Compositions and methods for the treatment of disorders related to ataxin-2

Abstract

The disclosure relates to compositions and methods for modulating, e.g., reducing or eliminating, the expression of ATXN2 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 spinocerebellar ataxia type 2 or another ATXN2-related disorder.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF DISORDERSRELATED TO ATAXIN-2RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to US Provisional Application Serial No. 63 / 606,202, 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_0103-00304_SL.xml. was created on September 18, 2024, and is 2,002.224 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety7.FIELD

[0003] Described 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 Ataxin-2 (ATXN2) mRNA, thereby reducing or eliminating ATXN2 protein, and their use in the treatment of spinocerebellar ataxia type 2 (SCA2) and other disorders associated with aberrant ATXN2 protein expression or activity or mutated ATXN2 protein. In some embodiments, compositions described herein may be used to treat a subject in need thereof, such as a human subject having SCA2 or another condition resulting from or associated with mutant ATXN2 protein or aberrant ATXN2 protein expression or activity7.BACKGROUND

[0004] The Ataxin-2 protein (also referred to herein as ATXN2 protein) is encoded by the ATXN2 gene (Ensembl Gene ID No. ENSG00000204842), which is also known as ATX2 and TNRC13. It is located on chromosome 12 of the human genome.

[0005] The function of Ataxin-2 is not known, but it is found in the cytoplasm and is thought to interact with the endoplasmic reticulum.

[0006] Mutations in ATXN2 cause disease in human subjects. ATXN2 mutations are known to cause spinocerebellar ataxia type 2 (SCA2). SCA2 is an autosomal dominant cerebellar ataxia. ATXN2 mutations also play a role in other ATXN2-related disorders, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD). and frontotemporal lobar degeneration (FTLD).

[0007] SC A2 is typically caused by CAG trinucleotide repeat expansions in the ATXN2 gene on 12q24.1. Patients with SCA2 have ATXN2 alleles with 32 or more trinucleotide repeats, resulting in ATXN2 protein with polyQ expansions. PolyQ expansions in ATXN2 (e.g.. intermediate length polyQ expansions) have been associated with other diseases, like ALS, as well.

[0008] The age of onset of SCA2 is thought to correlate with the number of C AG trinucleotide repeats. Onset is typically later than age 60 in subjects with 32-34 CAG repeats (SEQ ID NO: 6432). Onset is typically between ages 20 and 60 in subjects with 35-40 CAG repeats (SEQ ID NO: 6433). Onset is typically before age 20 for patients with 45 or more CAG repeats (SEQ ID NO: 6434).

[0009] Mutated ATXN2 is thought to produce an Ataxin-2 protein with an abnormally long stretch of glutamine residues. The cellular impact of mutant Ataxin-2 protein is not confinned. Without being bound by theory, mutant Ataxin-2 (e.g.. Ataxin-2 with a polyQ expansion) may accumulate in cells and cause cell death.

[0010] SCA2 is characterized by progressive cerebellar ataxia, including nystagmus, and slow saccadic eye movements. It may also present with ophthalmoparesis and / or parkinsonism. Later stages of SCA2 mainly involve the brainstem and cerebellum.[OH] There are no specific treatments for SCA2. Trials have considered riluzole, which inhibits glutamic acid release, for treating SCA2. However, there are no treatments targeting the cause of SCA2. Thus, there remains a long-felt need to develop pharmaceutical compositions and methods for the treatment of SCA2. In particular, a need exists for treatments of SCA2 targeting the brainstem and cerebellum.

[0012] 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 a modulatory polynucleotide for reducing or eliminating expression of ATXN2 mRNA (thereby reducing or eliminating ATXN2 protein), to a target cell or tissue, e.g., a CNS cell or tissue.SUMMARY

[0013] In some aspects, the present disclosure provides 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 Ataxin-2 (ATXN2) 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); [Nl] comprises amino acids Xi. X . Xs, and X4, wherein X4 is Q, K. E, S, P, R, N, or H; and / or [N3] comprises amino acids Xs. Xg. and X?, wherein Xs is 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.

[0014] In some embodiments, Xi is T, S, R, A, I, C, N. K, L, or Q; X2is N, T, G, V, S. Y, K, I, H, D, or F; and X3is T, N, K, D, I, S, P, A, Y, E, V, L. M, R, H, Q, or C. In some embodiments, X6is A, Y, P, N, S, T, G, E, V, W, F, or Q; and X- is Q, G, N, K, H, R, E, L, P, or M.

[0015] 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).

[0016] In some embodiments, [Nl] is present at amino acids comprising those corresponding to positions 580-599 of the amino acid sequence of SEQ ID NO: 981, [N2] is present at amino acids corresponding to positions 586-589 of the amino acid sequence of SEQ ID NO: 981, and [N3] is present at amino acids corresponding to positions 590-592 of the amino acid sequence of SEQ ID NO: 981.

[0017] In some embodiments, the AAV capsid variant further comprises [N4] comprising amino acids X8, X9, Xio, and Xu, wherein: X8is T. S, N, P. A, or I; X9is G, N, D, R. V, A. S, or Q: Xio is W, S, C, R, L. or G; and / or 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.

[0018] In some embodiments, [N1]-[N2]-[N3]-[N4] comprises the amino acid sequence of TNTQDWHRIAQTGWV (SEQ ID NO: 201).

[0019] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulator}' polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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.

[0020] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and an AAV capsid variant comprising an amnio acid sequence that is at least 95% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981. In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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.

[0021] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and an AAV capsid variant comprising an ammo acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 981. In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and an AAV capsid variant comprising an ammo 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; and I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 981.

[0022] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and an AAV capsid variant, 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. In some embodiments, the AAV capsid variant comprises the ammo acid sequence of TQDWHRI (SEQ ID NO: 941).

[0023] 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.

[0024] 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 ammo acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981.

[0025] In some embodiments, the modulatory polynucleotide comprises a molecular scaffold, wherein the molecular scaffold comprises: (a) a 5’ flanking region comprising any one of SEQ ID NOs: 6413-6416 or a nucleic acid sequence that is at least 95% identical thereto; (b) a loop region comprising any one of SEQ ID NOs: 6417-6421 or a nucleic acid sequence that is at least 95% identical thereto; and(c) a 3’ flanking region comprising any one of SEQ ID NOs: 6422-6427 or a nucleic acid sequence that is at least 95% identical thereto. In some embodiments, the (a) the 5 ’ flanking region comprises SEQ ID NO: 6414 or SEQ ID NO: 641 , or a nucleic acid sequence that is at least 95% identical thereto; (b) the loop region comprises SEQ ID NO: 6417. SEQ ID NO: 6418, or SEQ ID NO: 6421. or a nucleic acid sequence that is at least 95% identical thereto: and (c) the 3’ flanking region comprises SEQ ID NO: 6423, SEQ ID NO: 6424, or SEQ ID NO: 6425, or a nucleic acid sequence that is at least 95% identical thereto. In some embodiments, the 5’ flanking region comprises SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, the loop region comprises SEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and the 3’ flanking region comprises SEQ ID NO: 6423 or a nucleic acid sequence that is at least 95% identical thereto. In some embodiments, the 5’ flanking region comprises SEQ ID NO: 6415 or a nucleic acid sequence that is at least 95% identical thereto, the loop region comprises SEQ ID NO: 6421 or a nucleic acid sequence that is at least 95% identical thereto, and the 3’ flanking region comprises SEQ ID NO: 6425 or a nucleic acid sequence that is at least 95% identical thereto. In some embodiments, the 5’ flanking region comprises SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, the loop region comprises SEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and the 3’ flanking region comprises SEQ ID NO: 6424 or a nucleic acid sequence that is at least 95% identical thereto. In some embodiments, the 5’ flanking region comprises SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, the loop region comprises SEQ ID NO: 6418 or a nucleic acid sequence that is at least 95% identical thereto, and the 3’ flanking region comprises SEQ ID NO: 6423 or a nucleic acid sequence that is at least 95% identical thereto.

[0026] In some embodiments, the modulatory polynucleotide comprises an siRNA or shRNA.

[0027] 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 ATXN2 mRNA transcripts (c.g., one or more mutated ATXN2 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.

[0028] In some embodiments, the passenger strand is 15-30 nucleotides in length. In some embodiments, the guide strand is 15-30 nucleotides in length. In some embodiments, the guide strand is 21-25 nucleotides in length and / or the passenger strand is 21-25 nucleotides in length. In some embodiments, the passenger strand is at least 70%, at least 80%, at least 90%. at least 95%, or is 100%. complementary to the guide strand.

[0029] In some embodiments, the one or more ATXN2 mRNA transcripts comprises SEQ ID NO: 6428. SEQ ID NO: 6429, SEQ ID NO: 6430, SEQ ID NO: 6431. SEQ ID NO: 6434, SEQ ID NO: 6435, SEQ ID NO: 6436, and / or SEQ ID NO: 6437. or a trinucleotide repeat expansion thereof.

[0030] In some embodiments, the viral genome comprises a promoter operably linked to the nucleic acid sequence encoding the modulatory polynucleotide. In some embodiments, the viral genome further comprises an inverted terminal repeat (ITR) sequence. In some embodiments, the viral genome comprises an ITR sequence positioned 5’ relative to the nucleic acid sequence encoding the modulator,' polynucleotide. In some embodiments, the viral genome comprises an ITR sequence positioned 3’ relative to the nucleic acid sequence encoding the modulatory polynucleotide. In some embodiments, the viral genome comprises an ITR sequence positioned 5 ’ relative to the nucleic acid sequence encoding the modulatory polynucleotide, and an ITR sequence positioned 3’ relative to the nucleic acid sequence encoding the modulatory polynucleotide.

[0031] In some aspects, the present disclosure provides a cell comprising an AAV particle of any one of the embodiments disclosed herein. In some embodiments, the cell is a mammalian cell (e.g., an HEK.293 cell), an insect cell (e.g., an S19 cell), or a bacterial cell.

[0032] In some aspects, the present disclosure provides a method of making an AAV particle provided herein, comprising: (i) providing a host cell comprising the viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of ATXN2 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.

[0033] In some embodiments of a method of making an AAV particle, the viral genome comprises: (i) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6423 or a nucleic acid sequence that is at least 95% identical thereto; (ii) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6415 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6421 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6425 or a nucleic acid sequence that is at least 95% identical thereto; (iii) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6424 or a nucleic acid sequence that is at least 95% thereto; or (iv) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6418 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6423 or a nucleic acid sequence that is at least 95% thereto; and 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 least95%, 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.

[0034] In some embodiments of a method of making an AAV particle, the viral genome comprises: (i) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising die nucleic acid sequence of SEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6423 or a nucleic acid sequence that is at least 95% identical thereto; (ii) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6415 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6421 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6425 or a nucleic acid sequence that is at least 95% identical thereto; (iii) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6424 or a nucleic acid sequence that is at least 95% thereto; or (iv) a 5’ flanking region comprising tire nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6418 or a nucleic acid sequence that is at least 95% identical thereto, and a 3' flanking region comprising the nucleic acid sequence of SEQ ID NO: 6423 or a nucleic acid sequence that is at least 95% thereto; and the AAV capsid variant comprises SEQ ID NO: 981, tire ammo 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.

[0035] In some embodiments of a method of making an AAV particle, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981. In some embodiments, the method further comprises, prior to step (i). introducing a nucleic acid comprising the viral genome into the cell. In some embodiments, the method further comprises, prior to step (i). introducing the nucleic acid molecule encoding the AAV capsid variant into the cell. In some embodiments, the cell comprises a mammalian cell (e.g., an HEK.293 cell), an insect cell (e.g., an SI9 cell), or a bacterial cell.

[0036] In some aspects, the present disclosure provides a pharmaceutical composition comprising an AAV particle provided herein and a pharmaceutically acceptable excipient.

[0037] In some aspects, the present disclosure provides a method of delivering an AAV particle encoding a modulatory polynucleotide for reducing or eliminating expression of ATXN2 mRNA to a cell, comprising administering an effective amount of a pharmaceutical composition or AAV particle provided herein. In some embodiments, the cell is in a subject. In some embodiments, the method reduces or eliminates ATXN2 protein in the subject. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having an ATXN2-related disorder. In some embodiments, the ATXN2-related disorder is spinocerebellar ataxia type 2 (SCA2).

[0038] In some aspects, the present disclosure provides a method of treating an ATXN2-related disorder in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle provided herein. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having the ATXN2-related disorder.

[0039] In some embodiments, the subject has one or more mutations in the ATXN2 gene. In some embodiments, the one or more mutations in the ATXN2 gene comprises a trinucleotide repeat expansion. In some embodiments, the trinucleotide repeat expansion in the ATXN2 gene comprises 32 or more CAG repeats (SEQ ID NO: 6435).

[0040] In some embodiments, a method of treating provided herein results in prevention of progression of the ATXN2-related disorder in the subject. In some embodiments, a method of treating provided herein results in amelioration of at least one symptom of the ATXN2-related disorder in the subject. In some embodiments, the at least one symptom comprises progressive cerebellar ataxia, nystagmus, slow saccadic eye movements, ophthalmoparesis. parkinsonism, or a combination thereof. In some embodiments, the ATXN2-related disorder is SCA2.

[0041] In some aspects, the present disclosure provides a method of treating an ATXN2-related disorder in a subject, wherein the ATXN2-related disorder is SCA2, comprising administering to the subject an effective amount of a pharmaceutical composition or an AAV particle provided herein. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having SCA2.

[0042] In some embodiments, the subject is a human.

[0043] In some embodiments, an AAV particle or the pharmaceutical composition provided herein is delivered to a cell or tissue of the central nervous system (CNS) in the subject. In some embodiments, the cell or tissue of the CNS is a cell or tissue of tire 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). In some embodiments, the AAV particle or the pharmaceutical composition is delivered via intravenous administration.

[0044] In some embodiments, a method of deliver}' or treating provided herein further comprises evaluating, e.g., measuring, the level of modulatory polynucleotide expression, and / or the level of ATXN2 expression, e.g., ATXN2 gene expression, ATXN2 mRNA expression, and / or ATXN2 protein expression, in the subject, e.g., in a cell, tissue, or fluid, of the subject. In some embodiments, the level of ATXN2 protein is measured by an enzyme-linked immunosorbent assay (ELISA), a Western blot, or an immunohistochemistry assay. In some embodiments, the evaluating of the subject’s level of modulatory polynucleotide expression and / or level of ATXN2 expression is performed prior to and / or subsequent to administration of a pharmaceutical composition or AAV particle provided herein. In some embodiments, the subject’s level of modulatory polynucleotide expression and / or the subject’s level of ATXN2 expression prior to administration is compared to the subject’s level of modulatory polynucleotide expression and / or the subject’s level of ATXN2 expression subsequent to administration. In some embodiments the subject’s level of ATXN2 protein expression subsequent to administration is decreased relative to the subject’s level of ATXN2 protein expression prior to administration. In some embodiments, the method further comprises evaluating, e.g., measuring, the level of modulatory polynucleotide activity and / or ATXN2 activity in the subject, e.g., in a cell or tissue of the subject.

[0045] In some embodiments, administering the pharmaceutical composition or the AAV particle to the subject results in: (i) a decrease in ATXN2 protein expression in a cell, 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, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)), and / or fluid (e.g., CSF and / or serum) of the subject relative to baseline and / or relative to ATXN2 protein expression in a cell, tissue, or fluid of an individual with an ATXN2- rclatcd disorder who has not been administered the pharmaceutical composition or AAV particle; (ii) 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; and / or (iii) a decrease in ATXN2 mRNA expression in a cell or tissue (e.g., a cell or tissue of the CNS, e.g.. 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) of the subject relative to baseline and / or relative to ATXN2 mRNA expression in a cell or tissue of an individual with an ATXN2-related disorder who has not been administered the pharmaceutical composition or AAV particle.

[0046] In some aspects, the present disclosure provides a method of treating a subject having or diagnosed with having an ATXN2-related disorder, wherein the ATXN2-related disorder is amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), or frontotemporal lobar degeneration (FTLD), comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle provided herein.

[0047] In some embodiments, a method of delivery or treating provided herein further comprises administering to the subject at least one additional agent and / or therapy. In some embodiments, the at least one additional agent and / or therapy comprises an agent and / or therapy suitable for treating the ATXN2-related disorder. In some embodiments, the at least one additional agent and / or therapy comprises one or more of: growth and trophic factors, cytokines, hormones, neurotransmitters, enzymes, anti-apoptotic factors, angiogenic factors, modulatory polynucleotides, and any protein known to be mutated in pathological disorders such as ATXN2-related disorders. In some embodiments, the ATXN2- related disorder is SCA2. ALS, PD, or FTLD. In some embodiments, the ATXN2-related disorder is SCA2.

[0048] In some embodiments, a method of delivery or treating provided herein further comprises administering an immunosuppressant to the subject. In some embodiments, the immunosuppressant comprises a corticosteroid (e.g., prednisone, prednisolone, methylprednisolone, and / or dexamethasone), rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and / or eculizumab hydroxy chloroquine.

[0049] In some aspects, the present disclosure provides a pharmaceutical composition or AAV particle provided herein for use in a method of treating a disorder provided herein.

[0050] In some aspects, the present disclosure provides a pharmaceutical composition or AAV particle provided herein for use in treating an ATXN2-related disorder in a subject. In some embodiments, the ATXN2-related disorder is SCA2. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having the ATXN2-related disorder (e.g., SCA2).

[0051] In some aspects, the present disclosure provides a use of an effective amount of a pharmaceutical composition or AAV particle provided herein in the manufacture of a medicament for treatment of an ATXN2-related disorder in a subject. In some embodiments, the ATXN2-related disorder is SCA2. In some embodiments, the subject has. has been diagnosed with having, or is at risk of having the ATXN2-related disorder (e.g., SCA2).Enumerated Embodiments1. An adeno-associated virus (AAV) particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and an AAV capsid variant (e.g., an AAV9 capsid variant), wherein 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) | N 11 comprises amino acids Xi, X2, X3, and X4. wherein X4is Q, K, E. S, P, R. N, H; and / or(ii) [N3] comprises amino acids Xs. Xe. and X?, wherein Xs is I, V. T. M. S. N, L. F; and wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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, tw o. 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 X4is Q. K, E, S. P, R, N. or H.7. The AAV particle of embodiment 6, wherein X4is Q or K.8. The AAV particle of any one of embodiment 7, wherein X4is Q.9. The AAV particle of embodiment 7, wherein X 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), TVKQDWHR (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 ID NO: 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 the 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. Xe, and X7, wherein X5is 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) X7is 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), DWHR1SG (SEQ ID NO: 5036), DWHR1SQ (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), DWHRIAK (SEQ ID NO: 5048), DWHRIAH (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 ID NO: 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 [N1]-[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, 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).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 Ataxin-2 (ATXN2) mRNA and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising an amino acid sequence having thefollowing fonnula: [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 X4 is Q, P, or a conservative substitution thereof; and / or(ii) [N3] comprises amino acids X5, Xe, and X?, wherein X, is I, V, or a conservative substitution thereof; and wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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 X4 is 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, 1, 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, NI, 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, 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).64. The AAV particle of any one of embodiments 51-63, wherein [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 X,, Xe, and X7, wherein X5is I or V.73. The AAV particle of embodiment 72, wherein Xs 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 [N 1 ]-[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, die amino acid N at position 594, the amino acid W at position 595, and die amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981:(v) the amino acid T at position 593, die 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 the 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 [N 1 ]-[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, 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, 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, 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. 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, 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, 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. R589) of SEQ ID NO: 981.106. The AAV particle of any one of embodiments 1-105, wherein |N 1 HN2J 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 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, and 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, [N1]-[N2]-[N3]-[N4],130. An AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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, 9, 23, 24, or 25;(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, 9, 23, 24, or 25; 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, 9, 23, 24, or 25; 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. 9, 23. 24, or 25; and wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.131. Ail AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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; and wherein, optionally, the modulatory' polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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), NHQSAQ AQ (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 ofSEQ 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 TNTQDWHR (SEQ ID NO: 4898).146. The AAV particle of any one of embodiments 130-134 and 140-145, w herein 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 TQDWHR1 (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 tw o. 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 tire 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 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: 747.160. 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 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: 747.162. The AAV particle of any one of embodiments 130-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 9, 23, 24, or 25 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, 9, 23, 24. or 25 replaces amino 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, 9, 23, 24. or 25 is present at amino acids 584. 585, 586. 587, 588, 589. and / or 590, as numbered according to the 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, 9, 23, 24, or 25 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, 9, 23, 24. or 25 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, 9, 23, 24. or 25 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. 9. 23. 24, or 25 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, 9, 23. 24, or 25 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 viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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: and wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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 1 at position 590, as numbered according to SEQ ID NO: 981.173. An AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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 a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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 a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.183. An AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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 the AAV capsid variant an ammo 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: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 VP 1 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 two, or at least three modifications, but not morethan 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 a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising the amino acid sequence of SEQ ID NO: 981; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.212. An AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.213. Ail AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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 a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and an AAV capsid variant (e g., an AAV9 capsid variant) comprising amino acids 203-736 of SEQ ID NO: 981; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.216. An AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and 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; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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 a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising amino acids 138-736 of SEQ ID NO: 981; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.219. An AAV particle comprising a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising the amino acid sequence 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; wherein, optionally, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA.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 particlecomprising 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 tw o 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 4.227. The AAV particle of embodiment 225 or 226. wherein the at least two or at least three species comprise Macaca fascicularis, Chlorocebus 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 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 increase is 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.[Embodiments 233-311 are intentionally absent.]312. The AAV particle of any one of embodiments 1-232, wherein the viral genome is single-stranded.313. The AAV particle of any one of embodiments 1-232, wherein the viral genome is self- complementary.314. The AAV particle of any one of embodiments 1-232, embodiment 312, or embodiment 313, w herein die viral genome further comprises a nucleotide sequence encoding a Rep protein, e.g., a non-structural protein, wherein the Rep protein comprises a Rcp78 protein, a Rcp68 protein, a Rcp52 protein, and / or a Rep40 protein (e.g., a Rep78 protein and a Rep52 protein).315. The AAV particle of any one of embodiments 1-232, embodiment 312, or embodiment 313, w herein 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 1-232 and 312-316. w'herein the AAV particle is an isolated AAV particle.318. The AAV particle of any one of embodiments 1-232 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 1-232 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 pharmaceutical composition comprising the AAV particle of any one of embodiments 1-232 and 312-318, and a pharmaceutically acceptable excipient.327. A method of delivering a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA to a cell or tissue (e.g., a CNS cell or CNS tissue), comprising administering an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318.328. The method of embodiment 327, wherein the cell is a cell 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 neuron, a sensory neuron, and / or a motor neuron.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 neurological disorder.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 neurodegenerative 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 a muscular disorder or a neuromuscular disorder.335. A method of treating a subject having or diagnosed with having a genetic disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318.336. A method of treating a subject having or diagnosed with having a neurological disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318.337. A method of treating a subject having or diagnosed with having a muscular disorder or a neuromuscular disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318.338. A method of treating a subject having or diagnosed with having a neurodegenerative disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318.339. The method of any one of embodiments 327-338, wherein the genetic disorder, neurological disorder, neurodegenerative disorder, muscular disorder, or neuromuscular disorder is an ATXN2-related disorder, e.g., SCA2.340. 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 342-346, wherein administration of the AAV particle or pharmaceutical composition results in a decrease in expression of an ATXN2 gene, mRNA, protein, or combination thereof.348. The method of any one of embodiments 342-346, wherein administration of the AAV particle or pharmaceutical composition results in a decreased activity of an ATXN2 gene, mRNA, protein, or a combination thereof.349. The pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318, for use in a method of delivering a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA to a cell or tissue.350. The pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318, for use in a method of treating a genetic disorder, a neurological disorder, a neurodegenerative disorder, a muscular disorder, or a neuromuscular disorder.351. The pharmacal composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318, for use in the manufacture of a medicament.352. Use of the pharmacal composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318 in the manufacture of a medicament.353. Use of the pharmaceutical composition of embodiment 326 or the AAV particle of any one of embodiments 1-232 and 312-318 in the manufacture of a medicament for treating a genetic disorder, a neurological disorder, a neurode generative disorder, a muscular disorder, or a neuromuscular disorder.354. 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 Ataxin-2 (ATXN2) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA; and wherein the AAV capsid variant comprises an amino acid sequence having the formula [Nl]- [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 X?, wherein X5is I, V, T, M, S, N, L, or F.355. The AAV particle of embodiment 354, wherein the AAV capsid variant is an AAV9 capsid variant.356. The AAV particle of embodiment 354 or embodiment 355, 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: 981.357. The AAV particle of any one of embodiments 354-356, wherein loop VIII comprises amino acids 580-599 as numbered according to SEQ ID NO: 981.358. The AAV particle of any one of embodiments 354-357, 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.359. The AAV particle of any one of embodiments 354-358. 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.360. The AAV particle of any one of embodiments 354-359, wherein [Nl] comprises the amino acid sequence of TNTQ (SEQ ID NO: 4688).361. The AAV particle of any one of embodiments 354-360, wherein [N3] comprises the amino acid sequence of IAQ.362. The AAV particle of any one of embodiments 354-361, wherein [N1]-N2]-[N3] comprises the amino acid sequence of TNTQD WHRI AQ (SEQ ID NO: 343).363. The AAV particle of any one of embodiments 354-362, 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: 981.364. The AAV particle of any one of embodiments 354-363, further comprising [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.365. The AAV particle of embodiment 364, wherein [N4] comprises the amino acid sequence of TGWV (SEQ ID NO: 5066).366. The AAV particle of embodiment 364 or embodiment 365, wherein [N4] is present at amino acids 593-596 as numbered according to SEQ ID NO: 981.367. The AAV particle of any one of embodiments 354-366, wherein [N1]-[N2]-[N3]-[N4] comprises the amino acid sequence of TNTQD WHRIAQTGWV (SEQ ID NO: 201).368. An adeno-associated virus (AAV) particle comprising:(i) a viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 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; 981.369. The AAV particle of any one of embodiments 354-368, 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: 981.370. 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 Ataxin-2 (ATXN2) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 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 arc numbered according to SEQ ID NO; 981.371. The AAV particle of any one of embodiments 354-370, 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 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 Ataxin-2 (ATXN2) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA; and(ii) an AAV capsid variant comprising 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 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, and I at position 590; wherein the amino acids are numbered according to SEQ ID NO; 981.373. The AAV particle of embodiment 372. wherein the AAV capsid variant comprises;(i) a VP1 protein comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 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: 981.374. The AAV particle of any one of embodiments 368-373, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941).375. The AAV particle of any one of embodiments 368-374, 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).376. The AAV particle of any one of embodiments 368-375, 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: 981.377. The AAV particle of any one of embodiments 374-376, 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: 981.378. The AAV particle of any one of embodiments 354-377, 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 the amino acid sequence according to positions 203-736 of SEQ ID NO: 981.379. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, 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.380. The AAV particle of embodiment 379, 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.381. The AAV particle of embodiment 380, 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 die 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 die 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.382. The AAV particle of embodiment 380. 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 the 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 themolecular 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.383. The AAV particle of embodiment 380, 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: 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.384. The AAV particle of embodiment 380. 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.385. The AAV particle of any one of embodiments 1-232, 312-318. and 354-384, wherein the modulatory polynucleotide comprises a siRNA.386. The AAV particle of embodiment 385, wherein the modulatory polynucleotide further comprises a passenger strand and a guide strand, wherein the guide strand binds to and reduces or eliminates expression of one or more ATXN2 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 between the loop region and the 3 ’ flanking region.387. The AAV particle of embodiment 385, wherein the modulatory polynucleotide further comprises a passenger strand and a guide strand, wherein the guide strand binds to and reduces or eliminates expression of one or more ATXN2 mRNA transcripts, and wherein the guide strand and the passenger strand are located, respectively, on a 5’ ann and a 3’ arm of a stem loop structure, wherein the guide strand is located between the 5 ’ flanking region and the loop region and the passenger strand is located between the loop region and the 3 ’ flanking region.388. The AAV particle of embodiment 386 or embodiment 387, wherein the passenger strand is 1 -30 nucleotides in length.389. The AAV particle of any one of embodiments 386-388, wherein the guide strand is 15-30 nucleotides in length.390. The AAV particle of embodiment 389. wherein the guide strand is 21-25 nucleotides in length and / or wherein the passenger strand in 21-25 nucleotides in length.391. The AAV particle of any one of embodiments 386-390. wherein the passenger strand is at least 70% complementary to the guide strand.392. The AAV particle of any one of embodiments 386-391. wherein the one or more ATXN2 mRNA transcripts comprises SEQ ID NO: 6428, SEQ ID NO: 6429, SEQ ID NO: 6430, and / or SEQ ID NO: 6431, or a trinucleotide repeat expansion thereof.393. The AAV particle of any one of embodiments 1-232, 312-318. and 354-392, wherein the viral genome comprises a promoter operably linked to the nucleic acid sequence encoding the modulatory polynucleotide.394. The AAV particle of any one of embodiments 1-232, 312-318. and 354-393, wherein the viral genome further comprises an inverted terminal repeat (ITR) sequence.395. The AAV particle of embodiment 394, 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 modulatory polynucleotide.396. The AAV particle of embodiment 394 or embodiment 395, 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.397. The AAV particle of any one of embodiments 1-232, 312-318, and 354-396, wherein the viral genome further comprises a polyadenylation (poly A) sequence.398. A cell comprising the AAV particle of any one of embodiments 1-232. 312-318, and 354-397, optionally wherein the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an S19 cell), or a bacterial cell.399. A method of making the AAV particle of any one of embodiments 1-232, 312-318, and 354-397, 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 ATXN2 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.400. The method of embodiment 399, wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981.401. The method of embodiment 399 or embodiment 400, further comprising, prior to step (i), introducing a nucleic acid comprising the viral genome into the cell.402. The method of any one of embodiments 399-401, further comprising, prior to step (i) introducing the nucleic acid encoding the AAV capsid variant into the cell.403. The method of any one of embodiments 399-402, wherein the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an S19 cell), or a bacterial cell.404. A pharmaceutical composition comprising the AAV particle of any one of embodiments 1-232, 312- 318, and 354-397 and a pharmaceutically acceptable excipient.405. A medrod of delivering a modulatory polynucleotide for reducing or eliminating expression of ATXN2 mRNA to a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 404 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-397, thereby delivering the modulatory polynucleotide.406. The method of embodiment 405. wherein the method reduces or eliminates ATXN2 protein in the subject.407. The method of embodiment 405 or embodiment 406, wherein the subject has, has been diagnosed with having, or is at risk of having an ATXN2-related disorder.408. The method of embodiment 407, wherein the disorder is spinocerebellar ataxia type 2 (SCA2).409. A method of treating an ATXN2-related disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 404 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-402, thereby treating the ATXN2-related disorder.410. The method of embodiment 409, wherein the subject has, has been diagnosed with having, or is at risk of having the ATXN2-related disorder.411. The method of embodiment 409 or embodiment 410, wherein the subject has one or more mutations in the ATXN2 gene.412. The method of embodiment 411, wherein the one or more mutations in the ATXN2 gene comprises a trinucleotide repeat expansion.413. The method of embodiment 412, wherein the trinucleotide repeat expansion in the ATXN2 gene comprises 32 or more CAG repeats (SEQ ID NO: 6435).414. The method of any one of embodiments 409-413, wherein the treating results in prevention of progression of the ATXN2-related disorder in the subject.415. The method of any one of embodiments 409-414, wherein the treating results in amelioration of at least one symptom of the ATXN2-related disorder in the subject.416. The method of embodiment 415, wherein the at least one sy mptom comprises progressive cerebellar ataxia, nystagmus, slow saccadic eye movements, ophthahnoparcsis, parkinsonism, or a combination diereof.417. The method of any one of embodiments 409-416, wherein the ATXN2-related disorder is SCA2.418. A method of treating spinocerebellar ataxia ty pe 2 (SCA2) in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 404 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-397, thereby treating SCA2.419. The method of embodiment 418, wherein the subject has, has been diagnosed with having, or is at risk of having SCA2.420. The method of any one of embodiments 405-419. wherein the subject is a human subject.421. The method of any one of embodiments 405-420, wherein the AAV particle or the pharmaceutical composition is delivered to a cell or tissue of the CNS.422. The method of embodiment 421, wherein the cell or tissue of the CNS is a cell or tissue of 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 ambiguous, 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).423. The method of any one of embodiments 405-422, further comprising evaluating, e.g., measuring, the level of modulatory' polynucleotide expression, and / or the level of ATXN2 expression, e g., ATXN2 gene, ATXN2 mRNA, and / or ATXN2 protein expression, in the subject, e.g., in a cell, tissue, or fluid, of the subject.424. The method of embodiment 423, wherein the level of ATXN2 protein is measured by an ELISA, a Western blot, or an immunohistochemistry assay.425. The method of embodiment 423 or embodiment 424, wherein the evaluating of the subject’s level of modulatory polynucleotide expression and / or level of ATXN2 expression 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 and / or the subject’s level of ATXN2 expression prior to administration is compared to the subject’s level of modulatory polynucleotide expression and / or the subject’s level of ATXN2 expression subsequent to administration.426. The method of any one of embodiments 405-425, wherein the subject’s level of ATXN2 protein expression subsequent to administration is decreased relative to the subject’s level of ATXN2 protein expression prior to administration.427. The method of any one of embodiments 405-426, further comprising evaluating, e.g., measuring, the level of modulatory' polynucleotide activity and / or ATXN2 activity in the subject, e.g., in a cell or tissue of the subject.428. The method of any one of embodiments 405-427, wherein the administration results in:(i) a decrease in ATXN2 protein expression in a cell, 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 ambiguous, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)), and / or fluid (e g., CSF and / or serum) of the subject relative to baseline and / or relative to ATXN2 protein expression in a cell, tissue, or fluid of an individual with an ATXN2-related disorder who has not been administered the pharmaceutical composition or AAV particle;(ii) 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 ambiguous, 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; and / or(iii) a decrease in ATXN2 mRNA expression in a cell or tissue (e.g., a cell or tissue of the CNS, e.g.. 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 ambiguous, 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 ATXN2 mRNA expression in a cell or tissue of an individual with an ATXN2-rclatcd disorder who has not been administered the pharmaceutical composition or AAV particle.429. The method of any one of embodiments 405-428, further comprising administering to the subject at least one additional therapeutic agent and / or therapy.430. The method of embodiment 429. wherein the at least one additional therapeutic agent and / or therapy comprises an agent and / or therapy for treating the ATXN2-related disorder.431. The method of embodiment 429 or embodiment 430, wherein the at least one additional therapeutic agent and / or therapy comprises one or more of: growth and trophic factors, cytokines, hormones, neurotransmitters, enzymes, anti-apoptotic factors, angiogenic factors, modulatory polynucleotides, and any protein known to be mutated in pathological disorders such as ATXN2-related disorders.432. The method of embodiment 430 or embodiment 431, wherein the ATXN2-related disorder is SCA2, amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), or frontotemporal lobar degeneration (FTLD).433. The method of any one of embodiments 430-432. wherein the ATXN2-related disorder is SCA2.434. The method of any one of embodiments 405-433. further comprising administering an immunosuppressant to the subject.435. The method of embodiment 434. wherein the immunosuppressant comprises a corticosteroid (e.g., prednisone, prednisolone, methylprednisolone, and / or dexamethasone), rapamycin, mycophenolate mofetil. tacrolimus, rituximab, and / or eculizumab hydroxychloroquine.436. A method of treating a subject having or diagnosed with having amyotrophic lateral sclerosis (ALS). Parkinson’s disease (PD). or frontotemporal lobar degeneration (FTLD), comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 404 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-397.437. The method of embodiment 436, further comprising administering to the subject at least one additional therapeutic agent and / or therapy.438. The method of embodiment 437, wherein the at least one additional therapeutic agent and / or therapy comprises one or more of: growth and trophic factors, cy tokines, hormones, nemotransmitters, enzymes, anti-apoptotic factors, angiogenic factors, modulatory’ polynucleotides, and any protein known to be mutated in pathological disorders such as ATXN2-related disorders.439. The method of any one of embodiments 436-438, further comprising administering an immunosuppressant to the subject.440. The method of embodiment 439. wherein the immunosuppressant comprises a corticosteroid (e.g., prednisone, prednisolone, methylprednisolone, and / or dexamethasone), rapamycin, mycophenolatemofetil, tacrolimus, rituximab, and / or eculizumab hydroxy chloroquine.441. The pharmacal composition of embodiment 404 or the AAV particle of any one of embodiments 1-232. 312-318, and 354-397 for use in a method of treating a disorder according to any one of embodiments 409-440.442. The pharmaceutical composition of embodiment 404 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-397 for use in treating an ATXN2-related disorder in a subject, optionally wherein the ATXN2-related disorder is SCA2.443. The pharmacal composition or the AAV particle for use of embodiment 442, wherein the subject has, has been diagnosed with having, or is at risk of having the ATXN2-related disorder, optionally wherein the ATXN2-related disorder is SCA2.444. Use of an effective amount of the pharmacal composition of embodiment 404 or the AAV particle of any one of embodiments 1-232, 312-318. and 354-397 in the manufacture of a medicament for treatment of an ATXN2-related disorder in a subject, optionally wherein the ATXN2-related disorder is SCA2.445. The use of embodiment 444. wherein the subject has, has been diagnosed with having, or is at risk of having the ATXN2-related disorder, optionally wherein the ATXN2-related disorder is SCA2.

[0052] 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 forms also include the plural unless the context clearly dictates otherwise. Unless defined otherw ise, 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

[0053] Described herein, inter alia, are compositions comprising an AAV capsid variant for delivery of modulatory polynucleotides for reducing or eliminating expression of ATXN2 to treat SCA2 or other disorders associated with aberrant ATXN2 protein expression or activity or mutant ATXN2 protein. In some embodiments, the modulatory polynucleotides reduce or eliminate expression of ATXN2 mRNA. thereby reducing or eliminating ATXN2 protein. The AAV capsid variants described herein have enhanced tropism for a cell or tissue, e.g., for the delivery of a payload to said cell or tissue, for example a CNS tissue or a CNS cell.

[0054] 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. Engineered adeno-associated virus (AAV) capsids with improved brain tropism represent an attractive solution to the limitations of CNS delivery'. AAV-derived vectors are promising tools for clinical genetransfer because of their non-patliogenic 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 serotypes, or insertion of fully randomized short peptides at defined positions.

[0055] 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 desired pay load. The genome of the virus may encode a modulatory polynucleotide, e.g., a modulatory polynucleotide for reducing or eliminating expression of ATXN2 mRNA, thereby reducing or eliminating ATXN2 protein, and the viral particle comprising said genome may be delivered to a target cell, tissue, or organism. In some embodiments, the genome encodes a modulatory polynucleotide for reducing or eliminating expression of ATXN2 mRNA, thereby reducing or eliminating ATXN2 protein. In some embodiments, the target cell is a CNS cell. In some embodiments, the target tissue is a CNS tissue. In some embodiments, the target CNS tissue is brain tissue. In some embodiments, the target CNS tissue comprises 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). In some embodiments, the target CNS tissue is in the forebrain. In some embodiments, the target CNS tissue is brainstem. In some embodiments, the target CNS tissue is cerebellum. In some embodiments, tire target CNS tissues are brainstem and cerebellum.

[0056] Gene therapy presents an alternative approach for SCA2 and related diseases sharing singlegene 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.

[0057] Provided herein are compositions and methods which may provide for improved features compared to prior AAV-mediated approaches, including (i) increased biodistribution throughout the CNS (e.g., amygdala, brainstem, caudate, central grey, cerebellum (e.g.. Purkinje cell layer and deep cerebellarnuclei), 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)), (ii) elevated modulatory polynucleotide expression in multiple CNS regions (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)); and (iii) preferential biodistribution in tire CNS over the liver and / or DRG, of the subject.

[0058] 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 CNS regions, 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), (iii) elevated modulatory polynucleotide expression in multiple CNS regions, (iv) wider distribution in one or more peripheral tissues, e.g., the heart, kidney, muscle (e.g., quadriceps muscle), pancreas, and / or liver, and / or (v) elevated modulatory polynucleotide expression in one or more peripheral tissues. In some embodiments, the AAV capsids described herein enhance the delivery of a modulatory polynucleotide to multiple regions of the CNS including, for example, 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). In some embodiments, the AAV capsids described herein enhance the delivery of a modulatory' polynucleotide to the spinal cord. In some embodiments, the AAV capsid variants enhance delivery of a modulatory polynucleotide to the dentate nucleus. In some embodiments, the AAV capsid variants enhance delivery of a modulatory polynucleotide to the somatosensory cortex. In some embodiments, the AAV capsid variants with enhanced brain tropism described herein increase modulatory polynucleotide expression indie brain. In some embodiments, the AAV capsid variants with enhanced brain tropism reduce or eliminate expression of ATXN2 mRNA in the brain.

[0059] The modulatory polynucleotides provided herein modulate expression of mutated ATXN2 by altering levels of the mutated protein and / or mRNA in cells and / or tissues. In some embodiments, the modulatory polynucleotides reduce or eliminate expression of mutated ATXN2 mRNA, e.g.. by binding to mutated ATXN2 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 ATXN2 mRNA. In some embodiments, the modulatory polynucleotides reduce or eliminate expression of mutated ATXN2 by reducing or eliminating translation of the mutated mRNA into protein. In some embodiments, the modulatory polynucleotide may reduce or eliminate formation of 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.

[0060] Thus, the compositions and methods described herein can be used in the treatment of ATXN2-related disorders (e.g., SCA2). In some embodiments, the disclosure provides an AAV particle comprising a AAV capsid variants 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 an ATXN2-related disorder. In some embodiments, the modulatory polynucleotide reduces or eliminates expression of Ataxin-2 (ATXN2) mRNA. In some embodiments, the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA. In some embodiments, the RNAi agent comprises siRNA. In some embodiments, the ATXN2-related disorder is SCA2.I. CompositionsAdeno-associated viral (AA V) Particles

[0061] AAVs have a genome of about 5,000 nucleotides in length and contains two 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 seroty pe, as a non-limiting example, for AAV9 / 11U.14 (SEQ ID NO: 123 of US 7,906,111, the contents of which are herein incorporated byreference 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, VP1 is the full-length capsid protein sequence, while VP2 and VP3 are shorter components of the whole. As a result, changes in the sequence in the VP3 region are also changes to VP1 and VP2, however, the percent difference as compared to the parent sequence will be greatest for VP3 since it is the shortest sequence of the three. Though described here in relation to the amino acid sequence, the nucleic acid sequence encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid. Without being bound by theory, the AAV capsid typically comprises a molar ratio of 1: 1: 10 of VP1:VP2:VP3.

[0062] The AAV particle typically requires a co-helper (e.g.. adenovirus) to undergo productive infection in cells. In the absence of such helper functions, the AAV virions essentially enter host cells but do not integrate into the cells’ genome.

[0063] 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).

[0064] 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 eases, 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 ATXN2, e g., mutant ATXN2 protein).

[0065] hi some embodiments, the AAV particles of the present disclosure may be introduced into mammalian cells.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] In some embodiments, an AAV capsid variant comprises one or more modifications 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 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, 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, 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.

[0070] The AAV particles and pay loads of the disclosure may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles demonstrate enhanced tropism for a target cell type, tissue, or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively). In some embodiments, an AAV particle may, in addition, or alternatively, have decreased tropism for a cell-type, tissue or organ.

[0071] 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 immunogenicprofile. 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 witii or engineered to target a particular tissue and express or deliver a desired pay load.

[0072] 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-com piemen tary region (145nt in wild-type AAV) at the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region. In some embodiments, the double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.

[0073] 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 (scAAVs) 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.

[0074] 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 content of each of which is incorporated herein by reference in its entirety ).

[0075] 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.AAV Capsid Variants

[0076] Disclosed herein are AAV particles comprising an AAV capsid variant comprising one or more modifications (e.g., comprising one or more substitutions relative to a wildtype AAV capsid) for enhanced or improved transduction of a target tissue (e.g., cells, regions, and / or tissues of the CNS and / or PNS). In some embodiments, the peptide (e.g., comprising one or more substitutions relative to a wildtype AAV capsid) is present in VP1, VP2, and / or VP3 proteins of the AAV capsid variant. In some embodiments, 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.

[0077] 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.

[0078] In some embodiments, the one or more substitutions in the AAV capsid 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. 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, visual cortex), hilus of the dentate gyrus, 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.

[0079] 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.

[0080] 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., 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, visual cortex), hilus of the dentate gyrus, 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) following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI- guided FUS coupled with intravenous administration.

[0081] hi 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 FU S coupled with intravenous administration.

[0082] 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.

[0083] hi some embodiments, tire 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) or a quadriceps 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.

[0084] 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.

[0085] 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 9. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 23. In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) a sequence as set forth in Table 24. In some embodiments, the AAV capsid variant is an AAV9 capsid variant.Table 1. Exemplary SequencesTable 2A. Exemplary SequencesTable 2B. Exemplary Sequences

[0086] 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: 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 X 1 is Q, K, E, S, P, R, N, H, or a conservative substitution thereof and / or [N3] comprises X5, Xe, and X7, wherein Xs is 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, X, 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 [Nl] is T, N. K, D. I. 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, [N 1]-[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, [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343). In some embodiments, [N1]-[N2]-[N3] is or comprises TNTKDWHRIAQ (SEQ ID NO: 344).

[0087] 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).

[0088] 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 X 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] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); [Nl] comprises amino acids Xi, X2, X3, and X4. wherein X i is Q or P; and / or [N3] comprises amino acids Xs. Xe. and X?. wherein X5 is I or V. In someembodiments, X4of [Nl] is Q. In some embodiments, X5of [N3] is I. In some embodiments, X5of [N3] is V. In some embodiments, Xi of [Nl] is T or S. In some embodiments, X2 of [Nl] is N, T, G, S, I, or V. In some embodiments, X3 of [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, X6of [N3] is A, P, S, Y, or N. In some embodiments, X7of [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 [N1]-[N2]-[N3] further comprises [N4], wherein [N4] comprises amino acids X8, X9, Xw, and Xu. wherein X10 is W. In some embodiments. Xxof [N4] is T, S. or N. In some embodiments, Xg of [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).

[0089] 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.

[0090] 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, X2 of [Nl] is present at an amino acid corresponding to position 583 of the amino acid sequence of SEQ ID NO: 138 or 981, X3 of [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.

[0091] 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).

[0092] 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 with T582, N583, T584, Q585, D586, W587, H588, and R589).

[0093] 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.

[0094] 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).

[0095] 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).

[0096] 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, X9of [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.

[0097] 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).

[0098] In some embodiments, [N 1]-[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 someembodiments, [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).

[0099] hi 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, [N1]-[N2]-[N3]-[N4],

[0100] 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, 9, 23, 24, or 25. 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.

[0101] 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, the at least 6 consecutive amino acids comprise TQDWHR (SEQ ID NO: 4686). In some embodiments, the at least 7 consecutive amino acids comprise TQDWHRI (SEQ ID NO: 941).

[0102] 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, die at least 5 consecutive amino acids comprise TNTQD (SEQ ID NO: 5119). In some embodiments, die 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).

[0103] 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. 9. 23. 24, or 25. 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 the sequences provided in Table 1. 2A. 2B. 9, 23. 24, or 25. In some embodiments, the AAV capsid variant comprises (e.g.. in loop VIII) anamino acid sequence comprising at least one, at least tw o. 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.

[0104] 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).

[0105] In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of the sequences provided in Table 1, 2 A, 2B, 9, 23, 24, or 25. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NOs: 201. 205-209, 211-214. 21 , 219, 220. 230, 232, 237. 238, 255, 262-265, 274, 283. 286, 290, 291, 293, 301. 306, 307, 308. 309, 314, and 336.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, or a portion thereof that is modified relative to a wildty pe 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.

[0110] 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. In some embodiments, the AAV capsid variant further comprises the amino acid T at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981. In some- TI -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, die 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.[oni] In some embodiments, an AAV capsid variant described herein comprises one, two, Uiree, 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 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; an amino acid other than and 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 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.

[0112] 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 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 amino acid I at a position corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0113] 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) correspond to position(s) 584, 586, 587, 588, 589, and 590 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0114] 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.

[0115] 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 ammo acid sequence of SEQ ID NO: 138.

[0116] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than Q at an amino acid corresponding to position 590 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.

[0117] 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 theAAV 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).

[0118] 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.

[0119] 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 an 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.

[0120] 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.

[0121] 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).

[0122] 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%, atleast 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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

[0127] 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% (c.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.

[0128] 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 threemodifications 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 tw o. 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.

[0129] 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.

[0130] 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.

[0131] 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 protein 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%, 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 1-736. e.g.. a VP1, of the amino acidsequence 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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 non- human primate and rodent (e.g., mouse) species, compared to enrichment in the brain of an AAV capsidcomprising 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).

[0137] 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.

[0138] 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.

[0139] In some embodiments, an AAV capsid variant delivers an increased level of a pay load 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.

[0140] In some embodiments, an AAV capsid variant described herein is enriched at least 5, at least 10, at least 0, 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.

[0141] In 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 modifications are 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.

[0142] In some embodiments, the 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 Til (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 aminoacid 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.

[0143] In 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 its 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.

[0144] 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.

[0145] Also provided herein are polynucleotide sequences encoding any of the AAV capsid variants described above and AAV particles, vectors, and cells comprising the same.AAV serotypes and capsids

[0146] In some embodiments, the AAV particle may comprise a capsid protein or variant of any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to, packaging, tropism, transduction and immunogenic 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.

[0147] In some embodiments, the AAV capsid variant allows for blood brain barrier penetration following intravenous administration. In some embodiments, the AAV capsid variant allows for blood brain barrier penetration following intravenous administration, focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration. In some embodiments, the AAV capsid variant allows for increased distribution to a CNS region. In some embodiments, the CNS region comprises 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). 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-neuronalcell, e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination thereof). In some embodiments, the AAV capsid variant allows for transduction in both neuronal cells and non-neuronal cell, e.g., a glial cell (e.g., an astrocyte, an oligodendrocyte, or a combination drereof).

[0148] 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.

[0149] In some embodiments, the AAV capsid variant allows for increased distribution to a heart region.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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-mcthioninc (Mctl) 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.

[0154] 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.

[0155] 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-).

[0156] 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).

[0157] 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.

[0158] 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-).

[0159] 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

[0160] In some embodiments, the AAV capsid variant comprises at amino acids corresponding to 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, 9, 23, 24, or 25.

[0161] 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. 2 A, 2B. 9, 23. 24. or 25. 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. 9. 23, 24, or 25 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 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).

[0162] 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

[0163] 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 poly nucleotide 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.

[0164] hi 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 ATXN2 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 nucleotide sequence encoding a miRNA binding site.Viral Genome Component: Inverted Terminal Repeats (ITRs)

[0165] In some embodiments, tire 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 modulatory polynucleotide for reducing or eliminating expression of ATXN2, e.g., mutant ATXN2 protein. In some embodiments, the viral genome has two ITRs. These two ITRs flank the region comprising the modulatory polynucleotide at the 5’ and 3’ ends. In some embodiments, the 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.

[0166] 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 seroty pe as one another. In some embodiments, the ITRs are of different serotypes. Non-limiting examples include zero, one, or both of the ITRs having the same serotype as the capsid.Viral Genome Component: Promoters and Enhancers

[0167] 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), poly adenylation (poly A) region, upstream enhancers (USEs), CMV enhancers, and introns.

[0168] hi 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 ).

[0169] 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 ATXN2, e.g., mutant ATXN2 protein. 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.

[0170] 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.

[0171] 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 promoters may be human promoters. In some embodiments, the promoter may be truncated.

[0172] 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 which drives or promotes expression in most mammalian tissues includes, but is not limited to, human elongation factor la-subunit (EFla) promoter, a cytomegalovirus (CMV) immediate-early enhancer and / or promoter, a chicken 0-actin (CBA) promoter, a CAG promoter, a 0 glucuronidase (GUSB) promoter, and a ubiquitin C (UBC) promoter.

[0173] In some embodiments, the viral genome comprises a nervous system specific promoter, e.g., 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.

[0174] In some embodiments, the promoter may be less than 1 kb.

[0175] In some embodiments, the promoter may be a combination of two or more components of the same or different starting or parental promoters.

[0176] In some embodiments, the viral genome comprises an enhancer.

[0177] In some embodiments, the viral genome comprises an engineered promoter.Viral Genome Component: Introns

[0178] In some embodiments, the viral genome comprises at least one intron or a fragment or derivative thereof. In some embodiments, the viral genome comprises at least one exon or a fragment or derivative thereof.

[0179] In some embodiments, the intron may be 100-600 nucleotides in length.

[0180] In some embodiments, the modulatory polynucleotide-encoding 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 polyadenylation 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 modulatory polynucleotide-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.

[0181] In certain embodiments, the intron sequence is not an enhancer sequence. In some embodiments, the intron sequence is not a sub-component of a promoter sequence. In some embodiments, the intron sequence is a sub-component of a promoter sequence.Viral Genome Component: Untranslated Regions (UTRs)

[0182] 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 the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.

[0183] Features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production.

[0184] In some embodiments, the viral genome encoding a modulatory polynucleotide described herein (e.g., a modulatory polynucleotide for reducing or eliminating expression of ATXN2 mRNA or protein) comprises a Kozak sequence. Without being bound by theory, wild-type 5’ untranslated regions (UTRs) include features that play roles in translation initiation. Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another “G”.

[0185] In some embodiments, the 5 ’UTR in the viral genome includes a Kozak sequence.

[0186] In some embodiments, the 5 ’UTR in the viral genome docs not include a Kozak sequence.

[0187] Without being bound by theory, wild-type 3’ UTRs are known to have stretches of adenosines and uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al. 1995. the contents of which are herein incorporated by reference in their entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs, such as, but not limited to, GM-CSF and TNF-a, possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Class III ARES, such as. but not limited to, c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR. have been documented to increase the stability' of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding anddius, stabilization of the message in vivo.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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

[0193] 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.

[0194] 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-ty pe sequence.

[0195] hi some embodiments, the viral genome comprises one or more filler sequences in order to have the length of the viral genome be the optimal size for packaging. In some embodiments, the viral genome comprises at least one filler sequence in order to have the length of the viral genome be about 2.3 kb. In some embodiments, the viral genome comprises at least one filler sequence in order to have the length of the viral genome be about 4.6 kb.Viral Genome Component: Modulatory polynucleotide-encoding sequences

[0196] In some embodiments, the disclosure provides an AAV particle comprising a viral genome encoding a modulatory polynucleotide for reducing or eliminating expression of ATXN2, thereby reducing or eliminating ATXN2 protein. In some embodiments, the modulatory polynucleotidecomprises 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.

[0197] In some embodiments, the modulatory polynucleotides of the disclosure reduce or eliminate ATXN2 mRNA. thereby reducing or eliminating ATXN2 protein. Exemplary ATXN2 mRNA sequences are provided in Table 7. In some embodiments, the modulatory polynucleotide of the disclosure reduces or eliminates expression of one or more of (e.g.. one, two, three or all four of) SEQ ID NOs: 6428, 6429, 6430, and 6431, or a trinucleotide repeat expansion of any one of the foregoing. The exemplary ATXN2 sequence information from the National Center for Biotechnology Information cited in Table 7 is hereby incorporated by reference in its entirety.Table 7. Exemplary ATXN2 mRNA transcripts

[0198] hi 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 more of (e.g., one, two, three or all four of) SEQ ID NOs: 6428-6431 or a trinucleotide repeat expansion thereof. In some embodiments, the guide strand targeting any one of SEQ ID NOs: 6428-6431 or a trinucleotide repeat expansion thereof is fully complementary to a region of that ATXN2 mRNA sequence. In some embodiments, the guide strand targeting any one of SEQ ID NOs: 6428-6431 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% identical 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 ATXN2 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 ATXN2 mRNA, e.g.. to a region of any one of SEQ ID NOs: 6428-6431 or a trinucleotide repeat expansion thereof.

[0199] 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%, at least 99%, or 100%) complementary to the guide strand. In some embodiments, the passenger strand has up to 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 theguide strand.

[0200] In some embodiments, the modulatory polynucleotide binds to a coding region of ATXN2 mRNA. In some embodiments, the modulatory polynucleotide binds to a noncoding region of ATXN2 mRNA.

[0201] hi some embodiments, the disclosure herein provides constructs that allow for improved expression of a modulatory polynucleotide delivered by gene therapy vectors.

[0202] In some embodiments, the disclosure provides constructs that allow for improved biodistribution of a modulatory polynucleotide delivered by gene therapy vectors.

[0203] 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.

[0204] In some embodiments, the disclosure provides constructs that allow for improved trafficking of a modulatory polynucleotide to lysosomal membranes delivered by gene therapy vectors.

[0205] In some embodiments, the present disclosure relates to a composition containing or comprising a nucleic acid sequence encoding modulatory polynucleotide for reducing or eliminating expression of ATXN2 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.. an ATXN2-related disorder.

[0206] In some embodiments, the disclosure provides a nucleotide sequence encoding a modulatory polynucleotide for use in an AAV genome. In some embodiments, the nucleotide sequence further comprises one or more, e.g., all of, a 5’ ITR sequence, an enhancer sequence, a promoter sequence, an intron sequence, a poly A sequence, and a 3’ ITR sequence.

[0207] In some embodiments, the AAV genome encodes a payload construct that comprises a combination of coding and non-coding nucleic acid sequences.

[0208] 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 pay load may express each of the pay loads in a single cell.

[0209] In some embodiments, tire 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.

[0210] A pay load 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.

[0211] In some embodiments, a pay load 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.

[0212] In some embodiments, a pay load construct encoding a selectable marker may comprise a fluorescent protein. A fluorescent protein as herein described may comprise any fluorescent marker including but not limited to green, yellow, and / or red fluorescent protein (GFP, YFP. and / or RFP). In some embodiments, a payload construct encoding a selectable marker may comprise a human influenza hemagglutinin (HA) tag.

[0213] 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

[0214] In some embodiments, the molecular scaffold of the modulatory polynucleotide is a known or wild type pri- or pre-microRNA. In other embodiments, the molecular scaffold of the modulatory polynucleotide is designed ab initio. In some embodiments, the molecular scaffold along with the pay load (e.g., passenger strand and guide strand) form a stem loop structure.

[0215] In some embodiments, the molecular scaffold comprises a 5’ flanking region. In some embodiments, the molecular scaffold comprises a 3’ flanking region. In some embodiments, the molecular scaffold comprises a loop region, wherein the loop region is present between the passenger and guide strand of the stem loop structure. In some embodiments, the molecular scaffold comprises one or more spacers between one or more modules of the modulatory poly nucleotide. 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.

[0216] 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.

[0217] In some embodiments, an encoded modulatory polynucleotide described herein comprises from 5’ to 3’: a 5’ flanking region, a passenger strand, a loop region, a guide strand, and a 3‘ flanking region. In some embodiments, an encoded modulatory polynucleotide described herein comprises from 5’ to 3’: a 5' flanking region, a guide strand, a loop region, a passenger strand, and a 3’ flanking region.

[0218] 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 8A-8C.Table 8A. Exemplary 5’ Flanking Regions for Molecular ScaffoldTable 8B. Exemplary Loop Regions for Molecular ScaffoldTable 8C. Exemplary 3’ Flanking Regions for Molecular Scaffold

[0219] 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%, 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 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%, or at least 99% identical to any one of SEQ ID NOs; 6422-6427.

[0220] 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.

[0221] 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’ 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: 6423, SEQ ID NO: 6424, or SEQ ID NO: 6425.

[0222] 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.

[0223] 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.

[0224] 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 least75%, 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.

[0225] 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%. 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.

[0226] 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

[0227] 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.

[0228] 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 as numbered according to SEQ ID NO: 981. In some embodiments, the AAV capsid variant comprises: (i) a VP1 protein comprising orconsisting 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.

[0229] 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 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.

[0230] 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 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.

[0231] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 981.

[0232] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the ammo acid sequence of amino acids 138-736 of SEQ ID NO: 981.

[0233] In some embodiments, the AAV particle comprises a viral genome that is packaged in a capsid comprising the amino acid sequence of SEQ ID NO: 981.

[0234] The present disclosure provides in some embodiments, vectors, cells, and / or AAV particles comprising the above identified viral genomes.Self-Complementary and Single Stranded Viral Genomes

[0235] In some embodiments, the AAV viral genome used in the present disclosure is singlestranded.

[0236] In some embodiments, the AAV viral genome is capable of forming double-stranded DNA. In some embodiments, the AAV viral genome is self-complementary. See, e.g.. US Patent No. 7,465,583. scAAV particles contain both DNA strands that anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell. In some embodiments, the viral genome is self-complementary.

[0237] Methods for producing and / or modifying AAV viral genome and particles are disclosed in the art such as pseudotyped AAV particles (International Patent Publication Nos. W0200028004;W0200123001; W02004112727; WO 2005005610 and WO 2005072364, the content of each of which are incorporated herein by reference in their entirety).II. AAV Production

[0238] Viral production disclosed herein describes processes and methods for producing AAV particles (with enhanced, improved and / or increased tropism for a target tissue), e g., an AAV particle comprising an AAV capsid variant that may be used to contact a target cell to deliver a modulatory polynucleotide for reducing or eliminating expression of ATXN2.

[0239] In some embodiments, disclosed herein is a method of making an AAV particle of the present disclosure, e.g., an AAV particle comprising an AAV capsid variant disclosed herein, wherein the method comprises: (i) providing a cell comprising a viral genome comprising a nucleotide sequence encoding a modulatory polynucleotide for reducing or eliminating expression of ATXN2 (e.g.. mutated ATXN2 mRNA or protein) 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. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981, and the viral genome comprises a molecular polynucleotide comprising: (a) 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; (b) 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%, or at least 99% identical to any one of SEQ ID NOs: 6422-6427. In some embodiments, the method comprises, prior to step (i), introducing a nucleic acid comprising the viral genome into the cell. In some embodiments, the method comprises, prior to step (i), introducing the nucleic acid encoding the AAV capsid variant into the cell. 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. In some embodiments, the AAV particle described herein is an isolated AAV particle. In some embodiments, the AAV particle described herein is a recombinant AAV particle.

[0240] Any method known in the art may be used for the preparation of AAV particles. In some embodiments. AAV particles are produced in mammalian cells (e.g., HEK293 cells). In some embodiments. AAV particles are produced in insect cells (e.g.. Sf9 cells).

[0241] Methods of making AAV particles are well known in the art and are described in e.g., U.S. Patent Nos. US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769. US6482634. US6485966, US6943019, US6953690, US7022519. US7238526, US7291498 and US7491508. US5064764. US6194191, US6566118, US8137948; or International Publication Nos. WO1996039530. W01998010088, WO1999014354. WO1999015685, WO1999047691.W02000055342, W02000075353 and W02001023597; Methods In Molecular Biology’, ed. Richard, Humana Press. NJ (1995); O'Reilly’ et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Or.63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Set. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir., 219:37-44 (1996); Zhao et al., Vir.272:382-93 (2000); the contents of each of which are herein incorporated by reference in their entirety’. In some embodiments, the AAV particles are made using the methods described in International Patent Publication WO2015191508, the contents of which are herein incorporated by reference in their entirety .III. Pharmaceutical Compositions

[0242] In some embodiments, the present disclosure provides pharmaceutical compositions of an adeno-associated virus (AAV) particle comprising a viral genome encoding a modulatory polynucleotide for reducing or eliminating expression of ATXN2 and an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 981.10243] In some embodiments, a composition described herein comprises an AAV polynucleotide or AAV genome or AAV particle, and at least one excipient.

[0244] Although pharmaceutical compositions provided herein, e.g., comprising AAV particles comprising a modulatory polynucleotide-encoding sequence, are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions may be suitable for administration to any other animal, e.g., non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order torender the compositions suitable for administration to various non-human animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary , if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0245] In some embodiments, compositions are administered to humans, e.g., human patients or human subjects.

[0246] In some embodiments, the AAV particle formulations described herein may contain a nucleic acid encoding at least one modulatory polynucleotide for reducing or eliminating expression of ATXN2 (e.g., aberrantly expressed, aberrantly active, or mutant ATXN2). In some embodiments, the formulations may contain a nucleic acid encoding 1. 2, 3, 4, or 5 modulatory polynucleotide(s) for reducing or eliminating expression of ATXN2 (e.g.. aberrantly expressed, aberrantly active, or mutant ATXN2). In some embodiments, the modulatory polynucleotide is a modulatory polynucleotide for reducing or eliminating expression of ATXN2 mRNA, thereby reducing or eliminating ATXN2 protein.

[0247] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.IV. Formulations

[0248] Formulations of the AAV pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory' ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0249] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmacal composition in accordance with the disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered.

[0250] For example, the composition may comprise about 0.1% to about 99% (w / w) of the active ingredient. By way of example, the composition may comprise about 0.1% to about 100%, e.g., about 0.5% to about 50%, about 1% to about 30%. about 5% to about 80%, or at least 80% (w / w) active ingredient.

[0251] The AAV particles of the disclosure can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayedrelease; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase tire translation of encoded protein in vivo'. (6) alter the release profile of the modulatory polynucleotide in vivo and / or (7) allow for regulatable expression of the modulatory polynucleotide.

[0252] Formulations of the present disclosure can include, without limitation, saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics and combinations thereof. Further, the viral vectors of the present disclosure may be formulated using selfassembled nucleic acid nanoparticles.

[0253] In some embodiments, the viral vectors encoding a modulatory polynucleotide may be formulated to optimize baricity and / or osmolality. In some embodiments, the baricity and / or osmolality of the formulation may be optimized to ensure optimal drug distribution in the central nervous system or a region or component of the central nervous system.Excipients

[0254] The formulations of the disclosure can include one or more excipients, each in an amount that together increases the stability of the AAV particle, increases cell transfection or transduction by the viral particle, increases the expression of viral particle encoded protein, and / or alters the release profile of AAV particle encoded proteins. In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary’ use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0255] Excipients, which, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; the contents of which are herein incorporated by reference in their entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.Inactive Ingredients

[0256] In some embodiments, AAV formulations may comprise at least one excipient which is an inactive ingredient. As used herein, the term “inactive ingredient ’ refers to one or more agents that do not- I l l -contribute to the activity of the pharmaceutical composition included in formulations. In some embodiments, all, none, or some of the inactive ingredients which may be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA).

[0257] Formulations of AAV particles may include cations or anions. In one embodiment, the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+, or combinations thereof. In some embodiments, formulations may include polymers or polynucleotides complexed with a metal cation See, e.g., U.S. Pat. Nos. 6.265,389 and 6,555,525, the contents of each of which are herein incorporated by reference in their entirety).V. Uses and Applications

[0258] The compositions of the disclosure may be administered to a subject or used in the manufacture of a medicament for administration to a subject having an ATXN2-related disorder. The ATXN2-related disorder may be a disorder of the central nervous system, and / or a neurological and / or neuromuscular disorder. Also specifically7contemplated herein is spinocerebellar ataxia type 2 (SCA2) and other disorder that arise from expression of an ATXN2 gene product with trinucleotide repeat expansions, e.g., ATXN2 with 32 or more CAG repeats (SEQ ID NO: 6435) (polyQ expansions).

[0259] The present disclosure addresses the need for new technologies by providing ATXN2-related treatment deliverable by AAV-based compositions and complexes for the treatment of ATXN2-related disorders.

[0260] The compositions of the disclosure may be administered to a subject, e.g., to deliver a modulatory’ poly nucleotide, e.g., to a subject who has, has been diagnosed with having, or is at risk of having an ATXN2-related disorder (e.g., SCA2). The compositions may similarly be used in the manufacture of a medicament for administration to a subject having an ATXN2-related disorder (e g., SCA2).

[0261] hi some embodiments, tire disclosure provides a method of delivering a modulatory polynucleotide to a subject comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding a modulatory polynucleotide, thereby delivering the modulatory polynucleotide. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having an ATXN2-related disorder. In some embodiments, the ATXN2-related disorder is SCA2.

[0262] In some embodiments, the disclosure provides a method for treating an ATXN2-related disorder (e.g., SCA2). In certain embodiments, a pharmaceutical composition or AAV particle disclosed herein, comprising a viral genome encoding a modulatory polynucleotide, may be administered to a subject to treat an ATXN2-related disorder (e.g., SCA2), thereby treating the disorder (e.g., SCA2). In some embodiments, the subject has. has been diagnosed with having, or is at risk of having an ATXN2- related disorder. In some embodiments, the treatment results in a reduction in the subject’s ATXN2 protein level (e.g., levels of aberrantly expressed, aberrantly active, and / or mutant ATXN2 protein) as compared to baseline.

[0263] In some embodiments, the disclosure provides an AAV particle or pharmaceutical composition according to any one of the embodiments disclosed herein for treating an ATXN2-related disorder, such as SCA2. In some embodiments, the present disclosure provides the pharmaceutical composition or the AAV particle of any one the embodiments disclosed herein for use in a method of treating a disorder as disclosed herein, such as SCA2.

[0264] In some embodiments, the disclosure provides a method of treating SCA2 in a subject. In certain embodiments, a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding a modulatory polynucleotide may be administered to a subject to treat SCA2. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having SCA2.

[0265] In some embodiments, the disclosure provides a method of treating a subject having or diagnosed with having amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD). or frontotemporal lobar degeneration (FTLD).

[0266] In some embodiments, a pharmaceutical composition or AAV particle may be administered to a subject may have one or more mutations in the ATXN2 gene. In some embodiments, the one or more mutations in the ATXN2 gene comprises a trinucleotide repeat expansion. In some embodiments, the trinucleotide repeat expansion in the ATXN2 gene is 32 or more CAG repeats (SEQ ID NO: 6435).

[0267] In some embodiments, the treatment may result in prevention of progression of an ATXN2- related disorder. For example, the treatment may result in amelioration of at least one symptom of the disease associated with aberrant ATXN2 protein expression or activity or with a mutated ATXN2 protein in the subject (e.g.. SCA2). In some embodiments, at least one symptom comprises progressive cerebellar ataxia, nystagmus, slow saccadic eye movements, ophthahnoparesis, parkinsonism, or a combination thereof.

[0268] In some embodiments, the methods disclosed herein further comprise evaluating, e.g., measuring, the level of modulatory polynucleotide expression, and / or the level of ATXN2 expression, e.g., ATXN2 gene, ATXN2 mRNA, and / or ATXN2 protein expression, in the subject, e.g., in a cell, tissue, or fluid of the subject. In some embodiments, the level of ATXN2 protein is measured by an ELISA, a Western blot, or an immunohistochemistry assay. In some embodiments, evaluating the subject's level of modulatory polynucleotide expression and / or the level of ATXN2 expression is performed prior to and / or subsequent to administration of the pharmaceutical composition or AAV particle, optionally wherein the subject's level of the modulatory polynucleotide expression and / or the level of ATXN2 expression prior to administration is compared to the subject’s level of ATXN2 expression subsequent to administration. In some embodiments, the level of modulatory polynucleotide expression may be evaluated in a cell or tissue of the central nervous system. In some embodiments, subject’s level of ATXN2 protein expression subsequent to administration is decreased relative to the subject’s level of ATXN2 protein expression prior to administration.

[0269] In some embodiments, the administration of the effective amount of a pharmaceutical composition or AAV particle disclosed herein that comprises a viral genome encoding a modulatorypolynucleotide may be a treatment that results in: (i) a decrease in ATXN2 protein expression in a cell, tissue, (e.g., 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 co...

Claims

CLAIMS1. 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 Ataxin-2 (ATXN2) mRNA. optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 mRNA; and wherein the AAV capsid variant comprises an amino acid sequence having the formula [N 1]- [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 X-, wherein X5is I, 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, 1, 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 of IAQ.

9. 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 nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 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: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 nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of Ataxin-2 (ATXN2) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 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 tire 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. 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 Ataxin-2 (ATXN2) mRNA, optionally wherein the modulatory polynucleotide comprises an RNAi agent targeting ATXN2 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 adeno-associated virus (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 die 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:(a) a 5’ flanking region comprising any one of SEQ ID NOs: 6413-6416 or a nucleic acid sequence that is at least 95% identical thereto;(b) a loop region comprising any one of SEQ ID NOs: 6417-6421 or a nucleic acid sequence that is at least 95% identical thereto: and(c) a 3’ flanking region comprising any one of SEQ ID NOs: 6422-6427 or a nucleic acid sequence that is at least 95% identical thereto.

1. The AAV particle of claim 26, wherein:(a) the 5’ flanking region comprises SEQ ID NO: 6414 or SEQ ID NO: 6415, or a nucleic acid sequence that is at least 95% identical thereto;(b) the loop region comprises SEQ ID NO: 6417, SEQ ID NO: 6418, or SEQ ID NO: 6421, or a nucleic acid sequence that is at least 95% identical thereto; and(c) the 3’ flanking region comprises SEQ ID NO: 6423, SEQ ID NO: 6424, or SEQ ID NO: 6425, or a nucleic acid sequence that is at least 95% identical thereto.

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

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

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

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

32. The AAV particle of any one of claims 1-31, wherein the modulatory polynucleotide comprises an 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 ATXN2 mRNA transcripts (e.g., one or more mutated ATXN2 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 ATXN2 mRNA transcripts comprises SEQ ID NO: 6428, SEQ ID NO: 6429, SEQ ID NO: 6430, SEQ ID NO: 6431, SEQ ID NO: 6434, SEQ ID NO: 6435, SEQ ID NO: 6436, and / or SEQ ID NO: 6437, 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 S19 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 host cell comprising the viral genome comprising a nucleic acid sequence encoding a modulatory polynucleotide for reducing or eliminating expression of ATXN2 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 nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6423 or a nucleic acid sequence that is at least 95% identical thereto;(ii) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6415 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6421 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6425 or a nucleic acid sequence that is at least 95% identical thereto;(iii) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6424 or a nucleic acid sequence that is at least 95% thereto; or(iv) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6418 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6423 or a nucleic acid 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 nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence ofSEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6423 or a nucleic acid sequence that is at least 95% identical thereto;(ii) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6415 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6421 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6425 or a nucleic acid sequence that is at least 95% identical thereto;(iii) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6417 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6424 or a nucleic acid sequence that is at least 95% thereto; or(iv) a 5’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6414 or a nucleic acid sequence that is at least 95% identical thereto, a loop region comprising the nucleic acid sequence of SEQ ID NO: 6418 or a nucleic acid sequence that is at least 95% identical thereto, and a 3’ flanking region comprising the nucleic acid sequence of SEQ ID NO: 6423 or a nucleic acid 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.

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 Sf9 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 ATXN2 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 method reduces or eliminates ATXN2 protein in the subject.

58. The method of claim 57, wherein the subject has, has been diagnosed with having, or is at risk of having an ATXN2-related disorder.

59. The method of claim 58, wherein the ATXN2-related disorder is spinocerebellar ataxia type 2 (SCA2).

60. A method of treating an ATXN2-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.

61. The method of claim 60, wherein the subject has, has been diagnosed with having, or is at risk of having the ATXN2-related disorder.

62. The method of claim 60 or claim 61, wherein the subject has one or more mutations in the ATXN2 gene.

63. The method of claim 62, wherein the one or more mutations in the ATXN2 gene comprises a trinucleotide repeat expansion.

64. The method of claim 63, wherein the trinucleotide repeat expansion in the ATXN2 gene comprises 32 or more CAG repeats.

65. The method of any one of claims 60-64. wherein the treating results in prevention of progression of the ATXN2-related disorder in the subject.

66. The method of any one of claims 60-65. wherein the treating results in amelioration of at least one symptom of the ATXN2-related disorder in the subject.

67. The method of claim 66, wherein the at least one symptom comprises progressive cerebellar ataxia, nystagmus, slow saccadic eye movements, ophthahnoparesis, parkinsonism, or a combination thereof.

68. The method of any one of claims 60-67. wherein the ATXN2-related disorder is spinocerebellar ataxia type 2 (SCA2).

69. A method of treating an ATXN2-related disorder in a subject, wherein the ATXN2-related disorder is spinocerebellar ataxia type 2 (SCA2), 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.

70. The method of claim 69, wherein the subject has, has been diagnosed with having, or is at risk of having SCA2.

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

72. The method of any one of claims 57-71, wherein the AAV particle or the pharmaceutical composition is delivered to a cell or tissue of the central nervous system (CNS) in the subject.

73. The method of claim 72, wherein the cell or tissue of the CNS is a cell or tissue of 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).

74. The method of any one of claims 57-73. wherein the AAV particle or the pharmaceutical composition is delivered to the subject via intravenous administration.

75. The method of any one of claims 57-74. further comprising evaluating, e.g.. measuring, the level of modulatory polynucleotide expression, and / or the level of ATXN2 expression, e.g.. ATXN2 gene expression, ATXN2 mRNA expression, and / or ATXN2 protein expression, in the subject, e.g.. in a cell, tissue, or fluid, of the subject.

76. The method of claim 75. wherein the level of ATXN2 protein is measured by an enzyme-linked immunosorbent assay (ELISA), a Western blot, or an immunohistochemistry assay.

77. The method of claim 75 or claim 76, wherein the evaluating of the subject’s level of modulatory polynucleotide expression and / or level of ATXN2 expression 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 and / or the subject’s level of ATXN2 expression prior to administration is compared to the subject's level of modulatory polynucleotide expression and / or the subject’s level of ATXN2 expression subsequent to administration.

78. The method of any one of claims 57-77. wherein the subject’s level of ATXN2 protein expression subsequent to administration is decreased relative to the subject’s level of ATXN2 protein expression prior to administration.

79. The method of any one of claims 57-78. further comprising evaluating, e.g.. measuring, the level of modulatory polynucleotide activity and / or ATXN2 activity in the subject, e.g., in a cell or tissue of the subject.

80. The method of any one of claims 57-79, wherein administering the pharmaceutical composition or the AAV particle to the subject results in:(i) a decrease in ATXN2 protein expression in a cell, 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, spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, or thoracic spinal cord region)), and / or fluid (e.g., CSF and / or scrum) of the subject relative to baseline and / or relative to ATXN2 protein expression in a cell, tissue, or fluid of an individual with an ATXN2-related disorder who has not been administered the pharmaceutical composition or AAV particle;(ii) 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; and / or(iii) a decrease in ATXN2 mRNA expression in a cell or tissue (e.g., a cell or tissue of the CNS, e.g., tire amygdala, brainstem, caudate, central grey, cerebellum (e.g.. Purkinje cell layer and deepcerebellar 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 ATXN2 mRNA expression in a cell or tissue of an individual with an ATXN2-related disorder who has not been administered the pharmaceutical composition or AAV particle.

81. A method of treating a subject having or diagnosed with having an ATXN2-related disorder, wherein the ATXN2-related disorder is amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), or frontotemporal lobar degeneration (FTLD), 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.

82. The method of any one of claims 57-81. further comprising administering to the subject at least one additional agent and / or therapy.

83. The method of claim 82, wherein the at least one additional agent and / or therapy comprises an agent and / or therapy suitable for treating the ATXN2-related disorder.

84. The method of claim 82 or claim 83, wherein the at least one additional agent and / or therapy comprises one or more of: growth and trophic factors, cytokines, hormones, nemotransmitters, enzymes, anti-apoptotic factors, angiogenic factors, modulatory polynucleotides, and any protein known to be mutated in pathological disorders such as ATXN2-related disorders.

85. The method of claim 83 or claim 84, wherein the ATXN2-rclatcd disorder is spinocerebellar ataxia 2 (SCA2), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), or frontotemporal lobar degeneration (FTLD).

86. The method of any one of claims 83-85. wherein the ATXN2-related disorder is spinocerebellar ataxia type 2 (SCA2).

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 (e.g., prednisone, prednisolone, methylprednisolone, and / or dexamethasone), rapamycin, mycophenolate mofetil. tacrolimus, rituximab, and / or eculizumab hydroxychloroquine.

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 60-88.

90. The pharmaceutical composition of claim 55 or the AAV particle of any one of claims 1-46 for use in treating an ATXN2-related disorder in a subject, optionally wherein the ATXN2-related disorder is spinocerebellar ataxia type 2 (SCA2).

91. The pharmaceutical composition or the AAV particle of claim 90, wherein the subject has. has been diagnosed with having, or is at risk of having the ATXN2-related disorder, optionally wherein the ATXN2-related disorder is SCA2.

92. 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 treatment of an ATXN2-related disorder in a subject, optionally wherein the ATXN2-related disorder is spinocerebellar ataxia type 2 (SCA2).

93. The use of claim 92, wherein the subject has, has been diagnosed with having, or is at risk of having the ATXN2-related disorder, optionally wherein the ATXN2-related disorder is SCA2.

Images