WO2025122530A1

WO2025122530A1 - Compositions and methods for the treatment of disorders related to glucosylceramidase beta 1 deficiency

Info

Publication number: WO2025122530A1
Application number: PCT/US2024/058328
Authority: WO
Inventors: Mathieu Emmanuel NONNENMACHER; Jing Lin; Hongxing Wang; Jinzhao Hou; Wei Wang; Jiangyu LI; Todd Carter; Hiu Yan CHUNG; Elisabeth KNOLL
Original assignee: Voyager Therapeutics Inc
Current assignee: Voyager Therapeutics Inc
Priority date: 2023-12-05
Filing date: 2024-12-04
Publication date: 2025-06-12
Anticipated expiration: 2026-06-05

Abstract

The disclosure relates to compositions and methods for, inter alia, altering, e.g., enhancing, the level of GBA1 protein via delivery using an adeno-associated viral (AAV) capsid variant. The compositions and methods of the present disclosure are useful, inter alia, for the treatment of subjects who have, have been diagnosed with having, or are at risk of having a GBA1-related disorder, e.g., Parkinson's Disease (PD), Gaucher Disease (GD), Parkinson's Disease Dementia (PDD), Dementia with Lewy Bodies (DLB), or Lewy Body Dementia (LBD).

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF DISORDERS

RELATED TO GLUCOSYLCERAMIDASE BETA 1 DEFICIENCY

RELATED APPLICATIONS

[01] This application claims the benefit of and priority to US Provisional Application Serial No. 63/606,162, filed December 5, 2023, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[02] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 14640_0101 -00304_SL.xml. was created on November 18, 2024, and is 1,936.273 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety⁷.

FIELD

[03] Described herein are compositions and methods relating to adeno-associated virus (AAV) viral particles for the delivery of polynucleotides, e.g.. polynucleotides encoding glucosylceramidase beta 1 (GBA1) proteins and peptides for use in the treatment of GBAl-related disorders, which include Parkinson’s Disease (PD) and other GBAl-related disorders, including Gaucher Disease, Parkinson’s Disease Dementia (PDD), Dementia with Lewy Bodies (DLB), and Lewy Body Dementia (LBD). In some embodiments, compositions described herein may be used to treat a subject in need thereof, such as a human subject diagnosed with a GBAl-related disorder or other condition resulting from a deficiency in the quantity and/or function of GBA1 protein.

BACKGROUND

[04] Lysosomal acid glucosylceramidase, commonly called glucosylcerebrosidase or Gcase, a D- glucosyl-N-acylsphingosine glucohydrolase, is a lysosomal membrane protein important in glycolipid metabolism. The enzyme is encoded by the glucosylceramidase beta 1 (GBA1) gene (Ensembl Gene ID No. ENSG00000177628). This enzyme, together with Saposin A and Saposin C, catalyzes the hydrolysis of glucosylceramide to ceramide and glucose. See Vaccaro, Anna Maria, et al. Journal of Biological Chemistry m.Tl (1997): 16862-16867.

[05] Mutations in GBA are known to cause disease in human subjects. Homozygous or compound heterozygous GBA1 mutations lead to Gaucher disease ("GD”). See Sardi, S. Pablo, Jesse M.

Cedarbaum. and Patrik Brundin. Movement Disorders 33.5 (2018): 684-696. Gaucher disease is one of the most prevalent lysosomal storage disorders, with an estimated standardized birth incidence in the general population of 0.4 to 5.8 individuals per 100,000. Heterozygous GBA1 mutations can lead to PD. Indeed, GBA1 mutations occur in 7-10% of total PD patients, making GBA1 mutations the most important genetic risk factor of PD. PD-GBA patients have reduced levels of the lysosomal enzyme beta- glucocerebrosidase (Gcase), which results in increased accumulations of glycosphingolipid glucosylceramide (GluCer), which in turn is correlated with exacerbated a-Synuclein aggregation and concomitant neurological symptoms. GD and PD, as well as other lysosomal storage disorders or Lewy body diseases such as Lewy Body Dementia (LBD). See Sidransky, E. and Lopez, G. Lancet Neurol. 2012 November; 11(11): 986-998.

[06] To date, there are limited available treatments for GBAl-related disorders such as PD, and delivery to the adult central nervous system (CNS) remains a significant challenge in the development of new and effective therapies. Thus, there remains a long-felt need to develop pharmaceutical compositions and methods that can be delivered to the CNS for the treatment of PD and other GBAl-related disorders and to ameliorate deficiencies of GBA1 protein in subjects, e.g., human subjects, afflicted with GBAl- related disorders.

[07] Adeno-associated viruses (AAVs) have emerged as a widely studied and utilized viral particles for delivery of therapeutically effective polypeptides to mammalian cells. See, e.g.. Tratschin et al.. Mol. Cell Biol., 5(11):3251-3260 (1985) and Grimm et al.. Hum. Gene Then, 10(15):2445-2450 (1999).

[08] 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 GBA1 to a target cell or tissue, e.g., a CNS cell or tissue.

SUMMARY

[09] 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 glucosylceramidase beta 1 (GBAl)-encoding sequence and 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); [N 1] comprises amino acids Xi, X₂, X₃. and X₄, wherein X₄ is Q. K, E, S. P. R, N. or H; and/or [N3] comprises amino acids Xs, X₆, and X₇. 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.

[010] In some embodiments. X] is T. S, R, A, I. C, N. K, L, or Q; X₂ is N, T, G, V, S. Y, K, I, H, D, or F; and X₃ is T, N, K. D, I, S, P. A, Y, E, V, L. M, R. H, Q, or C. In some embodiments, X₆ is A, Y. P, N, S, T, G, E, V, W, F, or Q; and X₇ is Q, G, N, K. H, R, E, L, P, or M.

[OH] 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). [012] In some embodiments, [Nl] is present at amino acids corresponding to positions 582-585 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 981.

[013] hi some embodiments, the AAV capsid variant further comprises [N4] comprising amino acids X₈, X₉, Xio, and Xu, wherein: X₈ is T. S, N, P, A, or I; X₉ is G, N, D, R. V, A, S, or Q; Xiois 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 ID NO: 981.

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

[015] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a GBA1 -encoding sequence 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. In some embodiments, the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981.

[016] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a GBA1 -encoding sequence 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. In some embodiments, the AAV capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 981.

[017] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a GBA1 -encoding sequence and 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 die 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.

[018] In some aspects, the present disclosure provides an AAV particle comprising a viral genome comprising a GBA1 -encoding sequence 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 amino acid sequence of TQDWHRI (SEQ ID NO: 941).

[019] In some embodiments, the AAV capsid variant comprises at least 8, at least 9, or at least 10 consecutive amino acids from the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343). In some embodiments, the AAV capsid variant comprises the amino acid sequence of TNTQDWHRIAQ (SEQ ID NO: 343) present at amino acids corresponding to positions 582-592 of the amino acid sequence of SEQ ID NO: 981. In some embodiments, the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or TNTQDWHRIAQ (SEQ ID NO: 343) is present in loop VIII, wherein loop VIII comprises amino acids 580-599 of the amino acid sequence of SEQ ID NO: 981.

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

[021] In some embodiments, the viral genome of the AAV particle encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1775, 1740, 1742, 1744, 1746, or 1748 or an amino acid sequence that is 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) identical thereto. In some embodiments, the viral genome of the AAV particle encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1775 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. In some embodiments, the viral genome encodes a wildtype GBA1 protein. In some embodiments, the viral genome encodes a human GBA1 protein, a dog GBA1 protein, an equine GBA1 protein, or a monkey GBA1 protein. In some embodiments, the viral genome encodes a wildtype human GBA1 protein. In some embodiments, the viral genome does not encode a hemagglutinin (HA) tag. In some embodiments, the GBA1 -encoding sequence comprises SEQ ID NO: 2002 or SEQ ID NO: 1773, or a nucleotide 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. In some embodiments, the viral genome further comprises a signal sequence-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2005 or SEQ ID NO: 1850, or a nucleotide 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. In some embodiments, the viral genome comprises the nucleotide sequence of 2001 or SEQ ID NO: 1772, or a nucleotide 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. In some embodiments, the viral genome encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1774 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.

[022] In some embodiments, the viral genome comprises a promoter operably linked to the GBA1- encoding sequence. In some embodiments, the promoter comprises a human elongation factor 1 alpha (EF-la) promoter, a chicken f> -actin (CBA) promoter, a CAG promoter, a CAG derivative promoter, a CMV immediate-early enhancer and/or promoter, a CMV promoter, a glucuronidase (GUSB) promoter, a ubiquitin C (UBC) promoter, a neuron-specific enolase (NSE) promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF-0) promoter, an intercellular adhesion molecule 2 (ICAM-2) promoter, a synapsin (Syn) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light chain (NFL) promoter, a neurofilament heavy chain (NFH) promoter, a 0-globin minigene n02 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) and excitatory' amino acid transporter 2 (EAAT2) promoter, a glial fibrillary' acidic protein (GFAP) promoter, a myelin basic protein (MBP) promoter, a cardiovascular promoter (e.g., aMHC, cTnT, and CMV-MLC2k), a liver promoter (e.g., hAAT, TBG), a skeletal muscle promoter (e.g., desmin, MCK, C512), or a fragment, e.g., a truncation, or a functional variant thereof. In some embodiments, the promoter comprises a CBA promoter. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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.

[023] In some embodiments, the viral genome further comprises an enhancer. In some embodiments, the enhancer comprises a CMV immediate-early (CMVie) enhancer. In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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.

[024] In some embodiments, the viral genome further comprises an intron. In some embodiments, the intron comprises the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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.

[025] In some embodiments, the viral genome further comprises a polyadenylation (poly A) region. In some embodiments, the polyA region comprises the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide 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.

[026] In some embodiments, the viral genome further comprises an inverted terminal repeat (ITR). In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 1829 or SEQ ID NO: 1830 or a nucleotide 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. In some embodiments, the viral genome comprises a 5’ ITR and a 3’ ITR. wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide 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 the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide 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.

[027] In some embodiments, the viral genome further comprises a nucleotide sequence encoding one or more miR183 binding sites. In some embodiments, the viral genome encodes at least four miR183 binding sites. In some embodiments, each of the at least four miR183 binding sites is separated by a spacer. In some embodiments, each of the miR183 binding sites is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 1847 or a nucleotide sequence that has up to three modifications relative thereto.

[028] In some embodiments, the viral genome further comprises a nucleotide sequence encoding a miR183 binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide 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.

[029] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) a promoter; (iii) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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 (iv) a 3 ’ ITR.

[030] In some embodiments, the viral genome comprises (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer; (iii) a promoter: (iv) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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 (v) a 3‘ ITR. [031] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer; (iii) a promoter: (iv) an intron; (v) the GBAl-encoding sequence, wherein the GBA1- encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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 (vi) a 3 ’ ITR.

[032] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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; (iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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; (iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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; (v) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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 (vi) a 3’ ITR.

[033] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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; (iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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; (iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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; (v) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (vi) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide 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 (vii) a 3’ ITR.

[034] In some embodiments, the viral genome comprises: (i) a 5' inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide 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) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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; (iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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; (iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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; (v) the GBAl-encoding sequence, wherein the GBA1- encoding sequences comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (vi) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide 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 (viii) a 3 ’ ITR comprising the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide 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.

[035] In some embodiments, (i) the 5' ITR comprises the nucleotide sequence of SEQ ID NO: 1829; (ii) the enhancer comprises the nucleotide sequence of SEQ ID NO: 1831; (iii) the promoter comprises the nucleotide sequence of SEQ ID NO: 1834; (iv) the intron comprises tire nucleotide sequence of SEQ ID NO: 1842; (v) the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001; (vi) the poly A region comprises the nucleotide sequence of SEQ ID NO: 1846; and (viii) the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 1830.

[036] In some embodiments, the viral genome comprises the nucleotide sequence of SEQ ID NO: 2006 or a nucleotide 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. In some embodiments, the viral genome comprises the nucleotide sequence of SEQ ID NO: 2006. In some embodiments, the viral genome consists of the nucleotide sequence of SEQ ID NO: 2006. [037] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) a promoter; (iii) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises tire nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (iv) a nucleotide sequence encoding at least one miR183 binding site; and (v) a 3’ ITR.

[038] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) a promoter; (iii) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (iv) a nucleotide sequence encoding a miR183 binding site series, wherein the miR183 binding site series comprises at least one miR183 binding site and at least one spacer sequence; and (v) a 3’ ITR.

[039] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer; (iii) a promoter: (iv) the GBAl-encoding sequence, wherein the GBA1 -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (v) a nucleotide sequence encoding a miR183 binding site series, wherein the miR183 binding site series comprises at least one miR183 binding site and at least one spacer sequence; and (vi) a 3’ ITR.

[040] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer; (iii) a promoter: (iv) an intron; (v) the GBAl-encoding sequence, wherein the GBAl- encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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; (vi) a nucleotide sequence encoding a miR183 binding site series, wherein the miR183 binding site series comprises at least one miR183 binding site and at least one spacer sequence; and (vii) a 3’ ITR.

[041] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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; (iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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; (iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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; (v) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (vi) a nucleotide sequence encoding a miR183 binding site series, wherein the miR183 binding site series comprises at least one miR183 binding site and at least one spacer sequence; and (vii) a 3‘ ITR.

[042] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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; (iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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; (iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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; (v) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (vi) a nucleotide sequence encoding a miR183 binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide 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 (vii) a 3’ ITR.

[043] In some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide 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) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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; (iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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; (iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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; (v) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (vi) a nucleotide sequence encoding at least one miR183 binding site, wherein the nucleotide sequence encoding the at least one miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 1847; (vii) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide 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 (viii) a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide 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.

[044] hi some embodiments, the viral genome comprises: (i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide 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) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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; (iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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; (iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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; (v) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (vi) a nucleotide sequence encoding a miR183 binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide 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; (vii) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide 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 (viii) a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 1830 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.

[045] In some embodiments, (i) the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1829; (ii) the enhancer comprises the nucleotide sequence of SEQ ID NO: 1831; (iii) the promoter comprises the nucleotide sequence of SEQ ID NO: 1834; (iv) the intron comprises the nucleotide sequence of SEQ ID NO: 1842; (v) the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001; (vi) the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 1849; (vii) the polyA region comprises the nucleotide sequence of SEQ ID NO: 1846; and (viii) the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 1830.

[046] In some embodiments, the viral genome comprises viral genome comprises the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide sequence 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. In some embodiments, the viral genome comprises the nucleotide sequence of SEQ ID NO: 2007. In some embodiments, the viral genome consists of the nucleotide sequence of SEQ ID NO: 2007.

[047] In some aspects, the present disclosure provides a cell comprising an AAV particle provided herein. In some embodiments, the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.

[048] In some aspects, the present disclosure provides a method of making an AAV particle provided herein, the method comprising: (i) providing a cell comprising viral genome comprising a GBAl-encoding sequence 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.

[049] In some embodiments of a method of making an AAV particle, the viral genome comprises (i) the nucleotide sequence of SEQ ID NO: 2006 or a nucleotide 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; or (ii) the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide sequence at that is 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 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.

[050] In some embodiments of a method of making an AAV particle, the viral genome comprises: (i) the nucleotide sequence of SEQ ID NO: 2006 or a nucleotide 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; or (ii) the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide 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 the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981, the amino acid sequence according to positions 138-736 of the amino acid sequence of SEQ ID NO: 981, and/or the amino acid sequence according to positions 203-736 of the amino acid sequence of SEQ ID NO: 981. In some embodiments of a method of making an AAV particle, the viral genome comprises the nucleotide sequence of SEQ ID NO: 2006 or SEQ ID NO: 2007 and the AAV capsid variant comprises the amino acid sequence of 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.

[051] In some embodiments, the method of making an AAV particle comprises, prior to step (i), introducing a nucleic acid molecule comprising the viral genome into the cell. In some embodiments, the method of making an AAV particle 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 S19 cell), or a bacterial cell.

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

[053] In some aspects, the present disclosure provides a method of delivering an AAV particle encoding a GBA1 protein to 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 subject, has, has been diagnosed with having, or is at risk of having a GBAl-related disorder.

[054] In some aspects, the present disclosure provides a method of treating a GBAl-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 GBAl-related disorder.

[055] In some embodiments, the GBAl-related disorder is a GBAl-related neurodegenerative or neuromuscular disorder. In some embodiments, the GBAl-related disorder is Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g., GD type 1, GD type 2, or GD type 3), Dementia with Lewy Bodies (DLB). Lewy Body Dementia (LBD), Multiple System Atrophy (MSA). Alzheimer’s Disease (AD). Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden- Spatz Syndrome. In some embodiments, the GBAl-related disorder is PD. In some embodiments, the GBA-1 related disorder is LBD. In some embodiments, the GBAl-related disorder is DLB. In some embodiments, the GBAl- related disorder is GD (e.g.. GD type 1, GD type 2. or GD type 3).

[056] In some aspects, the present disclosure provides a method of treating a GBAl-related disorder in a subject, wherein the GBAl-related disorder is Parkinson’s Disease (PD), 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 PD.

[057] In some aspects, the present disclosure provides a method of treating a GBAl-related disorder in a subject, wherein the GBAl-related disorder is Lewy Body Dementia (LBD), comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle provided herein. In some embodiments, tire subject has, has been diagnosed with having, or is at risk of having LBD.

[058] In some aspects, the present disclosure provides a method of treating a GBAl-related disorder in a subject, wherein the GBAl-related disorder is Dementia with Lewy Bodies (DLB), comprising administering to the subject an effective amount of the pharmaceutical composition or AAV particle provided herein. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having DLB.

[059] In some aspects, the present disclosure provides a method of treating a GBAl-related disorder in a subject, wherein the GBAl-related disorder is Gaucher Disease (GD), 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 GD (e.g.. GD type 1, GD type 2. or GD type 3). In some embodiments, the GD is GD type 1. In some embodiments, the GD is GD type 2. In some embodiments, the GD is GD type 3.

[060] In some embodiments, the subject has one or more mutations in the GBA1 gene. [061] In some embodiments, the subject has lower GCase activity prior to administration of the pharmaceutical composition or AAV particle as compared to GCase activity in an individual who does not have a GBA1 -related disorder. In some embodiments, the level of GCase activity' is measured by a 4- MUG assay or a SensoLyte Blue Glucocerebrosidase assay.

[062] hi some embodiments, the treating results in the prevention of progression of a GBA-related disorder in the subject. In some embodiments, the treating results in amelioration of at least one symptom of the GBA1 -related disorder in the subject. In some embodiments, the at least one symptom comprises developmental delay, progressive encephalopathy, progressive dementia, ataxia, myoclonus, oculomotor dysfunction, bulbar palsy, generalized weakness, trembling of a limb, depression, visual hallucinations, cognitive decline, or a combination thereof. In some embodiments, the treating results in a change in one or more biomarkers comprising a GCase activity, a level of glucocerebroside and other glycolipids, (e.g.. within immune cells such as macrophages), a level of synuclein aggregates (e.g., Lewy bodies), or a combination thereof.

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

[064] In some embodiments, the AAV particle or the pharmaceutical composition 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 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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).

[065] In some embodiments, the AAV particle or the pharmaceutical composition is delivered to a peripheral cell or tissue in the subject. In some embodiments, the peripheral cell or tissue is a cell or tissue of the heart, skeletal muscle, sympathetic ganglia, and/or plasma.

[066] In some embodiments, the AAV particle or pharmaceutical composition is delivered to the subject via intravenous administration.

[067] hi some embodiments, a method of delivery or treating provided herein further comprises evaluating, e.g., measuring, the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and/or GBA1 protein expression) in the subject, e.g., in a cell, tissue, or fluid of the subject. In some embodiments, the level of GBA1 protein expression is measured by an enzy me-linked immunosorbent assay (ELISA), a Western blot, or an immunohistochemistry assay. In some embodiments, evaluating the subject’s level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and/or GBA1 protein expression) is performed prior to and/or subsequent to administration of the pharmaceutical composition or AAV particle. In some embodiments, the subject’s level of GBA1 expression (e.g., GBA1 gene expression. GBA1 mRNA expression, and/or GBA1 protein expression) prior to administration is compared to the subject’s level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and/or GBA1 protein expression) subsequent to administration. In some embodiments, the method comprises evaluating the level of GBA1 expression in a cell or tissue of the central nervous system. In some embodiments, the subject’s level of GBA1 protein expression subsequent to administration is increased relative to the subject's level of GBA1 protein expression prior to administration. In some embodiments, the method further comprises evaluating, e.g., measuring, the level of GCase activity in the subject.

[068] In some embodiments, administering to the subject a phannaceutical composition or AAV particle provided herein results in an increase in: (i) GCase activity 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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/or fluid (e.g.. CSF and/or serum) of the subject relative to baseline and/or relative to GCase activity in a cell, tissue, or fluid of an individual with a GBA1 -related disorder who has not been administered the pharmaceutical composition or AAV particle; (ii) 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gy rus, hippocampus, inferior colliculus, inferior olivary⁷ 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) GBA1 mRNA expression in a cell or 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, sensory⁷ cortex, temporal cortex, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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 GBA1 mRNA expression in a cell or tissue of an individual with a GBAl-related disorder who has not been administered the pharmaceutical composition or AAV particle.

[069] In some embodiments, a method of delivery or treatment 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 a GBA1- related disorder. In some embodiments, the at least one additional agent and/or therapy comprises enzy me replacement therapy (ERT) (e.g., imiglucerase, velaghicerase alfa, or taliglucerase alfa); substrate reduction therapy (SRT) (e.g., eliglustat or miglustat), levodopa, carbidopa, Safinamide, a dopamine agonist (e.g., pramipexole, rotigotine, or ropinirole), a dopamine antagonist (e.g., quetiapine, clozapine), an anticholinergic (e.g., benztropine or trihexyphenidyl), a cholinesterase inhibitor (e.g., rivastigmine, donepezil, or galantamine), an N-methyl-d-aspartate (NMD A) receptor antagonist (e.g., memantine), or a combination thereof. In some embodiments, the at least one additional agent and/or therapy is suitable for treating Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g., GD type 1. GD type 2, or GD type 3), Dementia with Lewy Bodies (DLB), Lewy Body Dementia (LBD), Multiple System Atrophy (MSA), Alzheimer’s Disease (AD). Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation type 1 (NBIA 1). or Hallervorden-Spatz Syndrome. In some embodiments, the at least one additional agent and/or therapy is suitable for treating PD, LBD, GD. or DLB. In some embodiments, the GD is GD type 1, GD type 2, or GD ty pe 3.

[070] In some embodiments, a method of delivery or treatment 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. In some embodiments, a method of delivery or treatment provided herein further comprises administering a blood transfusion to the subject.

[071] 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.

[072] In some aspects, the present disclosure provides a pharmaceutical composition or AAV particle provided herein for use in the treatment of a GBAl-rclatcd disorder in a subject. In some embodiments, the GBAl-related disorder is Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g., GD type 1, GD type 2, or GD type 3), Dementia with Lewy Bodies (DLB), Lewy Body Dementia (LBD), Multiple System Atrophy (MSA), Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden- Spatz Syndrome. In some embodiments, the GBAl- related disorder is PD. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having PD. In some embodiments, the GBAl-related disorder is LBD. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having LBD. In some embodiments, the GBAl-related disorder is DLB. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having DLB. In some embodiments, the GBAl-related disorder is GD (e.g.. GD ty pe 1, GD type 2, or GD type 3). In some embodiments, the subject has, has been diagnosed with having, or is at risk of having GD (e.g., GD type 1, GD type 2, or GD ty pe 3). [073] In some aspects, the present disclosure provides a use of a pharmaceutical composition or AAV particle provided herein in the manufacture of a medicament for the treatment of a GBA1 -related disorder in a subject. In some embodiments, the GBA1 -related disorder is Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g., GD type 1, GD type 2, or GD type 3), Dementia with Lewy Bodies (DLB). Lewy Body Dementia (LBD), Multiple System Atrophy (MSA), Alzheimer’s Disease (AD). Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden- Spatz Syndrome. In some embodiments, the GBAl-related disorder is PD. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having PD. In some embodiments, the GBAl-related disorder is GD. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having GD. In some embodiments, the GD is GD type 1. In some embodiments, the GD is GD type 2. In some embodiments, the GD is GD type 3. In some embodiments, the GBAl-related disorder is LBD. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having LBD. In some embodiments, the GBAl-related disorder is DLB. In some embodiments, the subject has, has been diagnosed with having, or is at risk of having DLB.

Enumerated Embodiments

1. An adeno-associated virus (AAV) particle comprising a viral genome comprising a P- glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant), wherein tire AAV capsid variant comprises an amino acid sequence having the following formula: [N1]-[N2]-[N3] (SEQ ID NO: 4681), wherein [N2] comprises die amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein:

(i) [Nl] comprises amino acids Xi, X2, X3, and X4, wherein X4 is Q, K, E, S, P, R, N, H; and/or

(ii) [N3] comprises amino acids X5, Xg, and X-, wherein X, is I, V, T, M, S, N, L, F.

2. The AAV particle of embodiment 1, wherein the AAV capsid variant comprises the amino acid Q at position 585, as numbered according to SEQ ID NO: 138 or 981.

3. The AAV particle of embodiment 1, wherein the AAV capsid variant comprises an amino acid other than Q at position 585. as numbered according to SEQ ID NO: 138 or 981.

4. The AAV particle of embodiment 1 or 3, wherein the AAV capsid variant comprises the amino acid K at position 585, as numbered according to SEQ ID NO: 138 or 981.

5. The AAV particle of any one of embodiments 1-4. wherein the AAV capsid variant comprises one. two. three, or all of:

(i) an amino acid other than T at position 582 (e.g.. S, R, A. I. C, N, K, L, or Q);

(ii) an amino acid other than N at position 583 (e.g., T, G, V, S. Y, K, I, H, D, or F); (iii) an amino acid other than H at position 584 (e.g., T, N, K, D, I, S, P, A, Y, E, V, L. M, R, Q, or C); and/or

(iv) an amino acid other than Q at position 585 (e.g., K, E, S, P, R, N, or H); wherein the amino acids are numbered according to SEQ ID NO: 138.

6. The AAV particle of any one of embodiments 1-5, wherein [Nl] comprises amino acids Xi, X₂, X₃, and X₄, wherein X₄ is Q, K, E, S, P, R, N. or H.

7. The AAV particle of embodiment 6, wherein X₄ is Q or K.

8. The AAV particle of any one of embodiment 7. wherein X₄ is Q.

9. The AAV particle of embodiment 7, wherein X₄ is K.

10. The AAV particle of any one of embodiments 1-9, wherein the AAV capsid variant comprises an amino acid other than H at position 584 (e.g., T). as numbered according to SEQ ID NO: 138.

11. The AAV particle of any one of embodiments 1-10, wherein the AAV capsid variant comprises the amino acid T at position 584, as numbered according to SEQ ID NO: 138 or 981.

12. The AAV particle of any one of embodiments 1-11, wherein:

(i) Xj is T, S, R, A. I. C, N, K, L, or Q;

(ii) X₂ is N, T, G, V, S, Y, K, I, H, D, or F; and/or

(iii) X₃ is 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, Nl. 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, TTL 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 VTQ

15. 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, tw o. or three but no more than four modifications relative to any one of the amino acid sequences in (i); or

(iv) an amino acid sequence comprising one, two, or three but no more than four substitutions relative to any one of the amino acid sequences in (i).

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), RN1QDWHR (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;

(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. 1. 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 Xs, Xe, and X?, wherein Xs is I, V, T. M, S, N, L, or F.

29. The AAV particle of any one of embodiments 1-28, wherein Xs is I or V.

30. The AAV particle of embodiment 29, wherein Xs is I.

31. The AAV particle of any one of embodiments 1-30, wherein:

(i) X₆ is A, Y, P, N, S, T, G, E, V. W, F, or Q; and/or

(ii) X₇ is Q, G, N, K, H, R, E, L, P, or M.

32. The AAV particle of any one of embodiments 1-31, wherein [N3] comprises IA, IY, VP, IN, VN, VY, VA, IS, IT, TA, MA, SA, IG, IE, IV, NA, LA, IP, FA, VS, VT, IW, IF, IQ, VQ, AQ, AG, YQ, PQ, AN, NQ, SG, SQ, TQ, GQ, EQ, AK, AH, AR, AE, AL, AP, TM, SM, WQ, FQ, QQ, FM, AM. or SN.

33. The AAV particle of any one of embodiments 1-32, wherein [N3] is or comprises IAQ, IAG. IYQ, VPQ, IAN, INQ, VNQ, VYQ, VAN, ISG, ISQ, VAQ, ITQ, TAQ, MAQ, SAQ, IGQ, IEQ. IVQ, NAQ, LAQ. IAK, IAH, IPQ, IAR, IAE. IAL, IAP. FAQ. VSQ. VTM, ISM. IWQ, IFQ, IQQ. VQQ, IFM. IAM, or ISN.

34. The AAV particle of any one of embodiments 1-33, wherein [N3] is or comprises IAQ. 35. The AAV particle of any one of embodiments 1-34, wherein [N2]-[N3] comprises:

(i) DWHRIA (SEQ ID NO: 5002), DWHRIY (SEQ ID NO: 5003), DWHRVP (SEQ ID NO: 5004), DWHRIN (SEQ ID NO: 5005), DWHRVN (SEQ ID NO: 5006), DWHRVY (SEQ ID NO: 5007), DWHRVA (SEQ ID NO: 5008), DWHRIS (SEQ ID NO: 5009), DWHRIT (SEQ ID NO: 5010), DWHRTA (SEQ ID NO: 5011), DWHRMA (SEQ ID NO: 5012), DWHRSA (SEQ ID NO: 5013), DWHRIG (SEQ ID NO: 5014), DWHRIE (SEQ ID NO: 5015), DWHRIV (SEQ ID NO: 5016), DWHRNA (SEQ ID NO: 5017), DWHRLA (SEQ ID NO: 5018), DWHRIP (SEQ ID NO: 5019), DWHRFA (SEQ ID NO: 5020), DWHRVS (SEQ ID NO: 5021). DWHRVT (SEQ ID NO: 5022), DWHRIW (SEQ ID NO: 5023), DWHRIF (SEQ ID NO: 5024), DWHRIQ (SEQ ID NO: 5025). or DWHRVQ (SEQ ID NO: 5026);

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i), e.g., any 2, 3. 4, or 5 amino acids, e.g.. consecutive amino acids, thereof;

(iii) an amino acid sequence comprising one, two. or three but no more than four modifications relative to any one of the amino acid sequences in (i); or

36. The AAV particle of any one of embodiments 1-35, wherein [N2]-[N3] is or comprises:

(i) DWHRIAQ (SEQ ID NO: 5027), DWHRIAG (SEQ ID NO: 5028), DWHRIYQ (SEQ ID NO: 5029), DWHRVPQ (SEQ ID NO: 5030), DWHRIAN (SEQ ID NO: 5031). DWHRINQ (SEQ ID NO: 5032), DWHRVNQ (SEQ ID NO: 5033). DWHRVYQ (SEQ ID NO: 5034), DWHRVAN (SEQ ID NO: 5035), DWHRISG (SEQ ID NO: 5036), DWHRISQ (SEQ ID NO: 5037), DWHRVAQ (SEQ ID NO: 5038), DWHRITQ (SEQ ID NO: 5039), DWHRTAQ (SEQ ID NO: 5040), DWHRMAQ (SEQ ID NO: 5041), DWHRSAQ (SEQ ID NO: 5042), DWHRIGQ (SEQ ID NO: 5043), DWHRIEQ (SEQ ID NO: 5044), DWHRIVQ (SEQ ID NO: 5045), DWHRNAQ (SEQ ID NO: 5046), DWHRLAQ (SEQ ID NO: 5047), 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);

(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;

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 tire 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) X_s is T, S, N, P. A, or I;

(ii) X₉ is G, N. D, R. V, A, S. or Q;

(iii) X₁₀is W, S, C. R, L. or G; and/or

(iv) X_n 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, GC, 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;

(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 P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising an amino acid sequence having the following fonnula: [N1]-[N2]-[N3] (SEQ ID NO: 4683), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein:

(i) LN 1 J comprises amino acids Xi, X₂, X₃, and X₄. wherein X₄ is Q, P, or a conservative substitution thereof; and/or

(ii) [N3] amino acids X₅, X«, and X?, wherein Xs is I. V, or a conservative substitution thereof.

52. The AAV particle of embodiment 51, wherein the AAV capsid variant comprises the amino acid Q at position 585. as numbered according to SEQ ID NO: 138 or 981.

53. The AAV particle of embodiment 51 or 52, wherein the AAV capsid variant comprises one, tw o, 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 arc 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 X₄ is 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) X2 is N, T, G, S, I, or V; and/or

(iii) X₃ is T, N, I, S, A, V, or L.

59. The AAV particle of any one of embodiments 51-58, wherein [Nl] comprises TN, TT. TG, ST, TS, TI, TV, TQ, NQ, IQ. SQ, AQ, VQ, TP, LQ, NT, TA, 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;

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;

65. The AAV particle of any one of embodiments 51-64, wherein [N1]-[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;

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, w herein the AAV capsid variant comprises one. two, or all of: an amino acid other than Q at position 590 (e.g., I or V), an amino acid other than A at position 591 (e.g., P. S, Y, or N), and/or an amino acid other than Q at position 592 (e g., G or N), as numbered according to SEQ ID NO: 138 or 981.

68. The AAV particle of any one of embodiments 51-67, wherein the AAV capsid variant comprises an amino acid other than Q at position 590 (e.g., I or V), as numbered according to SEQ ID NO: 138 or 981.

69. The AAV particle of any one of embodiments 51-68, wherein the AAV capsid variant comprises the amino acid I at position 590. as numbered according to SEQ ID NO: 138 or 981. 70. The AAV particle of any one of embodiments 51-68, wherein the AAV capsid variant comprises the amino acid V at position 590, as numbered according to SEQ ID NO: 138 or 981.

71. The AAV particle of any one of embodiments 51-70, wherein the AAV capsid variant comprises the amino acid A at position 591 and/or the amino acid Q at position 592, as numbered according to SEQ ID NO: 138 or 981.

72. The AAV particle of any one of embodiments 51-71. wherein [N3] comprises amino acids X₅, X₆, and X₇, wherein X₅ is 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) X₆ is A. P. S, Y. or N; and/or

(ii) X? is 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);

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);

80. The AAV particle of any one of embodiments 51-79, wherein IN2J-[N3J is or comprises DWHRIAQ (SEQ ID NO: 5027).

81. The AAV particle of any one of embodiments 51-80, wherein [N1]-[N2]-[N3] is or comprises:

(i) the amino acid sequence of any one of SEQ ID NOs: 343, 350. 352, 355, 359. 361, 364, 367. 370. 371, 373, 374. 376, 377, 378, 381, 395. 420, 454, 457. 460, 464, 481. 482, 488, 493, 494, 516. 525, 536;

(ii) an amino acid sequence comprising any portion of an amino acid sequence in (i). e.g., any 2, 3, 4. 5, 6, 7, 8, 9. or 10 amino acids, e.g., consecutive amino acids, thereof;

82. The AAV particle of any one of embodiments 51-81, wherein [N1]-[N2]-[N3] is or comprises TNTQDWHRIAQ (SEQ ID NO: 343).

83. The AAV particle of any one of embodiments 51-82, wherein the AAV capsid variant comprises the amino acid W at position 595, as numbered according to SEQ ID NO: 138 or 981.

84. The AAV particle of any one of embodiments 51-83, wherein the AAV capsid variant comprises one. two or all of: an amino acid other than T (e.g.. S or N) at position 593, an amino acid other than G (e.g., N) a position 594, and/or an amino acid other than V (e.g.. A, I, or S) at position 596, as numbered according to SEQ ID NO: 138 or 981.

85. The AAV particle of any one of embodiments 51-84, wherein the AAV capsid variant comprises: (i) the amino acid T at position 593. the amino acid G at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981;

(ii) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid A at position 596, as numbered according to SEQ ID NO: 138 or 981:

(iii) the amino acid S at position 593, the amino acid N at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981:

(iv) the amino acid N at position 593, the amino acid N at position 594, the amino acid W at position 595, and the amino acid V at position 596, as numbered according to SEQ ID NO: 138 or 981:

(v) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and the amino acid I at position 596. as numbered according to SEQ ID NO: 138 or 981; or

(vi) the amino acid T at position 593, the amino acid G at position 594, the amino acid W at position 595, and 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) X₈ is T, S, or N;

(ii) X₉ is G or N; and/or

(iv) Xu is V, A, I, or S.

89. The AAV particle of embodiment 87 or 88, wherein [N4] comprises TG, SN, NN, WV, WA, WI, WS, GW, or NW.

90. The AAV particle of any one of embodiments 87-89, wherein [N4] comprises TGW. SNW, NNW, GWV, GW A, NWV, GWI. or GWS.

91. The AAV particle of any one of embodiments 87-90. wherein [N4] is or comprises TGWV (SEQ ID NO: 5066), TGWA (SEQ ID NO: 5067), SNWV (SEQ ID NO: 5069), NNWV (SEQ ID NO: 5072), TGWI (SEQ ID NO: 5074), or TGWS (SEQ ID NO: 5073).

92. The AAV particle of any one of embodiments 87-91. wherein [N4] is or comprises TGWV (SEQ ID NO: 5066). 93. The AAV particle of any one of embodiments 87-92, wherein [N1]-[N2]-[N3]-[N4] is or comprises:

(i) the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336;

(ii) an ammo 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;

94. The AAV particle of any one of embodiments 87-93, wherein [N 1 J-[N2J-IN3 J-LN4J 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 [N1]-[N2] is present at amino acids 582-589, as numbered according to SEQ ID NO: 138 or 981.

107. The AAV particle of any one of embodiments 1-106, wherein [N3] replaces amino acids 590-592 (e.g., Q590, A591, and Q592) of SEQ ID NO: 138.

108. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, and 101-107, wherein [N3] corresponds to amino acids 590-592 (e.g., 1590, A591, and Q592) of SEQ ID NO: 981.

109. The AAV particle of any one of embodiments 1-108, wherein [N3] is present at amino acids 590- 592, as numbered according to SEQ ID NO: 138 or 981.

110. The AAV particle of any one of embodiments 1-109, wherein [N2]-[N3] replaces amino acids 586- 592 (e.g., S586, A587, Q588, A589, Q590, A591, and Q592) of SEQ ID NO: 138.

111. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-110, wherein [N2]-[N3] corresponds to amino acids 586-592 (e.g., D586, W587, H588, R589. 1590, A591. and Q592) of SEQ ID NO: 981.

112. The AAV particle of any one of embodiments 1-111, wherein [N2]-[N3] is present at amino acids 586-592, as numbered according to SEQ ID NO: 138 or 981. 113. The AAV particle of any one of embodiments 1-112, wherein [N1]-[N2]-[N3] replaces amino acids 582-592 (e.g., T582, N583, H584, Q585, S586, A587, Q588, A589, Q590, A591, Q592) of SEQ ID NO: 138.

114. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-113, wherein [N1]-[N2]-[N3] corresponds to amino acids 582-592 (e.g., T582, N583, T584. Q585, D586, W587, H588, R589, 1590. A591, Q592) of SEQ ID NO: 981.

115. The AAV particle of any one of embodiments 1-114, wherein [N1]-[N2]-[N3] is present at amino acids 582-592, as numbered according to SEQ ID NO: 138 or 981.

116. The AAV particle of any one of embodiments 43-50 and 87-115, wherein [N4] replaces amino acids 593-596 (e.g., T593, G594, W595, and V596) of SEQ ID NO: 138.

117. The AAV particle of any one of embodiments 43-49 and 87-115. wherein [N4] corresponds to amino acids 593-596 (e g., T593, G594, W595, and V596) of SEQ ID NO: 138 or 981.

11 . 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 |N2J.

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 P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) 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, 14, 27, 28, or 29;

(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, 14, 27, 28, or 29; 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, 14, 27, 28, or 29; 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. 14. T1 , 28, or 29. 131. An AAV particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) 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 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:

(c) an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions, relative to the amino acid sequence of any one of SEQ ID NOs: 201. 205-209, 211-214, 216. 219, 220, 230. 232, 237, 238. 255, 262-265. 274, 283, 286. 290, 291, 293. 301, 306. 307, 308, 309, 314. and 336; or

(d) an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214. 216, 219. 220, 230, 232. 237, 238, 255. 262-265, 274. 283, 286, 290. 291, 293, 301. 306, 307. 308. 309, 314, and 336.

132. The AAV particle of embodiment 130 or 131. wherein the AAV capsid variant does not comprise at least 3, at least 4, at least 5. at least 6, at least 7, at least 8, or at least 9 consecutive amino acids from TNHQSAQAQ (SEQ ID NO: 5100), optionally wherein the TNHQSAQAQ (SEQ ID NO: 5100) corresponds to amino acids 582-592 of SEQ ID NO: 138.

133. The AAV particle of any one of embodiments 130-132, wherein the AAV capsid variant does not comprise: TNH, TNHQ (SEQ ID NO: 4760), TNHQS (SEQ ID NO: 5101), TNHQSA (SEQ ID NO: 5102), TNHQSAQ (SEQ ID NO: 5103), TNHQSAQA (SEQ ID NO: 5104), TNHQSAQAQ (SEQ ID NO: 5100), NHQ, NHQS (SEQ ID NO: 5105), NHQSA (SEQ ID NO: 5106), NHQSAQ (SEQ ID NO: 5107), NHQSAQA (SEQ ID NO: 5108), 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 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. 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, wherein the at least 9 consecutive amino acids comprise TNTQDWHRI (SEQ ID NO: 746). 147. The AAV particle of any one of embodiments 130-146, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four modifications relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336.

148. The AAV particle of any one of embodiments 130-147, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one. at least two. or at least three but no more than four modifications relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941).

149. The AAV particle of any one of embodiments 130-147. wherein the AAV capsid variant comprises an amino acid sequence comprising at least one. at least two. or at least three but no more than four modifications relative to the amino acid sequence of TNTQDWHRI (SEQ ID NO: 746).

150. The AAV particle of any one of embodiments 130-149, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two. or at least three but no more than four modifications relative to the amino acid sequence of ATNTQDWHRIAQT (SEQ ID NO: 744).

151. The AAV particle of any one of embodiments 130-150, w herein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291. 293, 301, 306, 307, 308, 309, 314, and 336.

152. The AAV particle of any one of embodiments 130-149 and 151, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least tw o, or at least three but no more than four substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941).

153. The AAV particle of any one of embodiments 130-149, 151, and 152, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two, or at least three but no more than four substitutions relative to the amino acid sequence of TNTQDWHRI (SEQ ID NO: 746).

154. The AAV particle of any one of embodiments 130-153, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one. at least two. or at least three but no more than four substitutions relative to the amino acid sequence of ATNTQDWHRIAQT (SEQ ID NO: 744). 155. The AAV particle of any one of embodiments 1-154, wherein the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NOs: 201, 205-209, 211-214, 216, 219, 220, 230, 232, 237, 238, 255, 262-265, 274, 283, 286, 290, 291, 293, 301, 306, 307, 308, 309, 314, and 336.

156. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-155, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941), optionally wherein the amino acid sequence replaces amino acids 584-590 of SEQ ID NO: 138.

157. The AAV particle of any one of embodiments 1-3, 5-8, 10-16. 18-21, 23-39, 42-49. 51-69, 71-83, 85-99, and 101-156. wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941), optionally wherein the amino acid sequence corresponds to amino acids 584-590 of SEQ ID NO: 981.

158. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21. 23-39, 42-49, 51-69. 71-83, 85-99. 101-149, 151-153. and 155-157, wherein the AAV capsid variant comprises an amino acid sequence encoded by:

(i) the nucleotide sequence of SEQ ID NO: 942;

(ii) a nucleotide sequence comprising at least one, at least 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; 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;

(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, 14, 27, 28, or 29 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, 14. 27, 28, or 29 replaces ammo acids 584, 585, 586, 587, 588, 589, and/or 590 (e.g., H584, Q585, S586, A587, Q588, A589, and/or Q590), as numbered according to the amino acid sequence of SEQ ID NO: 138.

164. The AAV particle of any one of embodiments 130-157, wherein the amino acid sequence provided in Table 1, 2A, 2B, 14. 27, 28, or 29 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, 14. 27. 28, or 29 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, 14. 27, 28, or 29 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, 14. 27, 28, or 29 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, 14. 27. 28, or 29 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. 14, 27, 28, or 29 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 P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) 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.

172. The AAV particle of any one of embodiments 1-171, wherein the AAV capsid variant comprises one, two, three, four, five, or all of: T at position 584, D at position 586, W at position 587, H at position 588, R at position 589, and/or I at position 590, as numbered according to SEQ ID NO: 981.

173. An AAV particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) 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.

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 P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g.. encoding a human GBA1 protein) 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.

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 Q5901. as numbered according to SEQ ID NO: 138.

182. An AAV particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g.. encoding a human GBA1 protein) 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.

183. An AAV particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g.. encoding a human GBA1 protein) 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.

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 amino acid sequence with at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 138.

193. The AAV particle of embodiment 192, wherein the AAV capsid variant an amino acid sequence with at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 138.

194. The AAV particle of any one of embodiments 1-193, wherein the AAV capsid variant comprises an amino acid sequence encoded by a sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 137.

195. The AAV particle of embodiment 194. wherein the AAV capsid variant comprises an amino acid sequence encoded by a sequence with at least 90% (e.g.. at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 137.

196. The AAV particle of any one of embodiments 1-195, wherein the AAV capsid variant comprises a VP1 protein, a VP2 protein, and a VP3 protein in an about 1:1 :10 ratio.

197. The AAV particle of any one of embodiments 1-196, wherein the AAV capsid variant comprises amino acids 138-736, e.g., a VP2, of SEQ ID NO: 981, or a sequence with at least 80% (e.g., at least 80%, at least 85%. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%. at least 95%, at least 96%, at least 97%, at least 98%. or at least 99%) sequence identity thereto.

198. The AAV particle of any one of embodiments 1-197, wherein the AAV capsid variant comprises amino acids 203-736, e.g.. a VP3, of SEQ ID NO: 981, or a sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99%) sequence identity' thereto.

199. The AAV particle of any one of embodiments 1-198, wherein the AAV capsid variant comprises an amino acid sequence that is at least 80% (e.g., at least 80%. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%. at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 138-736, e.g.. a VP2, of SEQ ID NO: 138.

200. The AAV particle of any one of embodiments 1-199, wherein the AAV capsid variant comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 203-736, e.g., a VP3, of SEQ ID NO: 138.

201. The AAV particle of any one of embodiments 1-3, 5-8, 10-16, 18-21, 23-39, 42-49, 51-69, 71-83, 85-99, 101-186, and 189-200, wherein the AAV capsid variant comprises an amino acid sequence comprising at least 3, at least 4, at least 5, at least 6, or all 7 consecutive amino acids from the amino acid sequence of TQDWHRI (SEQ ID NO: 941), wherein:

(i) the at least 3 consecutive amino acids comprise TQD:

(ii) the at least 4 consecutive amino acids comprise TQDW (SEQ ID NO: 4684);

(iii) the at least 5 consecutive amino acids comprise TQDWH (SEQ ID NO: 4685);

(iv) the at least 6 consecutive amino acids comprise TQDWHR (SEQ ID NO: 4686); or

(v) the 7 consecutive amino acids comprise TQDWHRI (SEQ ID NO: 941): wherein the AAV capsid variant comprises: (a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 981; (b) a VP2 protein comprising amino acids 138-736 of SEQ ID NO: 981: (c) a VP3 protein comprising amino acids 203-736 of SEQ ID NO: 981; or (d) an amino acid sequence with at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to any one of tire 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 more than 30, not more than 20 or not more than 10 modifications relative to the amino acid sequence of SEQ ID NO: 981.

206. The AAV particle of any one of embodiments, 1-205, wherein the AAV capsid variant comprises an amino acid sequence comprising at least one, at least two or at least three, but not more than 30, not more than 20 or not more than 10 substitutions, relative to the amino acid sequence of SEQ ID NO: 981.

207. The AAV particle of any one of embodiments 1-206, wherein the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983, or a nucleotide sequence with at least 80% (e.g.. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99%) sequence identity thereto. 208. The AAV particle of embodiment 207, wherein the AAV capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 983.

209. The AAV particle of embodiment 207, wherein the AAV capsid variant is encoded by a codon- optimized nucleotide sequence.

210. An AAV particle comprising an AAV capsid variant (e.g.. an AAV9 capsid variant) comprising the amino acid sequence of any one of embodiments 1-3. 5-8, 10-16, 18-21. 23-39, 42-49, 51-69. 71-83, 85- 99, 101-186. and 189, and further comprising an amino acid sequence at least 95% (e.g., at least 95%. at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 981.

211. An AAV particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g.. encoding a human GBA1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising the amino acid sequence of SEQ ID NO: 981.

212. An AAV particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) 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.

213. An AAV particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) 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.

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 P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g.. encoding a human GBA1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising amino acids 203-736 of SEQ ID NO: 981. 216. An AAV particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) 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.

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 P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g., encoding a human GBA1 protein) and an AAV capsid variant (e.g., an AAV9 capsid variant) comprising amino acids 138-736 of SEQ ID NO: 981.

219. An AAV particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence (e.g.. encoding a human GBA1 protein) 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.

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 immuiiohistochcmistry assay or a qPCR or ddPCR assay, e.g., as described in Example 2.

223. The AAV particle of any one of embodiments 1-222, which is enriched at least 10, at least 14, at least 20, at least 24, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1 or 3.

224. The AAV particle of any one of embodiments 1-223, which is enriched at least 200, at least 300, at least 400, at least 425, at least 450, or at least 460-fold in the brain compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g.. when measured by an assay as described in Example 1.

225. The AAV particle of any one of embodiments 1-224, which is enriched in the brain of at least two or at least three species, e.g.. a non-human primate and rodent (e.g., mouse), e.g.. as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.

226. The AAV particle of any one of embodiments 1-225, which is enriched at least 2. at least 3, at least 5, at least 10, at least 15, at least 20. at least 25, at least 30, at least 35, at least 40, at least 45. at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170. at least 175. at least 180, at least 190, at least 200, at least 205, or at least 210-fold in the brain of at least 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, CMorocebus sabaeus, Callithrix jacchus, and/or mouse (e.g., outbred mice).

228. The AAV particle of any one of embodiments 1-227, which is enriched at least 2, at least 3. at least 4. at least 5. at least 10, at least 15, at least 17. at least 20, at least 50, at least 75, at least 100, at least 103, at least 107. at least 125. at least 150. at least 200. at least 250. at least 300, at least 350, at least 400, at least 450, at least 500, at least 750, at least 1000, at least 1200-fold in the brain compared to an AAV particle comprising an AAV capsid 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 GBA1 to a brain region, optionally wherein the GBA1 level is increased by at least 39, at least 50. at least 100. at least 120, at least 132, at least 146, at least 150, at least 161, at least 174, at least 175, at least 200, at least 225, at least 250, at least 275, at least 283, at least 300, at least 350, at least 400, at least 450, at least 500. at least 525, at least 528, or at least 550-fold as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay, e.g., a qRT- PCR, a ddPCR, or a qPCR assay (e.g., as described in Example 2).

230. The AAV particle of any one of embodiments 1-229, which delivers an increased level of viral genomes to a brain region, optionally wherein the level of viral genomes is increased by at least 2. at least 5, at least 7, at least 10, at least 15, at least 19, at least 20, at least 22, or at least 25-fold as compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g.. when measured by an assay, e.g.. a qRT-PCR or a qPCR assay (e.g., as described in Example 2).

231. The AAV particle of embodiment 229 or 230. wherein the brain region is a sensory’ cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and/or cerebellum.

232. The AAV particle of any one of embodiments 1-231, which is enriched at least 5. at least 10, at least 50, at least 100, at least 115, at least 120, at least 150, at least 175, at least 200, at least 207, at least 225, at least 250, or at least 275-fold in the spinal cord compared to an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138, e.g., when measured by an assay as described in Example 1 or 2.

[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, wherein the viral genome further comprises a nucleotide sequence encoding a Rep protein, e.g., a non-structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68 protein, a Rep52 protein, and/or a Rep40 protein (e.g., a Rep 78 protein and a Rep52 protein). 315. The AAV particle of any one of embodiments 1-232, embodiment 312, or embodiment 313, wherein die AAV particle furdier 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. wherein 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 arc 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 GBA1 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, neurode generative disorder, muscular disorder, or neuromuscular disorder is Parkinson’s Disease (PD) (e.g., a PD associated with a GBA1 mutation). Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g., GDI, GD2, or GD3), Dementia with Lewy Bodies (DLB), Lewy Body Dementia (LBD), Spinal muscular atrophy (SMA), Multiple System Atrophy (MSA), or Multiple sclerosis (MS).

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 an increased expression of a GBA1 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 an increased activity of a GBA1 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 GBA1 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 phannaceutical 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 phannaceutical 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 neurodegenerative 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 P-glucocerebrosidase 1 (GBAl)-encoding sequence and the AAV capsid variant comprises an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4681) in loop VIII: wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682); and wherein:

(i) [Nl] comprises amino acids Xi, X₂, X₃, and X₄, wherein X₄ is Q, K, E, S, P, R, N, or H; and/or

(ii) [N3] comprises amino acids X5, Xg, and X-, wherein X, is 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) X₂ is N, T, G, V, S, Y, K, I, H, D, or F; and

(iii) X₃ is 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) X₆ is A, Y, P, N, S, T, G, E, V, W, F, or Q; and

(ii) X₇ is 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 LN3 J 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 TNTQDWHRI 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) X₈ is T, S, N, P, A, or I;

(ii) X₉ is 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 TNTQDWHRIAQTGWV (SEQ ID NO: 201).

368. An adeno-associated virus (AAV) particle comprising:

(i) a viral genome comprising a P-glucocerebrosidase 1 (GBAl)-encoding sequence: 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, and

I 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 P-glucocerebrosidase 1 (GBAl)-encoding sequence; 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, and

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 -glucocerebrosidase 1 (GBAl)-encoding sequence; 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

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 viral genome encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1775 or an amino acid sequence at least 90% identical thereto.

380. The AAV particle of any one of embodiments 1-232, 312-318, and 354-379, wherein the viral genome encodes a wildtvpc GBA1 protein.

381. The AAV particle of any one of embodiments 1-232, 312-318, and 354-380, wherein the viral genome encodes a human GBA1 protein, a dog GBA1 protein, or an equine GBA1 protein.

382. The AAV particle of any one of embodiments 1-232, 312-318. and 354-381, wherein the viral genome encodes a wildty pe human GBA1 protein.

383. The AAV particle of any one of embodiments 1-232, 312-318. and 354-382, wherein the viral genome does not encode a hemagglutinin (HA) tag.

384. The AAV particle of any one of embodiments 1-232, 312-318, and 354-383, wherein the GBA1- encoding sequence comprises SEQ ID NO: 2002 or a nucleotide sequence 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.

385. The AAV particle of any one of embodiments 1-232, 312-318, and 354-384, where the viral genome further comprises a signal sequence comprising the nucleotide sequence of SEQ ID NO: 2005 or a nucleotide sequence 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.

386. The AAV particle of any one of embodiments 1-232, 312-318, and 354-385, wherein the viral genome comprises the nucleotide sequence of 2001 or a nucleotide sequence 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. 387. The AAV particle of any one of embodiments 1-232, 312-318, and 354-386, wherein the viral genome encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1774 or an amino acid sequence 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.

388. The AAV particle of any one of embodiments 1-232, 312-318, and 354-387, wherein the viral genome comprises a promoter operably linked to the GBAl-encoding sequence.

389. The AAV particle of embodiment 388. wherein the promoter comprises an EF-la promoter, a CBA promoter, a chicken |3-actin (CBA) promoter, a CAG promoter, a CAG derivative promoter, a CMV immediate-early enhancer and/or promoter, a CMV promoter, a P glucuronidase (GUSB) promoter, a ubiquitin C (UBC) promoter, a neuron-specific enolase (NSE) promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF- ) promoter, an intercellular adhesion molecule 2 (ICAM-2) promoter, a synapsin (Syn) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light (NFL) or heavy (NFH) promoter, a -globin minigene n 2 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoter, a glial fibri 1 lan acidic protein (GFAP) promoter, a myelin basic protein (MBP) promoter, a cardiovascular promoter (e.g., aMHC. cTnT, and CMV-MLC2k). a liver promoter (e.g.. hAAT, TBG). a skeletal muscle promoter (e.g., desmin, MCK. C512), or a fragment, e.g., a truncation, or a functional variant thereof.

390. The AAV particle of embodiment 388 or embodiment 389, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide sequence 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.

391. The AAV particle of any one of embodiments 1-232, 312-318, and 354-390, wherein the viral genome further comprises an enhancer.

392. The AAV particle of embodiment 391. wherein the enhancer comprises a CMV immediate-early (CMVie) enhancer; optionally wherein the CMVie enhancer comprises the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide sequence 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. 393. The AAV particle of any one of embodiments 1-232, 312-318, and 354-392, wherein the viral genome further comprises an intron; optionally wherein the intron comprises die nucleotide sequence of SEQ ID NO: 1842 or a nucleotide sequence 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.

394. The AAV particle of any one of embodiments 1-232, 312-318, and 354-393, wherein the viral genome further comprises a polyadenylation (poly A) region; optionally wherein the polyA region comprises the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide sequence 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.

395. The AAV particle of any one of embodiments 1-232. 312-318, and 354-394. wherein the viral genome further comprises an inverted terminal repeat (ITR); optionally wherein the ITR comprises the nucleotide sequence of SEQ ID NO: 1829 or SEQ ID NO: 1830 or a nucleotide sequence 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) to the nucleotide sequence of SEQ ID NO: 1829 or SEQ ID NO: 1830.

396. The AAV particle of embodiment 395, wherein the viral genome comprises a 5’ ITR and a 3’ ITR. wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide sequence 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 the 3‘ ITR comprises the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide sequence 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.

397. The AAV particle of any one of embodiments 1-232, 312-318, and 354-396, wherein the viral genome further comprises one or more miR183 binding sites.

398. The AAV particle of any one of embodiments 1-232, 312-318, and 354-397, wherein the viral genome comprises at least four miR183 binding sites.

399. The AAV particle of embodiment 398. wherein each of the at least four miR183 binding sites is separated by a spacer. 400. The AAV particle of any one of embodiments 396-399, wherein each of the miR183 binding sites comprises the nucleotide sequence of SEQ ID NO: 1847 or a nucleotide sequence that has up to three modifications relative to SEQ ID NO: 1847.

401. The AAV particle of any one of embodiments 1-232, 312-318, and 354-396, wherein the viral genome further comprises a miR183 binding site series comprising the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide sequence 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.

402. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) a promoter;

(iii) a GBA1 -encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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

(iv) a 3’ ITR.

403. The AAV particle of any one of embodiments 1-232, 312-318. and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer;

(iii) a promoter;

(iv) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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

(v) a 3’ ITR.

404. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer;

(iii) a promoter;

(iv) an intron; (v) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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

(vi) a 3‘ ITR.

405. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) a CMV immediate-early (CM Vie) enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 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;

(iii) a chicken beta actin (CBA) promoter comprising the nucleotide sequence of SEQ ID NO: 1834 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;

(iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 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;

(v) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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

(vi) a 3’ ITR.

406. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) a CMV immediate-early (CMVie) enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 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) to thereto;

(iii) a chicken beta actin (CBA) promoter comprising the nucleotide sequence of SEQ ID NO: 1834 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;

(iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 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; (v) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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;

(vi) a poly adenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 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; and

(vii) a 3’ ITR.

407. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5' inverted tenninal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 1829 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;

(ii) a CMV immediate-early (CM Vie) enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 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;

(v) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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;

(vi) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 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; and

(viii) a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 1830 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.

408. The AAV particle of embodiment 407. wherein:

(i) the 5' ITR comprises the nucleotide sequence of SEQ ID NO: 1829;

(ii) the CMVie enhancer comprises the nucleotide sequence of SEQ ID NO: 1831; (iii) the CBA promoter comprises the nucleotide sequence of SEQ ID NO: 1834;

(iv) the intron comprises the nucleotide sequence of SEQ ID NO: 1842;

(v) the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001;

(vi) the poly A region comprises the nucleotide sequence of SEQ ID NO: 1846; and (viii) the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 1830.

409. The AAV particle of any one of embodiments 1-232, 312-318, and 354-407, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 2006 or a nucleotide sequence 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.

410. The AAV particle of embodiment 409. wherein the viral genome comprises or consists of the nucleotide sequence of SEQ ID NO: 2006.

411. The AAV particle of any one of embodiments 1-232, 312-318. and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) a promoter;

(iii) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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;

(iv) at least one miR183 binding site; and

(v) a 3’ ITR.

412. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) a promoter;

(iii) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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;

(iv) at least one miR183 binding site series comprising at least one miR183 binding site and at least one spacer sequence; and

(v) a 3’ ITR. 413. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer;

(iii) a promoter;

(iv) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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;

(v) at least one miR183 binding site series comprising at least one miR183 binding site and at least one spacer sequence; and

(vi) a 3’ lTR.

414. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer;

(iii) a promoter;

(iv) an intron;

(v) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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;

(vi) at least one miR183 binding site series comprising at least one miR183 binding site and at least one spacer sequence; and

(vii) a 3’ ITR.

415. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) a CMV immediate-early (CMVie) enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 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;

(iii) a chicken beta actin (CBA) promoter comprising the nucleotide sequence of SEQ ID NO: 1834 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; (iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 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;

(v) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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:

(vi) at least one miR183 binding site; and

(vii) a 3’ ITR.

416. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(vi) a miR183 binding site series comprising the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide sequence 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

(vii) a 3’ ITR.

417. The AAV particle of any one of embodiments 1-232, 312-318, and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 1829 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; (ii) a CMV immediate-early (CMVie) enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 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;

(iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 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;

(v) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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;

(vi) at least one miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847;

(vii) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 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; and

(viii) a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 1830 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.

418. The AAV particle of any one of embodiments 1-232, 312-318. and 354-378, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 1829 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;

(ii) a CMV immediate-early (CMVie) enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 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;

(v) a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence 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;

(vi) a miR183 binding site series comprising the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide sequence 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;

(vii) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 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; and

419. The AAV particle of embodiment 418. wherein:

(i) the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1829;

(ii) the CMVie enhancer comprises the nucleotide sequence of SEQ ID NO: 1831;

(iii) the CBA promoter comprises the nucleotide sequence of SEQ ID NO: 1834;

(iv) the intron comprises the nucleotide sequence of SEQ ID NO: 1842;

(vi) the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 1849;

(vii) the poly A region comprises the nucleotide sequence of SEQ ID NO: 1846; and

(viii) the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 1830.

420. The AAV particle of any one of embodiments 1-232, 312-318, and 354-418, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide sequence 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.

421. The AAV particle of embodiment 420, wherein the viral genome comprises or consists of the nucleotide sequence of SEQ ID NO: 2007.

422. A cell comprising the AAV particle of any one of embodiments 1-232. 312-318, and 354-421, optionally wherein the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an SI9 cell), or a bacterial cell.

423. A method of making the AAV particle of any one of embodiments 1-232, 312-318. and 354-421, the method comprising: (i) providing a cell comprising a viral genome comprising a GBA1 -encoding sequence 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.

424. The method of embodiment 423. wherein the viral genome comprises:

(a) the nucleotide sequence of SEQ ID NO: 2006 or a nucleotide sequence 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; or

(b) the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide sequence 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 wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981.

425. The method of embodiment 423 or embodiment 424, further comprising, prior to step (i), introducing a nucleic acid comprising the viral genome into the cell.

426. The method of any one of embodiments 423-425, further comprising, prior to step (i). introducing the nucleic acid encoding the AAV capsid variant into the cell.

427. The method of any one of embodiments 423-426, wherein the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an S19 cell), or a bacterial cell.

428. A pharmaceutical composition comprising the AAV particle of any one of embodiments 1-232, 312- 318, and 354-421 and a pharmaceutically acceptable excipient.

429. A method of delivering a GBA1 protein to a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 428 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-421, thereby delivering the GBA1 protein.

430. The method of embodiment 429, wherein the subject has, has been diagnosed with having, or is at risk of having a GBAl-related disorder.

431. A method of treating a GBAl-related disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 428 or the AAV particle of any one of embodiments 1-232. 312-318, and 354-421. thereby treating the GBAl-related disorder. 432. The method of embodiment 431, wherein the subject has, has been diagnosed with having, or is at risk of having a GBA1 -related disorder.

433. The method of any one of embodiments 430-432, wherein the GBA1 -related disorder is a GBA1- related neurodegenerative or neuromuscular disorder.

434. The method of any one of embodiments 430-433. wherein the GBAl-related disorder is Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g.. GD type 1, 2, or 3), Dementia with Lewy Bodies (DLB), Lewy Body Dementia (LBD). Multiple System Atrophy (MSA), Alzheimer’s Disease (AD). Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden- Spatz Syndrome.

435. The method of any one of embodiments 430-434, wherein the GBAl-related disorder is PD.

436. The method of any one of embodiments 430-434, wherein the GBAl-related disorder is LBD.

437. The method of any one of embodiments 430-434, wherein the GBAl-related disorder is GD (e g.,

GD type 1 (GDI) or GD type 3 (GD3)).

438. The method of any one of embodiments 430-434, wherein the GBAl-related disorder is DLB.

439. A method of treating Parkinson’s Disease (PD) in a subject, comprising administering to the subject an effective amount of die pharmaceutical composition of embodiment 428 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-421, thereby treating the PD.

440. The method of embodiment 439, wherein the subject has, has been diagnosed with having, or is at risk of having PD.

441. A method of treating Lewy Body Dementia (LBD) in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 428 or the AAV particle of any one of embodiments 1-232, 312-318. and 354-421, thereby treating the LBD.

442. The method of embodiment 441, wherein the subject has, has been diagnosed with having, or is at risk of having LBD. 443. A method of treating Gaucher Disease (GD) in a subject, comprising administering to tire subject an effective amount of the pharmaceutical composition of embodiment 428 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-421, thereby treating the GD.

444. The method of embodiment 443, wherein the subject has, has been diagnosed with having, or is at risk of having GD (e.g., GDI or GD3).

445. A method of treating Dementia with Lewy Bodies (DLB) in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 428 or the AAV particle of any one of embodiments 1-232, 312-318. and 354-421, thereby treating the DLB.

446. The method of embodiment 445, wherein the subject has. has been diagnosed with having, or is at risk of having DLB.

447. The method of any one of embodiments 429-446, wherein the subject has one or more mutations in the GBA1 gene.

448. The method of any one of embodiments 429-447, wherein, prior to the administration, the subject has lower GCase activity’ as compared to GCase activity in an individual who does not have a GBA1- related disorder, optionally wherein the level of GCase activity’ is measured by a 4-MUG assay or a SensoLyte Blue Glucocerebrosidase assay.

449. The method of any one of embodiments 431-438, wherein the treating results in prevention of progression of the disorder in the subject.

450. The method of any one of embodiments 431-438 or embodiment 449, wherein the treating results in amelioration of at least one symptom of the disorder in the subject.

451. The method of embodiment 450, wherein the at least one symptom comprises developmental delay, progressive encephalopathy, progressive dementia, ataxia, myoclonus, oculomotor dysfunction, bulbar palsy, generalized weakness, trembling of a limb, depression, visual hallucinations, cognitive decline, or a combination thereof.

452. The method of embodiment 449 or embodiment 450, wherein the treating results in a change in one or more biomarkers comprising a GCase activity, a level of glucocerebroside and other glycolipids, (e.g.. within immune cells such as macrophages), a level of synuclein aggregates (e.g., Lewy bodies), or a combination thereof. 453. The method of any one of embodiments 429-452, wherein the subject is a human subject.

454. The method of any one of embodiments 429-453, wherein the AAV particle or the pharmaceutical composition is delivered to a cell or tissue of the CNS.

455. The method of embodiment 454, 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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).

456. The method of any one of embodiments 429-453, wherein the AAV particle or the pharmaceutical composition is delivered to a peripheral cell or tissue.

457. The method of embodiment 456, wherein the peripheral cell or tissue is a cell or tissue of the liver, heart, skeletal muscle, sympathetic ganglia, and/or plasma.

458. The method of any one of embodiments 429-457, further comprising evaluating, e.g., measuring, the level of GBA1 expression (e.g.. GBA1 gene expression, GBA1 mRNA expression, and/or GBA1 protein expression) in the subject, e.g., in a cell, tissue, or fluid of the subject.

459. The method of embodiment 458, wherein the level of GBA1 protein expression is measured by an ELISA, a Western blot, or an immunohistochemistry assay.

460. The method of embodiment 458 or embodiment 459, wherein evaluating the subject’s level of GBA1 expression is performed prior to and/or subsequent to administration of the pharmaceutical composition or AAV particle, optionally wherein the subject’s level of GBA1 expression prior to administration is compared to the subject’s level of GBA1 expression subsequent to administration.

461. The method of any one of embodiments 458-460. comprising evaluating the level of GBA1 expression in a cell or tissue of the central nervous system. 462. The method of any one of embodiments 458-461, wherein the subject’s level of GBA1 protein expression subsequent to administration is increased relative to the subject’s level of GBA1 protein expression prior to administration.

463. The method of any one of embodiments 429-462, further comprising evaluating, e.g., measuring, the level of GCase activity in the subject.

464. The method of any one of embodiments 429-463. wherein the administration results in an increase in:

(i) GCase activity 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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/or fluid (e g., CSF and/or serum) of the subject relative to baseline and/or relative to GCase activity in a cell, tissue, or fluid of an individual with a GBA1 -related disorder who has not been administered the pharmaceutical composition or AAV particle;

(ii) 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gy rus, 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) GBA1 mRNA expression in a cell or 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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 GBA1 mRNA expression in a cell or tissue of an individual with a GBA1 -related disorder who has not been administered the pharmaceutical composition or AAV particle. 465. The method of any one of embodiments 429-464, further comprising administering to the subject at least one additional therapeutic agent and/or therapy.

466. The method of embodiment 465. wherein the at least one additional therapeutic agent and/or therapy comprises an agent and/or therapy for treating a GBA1 -related disorder.

467. The method of embodiment 466. wherein the at least one additional therapeutic agent and/or therapy for treating the GBAl-related disorder comprises enzyme replacement therapy (ERT) (e.g., imiglucerase. velaglucerase alfa. or taliglucerase alfa); substrate reduction therapy (SRT) (e.g., eliglustat or miglustat), levodopa, carbidopa. Safinamide. a dopamine agonist (e.g., pramipexole, rotigotine. or ropinirole). a dopamine antagonist (e.g., quetiapine, clozapine), an anticholinergic (e.g., benztropine or trihexyphenidyl), a cholinesterase inhibitor (e.g., rivastigmine, donepezil. or galantamine), an N-methyl- d-aspartate (NMDA) receptor antagonist (e.g.. memantine), or a combination thereof.

468. The method of embodiment 466 or embodiment 467, wherein the at least one additional therapeutic agent and/or therapy is for treating Parkinson’s Disease (PD). Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g., GD type 1. 2. or 3). Dementia with Lewy Bodies (DLB), Lewy Body Dementia (LBD), Multiple System Atrophy (MSA), Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Nemodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden- Spatz Syndrome.

469. The method of embodiment 468, wherein the GBAl-related disorder is PD, LBD, GD, or DLB, wherein, optionally, the GD is GD type 1 (GDI) or GD ty pe 3 (GD3).

470. The method of any one of embodiments 429-469, further comprising administering an immunosuppressant to the subject.

471. The method of embodiment 470. wherein the immunosuppressant comprises a corticosteroid (e.g., prednisone, prednisolone, methylprednisolone, and/or dexamethasone), rapamycin, my cophenolate mofetil, tacrolimus, rituximab, and/or eculizumab hydroxychloroquine.

472. The method of any one of embodiments 429-471. further comprising administering a blood transfusion to the subject.

473. The phannaceutical composition of embodiment 428 or the AAV particle of any one of embodiments 1-232. 312-318, and 354-421 for use in a method of treating a disorder according to any one of embodiments 431-472. 474. The pharmaceutical composition of embodiment 428 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-421 for use in the treatment of a GBAl-related disorder in a subject.

475. The phannaceutical composition or AAV particle for use of embodiment 474, wherein the GBAl- related disorder is Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g., GD type 1, 2. or 3). Dementia with Lewy Bodies (DLB), Lewy Body Dementia (LBD). Multiple System Atrophy (MSA), Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure. Neurodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden-Spatz Syndrome.

476. The pharmaceutical composition or AAV particle for use of embodiment 474 or embodiment 475. wherein the GBAl-related disorder is PD.

477. The pharmaceutical composition or AAV particle for use of embodiment 476. wherein the subject has, has been diagnosed with having, or is at risk of having PD.

478. The pharmaceutical composition or AAV particle for use of embodiment 474 or embodiment 475, wherein the GBAl-related disorder is LBD.

479. The pharmaceutical composition or AAV particle for use of embodiment 478, wherein the subject has, has been diagnosed with having, or is at risk of having LBD.

480. The pharmaceutical composition or AAV particle for use of embodiment 474 or embodiment 475, wherein the GBAl-related disorder is GD (e.g., GDI, GD2, or GD3).

481. The phannaceutical composition or AAV particle for use of embodiment 480, wherein the subject has. has been diagnosed with having, or is at risk of having GD (e.g., GDI or GD3).

482. The pharmaceutical composition or AAV particle for use of embodiment 474 or embodiment 475. wherein the GBAl-related disorder is DLB.

483. The phannaceutical composition or AAV particle for use of embodiment 482, wherein the subject has. has been diagnosed with having, or is at risk of having DLB. 484. Use of the pharmaceutical composition of embodiment 428 or the AAV particle of any one of embodiments 1-232, 312-318, and 354-421 in the manufacture of a medicament for the treatment of a GBA1 -related disorder in a subject.

485. The use of embodiment 484, wherein the GBA1 -related disorder is Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g., GD type 1, 2, or 3), Dementia with Lewy Bodies (DLB), Le Body Dementia (LBD), Multiple System Atrophy (MSA). Alzheimer’s Disease (AD). Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden-Spatz Syndrome.

486. The use of embodiment 484 or embodiment 485, wherein the GBAl-related disorder is PD.

487. The use of embodiment 486, wherein the subject has. has been diagnosed with having, or is at risk of having PD.

488. The use of embodiment 484 or embodiment 485, wherein the GBAl-related disorder is GD (e.g., GDI or GD3).

489. The use of embodiment 488. wherein the subject has, has been diagnosed with having, or is at risk of having GD (e.g., GDI or GD3).

490. The use of embodiment 484 or embodiment 485, wherein the GBAl-related disorder is LBD.

491. The use of embodiment 490. wherein the subject has, has been diagnosed with having, or is at risk of having LBD.

492. The use of embodiment 484 or embodiment 485, wherein the GBAl-related disorder is DLB.

493. The use of embodiment 492, wherein the subject has, has been diagnosed with having, or is at risk of having DLB.

[074] 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 otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of this disclosure. In the case of conflict, the present description will control. BRIEF DESCRIPTION OF THE DRAWINGS

[075] FIG. 1 depicts the biodistribution (VG/cell) hi the cortex, striatum, and brainstem of wildtype mice at 28 days post-IV injection of VOY101.GBA VG17 (SEQ ID NO: 1812), VOY101.GBA VG35 (SEQ ID NO: 2006) or VOY101.GBA VG36 (SEQ ID NO: 2007).

[076] FIG. 2A and FIG. 2B depict the GCase activity in the cortex, striatum, and brainstem of wild-type mice at 28 days post-IV injection of VOY101.GBA VG17 (SEQ ID NO: 1812), VOY101.GBA VG35 (SEQ ID NO: 2006) or VOY101.GBA VG36 (SEQ ID NO: 2007).

[077] FIG. 3A and FIG. 3B depict the biodistribution, mRNA expression, and Gcase activities in the brainstem and DRGs of wild-type mice at 28 days post-IV injection of VOY101.GBA VG17 (SEQ ID NO: 1812), VOY101.GBA_VG35 (SEQ ID NO: 2006) or VOY101.GBA_VG36 (SEQ ID NO: 2007).

[078] FIG. 4A and FIG. 4B depict the substrate quantification of glucosylceramide and glucosylsphingosine by LC-MS/MS in the brainstem, striatum, and DRGs of wild-ty pe mice at 28 days post-IV injection of VOY101.GBA VG17 (SEQ ID NO: 1812), VOY101.GBA VG35 (SEQ ID NO: 2006) or VOY101.GBA VG36 (SEQ ID NO: 2007).

[079] FIG. 5A and FIG. 5B depict biodistribution (VG/cell) in the cortex and GCase activities in the cortex, striatum, and brainstem of wild-type mice at 28 days post-IV injection of VOY101.GBA VG17 (SEQ ID NO: 1812) or VOY101.GBA VG17-HA.

[080] FIG. 6A depicts immunohistochemical analysis of HA expression in the cortex, striatum, and brainstem of wild-type mice at 28 days post-IV injection of VOY101.GBA VG17 (SEQ ID NO: 1812) or VOY101.GBA VG17-HA. FIG. 6B depicts immunohistochemical analysis of HA expression in the cerebellum, thalamus, and hippocampus of wild-type mice at 28 days post-IV injection of VOY101.GBA VG17 (SEQ ID NO: 1812) or VOY101.GBA VG17-HA.

DETAILED DESCRIPTION

Overview

[081] Described herein, inter alia, are compositions comprising an AAV capsid variant for deliver}' of a sequence encoding GBA1 protein, e.g., a wildtype GBA1 protein, e.g., a wildtype human GBA1 protein. In some embodiments, the present disclosure provides a method for delivering the AAV capsid variant comprising the sequence encoding the GBA1 protein to a cell or tissue in a subject. In some embodiments, the present disclosure provides a method for delivering the AAV capsid variant, thereby providing a GBA1 protein, e.g., wildtype GBA1 protein, e.g., a wildty pe human GBA1 protein, to a cell or tissue in a subject. The AAV capsid variants described herein have enhanced CNS tropism compared to other cells or tissues in the body, e g., liver and/or the DRG.

[082] 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 gene transfer because of their non-pathogenic nature, their low immunogenic profile, low rate of integration into the host genome, and long-term transgene expression in non-dividing cells. However, the transduction efficiency of naturally occurring AAVs in certain organs is too low for clinical applications, and capsid neutralization by pre-existing neutralizing antibodies may prevent treatment of a large proportion of patients. For these reasons, considerable efforts have been devoted to obtaining capsid variants with enhanced properties. Of many approaches tested so far, significant advances have resulted from directed evolution of AAV capsids using in vitro or in vivo selection of capsid variants created by capsid sequence randomization using either error-prone PCR, shuffling of various parent serotypes, or insertion of fully randomized short peptides at defined positions.

[083] 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 payload. The genome of the virus may encode a GBA1 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 human GBA1 protein, e.g., a wildtype human GBA1 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.

[084] In some embodiments, the genome encodes a wildtype GBA1 protein. In some embodiments, the genome comprises a codon-optimized. CpG-reduced (e.g., CpG-depleted) nucleotide sequence encoding a wildtype GBA1 protein, e.g., as compared to a wildtype nucleotide sequence encoding a wildtype GBA1 protein (e.g., comprising the nucleotide sequence of SEQ ID NO: 1776 or 1777). In some embodiments, the target cell is a CNS cell. In some embodiments, the target tissue is a CNS tissue. The target CNS tissue may be brain tissue. In some embodiments, the 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, 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).

[085] Gene therapy presents an alternative approach for treating a GBAl-related disorder, e.g., Parkinson’s Disease (PD) or Gaucher Disease (GD). 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, an AAV particle described herein can be used to administer and/or deliver a gene encoding GBA1 (e.g., a human GBA1 protein) preferentially to the CNS (e.g., the brain or the spinal cord).

[086] Provided herein are compositions and methods which may provide for improved features compared to prior AAV-mediated enzyme replacement approaches, including (i) increased biodistribution throughout 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), (ii) elevated GBA1 expression in multiple brain regions (e.g., cortex, thalamus, and brain stem); and (iii) preferential biodistribution in the CNS over the liver and/or DRG. of the subject. In some embodiments, an AAV viral genome comprising a codon-optimized, CpG- reduced (e.g., CpG-depleted) nucleotide sequence encoding a GBA1 protein (e.g.. SEQ ID NO: 2001 or SEQ ID NO: 2002) results in high biodistribution in the CNS; increased GCase activity in the CNS, peripheral tissues, and/or fluid; and successful transgene transcription and expression.

[087] Also provided herein are AAV capsid variants (e.g., AAV9 capsid variants) with improved properties compared to wildtype AAV9, such as (i) increased penetrance through the blood brain barrier following intravenous administration, (ii) wider distribution throughout the multiple brain regions, e.g., 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, (iii) elevated GBA1 expression in multiple brain regions, (iv) wider distribution in one or more peripheral tissues, e.g., die heart, kidney, muscle (e.g., quadriceps muscle), pancreas, and/or liver, and/or (v) elevated GBA1 expression in one or more peripheral tissues. In some embodiments, the AAV capsids described herein enhance the delivery of GBA1 to multiple regions of the brain including, for example, die 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). In some embodiments, the AAV capsids described herein enhance the deliver}' of GBA1 to the spinal cord. In some embodiments, the AAV capsid variants with enhanced brain tropism described herein are capable of significantly increasing GBA1 mRNA expression in the brain. In some embodiments, the AAV capsid variants described herein enhance the delivery of GBA1 to the dentate nucleus. In some embodiments, the AAV capsid variants described herein enhance the delivery of GBA1 to the somatosensory cortex. [088] Thus, the compositions and methods described herein can be used in the treatment of GBA1- related disorders. In some embodiments, GBA1 -related disorders are disorders associated with a lack of GBA1 protein expression and/or GCase activity and include neuronopathic (affects the CNS) and non- neuronopathic (affects non-CNS) Gaucher disease (e.g., Type 1 GD (GDI), Type 2 GD (GD2), or Type 3 GD (GD3)), PD associated with at least one mutation in a GBA gene. Parkinson’s disease dementia (PDD), Dementia with Lewy Bodies (DLB), and Lew Body Dementia (LBD). In some embodiments, the disclosure provides an AAV particle comprising one of the AAV capsid variants disclosed herein and an AAV viral genome comprising a codon-optimized, CpG-reduced (e.g.. CpG-depleted) nucleotide sequence encoding a GBA1 protein (e.g., comprising the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2001 or SEQ ID NO: 2002). In some embodiments, the codon-optimized. CpG-reduced e.g.. CpG-depleted) nucleotide sequence has reduced immunogenicity compared to a codon-optimized sequence comprising one or more or all CpG motifs. In some embodiments, the codon-optimized, CpG-reduced e.g., CpG-depleted) nucleotide sequence is suitable for use in treating disorders associated with a lack of a GBA1 protein expression and/or GCase activity, such as neuronopathic (affects the CNS) and non -neuronopathic (affects non-CNS). Gaucher disease (e.g., GDI, GD2, or GD3). PD associated with at least one mutation in a GBA gene, Parkinson’s disease dementia (PDD), Dementia with Lewy Bodies (DLB), and I.ewy Body Dementia (LBD).

I. Compositions

Adeno-associated viral (AA V) Particles

[089] 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/hu. l 4 (SEQ ID NO: 123 of US 7,906,11 1, the contents of which are herein incorporated by reference in their entirety), VP1 refers to amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. In some embodiments, with reference to the amino acid sequence of SEQ ID NO: 981, VP1 comprises amino acids 1-736, VP2 comprises amino acids 138-736, and VP3 comprises amino acids 203-736. In other words, 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 die 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.

[090] 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.

[091] 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 el al., Biotechniques, 2003, 34, 148, the contents of which are herein incorporated by reference in their entirety).

[092] Typically, AAV particles for GBA1 delivery may be recombinant viral particles which are replication defective as they lack sequences encoding functional Rep and Cap proteins within the viral genome. In some cases, the replication defective AAV particles may lack most or all coding sequences and essentially only contain one or two AAV ITR sequences and a nucleic acid sequence encoding a GBA1 protein (e.g., human GBA1 protein).

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

[094] 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'.

[095] In other embodiments, AAV particles of the present disclosure may be used to deliver GBA1 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.

[096] 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.

[097] 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., 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.

[098] The AAV particles and payloads of the disclosure may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles demonstrate enhanced tropism for a target cell type, tissue, or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral 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.

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

[0100] 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 tw o ITR sequences. In some embodiments, the ITRs have a characteristic T-shaped hairpin structure defined by a sclf-complcnicntary 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.

[0101] 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.

[0102] 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 w hich is incorporated herein by reference in its entirety).

[0103] 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

[0104] 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 wildly pc 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 wildly pc 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.

[0105] 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.

[0106] 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. [0107] 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.

[0108] 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.

[0109] In some embodiments, the one or more substitutions may increase distribution of an AAV particle to the PNS (e.g., DRG) after intravenous administration. In some embodiments, the one or more substitutions may increase distribution of an AAV particle to non-DRG cells of the PNS following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration. In some embodiments, the one or more substitutions may decrease distribution of an AAV particle to the DRG following focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.

[0110] In 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 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.

[oni] In some embodiments, the one or more modifications, e.g., substitutions, may increase distribution of an AAV particle to a cell, region, or tissue of a muscle. In some embodiments, the muscle is a heart muscle (e.g., a heart atrium or a heart ventricle) 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. [0112] 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. [0113] 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 14. In some embodiments, the AAV capsid variant comprises (e.g.. in loop VIII) a sequence as set forth in Table 27. In some embodiments, the AAV capsid variant comprises (e.g.. in loop VIII) a sequence as set forth in Table 28. In some embodiments, the AAV capsid variant is an AAV9 capsid variant.

Table 1. Exemplary Sequences

Table 2A. Exemplary Sequences

Table 2B. Exemplary Sequences

[0114] 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 X X₂, X3, and X4, wherein X4 is Q, K, E, S, P, R, N, H, or a conservative substitution thereof and/or [N3] comprises X5. X₆, and X₇, wherein X₅ 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 X₄ is Q, K, E, S, P, R. N, or H, and/or [N3] comprises X₅. X₆, and X₇, wherein X5 is I, V. T, M. S, N, L. or F. In some embodiments, X4 of [Nl] is Q. In some embodiments, X4 of [Nl] is K. In some embodiments, X5 of [N3] is I. In some embodiments, Xi of [Nl] is T, S, R. A, I, C. N, K, L, or Q. In some embodiments, X₂ of [Nl] is N, T, G, V, S, Y, K, I, H, D, or F. In some embodiments, X₃ of [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, [N1]-[N2] is or comprises TNTQDWHR (SEQ ID NO: 4898). In some embodiments, [N1]-[N2] is or comprises TNTKDWHR (SEQ ID NO: 4899). In some embodiments, X₆ of [N3] is A, Y, P, N, S, T, G, E, V, W, F, or Q. In some embodiments, X₇ 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).

[0115] 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 Xn, wherein X₈ is T, S. N, P, A. or I; X₉ is G. N, D. R, V. A, S, or Q; X10 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] 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).

[0116] In some embodiments, the AAV capsid variant comprises (e.g., in loop VIII) an amino acid sequence having the formula [N1]-[N2]-[N3] (SEQ ID NO: 4683), wherein [N2] comprises the amino acid sequence of DWHR (SEQ ID NO: 4682) and wherein [Nl] comprises amino acids Xi, X₂, X₃, and X₄, wherein X₄ is Q, P, or a conservative substitution thereof; and/or [N3] comprises amino acids X₅, X₆, and X?, wherein X₃ is 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 X₄ is Q or P; and/or [N3] comprises amino acids X5. Xe, and X₇, wherein X, is I or V. In some embodiments, X₄ of [Nl] is Q. In some embodiments, X, of [N3] is I. In some embodiments, X5 of [N3] is V. In some embodiments, Xi of [Nl] is T or S. In some embodiments, 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, Xe of [N3] is A, P, S. Y, or N. In some embodiments, X7 of [N3] is Q. G, or N. In some embodiments. [N3] is or comprises IAQ. In some embodiments. [N2]-[N3] is or comprises DWHRIAQ (SEQ ID NO: 5027). In some embodiments, [N 1 ]-[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, X10. and Xii, wherein Xio is W. In some embodiments, X_x of [N4] is T, S, or N. In some embodiments, X₉ 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).

[0117] 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.

[0118] 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 X, of [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 at positions corresponding to positions 582-585 (e.g., T582. N583. H584, and Q585) of the ammo acid sequence of SEQ ID NO: 138.

[0119] 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 at positions 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).

[0120] In some embodiments, [N1]-[N2] is present at ammo 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 at positions corresponding to positions 582-589 of the 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).

[0121] 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, X₅ of [N3] is present at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138 or 981, X₆ of [N3] is present at an amino acid corresponding to position 591 of the amino acid sequence of SEQ ID NO: 138 or 981, and X₇ of [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 at positions corresponding to positions 590-592 (e.g., 1590, A591, and Q592) of die amino acid sequence of SEQ ID NO: 138.

[0122] 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 at positions 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).

[0123] 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 at positions 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).

[0124] 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, X§ of [N4] is present at an amino acid corresponding to position 593 of the amino acid sequence of SEQ ID NO: 138 or 981, X₉ of [N4] is present at an amino acid corresponding to position 594 of the amino acid sequence of SEQ ID NO: 138 or 981. Xin 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 at positions corresponding to positions 593-596 (e.g., T593. G594. W595, and V596) of the amino acid sequence of SEQ ID NO: 138.

[0125] 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 at positions 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).

[0126] hi some embodiments, [N1]-[N2]-[N3]-[N4] is present at amino acids corresponding to positions 582-596 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, [N1]-[N2]-[N3]-[N4] replaces amino acids at positions 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).

[0127] In some embodiments, [N2] is present immediately subsequent to [Nl], In some embodiments. [N3] is present immediately subsequent to [N2], In some embodiments. [N4] is present immediately subsequent to [N3] . In some embodiments, loop VIII of the AAV capsid variant comprises an amino acid sequence comprising, from N-terminus to C-tenninus, [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],

[0128] 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, 14, 27, 28, or 29. 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.

[0129] 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).

[0130] In some embodiments, the at least 3 consecutive amino acids comprise TNT. In some embodiments, the at least 4 consecutive amino acids comprise TNTQ (SEQ ID NO: 4688). In some embodiments, the at least 5 consecutive amino acids comprise TNTQD (SEQ ID NO: 5119). In some embodiments, the at least 6 consecutive amino acids comprise TNTQDW (SEQ ID NO: 5120). In some embodiments, the at least 7 consecutive amino acids comprise TNTQDWH (SEQ ID NO: 5121). In some embodiments, the at least 8 consecutive amino acids comprise TNTQDWHR (SEQ ID NO: 4898). In some embodiments, the at least 9 consecutive amino acids comprise TNTQDWHRI (SEQ ID NO: 746). [0131] 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, 14, 27, 28, or 29. 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, 14, 27, 28, or 29. 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 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.

[0132] 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).

[0133] In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of the sequences provided in Table 1. 2A. 2B. 14, 27, 28, or 29. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NOs: 201, 205-209. 211-214, 216, 219. 220, 230, 232. 237, 238, 255. 262-265, 274. 283, 286, 290. 291, 293. 301, 306, 307. 308, 309, 314. and 336.

[0134] 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., 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 tire 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.

[0135] 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 tw o. 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.

[0136] 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.

[0137] 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 the nucleotide sequence of SEQ ID NO: 942, or a nucleotide sequence comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven modifications, but no more than ten modifications, relative to the nucleotide sequence of SEQ ID NO: 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.

[0138] 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 embodiments, the AAV capsid variant further comprises the amino acid V at a position corresponding to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variant comprises the substitution A581T or A581V, wherein the substitution is at a position that corresponds to position 581 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0139] In some embodiments, an AAV capsid variant described herein comprises one. two, three, four, five, or all of an amino acid other than H at a position corresponding to position 584 (e.g., comprises T at an amino acid corresponding to position 584) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than S at a position corresponding to position 586 (e.g., comprises D at an amino acid corresponding to position 586) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 587 (e.g., comprises W at an amino acid corresponding to position 587) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than Q at a position corresponding to position 588 (e.g., comprises H at an amino acid corresponding to position 588) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 589 (e.g.. comprises R at an amino acid corresponding to position 589) of the amino acid sequence of SEQ ID NO: 138; and/or an amino acid other than Q at a position corresponding to position 590 (e.g.. comprises I at an amino acid corresponding to position 590) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises an amino acid other than H at a position corresponding to position 584 (e.g., comprises T at an amino acid corresponding to position 584) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than S at a position corresponding to position 586 (e.g., comprises D at an amino acid corresponding to position 586) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 587 (e.g., comprises W at an amino acid corresponding to position 587) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than Q at a position corresponding to position 588 (e.g., comprises H at an amino acid corresponding to position 588) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than A at a position corresponding to position 589 (e.g., comprises R at an amino acid corresponding to position 589) of the amino acid sequence of SEQ ID NO: 138; and an amino acid other than Q at a position corresponding to position 590 (e.g., comprises I at an amino acid corresponding to position 590) of die 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.

[0140] 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.

[0141] In some embodiments, an AAV capsid variant described herein comprises one. two, three, four, five, or all of the substitutions H584T. S586D, A587W, Q588H, A589R, and/or Q590I, wherein the substitution(s) is/are at position(s) that correspond to position/ s) 584. 586, 587, 588. 589, and/or 590 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the substitutions H584T, S586D, A587W, Q588H, A589R, and Q590I, wherein the substitution(s) is/are at position(s) that correspond to position(s) 584, 586. 587, 588, 589. and/or 590 of the amino acid sequence of SEQ ID NO: 138 or 981.

[0142] 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. [0143] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than Q at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid K at an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138.

[0144] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than Q at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid I at an amino acid corresponding to position 590 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, die 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.

[0145] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941), wherein TQDWHRI (SEQ ID NO: 941) is present in the AAV capsid variant at amino acids corresponding to positions 584-590 of the amino acid sequence of SEQ ID NO: 138 or SEQ ID NO: 981. In some embodiments, TQDWHRI (SEQ ID NO: 941) is present at amino acids corresponding to positions 584-590 of the amino acid sequence of SEQ ID NO: 138 (e.g., H584, S586, A587, Q588, A589, and Q590 may be replaced with T584, D586, W587. H588, R589, and 1590).

[0146] 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. [0147] In some embodiments, the AAV capsid variant further comprises a substitution corresponding to amino acid K449 (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 corresponding to position 449) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises an R at amino acid corresponding to position 449 of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a modification in loop I, II, IV. and/or VI. [0148] 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.

[0149] 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 tw o, 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 tire amino acid sequences in (a)-(c), or an amino acid sequence comprising at least one, at least tw o. or at least three modifications, but not more than 30, not more than 20, or not more than 10 such modifications relative to any one of the amino acid sequences in (a)-(c).

[0150] In some embodiments, the AAV capsid variant comprises (a) a VP1 protein comprising an amino acid sequence that is at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 981; (b) a VP2 protein comprising an amino acid sequence that is at least 90% (e.g.. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to amino acids 138-736 of SEQ ID NO: 981; and/or (c) a VP3 protein comprising an amino acid sequence that is at least 90% (e.g.. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99%) identical to amino acids 203-736 of SEQ ID NO: 981, wherein the AAV capsid variant comprises the amino acid sequence of TQDWHRI (SEQ ID NO: 941) or a sequence comprising one. two, or three but not more than three substitutions relative to the amino acid sequence of TQDWHRI (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises the amino acid sequence comprising one, tw o, 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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 sequences

Table 4. Exemplary full length capsid amino acid sequences

Table 5. Exemplary full length capsid nucleic acid sequences

[0155] In some embodiments, the polynucleotide encoding an AAV capsid variant comprises the nucleotide sequence of SEQ ID NO: 983, or a nucleotide sequence with at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.

[0156] In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, at least two, or at least three modifications but not more than 30, not more than 20, or not more than 10 modifications, relative to the nucleotide sequence of SEQ ID NO: 983. In some embodiments, the nucleotide sequence encoding an AAV capsid variant described herein, comprises a nucleotide sequence comprising at least one, at least two, or at least three substitutions, but not more than 30. not more than 20, or not more than 10 substitutions relative to the amino acid sequence of SEQ ID NO: 983. In some embodiments, the nucleic acid sequence encoding an AAV capsid variant described herein is codon optimized.

[0157] 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.

[0158] 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 two, or at least three substitutions, but not more than 30, not more than 20, or not more than 10 substitutions, relative to the amino acid sequence of SEQ ID NO: 983. In some embodiments, 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.

[0159] 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 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. [0160] 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 that is 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. 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 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. 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 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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 capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the at least two to three species are Macaca fascicularis, Chlorocebus sabaeus, Callithrix jacchus, and/or mouse (e.g., outbred mice).

[0165] 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. [0166] 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.

[0167] In some embodiments, an AAV capsid variant delivers an increased level of a payload to a brain region. In some embodiments, the level of the payload is increased by at least 39. at least 50, at least 100, at least 120, at least 132, at least 146, at least 150, at least 161, at least 174, at least 175, at least 200. at least 225. at least 250. at least 275. at least 283. at least 300. at least 350. at least 400, at least 450, at least 500, at least 525, at least 528, or at least 550-fold, as compared to the level of a payload delivered by an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the brain region comprises a sensory cortex, motor cortex, putamen, thalamus, caudate, hippocampus, and/or cerebellum.

[0168] In some embodiments, an AAV capsid variant described herein is enriched at least 5, at least 10, at least 50, at least 100, at least 115, at least 120, at least 150, at least 175, at least 200, at least 207, at least 225, at least 250, or at least 275-fold in the spinal cord compared to enrichment in the spinal cord of an AAV capsid comprising the amino acid sequence of SEQ ID NO: 138.

[0169] 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. [0170] 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 272 (e.g., comprises A at an amino acid corresponding to position 272) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than Y at a position corresponding to position 446 (e.g., comprises A at an amino acid corresponding to position 446) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than N at a position corresponding to position 498 (e.g., comprises Y or I at an amino acid corresponding to position 498) of the amino acid sequence of SEQ ID NO: 138; an amino acid other than W at a position corresponding to position 503 (e.g., comprises R or A at an amino acid corresponding to position 503) of the amino acid sequence of SEQ ID NO: 138; and an amino acid other than L at a position corresponding to position 620 (e.g., comprises F at an amino acid corresponding to position 620) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, die 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 D271 A at an amino acid corresponding to D271) of the amino acid sequence of SEQ ID NO: 138; corresponding to amino acid N272 (e.g., comprises N272A at an amino acid corresponding to N272) of the amino acid sequence of SEQ ID NO: 138; corresponding to amino acid Y446 (e.g., comprises Y446A at an amino acid corresponding to Y446) of the amino acid sequence of SEQ ID NO: 138; and corresponding to amino acid W503 (e.g., comprises W503R or W503A at an amino acid corresponding to W503) of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises a modification corresponding to amino acid N498 (e.g.. comprises N498Y at an amino acid corresponding to N498) of the amino acid sequence of SEQ ID NO: 138 and corresponding to amino acid L620 (e.g., comprises L620F at an amino acid corresponding to L620) of the amino acid sequence of SEQ ID NO: 138.

[0171] 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.

[0172] 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.

[0173] 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

[0174] In some embodiments, the AAV particle may comprise a capsid protein or variant of any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to, packaging, tropism, transduction and immunogenic profdes. Without being bound by theory, it is believed in some embodiments that the AAV capsid protein, e.g.. an AAV capsid variant, can modulate AAV particle tropism in a particular tissue.

[0175] In some embodiments, the AAV capsid variant allow s for blood brain barrier penetration following intravenous administration. In some embodiments, the AAV capsid variant allows for blood brain barrier penetration following intravenous administration, focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration. In some embodiments, the AAV capsid variant allows for increased distribution to a brain region. In some embodiments, the brain region comprises the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and/or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, visual cortex, and/or temporal 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, and/or vestibular nucleus. In some embodiments, the AAV capsid variant allows for preferential transduction in a brain region relative to the transduction in the dorsal root ganglia (DRG). In some embodiments, the AAV capsid variant allows for preferential transduction in a brain region relative to the transduction in the liver. In some embodiments, the AAV capsid variant allows for transduction in neuronal cells. In some embodiments, the AAV capsid variant allows for transduction in a non-neuronal cell, e.g.. a glial cell (e.g.. an astrocyte, an oligodendrocyte, or a combination thereof). In 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 thereof).

[0176] 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.

[0177] In some embodiments, the AAV capsid variant allows for increased distribution to a heart region.

[0178] 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.

[0179] 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. [0180] 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.

[0181] The present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (e.g., capsid) of a viral vector such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Metl), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for a first-methionine (Metl) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzy mes such as Mct-aminopcptidascs. 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.

[0182] 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 entirety⁷. [0183] 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-).

[0184] 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).

[0185] 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 “AAl” 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 “AAl” amino acid (AA1-) of the 736 amino acid AA1+ sequence.

[0186] References to viral capsids formed from VP capsid proteins (such as reference to specific AAV capsid serotypes), can incorporate VP capsid proteins which include a Metl/AAl ammo acid (Met+/AA1+). corresponding VP capsid proteins which lack the Metl/AAl amino acid as a result of Met/ AAl -clipping (Mct-/AA1-), and combinations thereof (Mct+/AA1+ and Mct-/AA1-).

[0187] 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

[0188] 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 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, 14, 27, 28, or 29.

[0189] In some embodiments, the AAV capsid variant comprises, immediately subsequent to an amino acids corresponding to position 582, 583, 584, 585, 586. 587, 588, 589, and/or 590 of the amino acid sequence of SEQ ID NO: 138 (e.g., corresponding to equivalent positions in any other AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrhlO, AAVrh32.33. AAVrh74, PHP.N, PHP.B. or an AAV serotype as provided in Table 6 of WO 2021/230987 (the contents of which are hereby incorporated by reference in their entirety))), at least 3, at least 4, at least 5, at least 6, at least 7. at least 8, at least 9, at least 10. at least 11, at least 12, at least 13. at least 14, or at least 15 consecutive amino acids of any one of the amino acid sequences provided in Table 1. 2A, 2B. 14. 27, 28, or 29. 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. 14, 27, 28. or 29 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)). 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). 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).

[0190] In some embodiments, the AAV capsid variant may comprise a variant of a VOY101 capsid polypeptide, an AAVPHP.B (PHP.B) capsid polypeptide, a 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 some embodiments, 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 die 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 Sequences

AAV Viral Genome

[0191] In some embodiments, the AAV particle of the present disclosure serves as an expression vector comprising a viral genome that encodes a GBA1 protein (e.g., a wildtype GBA1 protein, e.g., a wildtype human GBA1 protein).

[0192] In some embodiments, an AAV particle, e.g., an AAV particle for the vectorized deliver}' of GBA1 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, tire 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 transgene encoding a GBA1 protein, a nucleotide sequence encoding at least one miR binding site (e.g., at least one miR183 binding site), a polyA region, or a combination thereof.

Viral Genome Component: Inverted Terminal Repeats (ITRs)

[0193] In some embodiments, the viral genome may comprise at least one inverted terminal repeat (ITR) region. The AAV particles of the present disclosure comprise a viral genome with at least one ITR region and a region comprising a GBAl-encoding sequence. In some embodiments, the viral genome has two ITRs. These two ITRs flank the region comprising the GBAl-encoding sequence 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.

[0194] In some embodiments, the ITR is of the same serotype as the capsid, selected from any one of the serotypes known in the art, or a derivative thereof. In some embodiments, the ITR is of a different serotype than the capsid. In some embodiments, the AAV particle has more than one ITR. In some embodiments, the AAV particle comprises a viral genome comprising two ITRs. In some embodiments, the ITRs are of the same serotype as one another. In some embodiments, the ITRs are of different serotypes. Non-limiting examples include zero, one. or both of the ITRs having the same serotype as the capsid. In some embodiments, both ITRs of the viral genome of the AAV particle are AAV2 ITRs.

[0195] Independently, each ITR may be about 100 to about 150 nucleotides in length. In some embodiments, the ITR is about 120-140 nucleotides in length, e.g.. about 130 nucleotides in length.

[0196] In some embodiments, each ITR is 141 nucleotides in length. In some embodiments, each ITR is 130 nucleotides in length. In some embodiments, the AAV particles comprise two ITRs and one ITR is 141 nucleotides in length and the other ITR is 130 nucleotides in length.

[0197] In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 1829 or 1830, or a nucleotide sequence that is at least 70%. at least 75%, at least 80%, at least 85%, at least 90%, at least 91%. at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1829 or 1830. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 1829 or 1830, or a nucleotide sequence having one, two. three, four, but no more than four modifications, e.g., substitutions, relative to any one of SEQ ID NOs: 1829 or 1830. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 1829 or 1830. In some embodiments, the viral genome comprises a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 1829 and a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 1830.

Viral Genome Component: Promoters and Enhancers

[0198] 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; tire contents of which are herein incorporated by reference in their entirety. Non-limiting examples of elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (poly A) sequences, upstream enhancers (USEs), CMV enhancers, and introns.

[0199] In some embodiments, expression of the polypeptides in a target cell may be driven by a specific promoter, including but not limited to, a promoter that is species specific, inducible, tissuespecific, or cell cycle-specific (Parr et al.. Nat. Med.3'.1145-9 (1997); the contents of which are herein incorporated by reference in their entirety). [0200] In some embodiments, the viral genome provides expression of a GBA1 protein encoded by a transgene for expression in a target tissue. In some embodiments, the promoter is deemed to be efficient when it drives expression of the GBA1 encoded in the viral genome of the AAV particle.

[0201] In some embodiments, the promoter is a promoter deemed to be efficient when it drives expression in the cell or tissue being targeted (e.g., the CNS).

[0202] 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.

[0203] In some embodiments, the viral genome comprises a promoter that results in expression in one or more cells and/or tissues. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, a promoter that drives or promotes expression in most mammalian tissues includes, but is not limited to. a human elongation factor la-subunit (EFla) promoter, a cytomegalovirus (CMV) immediate-early enhancer and/or promoter, a chicken P-actin (CBA) promoter, a CAG promoter, a CAG derivative promoter, a glucuronidase (GUSB) promoter, and a ubiquitin C (UBC) promoter. A CAG promoter typically comprises: (C) the cytomegalovirus early enhancer element; (A) the promoter, the first exon, and the first intron of the chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene. In some embodiments, a derivative of a CAG promoter may comprise (i) a CMVie enhancer and a beta-actin promoter or (ii) a beta-actin promoter and an intron sequence.

[0204] In some embodiments, the viral genome comprises a native GBA1 promoter.

[0205] In some embodiments, the viral genome comprises a nervous system-specific promoter, e.g., a promoter that results in expression of GBA1 in a neuron, an astrocyte, and/or an oligodendrocyte. Nonlimiting examples of tissue-specific expression elements for neurons include synapsin (Syn) or synapsin 1 (Synl), e.g., human synapsin or synapsin 1.

[0206] In some embodiments, the promoter may be less than 1 kb.

[0207] In some embodiments, the promoter may be a combination of tw o or more components of the same or different starting or parental promoters such as, but not limited to, CMV and CBA. Each component may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381. 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450,

460, 470, 480, 490, 500, 510. 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660,

670, 680, 690, 700, 710, 720. 730, 740, 750, 760, 770, 780, 790, 800. or more than 800 nucleotides. Each component may have a length of 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, SOO- SOO, 300-600, 300-700, 300-800, 400-500, 400-600. 400-700, 400-800. 500-600, 500-700. 500-800, 600- 700, 600-800 or 700-800 nucleotides. In some embodiments, the promoter is a combination of a 380 nucleotide CMV-enhancer sequence and a 260 nucleotide CBA-promoter sequence.

[0208] In some embodiments, the viral genome comprises an enhancer. In some embodiments, the enhancer comprises a CMVie enhancer.

[0209] In some embodiments, the viral genome comprises a CMVie enhancer and a CBA promoter. In some embodiments, the viral genome comprises a CMVie enhancer and a CMV promoter (e.g., a CMV promoter region). In some embodiments, the viral genome comprises a CMVie enhancer, a CBA promoter or functional variant thereof, and an intron.

[0210] In some embodiments, the viral genome comprises an engineered promoter.

[0211] hi some embodiments, a CBA promoter is used in a viral genome of an AAV particle described herein. In some embodiments, a CBA promoter is used in an AAV particle comprising a viral genome encoding a GBA1 protein.

[0212] In some embodiments, the intron may be 100-600 nucleotides in length. In some embodiments, the intron is 566 nucleotides in length.

[0213] In some embodiments, the AAV vector may comprise a beta-globin intron or a fragment or variant thereof. In some embodiments, the intron comprises one or more human beta-globin sequences (e.g., including fragments/variants thereol). In some embodiments, the promoter may be a CBA promoter. In some embodiments, the promoter comprises a CMV promoter. In some embodiments, the promoter comprises a minimal CBA promoter.

[0214] In some embodiments, the GBAl-encoding sequence may be located downstream of an intron in an expression vector such as a beta globin intron or others known in the art.

[0215] 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: miR Binding Site-encoding sequence

[0216] Tissue- or cell-specific expression of the AAV viral particles of the disclosure can be enhanced by introducing tissue- or cell-specific regulatory sequences, e.g., promoters, enhancers. microRNA binding sites, e.g., a detargeting site. Without being bound by theory , it is believed that an encoded miR binding site can modulate, e.g., prevent, suppress, or otherwise inhibit, the expression of a gene of interest in the viral genome of the disclosure, based on the expression of the corresponding endogenous microRNA (miRNA) or a corresponding controlled exogenous miRNA in a tissue or cell, e.g., a non-targeting cell or tissue. In some embodiments, a miR binding site modulates, e.g., reduces, expression of GBA1 in a cell or tissue where the corresponding mRNA is expressed. In some embodiments, the miR binding site modulates, e.g., reduces, expression of GBA1 in a cell or tissue of tire DRG, liver, heart, hematopoietic lineage, or a combination thereof.

[0217] In some embodiments, the viral genome of an AAV particle described herein comprises a nucleotide sequence encoding a microRNA binding site, e.g., a detargeting site. In some embodiments, the viral genome of an AAV particle described herein comprises a nucleotide sequence encoding a miR binding site, a microRNA binding site series (miR BSs). or a reverse complement thereof.

[0218] In some embodiments, the encoded miR binding site series comprise at least 1-5 copies, e.g., 1-3. 2-4, or 3-5 copies, or at least 1. at least 2. at least 3, at least 4, at least 5 or more copies of a miR binding site (miR BS). In some embodiments, the encoded miR binding site series comprises 4 copies of

- I l l - a miR binding site. In some embodiments, all copies are identical, e.g., comprise the same miR binding site. In some embodiments, the miR binding sites within the encoded miR binding site series are continuous and not separated by a spacer (also referred to herein as a spacer sequence). In some embodiments, the miR binding sites within an encoded miR binding site series are separated by a spacer, e.g., a non-coding sequence. In some embodiments, the spacer is 1 to 6 nucleotides or 5 to 10 nucleotides, e.g., 7-8 nucleotides in length. In some embodiments, the spacer is 8 nucleotides in length. In some embodiments, the spacer sequence is encoded by a nucleotide sequence that comprises one or more of (i) GGAT; (ii) CACGTG; (iii) GCATGC. or a repeat of one or more of (i)-(iii). In some embodiments, the spacer is encoded by the nucleotide sequence of GATAGTTA or a nucleotide sequence having at least one. at least two. or at least three modifications, but no more than four modifications of GATAGTTA.

[0219] In some embodiments, the encoded miR binding site is substantially complementary (e.g., at least 70%, at least 75%, at least 80%. at least 85%, at least 90%, at least 95%. at least 99% or 100% complementary) to the miR in the host cell. In some embodiments, the encoded miR binding site comprises at least 1, at least 2, at least 3. at least 4. or at least 5 mismatches or no more than 6, no more than 7. no more than 8, no more than 9, or no more than 10 mismatches relative to tire corresponding miR in the host cell. In some embodiments, the mismatched nucleotides are contiguous. In some embodiments, the mismatched nucleotides are non-contiguous. In some embodiments, the mismatched nucleotides occur outside the seed region-binding sequence of the miR binding site, such as at one or both ends of tire miR binding site. In some embodiments, the encoded miR binding site is 100% complementary to the miR in the host cell. In some embodiments, the encoded miR binding site is capable of hybridizing to tire miR in the host cell.

[0220] In some embodiments, the encoded miR binding site or the encoded miR binding site series is about 10 to about 125 nucleotides in length, e.g., about 10 to about 50 nucleotides in length, e.g., 22 nucleotides in length. In some embodiments, an encoded miR binding site comprises at least one consecutive region (e.g., 7 or 8 nucleotides) complementary (e.g., full complementary or partially complementary) to the seed sequence of a miRNA (e.g., a miR183).

[0221] In some embodiments, tire encoded miR binding site is complementary (e.g., fully complementary, or partially complementary) to a miR expressed in a DRG (dorsal root ganglion) neuron. e.g, a miR183. In some embodiments, the encoded miR binding site is complementary (e.g.. fully complementary, or partially complementary) to a miR expressed in expressed in a DRG neuron. In some embodiments, the encoded miR binding site comprises a nucleotide sequence disclosed, e.g.. in WO2020/132455, the contents of which are incorporated by reference herein in its entirety.

[0222] In some embodiments, the encoded miR binding site or encoded miR binding site series comprises a miR183 binding site sequence. In some embodiments, the encoded miR183 binding site is encoded by a nucleotide sequence that comprises the nucleotide sequence of AGTGAATTCTACCAGTGCCATA (SEQ ID NO: 1847). or a nucleotide sequence having 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 99%, or 100% sequence identity, or having 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 to SEQ ID NO: 1847, e.g., wherein the modification can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the sequence complementary (e.g., fully complementary, or partially complementary) to the seed sequence corresponds to the double underlined of the miR183 binding site sequence. In some embodiments, the viral genome encodes at least 3, at least 4. or at least 5 copies (e.g., 4 copies) of the miR183 binding site, e.g., an encoded miR183 binding site. In some embodiments, the viral genome encodes at least 4 copies of the miR183 binding site, e.g., an encoded miR183 binding site comprising 4 copies of a miR183 binding site. In some embodiments, the at least 3, at least 4, or at least 5 copies (e.g., 4 copies) of the encoded miR183 binding site are continuous (e.g.. not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is 1 to 6 nucleotides or 5 to 10 nucleotides, e.g., 7-8 nucleotides or 8 nucleotides, in length. In some embodiments, the spacer is encoded by a nucleotide sequence that comprises the nucleotide sequence of GATAGTTA, or a nucleotide sequence having at least one. two, three, four modifications, but no more than four modifications of GATAGTTA. In some embodiments, the encoded miRl 83 binding site series is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide sequence having 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 99%. or 100% sequence identity, or having at least one. two, three, four, five, six, or seven modifications but no more than ten modifications to SEQ ID NO: 1849. In some embodiments, the encoded miRl 83 binding site series is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 1849.

Viral Genome Component: Poly adenylation Region

[0223] In some embodiments, the viral genome of the AAV particles of the present disclosure comprises at least one poly adenylation (poly A) sequence. In some embodiments, the polyadenylation sequence is positioned 3" relative to the nucleic acid comprising the GBAl-encoding sequence.

[0224] In some embodiments, the poly A region comprises a length of about 100 to about 150 nucleotides, e.g., about 127 nucleotides. In some embodiments, the polyA region comprises a length of 127 nucleotides.

[0225] In some embodiments, the viral genome comprises a human growth hormone (hGH) polyA sequence. In some embodiments, the viral genome comprises an hGH polyA as described above and a GBAl-encoding sequence.

Viral Genome Component: GBAl-encoding sequence

[0226] In some embodiments, the disclosure provides an AAV particle comprising a viral genome encoding a GBA1 protein, wherein the viral genome comprises a nucleotide sequence as disclosed in PCT/US2021/043216. PCT/US2022/079964, and/or PCT/US2023/061837. the contents of each of which are hereby incorporated by reference in their entirety.

[0227] In some embodiments, the disclosure provides an AAV particle comprising a viral genome encoding a GBA1 protein, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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) to SEQ ID NO: 2001 or SEQ ID NO: 2002. In some embodiments, the viral genome comprises a promoter operably linked to a nucleotide sequence encoding a GBA1 protein.

[0228] In some embodiments, the viral genome comprises a nucleotide sequence encoding a nonhuman, animal GBA1 protein. In some embodiments, the viral genome comprises a nucleotide sequence encoding a canine GBA1 protein. In some embodiments, the viral genome comprises a nucleotide sequence encoding an equine GBA1 protein.

[0229] In some embodiments, the disclosure herein provides constructs that allow for improved expression of GBA1 protein delivered by gene therapy vectors.

[0230] In some embodiments, the disclosure provides constructs that allow for improved biodistribution of GBA1 protein delivered by gene therapy vectors.

[0231] In some embodiments, the disclosure provides constructs that allow for improved sub-cellular distribution or trafficking of GBA1 protein delivered by gene therapy vectors.

[0232] In some embodiments, the disclosure provides constructs that allow for improved trafficking of GBA1 protein to lysosomal membranes delivered by gene therapy vectors.

[0233] In some embodiments, the present disclosure relates to a composition containing or comprising a nucleic acid sequence encoding a GBA1 protein or a functional fragment or variant thereof and methods of administering the composition in vitro or in vivo in a subject, e.g., a human subject and/or an animal model of disease, e.g., a disease related to expression of GBA. In some embodiments, the subject is a human subject. In some embodiments, the subject is a veterinary' subject (e.g., dog, horse). [0234] In some embodiments, the disclosure provides a nucleotide sequence encoding a GBA1 protein for use in an AAV genome, wherein the nucleotide sequence comprises a codon-optimized, CpG- reduced (e.g., CpG-depleted) GBAl-encoding sequence. In some embodiments, the CpG-reduced (e.g., CpG-depleted) GBAl-encoding sequence provides decreased immunogenicity in vivo, e.g., reduced immunogenicity in a human or animal subject. In some embodiments, the nucleotide sequence further comprises one or more, e.g., all of, a 5’ ITR sequence, a CMVie sequence, a CBA promoter sequence, an intron sequence, a signal sequence-encoding sequence, a poly A sequence, and a 3’ ITR sequence. In some embodiments, the GBA1 protein encoded by die nucleotide sequence has an amino acid sequence that is 100% identical to a wildtype GBA1 protein. In some embodiments, the wildtype GBA-encoding sequence is as provided by NCBI Reference Sequence NCBI Reference Sequence NP 000148.2 (SEQ ID NO: 14 of Int’l Pub. No. W02019070893, incorporated by reference herein).

[0235] In some embodiments, the AAV genome encodes a payload construct that comprises a combination of coding and non-coding nucleic acid sequences. [0236] 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.

[0237] hi some embodiments, tire pay load comprises a gene therapy product including, but not limited to. a polypeptide, protein. RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect. In some embodiments, a gene therapy product may comprise a substitute for a non-functional gene or a gene that is absent, expressed in insufficient amounts, or mutated. In some embodiments, a gene therapy product may comprise a substitute for a non-functional protein or polypeptide or a protein or polypeptide that is absent, expressed in insufficient amounts, misfolded, degraded too rapidly, or mutated. For example, a gene therapy product may comprise a polynucleotide encoding a GBA1 protein to treat GCase deficiency or a GBAl-related disorder. In some embodiments, the gene therapy product comprises a polynucleotide sequence encoding a GBA1 protein. [0238] In some embodiments, the payload construct encodes a messenger RNA (mRNA). Certain embodiments provide the mRNA as encoding GBA1 or a variant thereof.

[0239] A pay load construct encoding GBA1 may 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.

[0240] In some embodiments, a payload construct encoding GBA1 may comprise a selectable marker including, but not limited to, (3-lactamasc, luciferase, P-galactosidasc, or any other reporter gene as that term is understood in the art, including cell-surface markers, such as CD4 or the truncated nen e growth factor (NGFR) (for GFP, see WO 96/23810; Heim et al., Current Biology 2: 178-182 (1996); Heim et al., Proc. Natl. Acad. Sci. USA (1995); or Heim et al., Science 373:663-664 (1995); for P-lactamase, see WO 96/30540); the contents of each of which are herein incorporated by reference in their entirety.

[0241] In some embodiments, a payload construct encoding a selectable marker may comprise a fluorescent protein. A fluorescent protein as herein described may comprise any fluorescent 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.

[0242] In certain embodiments, a nucleic acid for expression of GBA1 in a target cell may be incorporated into the viral genome and located between two ITR sequences. Viral Genome Component: Signal Sequence-encoding sequence

[0243] In some embodiments, the disclosure provides a viral genome encoding a GBA1 protein that comprises a nucleic acid sequence encoding a signal peptide (e.g., a signal sequence region described herein).

[0244] hi some embodiments, tire viral genome encoding a GBA1 protein further comprises a nucleotide sequence encoding a signal sequence (also referred to herein as a signal sequence-encoding sequence). In some embodiments, the nucleotide sequence encoding the signal sequence is located 5’ relative to the nucleotide sequence encoding the GBA1 protein. In some embodiments, the encoded GBA1 protein comprises a signal sequence at the N-terminus, wherein the signal sequence is optionally cleaved during cellular processing and/or localization of the GBA1 protein.

[0245] In some embodiments, the signal sequence-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2005 or a nucleotide 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. In some embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO: 1853 or an amino acid sequence 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. In some embodiments, the signal sequence-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2005. In some embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO: 1853.

Exemplary GCase (GBA) Protein

[0246] In some embodiments, the encoded GBA1 protein is a wildtype GBA1 protein, e.g., a wildtype human GBA1 protein.

[0247] Tables 7A and 7B provide exemplary nucleotide sequences encoding a GBA1 protein that may be used in viral genomes disclosed herein and exemplary’ GBA1 amino acid sequences that may be encoded by viral genomes disclosed herein. In some embodiments, the GBA1 protein is encoded by the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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) to SEQ ID NO: 2001 or SEQ ID NO: 2002. In some embodiments, the GBA1 protein is encoded by the nucleotide sequence of SEQ ID NO:

2001. In some embodiments, the GBA1 protein is encoded by the nucleotide sequence of SEQ ID NO:

2002.

[0248] The exemplary GBA1 sequence information from the National Center for Biotechnology Information cited in Table 7A is hereby incorporated by reference in its entirety.

Table 7A. Exemplary GCase Sequences

Table 7B. Exemplary GCase Sequences

Exemplary GBA1 AAV Viral Genome Sequence Regions and ITR-to-ITR Sequences

[0249] In some embodiments, a viral genome, e.g., an AAV viral genome or vector genome, described herein, comprises a promoter operably linked to a transgene encoding a GBA1 protein. In some embodiments, the viral genome further comprises an inverted terminal repeat region, an enhancer, an intron, a nucleotide sequence encoding a miR binding site, a polyA region, or a combination thereof.

Exemplar}' sequence regions within ITR-to-ITR sequences for viral genomes according to the description are provided in Table 8 A.

Table 8A. Exemplary Viral Genome Sequence Regions in ITR-to-ITR Constructs

[0250] In some embodiments, the viral genome comprises a nucleotide sequence encoding a GBA1 protein, wherein the GBA1 -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or a nucleotide sequence having 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. In some embodiments, the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2002 or a nucleotide sequence having 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. In some embodiments, the viral genome comprises a promoter comprising the nucleotide sequence SEQ ID NO: 1834 or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0251] In some embodiments, the viral genome of an AAV particle described herein comprises tire nucleotide sequence of SEQ ID NO: 2006 or a nucleotide 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. In some embodiments, the viral genome of an AAV particle described herein comprises the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide 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.

[0252] In some embodiments, the present disclosure also provides a GBA1 protein encoded by SEQ ID NO: 2001 or SEQ ID NO: 2002 or a 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%) thereto.

[0253] In some embodiments, the viral genome comprises a codon-optimized nucleotide sequence encoding a wildtype GBA1 protein, wherein the GBA1 -encoding nucleotide sequence comprises a reduced number of CpG motifs (e.g., lacking all CpG motifs), as compared to a wildtype nucleotide sequence encoding a wild-type GBA1 protein (e.g., a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1776 or 1777). In some embodiments, the GBAl-encoding nucleotide sequence does not comprise any CpG motifs.

Table 8B. Exemplary Viral Genome (ITR-to-ITR) Sequences

Table 9. Exemplary ITR to ITR sequences encoding a GBA1 protein

[0254] In some embodiments, the viral genome of an AAV particle described herein comprises one or more, e.g.. all of. the nucleotide sequences provided in Table 12 or Table 13, or nucleotide sequences having at least 70%, at least 75%. at least 80%, at least 85%, at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%. at least 97%, at least 98%, or at least 99% sequence identity thereto. [0255] In some embodiments, the viral genome of an AAV particle described herein comprises a GBA1 -encoding nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 2002 or a nucleotide sequence having at least 93% (e.g., at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, said AAV particle comprises improved (e.g., reduced) GC content and reduced immunogenicity compared to an AAV particle comprising a GBA1 -encoding nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1773.

[0256] In some embodiments, the viral genome of an AAV particle described herein comprises a signal sequence-encoding sequence and a GBA1 -encoding sequence. In some embodiments, the viral genome comprises the nucleotide sequence of SEQ ID NO: 2001 or a nucleotide sequence that is at least 94% (e.g.. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99%) identical thereto. In some embodiments, an AAV particle disclosed herein may comprise improved (e.g., reduced) GC content and reduced immunogenicity compared to an AAV particle comprising the nucleotide sequence of SEQ ID NO: 1772.

[0257] In some embodiments, the viral genome of an AAV particle described herein comprises a nucleotide sequence comprising SEQ ID NO: 2006 or a sequence that is at least 97% (e.g., at least 97%, at least 98%. or at least 99%) identical thereto. In some embodiments, said AAV particle comprises improved (e.g., reduced) GC content and reduced immunogenicity compared to an AAV particle comprising a nucleotide sequence comprising SEQ ID NO: 1812.

Table 10. Sequence Regions in ITR-to-ITR Sequences

Table 11. Sequence Regions in ITR-to-ITR Sequences

Table 12. Sequence Regions in ITR-to-ITR Sequences

[0258] In some embodiments, the AAV particle comprises a nucleotide sequence encoding a wildtype GBA1 protein, wherein the nucleotide sequence comprises the sequence of SEQ ID NO: 2002 or a 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. In some embodiments, the GBAl-encoding sequence comprises SEQ ID NO: 2002.

[0259] In some embodiments, the AAV particle comprising the GBAl-encoding sequence of SEQ ID NO: 2002 further comprises at least one 1TR sequence that is 130 nucleotides in length, optionally comprising two ITR sequences that are each 130 nucleotides in length. In some embodiments, the at least one ITR sequence comprises the nucleotide sequence of SEQ ID NO: 1829 or SEQ ID NO: 1830 or a 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. In some embodiments, the AAV particle comprises a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 1829 or a 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 a 3’ITR comprising the nucleotide sequence of SEQ ID NO: 1830 or a 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. In some embodiments, the 5 ‘ ITR sequence comprises SEQ ID NO: 1829. In some embodiments, the 3‘ ITR sequence comprises SEQ ID NO: 1830. In some embodiments, the 5’ ITR sequence comprises SEQ ID NO: 1829 and the 3’ ITR sequence comprises SEQ ID NO: 1830.

[0260] In some embodiments, the AAV particle comprising the GBA1 -encoding sequence of SEQ ID NO: 2002 further comprises a CMVie sequence and/or CBA promoter operably linked to the GBA1- encoding sequence. In some embodiments, the CMVie sequence comprises the nucleotide sequence of SEQ ID NO: 1831 or a 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 the CBA promoter comprises the nucleotide sequence of SEQ ID NO: 1834 or a 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. In some embodiments, the CMVie sequence comprises the nucleotide sequence of SEQ ID NO: 1831 and the CBA promoter comprises the nucleotide sequence of SEQ ID NO: 1834.

[0261] In some embodiments, the AAV particle comprising the GBAl-encoding sequence of SEQ ID NO: 2002 further comprises a sequence encoding a signal peptide, wherein the sequence encoding the signal peptide comprises the nucleotide sequence of 2005 or a 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, wherein the sequence encoding the signal peptide is 5’ to the GBAl-encoding sequence. In some embodiments, the sequence encoding the signal peptide comprises the nucleotide sequence of 2005. In some embodiments, the AAV particle comprising the GBA1 -encoding sequence of SEQ ID NO: 2002 further comprises an intron region comprising the nucleotide sequence of SEQ ID NO: 1842 or a 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%) thereto. In some embodiments, the intron region comprises the nucleotide sequence of SEQ ID NO: 1842.

[0262] In some embodiments, the AAV particle comprising the GBAl-encoding sequence of SEQ ID NO: 2002 further comprises a polyA sequence comprising the nucleotide sequence of SEQ ID NO: 1846 or a 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. In some embodiments, the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 1846.

[0263] In some embodiments, the AAV particle comprising the GBAl-encoding sequence of SEQ ID NO: 2002 further comprises, from 5’ to 3’, one or more of. e.g., all of, an ITR comprising the nucleotide sequence of SEQ ID NO: 1829 or a 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; a CMVie sequence comprising the nucleotide sequence of SEQ ID NO: 1831 or a 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; a CBA promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a 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; an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a 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; a signal sequence-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2005 or a 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; a poly A sequence comprising the nucleotide sequence of SEQ ID NO: 1846; and/or an ITR comprising the nucleotide sequence of SEQ ID NO: 1830 or a 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, wherein the GBAl-encoding sequence is 3’ to the signal sequence-encoding sequence and 5’ to the polyA sequence.

[0264] In some embodiments, the AAV particle comprises a viral genome comprising a GBAl- encoding sequence comprising SEQ ID NO: 2002 or a 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. In some embodiments, the AAV particle comprises a viral genome comprising the nucleotide sequence of SEQ ID NO: 2006 (GBA VG35) or a nucleotide 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. In some embodiments, die viral genome comprising the nucleotide sequence of SEQ ID NO: 2006 comprises in 5’ to 3’ order: a 5’ ITR sequence region comprising the nucleotide sequence of SEQ ID NO: 1829, or a nucleotide sequence 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; a CMVie enhancer comprising the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence 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: a CBA promoter comprising the nucleotide sequence of SEQ ID NO: 1834, or a nucleotide sequence 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; an intron comprising the nucleotide sequence of SEQ ID NO: 1842, or a nucleotide sequence 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; a nucleotide sequence encoding a signal peptide comprising the nucleotide sequence of SEQ ID NO: 2005, or a nucleotide sequence 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; a nucleotide sequence encoding a GBA1 protein comprising the nucleotide sequence of SEQ ID NO: 2002 or a nucleotide sequence 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; a polyadenylation (poly A) sequence comprising the nucleotide sequence of SEQ ID NO: 1846. or a nucleotide sequence 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 a 3’ ITR sequence region comprising the nucleotide sequence of SEQ ID NO: 1830. or a nucleotide sequence 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. [0265] In some embodiments, the AAV viral genome does not encode a miR183 binding site.

[0266] In some embodiments, the viral genome comprising the nucleotide sequence of SEQ ID NO:

2006, or a nucleotide 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, encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1775 or an amino acid sequence 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. In some embodiments, the AAV particle comprises a viral genome comprising the nucleotide sequence of SEQ ID NO: 2006.

Table 13. Sequence Regions in ITR-to-ITR Sequences

|0267| In some embodiments, the AAV particle comprises a viral genome comprising a nucleotide sequence encoding a wildtype GBA1 protein, wherein the nucleotide sequence comprises the sequence of SEQ ID NO: 2002 or a 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.

[0268] In some embodiments, the viral genome further comprises a nucleotide sequence encoding at least one miR183 binding site. In some embodiments, the viral genome further comprises a nucleotide sequence encoding four miR183 binding sites, wherein each miR183 binding site is encoded by a nucleotide sequence that comprises the sequence of SEQ ID NO: 1847 or a nucleotide sequence that has up to three modifications relative to SEQ ID NO: 1847.

[0269] In some embodiments, the nucleotide sequence encoding at least one miR183 binding site further encodes at least one spacer sequence between two miR183 binding sites, wherein each spacer sequence is encoded by a nucleotide sequence comprising GATAGTTA or a nucleotide sequence that has up to three modifications relative to GATAGTTA.

[0270] In some embodiments, the viral genome comprises a nucleotide sequence encoding a miR binding site series, wherein the nucleotide sequence encoding the miR binding site series comprises the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide sequence 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. In some embodiments, the nucleotide sequence encoding tire miR binding site series comprises the nucleotide sequence of SEQ ID NO: 1849.

[0271] In some embodiments, the viral genome comprises, from 5’ to 3’, one or more of, e.g., all of, an ITR comprising the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide sequence 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; a CMVie sequence comprising the nucleotide sequence of SEQ ID NO: 1831 or a sequence 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; a CBA promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a sequence 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: an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a sequence 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: a signal sequence-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2005 or a sequence 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; a GBA1 -encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2002 or a sequence that is at least 93% identical (e.g., 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 nucleotide sequence encoding a miR183 binding site series, wherein the nucleotide sequence encoding the miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 1849 or a sequence 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; a polyA sequence comprising the nucleotide sequence of SEQ ID NO: 1846 or a sequence 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 an ITR comprising the nucleotide sequence of SEQ ID NO: 1830 or a sequence 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.

[0272] In some embodiments, the AAV particle comprises a viral genome comprising a GBA1- encoding sequence comprising SEQ ID NO: 2002 or a sequence that is at least 93% (e.g., at least 93%, at least 94%, at least 95%. at least 96%, at least 97%. at least 98%, or at least 99%) identical thereto.

[0273] In some embodiments, the AAV particle comprises a viral genome comprising the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide 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. In some embodiments, the viral genome comprises a nucleotide sequence that is at least 97% identical (e.g., at least 97%. at least 98%, or at least 99% identical) to SEQ ID NO: 2007. In some embodiments, the AAV particle comprises a viral genome comprising the nucleotide sequence of SEQ ID NO: 2007. In some embodiments, the viral genome encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1775 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.

[0274] In some embodiments, the AAV particle comprises a viral genome 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.

[0275] 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 or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; and/or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

[0276] 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 1 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.

[0277] 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.

[0278] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 1773, or a sequence having 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, comprises a capsid comprising (i) a VP 1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; and/or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises T at amino acid 584, D at amino acid 586, W at amino acid 587, H at amino acid 588, R at amino acid 589, and/or I at amino acid 590, numbered according to SEQ ID NO: 981.

[0279] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 1773, or a sequence having 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, comprises a capsid comprising (i) a VP 1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; and/or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises 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.

[0280] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 2002, or a sequence having 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, comprises a capsid comprising (i) a VP 1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g.. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%. at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; and/or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises T at amino acid 584, D at amino acid 586, W at amino acid 587, H at amino acid 588, R at amino acid 589, and/or I at amino acid 590, numbered according to SEQ ID NO: 981.

[0281] hi some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 2002, or a sequence having 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, comprises a capsid comprising (i) a VP 1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g.. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%. at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; and/or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%. at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises 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.

[0282] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 1812 or SEQ ID NO: 1828, or a sequence having 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, comprises a capsid comprising (i) a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; and/or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g.. at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises T at amino acid 584, D at amino acid 586, W at amino acid 587. H at amino acid 588, R at amino acid 589. and/or I at amino acid 590. numbered according to SEQ ID NO: 981.

[0283] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 1812 or SEQ ID NO: 1828. or a sequence having 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, comprises a capsid comprising (i) a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; and/or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g.. at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises 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.

[0284] In some embodiments, the AAV particle comprises a viral genome comprising the nucleotide sequence of SEQ ID NO: 2006 or SEQ ID NO: 2007, or a sequence having 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.

[0285] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 2006 or SEQ ID NO: 2007, or a 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, comprises a capsid comprising an amino acid sequence selected from Table 3 or Table 4.

[0286] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 2006 or SEQ ID NO: 2007, or a sequence having 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, comprises a capsid comprising (i) a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g.. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; and/or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g.. at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises T at amino acid 584, D at amino acid 586, W at amino acid 587. H at amino acid 588, R at amino acid 589. and/or I at amino acid 590. numbered according to SEQ ID NO: 981. [0287] In some embodiments, the AAV particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 2006 or SEQ ID NO: 2007, or a sequence having 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, comprises a capsid comprising (i) a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; (ii) a VP2 protein comprising or consisting of the amino acid sequence according to positions 138-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g.. at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% identical) thereto; and/or (iii) a VP3 protein comprising or consisting of the amino acid sequence according to positions 203-736 of SEQ ID NO: 981 or an amino acid sequence that is at least 95% identical (e.g.. at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% identical) thereto, wherein the capsid in (i), (ii), or (iii) comprises 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.

[0288] 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

[0289] In some embodiments, the AAV viral genome used in the present disclosure is singlestranded.

[0290] 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.

[0291] 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).

IL AAV Production

[0292] 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 GBA1.

[0293] 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 GBA1 -encoding sequence 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 viral genome comprises: (a) the nucleotide sequence of SEQ ID NO: 2006 or a nucleotide sequence 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; or (b) the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide sequence 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 wherein the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 981. 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.

[0294] 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., Sf? cells).

[0295] 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 ct al., Baculovirus Expression Vectors, A Laboratory' Manual, Oxford Univ. Press (1994); Samulski et al., J. F7r.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 W02015191508, the contents of which are herein incorporated by reference in their entirety.

III. Pharmaceutical Compositions

[0296] In some embodiments, the present disclosure provides pharmaceutical compositions of an adeno-associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 2006 and an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 981. In some embodiments, the present disclosure provides pharmaceutical compositions of an adeno- associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 2007 and an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 981.

[0297] In some embodiments, a composition described herein comprises an AAV polynucleotide or AAV genome or AAV particle, and at least one excipient.

[0298] Although pharmaceutical compositions provided herein, e.g., comprising AAV particles comprising a GBA1 -encoding sequence, are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions may be suitable for administration to any other animal, e.g., non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various non-human 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.

[0299] In some embodiments, compositions are administered to humans, e.g., human patients or human subjects.

[0300] In some embodiments, the AAV particle formulations described herein may contain a nucleic acid encoding at least one GBA1 -encoding sequence. In some embodiments, the formulations may⁷ contain a nucleic acid encoding 1, 2, 3, 4, or 5 GBA1 -encoding sequences. In some embodiments, the formulation may contain GBAl-encoding sequences encoding GBA1 proteins selected from categories such as, but not limited to, human proteins, veterinary proteins, bacterial proteins, biological proteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secreted proteins, plasma membrane proteins, cytoplasmic proteins, cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, and/or proteins associated with non-human diseases. In some embodiments, the AAV formulation comprises at least one sequence encoding human GBA1. In some embodiments, the AAV formulation comprises at least one sequence encoding wildty pe human GBA1.

[0301] 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

[0302] Formulations of the AAV pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology⁷. In general, such preparatory⁷ 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.

[0303] Relative amounts of the active ingredient, tire pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical 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.

[0304] 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.

[0305] The AAV particles of the disclosure can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; (6) alter the release profile of encoded protein in vivo and/or (7) allow for regulatable expression of GBA1.

[0306] 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.

[0307] In some embodiments, the viral vectors encoding GBA1 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

[0308] 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. [0309] 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

[0310] In some embodiments. AAV formulations may comprise at least one excipient which is an inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more agents that do not contribute to the activity of the pharmaceutical composition included in formulations. In some embodiments, all, none, or some of the inactive ingredients which may be used in the formulations of the present disclosure may be approved by the US Food and Drug Administration (FDA).

[0311] 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

[0312] The compositions of the disclosure may be administered to a subject or used in the manufacture of a medicament for administration to a subject having a GBAl-related disorder. In some embodiments, the present disclosure provides a method for treating Parkinson’s Disease (PD) and related disorders arising from expression of defective GBA1 gene product, e.g., a PD associated with a GBA1 mutation. Such a disease or condition may be a neuromuscular or a neurological disorder or condition. In some embodiments, a disease associated with decreased GBA1 protein levels may be Parkinson’s Disease (PD) (e.g.. a PD associated with a GBA1 mutation), Parkinson’s Disease Dementia (PDD). Gaucher Disease (GD) (e.g., GD type 1 (GDI), GD type 2 (GD2). or GD type 3 (GD3)), Dementia with Lewy Bodies (DLB). Lewy Body Dementia (LBD), Spinal muscular atrophy (SMA), Multiple System Atrophy (MSA), or Multiple sclerosis (MS).

[0313] The present disclosure addresses the need for new technologies by providing GBA1 protein- related treatment deliverable by AAV-based compositions and complexes for the treatment of GBAl- related disorders.

[0314] In some embodiments, the disclosure provides an AAV particle or pharmaceutical composition according to any one of the embodiments disclosed herein for treating a GBA1 -related disorder, such as PD, GD, PDD, DLB, or LBD. 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 PD, GD, PDD, DLB, or LBD.

[0315] hi some embodiments, tire disclosure provides a method for treating Parkinson’s Disease (PD), e.g.. a PD with one or more mutations in a GBA1 gene. In certain embodiments, the AAV particles including GBA1 protein may be administered to a subject to treat Parkinson’s Disease, e.g., as PD associated with one or more mutations in a GBA1 gene.

[0316] In some embodiments, the disclosure provides a method for treating Gaucher Disease (GD) (e.g., GDI, GD2, or GD3). In some embodiments, the GD is GDI. In some embodiments, the GD is GD3.

[0317] In some embodiments, the disclosure provides a method for treating Lewy Body Dementia (LBD).

[0318] In some embodiments, administration of the AAV particles comprising viral genomes that encode GBA1 protein may protect central nervous system pathways from degeneration. The compositions and methods described herein are also useful for treating Gaucher disease (such as Type 1 or 3 GD), PDD, Lewy Body Dementia (LBD). or other GBA1 -related disorders.

[0319] In some embodiments, the delivery of the AAV particles may halt or slow progression of GBA1 -related disorders as measured by cholesterol accumulation in CNS cells (as determined, for example, by filipin staining and quantification). In certain embodiments, the delivery of the AAV particles improves symptoms of GBA1 -related disorders, including, for example, cognitive, muscular, physical, and sensory symptoms of GBA1 -related disorders.

[0320] In some embodiments, the present disclosure encompasses the delivery' of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, and/or modify their distribution within the body. [0321] In certain embodiments, the pharmaceutical compositions described herein are used as research tools, particularly in in vitro investigations using human cell lines such as HEK293T and in vivo testing in nonhuman primates which will occur prior to human clinical trials.

CNS diseases

[0322] The present disclosure provides a method for treating a disease, disorder and/or condition in a mammalian subject, including a human subject, comprising administering to the subject any of the viral particles e.g., AAV, AAV particle, or AAV viral genome that produces GBA1 protein described herein or administering to the subject a particle comprising said AAV particle or AAV genome, or administering to the subject any of the described compositions, including pharmaceutical compositions.

[0323] In some embodiments, AAV particles of the present disclosure, through delivery of a functional payload that is a therapeutic product comprising a GBA1 protein or variant thereof that can modulate the level or function of a gene product in the CNS. [0324] A functional payload may alleviate or reduce symptoms that result from abnormal level and/or function of a gene product (e.g., an absence or defect in a protein) in a subject in need thereof or that otherwise confers a benefit to a CNS disorder in a subject in need thereof.

[0325] As non-limiting examples, companion or combination therapeutic products delivered by AAV particles of the present disclosure may include, but are not limited to, growth and trophic factors. cytokines, hormones, neurotransmitters, enzymes, anti-apoptotic factors, angiogenic factors, GBA1 proteins, and any protein known to be mutated in pathological disorders such as GBA1 -related disorders. [0326] In some embodiments, AAV particles of the present disclosure may be used to treat diseases that are associated with impairments of the growth and development of the CNS, e.g., neurodevelopmental disorders. In some embodiments, such neurodevelopmental disorders may be caused by genetic mutations.

[0327] In some embodiments, the neurological disorders may be functional neurological disorders with motor and/or sensory symptoms which have neurological origin in the CNS. As non-limiting examples, functional neurological disorders may be chronic pain, seizures, speech problems, involuntary movements, or sleep disturbances.

[0328] In some embodiments, the neurological or neuromuscular disease, disorder, and/or condition is GBA1 -related disorders. In some embodiments, the delivery of the AAV particles may halt or slow the disease progression of GBAl-related disorders by 10%, 20%. 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% using a known analysis method and comparator group for GBAl-related disorders. As a non-limiting example, the deliver}' of the AAV particles may halt or slow progression of GBAl-related disorders as measured by cholesterol accumulation in CNS cells (as determined, for example, by filipin staining and quantification).

[0329] In some embodiments, delivery' of an AAV particle described herein increases the amount of GBA1 protein in a tissue by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more than 100%. In some embodiments, delivery of an AAV particle described herein increases the amount of GBA1 protein in a tissue to be comparable to the amount of GBA1 protein in the corresponding tissue of a healthy individual. In some embodiments, delivery' of the AAV particle increases the amount of GBA1 protein in a tissue effective to reduce one or more symptoms of a disease associated with decreased GBA1 protein expression or a deficiency in the quantity' and/or function of GBA1 protein.

[0330] In some embodiments, the AAV particles and AAV vector genomes described herein, upon administration to subject or introduction to a target cell, increase GBA activity 2-3 fold over baseline GBA activity. In the case of subjects or target cells with deficient GBA activity, as in the case of subjects having a GBAl-related disorder or cells or tissues harboring at least one mutation in a GBA1 gene, the AAV particles and AAV vector genomes described herein restore GBA activity to normal levels, as defined by GBA activity levels in subjects, tissues, and cells not afflicted with a GBAl-related disorder or not harboring a GBA1 gene mutation. In some embodiments, the AAV particles and AAV vector genomes described herein effectively reduce a-synuclein levels in subjects having a GBAl-related disorder or cells or tissues harboring at least one mutation in a GBA1 gene. In some embodiments, the AAV particles and AAV vector genomes described herein effectively prevent a-synuclein mediated pathology.

Therapeutic applications

[0331] The present disclosure additionally provides methods for treating non-infectious diseases and/or disorders in a mammalian subject, including a human subject, comprising administering to the subject any one of the AAV particles or pharmaceutical compositions described herein. In some embodiments, non-infectious diseases and/or disorders treated according to the methods described herein include, but are not limited to. Parkinson’s Disease (PD) (e.g., PD associated with one or more mutations in a GBA1 gene); Parkinson’s Disease Dementia (PDD); Dementia with Lewy Bodies (DLB); Lewy Body Dementia (LBD); Gaucher Disease (GD); Multiple System Atrophy (MSA); decreased muscle mass; Spinal muscular atrophy (SMA); Alzheimer’s disease (AD); Amyotrophic lateral sclerosis (ALS); Huntington’s Disease (HD); Multiple sclerosis (MS); stroke; migraine; pain; neuropathies; psychiatric disorders including schizophrenia, bipolar disorder, and autism; ocular diseases; systemic diseases of the blood, heart, and bone; immune system and autoimmune diseases; and inflammatory diseases.

[0332] The present disclosure provides a method for administering to a subject in need thereof, including a human subject, a therapeutically effective amount of the AAV particles of the disclosure to slow, stop or reverse disease progression. As a non-limiting example, disease progression may be measured by tests or diagnostic tool(s) know n to those skilled in the art. As another non-limiting example, disease progression may be measured by change in the pathological features of the brain, CSF, or other tissues or fluids of the subject.

Gaucher Disease

[0333] Homozygous or compound heterozygous GBA1 mutations lead to Gaucher Disease (GD). See Sardi. S. Pablo, Jesse M. Cedarbaum. and Patrik Brundin. Movement Disorders 33.5 (2018): 684- 696, the contents of which are incorporated by reference in their entirety. Gaucher Disease is one of the most prevalent lysosomal storage disorders, with an estimated standardized birth incidence in the general population of 0.4 to 5.8 individuals per 100,000.

[0334] Gaucher Disease can present as GDI (Type 1 GD (GDI)), which is the most common type of Gaucher disease among Ashkenazi Jewish populations. In some embodiments, a Type 1 GD is a non- neuronopathic GD (e.g., does not affect the CNS, e g., impacts cells and tissues outside of the CNS. e.g., a peripheral cell or tissue, e.g., a heart tissue, a liver tissue, a spleen tissue, or a combination thereof). The carrier frequency among Ashkenazi Jewish populations is approximately 1 in 12 individuals. GD2 (Type 2 GD (GD2)) is characterized by acute neuronopathic GD (e.g., affects the CNS, e.g., cells and tissues of the brain, spinal cord, or both), and has an estimated incidence of 1 in 150,000 live births. GD2 is an early onset disease, typically presenting at about 1 year of age. Visceral involvement is extensive and severe, with numerous attributes of CNS disease, including oculomotor dysfunction, and bulbar palsy and generalized weakness, and progressive development delay. GD2 progresses to severe hypertonia, rigidity, opisthotonos, dysphagia, and seizures, typically resulting in death before age 2. GD3 (type 3 GD (GD3)) is characterized by sub-acute neuropathic GD and as an estimated incidence of 1 in 200,000 live births. GD3 typically presents with pronounced neurologic signs, including a characteristic mask-like face, strabismus, supranuclear gaze palsy, and poor upward gaze initiation. GD2 and GD3 are each further characterized as associated with progressive encephalopathy, with developmental delay, cognitive impairment, progressive dementia, ataxia, myoclonus, and various gaze palsies. GDI, on the other hand, can have variable etiology, with visceromegaly, marrow and skeletal and pulmonary pathology, bleeding diatheses, and developmental delay. GD is further associated with increased rates of hematologic malignancies.

[0335] Deficiency of glucocerebrosidase (GCase) is the underlying mechanism of GD. Low GCase activity leads to accumulation of glucocerebroside and other glycolipids within the lysosomes of macrophages. Accumulation can amount to about 20-fold to about 100-fold higher than in control cells or subjects without GCase deficiency. Pathologic lipid accumulation in macrophages accounts for < 2% of additional tissue mass observed in the liver and spleen of GD patients. Additional increase in organ weight and volume is attributed to an inflammatory and hyperplastic cellular response.

[0336] Current treatments of GD include administration of recombinant enzymes, imiglucerase, taliglucerase alfa, and velaglucerase alfa. However, these intravenous enzyme therapies do not cross the blood brain barrier (BBB), and are not suitable for treatment of GD with Parkinson’s disease or other neuronopathic forms of GD.

Parkinson ’s Disease

[0337] Heterozygous GBA mutations can lead to PD. Indeed, GBA1 mutations occur in 7-10% of total PD patients, making GBA1 mutations the most important genetic risk factor of PD. PD-GBA patients have reduced levels of lysosomal enzyme beta-glucocerebrosidase (GCase), which results in increased accumulations of glycosphingolipid glucosylceramide (GluCer), which in turn is correlated with exacerbated a-Synuclein aggregation and concomitant neurological symptoms. Gaucher disease and PD. as well as other disorders including Lewy body diseases such as LBD and PDD, and related diseases, in some cases, share common etiology in the GBA gene. See Sidransky, E. and Lopez, G. Lancet Neurol. 2012 November; 11(11): 986-998, the contents of which are incorporated by reference in their entirety. |0338| Parkinson’s Disease (PD) is a progressive disorder of the nervous system affecting especially the substantia nigra of the brain. PD develops as a result of the loss of dopamine producing brain cells. Typical early symptoms of PD include shaking or trembling of a limb, e.g., hands, arms, legs, feet, and face. Additional characteristic symptoms are stiffness of the limbs and torso, slow movement, or an inability to move, impaired balance and coordination, cognitional changes, and psychiatric conditions e.g.. depression and visual hallucinations. PD has both familial and idiopathic forms and it is suggestion to be involved with genetic and environmental causes. PD affects more than 4 million people worldwide. In the US, approximately 60, 000 cases are identified annually. Generally , PD begins at the age of 50 or older. An early -onset form of the condition begins at age younger than 50, and juvenile-onset PD begins before age of 20.

[0339] Death of dopamine producing brain cells related to PD has been associated with aggregation, deposition and dysfunction of alpha-synuclein protein (see, e.g., Marques and Outeiro, 2012, Cell Death Dis. 3:e350, Jenner, 1989, J Neurol Neurosurg Psychiatry. Special Supplement, 22-28, and references therein). Studies have suggested that alpha-synuclein has a role in presynaptic signaling, membrane trafficking and regulation of dopamine release and transport. Alpha-synuclein aggregates, e.g.. in fonns of oligomers, have been suggested to be species responsible for neuronal dysfunction and death.

Mutations of the alpha-synuclein gene (SNCA) have been identified in the familial forms of PD, but also environmental factors, e.g., neurotoxin affect alpha-synuclein aggregation. Other suggested causes of brain cell death in PD are dysfunction of proteasomal and lysosomal systems, reduced mitochondrial activity.

[0340] PD is related to other diseases related to alpha-synuclein aggregation, referred to as “synucleinopathies.” Such diseases include, but are not limited to. Parkinson's Disease Dementia (PDD), multiple system atrophy (MSA), Lewy Body Dementia (LBD), juvenile-onset generalized neuroaxonal dystrophy (Hallervorden-Spatz disease), pure autonomic failure (PAF), neurodegeneration with brain iron accumulation type-1 (NBIA-1) and combined Alzheimer’s and Parkinson’s disease.

[0341] As of today, no erne or preventative therapy for PD has been identified. A variety of drug therapies available provide relief to the symptoms. Non-limiting examples of symptomatic medical treatments include carbidopa and levodopa combination reducing stiffness and slow movement, and anticholinergics to reduce trembling and stiffness. Other optional therapies include, e.g., deep brain stimulation and surgery. There remains a need for therapy affecting the underlying pathophysiology. For example, antibodies targeting alpha-synuclein protein, or other proteins relevant for brain cell death in PD, may be used to prevent and/or treat PD.

[0342] In some embodiments, methods of the present disclosure may be used to treat subjects suffering from PD (e.g., PD associated with one or more mutations in a GBA1 gene) and other synucleinopathies. In some cases, methods of the present disclosure may be used to treat subjects suspected of developing PD (e.g., PD associated with one or more mutations in a GBA1 gene) and other synucleinopathies.

[0343] AAV Particles and methods of using the AAV particles described herein may be used to prevent, manage and/or treat PD, e.g., a PD associated with one or more mutations in a GBA1 gene. [0344] Approximately 5% of PD patients carry at least one GBA mutation: 10% of patients with type 1 GD develop PD before the age of 80 years, compared to about 3-4% in the normal population.

Additionally, heterozygous, or homozygous GBA mutation(s) have been shown to increase the risk of PD 20-30 fold. Lewy Body Dementia (LBD)

[0345] Lew Body Dementia (LBD), also known as dementia with Lewy bodies (DLB) or diffuse Lewy body disease, is a form of progressive dementia, characterized by cognitive decline, fluctuating alertness and attention, visual hallucinations, and parkinsonian motor symptoms. LBD may be inherited by an autosomal dominant pattern. LBD affects more than 1 million individuals in the US. The condition typically shows symptoms at the age of 50 or older.

10346| LBD is caused by the abnormal build-up of Lewy bodies, aggregates of the alpha-synuclein protein, in the cytoplasm of neurons in the brain areas controlling memory and motor control. The pathophysiology of these aggregates is very similar to aggregates observed in Parkinson’s disease and LBD also has similarities to Alzheimer’s disease. Inherited LBD has been associated with gene mutations in GBAs.

[0347] As of today, there is no cure or prevention therapy for LBD. A variety of drug therapies available are aimed at managing the cognitive, psychiatric, and motor control symptoms of the condition. Non-limiting examples of symptomatic medical treatments include, e.g., acetylcholinesterase inhibitors to reduce cognitive symptoms, and levodopa to reduce stiffness and loss of movement. There remains a need for therapy affecting the underlying pathophysiology.

[0348] In some embodiments, methods of the present disclosure may be used to treat subjects suffering from LBD (e.g., LBD associated with one or more mutations in a GBA1 gene). In some cases, the methods may be used to treat subjects suspected of developing LBD (e.g., LBD associated with one or more mutations in a GBA1 gene).

[0349] AAV particles and methods of using die AAV particles described in the present disclosure may be used to prevent, manage and/or treat LBD (e.g., LBD associated with one or more mutations in a GBA1 gene).

VI. Delivery of AAV Particles

Delivery to Cells

[0350] In some aspects, the present disclosure provides a method of delivering to a cell or tissue any of the above-described AAV particles, comprising contacting the cell or tissue with said AAV particle or contacting the cell or tissue with a formulation comprising said AAV particle, or contacting the cell or tissue with any of the described compositions, including pharmaceutical compositions. The method of delivering the AAV particle to a cell or tissue can be accomplished in vitro, ex vivo, or in vivo.

[0351] In some embodiments, the AAV particles are delivered to a cell, tissue, or region of the CNS. In some embodiments, the AAV particles are delivered to a cell or tissue of the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and/or deep cerebellar nuclei), cortex (e.g.. frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex and/or visual cortex), hilus of the dentate gy rus, external cuncatc nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hippocampus, inferior colliculus, inferior olivary' complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and/or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and/or thoracic spinal cord region).

Delivery to Subjects

[0352] In some aspects, the present disclosure additionally provides a method of delivering to a subject, including a mammalian subject, any of the above-described AAV particles comprising administering to the subject said AAV particle, or administering to the subject a formulation comprising said AAV particle, or administering to the subject any of the described compositions, including pharmaceutical compositions.

[0353] In some embodiments, the AAV particles may be delivered to bypass anatomical blockages (e.g., the blood brain barrier).

[0354] In some embodiments, the AAV particles may be formulated and delivered to a subject by a route which increases the speed of drug effect as compared to oral delivery.

[0355] In some embodiments, the AAV particles may be delivered using intrathecal infusion.

[0356] In some embodiments, a subject may be administered the AAV particles described herein using a bolus infusion.

[0357] In some embodiments, the AAV particles may be delivered in a continuous and/or bolus infusion. Each site of delivery may use a different dosing regimen or the same dosing regimen may be used for each site of delivery'. As a non-limiting example, the sites of delivery may be in the cervical and the lumbar region. As another non-limiting example, the sites of delivery’ may be in the cervical region. As another non-limiting example, the sites of delivery may be in the lumbar region.

[0358] In some embodiments, the AAV particles may be delivered to a subject via a single route of administration.

[0359] In some embodiments, the AAV particles may be delivered to a subject via a multi-site route of administration. For example, a subject may be administered the AAV particles at 2, 3, 4, 5, or more than 5 sites.

[0360] In some embodiments, a subject may be administered the AAV particles described herein using sustained delivery over a period of minutes, hours, or days. The infusion rate may be changed depending on the subject, distribution, formulation, or another delivery parameter known to those in the art.

[0361] In some embodiments, if continuous delivery (continuous infusion) of the AAV particles is used, the continuous infusion may be for 1 horn, 2, hours, 3 hours, 4 hours, 5 hours. 6 hours. 7 hours. 8 hours, 9 hours, 10 horns. 11 hours, 12 hours, 13 hours. 14 hours. 15 hours, 16 hours, 17 horns. 18 horns, 19 horns, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or more than 24 hours.

[0362] In some embodiments, the intracranial pressure may be evaluated prior to administration. The route, volume, AAV particle concentration, infusion duration and/or vector titer may be optimized based on the intracranial pressure of a subject. [0363] In some embodiments, the AAV particles may be delivered by systemic delivery. In some embodiments, the systemic delivery' may be by intravascular administration. In some embodiments, the systemic delivery may be by intravenous administration.

[0364] In some embodiments, the AAV particles may be delivered by injection into the CSF pathway. Non-limiting examples of delivery to the CSF pathway include intrathecal and intracerebroventricular administration.

[0365] In some embodiments, an AAV particle described herein is administered intravenously.

[0366] In some embodiments, the AAV particles may be delivered by direct (intraparenchymal) injection into the substance of an organ, e.g, one or more regions of the brain.

[0367] In some embodiments, the AAV particles may be delivered by subpial injection into the spinal cord. For example, subjects may be placed into a spinal immobilization apparatus. A dorsal laminectomy may be performed to expose the spinal cord. Guiding tubes and XYZ manipulators may be used to assist catheter placement. Subpial catheters may be placed into the subpial space by advancing the catheter from the guiding tube and AAV particles may be injected through the catheter (Miyanohara et al., Mol Ther Methods Clin Dev. 2016; 3: 16046). In some cases, the AAV particles may be injected into the cervical subpial space. In some cases, the AAV particles may be injected into the thoracic subpial space.

[0368] In some embodiments, the AAV particles may be delivered by direct injection to the CNS of a subject. In some embodiments, direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intra-cistema magna injection, or any combination thereof. In some embodiments, direct injection to the CNS of a subject comprises convection enhanced delivery (CED). In some embodiments, administration comprises peripheral injection. In some embodiments, peripheral injection is intravenous injection.

[0369] In some embodiments, the AAV particles may be delivered to a subject in order to increase a GBA1 protein level in the CNS (c.g., the amy gdala, brainstem, caudate, central grey, cerebellum (c.g., Purkinje cell layer and/or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory' cortex, temporal cortex, and/or visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and/or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and/or thoracic spinal cord region)) as compared to a baseline level in the subject.

[0370] In some embodiments, the AAV particles may be delivered to a subject in order to increase a GBA1 protein level in the CNS (e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and/or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex, and/or visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus. gracile nucleus, hilus of the dentate gyrus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen. substantia nigra, thalamus. ventral palladium, vestibular nucleus, and/or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and/or thoracic spinal cord region)) by transducing cells in these CNS regions. Transduction may also be referred to as the number of cells that are positive for GBA1 protein.

[0371] In some embodiments, delivery of AAV particles comprising a viral genome encoding GBA1 as described herein to neurons in the brain (e.g., the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and/or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex, and/or visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus. gracile nucleus, hilus of the dentate gyrus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus, oculomotor nucleus, putamen. substantia nigra, thalamus, ventral palladium, and/or vestibular nucleus) may lead to an increased expression of GBA1 protein in one or more of those neurons. In some embodiments, the increased GBA1 protein expression may lead to improved survival and/or function of various cell types in these CNS regions and/or improvement of at least one symptom of a GBAl-related disorder (e.g.. PD or GD).

[0372] In some embodiments, the AAV particles may be delivered to a subject in order to establish widespread distribution of GBA1 throughout the CNS, e.g.. by administering the AAV particles to the thalamus of the subject.

[0373] In some embodiments, the increased expression of GBA1 protein may lead to improved gait, improved ataxia-associated heart conditions, decreased feeling of exhaustion, improved symptoms relating to metabolic disorders such as diabetes, and/or improved quality of life.

Administration

[0374] In some embodiments, the present disclosure provides methods comprising administering a viral vector comprising a payload construct in accordance with the disclosure to a subject in need thereof. Viral vector pharmaceutical, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any amount and any route of administration effective for treating, or diagnosing a disease, disorder, and/or condition associated with decreased GBA1 expression or GBA1 deficiency. In some embodiments, the disease, disorder, and/or condition is a GBAl-related disorder (e.g., PD or GD).

[0375] Compositions in accordance with the disclosure may be formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophy tactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific protein employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. [0376] In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic composition administered in one dose/at one time/single route/single point of contact, e.g. , single administration event. In some embodiments, a single unit dose is provided as a discrete dosage form (e.g.. a tablet, capsule, patch, loaded syringe, vial, etc ). As used herein, a “total daily dose” is an amount given or prescribed in 24-hour period. It may be administered as a single unit dose. The viral particles may be formulated in buffer only or in a formulation described herein.

[0377] In some embodiments, a pharmaceutical composition described herein can be formulated into a topical, intranasal, pulmonary’, intratracheal, or injectable dosage form. In some embodiments, a pharmaceutical composition described herein can be formulated in a dosage form suitable for intravenous, intraocular, intravitreal. intramuscular, intracardiac, intraperitoneal, and/or subcutaneous administration.

[0378] In some embodiments, an AAV particle described herein is administered via intravenous administration. In some embodiments, an AAV particle described herein is formulated for intravenous administration.

[0379] In some embodiments, delivery of the AAV particles described herein results in minimal serious adverse events (SAEs) as a result of the delivery of the AAV particles.

Combinations

[0380] The AAV particles may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. The phrase “in combination with.” is not intended to require that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, and/or modify their distribution within the body.

[0381] The therapeutic agents may be approved by the US Food and Drug Administration or may be in clinical trial or at the preclinical research stage. The therapeutic agents may utilize any therapeutic modality known in the art, with non-limiting examples including gene silencing or interference (e.g., miRNA. siRNA, RNAi, shRNA), gene editing (e.g., TALEN. CRISPR/Cas9 systems, zinc finger nucleases), and gene, protein, or enzy me replacement. [0382] In some embodiments, an AAV particle described herein, or a pharmaceutical composition comprising the AAV particle, may be administered in combination with at least one additional therapeutic agent and/or therapy.

[0383] In some embodiments, the at least one additional therapeutic agent and/or therapy comprises an agent and/or therapy for treating the GBAl-related disorder. In some embodiments, the at least one additional therapeutic agent and/or therapy for treating the GBAl-related disorder comprises enzyme replacement therapy (ERT) (e.g., imiglucerase, velaglucerase alfa, or taliglucerase alfa); substrate reduction therapy (SRT) (e.g., eliglustat or miglustat), levodopa, carbidopa. Safinamide. a dopamine agonist (e.g., pramipexole, rotigotine. or ropinirole). a dopamine antagonist (e.g., quetiapine, clozapine), an anticholinergic (e.g., benztropine or trihexyphenidyl), a cholinesterase inhibitor (e.g.. rivastigmine, donepezil. or galantamine), an N-methyl-d-aspartate (NMD A) receptor antagonist (e.g., memantine), or a combination thereof.

[0384] In some embodiments, the at least one additional therapeutic agent and/or therapy comprises an immunosuppressant. In some embodiments, the immunosuppressant may be administered to the subject prior to administration of an AAV particle or pharmaceutical composition described herein. In some embodiments, the immunosuppressant may be administered to the subject simultaneously with administration of an AAV particle or pharmaceutical composition described herein. In some embodiments, the immunosuppressant may be administered to the subject after administration of an AAV particle or pharmaceutical composition described herein. In some embodiments, the AAV particle or pharmaceutical composition is administered to a subject who is receiving or has received an immunosuppressant. In some embodiments, the immunosuppressant comprises a corticosteroid (for example, and without limitation, prednisone, prednisolone, methylprednisolone, and/or dexamethasone), rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and/or eculizumab hydroxy chloroquine. In some embodiments, the corticosteroid comprises prednisone, prednisolone, methy lprednisolone, and/or dexamethasone.

Measurement of Expression

[0385] Expression of GBA1 from viral genomes may be determined using various methods known in the art such as, but not limited to immunochemistry (e.g., IHC), enzyme-linked immunosorbent assay (ELISA), affinity ELISA, ELISPOT, flow cytometry', immunocytology, surface plasmon resonance analysis, kinetic exclusion assay, liquid chromatography -mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), BCA assay, immunoelectrophoresis. Western blot, SDS-PAGE. protein immunoprecipitation, PCR, and/or in situ hybridization (ISH). In some embodiments, transgenes encoding GBA1 protein delivered in different AAV capsids may have different expression levels in dorsal root ganglion (DRG).

[0386] In certain embodiments, the GBA1 protein is detectable by an enzyme-linked immunosorbent assay (ELISA).

[0387] In certain embodiments, the GBA1 protein is detectable by an immunohistochemistry assay. [0388] In certain embodiments, the GBA1 protein is detectable by Western blot.

[0389] In certain embodiments, expression of a GBA1 gene, mRNA, and/or protein is measured in a cell or tissue of a subject who is receiving or has received an AAV particle described herein. In certain embodiments, the GBA1 gene, mRNA, and/or protein expression is measured in a cell or tissue of the CNS comprising the amygdala, brainstem, caudate, central grey, cerebellum (e.g., Purkinje cell layer and/or deep cerebellar nuclei), cortex (e.g., frontal cortex, motor cortex, perirhinal cortex, sensory cortex, temporal cortex, and/or visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, hippocampus, inferior colliculus, inferior olivary complex, nucleus ambiguus. oculomotor nucleus, putamen, substantia nigra, thalamus, ventral palladium, vestibular nucleus, and/or spinal cord (e.g., cervical spinal cord region, lumbar spinal cord region, and/or thoracic spinal cord region). In certain embodiments, the GBA1 gene, mRNA, and/or protein expression is measured in a peripheral cell or tissue, such as the heart, kidney, muscle (e.g.. quadriceps muscle), pancreas, and/or liver.

VII. Kits and Devices

Kits

[0390] In some aspects, the present disclosure provides a variety of kits for conveniently and/or effectively carrying out methods of die present disclosure. Typically, kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.

[0391] Any of the vectors, constructs, or GBA1 sequences of the present disclosure may be comprised in a kit. In some embodiments, kits may further include reagents and/or instructions for creating and/or synthesizing compounds and/or compositions of the present disclosure. In some embodiments, kits may also include one or more buffers. In some embodiments, kits of the disclosure may include components for making protein or nucleic acid arrays or libraries and thus, may include, for example, solid supports.

[0392] In some embodiments, kit components may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and suitably aliquoted. Where there is more than one kit component, (labeling reagent and label may be packaged together), kits may also generally contain second, third or other additional containers into which additional components may be separately placed. In some embodiments, kits may also comprise second container means for containing sterile, pharmaceutically acceptable buffers and/or other diluents. In some embodiments, various combinations of components may be comprised in one or more vial. Kits of the present disclosure may also typically include means for containing compounds and/or compositions of the present disclosure, e.g., proteins, nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which desired vials are retained.

[0393] In some embodiments, kit components are provided in one and/or more liquid solutions. In some embodiments, liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly used. In some embodiments, kit components may be provided as dried powder(s). When reagents and/or components are provided as dry powders, such powders may be reconstituted by the addition of suitable volumes of solvent. In some embodiments, it is envisioned that solvents may also be provided in another container means. In some embodiments, labeling dyes are provided as dried powders. In some embodiments, it is contemplated that 10, 20, 30. 40, 50, 60, 70, 80. 90, 100. 120, 120, 130, 140, 150, 160, 170. 180, 190, 200. 300, 400, 500, 600, 700. 800, 900, 1000 micrograms or at least or at most those amounts of dried dye are provided in kits of the disclosure. In such embodiments, dye may then be resuspended in any suitable solvent, such as DMSO.

[0394] In some embodiments, kits may include instructions for employing kit components as well the use of any other reagent not included in the kit. Instructions may include variations that may be implemented.

Devices

[0395] In some embodiments, compounds and/or compositions of the present disclosure may be combined with, coated onto or embedded in a device. Devices may include, but are not limited to, dental implants, stents, bone replacements, artificial joints, valves, pacemakers and/or other implantable therapeutic device.

[0396] The present disclosure provides for devices which may incorporate viral vectors that encode one or more GBA1 molecules. These devices contain in a stable formulation the viral vectors which may be immediately delivered to a subject in need thereof, such as a human patient.

[0397] Devices for administration may be employed to deliver the viral vectors encoding GBA1 of the present disclosure according to single, multi- or split-dosing regimens taught herein.

[0398] Method and devices known in the art for multi-administration to cells, organs and tissues are contemplated for use in conjunction with the methods and compositions disclosed herein as embodiments of the present disclosure.

VIII. Definitions

[0399] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every' individual sub-combination of the members of such groups and ranges. The following is a non-limiting list of term definitions.

[0400] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. [0401] The articles “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherw ise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from die context. The disclosure includes embodiments in which exactly one member of die group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

[0402] The term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps.

[0403] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art. values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0404] Adeno-associated virus: As used herein, the term “adeno-associated virus” or “AAV” refers to members of the dependovirus genus or a functional variant thereof. Unless stated otherwise, “AAV” may refer to wildtype (i.e., naturally occurring) AAV or recombinant AAV.

[0405] AAV Particle: As used herein, an “AAV particle” refers to a particle comprising an AAV capsid, e.g., an AAV capsid variant (such as a parent capsid sequence with at least one peptide insertion and/or with at least one substitution), and a polynucleotide, e.g., a viral genome or a vector genome. The AAV particle may be capable of delivering a GBA1 polynucleotide to cells. The cells may be mammalian cells, e.g., human cells. In some embodiments, an AAV particle of the present disclosure may be produced recombinantly. In some embodiments, an AAV particle may be derived from any serotype, described herein or known in the art. including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary). In some embodiments, the AAV particle may be replication defective and/or targeted. In some embodiments, the AAV particle may comprise a peptide present in, e.g., inserted into and/or replacing a wildtype amino acid of, the capsid to enhance tropism for a desired target tissue. It is to be understood that reference to the AAV particle of the disclosure also includes pharmaceutical compositions thereof, even if not explicitly recited.

[0406] Administering: As used herein, the term “administering” refers to providing a pharmaceutical agent or composition to a subject.

[0407] Amelioration: As used herein, the term “amelioration” or “ameliorating” refers to a lessening of severity of at least one indicator of a condition or disease. For example, in the context of a neurodegenerative disorder, amelioration includes the reduction or stabilization of neuron loss.

[0408] Approximately: As used herein, the term “approximately” or "about.” as applied to one or more values of interest, refers to a value that is similar to, i.e., within 10% of. a stated reference value. [0409] Baseline: The term "baseline,” when used to describe a measurement in a subject receiving or about to receive a treatment, refers to a measurement made before starting the treatment. [0410] Capsid: As used herein, the term “capsid” refers to the exterior, e.g., a protein shell, of a virus particle, e.g., an AAV particle, that is substantially (e.g., >50%, >60%, >70%, >80%, >90%, >95%, >99%, or 100%) protein. In some embodiments, the capsid is an AAV capsid comprising an AAV capsid protein described herein, e.g., a VP1, VP2, and/or VP3 polypeptide. The AAV capsid protein can be a wild-type AAV capsid protein or a variant, e.g., a structural and/or functional variant from a wild-type or a reference capsid protein, referred to herein as an “AAV capsid variant.” For example, and without limitation, an AAV capsid variant may refer to at least a VP1 protein, a VP2 protein, or a VP3 protein (e.g., all of the VP1, VP2, and VP3 proteins forming the AAV capsid) as will be clear from context. In some embodiments, the AAV capsid variant described herein may comprise a peptide and/or amino acid insertion and/or substitution. The terms “substitution” and “replacement” are used interchangeably in this context. In some embodiments, the AAV capsid variant described herein has the ability to encapsulate a viral genome and/or is capable of entry into a cell, e.g., a mammalian cell. In some embodiments, the AAV capsid variant described herein may have modified tropism compared to that of a wild-type AAV capsid, e.g., the corresponding wild-type capsid.

[0411] CNS Cells: As used herein, “CNS cells” refers to cells of the central nervous system and substructures thereof. Non-limiting examples of CNS cells include neurons and sub-types thereof, glia, microglia, oligodendrocytes, ependymal cells, and astrocytes. Non-limiting examples of neurons include sensory neurons, motor neurons, interneurons, unipolar cells, bipolar cells, multipolar cells, pseudounipolar cells, pyramidal cells, basket cells, stellate cells, Purkinje cells, Betz cells, amacrine cells, granule cell, ovoid cell, medium aspiny neurons and large aspiny neurons, GABAergic neurons and/or glutamatergic neurons.

[0412] Codon optimization: As used herein, the term “codon optimization” refers to a process of changing codons of a given gene in such a manner that the polypeptide sequence encoded by the gene remains the same.

[0413] Complementary and substantially complementary: As used herein, the tcnn “complementary” refers to the ability of polynucleotides to form base pairs with one another. Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can form a hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can form hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can form a hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can form hydrogen bonds with each other, the polynucleotide strands exhibit 90% complementarity. The term “complementary” as used herein can encompass fully complementary or partially (e.g., substantially) complementary. “Fully complementary”, “perfect complementarity”, or “100% complementarity” refers to the situation in which each nucleotide unit of one polynucleotide or oligonucleotide strand can base-pair with a nucleotide unit of a second polynucleotide or oligonucleotide strand. As used herein, the term “substantially complementary” means that >50% of the nucleotide units of a first polynucleotide strand can base pair with nucleotide units on a second polynucleotide strand. When used in the context of RNA silencing, “substantially complementary’' refers to an siRNA that has a sequence (e.g., in the antisense strand) that is sufficient to bind the desired target mRNA and to trigger the RNA silencing of the target mRNA.

[0414] Conservative substitution'. As used herein, a conservative substitution, as applied to an amino acid sequence, also referred to as a “conservative amino acid substitution.” is one in which the amino acid residue is replaced with an amino acid residue having similar biochemical properties. When used in reference to a nucleic acid sequence, the tenn “conservative substitution” refers to a nucleotide replacement that results in an amino acid residue having similar biochemical properties compared to a reference sequence. Families of amino acid residues having similar biochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g.. lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.. tyrosine, phenylalanine, tryptophan, histidine).

[0415] Corresponding to: As used herein, the phrase “corresponding to” in the context of an amino acid sequence refers to the location of an amino acid in a reference sequence or the equivalent position in a modified sequence when aligned. For example, an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138 refers to the amino acid at the 585th position from the N- terminus in the amino acid sequence of SEQ ID NO: 138 or the equivalent position in an aligned sequence. As used herein, an amino acid at a position corresponding to that in a designated sequence may also be referred to as an amino acid at a particular position, numbered according or numbered relative to die designated sequence. For instance, an amino acid corresponding to position 585 of the amino acid sequence of SEQ ID NO: 138 may also be referred to as an amino acid at position 585 (or amino acid 585), numbered according to the amino acid sequence of SEQ ID NO: 138 or as numbered according to a sequence corresponding to the amino acid sequence of SEQ ID NO: 138 (i.e., relative to a reference sequence of the amino acid sequence of SEQ ID NO: 138).

[0416] Derivative: As used herein, the tenn “derivative” refers to a composition (e.g., sequence, compound, formulation, etc.) that is derived from, or finds its basis in, a parent composition. Nonlimiting examples of a parent composition include a wild-type or original amino acid or nucleic acid sequence, or an undiluted fonnulation. In some embodiments, a derivative is a variant of a parent composition. A derivative may differ from the parent composition by less than about 1%, less than about 5%. less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%. less than about 45%. or less than about 50%. In certain embodiments, a derivative may differ from a parent composition by more than about 50%. In certain embodiments, a derivative may differ from a parent composition by more than about 75%. In some embodiments, a derivative may be a fragment or truncation of a parent amino acid or nucleotide sequence. As a non-limiting example, a derivative may be a sequence with a nucleotide, amino acid, or peptide substitution and/or insertion as compared to a parent nucleic acid or amino acid sequence (e.g., as compared to AAV9).

[0417] Effective amount: As used herein, the term “effective amount" or “therapeutically effective amount" of an agent is that amount sufficient to effect beneficial or desired results. An effective amount is provided in a single dose or multiple doses to treat, improve symptoms of, delay progression of symptoms, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition. [0418] Fragment: A “fragment," as used herein, refers to a contiguous portion of a reference sequence. A fragment may comprise a functional fragment that retains at least one activity of the reference sequence. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells. A fragment may also refer to a truncation (e.g.. an N- terminal and/or C-terminal truncation) of a protein or a truncation (e.g.. at the 5’ and/or 3’ end) of a nucleic acid. A protein fragment may be obtained by expression of a truncated nucleic acid, such that the nucleic acid encodes a portion of the full-length protein.

[0419] GBA GCase protein: As used herein, the terms “GCase”, “GCase protein,” “GBA protein”, and “GBA1 protein” are used interchangeably to refer to a protein product or a functional portion thereof of the GBA1 gene (Ensemble gene ID: ENSG00000177628).

[0420] GBAl-related disorder: As used herein, a “GBAl-related disorder” refers to a disease, disorder, or condition in which one or more symptoms is caused by or associated with a deficiency of GBA1 in a subject.

[0421] Healthy’ individual: As used herein, the term “healthy individual” refers to an individual who does not have a disease or disorder associated with GBA1 protein deficiency, e.g., an individual who does not have a GBAl-related disorder.

[0422] Identity’: As used herein, the term “identity " refers to the overall rclatcdncss between polymeric molecules, e.g., between oligonucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or betw een polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the tw o sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.. eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York. 1991; each of which is incorporated herein by reference in its entirety. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Myers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity betw een two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H.. and Lipman, D.. SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference in its entirety. Techniques for determining identity are codified in publicly available computer programs. Computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al.. Nucleic Acids Research. 12(1). 387 (1984)), the Basic Local Alignment Search Tool (BLAST, which includes, e.g., BLASTP for protein sequences and BLASTN for nucleic acid sequences), and FASTA Altschul, S. F. et al., J. Molecular Biol.. 215, 403 (1990)), EMBOSS Needle, Clustal Omega, Benchling, and Geneious. In preferred embodiments, sequence identity⁷ may be determined using BLAST. Clustal Omega, or EMBOSS Needle.

[0423] Inverted terminal repeat'. As used herein, the term “inverted terminal repeat” or “ITR” refers to a cis-regulatory element for the packaging of polynucleotide sequences into viral capsids.

[0424] Isolated. As used herein, the term “isolated” refers to a substance or entity that is altered or removed from the natural state, e.g., altered or removed from at least some of component w ith which it is associated in the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated.” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature. In some embodiments, an isolated nucleic acid is recombinant, e.g., incorporated into a vector.

[0425] miRNA binding site: As used herein, a “miRNA binding site” or “miR binding site” refers either to a DNA sequence corresponding to an RNA sequence that is bound by a microRNA. or to the RNA sequence that is bound by the microRNA. The miR binding site is capable of binding, or binds, in whole or in part to a microRNA (miRNA. miR) through complete or partial hybridization. A miR binding site may be encoded or transcribed in series, also referred to as a “miR binding site series” or “miR BSs”, which includes two or more miR binding sites having the same or a different nucleic acid sequence.

[0426] Modification-. As used herein, the term “modification” or “modified,” refers to any substance, compound, or molecule that has been changed in any way. For example, a modification in an amino acid sequence may comprise a substitution (e.g., a conservative substitution), an insertion, and/or a deletion of one or more amino acids in the sequence.

[0427] Neurological disease: As used herein, a “neurological disease” is any disease associated with the central or peripheral nervous system and components thereof (e.g., neurons).

[0428] Operably linked: As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.

[0429] Payload. As used herein, “pay load,” “payload sequence,” or "payload region” refers to one or more polynucleotides or polynucleotide regions encoded by or within a viral genome or an expression product of such polynucleotide or polynucleotide region, e.g., a transgene, a polynucleotide encoding a polypeptide.

[0430] Pharmaceutically acceptable'. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are suitable for use in contact with the tissues of human beings and animals.

[0431] Pharmaceutical Composition: As used herein, the term “pharmaceutical composition” or pharmaceutically acceptable composition” comprises AAV polynucleotides, AAV genomes, or AAV particle and one or more pharmaceutically acceptable excipients, solvents, adjuvants, and/or the like. [0432] Position-. The term “position,” as used herein in the context of an amino acid sequence, refers to the location of a particular amino acid or set of amino acids relative to a larger sequence. A position or positions of amino acids may interchangeably be referred to by an amino acid number or numbers of a reference sequence. For example, and unless otherwise specified, “positions 1-736, as numbered according to SEQ ID NO: 981” is interchangeable with “amino acids 1-736, as numbered according to SEQ ID NO: 981” and “one or more modifications at one or more of positions 580-599, as numbered according to SEQ ID NO: 138” is interchangeable with “one or more modifications at one or more of amino acids 580-599, as numbered according to SEQ ID NO: 138.” Within a sequence, an amino acid position is counted from the A'-lcnninus.

[0433] Preventing-. As used herein, the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition: partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition: and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition. The term “prevention” or “preventing" of an infection, disease, disorder and/or condition may be considered a subset within the meaning with the term “treatment” or “treating” of the infection, disease, disorder and/or condition. [0434] Region: As used herein, the term “region” refers to a zone or general area. In some embodiments, when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three-dimensional area. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region" refers to regions located at the ends or termini of a given agent. When referring to proteins, terminal regions may comprise N- and/or C-termini. N-termini refer to the end of a protein comprising an amino acid with a free amino group. C-termini refer to the end of a protein comprising an amino acid with a free carboxyl group. N- and/or C-tenninal regions may comprise the N- and/or C-termini as well as surrounding amino acids. When referring to a polynucleotide, a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three-dimensional area, secondary structure, or tertiary structure. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to polynucleotides, terminal regions may comprise 5 ’ and/or 3 ’ termini.

[0435] Sample: As used herein, the term “sample” or “biological sample” refers to a subset of tissues, cells, nucleic acids, or a component or part of the body e.g., a body fluid, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen).

[0436] Self-complementary AA V; As used herein, the term “self-complementary AAV” refers to an AAV comprising at least a protein capsid and a self-complementary viral genome.

[0437] Serotype: As used herein, the term “serotype” refers to distinct variations in a capsid of an AAV based on surface antigens which allow epidemiologic classifications of the AAVs at the subspecies level.

[0438] Silent Mutation: As used herein, a “silent mutation” or “silent substitution” refers to a nucleotide replacement that results in the same amino acid residue as a reference sequence.

[0439] Signal Sequence: As used herein, the phrase “signal sequence” refers to a sequence which can direct the transport or localization.

[0440] Similarity; As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g.. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.

[0441] Spacer: As used herein, a “spacer” is generally any selected nucleic acid sequence of, e.g., 1, 2. 3, 4, 5, 6. 7. 8, 9, or 10 nucleotides in length, which is located between two or more consecutive miR binding site sequences. In some embodiments, spacers may also be more than 10 nucleotides in length, e.g., 20, 30, 40, or 50 or more than 50 nucleotides.

[0442] Subject: As used herein, the term “subject’’ or “patient” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Similarly, “subject” or “patient” refers to an organism who may seek, who may require, who is receiving, or who will receive treatment or who is under care by a trained professional for a particular disease or condition. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). As used herein, a subject or patient may be susceptible to, suspected of having, or have a deficiency in GBA1 protein, e.g.. human GBA1 protein, and, e.g.. may be susceptible to, suspected of having, or have a GBAl-related disorder. [0443] Substantially. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0444] Suffering from-. An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition. [0445] Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition but harbors a propensity to develop a disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifesty les associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

[0446] Target Cells: As used herein, “target cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a human patient. [0447] Target Tissue: As used herein, “target tissue” refers to a tissue of interest that may be found in vitro, in situ, or as part of an animal, preferably a mammal, more preferably a human and most preferably a human patient.

[0448] Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject, elicits a desired biological and/or pharmacological effect.

[0449] Therapeutically Effective Outcome'. As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, delay progression of symptoms, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

[0450] Treating'. As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of. reducing severity of. reducing incidence of, and/or preventing one or more symptoms or features of a particular infection, disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

[0451] Unmodified'. As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may. but does not always, refer to the wild-type or native form of a biomolecule or entity. Molecules or entities may undergo a series of modifications whereby each modified product may serve as the “unmodified” starting molecule or entity for a subsequent modification.

[0452] Variant: The term “variant” refers to a polypeptide or polynucleotide that has an amino acid or a nucleotide sequence that has 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 a reference sequence. The variant may be a functional variant. As used herein, the term “functional variant” refers to a polypeptide variant or a polynucleotide variant that has at least one activity of the reference sequence.

[0453] Vector. As used herein, a “vector” is any molecule or moiety' which transports, transduces, or otherwise acts as a carrier of a heterologous molecule. Vectors of the present disclosure may be produced recombinantly and may be based on and/or may comprise adeno-associated virus (AAV) parent or reference sequence(s).

[0454] Viral genome: As used herein, a “viral genome.” “vector genome,” or “VG” is a polynucleotide comprising at least one inverted terminal repeat (ITR) and at least one nucleic acid sequence encoding a pay load. A viral genome encodes at least one copy of the pay load. EXAMPLES

[0455] The present disclosure is further illustrated by the following non-limiting examples.

Example 1. High-throughput screen of TRACER AAV library in non-human primates (NHPs) [0456] A TRACER based method as described in W02020072683. WO 2021/202651, and

WO2021230987, the contents of which are herein incorporated by reference in their entirety, was used to generate the AAV capsid variants described herein. An orthogonal evolution approach was combined with a high throughput screening by NGS. Briefly, the library of AAV capsid variants was generated using a sliding window approach, where amino acid modifications, e.g., substitutions, were randomly introduced into various positions across loop VIII of AAV9, including between positions 581-593, relative to a reference sequence numbered according to SEQ ID NO: 138. The initial library (passage 1) w as passed first through cynomolgus macaques (Macaca fascicularis. n=2, 2-4 years of age) or human brain microvascular endothelial cells (hBMVECs) followed by cynomolgus macaques (n=2). These first passage libraries w ere then pooled and screened a second time through cynomolgus macaques (passage 2). After the second passage (e.g.. 28 days post injection into two NHPs), RNA was extracted from multiple brain and spinal cord regions. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate fold enrichment relative to an AAV9 wild-type control. The top variants were selected for the creation of a synthetic library.

[0457] After creation of the synthetic library with the sub-selected variants, the synthetic library w as screened (passage 3) in two NHPs (2-4 years of age). The animals were injected intravenously with the synthetic library. After a period in vivo, (e.g., 28-days) RNA was extracted from nervous tissue, e.g., brain, spinal cord, and DRG of the NHPs. Following RNA recovery' and RT-PCR amplification, a systematic NGS enrichment analysis was performed, and the peptides comprised within the variants were identified and the capsid enrichment ratio for each variant compared to the wild-ty pe AAV9 control was calculated (fold enrichment relative to wild-type AAV9). Values above 1 indicate an increase in expression relative to AAV9. All animals were dosed intravenously at 2el3 VG/kg across the screen. [0458] Following these three passages, approximately 1542 variants were identified with an average fold change greater than wild-type AAV9 in the brain of the NHPs (Macaca fascicularis). Of the 1542 variants, 422 demonstrated a fold-change of greater than 5 and 118 demonstrated a fold-change greater than 10 compared to wild-type and were detected across all brain regions investigated. The capsid variant that demonstrated the greatest fold-change in expression relative to AAV9 in the brain of the NHP comprised the amino acid sequence TQDWHR1 (SEQ ID NO: 941), resulting in a fold-change in expression of 460 relative to the AAV9 control (Table 14).

Table 14. NGS fold-enrichment of AAV capsid variants in NHPs (Macaca fascicularis)

[0459] Taken together, these results demonstrate that after 3 rounds of screening of this AAV9 variant library with loop VIII modifications in NHPs (cynomolgus macaques (Macaca fascicularis) , many AAV capsid variants outperformed the wild-type AAV9, for example, in penetration of the blood brain barrier (BBB) and spinal cord expression.

Example 2. Individual Capsid Characterization in NHPs

[0460] The goal of these experiments was to determine the transduction level, tropism, ability to cross the blood brain barrier, and overall spatial distribution in the central nervous system (CNS) of a capsid variant selected from the study described in Example 1 relative to AAV9 following intravenous injection in NHPs (cynomolgus macaques (Macaca fascicularis)) and marmosets (Callithrix jacchus). The capsid variant was TTJ-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941 or 201), as outlined in Table 3 above. The amino acid and DNA sequences of TTJ-001 are provided, e.g.. in Tables 4 and 5, respectively.

A. Evaluation of TTJ-001 in Cynomolgus macaques

[0461] AAV particles were generated with this capsid variant encapsulating a sequence encoding a cynomolgus frataxin protein tagged with hemagglutinin (cyno-FXN-HA) driven by a CBA promoter. The capsid variant and AAV9 control were tested by intravenously administering the AAV particle formulation at 2el3 VG/kg to NHPs (n=2). The in-life period was 14 days and the brain, spinal cord, and peripheral tissues including the heart, liver, and quadriceps were collected for measuring transgene mRNA, transgene protein, and viral DNA (biodistribution). The AAV particles administered to the NHPs comprised single stranded viral genomes.

[0462] The brains isolated from the NHPs injected with the AAV particles encapsulated in the TTJ- 001 capsid variant (AAV TTJ-001) were assayed by ddPCR for the presence of transgene RNA as a measure of transgene expression, and the presence of viral DNA as a measure of viral genome levels. Data were provided as average mRNA fold change for the transgene relative to a housekeeping gene as well as the fold change relative to the AAV9 control (Tabic 15). As shown in Tabic 15, mRNA transgcnc expression from the TTJ-001 capsid variant, which is an AAV9 capsid variant, was significantly higher in the brain of NHPs relative to the wild-type AAV9 control. More specifically, mRNA expression was approximately 40-280-fold higher from the TTJ-001 capsid variant compared to wild-type AAV9 in the brain of NHPs. Additionally, mRNA expression was approximately 3-274-fold higher from the TTJ-001 capsid variants compared to wild-type AAV9 in the spinal cord of the NHPs. Comparable results were obtained when mRNA levels were measured by qPCR.

Table 15. Transgene mRNA expression with the TTJ-001 capsid variant in NHPs

[0463] The brains, spinal cord, and peripheral tissues isolated from the NHPs were also assayed for the presence of viral DNA as a measure of viral genome levels by ddPCR. Data are provided in Table 16 as average DNA (viral genome (VG)) copies per diploid genome as well as fold change relative to the AAV9 control. As shown in Table 16. biodistribution of the AAV9 capsid variant. TTJ-001, was significantly higher in the NHP brain relative to the wild-type AAV9 control. Biodistribution of TTJ-001 was lower in the NHP liver relative to the wild-type AAV9 control. Comparable results were obtained when average DNA levels were measured by qPCR.

Table 16. Viral DNA biodistribution with the TTJ-001 capsid variant in NHPs

[0464] The brain tissues and spinal cords of the mice were also subjected to immunohistochemistry staining to evaluate overall CNS tropism and biodistribution in various regions. Immunohistochemical staining correlated with the qPCR analysis, as TTJ-001 showed significantly stronger staining and more widespread payload expression in the brain and spinal cord, as compared to the AAV9 control. More specifically, TTJ-001 demonstrated moderately to strongly positive staining in the globus pallidus, thalamus, substantia nigra, oculomotor nucleus, geniculate nucleus, central grey, inferior colliculus, external cuneate nucleus, gracile nucleus, nucleus ambiguus, ventral horn of the spinal cord, vestibular nucleus, inferior olivary complex, deep cerebellar nuclei, and the DRG. TTJ-001 demonstrated weakly positive to moderately positive staining in the ventral pallidum, amygdala, caudate, putamen, hippocampus, temporal cortex, auditory cortex, and somatosensory cortex.

[0465] The peripheral tissues were also subjected to immunohistochemistry staining to evaluate tropism and biodistribution. TTJ-001 resulted in widespread transduction of multiple peripheral tissues, as compared to the AAV9 control. TTJ-001 demonstrated weakly positive to moderately positive staining in the pancreas and quadriceps and moderately to strongly positive staining in the heart, kidney, and liver.

[0466] Taken together, these data demonstrate that TTJ-001 is an enhanced CNS-tropic capsid variant in NHPs. Additionally, this capsid variant was able to successfully penetrate the blood brain barrier following intravenous injection. B. Evaluation ofTTJ-001 in Marmosets (Callithrix iacchus)

[0467] AAV particles were generated with the TTJ-001 capsid variant or the AAV9 capsid control which comprised a self-complementary viral genome encoding a histone H2b protein with a VSV tag (TTJ-001 capsid variant) or HA tag (AAV9 control capsid) driven by a ubiquitous CAG promoter. The AAV particles comprising the TTJ-001 capsid variant or the AAV9 capsid control were administered to the marmosets (Callithrix jacchus) (n=3) intravenously in a single solution, at the doses indicated in Table 17. The in-life period was 28 days and then various CNS and peripheral tissues were collected for measuring transgene mRNA (expression) by RT-qPCR, protein expression by IHC, and viral DNA (biodistribution) by ddPCR. Data were then normalized to the dose of each viral vector in the dosing solution.

Table 17. Titer of the AAV particles comprising the TTJ-001 or AAV9 capsid in the solution dosed in marmosets

|0468| As shown in Table 18, the TTJ-001 capsid variant demonstrated increased biodistribution in the caudate and motor cortex in the brain of the marmosets relative to the AAV9 control. The TTJ-001 capsid variant also led to increased transgene expression (Table 19) in the caudate and motor cortex in the brain of the marmosets. In fact, biodistribution and transgene expression were increased over 5-7 fold and 15-17 fold, respectively, for TTJ-001 in the brain of marmosets relative to AAV9. Similar expression and biodistribution was observed by immunohistochemistry. More specifically, staining for TTJ-001 was detected in the mid-brain, caudate, putamen. thalamus, and cerebellum, and this staining was increased relative to AAV9. Staining for TTJ-001 was also observed in the molecular and granule layer of the cerebellum.

[0469] Biodistribution and transgene expression were also measured in the peripheral tissues of the liver, heart, and quadriceps. In the liver, the TTJ-001 capsid variant exhibited lower biodistribution (Table 18) and transgene expression (Table 19) relative to AAV9, indicating that the TTJ-001 capsid variant was detargeted in the liver relative to AAV9 in marmosets. Additionally, the TTJ-001 capsid variant resulted in increased transduction of the heart and muscle, as compared to the AAV9 control (Tabic 18 and Tabic 19).

Table 18. Quantification of viral genome copies per diploid genome (biodistribution) by ddPCR following intravenous administration of AAV particles comprising the TTJ-001 capsid variant normalized to the actual titer of the viral vector in the dosing solution (vg/dg = viral genome copies/ diploid genome)

Table 19. Quantification of transgene mRNA by RT-qPCR following intravenous administration of AAV particles comprising the TTJ-001 capsid variant normalized to the actual titer of the viral vector in the dosing solution (mRNA = transgene mRNA fold over housekeeping gene; rel. to AAV9= transgene mRNA fold over housekeeping gene relative to AAV9)

[0470] These data in marmosets for TTJ-001 were similar to those observed in cynomolgus macaques, demonstrating cross-species compatibility of the TTJ-001 capsid variant.

[0471] Taken together, these data demonstrate that TTJ-001 is an enhanced CNS tropic capsid in marmosets. Additionally, the TTJ-001 capsid variant was able to successfully penetrate the blood brain barrier following intravenous injection in the mannosets.

C. Evaluation of the TTJ-001 Capsid Variant to deliver a GBA1 protein in Cynomolgus Macaques (Macaca fascicularis)

[0472] The goal of these experiments was to determine the ability of AAV particles comprising TTJ- 001 capsid variant (SEQ ID NO: 981 (amino acid) and SEQ ID NO: 983 (DNA), comprising SEQ ID NO: 941 or SEQ ID NO: 201) to deliver a human GBA1 protein. AAV particles were generated with the TTJ-001 capsid variant or an AAV9 capsid control comprising a single-stranded viral genome encapsulating codon-optimized nucleotide sequence encoding GBA1 (SEQ ID NO: 1773). which was tagged with hemagglutinin (GBA1-HA). The human GBA1-HA payload was driven by a CBA promoter. The AAV particles comprising the viral genome encoding the human GBA1-HA protein and the TTJ-001 capsid variant or AAV9 control were intravenously administered to the NHPs at le 13 vg/kg (n=3) or 3el2 vg/kg (n=3). The in-life period was 28 days and the brain, spinal cord, and peripheral tissues including the heart, liver, and quadriceps were collected for measuring exogenous human GBA1-HA mRNA. exogenous human GBA1-HA protein, and viral DNA (biodistribution).

[0473] As shown in Tables 20, 21 A, and 21B, the AAV particles comprising the TTJ-001 capsid variant encoding the GBA1-HA protein demonstrated increased biodistribution and transduction as well as increased GBA1-HA mRNA expression in the brain (putamen, motor cortex, frontal cortex, substantia nigra, and dentate nucleus) and spinal cord (cervical C3 ventral horn) relative to AAV9. When dosed at 1 e 13 vg/kg, TTJ-001 led to at least a 6-9-fold increase in biodistribution in the brain and at least an 8-fold increase in biodistribution in the spinal cord relative to AAV9. With respect to GBA1-HA mRNA expression, when dosed at lel3 kg/vg, the AAV particles comprising the TTJ-001 capsid variant encoding the GBA1-HA protein demonstrated at least a 25-61-fold increase in GBA1-HA mRNA expression in the brain and at least a 20-fold increase in the spinal cord relative to AAV particles comprising the AAV9 capsid control encoding the GBA1-HA protein. The AAV particles comprising the TTJ-001 capsid variant encoding the GBA1-HA protein also demonstrated comparable biodistribution and GBA1-HA expression in the DRG relative to AAV particles comprising the AAV9 capsid control encoding the GBA1-HA protein (Tables 20, 21A, and 21B).

[0474] By immunohistochemistry (IHC), when dosed at lel3 vg/kg, TTJ-001 demonstrated largely neuronal tropism, as evidenced by positive co-staining for HA (GBA1 protein marker) and SMI 331 (neuronal marker) at least in the putamen and substantia nigra. No staining was observed in astrocytes in the putamen and substantia nigra. Additionally, by IHC, increased staining and transduction by AAV particles comprising the TTJ-001 capsid variant was also observed in multiple brain regions (substantia nigra, putamen, motor cortex, frontal cortex) and the spinal cord regions (cervical region, thoracic region, Clarke’s column) of the NHPs, relative to AAV9, when administered at a dose of le 13 vg/kg, indicating increased GBA1 expression in these regions. Staining and therefore transduction and GBA1-HA expression was also observed in the brain and spinal cord regions even when the AAV particles comprising the TTJ-001 capsid variant were administered at the lower dose of 3el2 vg/kg. Comparable transduction and GBA1-HA expression were observed by IHC in the DRG following administration of AAV particles comprising the TTJ-001 capsid variant and AAV particles comprising the AAV9 control capsid. IHC staining of the GBA1-HA positive cells of various brain regions following transduction with the AAV particles comprising the TTJ-001 capsid variant or AAV particles comprising an AAV9 control capsid was also quantified (Table 22). At a dose of lel3 vg/kg. TTJ-001 was capable of transducing greater than 2-9% of cells across the various brain regions investigated (Table 22). Even at the low dose of 3el2 vg/kg, TTJ-001 was capable of transducing greater than 0.9 to 5% cells in the brain regions tested (Table 22). The percent HA+ cells in Table 22 may underestimate true positivity for tire TTJ-001 capsid variant, as it demonstrated neuronal tropism.

[0475] Biodistribution and GBA1-HA expression were also measured in the peripheral tissues of the liver and heart. In the liver, at a dose of lel3 vg/kg, the TTJ-001 capsid variant exhibited lower biodistribution (Table 20) and GBA1-HA expression (Tables 21A and 21B) relative to AAV9, indicating that the TTJ-001 capsid variant was detargeted in the liver relative to AAV9. At the lcl3 vg/kg dose, the TTJ-001 demonstrated comparable biodistribution but decreased GBA1-HA expression in die heart relative to AAV9 (Tables 20, 21A, and 21B). Similar transduction and GBA1-HA expression were observed in the heart by IHC following transduction with AAV particles comprising the TTJ-001 capsid variant and AAV particles comprising the AAV9 capsid control at a dose of lel3 vg/kg.

Table 20. Quantification of viral genome copies per diploid genome (biodistribution) by ddPCR following intravenous administration of AAV particles comprising the TTJ-001 capsid variant (vg/dg = viral genome copies/ diploid genome)

Table 21A. Quantification of GBA1-HA mRNA by RT-qPCR following intravenous administration of AAV particles comprising the TTJ-001 capsid variant (mRNA = GBA1-HA mRNA fold over housekeeping gene; rel. to AAV9= GBA1-HA mRNA fold over housekeeping gene relative to AAV9)

Table 21B. Quantification of exogenous, human GBA1-HA mRNA relative to endogenous cynomolgus GBA1 mRNA by RT-qPCR follow ing intravenous administration of AAV particles comprising the TTJ-001 capsid variant (mRNA = exogenous, human GBA1-HA mRNA fold over endogenous cynomolgus GBA mRNA; rel. to AAV9= exogenous, human GBA1-H1 mRNA fold change over endogenous cynomolgus GBA mRNA relative to fold change over AAV9)

Table 22. Quantification of GBA1-HA positive cells by immunohistochemistry

[0476] These data further confirm that GBA1 expression was observed in NHPs following intravenous administration of AAV particles comprising the TTJ-001 variant, and further confirms that TTJ-001 is a CNS-tropic capsid, capable of crossing the blood brain barrier following intravenous administration in NHPs.

D. Further Evaluation of TTJ-001 in Cynomolgus Macaques (Macaca fascicularis)

[0477] AAV particles were generated with the TTJ-001 capsid variant or the AAV9 capsid control, each of which comprised a self-complementary viral genome encoding a histone H2b protein driven by a ubiquitous CAG promoter. The AAV particles comprising the TTJ-001 capsid control were administered to a first group of male cynomolgus macaques (Macaca fascicularis', 4-6 kg body weight; over 2 years old) intravenously at a dose per capsid of 4el2 VG/kg. The AAV particles comprising the AAV9 capsid control were administered to a second group of male cynomolgus macaques (Macaca fascicularis,' 4-6 kg body weight; over 2 years old) intravenously at a dose per capsid of 4el2 VG/kg. The in-life period was 28 days for both groups, and then various CNS and peripheral tissues were collected for measuring transgene mRNA (expression) by RT-qPCR; protein expression by IHC/chromogenic staining (e.g., DAB staining for percent of DAB+ cells indicating the percent of cells transduced); percent positive cells (e.g., neurons, motor neurons, and astrocytes) in brain and spinal cord regions by immunofluorescence microscopy; and viral DNA (biodistribution) by ddPCR.

[0478] As shown in Table 23, TTJ-001 demonstrated increased CNS transduction and/or biodistribution in several regions of the brain (greater than 10-20% of cells transduced observed in several regions) and spinal cord of the cynomolgus macaques after intravenous administration at a relatively low dose of 4el2 vg/kg. More specifically, TTJ-001 was capable of transducing up to 11% of cells in the putamen; up to 19% of cells in the caudate, and up to 23% of cells in the putamen. TTJ-001 also showed improved delivery to the spinal cord relative to AAV9, at a dose of 4el2 vg/kg.

[0479] TTJ-001 also demonstrated increased transduction and/or biodistribution in the peripheral tissues of the muscle, specifically the vastus lateralis muscle and the gastrocnemius muscle relative to AAV9, and comparable transduction and/or biodistribution in the heart relative to AAV9 (Table 24 A and Table 24B). TTJ-001 also appeared to exhibit partial de-targeting in the liver (Table 24A and Table 24B). Table 23. Quantification of viral genome copies per diploid genome (vg/dg) (biodistribution) by ddPCR, transgene mRNA by RT-qPCR (mRNA = transgene mRNA fold over housekeeping gene), and percent of DAB+ cells in tissues of the CNS of cynomolgus macaques

Table 24A. Quantification of viral genome copies per diploid genome (vg/dg) (biodistribution) by ddPCR, transgene mRNA by RT-qPCR (mRNA = transgene mRNA fold over housekeeping gene), and percent of DAB+ cells in peripheral tissues of cynomolgus macaques

Table 24B. Ratio of viral genome copies per diploid genome (vg/dg) (biodistribution) or transgene mRNA fold over housekeeping gene (mRNA) in the muscle and motor cortex relative to the liver and/or heart

[0480] Taken together, these data demonstrate that TTJ-001 demonstrated increased CNS tropism but also increased tropism in the heart and muscle in cynomolgus macaques.

Example 3. Evaluation of TTJ-001 AAV9 capsid variant in Diverse Primate Species

[0481] This Example evaluates the tropism and cross-species compatibility of the TTJ-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941 or 201) capsid variant in two diverse primate species, marmosets (Callithrix jacchus) and African green monkeys (Chlorocebus sabaeus). as compared to their tropism in cynomolgus macaques (Macaca fascicularis) provided in Example 1 and 2.

[0482] To investigate tropism in African green monkeys. AAV particles comprising the TTJ-001 capsid variant or an AAV9 control, under the control of a synapsin promoter, were intravenously injected into NHPs (n=2, 3-12 years of age) at a dose of 2E13 vg/kg. After 14-days in life, the brains and tissues (liver, DRG, quadriceps, and heart) of the NHPs were collected and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the AAV9 wild-type control.

[0483] To investigate tropism in mannoset monkeys, AAV particles comprising the TTJ-001 capsid variant or an AAV9 control, were intravenously injected into the NHPs (n=2, >10 months of age) at a dose of 2E13 vg/kg (8.75E12 vg/mL). After 28-days in life, the brains and tissues (liver quadriceps, and heart) of the NHPs were collected and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was perfonned to calculate the fold enrichment ratio relative to the AAV9 wild-type control.

[0484] As provided in Table 25 (African green monkeys) and Table 26 (marmosets), the TTJ-001 capsid variant demonstrated increased CNS tropism in diverse primate species. The TTJ-001 capsid variant demonstrated a 460.2-fold increase in expression relative to AAV9 in the brain of cynomolgus macaques (Table 14. Example 1). a 14.67-fold increase in expression relative to AAV9 in the brain of African green monkeys, and a 24.2-fold increase in expression relative to AAV9 in the brain of marmosets.

Table 25. NGS fold-enrichment of TTJ-001 AAV capsid variant comprising SEQ ID NO: 941 in African green monkeys

Table 26. NGS-fold enrichment of TTJ-001 (comprises SEQ ID NO: 941) in marmosets

[0485] Taken together, these data demonstrate that the AAV9 capsid variant TTJ-001 demonstrated increased CNS tropism relative to the AAV9 control in the CNS across three diverse primate species, providing evidence of strong cross-species capacity.

Example 4. Maturation of TTJ-001 Capsid in NHPs and Mice

[0486] This Example describes maturation of the AAV9 capsid variant, TTJ-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941 (encoded by SEQ ID NO: 942)) or 201) in two species of NHPs, cynomolgus macaques (Macaca fascicularis) and marmosets (CaUithrix jacchus) to further enhance their transduction and biodistribution in the central nerv ous system as well as other tissues, and evolve the AAV capsid variants to provide further cross-spccics compatibility. The TTJ-001 capsid variant was also matured in mice. Two approaches were used to mature the TTJ-001 capsid sequence in order to randomize and mutate within and around the modifications comprised within loop VIII of the capsid variant. In die first maturation approach, sets of three contiguous amino acids were randomized across the mutagenesis region in die TTJ-001 sequence, which spanned from position 582 to position 593, numbered according to SEQ ID NO: 981. In the second maturation approach, mutagenic primers were used to introduce point mutations at a low frequency, scattered across the mutagenesis region in the TTJ-001 sequence ranging from position 582 to position 593, numbered according to SEQ ID NO: 981.

[0487] The library of pooled matured AAV capsid variants generated from TTJ-001 using the first maturation approach and the library of pooled matured AAV capsid variants generated from TTJ-001 using the second maturation approach were each injected into two cynomolgus macaques (Macaca fascicularis), two marmosets (CaUithrix jacchus). and three outbred mice. After a 14- or 28-day period in life, the brains of the NHPs and mice were isolated and RNA was extracted. Following RNA recovery and RT-PCR amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the TTJ-001 non-matured control or an AAV9 control, and the peptides comprised within the variants were identified.

[0488] Following the RNA recovery and NGS analysis from the second maturation approach, the matured capsid variants were filtered based on their coefficient of variation (CV), which was calculated for each peptide across the brain samples taken from the NHPs and mice. Those that had a CV value <1 were identified, as these were the peptides that were reliably detected in the majority of samples isolated from the brains of the two NHPs. The peptides were also filtered based on a read per million (RPM) greater than or equal to 1 in the original virus stock.

[0489] Table 27 provides the peptide sequences of the matured capsid variants from the first maturation approach having an RPM greater than or equal to 1 in the original virus stock, a CV of less than 1 for the brain samples isolated, and that also demonstrated a 24-fold or greater fold-increase in expression in the brain relative to the AAV9 control in the brain of cynomolgus macaques (Macaca fascicularis) and/or a 50-fold or greater fold-increase in expression in the brain relative to the AAV9 control in the brain of marmosets (Callithrix jacchus), that were also detected in the maturation screen in mice, as these variants demonstrate cross-species compatibility. For example, the TTJ-001 capsid variants comprising SEQ ID NOs: 205, 208, 212, 219, 286, or 307 generated using tire first maturation approach demonstrated a fold change greater than the non-matmed TTJ-001 control and the AAV9 control in both species of NHPs and mice. TTJ-001 capsid variants comprising any one of SEQ ID NOs: 262-265 generated using the first matmation approach demonstrated a fold change greater than the nonmatmed TTJ-001 control and the AAV9 control in both species of NHPs.

Table 27. NGS fold-enrichment of the TTJ-001 matured AAV capsid variants in the brain of cynomolgus macaques, marmosets, and mice generated using the first maturation approach

[0490] Table 28 provides the peptide sequences of the matured capsid variants from the second maturation approach having an RPM greater than or equal to 1 in the original virus stock, a C V of less than 1 for the brain samples isolated, and that also demonstrated a 36-fold or greater fold-increase in expression in the brain relative to the AAV9 control in the brain of cynomolgus macaques (Macaca fascicular) s)' and/or about a 10-fold or greater fold-increase in expression in the brain relative to the AAV9 control in the brain of marmosets (Callithrix jacchus), that were also detected in the maturation screen in mice, as these variants demonstrate cross-species compatibility. For example, the TTJ-001 capsid variants comprising SEQ ID NOs: 283, 286, 290, 291, and 293 generated using the second maturation approach demonstrated a fold change greater than the non-matured TTJ-001 control and the AAV9 control in both species of NHPs and mice. The TTJ-001 capsid variants comprising SEQ ID NOs: 205, 208, 212. 213, or 307 generated using the second maturation approach demonstrated a fold change greater than the non-matured TTJ-001 control in cynomolgus macaques and mice and a fold-change greater than the AAV9 control in both species of NHPs and mice. The TTJ-001 capsid variants comprising SEQ ID NOs: 219 and 265 generated using the second maturation approach demonstrated a fold change greater than the non-matured TTJ-001 control and the AAV9 control in both species of NHPs and the TTJ-001 capsid variant comprising SEQ ID NO: 263 generated using the second maturation approach lead to a fold-change greater than AAV9 in both species of NHPs and a fold-change greater than TTJ-001 in marmosets.

Table 28. NGS fold-enrichment of the TTJ-001 matured AAV capsid variants in the brain of cynomolgus macaques, marmosets, and mice generated using the second maturation approach

[0491] Tabic 29 provides the sequences of TTJ-001 capsid variants with an RPM greater than or equal to 1 in tire original virus stock, a CV value of less than or equal to 1 and a fold-change in the brain of cynomolgus macaques of greater than 50 relative to AAV9. Approximately 59 variants were identified using the first maturation approach with a fold change above the TTJ-001 non-matured control in the brain of cynomolgus macaques. Approximately 181 variants were identified using the second maturation approach with a fold change above the TTJ-001 non-matured control in the brain of cynomolgus macaques. Many of the variants with increased expression in the brain of cynomolgus macaques relative to AAV9 and the non-matured TTJ-001 controls comprised a K at position 585 numbered according to SEQ ID NO: 981 or 138.

Table 29. NGS fold-enrichment of TTJ-001 in the brain of cynomolgus macaques by both maturation approaches

[0492] These data demonstrate that following tw o matmation approaches, matured TTJ-001 capsid variants (AAV9 capsid variants) with loop VIII modifications were generated with significantly enhanced CNS tropism over wild-type AAV9 controls in two species of NHPs (cynomolgus macaques (Macaca fascicularis) and mannosets (CalUthrix jacchus)~) and mice, as well as cross-species compatibility.

Example 5. Individual Capsid Characterization in Mice

[0493] The goal of these experiments was to determine the transduction level, tropism, ability to cross the blood brain barrier, and overall spatial distribution in the central nervous system (CNS) of a capsid variant selected from the study described in Example 1 relative to AAV9 following intravenous injection in BALB/c mice. The capsid variant was TTJ-001 (SEQ ID NO: 981 (amino acid) and 983 (DNA), comprising SEQ ID NO: 941 or 201), as outlined in Table 3 above. The amino acid and DNA sequences of TTJ-001 are provided, e.g., in Tables 4 and 5. respectively.

[0494] AAV particles were generated with this capsid variant encapsulating a luciferase-EGFP-HA tagged reporter gene driven by a CBA promoter encoded by a single stranded viral genome. The TTJ-001 capsid variant and AAV9 control were tested by intravenously administering the AAV particle formulation at 2el3 VG/kg (5el l/dose) to mice (n=3). The in-life period was 28 days and the brain and tire liver were collected for measuring transgene mRNA, transgene protein, and viral DNA (biodistribution).

[0495] At 27 days post-injection of the AAV particles encapsulated in the TTJ-001 capsid variant, mice were injected with luciferin and imaged on the ventral and dorsal side by the IVIS imager. Robust luciferase signal was observed in mice injected with AAV particles encapsulated in the AAV9 control capsid but less luciferase signal was observed in mice injected with the AAV particles encapsulated in the TTJ-001 capsid variant.

[0496] The brains and livers were isolated on day 28 and subsequently assayed by qPCR for the presence of transgene mRNA, as a measure of transgene expression. Data were provided as average mRNA fold change for the transgene relative to a housekeeping gene as well as the fold change relative to the AAV9 control (Table 30). As shown in Table 30, mRNA expression from die TTJ-001 capsid variant was slightly increased in the brain of the mice relative to the AAV9 control. More specifically, mRNA expression was approximately 1.3-fold higher from the TTJ-001 capsid variant compared to wild- ty pe AAV9 in the brain of the mice. In the liver, mRNA expression from the TTJ-001 capsid variant was decreased relative to the AAV9 control.

Table 30. Transgene mRNA expression with the TTJ-001 capsid variant in the brain and liver of mice

[0497] The brains and livers isolated from the mice on day 28 were also assayed for the presence of viral DNA by qPCR as a measure of viral genome levels. Data are provided in Table 31 as average DNA (viral genome (VG) copies per diploid genome (dg) as well as fold change relative to the AAV9 control. As shown in Table 31, biodistribution of TTJ-001, was slightly higher in the mouse brain relative to the wild-type AAV9 control. Biodistribution of TTJ-001 was lower in the mouse liver relative to the wildtype AAV9 control.

Table 31. Viral DNA biodistribution with the TTJ-001 capsid variant in the brain and liver of mice

[0498] The brain, heart, liver, and muscle tissue isolated from the mice on day 28 were also subjected to immunohistochemistry staining to evaluate overall tropism in the CNS and peripheral tissues and transgene expression. TTJ-001 showed comparable staining in the brain, muscle, and liver, as compared to the AAV9 control. Additionally, TTJ-001 demonstrated decreased staining in the heart relative to AAV9.

Example 6. Evaluation of additional TTJ-001 capsid variants

[0499] Matured variants of the TTJ-001 capsid variant generated using the methods described in Example 4 w ere injected into cynomolgus macaques (Macaca fascicularis), marmosets (CalHthrix jacchus), or mice. The matured AAV capsid variants generated from the TTJ-001 non-matured capsid variant (SEQ ID NO: 981 (amino acid) and SEQ ID NO: 983 (DNA), comprising SEQ ID NO: 941 or SEQ ID NO: 201)) w ere injected into two NHPs (cynomolgus macaques or marmosets) or mice. After a period in life, the brains, heart, muscles, and/or livers of the NHPs or mice were isolated and DNA/RNA was extracted. Following DNA/RNA recovery' and amplification, a systematic NGS enrichment analysis was performed to calculate the fold enrichment ratio relative to the TTJ-001 control or an AAV9 control, and the peptides comprised within the AAV capsid variants were identified.

[0500] Table 32 provides the sequences of the matured capsid variants having a counts per million greater than or equal to 10. a CV of less than or equal to 1. and an average fold-change in cDNA levels in die brain relative to the cDNA levels of TTJ-001 that is greater than or equal to 1 in cynomolgus macaques. These variants demonstrated increased tropism in the brain of cynomolgus macaques.

Table 32. NGS fold-change in cDNA or viral DNA (vDNA) levels of TTJ-001 matured AAV capsid variants in the brains and livers of cynomolgus macaques (changes in the peptide sequence relative to TTJ-001 are bolded and underlined) (Fc rel. to AAV9= fold change relative to AAV9; Fc. rel. to TTJ-001 = fold change relative to TTJ-001)

[0501] Table 33 provides the sequences of the matured capsid variants having a counts per million greater than or equal to 10, a CV of less than or equal to 1, and an average fold-change in cDNA levels in the brain relative to the cDNA levels of TTJ-001 that is greater than or equal to 1 in mannosets. These variants demonstrated increased tropism in the marmoset brain. AAV capsid variants comprising SEQ ID NO: 216 or SEQ ID NO: 943, were able to cross the blood brain barrier and demonstrate increased expression in the brains of both cynomolgus macaques (Table 32) and marmosets (Table 33) relative to AAV9, and comparable or increased expression relative to TTJ-001 in the same tissues.

Table 33. NGS fold-change in cDNA levels of TTJ-001 matured AAV capsid variants in the brains, hearts, and muscles of marmosets (changes in the peptide sequence relative to TTJ-001 are bolded and underlined) (Fc rel. to AAV9= fold change relative to AAV9; Fc. rel. to TTJ-001= fold change relative to TTJ-001)

[0502] Table 34 provides the sequences of additional matured capsid variants having a counts per million greater than or equal to 10, a CV across the brain tissues of less than or equal to 1, and an average fold-change in cDNA levels in the brain relative to the cDNA levels of TTJ-001 that is greater than or equal to 1 in marmosets that were generated using two different maturation approaches. These variants demonstrated increased or comparable tropism in the marmoset brain relative to TTJ-001.

Table 34. NGS fold-change in cDNA levels of TTJ-001 matured AAV capsid variants in the brains, liver, hearts, and muscles of marmosets (changes in the peptide sequence relative to TTJ-001 are bolded and underlined)

[0503] The AAV capsid variants comprising either SEQ ID NO: 208 or SEQ ID NO: 232 were also isolated from the brains of mice following two maturation approaches, as they demonstrated increased tropism in the brain relative to TTJ-001 (Table 35). Table 35 provides data that was filtered by a counts per million greater than or equal to 10, a CV across the brain tissues of less than or equal to 1, and an average fold-change in cDNA levels in the brain relative to the cDNA levels of TTJ-001 that is greater than or equal to 1 in mice.

Table 35. NGS fold-change in cDNA levels of TTJ-001 matured AAV capsid variants in the brains, hearts, and muscles of mice (changes in the peptide sequence relative to TTJ-001 are bolded and underlined)

[0504] Table 36 provides the sequences of the matured capsid variants having a counts per million greater than or equal to 10, a CV of less than or equal to 1, and an average fold-change in cDNA levels in die heart relative to die cDNA levels of AAV9 that is greater than or equal to 2 in cynomolgus macaques. These variants demonstrated increased tropism in the heart and muscles (intercostal, diaphragm, and quadriceps) relative to AAV9 and comparable expression to TTJ-001 in diese same tissues in cynomolgus macaques.

Table 36. NGS fold-change in cDNA levels of TTJ-001 matured AAV capsid variants in the muscle, heart, and liver of cynomolgus macaques (changes in the peptide sequence relative to TTJ-001 are bolded and underlined)

[0505] The AAV capsid variants comprising SEQ ID NO: 241 or SEQ ID NO: 984. also demonstrated increased expression in the heart and brain relative to AAV9 and TTJ-001 and in the muscle relative to TTJ-001 in marmosets (Table 37). The AAV capsid variants comprising SEQ ID NO:

241 or SEQ ID NO: 984 also demonstrated detargeting in the liver relative to AAV9 and TTJ-001 (Table 37). Table 37 provides data that was filtered by a counts per million greater than or equal to 10, a CV of less than or equal to 1, and an average fold-change in cDNA levels in the heart relative to the cDNA levels of AAV9 that is greater than or equal to 2 in marmosets.

Table 37. NGS fold-change in cDNA levels of TTJ-001 matured AAV capsid variants in the brains, hearts, livers, and muscles of marmosets (changes in the peptide sequence relative to TTJ-001 are bolded and underlined) (Fc rel. to AAV9= fold change relative to AAV9; Fc. rel. to TTJ-001= fold change relative to TTJ-001)

[0506] These data demonstrate that following maturation, several matured TTJ-001 capsid variants with loop VIII modifications were generated with enhanced CNS, heart, and/or muscle tropism over wild-type AAV9 controls and/or the non-matured TTJ-001 capsid in two species of NHPs (cynomolgus macaques (Macaca fascicularis) and marmosets (Callithrix jacchus)) and mice.

Example 7. In vivo Evaluation of a Vectorized Viral Genome Comprising a Codon-optimized, CpG Depleted Nucleotide Sequence Encoding GBA in Wildtype Mice

[0507] This Example investigates the distribution and efficacy of two viral genome constructs: GBA VG35 (SEQ ID NO: 2006) and GBA VG36 (SEQ ID NO: 2007), each comprising a codon- optimized. CpG-depleted nucleotide sequence (SEQ ID NO: 2002) encoding a GBA protein and vectorized in a VOY101 capsid (VOY101.GBA VG35 and VOY101.GBA VG36) in wild-type C57BL/6 mice. In addition, the viral construct VOY101.GBA VG17, which is described in PCT/US2021/043216, was also assessed.

[0508] Mice were intravenously injected with 2el3 VG/kg of VOY101.GBA VG35, VOY101.GBA VG36, VOY101.GBA VG17, or a vehicle control, into the lateral tail vein. At 28-days post IV injection, various CNS tissues (e.g., cortex, striatum, hippocampus, thalamus, cerebellum, brainstem, and/or spinal cord) and peripheral tissues (e.g., heart, liver, and/or spleen) were harvested to measure viral genome (VG) biodistribution (VG/cell), GCase activity, and GBA mRNA expression (transgene specific and endogenous).

[0509] VG biodistributions in the cortex, brainstem, and striatum are shown in FIG. 1. All VG values are presented as mean and standard deviation and statistical analysis was done using 1-way ANOVA and Tukey HSD post-hoc test.

[0510] GCase activities (measured as nM 4mu pdet/mg protein) in the cortex, striatum, and brainstem are shown in FIG. 2A and FIG. 2B. Example 8. In vivo Evaluation of a Vectorized Viral Genome Comprising a Codon-optimized, CpG Depleted Nucleotide Sequence Encoding GBA in 4L-PS/NA Mice

[0511] This Example investigates the distribution, GCase activities, and substrate reduction of two viral genome constructs: GBA VG35 (SEQ ID NO: 2006) and GBA VG36 (SEQ ID NO: 2007), each comprising a codon-optimized, CpG-depleted nucleotide sequence (SEQ ID NO: 2002) encoding a GBA protein and vectorized in a single-stranded VOY101 capsid (ssVOY101.GBA_VG35 and ssVOY101.GBA_VG36) in 4L-PS/NA mice. In addition, single -stranded VOY101 comprising a codon- optimized nucleotide sequence (SEQ ID NO: 1773) encoding a GBA protein was also assessed (ssVOY101.GBA_VG17).

[0512] Mice were intravenously administered 2el3 VG/kg of one of the three viral genome constructs or a vehicle control via lateral tail injection. At 28-days post IV injection, various CNS tissues (e.g., cortex, striatum, hippocampus, thalamus, cerebellum, brainstem, and/or spinal cord) and peripheral tissues (e.g.. heart, liver, and/or spleen) were harvested to measure viral genome (VG) biodistribution (VG/cell), GCase activity, and substrate reduction.

[0513] Biodistributions and GCase activities in the brainstem and DRGs are shown in FIG. 3A and FIG. 3B. ssVOY101.GBA_VG36 showed significant reduction in GBA1 expression & GCase activities in DRGs but retain expression and activities in brain stem.

[0514] The substrates glucosylceramide and glucosylsphingosine were quantified in the brain stem, striatum, and DRGs by LC/MS-MS. Significant substrate reduction was observed in both brain stem and striatum with all three constructs (FIG. 4A and FIG. 4B). DRG substrates were not significantly changed. Also, the significantly reduced GBA mRNA levels obtained in DRGs using ssVOY101.GBA_VG36 resulted in reduced potential toxicity. These data demonstrate that both ssVOY101.GBA_VG35 and ssVOY101.GBA_VG36 exhibited a desired biodistribution profile and were effective in reducing substrate levels in brain tissues.

Example 9. In vivo Evaluation of a Vectorized Viral Genome Comprising an HA-taggcd Nucleotide Sequence Encoding GBA in Wildtype Mice

[0515] This Example investigates the distribution and efficacy of GBA VG17-VP and GBA VG17- VE comprising a codon-optimized nucleotide sequence (SEQ ID NO: 1773) encoding a GBA protein, and GBA VG17-HA comprising a codon-optimized nucleotide sequence (SEQ ID NO: 1773) encoding a GBA and further comprising an HA-tag. Both constructs were vectorized in a VOY101 capsid (VOY101.GBA VG17 and VOY101.GBA VG17-HA) for administration in wild-type C57BL/6 mice. [0516] Mice were intravenously injected with 2el3 VG/kg of VOY101.GBA VG17 and VOY101.GBA VG17-HA into the lateral tail vein. At 28-days post IV injection, various CNS tissues (e.g., cortex, striatum, hippocampus, thalamus, cerebellum, brainstem, and/or spinal cord) and peripheral tissues (e.g.. heart, liver, and/or spleen) were harvested to measure viral genome (VG) biodistribution (VG/cell). GCase activity, and GBA mRNA expression (transgene specific and endogenous).

[0517] Biodistribution in the cortex and GCase activities in the cortex, striatum, and brainstem are shown in FIG. 5A and FIG. 5B. VOY101.GBA VG17-HA showed comparable GCase activity as VOY101.GB-VG17.

[0518] Immunohistochemical assessments of GBA or GBA-HA expression in the cortex, striatum, brainstem, cerebellum, thalamus, and hippocampus are shown in FIG. 6A and FIG. 6B.

VOY101.GBA VG17-HA showed robust HA expression in all regions evaluated. No HA signal was detected in mice injected with VOY101.GBA VG17.

Equivalents and Scope

[0519] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the Detailed Description provided herein. The scope of the present disclosure is not intended to be limited to the above Detailed Description, but rather is as set forth in the appended claims.

[0520] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0521] In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any, composition, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0522] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

[0523] While the present disclosure has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the disclosure.

[0524] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

CLAIMS We claim:

1. An adeno-associated virus (AAV) particle comprising an AAV capsid variant and a viral genome, wherein the viral genome comprises a [3-ghicocerebrosidase 1 (GBAl)-encoding sequence and 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 X₄ is Q. K, E, S, P, R, N, or H; and/or

(ii) [N3] comprises amino acids X₅, X₆, and X?, wherein X₅ is 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, I, C, N. K, L, or Q;

(ii) X₂ is N, T. G, V. S. Y, K. 1. H, D. or F; and

(iii) X₃ is 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) X₆ is A. Y, P, N. S, T, G. E, V. W, F, or Q; and

(ii) X₇ is 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) X₈ is T, S. N, P, A. or I;

(ii) X₉ is G, N, D. R, V. A, S, or Q;

(iii) Xio is 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:

(ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to positions 203-736 of the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises:

T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID NO: 981;

D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO:

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.

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:

(ii) an AAV capsid variant comprising an amino acid sequence that is at least 95% identical to positions 138-736 of the amino acid sequence of SEQ ID NO: 981, wherein the AAV capsid variant comprises:

T at an amino acid corresponding to position 584 of the amino acid sequence of SEQ ID

NO: 981;

D at an amino acid corresponding to position 586 of the amino acid sequence of SEQ ID NO: 981;

W at an amino acid corresponding to position 587 of the amino acid sequence of SEQ ID NO: 981;

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:

(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:

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 tire 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 tire 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 viral genome encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1775. 1740. 1742. 1744, 1746, or 1748 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.

27. The AAV particle of any one of claims 1-26, wherein the viral genome encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1775 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.

28. The AAV particle of any one of claims 1-27. wherein the viral genome encodes a wildtype GBA1 protein.

29. The AAV particle of any one of claims 1-28, wherein the viral genome encodes a human GBA1 protein, a dog GBA1 protein, an equine GBA1 protein, or a monkey GBA1 protein.

30. The AAV particle of any one of claims 1-29, wherein the viral genome encodes a wildtype human GBA1 protein.

31. The AAV particle of any one of claims 1-30, wherein the viral genome docs not encode a hemagglutinin (HA) tag.

32. The AAV particle of any one of claims 1-31, wherein the GBAl-encoding sequence comprises SEQ ID NO: 2002 or SEQ ID NO: 1773, or a nucleotide 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.

33. The AAV particle of any one of claims 1-32, where the viral genome further comprises a signal sequence-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 2005 or SEQ ID NO: 1850. or a nucleotide 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.

34. The AAV particle of any one of claims 1-33, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 1772, or a nucleotide 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.

35. The AAV particle of any one of claims 1-34, wherein the viral genome encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 1774 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.

36. The AAV particle of any one of claims 1-35, wherein the viral genome comprises a promoter operably linked to the GBA1 -encoding sequence.

37. The AAV particle of claim 36, wherein the promoter comprises a human elongation factor 1 alpha (EF-la) promoter, a chicken [Lactin (CBA) promoter, a CAG promoter, a CAG derivative promoter, a CMV immediate-early enhancer and/or promoter, a CMV promoter, a [3 glucuronidase (GUSB) promoter, a ubiquitin C (UBC) promoter, a neuron-specific enolase (NSE) promoter, a platelet- derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF-0) promoter, an intercellular adhesion molecule 2 (ICAM-2) promoter, a synapsin (Syn) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light chain (NFL) promoter, aneurofilament heavy chain (NFH) promoter, a -globin minigene n 2 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoter, a glial fibrillary acidic protein (GFAP) promoter, a myelin basic protein (MBP) promoter, a cardiovascular promoter (e.g., aMHC, cTnT, and CMV-MLC2k), a liver promoter (e.g., hAAT, TBG), a skeletal muscle promoter (e.g., desmin, MCK, C512), or a fragment, e.g., a truncation, or a functional variant thereof.

38. The AAV particle of claim 36 or claim 37, wherein the promoter comprises a CBA promoter.

39. The AAV particle of any one of claims 36-38, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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.

40. The AAV particle of any one of claims 1-39, wherein the viral genome further comprises an enhancer.

41. The AAV particle of claim 40, wherein the enhancer comprises a CMV immediate-early (CMVie) enhancer; optionally wherein the enhancer comprises the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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.

42. The AAV particle of any one of claims 1-41, wherein the viral genome further comprises an intron; optionally wherein the intron comprises the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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.

43. The AAV particle of any one of claims 1-42. wherein the viral genome further comprises a polyadenylation (poly A) region; optionally wherein the polyA region comprises the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide 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.

44. The AAV particle of any one of claims 1-43, wherein the viral genome further comprises an in verted terminal repeat (ITR); optionally wherein the ITR comprises the nucleotide sequence of SEQ ID NO: 1829 or SEQ ID NO: 1830 or a nucleotide 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.

45. The AAV particle of claim 44, wherein the viral genome comprises a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide 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 the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide 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.

46. The AAV particle of any one of claims 1-45, wherein the viral genome further comprises a nucleotide sequence encoding one or more miR183 binding sites.

47. The AAV particle of any one of claims 1-46, wherein the viral genome encodes at least four miR183 binding sites.

48. The AAV particle of claim 47, wherein each of the at least four miR183 binding sites is separated by a spacer.

49. The AAV particle of any one of claims 45-48. wherein each of the miR183 binding sites is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 1847 or a nucleotide sequence that has up to three modifications relative thereto.

50. The AAV particle of any one of claims 1-45, wherein the viral genome further comprises a nucleotide sequence encoding a miR183 binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide 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.

51. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) a promoter;

(iii) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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

(iv) a 3’ ITR.

52. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer;

(iii) a promoter;

(iv) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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

(v) a 3’ ITR.

53. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer;

(iii) a promoter;

(iv) an intron;

(v) the GBA1 -encoding sequence, wherein the GBA1 -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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

(vi) a 3’ ITR.

54. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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;

(iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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;

(iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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;

(v) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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

(vi) a 3‘ ITR.

55. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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;

(iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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; (iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide sequence drat 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;

(v) the GBA1 -encoding sequence, wherein the GBA1 -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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;

(vi) a poly adenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide 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

(vii) a 3’ ITR.

56. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5' inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide 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) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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;

(iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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;

(iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide sequence tiiat 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;

(viii) a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide 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.

57. The AAV particle of any one of claims 51-56. wherein: (i) the 5' ITR comprises the nucleotide sequence of SEQ ID NO: 1829;

(ii) the enhancer comprises the nucleotide sequence of SEQ ID NO: 1831;

(iii) the promoter comprises the nucleotide sequence of SEQ ID NO: 1834;

(iv) the intron comprises the nucleotide sequence of SEQ ID NO: 1842;

58. The AAV particle of any one of claims 1-56, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 2006 or a nucleotide 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.

59. The AAV particle of any one of claims 1-45 or 51-58, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 2006.

60. The AAV particle of any one of claims 1-45 or 51-59, wherein the viral genome consists of the nucleotide sequence of SEQ ID NO: 2006.

61. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) a promoter;

(iii) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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;

(iv) a nucleotide sequence encoding at least one miR183 binding site; and

(v) a 3’ ITR.

62. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) a promoter;

(iii) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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; (iv) a nucleotide sequence encoding a miR183 binding site series, wherein the miR183 binding site series comprises at least one miR183 binding site and at least one spacer sequence; and

(v) a 3’ ITR.

63. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer;

(iii) a promoter;

(iv) the GBA1 -encoding sequence, wherein the GBA1 -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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;

(v) a nucleotide sequence encoding a miR183 binding site series, wherein the miR183 binding site series comprises at least one miR183 binding site and at least one spacer sequence; and

(vi) a 3’ ITR.

64. The AAV particle of any one of claims 1-25. wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(ii) an enhancer;

(iii) a promoter;

(iv) an intron;

(v) the GB Al -encoding sequence, wherein the GBA1 -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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;

(vi) a nucleotide sequence encoding a miR183 binding site series, wherein the miR183 binding site series comprises at least one miR183 binding site and at least one spacer sequence; and

(vii) a 3’ ITR.

65. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(vii) a 3’ ITR.

66. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR);

(v) the GBAl-encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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;

(vi) a nucleotide sequence encoding a miR183 binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide 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

(vii) a 3’ ITR.

67. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide 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) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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;

(iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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;

(iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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;

(v) the GBA1 -encoding sequence, wherein the GBA1 -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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;

(vi) a nucleotide sequence encoding at least one miR183 binding site, wherein the nucleotide sequence encoding the at least one miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 1847;

(vii) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide 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

(viii) a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide 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.

68. The AAV particle of any one of claims 1-25, wherein the viral genome comprises:

(i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide 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) an enhancer comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide 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;

(iii) a promoter comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide 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;

(iv) an intron comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide 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; (v) the GBA1 -encoding sequence, wherein the GBA1 -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 2001 or SEQ ID NO: 2002 or a nucleotide 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;

(vi) a nucleotide sequence encoding a miR183 binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 1849 or a nucleotide 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;

(vii) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide 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

(viii) a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide 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.

69. The AAV particle of claim 68, wherein:

(i) the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 1829;

(ii) the enhancer comprises the nucleotide sequence of SEQ ID NO: 1831 ;

(iii) the promoter comprises the nucleotide sequence of SEQ ID NO: 1834;

(iv) the intron comprises the nucleotide sequence of SEQ ID NO: 1842;

(vi) the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 1849;

(viii) the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 1830.

70. The AAV particle of any one of claims 1-68, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide 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.

71. The AAV particle of claim 70, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 2007.

72. The AAV particle of claim 71, wherein the viral genome consists of the nucleotide sequence of SEQ ID NO: 2007.

73. A cell comprising the AAV particle of any one of claims 1-72, optionally wherein the cell is a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.

74. A method of making the AAV particle of any one of claims 1-72, the method comprising:

(i) providing a cell comprising the viral genome comprising a GBAl-encoding sequence and a nucleic acid encoding the AAV capsid variant; and

75. The method of claim 74, wherein the viral genome comprises:

(i) the nucleotide sequence of SEQ ID NO: 2006 or a nucleotide 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;

(ii) the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide 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;

(iii) the nucleotide sequence of SEQ ID NO: 1812 or a nucleotide 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; or

(iv) the nucleotide sequence of SEQ ID NO: 1828 or a nucleotide 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 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.

76. The method of claim 74. wherein the viral genome comprises: (i) the nucleotide sequence of SEQ ID NO: 2006 or a nucleotide 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; or

(ii) the nucleotide sequence of SEQ ID NO: 2007 or a nucleotide 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 wherein the AAV capsid variant comprises the amino acid sequence of 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.

77. The method of claim 74, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 2006 or SEQ ID NO: 2007 and the AAV capsid variant comprises the amino acid sequence of 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.

78. The method of any one of claims 74-77, further comprising, prior to step (i). introducing a nucleic acid molecule comprising the viral genome into the cell.

79. The method of any one of claims 74-78, further comprising, prior to step (i). introducing the nucleic acid encoding the AAV capsid variant into the cell.

80. The method of any one of claims 74-79, wherein the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an SI9 cell), or a bacterial cell.

81. A pharmaceutical composition comprising the AAV particle of any one of claims 1-72 and a pharmaceutically acceptable excipient.

82. A method of delivering an AAV particle encoding a GBA1 protein to a cell, comprising administering an effective amount of the pharmaceutical composition of claim 81 or the AAV particle of any one of claims 1-72.

83. The method of claim 82, wherein the cell is in a subject, optionally where in subject has. has been diagnosed with having, or is at risk of having a GBA1 -related disorder.

84. A method of treating a GBA1 -related disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 81 or the AAV particle of any one of claims 1-72.

85. The method of claim 84, wherein the subject has, has been diagnosed with having, or is at risk of having the GB Al -related disorder.

86. The method of any one of claims 83-85. wherein the GBAl-related disorder is a GBAl-related neurodegenerative or neuromuscular disorder.

87. The method of any one of claims 83-86. wherein the GBAl-related disorder is Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g.. GD type 1, GD type 2. or GD type 3), Dementia with Lewy Bodies (DLB). Lewy Body Dementia (LBD), Multiple System Atrophy (MSA), Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden- Spatz Syndrome.

88. The method of any one of claims 83-87, wherein the GBAl-related disorder is PD.

89. The method of any one of claims 83-87, wherein the GBAl-related disorder is LBD.

90. The method of any one of claims 83-87, wherein the GBAl-related disorder is DLB.

91. The method of any one of claims 83-87, wherein the GBAl-related disorder is GD.

92. A method of treating a GBAl-related disorder in a subject, wherein the GBAl-related disorder is Parkinson’s Disease (PD), comprising administering to the subject an effective amount of the pharmaceutical composition of claim 81 or the AAV particle of any one of claims 1-72.

93. The method of claim 92, wherein the subject has, has been diagnosed with having, or is at risk of having PD.

94. A method of treating a GBAl-related disorder in a subject, wherein the GBAl-related disorder is Lewy Body Dementia (LBD), comprising administering to the subject an effective amount of the pharmaceutical composition of claim 81 or the AAV particle of any one of claims 1-72.

95. The method of claim 94, wherein the subject has, has been diagnosed with having, or is at risk of having LBD.

96. A method of treating a GBAl-related disorder in a subject, wherein the GBAl-related disorder is Dementia with Lewy Bodies (DLB), comprising administering to the subject an effective amount of the pharmaceutical composition of claim 81 or the AAV particle of any one of claims 1-72.

97. The method of claim 96, wherein the subject has, has been diagnosed with having, or is at risk of having DLB.

98. A method of treating a GBAl-related disorder in a subject, wherein the GBAl-related disorder is Gaucher Disease (GD), comprising administering to the subject an effective amount of the pharmaceutical composition of claim 81 or the AAV particle of any one of claims 1-72.

99. The method of claim 98, wherein the subject has. has been diagnosed with having, or is at risk of having GD.

100. The method of claim 98 or claim 99, wherein the GD is GD type 1.

101. The method of claim 98 or claim 99, wherein the GD is GD type 2.

102. The method of claim 98 or claim 99, wherein the GD is GD type 3.

103. The method of any one of claims 83-102, wherein the subject has one or more mutations in the

GBA1 gene.

104. The method of any one of claims 83-103, wherein the subject has lower GCase activity prior to administration of the pharmaceutical composition or AAV particle as compared to GCase activity in an individual who does not have a GBAl-related disorder, optionally wherein the level of GCase activity is measured by a 4-MUG assay or a SensoLyte Blue Glucocerebrosidase assay.

105. The method of any one of claims 84-104, wherein the treating results in prevention of progression of the GBAl-related disorder in the subject.

106. The method of any one of claims 84-105, wherein the treating results in amelioration of at least one symptom of the GBAl-related disorder in the subject.

107. The method of claim 106, wherein the at least one symptom comprises developmental delay, progressive encephalopathy, progressive dementia, ataxia, myoclonus, oculomotor dysfunction, bulbar palsy, generalized weakness, trembling of a limb, depression, visual hallucinations, cognitive decline, or a combination thereof.

108. The method of any one of claims 105-107, wherein the treating results in a change in one or more biomarkers comprising a GCase activity, a level of glucocerebroside and other glycolipids, (e.g., within immune cells such as macrophages), a level of synuclein aggregates (e.g., Lewy bodies), or a combination thereof.

109. The method of any one of claims 83-108, wherein the subject is a human.

110. The method of any one of claims 83-109, wherein the AAV particle or the pharmaceutical composition is delivered to a cell or tissue of the central nervous system (CNS) in the subject.

111. The method of claim 110. 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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).

112. The method of any one of claims 82-108, wherein the AAV particle or the pharmaceutical composition is delivered to a peripheral cell or tissue in the subject.

113. The method of claim 112, wherein the peripheral cell or tissue is a cell or tissue of the heart, skeletal muscle, sympathetic ganglia, and/or plasma.

114. The method of any one of claims 83-113, wherein the AAV particle or pharmaceutical composition is delivered to the subject via intravenous administration.

115. The method of any one of claims 83-114, further comprising evaluating, e.g., measuring, the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and/or GBA1 protein expression) in the subject, e.g.. in a cell, tissue, or fluid of the subject.

116. The method of claim 115, wherein the level of GBA1 protein expression is measured by an enzyme-linked immunosorbent assay (ELISA), a Western blot, or an immunohistochemistry assay.

117. The method of claim 115 or claim 116, wherein evaluating the subject’s level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and/or GBA1 protein expression) is performed prior to and/or subsequent to administration of the pharmaceutical composition or AAV particle, optionally wherein the subject’s level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and/or GBA1 protein expression) prior to administration is compared to the subject’s level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and/or GBA1 protein expression) subsequent to administration.

118. The method of any one of claims 115-117, comprising evaluating the level of GBA1 expression in a cell or tissue of the central nervous system.

119. The method of any one of claims 115-118. wherein the subject’s level of GBA1 protein expression subsequent to administration is increased relative to the subject’s level of GBA1 protein expression prior to administration.

120. The method of any one of claims 83-119, further comprising evaluating, e.g., measuring, the level of GCase activity in the subject.

121. The method of any one of claims 83-120, wherein administering the pharmaceutical composition or AAV particle to the subject results in an increase in:

(i) GCase activity 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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/or fluid (e.g., CSF and/or serum) of the subject relative to baseline and/or relative to GCase activity in a cell, tissue, or fluid of an individual with a GBA1 -related disorder who has not been administered the pharmaceutical composition or AAV particle;

(ii) 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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) GBA1 mRNA expression in a cell or 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, visual cortex), external cuneate nucleus, geniculate nucleus, globus pallidus, gracile nucleus, hilus of the dentate gyrus, 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 GBA1 mRNA expression in a cell or tissue of an individual with a GBA1 -related disorder who has not been administered the pharmaceutical composition or AAV particle.

122. The method of any one of claims 83-121, further comprising administering to the subject at least one additional agent and/or therapy.

123. The method of claim 122. wherein the at least one additional agent and/or therapy comprises an agent and/or therapy suitable for treating a GB Al -related disorder.

124. The method of claim 123, wherein the GB Al -related disorder is PD, LBD, GD, or DLB, wherein, optionally, the GD is GD type 1, GD type 2, or GD type 3.

125. The method of any one of claims 122-124, wherein the at least one additional agent and/or dicrapy is suitable for treating Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e g., GD type 1, GD type 2, or GD type 3), Dementia with Lewy Bodies (DLB), Lewy Body Dementia (LBD), Multiple System Atrophy (MSA), Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation ty pe

1 (NBIA 1), or Hallervorden-Spatz Syndrome.

126. The method of any one of claims 122-125, wherein the at least one additional agent and/or therapy comprises enzyme replacement therapy (ERT) (e.g., imiglucerase, velaglucerase alfa, or taliglucerase alfa); substrate reduction therapy (SRT) (e.g.. eliglustat or miglustat), levodopa, carbidopa, Safinamide. a dopamine agonist (e.g.. pramipexole, rotigotine, or ropinirole), a dopamine antagonist (e.g., quetiapine, clozapine), an anticholinergic (e.g., benztropine or trihexyphenidyl), a cholinesterase inhibitor (e.g.. rivastigmine, donepezil, or galantamine), an N-methyl-d-aspartate (NMDA) receptor antagonist (e.g.. memantine), or a combination thereof.

127. The method of any one of claims 83-126, further comprising administering an immunosuppressant to the subject.

128. The method of claim 127, wherein the immunosuppressant comprises a corticosteroid (e g., prednisone, prednisolone, methylprednisolone, and/or dexamethasone), rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and/or eculizumab hydroxychloroquine.

129. The method of any one of claims 83-128, further comprising administering a blood transfusion to the subject.

130. The pharmaceutical composition of claim 81 or the AAV particle of any one of claims 1-72 for use in a method of treating a disorder according to any one of claims 84-129.

131. The pharmaceutical composition of claim 81 or the AAV particle of any one of claims 1-72 for use in the treatment of a GBA1 -related disorder in a subject.

132. The pharmaceutical composition or AAV particle of claim 131. wherein the GBAl-related disorder is Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), Gaucher Disease (GD) (e.g.. GD t pe 1, GD type 2, or GD type 3), Dementia with Lewy Bodies (DLB), Lewy Body Dementia (LBD). Multiple System Atrophy (MSA). Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden-Spatz Syndrome.

133. The pharmaceutical composition or AAV particle of claim 131 or claim 132, wherein the GBAl- related disorder is PD.

134. The pharmaceutical composition or AAV particle of claim 133, wherein the subject has, has been diagnosed with having, or is at risk of having PD.

135. The pharmaceutical composition or AAV particle of claim 131 or claim 132, wherein the GBAl- related disorder is LBD.

136. The pharmaceutical composition or AAV particle of claim 135, wherein the subject has, has been diagnosed with having, or is at risk of having LBD.

137. The pharmaceutical composition or AAV particle of claim 131 or claim 132, wherein the GBA1- related disorder is DLB.

138. The pharmaceutical composition or AAV particle of claim 137, wherein the subject has, has been diagnosed with having, or is at risk of having DLB.

139. The pharmaceutical composition or AAV particle of claim 131 or claim 132, wherein the GBA1- related disorder is GD (e.g., GD type 1, GD type 2, or GD type 3).

140. The pharmaceutical composition or AAV particle of claim 139, wherein the subject has, has been diagnosed with having, or is at risk of having GD (e.g., GD type 1. GD type 2, or GD type 3).

141. Use of the pharmaceutical composition of claim 81 or the AAV particle of any one of claims 1- 72 in the manufacture of a medicament for the treatment of a GBAl-related disorder in a subject.

142. The use of claim 141. wherein the GBAl-related disorder is Parkinson’s Disease (PD). Parkinson’s Disease Dementia (PDD). Gaucher Disease (GD) (e.g.. GD type 1, GD type 2. or GD type 3), Dementia with Lewy Bodies (DLB), Lewy’ Body’ Dementia (LBD), Multiple System Atrophy’ (MSA), Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation type 1 (NBIA 1), or Hallervorden- Spatz Syndrome.

143. The use of claim 141 or claim 142, wherein the GBAl-related disorder is PD.

144. The use of claim 143, wherein the subject has, has been diagnosed with having, or is at risk of having PD.

145. The use of claim 141 or claim 142, wherein the GBAl-related disorder is GD.

146. The use of claim 145, wherein the subject has, has been diagnosed with having, or is at risk of having GD.

147. The use of claim 142, 145, or 146, wherein the GD is GD type 1.

148. The use of claim 142, 145, or 146, wherein the GD is GD type 2.

149. The use of claim 142, 145, or 146, wherein the GD is GD type 3.

150. The use of claim 141 or claim 142, wherein the GBAl-related disorder is LBD.

151. The use of claim 150, wherein the subject has, has been diagnosed with having, or is at risk of having LBD.

152. The use of claim 141 or claim 142, wherein the GBAl-related disorder is DLB.

153. The use of claim 152, wherein the subject has. has been diagnosed with having, or is at risk of having DLB.