multiple brain regions, e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, and / or vestibular nucleus, (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., quadriceps, vastus lateralis, and / or gastrocnemius), 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 modulatory polynucleotide to multiple regions of the brain. Exemplary' regions may comprise the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, and / or vestibular nucleus. In some embodiments, the AAV capsid variants described herein enhance the delivery of a modulatory polynucleotide 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, the 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 tire forebrain. In some embodiments, the AAV capsid variants are capableof increasing expression of a modulatory polynucleotide in the dentate nucleus. In some embodiments, die 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 comprises an 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, die DM1 is congenital DM1.I. CompositionsAdeno-associated viral (AA V) Particles

[0082] AAVs have a genome of about 5,000 nucleotides in length and which 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-type 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 serotype, as a non-limiting example, for AAV9 / hu. l4 (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 the amino acid sequence of SEQ ID NO: 981, VP1 comprises amino acids 1-736, VP2 comprises amino acids 138-736, and VP3 comprises amino acids 203-736. In other words, VP 1 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 similarlydescribed. 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 ty pically 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 and Samulski 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 poly nucleotide (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 (e.g., one or more substitutions) in loop VIII of AAV9, e.g., at one or more amino acids corresponding to positions 580-599 of VP1 of AAV9. In some embodiments, an AAV capsid variant comprises a substitution at one, two, three, four, five, or all of amino acids corresponding to position(s) 584, 586. 587, 588, 589. and / or 590 of the amino acid sequence of SEQ ID NO: 138 or 981. 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, loop VIII comprises amino acids corresponding topositions 580-599 (e.g., the positions of amino acids VATNHQSAQAQAQTGWVQNQ (SEQ ID NO: 5122)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII is present (is located) at amino acids comprising those corresponding to positions 580-599 (e g., amino acids VATNHQSAQAQAQTGWVQNQ (SEQ ID NO: 5122)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII comprises amino acids corresponding to positions 582-593 (e.g., the positions of amino acids TNHQSAQAQAQT (SEQ ID NO: 5123)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII comprises amino acids corresponding to positions 587-593 (e.g., the positions of amino acids AQAQAQT (SEQ ID NO: 4687)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII comprises amino acids corresponding to positions 587-590 (e.g.. the positions of amino acids AQAQ (SEQ ID NO: 5099)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 587-590 (e.g., the positions of amino acids AQAQ (SEQ ID NO: 5099)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII or variable region VIII (VR- VIII) is as described in DiMattia et al. “Structural Insights into the Unique Properties of the Adeno- Associated Virus Serotype 9,” Journal of Virology. 12(86):6947-6958 (the contents of which are hereby incorporated by reference in their entirety), e.g., comprising amino acids corresponding to positions 581 - 593 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 581-593 of the amino acid sequence of SEQ ID NO: 138.

[0091] The AAV particles and payloads 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 nerv ous systems (CNS and PNS, respectively ). In some embodiments, an AAV particle may, in addition, or alternatively, have decreased tropism for a ccll-ty pc, 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 inwild-type AAV) at the 5’ and 3’ ends of the ssDNA which fonn 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 anneal 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 modifying 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 arc 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 tire AAV capsid variant. In some embodiments, the modification (e.g., comprising one or more substitutions relative to a wildtype 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 AAV9 capsid variant.|0991 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 variantmay increase distribution of an AAV particle to a cell, tissue, or region of two or more (e.g., tw o 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 amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory' cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus. oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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.

[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. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to the CNS (e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory' cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary' complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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. 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 the intravenous 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. In 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.

[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 quadriceps muscle, a vastus lateralis muscle, and / or a gastrocnemius 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) a sequence as set forth in Table 1. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 2A. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 2B (e.g.. a sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219. 220, 230, 232. 237, 238, 255, 262-265, 274. 283, 286, 290, 291, 293, 301, 306, 307. 308. 309, 314, and 336). In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence set forth in Table 11. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 26. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 27. In some embodiments, the AAV capsid variant is an AAV9 capsid variant.Tabic 1. Exemplary SequencesTable 2A. Exemplary SequencesTable 2B. Exemplary Sequences

[0108] In some embodiments, the AAV capsid variant comprises (c.g., in loop VIII) an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4681), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein [Nl] comprises amino acids Xi, X2, X3, and X4, wherein X4 is Q, K, E, S, P, R, N, H, or a conservative substitution thereof and / or [N3] comprises X5, Xe, and X7, wherein X3is I, V, T, M, S, N, L, F, or a conservative substitution thereof. In some embodiments, [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682), [Nl] comprises Xi, X2, X3, and X4, wherein X4is Q, K, E, S, P, R. N, or H, and / or [N3] comprises X5, X6, and X7, wherein X5 is I, V. T, M. S, N, L. or F. In some embodiments, X4 of [Nl] is Q. In some embodiments, X4 of [Nl] is K. In some embodiments, X5 of [N3] is I. In some embodiments, Xi of [Nl] is T, S, R. A, I, C. N, K, L, or Q. In some embodiments. X2of [Nl] is N, T. G, V. S, Y, K. I. H, D. or F. In some embodiments. X3of [ N 11 is T, N. K, D. 1. S, P, A. Y, E, V. L, M. R, H. Q, or C. In some embodiments. [Nl] is or comprises TNTQ (SEQ ID NO: 4688). In some embodiments, [Nl] is or comprises TNTK (SEQ ID NO: 4689). In some embodiments, [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898). In some embodiments. [N 1]-[N2] is or comprises TNTKDWHR (SEQ ID NO: 4899). In some embodiments, Xe of [N3] is A, Y. P. N, S, T. G, E. V, W. F, or Q. In some embodiments. X- of [N3] is Q. G. N, K. H, R, E. L, P, or M. In some embodiments, [N3] is or comprises IAQ. In some embodiments, [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027). In some embodiments, [Nl ]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343). In some embodiments, [N1]-[N2]-[N3] is or comprises TNTKDWHRIAQ (SEQ ID NO: 344).

[0109] In some embodiments, the amino acid sequence having the formula [N1]-[N2]-[N3] further comprises [N4], wherein [N4] comprises amino acids X8, X9. X10, and Xu, wherein X8is T, S, N, P, A, or I; X9is G, N, D, R, V, A. S, or Q; Xj0is W, S, C, R, L, or G; and / or Xnis V, A, S, I, C, G, D, F, L, or T. In some embodiments, [N4] is or comprises TGWV (SEQ ID NO: 5066). In some embodiments, [Nl]- [N2]-[N3]-[N4] is or comprises any one of SEQ ID NOs: 201-245, 247-250, 253-255, 257-265, 268-274, 276-286, 288, 290-297, 299-303, 305-309, 311, 313-319, 323-328, 330-337, 339-342, 539-542, 544, 546, 547, 549-557, 559-589, 592, 593, 595, 596, 598, 599, 601-608, 610-614, 616-622, 625, 628, 630, 631, 633, 636, 638, 639-646, 649, 651-657, 667, 669, 670, 672, 673, 679-683, 685-690, 692, 693, 695, 697, 699-701, 703-705, 708-710, 712-717, 719-723, 728-731, 733-738, 740, and 742. In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises TNTQDWHRIAQTGWV (SEQ ID NO: 201). In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises TNTKDWHRIAQTGWV (SEQ ID NO: 202).

[0110] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4683), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein [Nl] comprises amino acids Xi, X2, X3. and X4, wherein X4is Q. P, or a conservative substitution thereof; and / or [N3] comprises amino acids X5. X6. and X7, wherein X5is I, V. or a conservative substitution thereof. In some embodiments. |N2| comprisestlie amino acid sequence of DWHR (SEQ ID NO: 4682); [Nl] comprises amino acids Xi, X2, X3, and X4, wherein X , is Q or P; and / or [N3] comprises amino acids X5, Xe, and X7, wherein Xs is I or V. In some embodiments, X 1 of [Nl] is Q. In some embodiments, X of [N3] is I. In some embodiments, X5 of [N3] is V. In some embodiments, Xi of [Nl] is T or S. In some embodiments, X2of [Nl] is N, T, G, S, I, or V. In some embodiments, X3of [Nl] is T, N, I, S, A, V, or L. In some embodiments, [Nl] is or comprises TNTQ (SEQ ID NO: 4688). In some embodiments, [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898). In some embodiments, Xe of [N3] is A, P, S. Y, or N. In some embodiments, X7 of [N3] is Q. G, or N. In some embodiments. [N3] is or comprises IAQ. In some embodiments, [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027). In some embodiments, [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343). In some embodiments, the amino acid sequence having the formula [Nl ]-[N2]-[N3] further comprises [N4], wherein [N4] comprises amino acids X8, X9, X10, and Xu. wherein X10 is W. In some embodiments. X8of [N4] is T, S. or N. In some embodiments, X9of [N4] is G or N. In some embodiments. Xu of [N4] is V, A, I, or S. In some embodiments, [N4] is or comprises TGWV (SEQ ID NO: 5066). In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises any one of SEQ ID NOs: 201. 205-209, 211-214. 216, 219, 220. 230, 232, 237. 238, 255, 262-265, 274, 283. 286, 290. 291, 293, 301. 306, 307, 308. 309, 314. and 336. In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises TNTQDWHRIAQTGWV (SEQ ID NO: 201).

[0111] In some embodiments. [N1]-[N2]-[N3] is present in loop VIII of the AAV capsid variant. In some embodiments, [N4] is present in loop VIII of the AAV capsid variant. In some embodiments, [Nl]- [N2]-[N3]-[N4] is present in loop VIII of the AAV capsid variant. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 581-593 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, loop VIII is present at amino acids comprising those corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981.

[0112] In some embodiments, [Nl] is present at amino acids corresponding to positions 582-585 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, Xi of [Nl] is present at an amino acid corresponding to position 582 of the amino acid sequence of SEQ ID NO: 138 or 981, X2of [Nl] is present at an amino acid corresponding to position 583 of the amino acid sequence of SEQ ID NO: 138 or 981, X3of [Nl] is present at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 138 or 981, and X4of [Nl] is present at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, [Nl] replaces amino acids corresponding to positions 582-585 (e g., T582, N583, H584, and Q585) of the amino acid sequence of SEQ ID NO: 138.

[0113] In some embodiments, [N2] is present at amino acids corresponding to positions 586-589 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N2] replaces amino acids corresponding to positions 586-589 of the amino acid sequence of SEQ ID NO: 138 (e.g., S586. A587, Q588, and A589 may be replaced with D586. W587, H588. and R589).

[0114] In some embodiments, [N1]-[N2] is present at amino acids corresponding to positions 582- 589 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [Nl]- [N2] replaces amino acids corresponding to positions 582-589 of die amino acid sequence of SEQ ID NO: 138 (e.g., T582. N583, H584, Q585, S586, A587, Q588, and A589 may be replaced witli T582, N583, T584, Q585, D586, W587, H588, and R589).

[0115] In some embodiments, [N3] is present at amino acids corresponding to positions 590-592 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, Xs of [N3] is present at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981, X6of [N3] is present at an amino acid corresponding to position 591 of the amino acid sequence of SEQ ID NO: 138 or 981, and X7of [N3] is present at an amino acid corresponding to position 592 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, [N3] replaces amino acids corresponding to positions 590-592 (e.g., 1590, A591, and Q592) of the amino acid sequence of SEQ ID NO: 138.

[0116] In some embodiments, [N2]-[N3] is present at amino acids corresponding to positions 586- 592 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N2]- [N3] replaces amino acids corresponding to positions 586-592 of the amino acid sequence of SEQ ID NO: 138 (e.g., S586, A587. Q588, A589, Q590, A591, and Q592 may be replaced with D586, W587. H588. R589. 1590, A591. and Q592).

[0117] In some embodiments, [N1]-[N2]-[N3] is present at amino acids corresponding to positions 582-592 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments. [N1]-[N2]-[N3] replaces amino acids corresponding to positions 582-592 of the amino acid sequence of SEQ ID NO: 138 (e.g., T582. N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, and Q592 may be replaced with T582, N583, T584, Q585, D586, W587, H588, R589, 1590, A591, and Q592).

[0118] In some embodiments, [N4] is present at amino acids corresponding to positions 593-596 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, X8of [N4] is present at an amino acid corresponding to position 593 of the amino acid sequence of SEQ ID NO: 138 or 981, Xg of [N4] is present at an amino acid corresponding to position 594 of the amino acid sequence of SEQ ID NO: 138 or 981, Xio of [N4] is present at an amino acid corresponding to position 595 of the amino acid sequence of SEQ ID NO: 138 or 981, and Xu of [N4] is present at an amino acid corresponding to position 596 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, [N4] replaces amino acids corresponding to positions 593-596 (e g., T593, G594, W595, and V596) of the amino acid sequence of SEQ ID NO: 138.

[0119] In some embodiments, [N2]-[N3]-[N4] is present at amino acids corresponding to positions 586-596 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N2]-[N3]-[N4] replaces amino acids corresponding to positions 586-596 of the amino acid sequence of SEQ ID NO: 138 (e.g.. S586, A587, Q588, A589. Q590. A591, Q592, T593, G594. W595, and V596 may be replaced with D586, W587. H588, R589, 1590, A591, Q592, T593. G594. W595, and V596).

[0120] In some embodiments, [N1]-[N2]-[N3]-[N4] is present at amino acids corresponding to positions 582-596 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N1]-[N2]-[N3]-[N4] replaces amino acids corresponding to positions 582-596 of the amino acid sequence of SEQ ID NO: 138 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, Q592, T593, G594, W595, and V596 may be replaced with T582, N583. T584, Q585, D586, W587, H588, R589, 1590, A591, Q592, T593, G594, W595, and V596).

[0121] In some embodiments, [N2] is present immediately subsequent to [Nl], In some embodiments, [N3] is present immediately subsequent to [N2], In some embodiments. [N4] is present immediately subsequent to [N3] . In some embodiments, loop VIII of the AAV capsid variant comprises an amino acid sequence comprising, from N-tenninus to C-terminus, [N1]-[N2]-[N3], In some embodiments, loop VIII of the AAV capsid variant comprises an amino acid sequence comprising, from N-terminus to C-terminus, [N 1]-[N2]-[N3]-[N4] .

[0122] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising 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 consecutive amino acids from any one of the sequences provided in Table 1, 2A, 2B, 11, 26. 27, or 28. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising 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 consecutive amino acids from any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220. 230, 232, 237. 238, 255, 262-265, 274. 283, 286, 290, 291, 293, 301, 306, 307. 308, 309, 314. and 336.

[0123] In some embodiments, the at least 3 consecutive amino acids comprise TQD. In some embodiments, the at least 4 consecutive amino acids comprise TQDW (SEQ ID NO: 4684). In some embodiments, the at least 5 consecutive amino acids comprise TQDWH (SEQ ID NO: 4685). In some embodiments, die at least 6 consecutive amino acids comprise TQDWHR (SEQ ID NO: 4686). In some embodiments, die at least 7 consecutive amino acids comprise TQDWHRI (SEQ ID NO: 941).

[0124] In some embodiments, the at least 3 consecutive amino acids comprise TNT. In some embodiments, die at least 4 consecutive amino acids comprise TNTQ (SEQ ID NO: 4688). In some embodiments, the at least 5 consecutive amino acids comprise TNTQD (SEQ ID NO: 5119). In some embodiments, the at least 6 consecutive amino acids comprise TNTQDW (SEQ ID NO: 5120). In some embodiments, the at least 7 consecutive amino acids comprise TNTQDWH (SEQ ID NO: 5121). In some embodiments, the at least 8 consecutive amino acids comprise TNTQDWHR (SEQ ID NO: 4898). In some embodiments, the at least 9 consecutive amino acids comprise TNTQDWHRI (SEQ ID NO: 746).

[0125] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one. at least two. or at least three modifications, but no more than four modifications, relative to the amino acid sequence of any one of the sequences provided in Table 1, 2A, 2B. 11, 26, 27, or 28. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one, at least two, or at least three substitutions, but no more thanfour substitutions, relative to the amino acid sequence of any one of the sequences provided in Table 1, 2A, 2B, 11, 26, 27, or 28. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an ammo acid sequence comprising at least one, at least two, or at least three modifications, but no more than four modifications, relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307,308, 309, 314, and 336. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one, at least two, or at least three substitutions, but no more than four substitutions, relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209. 211- 214, 216, 219. 220, 230, 232. 237, 238, 255, 262-265, 274. 283, 286, 290, 291, 293. 301, 306, 307. 308,309, 314, and 336.

[0126] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one. at least two. or at least three modifications, but no more than four modifications, relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence comprising at least one, at least two, or at least three substitutions, but no more than four substitutions, relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941).

[0127] In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of the sequences provided in Table 1, 2A, 2B, 11, 26, 27. or 28. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NOs: 201. 205-209, 211-214, 216, 219, 220. 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301. 306, 307, 308. 309, 314, and 336.

[0128] In some embodiments, the amino acid sequence is present in loop VIII. In some embodiments, loop VIII comprises amino acids corresponding to positions 581-593 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII comprises amino acids corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, loop VIII is present (is located) at amino acids comprising those corresponding to positions 580-599 (e.g., the positions of amino acids VATNHQSAQAQAQTGWVQNQ (SEQ ID NO: 5122)) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces 1, 2. 3, 4, 5, 6, 7, 8. 9, 10. 11, 12, 13, 14, or all of amino acids corresponding to position(s) 582 (e.g., T582). 583 (e.g., N583), 584 (e.g., H584), 585 (e.g., Q585), 586 (e.g.. S586), 587 (e.g., A587), 588 (e.g., Q588), 589 (e.g., A589), 590 (e.g., Q590), 591 (e.g., A591), 592 (e.g.. Q592), 593 (e.g., T593), 594 (e.g., G594), 595 (e.g.. W595), and / or 596 (e.g., V596) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence is present at 3. 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, or all of amino acids corresponding to position(s) 582. 583, 584, 585. 586, 587, 588, 589, 590, 591, 592, 593. 594, 595, and / or 596 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises one or more substitutions at amino acids corresponding to position(s) 582 (e.g.. T582), 583 (e.g., N583), 584 (e.g.. H584), 585 (e.g., Q585). 586 (e.g.. S586), 587 (e.g.. A587), 588 (e.g., Q588). 589(e.g., A589), 590 (e.g., Q590), 591 (e.g, A591), 592 (e.g, Q592), 593 (e.g., T593), 594 (e.g, G594), 595 (e.g., W595), and / or 596 (e.g., V596) of the amino acid sequence of SEQ ID NO: 138.

[0129] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence encoded by a nucleotide sequence described herein, e.g., a nucleotide sequence of Table 2A. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) 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: 942. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) 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 different substitutions, but not more than ten substitutions, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 942, 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.

[0130] In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, or a portion thereof that is modified relative to a wildtype AAV capsid (e.g., comprising one or more substitutions relative to a wildtype AAV capsid), comprises a nucleotide sequence described herein, e.g.. as described in Table 2A. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, or a portion thereof that is modified relative to a wildtype AAV capsid (e.g.. comprising one or more substitutions relative to a wildtype AAV capsid), is codon optimized. In some embodiments, the nucleotide sequence is an isolated nucleotide sequence. In some embodiments, the nucleotide sequence is a recombinant nucleotide sequence.

[0131] In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, or a portion thereof that is modified relative to a wildtypc AAV capsid (e.g., comprising one or more substitutions relative to a wildtype AAV capsid), comprises the nucleotide sequence of SEQ ID NO: 942, or 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 sequence of SEQ ID NO: 942. In some embodiments, the nucleotide sequence 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 substitutions, but no more than ten substitutions, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 942. 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.

[0132] In some embodiments, the AAV capsid variant further comprises an amino acid other than A at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981. Insome embodiments, the AAV capsid variant further comprises the ammo acid T at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant further comprises the amino acid V at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant comprises the substitution A581T or A581V, wherein the substitution is at a position that corresponds to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0133] In some embodiments, an AAV capsid variant described herein comprises one. two, three, four, five, or all of an amino acid other than H at a position corresponding to position 584 (e.g., comprises T at an amino acid corresponding to position 584) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than S at a position corresponding to position 586 (e.g., comprises D at an amino acid corresponding to position 586) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 587 (e.g., comprises W at an amino acid corresponding to position 587) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than Q at a position corresponding to position 588 (e.g., comprises H at an amino acid corresponding to position 588) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 589 (e.g., comprises R at an amino acid corresponding to position 589) of the amino acid sequence of SEQ ID NO: 138; and / or an amino acid other than Q at a position corresponding to position 590 (e.g., comprises I at an amino acid corresponding to position 590) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises an amino acid other than H at a position corresponding to position 584 (e.g., comprises T at an amino acid corresponding to position 584) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than S at a position corresponding to position 586 (e.g., comprises D at an amino acid corresponding to position 586) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 587 (e.g., comprises W at an amino acid corresponding to position 587) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than Q at a position corresponding to position 588 (e.g., comprises H at an amino acid corresponding to position 588) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 589 (e.g., comprises R at an amino acid corresponding to position 589) of the amino acid sequence of SEQ ID NO: 138; and an amino acid other than Q at a position corresponding to position 590 (e.g., comprises I at an amino acid corresponding to position 590) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid T at a position corresponding to position 584 of the amino acid sequence of SEQ ID NO: 138 or 981. amino acid D at a position corresponding to position 586 of the amino acid sequence of SEQ ID NO: 138 or 981. amino acid W at a position corresponding to position 587 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid H at a position corresponding to position 588 of the amino acid sequence of SEQ ID NO: 138 or 981. amino acid R at a position corresponding to position 589 of the amino acid sequence ofSEQ ID NO: 138 or 981, and amino acid I at a position corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0134] In some embodiments, an AAV capsid variant described herein comprises one, two, three, four, five, or all of the amino acid T at a position corresponding to position 584 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid D at a position corresponding to position 586 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid W at a position corresponding to position 587 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid H at a position corresponding to position 588 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid R at a position corresponding to position 589 of the amino acid sequence of SEQ ID NO: 138 or 981. and / or amino acid I at a position corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant comprises the amino acid T at a position corresponding to position 584 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid D at a position corresponding to position 586 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid Wat a position corresponding to position 587 of the amino acid sequence of SEQ ID NO: 138 or 981, amino acid H at a position corresponding to position 588 of the amino acid sequence of SEQ ID NO: 138 or 981. amino acid R at a position corresponding to position 589 of the amino acid sequence of SEQ ID NO: 138 or 981, and amino acid I at a position corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0135] In some embodiments, an AAV capsid variant described herein comprises one, two, three, four, five, or all of the substitutions H584T, S586D, A587W, Q588H. A589R. and / or Q590I, wherein the substitution(s) is / are at position(s) that correspond to position(s) 584, 586. 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the substitutions H584T, S586D, A587W, Q588H. A589R. and Q590I, wherein the substitution(s) is / are at position(s) that correspond to position(s) 584, 586, 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0136] In some embodiments, an AAV capsid variant described herein comprises the amino acid Q at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0137] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than Q at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid K at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138.

[0138] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than Q at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid V at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138.

[0139] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941), wherein TQDWHRI (SEQ ID NO: 941) is present in the AAV capsid variant at amino acids corresponding to positions 584-590 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, TQDWHRI (SEQ ID NO: 941) is present at amino acids corresponding to positions 584-590 of the amino acid sequence of SEQ ID NO: 138 (e.g., H584, S586, A587, Q588, A589, and Q590 may be replaced with T584, D586, W587, H588, R589, and 1590).

[0140] In some embodiments, an AAV capsid variant described herein comprises the amino acid W at an amino acid corresponding to position 595 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0141] In some embodiments, the AAV capsid variant further comprises a substitution corresponding to amino acid K.449. (e.g., K449R) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an amino acid other than K at a position corresponding to position 449 (e.g.. comprises an R at a position corresponding to position 449) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises an R at amino acid corresponding to position 449 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a modification in loop I, II, IV, and / or VI.

[0142] 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, relative to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence does not comprise more than 10 modifications. 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 amino acids that differ from the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 138, 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.

[0143] In some embodiments, the AAV capsid variant comprises (a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 981 (i.e., comprising amino acids 1-736 of SEQ ID NO: 981); (b) a VP2 protein comprising amino acids 138-736 of SEQ ID NO: 981; (c) a VP3 protein comprising amino acids 203-736 of SEQ ID NO: 981; or (d) 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 to any one of the amino acid sequences in (a)-(c), an amino acid sequence comprising at least one. at least two. or at least three substitutions, but not more than 30, not more than 20, or not more than 10 substitutions relative to any one of the amino acid sequences in (a)-(c), or 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 such modifications relative to any one of the amino acid sequences in (a)-(c).

[0144] In some embodiments, the AAV capsid variant comprises (a) a VP1 protein comprising an amino acid sequence that is 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: 981; (b) a VP2 protein comprising an amino acid sequence that is 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 amino acids 138-736 of SEQ ID NO: 981; and / or (c) a VP3 protein comprising an amino acid sequence that is 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 amino acids 203-736 of SEQ ID NO: 981, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or a sequence comprising one. two, or three but not more than three substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises the amino acid sequence comprising one. two, or three but not more than three substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941) in loop VIII. In some embodiments, the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941) in loop VIII.

[0145] In some embodiments, the AAV capsid variant comprises an amino acid sequence as described herein, e.g., an amino acid sequence of TTJ-001, e.g.. as described in Tables 3 and 4. In some embodiments, the AAV capsid variant is or comprises TTJ-001. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 941 in a loop VIII region.

[0146] In some embodiments, the AAV capsid variant comprises a VP1, VP2. and / or VP3 protein comprising an amino acid sequence described herein, e.g., an amino acid sequence of TTJ-001. e.g., as described in Tables 3 and 4.

[0147] In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence as described herein, e.g., a nucleotide sequence encoding TTJ-001, e.g., as described in Tables 3 and 5.

[0148] In some embodiments, the polynucleotide or nucleic acid encoding the AAV capsid variant comprises a nucleotide sequence described herein, e.g., a nucleotide sequence encoding TTJ-001, e.g., as described in Tables 3 and 5.Table 3. Exemplary full length capsid sequencesTable 4. Exemplary full length capsid amino acid sequencesTable 5. Exemplary full length capsid nucleic acid sequences

[0149] In some embodiments, the polynucleotide encoding an AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 983, 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.

[0150] In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises 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: 983. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, at least two, or at least three substitutions, but not more than 30, not more than 20, or not more than 10 substitutions relative to the amino acid sequence of SEQ ID NO: 983. In some embodiments, the nucleic acid sequence encoding an AAV capsid variant described herein is codon optimized.

[0151] In some embodiments, the AAV capsid variant comprises one or more substitutions in loop VIII and comprises the amino acid sequence of SEQ ID NO: 981, 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 described herein 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: 981. In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence comprising at least one, at least two. or at least three substitutions, but not more than 30, not more than 20, or not more than 10 substitutions, relative to the amino acid sequence of SEQ ID NO: 981.

[0152] In some embodiments, the AAV capsid variant comprises one or more substitutions in loop VIII and comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983. 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 described herein comprises an amino acid sequence encoded by a nucleotide sequence comprising at least one, at least Evo, or at least three substitutions, but not more than 30, not more than 20, or not more than 10 substitutions, relative to the amino acid sequence of SEQ ID NO: 983. In some embodiments, an AAV capsid variant described herein 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: 983.

[0153] In some embodiments, an AAV capsid variant described herein comprises a VP1, VP2, VP3 protein, wherein at least one of the VP1, VP2. and VP3 comprise one or more substitutions in loop VIII. In some embodiments, the AAV capsid variant comprises amino acids 138-736. e.g., a VP2, of the amino acid sequence of SEQ ID NO: 981, 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 amino acids 203-736, e.g.. a VP3, of the amino acid sequence of SEQ ID NO: 981, 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%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity’ thereto. In some embodiments, die AAV capsid variant comprises amino acids 1-736, e.g., a VP1, of the amino acid sequence of SEQ ID NO: 981, 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.

[0154] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 981. or an amino acid sequence at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 981, or an amino acid sequence at least 95%. at least 96%, at least 97%. at least 98%, or at least 99% identical thereto. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 981, or an amino acid sequence at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0155] In some embodiments, an AAV capsid variant described herein 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: 138.

[0156] In some embodiments, an AAV capsid variant described herein transduces a brain region, e.g.. a midbrain region (e.g., the hippocampus, or thalamus) or the brain stem. In some embodiments, the level of transduction is at least 39, at least 50, at least 100, at least 120, at least 132, at least 146, at least 150. at least 161. at least 174. at least 175. at least 200. at least 225. at least 250. at least 275, at least 283, at least 300, at least 350, at least 400, at least 450, at least 500, at least 525, at least 528, or at least 550- fold greater as compared to an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.

[0157] In some embodiments, an AAV capsid variant described herein is enriched at least 10, at least 14, at least 20, at least 24, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to enrichment in the brain of an AAV capsid comprising tire amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein is enriched at least 200, at least 250, at least 300, at least 350, at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to enrichment in the brain of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.

[0158] In some embodiments, an AAV capsid variant described herein is enriched in the brain of at least two to 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 sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein is enriched at least 2, at least 3. at least 5, at least 10, at least 15, at least 20. at least 25, at least 30, at least 35. at least 40, at least 45. at least 50, at least 55, at least 60, at least 65. at least 70, at least 75, at least 80. at least 85, at least 90. at least 95, at least 100, at least 105, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 190.at least 200, at least 205, or at least 210-fold in the brain of at least two to three species, e.g., a nonhuman primate and rodent (e g., mouse) species, compared to enrichment in the brain of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the at least tw o to three species are Macaca fascicularis, CMorocebus sabaeus, Callithrix jacchus, and / or mouse (e.g., outbred mice).

[0159] In some embodiments, an AAV capsid variant described herein is enriched at least 2, at least 3. at least 4, at least 5, at least 10. at least 15, at least 17, at least 20, at least 50, at least 75. at least 100. at least 103, at least 107, at least 125, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 750, at least 1000, or at least 1200-fold in the brain compared to enrichment in the brain of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 981.

[0160] In some embodiments, an AAV capsid variant described herein delivers an increased level of viral genomes to a brain region. In some embodiments, the level of viral genomes is increased by at least 2. at least 5. at least 7. at least 10. at least 15, at least 19. at least 20, at least 22, or at least 25-fold, as compared to the level of viral genomes delivered by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the brain region comprises a sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and / or cerebellum.

[0161] In some embodiments, an AAV capsid variant delivers an increased level of a payload to a brain region. In some embodiments, the level of the payload is increased by at least 39, at least 50, at least 100, at least 120, at least 132, at least 146, at least 150, at least 161, at least 174, at least 175, at least 200. at least 225. at least 250. at least 275. at least 283. at least 300. at least 350. at least 400, at least 450, at least 500, at least 525, at least 528, or at least 550-fold, as compared to the level of a payload delivered by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the brain region comprises a sensory’ cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and / or cerebellum.

[0162] In some embodiments, an AAV capsid variant described herein is enriched at least 5, at least 10, at least 50, at least 100, at least 115, at least 120, at least 150, at least 175, at least 200, at least 207, at least 225, at least 250, or at least 275-fold in the spinal cord compared to enrichment in the spinal cord of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.

[0163] hi some embodiments, an AAV capsid variant of the present disclosure has decreased tropism for the liver. In some embodiments, an AAV capsid variant comprises a modification that results in reduced tropism (e.g., de-targeting) and / or activity in the liver. In some embodiments, the reduced tropism in the liver is compared to an otherwise similar capsid that does not comprise the modification, e.g., a wild-type capsid polypeptide. In some embodiments, an AAV capsid variant comprises a modification that results in one or more of the following properties: (1) reduced tropism in the liver; (2) de-targeted expression in the liver; (3) reduced activity in the liver; and / or (4) reduced binding to galactose. In some embodiments, the reduction in any one or all of properties (l)-(3) is compared to an otherwise similar AAV capsid variant that does not comprise the modification. Exemplary modificationsare provided in WO 2018 / 119330; Pulicherla et al. (2011) Mol. Ther. 19(6): 1070-1078; Adachi et al. (2014) Nature Communications 5(3075), DOI: 10.1038 / ncomms4075; and Bell et al. (2012) J. Virol. 86(13): 7326-33; the contents of which are hereby incorporated by reference in their entirety.

[0164] In some embodiments, tire AAV capsid variant comprises a modification at an amino acid corresponding to N470 (e.g., comprises N470A at an amino acid corresponding to N470) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to D271 (e.g., comprises D271 A at an amino acid corresponding to D271) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to N272 (e.g., comprises N272A at an amino acid corresponding to N272) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to Y446 (e.g., comprises Y446A at an amino acid corresponding to Y446) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to N498 (e.g.. comprises N498Y or N498I at an amino acid corresponding to N498) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to W503 (e.g., comprises W503R or W503A at an amino acid corresponding to W503) of the amino acid sequence of SEQ ID NO: 138; an amino acid corresponding to L620 (e.g., comprises L620F at an amino acid corresponding to L620) of the amino acid sequence of SEQ ID NO: 138 or a combination thereof. In some embodiments, the AAV capsid variant comprises one, two. three, four, five, or all of an amino acid other than N at a position corresponding to position 470 (e.g.. comprises A at an amino acid corresponding to position 470) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than D at a position corresponding to position 271 (e.g., comprises A at an amino acid corresponding to position 271) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than N at a position corresponding to position 272 (e.g., comprises A at an amino acid corresponding to position 272) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than Y at a position corresponding to position 446 (e.g., comprises A at an amino acid corresponding to position 446) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than N at a position corresponding to position 498 (e.g., comprises Y or I at an amino acid corresponding to position 498) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than W at a position corresponding to position 503 (e.g., comprises R or A at an amino acid corresponding to position 503) of the amino acid sequence of SEQ ID NO: 138; and an amino acid other than L at a position corresponding to position 620 (e.g., comprises F at an amino acid corresponding to position 620) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a modification at positions corresponding to amino acid N470 (e.g., comprises N470A at an amino acid corresponding to N470) of the amino acid sequence of SEQ ID NO: 138; corresponding to amino acid D271 (e.g., comprises D271A at an amino acid corresponding to D271) of the amino acid sequence of SEQ ID NO: 138; corresponding to amino acid N272 (e.g., comprises N272A at an amino acid corresponding to N272) of the amino acid sequence of SEQ ID NO: 138; corresponding to amino acid Y446 (e.g., comprises Y446A at an amino acid corresponding to Y446) of the amino acid sequence of SEQ ID NO: 138; and corresponding to amino acid W503 (e.g., comprises W503R or W503A at an amino acid corresponding to W503) of the amino acid sequence of SEQ ID NO:138. In some embodiments, the AAV capsid variant comprises a modification corresponding to amino acid N498 (e.g., comprises N498Y at an amino acid corresponding to N498) of the amino acid sequence of SEQ ID NO: 138 and corresponding to amino acid L620 (e.g., comprises L620F at an amino acid corresponding to L620) of the amino acid sequence of SEQ ID NO: 138.

[0165] hi some embodiments, the AAV capsid variant comprises a modification as described in Adachi et al. (2014) Nature Communications 5(3075), DOI: 10.1038 / ncomms4075, the contents of which are hereby incorporated by reference in their entirety. Exemplary modifications that alter or do not alter tissue transduction in at least the brain, liver, heart, lung, and / or kidney can be found in Supplementary Data 2 showing the AAV Barcode-Seq data obtained with AAV9-AA-VBCLib of Adachi et al. (supra), the contents of which are hereby incorporated by reference in their entirety.

[0166] In some embodiments, the 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 an isolated and / or a recombinant AAV capsid polypeptide.

[0167] Also provided herein are polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same.

[0168] Exemplary Properties of AAV Capsidsln some embodiments, the AAV particle may comprise a capsid protein or variant of any natural or recombinant AAV serotype. AAV seroty pes may differ in characteristics such as. but not limited to, packaging, tropism, transduction and immunogenic profiles. 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.

[0169] 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 microbubblcs (FUS-MB), or MRI-guidcd 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 amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and / or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, and / or temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, and / or vestibular nucleus. 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 thereof). In someembodiments, 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 drereof).

[0170] 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.

[0171] In some embodiments, the AAV capsid variant allows for increased distribution to a heart region.

[0172] 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 diaphragm 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 a quadriceps muscle, a vastus lateralis muscle, and / or a gastrocnemius muscle.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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 mixture 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-).

[0178] 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).

[0179] 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 codon may 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 VP 1 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.

[0180] References to viral capsids formed from VP capsid proteins (such as reference to specific AAV capsid scrotj pcs), can incorporate VP capsid proteins which include a Metl / AAl ammo 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-).

[0181] 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+ / AA1) and VP2 (Met- / AA1-).Additional AAV Capsid Sequences

[0182] In some embodiments, the AAV capsid variant comprises amino acids corresponding positions 582, 583. 584, 585, 586. 587, 588, 589. and / or 590 of the amino acid sequence of SEQ ID NO: 138. 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 least12, at least 13, at least 14, or at least 15 consecutive amino acids of any one of the amino acid sequences provided in Table 1, 2A, 2B, 11, 26, 27, or 28.

[0183] In some embodiments, the AAV capsid variant comprises, immediately subsequent to an amino acid corresponding to position 582, 583, 584, 585, 586, 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138 (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))), 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, or at least 15 consecutive amino acids of any one of the amino acid sequences provided in Table 1. 2A, 2B. 11. 26, 27, or 28. In some embodiments, the 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, or at least 15 consecutive amino acids of any one of the amino acid sequences provided in Table 1. 2A. 2B. 11, 26, 27. or 28 replaces at least one, at least two. at least three, at least four, at least five, at least six, at least seven, at least eight, or all of amino acid(s) corresponding to position(s) 582. 583. 584, 585. 586, 587, 588. 589, and / or 590 (e.g., T582, N583. H584, Q585, S586, A587, Q588. A589. and / or Q590) of the amino acid sequence of SEQ ID NO: 138 (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, which is hereby incorporated by reference in its entirety)). In some embodiments, the AAV capsid variant comprises an amino acid other than the wild-type amino acid, at one, two, three, four, five, six, seven, eight, or all of amino acid(s) corresponding to position(s) 582, 583, 584, 585, 586, 587, 588, 589, and / or 590 of the amino acid sequence of SEQ ID NO: 138 (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, which is hereby incorporated by reference in its entirety). In some embodiments, the AAV capsid variant comprises a modification replacing one, two, three, four, five, six, seven, eight, or all of amino acid(s) corresponding to position(s) 582, 583, 584. 585, 586, 587, 588, 589, and / or 590 (e.g., T582, N583. H584, Q585, S586, A587, Q588, A589, and / or Q590) of the amino acid sequence of SEQ ID NO: 138 (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, which is hereby incorporated by reference in its entirety).

[0184] In some embodiments, the AAV capsid variant may comprise a variant of a VOY101 capsid polypeptide, an AAVPHP.B (PHP.B) capsid polypeptide, an AAVPHP.N (PHP.N) capsid polypeptide, an AAV1 capsid polypeptide, an AAV2 capsid polypeptide, an AAV5 capsid polypeptide, an AAV9 capsid polypeptide, an AAV9 K449R capsid polypeptide, or an AAVrhlO capsid polypeptide. In someembodiments, the AAV capsid polypeptide, e.g., AAV capsid variant, comprises an amino acid sequence of any one of the AAV capsid polypeptides in Table 6, or an amino acid 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 nucleotide sequence encoding the AAV capsid polypeptide comprises any one of the nucleotide sequences in Table 6, 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.Table 6. AAV SequencesAAV Viral Genome

[0185] 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.

[0186] 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, also referred to as an AAV genome, a vector genome, or an AAV vector 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 polyA region, or a combination thereof. In some embodiments, the viral genome, e.g.. the AAV viral genome, further comprises at least one miRNA binding site.Viral Genome Component: Inverted Terminal Repeats (ITRs)

[0187] 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., mutated DMPK mRNA). 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 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.

[0188] 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, the ITRs are of the same serotype 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 serotype as the capsid.Viral Genome Component: Promoters and Enhancers

[0189] In 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), polyadcnylation (Poly A) region, upstream enhancers (USEs), CMV enhancers, and introns.

[0190] 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 l 145-9 (1997): the contents of which are herein incorporated by reference in their entirety).

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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, a promoter that drives or promotes expression in most mammalian tissues includes, but is not limited to, a human elongation factor la-subunit (EFla) promoter, a cytomegalovirus (CMV) immediate-early enhancer and / or promoter, a chicken p-actin (CBA) promoter, a CAG promoter, a glucuronidase (GUSB) promoter, or a ubiquitin C (UBC) promoter.

[0195] 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.

[0196] In some embodiments, the promoter may be less than 1 kb.

[0197] In some embodiments, the promoter may be a combination of two or more components of the same or different starting or parental promoters.

[0198] In some embodiments, the viral genome comprises an enhancer.

[0199] In some embodiments, the viral genome comprises an engineered promoter.Viral Genome Component: Intron and Exon Sequences

[0200] In some embodiments, the 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.

[0201] In some embodiments, the intron may be 100-600 nucleotides in length.

[0202] In some embodiments, the modulatory polynuclcotidc-cncoding sequence may be located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides downstream from a promoter comprising an intron (e.g., 3’ relative to the promoter comprising an intron) and / or upstream of the poly adenylation sequence (e g., 5’ relative to the polyadeny lation sequence) in an expression vector. In some embodiments, the modulatory polynucleotide-encoding sequence may be located within 1-5, 1-10, 1-15. 1-20, 1-25, 1-30, 5-10. 5-15, 5- 20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20. 15-25, 15-30, 20-25. 20-30, or 25-30 nucleotides downstream from the intron (e.g., 3’ relative to the intron) and / or upstream of the polyadenylation sequence (e.g., 5’ relative to the polyadenylation sequence) in an expression vector. In some embodiments, the modulatory polynucleotide-encoding sequence may be located within the first 1%. 2%. 3%. 4%, 5%, 6%, 7%, 8%. 9%. 10%. 15%, 20%, 25%, or more than 25% of the nucleotides downstream from the intron (e.g.. 3’ relative to the intron) and / or upstream of the polyadenylation sequence (e.g.. 5’ relative to the polyadenylation sequence) in an expression vector. In some embodiments, the modulatorypolynucleotide-encoding sequence may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% of the sequence downstream from the intron (e.g., 3’ relative to the intron) and / or upstream of the polyadenylation sequence (e.g., 5’ relative to the poly adenylation sequence) in an expression vector.

[0203] hi 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)

[0204] 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 die 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.

[0205] Features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production.

[0206] 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. While not wishing to be 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 “G”.

[0207] In some embodiments, the 5 ’UTR in the viral genome includes a Kozak sequence.

[0208] In some embodiments, the 5 ’UTR in the viral genome does not include a Kozak sequence.

[0209] While not wishing to be bound by theory , wild-ty pe 3' UTRs arc known to have stretches of adenosines and uridines embedded therein. These AU rich signatures are particularly prevalent in genes w ith 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. Engineering the 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.

[0210] 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.

[0211] In some embodiments, the 3' UTR of the viral genome may include an oligo(dT) sequence for templated addition of a polyadenylation (poly A) sequence.

[0212] 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.

[0213] In some embodiments, the viral genome of the AAV particle comprises at least one artificial UTR, which is not a variant of a wild ty pe UTR.

[0214] 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

[0215] As used herein, the terms “stuffer sequence” and “filler sequence” are used interchangeably. In some embodiments, the AAV particle viral genome comprises at least one filler sequence.

[0216] 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.

[0217] In some embodiments, tire viral genome comprises one or more filler sequences in order to have the length of the viral genome be tire 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

[0218] 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., mutated DMPK mRNA. In some embodiments, reduction or elimination of DMPK expression thereby reduces or eliminates DMPK protein (e.g., mutated DMPK protein). In some embodiments, the modulatorypolynucleotide comprises or encodes an inhibitory polynucleotide, e.g., an RNAi agent or an ASO. In some embodiments, the modulatory polynucleotide comprises or encodes an RNAi agent. In some embodiments, the RNAi agent comprises an siRNA.

[0219] In some embodiments, the modulatory polynucleotides of the disclosure reduce or eliminate DMPK mRNA, thereby reducing or eliminating DMPK protein. Exemplary DMPK mRNA sequences are provided in Table 7. In some embodiments, the modulatory' polynucleotide of the disclosure reduces or eliminates expression of one or both of SEQ ID NO: 6428 or SEQ ID NO: 6429, or a trinucleotide repeat expansion of either of the foregoing. The exemplary DMPK sequence information from the NationalCenter for Biotechnology Infonnation cited in Table 7 is hereby incorporated by reference in its entirety.Table 7. Exemplary DMPK mRNA transcripts

[0220] In some embodiments, the modulatory polynucleotide comprises a guide strand and a passenger strand, wherein the guide strand targets and thus reduces or eliminates expression of one or both of) SEQ ID NO: 6428 or SEQ ID NO: 6429 or a trinucleotide repeat expansion thereof. In some embodiments, the guide strand targeting SEQ ID NO: 6428 or SEQ ID NO: 6429 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: 6428 or SEQ ID NO: 6429 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: 6428 or SEQ ID NO: 6429 or a trinucleotide repeat expansion thereof.

[0221] 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 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 the guide strand.

[0222] 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.

[0223] In some embodiments, the disclosure herein provides constructs that allow for improved expression of a modulatory polynucleotide delivered by gene therapy' vectors.

[0224] In some embodiments, the disclosure provides constructs that allow for improved biodistribution of a modulatory' polynucleotide delivered by gene therapy' vectors.

[0225] 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.

[0226] In some embodiments, the disclosure provides constructs that allow for improved trafficking of a modulatory' poly nucleotide to lysosomal membranes delivered by gene therapy vectors.

[0227] In some embodiments, the present disclosure relates to a composition containing or comprising a nucleic acid sequence encoding modulatory' polynucleotide for reducing or eliminatingexpression 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.

[0228] 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 polyA sequence, and a 3’ ITR sequence.

[0229] In some embodiments, the AAV genome encodes a payload construct that comprises a combination of coding and non-coding nucleic acid sequences.

[0230] In some embodiments, the viral genome encodes more than one payload. As a non-limiting example, a viral genome encoding more than one payload may be replicated and packaged into a viral particle. A target cell transduced with a viral particle comprising more than one payload may express each of the pay loads in a single cell.

[0231] 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.

[0232] A payload construct 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.

[0233] hi some embodiments, a payload construct encoding a modulatory polynucleotide may comprise a selectable marker including, but not limited to, |3-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 Set. USA (1995); or Heim et al., Science 373:663-664 (1995); for -lactamase, see WO 96 / 30540); the contents of each of which are herein incorporated by reference in their entirety.

[0234] In some embodiments, a payload construct encoding a selectable marker may comprise a fluorescent protein. A fluorescent protein as herein described may comprise any fluorescent markerincluding 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.

[0235] 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 between two ITR sequences.Viral Genome Component: Molecular Scaffolds

[0236] In some embodiments, the molecular scaffold of the modulatory polynucleotide is a know n or wild type pri- or pre-microRNA. In other embodiments, the molecular scaffold of the modulator,' 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.

[0237] 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 betw een 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.

[0238] In some embodiments, the molecular scaffold comprises a 5’ flanking region, a loop region, and a 3’ flanking region. In some embodiments, the modulatory polynucleotide comprises, in the 5' to 3' direction, a 5' flanking sequence, a 5' arm 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.

[0239] 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 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.

[0240] 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 8-10.Table 8. Exemplary 5’ Flanking Regions for Molecular ScaffoldTable 9. Exemplary Loop Regions for Molecular ScaffoldTable 10. Exemplary 3’ Flanking Regions for Molecular Scaffold

[0241] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising any one of SEQ ID NOs: 6413-6416, 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: 6413-6416; a loop region comprising any one of SEQ ID NOs: 6417-6421. 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: 6417-6421; and a 3’ flanking region comprising any one of SEQ ID NOs: 6422-6427, 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: 6422-6427.

[0242] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising any one of SEQ ID NOs: 6413-6416; a loop region comprising any one of SEQ ID NOs: 6417-6421; and a 3’ flanking region comprising any one of SEQ ID NOs: 6422-6427.

[0243] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 6414 or SEQ ID NO: 6415; a loop region comprising SEQ ID NO: 6417, SEQ ID NO: 6418, or SEQ ID NO: 6421; and a 3’ flanking region comprising SEQ ID NO: 6422, SEQ ID NO: 6423, SEQ ID NO: 6424, or SEQ ID NO: 6425. In some embodiments, the molecular scaffold comprises a 5’ flankingregion comprising SEQ ID NO: 6414 or SEQ ID NO: 6415; a loop region comprising SEQ ID NO: 6417. SEQ ID NO: 6418, or SEQ ID NO: 6421; and a 3' flanking region comprising SEQ ID NO: 6423, SEQ ID NO: 6424, or SEQ ID NO: 6425.

[0244] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 6414, 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: 6417, 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: 6423 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.

[0245] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 6415, 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: 6421, 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: 6425 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.

[0246] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 6414, 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: 6417, 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: 6424 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.

[0247] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprises SEQ ID NO: 6414, 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: 6418, 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: 6423 or a nucleotide sequence at least 75%, at least 80%, at least 85%. atleast 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.

[0248] In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 6414, a loop region comprising SEQ ID NO: 6417, and a 3’ flanking region comprising SEQ ID NO: 6423. In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 6415, a loop region comprising SEQ ID NO: 6421, and a 3’ flanking region comprising SEQ ID NO: 6425. In some embodiments, the molecular scaffold comprises a 5’ flanking region comprising SEQ ID NO: 6414, a loop region comprising SEQ ID NO: 6417, and a 3’ flanking region comprising SEQ ID NO: 6424. In some embodiments, the molecular scaffold comprises a 5’ flanking region comprises SEQ ID NO: 6414, a loop region comprising SEQ ID NO: 6418, and a 3’ flanking region comprising SEQ ID NO: 6423.Exemplary AAV Capsid Sequences

[0249] 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.

[0250] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising an amino acid sequence selected from Table 3 or Table 4. In some embodiments, the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or TNTQDWHRIAQ (SEQ ID NO: 343) is present in loop VIII, wherein loop VIII comprises amino acids 580-599 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises: (i) a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 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 the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 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 according to positions 203-736 of SEQ ID NO: 981 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 T at amino acid 584, D at amino acid 586, W at amino acid 587. H at amino acid 588, R at amino acid 589, and / or I at amino acid 590, numbered according to SEQ ID NO: 981.

[0251] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the amino acid T at position 584, D at position 586, W at position 587, H at position- I l l -588, R at position 589, and I at position 590, numbered according to SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 981, 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 138-736 of SEQ ID NO: 981, 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: 981, 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.

[0252] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the amino acid T at a position corresponding to position 584 of SEQ ID NO: 981, D at a position corresponding to position 586 of SEQ ID NO: 981, W at a position corresponding to position 587 of SEQ ID NO: 981. H at a position corresponding to position 588 of SEQ ID NO: 981. R at a position corresponding to position 589 of SEQ ID NO: 981, and I at a position corresponding to position 590 of SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 981, 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 138-736 of SEQ ID NO: 981, or an ammo 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 ammo acid sequence SEQ ID NO: 981, or an amino acid sequence that is at least ...

Claims

CLAIMSWe claim:

1. An adeno-associated virus (AAV) particle comprising an AAV capsid variant and a viral genome, wherein the viral genome comprises 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 wherein the AAV capsid variant comprises an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4681) in loop VIII; wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); and wherein:(i) [N 1 ] comprises amino acids Xi, X2, X3, and X4. wherein X4is Q, K, E. S, P, R. N, or H; and / or(ii) [N3J comprises amino acids X5. X6, and X?, wherein X5is 1, V, T. M, S. N, L, or F.

2. The AAV particle of claim 1. wherein the AAV capsid variant is an AAV9 capsid variant.

3. The AAV particle of claim 1 or claim 2, wherein the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to positions 203-736 of the amino acid sequence of SEQ ID NO: 981.

4. The AAV particle of any one of claims 1-3, wherein loop VIII is present at amino acids comprising those corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 981.

5. The AAV particle of any one of claims 1-4, wherein:(i) Xi is T, S, R, A. I. C, N, K, L, or Q;(ii) X2is N, T, G, V, S, Y, K, I, H, D, or F; and(iii) X3is T, N, K, D, I, S, P, A, Y, E, V, L, M, R, H, Q, or C.

6. The AAV particle of any one of claims 1-5, wherein:(i) X6is A, Y, P, N, S, T, G, E, V, W, F, or Q; and(ii) X7is Q, G. N, K, H, R, E. L, P, or M.

7. The AAV particle of any one of claims 1-6, wherein [Nl] comprises the amino acid sequence of TNTQ (SEQ ID NO: 4688).

8. The AAV particle of any one of claims 1-7, wherein [N3] comprises the amino acid sequence of9. The AAV particle of any one of claims 1-8, wherein [N1]-[N2]-[N3] comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343).

10. The AAV particle of any one of claims 1-9, wherein:(i) [Nl] is present at amino acids corresponding to positions 582-585 of the amino acid sequence of SEQ ID NO: 981:(ii) [N2] is present at amino acids corresponding to positions 586-589 of the amino acid sequence of SEQ ID NO: 981: and(iii) [N3] is present at amino acids corresponding to positions 590-592 of the amino acid sequence of SEQ ID NO: 981.

11. The AAV particle of any one of claims 1-10, wherein the AAV capsid variant further comprises [N4] comprising amino acids X8, X9, Xio, and Xu, wherein:(i) X8is T, S. N, P, A. or I;(ii) X9is G, N, D. R, V. A, S, or Q;(iii) Xiois W, S, C, R, L, or G; and / or(iv) Xu is V. A, S, I, C. G, D. F, L, or T.

12. The AAV particle of claim 11, wherein [N4] comprises the amino acid sequence of TGWV (SEQ ID NO: 5066).

13. The AAV particle of claim 11 or claim 12, wherein [N4] is present at amino acids corresponding to positions 593-596 of the amino acid sequence of SEQ ID NO: 981.

14. The AAV particle of any one of claims 11-13, wherein [N1]-[N2]-[N3]-[N4] comprises the amino acid sequence of TNTQDWHRIAQTGWV (SEQ ID NO: 201).

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 positions 203-736 of the amino acid sequence of SEQ ID NO:

981. wherein the AAV capsid variant comprises:T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO:981;D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981;W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981;H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981;R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; andI at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981.

16. The AAV particle of any one of claims 1-15, wherein the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981.

17. 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 positions 138-736 of the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises:T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981;D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981;W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981;H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981;R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; andI at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981.

18. The AAV particle of any one of claims 1-17, wherein the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 981.

19. Aii adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleotide sequence encoding a modulatory poly nucleotide 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:

981. wherein the AAV capsid variant comprises:T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981;D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981;W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981;H at an amino acid corresponding to position 588 of the amino acid sequence of SEQ ID NO: 981;R at an amino acid corresponding to position 589 of the amino acid sequence of SEQ ID NO: 981; andI at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981.

20. The AAV particle of claim 19, 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: 981;(ii) an amino acid sequence that is at least 99% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981; and / or(iii) an amino acid sequence that is at least 99% identical to positions 203-736 of the amino acid sequence of SEQ ID NO: 981.

21. The AAV particle of any one of claims 15-20, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941).

22. The AAV particle of any one of claims 15-21. wherein the AAV capsid variant comprises at least 8. at least 9. or at least 10 consecutive amino acids from the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343).

23. The AAV particle of any one of claims 15-22, wherein the AAV capsid variant comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343) present at amino acids corresponding to positions 582-592 of the amino acid sequence of SEQ ID NO: 981.

24. The AAV particle of any one of claims 21-23, wherein the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or TNTQDWHRIAQ (SEQ ID NO: 343) is present in loop VIII, wherein loop VIII comprises amino acids 580-599 of the amino acid sequence of SEQ ID NO: 981.

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: 981;(ii) the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981; and / or(iii) the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981.

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: 6413- 6416, 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: 6417-6421, 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: 6422- 6427, 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: 6414 or SEQ ID NO: 6415, or a nucleotide sequence that is at least 95% identical thereto;(ii) the loop region comprises the nucleotide sequence of SEQ ID NO: 6417, SEQ ID NO: 6418, or SEQ ID NO: 6421, 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: 6423, SEQ ID NO: 6424, or SEQ ID NO: 6425, or a nucleotide sequence that is at least 95% identical thereto.

28. The AAV particle of claim 27, wherein the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% identical thereto.

29. The AAV particle of claim 27, wherein die 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6415 or a nucleotide sequence that is at least 95% identical thereto, the loop region of the comprises the nucleotide sequence of SEQ ID NO: 6421 or a nucleotide sequence that is at least 95% identical diereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6425 or a nucleotide sequence that is at least 95% identical thereto.

30. The AAV particle of claim 27, wherein the 5’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical diereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6424 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: 6414 or a nucleotide sequence that is at least 95% identical thereto, the loop region comprises the nucleotide sequence of SEQ ID NO: 6418 or a nucleotide sequence that is at least 95% identical thereto, and the 3’ flanking region comprises the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% identical thereto.

32. The AAV particle of any one of claims 1-31, wherein the 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 claim 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 NOs: 6428-6434 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 claim 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 claim 43 or claim 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 43-45, 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 SI9 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: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising die nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% identical thereto;(ii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6415 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6421 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6425 or a nucleotide sequence that is at least 95% identical thereto;(iii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6424 or a nucleotide sequence that is at least 95% thereto; or(iv) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6418 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6423 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: 981 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 138-736 of the amino acid sequence of SEQ ID NO: 981 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 203-736 of the amino acid sequence of SEQ ID NO: 981 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: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising die nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% identical thereto;(ii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6415 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising die nucleotide sequence of SEQ ID NO: 6421 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6425 or a nucleotide sequence that is at least 95% identical thereto:(iii) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6417 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6424 or a nucleotide sequence that is at least 95% thereto; or(iv) a 5’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6414 or a nucleotide sequence that is at least 95% identical thereto, a loop region comprising the nucleotide sequence of SEQ ID NO: 6418 or a nucleotide sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleotide sequence of SEQ ID NO: 6423 or a nucleotide sequence that is at least 95% thereto; and wherein the AAV capsid variant comprises SEQ ID NO: 981, the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981, and / or the amino acid sequence according to positions 203-736 of the ammo acid sequence of SEQ ID NO: 981.

51. The method of claim 48, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981.

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 (e.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 the 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.

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 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.

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, smooth, and / or skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius).

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, e.g., the brain.

74. The method of claim 73, wherein the pharmacal composition or AAV particle is delivered to the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen. substantia nigra.thalamus, ventral palladium, vestibular nucleus, 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 57-76. 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.

78. The method of claim 77, wherein 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.

79. The method of claim 77 or claim 78, wherein the cell or tissue of the subject is a muscle cell or tissue (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius)).

80. The method of claim 77 or claim 78, wherein the cell or tissue of the subject is a cell or tissue of the central nervous system (CNS) (e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g.. Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus. oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and / or spinal cord (e.g.. cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)).

81. The method of any one of claims 57-80, 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 pharmacal composition or AAV particle.

82. The method of any one of claims 57-72 or any one of claims 76-79. 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 tissue (e.g., cardiac muscle, smooth muscle, and / or skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius)) 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 (e.g.. cardiac muscle, smooth muscle, and / or skeletal muscle (e.g.. quadriceps, vastus lateralis, and / or gastrocnemius)) 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 muscle, smooth muscle, and / or skeletal muscle (e.g., quadriceps, vastus lateralis, and / or gastrocnemius)) 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.

83. The method of any one of claims 57-70, any one of claims 73-78, or claim 80, 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., amygdala, brainstem, caudate, central grey, cerebellum (e.g.. Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen. substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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., amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex), external cuneate nucleus, geniculate nucleus, globus pallidus. gracile nucleus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, 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.

84. The method of claim 83, wherein the subject has, has been diagnosed with having, or is at risk of having congenital myotonic dystrophy type 1.

85. The method of any one of claims 57-84, further comprising administering to the subject at least one additional agent and / or therapy.

86. The method of claim 85, wherein the at least one additional agent and / or therapy comprises an agent and / or therapy suitable for treating a DMPK-related disorder, optionally wherein the at least one additional 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.

87. The method of any one of claims 57-86. further comprising administering an immuno suppressant to the subject.

88. The method of claim 87, 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.

89. 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 59-88.

90. 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).

91. The pharmaceutical composition or AAV particle of claim 90, 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.

92. The pharmaceutical composition or AAV particle of claim 90 or claim 91. wherein the DMPK- related disorder is congenital DM1.

93. 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).

94. The use of claim 93, 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.

95. The use of claim 93 or claim 94, wherein the DMPK-related disorder is congenital DM1.

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