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

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, in the treatment of subjects who have, have been diagnosed with, 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

Attorney Docket No. 14640.0112-00304COMPOSITIONS AND METHODS FOR THE TREATMENT OF DISORDERS RELATED TO GLUCOSYLCERAMIDASE BETA 1 DEFICIENCYRelated Applications

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 716,927, filed on November 6, 2024, the contents of which are incorporated herein by reference in their entirety.Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 14640_0112-00304_SL.xml, was created on August 28, 2025, and is 37,813 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.Field

[0003] 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 (“GBA1 protein”) and peptides (“GBA1 peptide”) for use in the treatment of GB Al -related disorders, which include Parkinson’s Disease (PD) and other GB Al -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 GB Al -related disorder or other condition resulting from a deficiency in the quantity and / or function of GBA1 protein.Background

[0004] 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 (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 272.27 (1997): 16862-16867.Attorney Docket No. 14640.0112-00304

[0005] 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.

[0006] To date, there are limited available treatments for GB Al -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 GB Al -related disorders and to ameliorate deficiencies of GBA1 protein in subjects, e.g., human subjects, afflicted with GB Al -related disorders.

[0007] 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). 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

[0008] In some embodiments, the present disclosure provides an adeno-associated virus (AAV) particle comprising a viral genome comprising a P-glucocerebrosidase 1 (GBA1)- encoding sequence and an AAV9 capsid variant, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 22 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 thereto; wherein the AAV9 capsidAttorney Docket No. 14640.0112-00304 variant comprises the amino acid sequence of HDSPHK (SEQ ID NO: 1) present at amino acids 252-257 numbered according to SEQ ID NO: 22 and comprises the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22.

[0009] In some embodiments, the AAV9 capsid variant comprises: (i) 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%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 20, wherein the amino acid sequence comprises HDSPHK (SEQ ID NO: 1) present at amino acids 454-459 numbered according to SEQ ID NO: 20 and comprises the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20; and / or (ii) 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%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 21, wherein the amino acid sequence comprises HDSPHK (SEQ ID NO: 1) present at amino acids 317-322 numbered according to SEQ ID NO: 21 and comprises the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21.

[0010] In some embodiments, the AAV9 capsid variant comprises: (i) an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 20; (ii) an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 21; and / or (iii) an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 22.

[0011] In some embodiments, the AAV9 capsid variant comprises: (i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 20; (ii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 21; and / or (iii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 22.Attorney Docket No. 14640.0112-00304

[0012] In some embodiments, the AAV9 capsid variant comprises: (i) the amino acid sequence of SEQ ID NO: 20; (ii) the amino acid sequence of SEQ ID NO: 21; and / or (iii) the amino acid sequence of SEQ ID NO: 22.

[0013] In some embodiments, the viral genome encodes a wildtype GBA1 protein. In some embodiments, the viral genome encodes a human GBA1 protein, a canine GBA1 protein, or an equine GBA1 protein. In some embodiments, the viral genome encodes a human GBA1 protein. In some embodiments, the human GBA1 protein comprises the amino acid sequence of SEQ ID NO: 3.

[0014] In some embodiments, the GBAl-encoding sequence comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the GBAl-encoding sequence comprises a nucleotide sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10.

[0015] In some embodiments, the GBAl-encoding sequence further comprises a signal sequence-encoding sequence, wherein the signal sequence-encoding sequence comprises a nucleotide 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%, at least 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the signal sequence-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 9.

[0016] In some embodiments, the GBAl-encoding sequence comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11.

[0017] In some embodiments, the viral genome further comprises a promoter operably linked to the GBAl-encoding sequence. In some embodiments, the promoter comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO: 7.Attorney Docket No. 14640.0112-00304

[0018] In some embodiments, the viral genome further comprises an enhancer. In some embodiments, the enhancer comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the enhancer comprises the nucleotide sequence of SEQ ID NO: 6.

[0019] In some embodiments, the viral genome further comprises an intron. In some embodiments, the intron comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the intron comprises the nucleotide sequence of SEQ ID NO: 8.

[0020] In some embodiments, the viral genome further comprises a polyadenylation (poly A) region. In some embodiments, the polyA region comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the polyA region comprises the nucleotide sequence of SEQ ID NO: 14.

[0021] In some embodiments, the viral genome further comprises an inverted terminal repeat (ITR). In some embodiments, the ITR comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 15. In some embodiments, the ITR comprises the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 15. 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: 5 and the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 15.

[0022] In some embodiments, the viral genome further comprises a nucleotide sequence encoding one or more microRNA183 (miR183) binding sites. In some embodiments, the nucleotide sequence encoding the one or more miR183 binding sites encodes four miR183 binding sites. In some embodiments, each of the four miR183 binding sites is encoded by a nucleotide sequence that has up to three modifications relative to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, each of the four miR183 binding sites is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 12.Attorney Docket No. 14640.0112-00304

[0023] 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 a nucleotide sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13.

[0024] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (i) a 5’ inverted terminal repeat (ITR); (ii) a promoter; (iii) the GB Al -encoding sequence, wherein the GBA1- encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence that is at least 90% identical (e.g., at least 93% identical) thereto; and (iv) a 3’ ITR.

[0025] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (i) a 5’ inverted terminal repeat (ITR); (ii) a promoter; (iii) the GB Al -encoding sequence, wherein the GBA1- encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 90% identical (e.g., at least 94% identical) thereto; and (iv) a 3’ ITR.

[0026] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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; and (vi) a 3’ ITR.

[0027] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 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 theAttorney Docket No. 14640.0112-00304 nucleotide sequence of SEQ ID NO: 7 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: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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; (vi) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 14 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.

[0028] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 5 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: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GBAl- encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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; (vi) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 14 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 comprising the nucleotide sequenceAttorney Docket No. 14640.0112-00304 of SEQ ID NO: 15 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.

[0029] In some embodiments, (i) the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 5; (ii) the enhancer comprises the nucleotide sequence of SEQ ID NO: 6; (iii) the promoter comprises the nucleotide sequence of SEQ ID NO: 7; (iv) the intron comprises the nucleotide sequence of SEQ ID NO: 8; (v) the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11; (vi) the polyA region comprises the nucleotide sequence of SEQ ID NO: 14; and (vii) the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 15.

[0030] In some embodiments, the viral genome comprises the nucleotide sequence of SEQ ID NO: 16 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.

[0031] In some embodiments, the viral genome comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the viral genome consists of the nucleotide sequence of SEQ ID NO: 16.

[0032] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (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: 10 or a nucleotide sequence that is at least 90% identical (e.g., at least 93% identical) thereto; (iv) a nucleotide sequence encoding at least one microRNA183 (miR183) binding site, e.g., encoding a miR183 binding site series; and (v) a 3’ ITR.

[0033] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (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: 11 or a nucleotide sequence that is at least 90% identical (e.g., at least 94% identical) thereto; (iv) a nucleotide sequence encoding at least one microRNA183 (miR183) binding site, e.g., encoding a miR183 binding site series; and (v) a 3’ ITR.

[0034] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 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: 7 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;Attorney Docket No. 14640.0112-00304(iv) an intron comprising the nucleotide sequence of SEQ ID NO: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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; (vi) a nucleotide sequence encoding at least one microRNA183 (miR183) binding site, e.g., encoding a miR183 binding site series; and (vii) a 3’ ITR.

[0035] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (i) a 5’ inverted terminal repeat (ITR); (ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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; (vi) a nucleotide sequence encoding a microRNA183 (miR183) binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13 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.

[0036] In some embodiments, the viral genome comprises, in 5’ to 3’ order: (i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 5 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 sequenceAttorney Docket No. 14640.0112-00304 of SEQ ID NO: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GBAl- encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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; (vi) a nucleotide sequence encoding a microRNA183 (miR183) binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13 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; (vii) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 14 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: 15 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.

[0037] In some embodiments, (i) the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 5; (ii) the enhancer comprises the nucleotide sequence of SEQ ID NO: 6; (iii) the promoter comprises the nucleotide sequence of SEQ ID NO: 7; (iv) the intron comprises the nucleotide sequence of SEQ ID NO: 8; (v) the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11; (vi) the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13; (vii) the polyA region comprises the nucleotide sequence of SEQ ID NO: 14; and (viii) the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 15.

[0038] In some embodiments, the viral genome comprises the nucleotide sequence of SEQ ID NO: 17 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.Attorney Docket No. 14640.0112-00304

[0039] In some embodiments, the viral genome comprises the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the viral genome consists of the nucleotide sequence of SEQ ID NO: 17.

[0040] In some embodiments, the present disclosure provides an adeno-associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 11 and an AAV9 capsid variant comprising: (i) the amino acid sequence of SEQ ID NO: 20; (ii) the amino acid sequence of SEQ ID NO: 21; and / or (iii) the amino acid sequence of SEQ ID NO: 22.

[0041] In some embodiments, the present disclosure provides an adeno-associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 10 and an AAV9 capsid variant comprising: (i) the amino acid sequence of SEQ ID NO: 20; (ii) the amino acid sequence of SEQ ID NO: 21; and / or (iii) the amino acid sequence of SEQ ID NO: 22.

[0042] In some embodiments, the present disclosure provides an adeno-associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 16 and an AAV9 capsid variant comprising: (i) the amino acid sequence of SEQ ID NO: 20; (ii) the amino acid sequence of SEQ ID NO: 21; and / or (iii) the amino acid sequence of SEQ ID NO: 22.

[0043] In some embodiments, the present disclosure provides an adeno-associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 17 and an AAV9 capsid variant comprising: (i) the amino acid sequence of SEQ ID NO: 20; (ii) the amino acid sequence of SEQ ID NO: 21; and / or (iii) the amino acid sequence of SEQ ID NO: 22.

[0044] In some embodiments, the present disclosure provides a cell comprising an AAV particle disclosed 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.

[0045] In some embodiments, the present disclosure provides a method of making an AAV particle disclosed herein, the method comprising: (i) providing a cell comprising the viral genome comprising a GB Al -encoding sequence and a nucleic acid encoding the AAV9 capsid variant; and (ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV9 capsid variant; thereby making the AAV particle.

[0046] In some embodiments of the method of making an AAV particle, the viral genome comprises (a) the nucleotide sequence of SEQ ID NO: 16 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%,Attorney Docket No. 14640.0112-00304 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: 17 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 AAV9 capsid variant comprises (i) the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 90% identity (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% identity) thereto; (ii) the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 90% identity (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% identity) thereto; and / or (iii) the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 90% identity (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% identity) thereto.

[0047] In some embodiments of the method of making an AAV particle, the viral genome comprises (a) the nucleotide sequence of SEQ ID NO: 16 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 (b) the nucleotide sequence of SEQ ID NO: 17 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 AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22.

[0048] In some embodiments of the method of making an AAV particle, the viral genome comprises the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17 and the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22.

[0049] In some embodiments, the method of making an AAV particle further comprises, prior to step (i), introducing into the cell a nucleic acid comprising the viral genome. In some embodiments, the method further comprises, prior to step (i), introducing into the cell the nucleic acid encoding the AAV9 capsid variant. 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.

[0050] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an AAV particle disclosed herein and a pharmaceutically acceptable excipient.Attorney Docket No. 14640.0112-00304

[0051] In some embodiments, the present disclosure provides a method of delivering an AAV particle encoding a GBA1 protein to a cell, comprising administering an effective amount of a pharmaceutical composition or an AAV particle disclosed herein.

[0052] In some embodiments, the cell is in a subject.

[0053] In some embodiments, the subject has, has been diagnosed with having, or is at risk of having a GB Al -related disorder, optionally wherein the GB Al -related disorder is a GB Al -related neurodegenerative or neuromuscular disorder.

[0054] In some embodiments, the subject has, has been diagnosed with having, or is at risk of having 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.

[0055] In some embodiments, the present disclosure provides a method of treating a subject having or diagnosed with having a GB Al -related disorder, or treating at least one symptom thereof, comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle disclosed herein, optionally wherein the GB Al -related disorder is a GB Al -related neurodegenerative or neuromuscular disorder.

[0056] In some embodiments, the GB Al -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. In some embodiments, the GB Al -related disorder is PD. In some embodiments, the GBAl-related disorder is GD (e.g., GD type 1, 2, or 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. In some embodiments, the GBAl-related disorder is DLB. In some embodiments, the GBAl-related disorder is LBD.

[0057] In some embodiments, the present disclosure provides a method of treating a subject having or diagnosed with having Parkinson’s Disease (PD), or treating at least one symptom thereof, comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle disclosed herein.

[0058] In some embodiments, the present disclosure provides a method of treating a subject having or diagnosed with having Gaucher Disease (GD) (e.g., GD type 1, 2, or 3), or treatingAttorney Docket No. 14640.0112-00304 at least one symptom thereof, comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle disclosed herein. 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.

[0059] In some embodiments, the present disclosure provides a method of treating a subject having or diagnosed with having Lewy Body Dementia (LBD), or treating at least one symptom thereof, comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle disclosed herein.

[0060] In some embodiments, the present disclosure provides a method of treating a subject having or diagnosed with having Dementia with Lewy Bodies (DLB), or treating at least one symptom thereof, comprising administering to the subject an effective amount of a pharmaceutical composition or AAV particle disclosed herein.

[0061] In some embodiments, the subject has one or more mutations in the GBA1 gene.

[0062] In some embodiments, the subject has lower GCase activity as compared to GCase activity in an individual who does not have a GB Al -related disorder, optionally wherein the level of GCase activity is measured by a 4-MUG assay or a SensoLyte Blue Glucocerebrosidase assay.

[0063] In some embodiments, the treating results in prevention of progression of the disorder or at least one symptom of the disorder in the subject. In some embodiments, the treating results in amelioration of at least one symptom of the disorder and / or a change in one or more biomarkers of the disorder. In some embodiments, the one or more biomarkers comprises 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), a level of neurofilament light chain, or a combination thereof. 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.

[0064] In some embodiments, the subject is a human.

[0065] In some embodiments, the AAV particle is delivered to a cell, tissue, or region of the CNS, e.g., of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord), or a combination thereof, of the subject. In some embodiments, the AAV particle or pharmaceutical composition is delivered to the subject via intravenous administration.Attorney Docket No. 14640.0112-00304

[0066] In some embodiments, the method 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 enzyme-linked immunosorbent assay (ELISA), a Western blot, or an immunohistochemistry assay.

[0067] In some embodiments, evaluating the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and / or GBA1 protein expression) is performed before and after administration of the AAV particle or pharmaceutical composition, optionally wherein the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and / or GBA1 protein expression) before administration is compared to the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and / or GBA1 protein expression) after administration.

[0068] In some embodiments, the method comprises evaluating the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and / or GBA1 protein expression) in a cell or tissue of the central nervous system (e.g., parenchyma).

[0069] In some embodiments, the subject’s level of GBA1 protein expression after administration is increased relative to the subject’s level of GBA1 protein expression before administration.

[0070] In some embodiments, the method further comprises evaluating, e.g., measuring, the level of GCase activity in the subject.

[0071] In some embodiments, administering the AAV particle or pharmaceutical composition results in an increase in: (i) GCase activity in a cell, tissue, (e.g., a cell or tissue of the CNS, e.g., putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord)), and / or fluid (e.g., CSF and / or serum) of the subject relative to baseline or relative to GCase activity in an individual with a GB Al -related disorder who has not been administered the AAV particle or pharmaceutical composition; (ii) the number or level of viral genomes (VG) per cell in a CNS tissue (e.g., of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord)) of the subject relative to the number 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., of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord)) of the subject relative to baseline or relative to GCase activity in anAttorney Docket No. 14640.0112-00304 individual with a GB Al -related disorder who has not been administered the AAV particle or pharmaceutical composition.

[0072] In some embodiments, a method of delivering or treating provided herein further comprises administering to the subject an additional agent and / or therapy suitable for treatment of the GB Al -related disorder or at least one symptom thereof. In some embodiments, the 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., quetiapine, clozapine, pramipexole, rotigotine, or ropinirole), 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.

[0073] In some embodiments, a method of delivering or treating provided herein further comprises administering a blood transfusion to the subject.

[0074] In some embodiments, a method of delivering or treating provided herein further comprises administering an immunosuppressant to the subject. In some embodiments, the immunosuppressant comprises a corticosteroid (e.g., prednisone, prednisolone, methylprednisolone, and / or dexamethasone), rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and / or eculizumab hydroxychloroquine.

[0075] In some embodiments, the present disclosure provides a pharmaceutical composition or AAV particle disclosed herein, for use in the treatment of a GB Al -related disorder or at least one symptom thereof in a subject. In some embodiments, the GB Al -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 (LBD), 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 GD (e.g., GD type 1, 2, or 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. In some embodiments, the GBAl-related disorder is PD. In some embodiments, the GBAl- related disorder is LBD. In some embodiments, the GBAl-related disorder is DLB.

[0076] In some embodiments, the present disclosure provides the use of a pharmaceutical composition, AAV particle, or the cell of the disclosure in the manufacture of a medicament for the treatment of a GBAl-related disorder or at least one symptom thereof in a subject. InAttorney Docket No. 14640.0112-00304 some embodiments, the GB Al -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 (LBD), 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 GB Al -related disorder is GD (e.g., GD type 1, 2, or 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. In some embodiments, the GB Al -related disorder is PD. In some embodiments, the GB Al -related disorder is LBD. In some embodiments, the GB Al -related disorder is DLB.

[0077] In some embodiments, the present disclosure provides a pharmaceutical composition or AAV particle for use in a method of treating a disorder as disclosed herein.Brief Description of the Drawings

[0078] FIG. l is a graph showing virus detected in the bottom chamber as a percentage to the load in a transcytosis assay in a transwell model, where AAV particles comprising an AAV9 or TTM-043 capsid were added to the top of MDCK cells that expressed ALPL from a single clone, and the ability of these particles to move from the top to the bottom chamber was measured (virus detected in the bottom chamber (% to the load)); data represents an average of n=2 measurements.Detailed DescriptionI. CompositionsA. Viral Genomes

[0079] In some embodiments, an AAV particle of the present disclosure serves as an expression vector comprising a viral genome (e.g., recombinant viral genome) that encodes a GBA1 protein. In some embodiments, the GBA1 protein is a wildtype GBA1 protein. In some embodiments, the GBA1 protein is a human GBA1 protein. In some embodiments, the GBA1 protein is a canine GBA1 protein. In some embodiments, the GBA1 protein is an equine GBA1 protein.

[0080] Exemplary GBA1 proteins that may be encoded by a viral genome (e.g., recombinant viral genome) of the present disclosure are provided in Table 1. In some embodiments, a viral genome (e.g., recombinant viral genome) further encodes a signal sequence. In some embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO: 2 (MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASG). In some embodiments, theAttorney Docket No. 14640.0112-00304 viral genome (e.g., recombinant viral genome) encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the viral genome (e.g., recombinant viral genome) encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 3.Table 1. Exemplary GBA1 Protein Sequences

[0081] In some embodiments, a viral genome, e.g., a recombinant viral genome, described herein, comprises a promoter operably linked to a transgene encoding a GBA1 protein. As used herein, a nucleotide sequence encoding a GBA1 protein may also be referred to as a GB Al -encoding sequence or a GBA1 protein-encoding sequence. In some embodiments, the viral genome (e.g., recombinant viral genome) further comprises an inverted terminal repeat region, an enhancer, an intron, a miR binding site, a polyA region, or a combination thereof. Exemplary sequence regions within ITR-to-ITR sequences for viral genomes according to the description are provided in Table 2.Attorney Docket No. 14640.0112-00304Table 2. Exemplary viral genome sequence regions in ITR-to-ITR constructsAttorney Docket No. 14640.0112-00304Attorney Docket No. 14640.0112-00304Table 3. Sequence Regions in Exemplary ITR-to-ITR (Viral Genome) SequenceTable 4. Sequence Regions in Exemplary ITR-to-ITR (Viral Genome) SequenceAttorney Docket No. 14640.0112-00304Table 5. Exemplary ITR-to-ITR (Viral Genome) SequencesAttorney Docket No. 14640.0112-00304Attorney Docket No. 14640.0112-00304

[0082] In some embodiments, the viral genome (e.g., recombinant viral genome) of an AAV particle is as described in any one of Tables 3-5.

[0083] In some embodiments, the viral genome (e.g., recombinant viral genome) of an AAV particle described herein comprises a GB Al -encoding sequence comprising the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having 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% identity) identity thereto. In some embodiments, the GBA1- encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10.

[0084] In some embodiments, the viral genome (e.g., recombinant viral genome) of an AAV particle described herein comprises a GB Al -encoding sequence comprising the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity thereto. In some embodiments, the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10.

[0085] In some embodiments, the viral genome (e.g., recombinant viral genome) comprises a nucleotide sequence encoding a signal sequence, also referred to as a signal sequenceencoding sequence. In some embodiments, the signal sequence-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the signal sequenceencoding sequence consists of the nucleotide sequence of SEQ ID NO: 9.Attorney Docket No. 14640.0112-00304

[0086] In some embodiments, the viral genome (e.g., recombinant viral genome) comprises a GB Al -encoding sequence comprising a signal sequence such that the viral genome (e.g., recombinant viral genome) comprises the nucleotide sequence of SEQ ID NO: 11 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, the GB A- encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the GB Al -encoding sequence consists of the nucleotide sequence of SEQ ID NO: 11.

[0087] In some embodiments, the viral genome (e.g., recombinant viral genome) comprises a GBAl-encoding sequence comprising a signal sequence such that the viral genome (e.g., recombinant viral genome) comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 95% (e.g., least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto. In some embodiments, the GBA-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the GBAl- encoding sequence consists of the nucleotide sequence of SEQ ID NO: 11.

[0088] In some embodiments, the viral genome (e.g., recombinant viral genome) further comprises at least one ITR sequence. In some embodiments, the at least one ITR sequence comprises the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 15 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: 5 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: 15 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 the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 5 and the 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the 5’ ITR sequence consists of the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the 3’ ITR sequence consists of the nucleotide sequence of SEQ ID NO: 15. In some embodiments,Attorney Docket No. 14640.0112-00304 the 5’ ITR sequence consists of the nucleotide sequence of SEQ ID NO: 5 and the 3’ ITR sequence consists of the nucleotide sequence of SEQ ID NO: 15.

[0089] In some embodiments, the viral genome (e.g., recombinant viral genome) further comprises a CMVie sequence and / or CBA promoter operably linked to the GBAl-encoding sequence. In some embodiments, the CMVie sequence comprises the nucleotide sequence of SEQ ID NO: 6 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: 7 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: 6 and the CBA promoter comprises the nucleotide sequence of SEQ ID NO: 7.

[0090] In some embodiments, the viral genome (e.g., recombinant viral genome) further comprises an intron region comprising the nucleotide sequence of SEQ ID NO: 8 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: 8. In some embodiments, the intron region consists of the nucleotide sequence of SEQ ID NO: 8.

[0091] In some embodiments, the viral genome (e.g., recombinant viral genome) further comprises a polyA sequence comprising the nucleotide sequence of SEQ ID NO: 14 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: 14. In some embodiments, the polyA sequence consists of the nucleotide sequence of SEQ ID NO: 14.

[0092] In some embodiments, the viral genome (e.g., recombinant viral genome) comprises, from 5’ to 3’, a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 5 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; a CMVie sequence comprising the nucleotide sequence of SEQ ID NO: 6 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 atAttorney Docket No. 14640.0112-00304 least 99% identical) thereto; a CB A promoter comprising the nucleotide sequence of SEQ ID NO: 7 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; an intron comprising the nucleotide sequence of SEQ ID NO: 8 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; a GB Al -encoding sequence comprising the nucleotide sequence of SEQ ID NO: 11 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; a polyA sequence comprising the nucleotide sequence of SEQ ID NO: 14 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 / or a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 15 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 GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

[0093] In some embodiments, the viral genome (e.g., recombinant viral genome) does not comprise a microRNA183 (miR183) binding site.

[0094] In some embodiments, the viral genome (e.g., recombinant viral genome) of an AAV particle described herein comprises a nucleotide sequence comprising SEQ ID NO: 16 or a nucleotide 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 thereto. In some embodiments, the viral genome (e.g., recombinant viral genome) of an AAV particle described herein comprises a nucleotide sequence comprising SEQ ID NO: 16 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, the viral genome (e.g., recombinant viral genome) comprises the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the viral genome (e.g., recombinant viral genome) consists of the nucleotide sequence of SEQ ID NO: 16.

[0095] In some embodiments, the viral genome (e.g., recombinant viral genome) further comprises a nucleotide sequence encoding at least one miR183 binding site. In someAttorney Docket No. 14640.0112-00304 embodiments, the viral genome (e.g., recombinant 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 nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence that has up to three modifications relative to the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the nucleotide sequence encoding at least one miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the at least one miR183 binding site is encoded by the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the viral genome (e.g., recombinant viral genome) encodes four miR183 binding sites, wherein each miR183 binding site is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the viral genome (e.g., recombinant viral genome) encodes four miR183 binding sites, wherein each miR183 binding site is encoded by the nucleotide sequence of SEQ ID NO: 12.

[0096] In some embodiments, the viral genome (e.g., recombinant viral genome) further comprises a nucleotide sequence encoding at least one spacer sequence between two miR binding sites, wherein each spacer sequence is encoded by the nucleotide sequence GATAGTTA or a nucleotide sequence that has up to three modifications relative to the nucleotide sequence GATAGTTA.

[0097] In some embodiments, the viral genome (e.g., recombinant viral genome) encodes a miR183 binding site series, wherein the series comprises miR183 binding sites (e.g., four miR183 binding sites) separated by a spacer. In some embodiments, the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO:13 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 nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding the miR183 binding site series consists of the nucleotide sequence of SEQ ID NO: 13.

[0098] In some embodiments, the viral genome (e.g., recombinant viral genome) comprises, from 5’ to 3’, a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 5 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: 6 or a nucleotide sequence at least 90% identical (e.g., at least 90%, at least 91%, at least 92%, atAttorney Docket No. 14640.0112-00304 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: 7 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: 8 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 GB Al -encoding sequence comprising the nucleotide sequence of SEQ ID NO: 11 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 miR183 binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13 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 polyA sequence comprising the nucleotide sequence of SEQ ID NO: 14 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 / or a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 15 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. In some embodiments, the GBA1- encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto.

[0099] In some embodiments, the AAV particle comprises a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 17 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 (e.g., recombinant 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 the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the AAV particle comprises a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the AAVAttorney Docket No. 14640.0112-00304 particle comprises a viral genome (e.g., recombinant viral genome) consists of the nucleotide sequence of SEQ ID NO: 17.

[0100] In some embodiments, the viral genome (e.g., recombinant viral genome) encodes a GBA1 protein comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 3 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 encoded GBA1 protein comprises or consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 3.B. Adeno-Associated Virus (AAV) Capsids and Particles

[0101] In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) in hypervariable loop IV and the amino acid sequence of RQTALQL (SEQ ID NO: 18) in hypervariable loop VIII. In some embodiments, the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 18 are present in VP1, VP2, and / or VP3. In some embodiments, the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 18 are present in VP1, VP2, and VP3.

[0102] In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) in hypervariable loop IV (e.g., present at positions 252-257 numbered according to the amino acid sequence of SEQ ID NO: 22) and three, four, or all of the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and / or the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22. In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) in hypervariable loop IV (e.g., present at positions 252-257 numbered according to the amino acid sequence of SEQ ID NO: 22) and the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22. In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) at amino acids 252-257 numbered according to the amino acid sequence of SEQ ID NO: 22; and the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22.Attorney Docket No. 14640.0112-00304

[0103] In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) in hypervariable loop IV (e.g., present at positions 317-322 numbered according to the amino acid sequence of SEQ ID NO: 21) and three, four, or all of the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and / or the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21. In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) in hypervariable loop IV (e.g., present at positions 317-322 numbered according to the amino acid sequence of SEQ ID NO: 21) and the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21. In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) at amino acids 317-322 numbered according to the amino acid sequence of SEQ ID NO: 21; and the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21.

[0104] In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) in hypervariable loop IV (e.g., present at positions 454-459 numbered according to the amino acid sequence of SEQ ID NO: 20) and three, four, or all of the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and / or the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20. In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) in hypervariable loop IV (e.g., present at positions 454-459 numbered according to the amino acid sequence of SEQ ID NO: 20) and the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20. In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) at amino acids 454-459 numbered according to the amino acidAttorney Docket No. 14640.0112-00304 sequence of SEQ ID NO: 20; and the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20.

[0105] In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 22 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 252-257 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 22 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 252-257 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 252-257 numbered according to SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 252-257 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acidAttorney Docket No. 14640.0112-00304 sequence of SEQ ID NO: 22) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 252-257 numbered according to SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 252-257 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 252-257 numbered according to SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 252-257 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 98%Attorney Docket No. 14640.0112-00304 identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 252-257 numbered according to SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 252-257 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 252-257 numbered according to SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 252-257 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 252-257 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the amino acid sequence of SEQ ID NO: 22 may be referred to as VP3 of TTM-043. In some embodiments, an AAV particle of the present disclosure comprises a VP3 comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, anAttorney Docket No. 14640.0112-00304AAV particle of the present disclosure comprises a VP3 consisting of the amino acid sequence of SEQ ID NO: 22.

[0106] In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 21 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 317-322 numbered according to SEQ ID NO: 21 and comprises the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 21 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 317-322 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 317-322 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 317-322 numbered according to SEQ ID NO: 21 and comprises the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 317-322 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 317-322 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96%Attorney Docket No. 14640.0112-00304 identical to SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 317-322 numbered according to SEQ ID NO: 21 and comprises the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 317-322 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 317-322 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 317-322 numbered according to SEQ ID NO: 21 and comprises the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 317-322 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 317-322 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 317-322 numbered according to SEQ ID NO: 21 and comprises the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsidAttorney Docket No. 14640.0112-00304 variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 317-322 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 317-322 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids 317-322 numbered according to SEQ ID NO: 21 and comprises the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 317-322 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 317-322 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the amino acid sequence of SEQ ID NO: 21 may be referred to as VP2 of TTM-043. In some embodiments, an AAV particle of the present disclosure comprises a VP2 comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, an AAV particle of the present disclosure comprises a VP2 consisting of the amino acid sequence of SEQ ID NO: 21.

[0107] In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 at amino acids 454-459 numbered according to SEQ ID NO: 20 and comprises the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ IDAttorney Docket No. 14640.0112-00304NO: 20 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 454-459 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 454-459 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 at amino acids 454-459 numbered according to SEQ ID NO: 20 and comprises the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 454-459 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 454-459 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 at amino acids 454-459 numbered according to SEQ ID NO: 20 and comprises the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 454-459 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 454-459 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acidsAttorney Docket No. 14640.0112-00304 corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 at amino acids 454-459 numbered according to SEQ ID NO: 20 and comprises the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 454-459 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 454-459 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 at amino acids 454-459 numbered according to SEQ ID NO: 20 and comprises the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 454-459 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 454-459 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 at amino acids 454-459 numbered according to SEQ ID NO: 20 and comprises the amino acid R at position 590, the amino acidAttorney Docket No. 14640.0112-00304T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 454-459 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 454-459 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the amino acid sequence of SEQ ID NO: 20 may be referred to as VP1 of TTM-043. In some embodiments, an AAV particle of the present disclosure comprises a VP1 comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, an AAV particle of the present disclosure comprises a VP1 consisting of the amino acid sequence of SEQ ID NO: 20.

[0108] In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of HDSPHK (SEQ ID NO: 1) in hypervariable loop IV and the amino acid sequence of RQTALQL (SEQ ID NO: 18) in hypervariable loop VIII. In some embodiments, the amino acid sequences of SEQ ID NO: 1 and RQTALQL (SEQ ID NO: 18) are present in VP1, VP2, and / or VP3. In some embodiments, the amino acid sequences of SEQ ID NO: 1 and RQTALQL (SEQ ID NO: 18) are present in VP1, VP2, and VP3.

[0109] In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 22 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ IDAttorney Docket No. 14640.0112-00304NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) andAttorney Docket No. 14640.0112-00304 comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the amino acid sequence of SEQ ID NO: 22 may be referred to as VP3 of TTM-043. In some embodiments, an AAV particle of the present disclosure comprises a VP3 comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, an AAV particle of the present disclosure comprises a VP3 consisting of the amino acid sequence of SEQ ID NO: 22.

[0110] In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 21 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In someAttorney Docket No. 14640.0112-00304 embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the amino acid sequence of SEQ ID NO: 21 may be referred to as VP2 of TTM-043. In some embodiments, an AAV particle of the present disclosure comprises a VP2 comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, an AAV particle of the present disclosure comprises a VP2 consisting of the amino acid sequence of SEQ ID NO: 21.

[0111] In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positionsAttorney Docket No. 14640.0112-00304449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence ofAttorney Docket No. 14640.0112-00304SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the amino acid sequence of SEQ ID NO: 20 may be referred to as VP1 of TTM-043. In some embodiments, an AAV particle of the present disclosure comprises a VP1 comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, an AAV particle of the present disclosure comprises a VP1 consisting of the amino acid sequence of SEQ ID NO: 20.

[0112] In some embodiments, an AAV particle of the present disclosure comprises an AAV9 capsid variant comprising the amino acid sequence of KTINGHDSPHKSGQNQQT (SEQ ID NO: 19) in hypervariable loop IV and the amino acid sequence of RQTALQL (SEQ ID NO: 18) in hypervariable loop VIII. In some embodiments, the amino acid sequences of SEQ ID NO: 19 and RQTALQL (SEQ ID NO: 18) are present in VP1, VP2, and / or VP3. In some embodiments, the amino acid sequences of SEQ ID NO: 19 and RQTALQL (SEQ ID NO: 18) are present in VP1, VP2, and VP3.

[0113] In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 22 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO:Attorney Docket No. 14640.0112-0030419 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 22, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 247-264 of the amino acid sequence of SEQ ID NO: 22) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acidsAttorney Docket No. 14640.0112-00304 corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22 (e.g., present at positions 388-394 of the amino acid sequence of SEQ ID NO: 22). In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the amino acid sequence of SEQ ID NO: 22 may be referred to as VP3 of TTM-043. In some embodiments, an AAV particle of the present disclosure comprises a VP3 comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, an AAV particle of the present disclosure comprises a VP3 consisting of the amino acid sequence of SEQ ID NO: 22.

[0114] In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 21 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97%Attorney Docket No. 14640.0112-00304 identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 21, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 312-329 of the amino acid sequence of SEQ ID NO: 21) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21 (e.g., present at positions 453-459 of the amino acid sequence of SEQ ID NO: 21). In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the amino acid sequence of SEQ ID NO: 21 may be referred to as VP2 of TTM-043. In some embodiments, an AAV particle of the present disclosure comprises a VP2 comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, an AAV particle of the present disclosure comprises a VP2 consisting of the amino acid sequence of SEQ ID NO: 21.

[0115] In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20 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 thereto, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 ofAttorney Docket No. 14640.0112-00304 the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO:19 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO:20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ IDAttorney Docket No. 14640.0112-00304NO: 20). In some embodiments, the AAV9 capsid variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 20, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 19 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 449-466 of the amino acid sequence of SEQ ID NO: 20) and comprises the amino acid sequence of RQTALQL (SEQ ID NO: 18) present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20 (e.g., present at positions 590-596 of the amino acid sequence of SEQ ID NO: 20). In some embodiments, the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the amino acid sequence of SEQ ID NO: 20 may be referred to as VP1 of TTM-043. In some embodiments, an AAV particle of the present disclosure comprises a VP1 comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, an AAV particle of the present disclosure comprises a VP1 consisting of the amino acid sequence of SEQ ID NO: 20.

[0116] In some embodiments, an AAV particle of the present disclosure comprises an AAV capsid variant that is encoded by the nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence that is 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%) identical thereto. In some embodiments, the AAV capsid variant is encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 23. In some embodiments, the AAV capsid variant is encoded by a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 23. In some embodiments, the AAV capsid variant is encoded by a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 23. In some embodiments, the AAV capsid variant is encoded by a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 23. In some embodiments, the AAV capsid variant is encoded by the nucleotide sequence of SEQ ID NO: 23.

[0117] In some embodiments, an AAV particle of the present disclosure comprises an amino acid sequence provided in Table 6. In some embodiments, an AAV particle of the present disclosure comprises amino acids 2-742 of an amino acid sequence provided in Table 6. The amino acids in the loop IV insert and loop VIII substitutions, relative to a wildtype AAV9 capsid sequence, are shown with underlined and bolded text.Attorney Docket No. 14640.0112-00304

[0118] In some embodiments, an AAV particle of the present disclosure comprises an amino acid sequence encoded by a nucleotide sequence of Table 7. In some embodiments, an AAV particle of the present disclosure is encoded by a nucleotide sequence of Table 7.

[0119] In some embodiments, the AAV particle comprises the AAV capsid variant TTM- 043.Table 6. Exemplary Capsid Amino Acid SequencesAttorney Docket No. 14640.0112-00304Table 7. Exemplary Full-length Capsid Nucleotide Sequence

[0120] It is common for a first amino acid (AA1), e.g., a first methionine (Metl), encoded by a capsid gene to be expressed in capsid proteins but cleaved during or after polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases. This “Metl / AAl-Attorney Docket No. 14640.0112-00304 clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Metl / AAl -clipping commonly occurs with VP1 and VP3 but can also occur with VP2.

[0121] Where the Metl / AAl -clipping is incomplete, a mixture of VP capsid proteins comprising the viral capsid may be produced, some of which include a Metl / AAl amino acid (Met+ / AA+) and some of which may lack a Metl / AAl amino acid as a result of Metl / AAl - clipping (Metl- / AA1-). For further discussion regarding Metl / AAl -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 relevant contents of each of which are incorporated herein by reference in their entirety.

[0122] According to the present disclosure, references to capsid proteins, e.g., of AAV capsid variants, are not limited to either clipped (Metl- / AA1-) or unclipped (Metl+ / AA1+) and may refer to a mixture of clipped and unclipped capsid proteins. A direct reference to a capsid protein whether by VP1, VP2, or VP3 designation or by SEQ ID NO may encompass VP capsid proteins that include Metl / AAl (Metl+ / AA1+) as well as corresponding VP capsid proteins without Metl / AAl as a result of clipping (Metl- / AA1-).

[0123] Accordingly, in some embodiments, an AAV particle of the present disclosure comprises a Metl / AAl -clipped VP 1, VP2, and / or VP3 of TTM-043.

[0124] In some embodiments, an AAV particle of the present disclosure comprises an AAV capsid variant comprising amino acids 2-742 of the amino acid sequence of SEQ ID NO: 20, wherein amino acid 2 of SEQ ID NO: 20 is optionally acetylated. In some embodiments, an AAV particle of the present disclosure comprises an AAV capsid variant comprising amino acids 2-605 of the amino acid sequence of SEQ ID NO: 21, wherein amino acid 2 of SEQ ID NO: 21 is optionally acetylated. In some embodiments, an AAV particle of the present disclosure comprises an AAV capsid variant comprising amino acids 2-540 of the amino acid sequence of SEQ ID NO: 22, wherein amino acid 2 of SEQ ID NO: 22 is optionally acetylated.C. Exemplary AAV Particles

[0125] In some embodiments, the present disclosure provides an AAV particle comprising (i) a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having at least 90% identity (e.g., at least 90%, atAttorney Docket No. 14640.0112-00304 least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) thereto and (ii) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 22 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 AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22.

[0126] In some embodiments, the present disclosure provides an AAV particle comprising (i) a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 90% identity (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% identity) thereto and (ii) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 22 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 AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 247-264 of the amino acid sequence of SEQ ID NO: 22 and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 388-394 of the amino acid sequence of SEQ ID NO: 22.

[0127] In some embodiments, the present disclosure provides an AAV particle comprising (i) a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having at least 90% identity (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% identity) thereto and (ii) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 21 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 AAV9 variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21.

[0128] In some embodiments, the present disclosure provides an AAV particle comprising (i) a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 90% identity (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%, atAttorney Docket No. 14640.0112-00304 least 98%, or at least 99% identity) thereto and (ii) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 21 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 AAV9 variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 312-329 of the amino acid sequence of SEQ ID NO: 21 and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 453-459 of the amino acid sequence of SEQ ID NO: 21.

[0129] In some embodiments, the present disclosure provides an AAV particle comprising (i) a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having at least 90% identity (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% identity) thereto and (ii) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 20 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 AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20.

[0130] In some embodiments, the present disclosure provides an AAV particle comprising (i) a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having at least 90% identity (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% identity) thereto and (ii) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 20 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 AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 1 present at amino acids corresponding to positions 449-466 of the amino acid sequence of SEQ ID NO: 20 and comprises the amino acid sequence of SEQ ID NO: 18 present at amino acids corresponding to positions 590-596 of the amino acid sequence of SEQ ID NO: 20.

[0131] In some embodiments, the AAV particle comprises an AAV capsid comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and a viral genome comprising the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the AAV particle comprises a TTM-043 capsid and a viral genome comprising the nucleotide sequence of SEQ ID NO: 10.Attorney Docket No. 14640.0112-00304

[0132] In some embodiments, the AAV particle comprises an AAV capsid comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and a viral genome comprising the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the AAV particle comprises a TTM-043 capsid and a viral genome comprising the nucleotide sequence of SEQ ID NO: 11.

[0133] In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, 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) to any of the foregoing and comprising the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 18, and wherein the viral genome (e.g., recombinant viral genome) comprises a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 5 or a nucleotide 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, an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 or a nucleotide 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, a promoter comprising the nucleotide sequence of SEQ ID NO: 7 or a nucleotide 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, an intron comprising the nucleotide sequence of SEQ ID NO: 8 or a nucleotide 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, a GB Al -encoding sequence comprising the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or a nucleotide 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, a polyA region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide 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 a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 15 or a nucleotide 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.

[0134] In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, or an amino acid sequence that is at least 95% identical (e.g., atAttorney Docket No. 14640.0112-00304 least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to any of the foregoing and comprising the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 18, and wherein the viral genome (e.g., recombinant viral genome) comprises a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 5, an enhancer comprising the nucleotide sequence of SEQ ID NO: 6, a promoter comprising the nucleotide sequence of SEQ ID NO: 7, an intron comprising the nucleotide sequence of SEQ ID NO: 8, a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11, a polyA region comprising the nucleotide sequence of SEQ ID NO: 14, and a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 15.

[0135] In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, and wherein the viral genome (e.g., recombinant viral genome) comprises a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 5, an enhancer comprising the nucleotide sequence of SEQ ID NO: 6, a promoter comprising the nucleotide sequence of SEQ ID NO: 7, an intron comprising the nucleotide sequence of SEQ ID NO: 8, a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11, a polyAregion comprising the nucleotide sequence of SEQ ID NO: 14, and a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 15.

[0136] In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, 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) to any of the foregoing and comprising the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 18, and wherein the viral genome (e.g., recombinant viral genome) comprises a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 5 or a nucleotide 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, an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 or a nucleotide 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, a promoter comprising the nucleotide sequence of SEQ ID NO: 7 or a nucleotide 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, an intron comprising the nucleotide sequence of SEQ ID NO: 8 or a nucleotide acidAttorney Docket No. 14640.0112-00304 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, a GB Al -encoding sequence comprising the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or a nucleotide 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, a nucleotide sequence encoding at least one microRNA183 (miR183) binding site, wherein the nucleotide sequence encoding at least one miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13 or a nucleotide sequence having up to three modifications relative thereto, a polyA region comprising the nucleotide sequence of SEQ ID NO: 14 or a nucleotide 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 a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 15 or a nucleotide 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.

[0137] In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, 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) to any of the foregoing, and wherein the viral genome (e.g., recombinant viral genome) comprises a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 5, an enhancer comprising the nucleotide sequence of SEQ ID NO: 6, a promoter comprising the nucleotide sequence of SEQ ID NO: 7, an intron comprising the nucleotide sequence of SEQ ID NO: 8, a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11, a nucleotide sequence encoding at least one microRNA183 (miR183) binding site, wherein the nucleotide sequence encoding at least one miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13, a polyA region comprising the nucleotide sequence of SEQ ID NO: 14, and a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 15.

[0138] In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, and wherein the viral genome (e.g., recombinant viral genome) comprises a 5’ ITR comprising the nucleotide sequence of SEQ ID NO: 5, an enhancer comprising the nucleotide sequence of SEQ ID NO: 6, a promoter comprising the nucleotideAttorney Docket No. 14640.0112-00304 sequence of SEQ ID NO: 7, an intron comprising the nucleotide sequence of SEQ ID NO: 8, a GBAl-encoding sequence comprising the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11, a nucleotide sequence encoding at least one microRNA183 (miR183) binding site, wherein the nucleotide sequence encoding at least one miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13, a polyA region comprising the nucleotide sequence of SEQ ID NO: 14, and a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 15.

[0139] In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, 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) to any of the foregoing, and wherein the viral genome (e.g., recombinant viral genome) comprises the nucleotide sequence of SEQ ID NO: 16 or a sequence having at least 90% identity (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% identity) thereto. In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, and wherein the viral genome (e.g., recombinant viral genome) comprises the nucleotide sequence of SEQ ID NO: 16 or a sequence having at least 90% identity (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% identity) thereto.

[0140] In some embodiments, the present disclosure provides an AAV particle comprising (i) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and (ii) a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the present disclosure provides an AAV particle comprising (i) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and (ii) a viral genome (e.g., recombinant viral genome) consisting of the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the AAV particle comprises a TTM-043 capsid and a viral genome comprising the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the AAV particle comprises a TTM-043 capsid and a viral genome consisting of the nucleotide sequence of SEQ ID NO: 16.Attorney Docket No. 14640.0112-00304

[0141] In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, 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) to any of the foregoing, and wherein the viral genome (e.g., recombinant viral genome) comprises the nucleotide sequence of SEQ ID NO: 17 or a sequence having at least 90% identity (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% identity) thereto. In some embodiments, the present disclosure provides an AAV particle comprising an AAV9 capsid variant and a viral genome (e.g., recombinant viral genome), wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22, and wherein the viral genome (e.g., recombinant viral genome) comprises the nucleotide sequence of SEQ ID NO: 17 or a sequence having at least 90% identity (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% identity) thereto.

[0142] In some embodiments, the present disclosure provides an AAV particle comprising (i) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and (ii) a viral genome (e.g., recombinant viral genome) comprising the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the present disclosure provides an AAV particle comprising (i) an AAV9 capsid variant comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and (ii) a viral genome (e.g., recombinant viral genome) consisting of the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the AAV particle comprises a TTM-043 capsid and a viral genome comprising the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the AAV particle comprises a TTM-043 capsid and a viral genome consisting of the nucleotide sequence of SEQ ID NO: 17.D. Tropism and Biodistribution Properties

[0143] In some embodiments, an AAV particle (e.g., comprising TTM-043) described herein exhibits central nervous system (CNS) tropism. In some embodiments, the AAV particle (e.g., comprising TTM-043) has increased tropism for a cell, tissue, or region of the CNS relative to an AAV9 capsid. In some embodiments, an AAV particle described herein, e.g., comprising the amino acid sequence of SEQ ID NO: 20, 21, and / or 22, demonstrates increased tropism for a cell, tissue, or region of the CNS relative to an AAV9 capsid. In some embodiments, theAttorney Docket No. 14640.0112-00304AAV particle comprises the amino acid sequence of SEQ ID NO: 20, 21, and / or 22. In some embodiments, the AAV particle comprises TTM-043. In some embodiments, the cell, tissue, or region of the CNS comprises a cell, tissue, or region of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord). In some embodiments the cell is a neuron. In some embodiments, the neuron is a dopaminergic neuron in the substantia nigra. In some embodiments, the neuron is a Purkinje neuron in the cerebellum. In some embodiments, the cell is an astrocyte.

[0144] In some embodiments, an AAV particle (e.g., comprising TTM-043) described herein preferentially delivers GBA1 to a cell, tissue, or region of the CNS as compared to delivery to a peripheral cell, tissue, or region. In some embodiments, an AAV particle described herein, e.g. comprising the amino acid sequence of SEQ ID NO: 20, 21, and / or 22, preferentially delivers GBA1 to a cell, tissue, or region of the CNS as compared to delivery to a peripheral cell, tissue, or region. In some embodiments, the AAV particle comprises the amino acid sequence of SEQ ID NO: 20, 21, and / or 22. In some embodiments, the AAV particle comprises TTM-043. In some embodiments, the cell, tissue, or region of the CNS comprises a cell, tissue, or region of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord). In some embodiments the cell is a neuron. In some embodiments, the neuron is a dopaminergic neuron in the substantia nigra. In some embodiments, the neuron is a Purkinje neuron in the cerebellum. In some embodiments, the cell is an astrocyte. In some embodiments, the peripheral cell, tissue, or region is in the liver, heart, muscle (e.g., vastus lateralis, gastrocnemius), and / or dorsal root ganglia.

[0145] In some embodiments, an AAV capsid variant described herein (e.g., TTM-043) demonstrates increased tropism for a cell, tissue, or region of the CNS relative to an AAV capsid comprising a wild-type AAV9 capsid protein. In some embodiments, an AAV capsid variant described herein, e.g., an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 20, 21, and / or 22, demonstrates increased tropism for a cell, tissue, or region of the CNS relative to an AAV capsid comprising a wild-type AAV9 capsid protein. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 20, 21, and / or 22. In some embodiments, the AAV capsid variant is TTM-043. In some embodiments, the cell, tissue, or region of the CNS comprises a cell, tissue, or region of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord). In someAttorney Docket No. 14640.0112-00304 embodiments the cell is a neuron. In some embodiments, the neuron is a dopaminergic neuron in the substantia nigra. In some embodiments, the neuron is a Purkinje neuron in the cerebellum. In some embodiments, the cell is an astrocyte.

[0146] In some embodiments, an AAV capsid variant described herein (e.g., TTM-043) preferentially delivers GBA1 to a cell, tissue, or region of the CNS as compared to delivery to a peripheral cell, tissue, or region. In some embodiments, an AAV capsid variant described herein, e.g., an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 20, 21, and / or 22, preferentially delivers GBA1 to a cell, tissue, or region of the CNS as compared to delivery to a peripheral cell, tissue, or region. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 20, 21, and / or 22. In some embodiments, the AAV capsid variant is TTM-043. In some embodiments, the cell, tissue, or region of the CNS comprises a cell, tissue, or region of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord). In some embodiments the cell is a neuron. In some embodiments, the neuron is a dopaminergic neuron in the substantia nigra. In some embodiments, the neuron is a Purkinje neuron in the cerebellum. In some embodiments, the cell is an astrocyte. In some embodiments, the peripheral cell, tissue, or region is in the liver, heart, muscle (e.g., vastus lateralis, gastrocnemius), and / or dorsal root ganglia.II. AAV Particle Production

[0147] 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.

[0148] 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 (e.g., AAV9 capsid variant) disclosed herein, wherein the method comprises: (i) providing a cell comprising a viral genome (e.g., recombinant viral genome) comprising a GB Al -encoding sequence and a nucleic acid encoding an AAV capsid variant; and (ii) incubating the cell under conditions suitable to encapsulate the viral genome(e.g., recombinant viral genome) in the AAV capsid variant; thereby making the AAV particle. In some embodiments, the viral genome (e.g., recombinant viral genome) comprises: (a) the nucleotide sequence of SEQ ID NO: 16 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: 17 or aAttorney Docket No. 14640.0112-00304 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: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22. In some embodiments, the viral genome (e.g., recombinant viral genome) comprises the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17 and the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22. In some embodiments, the method comprises, prior to step (i), introducing a nucleic acid comprising the viral genome (e.g., recombinant 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.

[0149] Any method known in the art may be used for the preparation of AAV particles. In some embodiments, AAV particles are produced in mammalian cells (e.g., HEK293 cells). In some embodiments, AAV particles are produced in insect cells (e.g., Sf9 cells).

[0150] Methods of making AAV particles are well known in the art and are described in e.g., U.S. Patent Nos. US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, US5064764, US6194191, US6566118, US8137948; or International Publication Nos. WO1996039530, W01998010088, WO1999014354, WO1999015685, WO1999047691, W02000055342, W02000075353 and W02001023597; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O’Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir.63:3822-8 (1989); Kajigaya et al., Proc. Nat’l. Acad. Sci. 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., I7r.272:382-93 (2000); the relevant 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 relevant contents of which are herein incorporated by reference in their entirety.III. Pharmaceutical CompositionsAttorney Docket No. 14640.0112-00304

[0151] The present disclosure additionally provides a method for treating GB Al -related disorders and disorders related to deficiencies in the function or expression of GBA1 protein(s) in a subject, e.g., in a human subject, comprising administering to the subject any of the AAV polynucleotides or AAV genomes described herein or administering to the subject a particle comprising said AAV polynucleotide or AAV genome, or administering to the subject any of the described compositions, including pharmaceutical compositions.

[0152] In some embodiments, a composition described herein comprises an AAV polynucleotide or AAV genome or AAV particle and at least one excipient.

[0153] Although pharmaceutical compositions provided herein, e.g., comprising AAV particles comprising a GB Al -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 such as dogs or horses. 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.

[0154] In some embodiments, compositions are administered to humans, e.g., human patients or human subjects.

[0155] In some embodiments, the AAV particle formulations described herein may contain a nucleic acid encoding at least one GBA1 protein. In some embodiments, the formulations may contain a nucleic acid encoding 1, 2, 3, 4, or 5 GBA1 proteins.

[0156] 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.

[0157] In some embodiments, the pharmaceutical composition comprises an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the pharmaceuticalAttorney Docket No. 14640.0112-00304 composition comprising an AAV particle comprising a TTM-043 capsid and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 16.

[0158] In some embodiments, the pharmaceutical composition comprises an AAV particle comprising an AAV capsid comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the pharmaceutical composition comprising an AAV particle comprising a TTM-043 capsid and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 17.IV. Formulations

[0159] Formulations of the AAV pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0160] Relative amounts of the active ingredient, the 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.

[0161] 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.

[0162] 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.

[0163] 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 self-assembled nucleic acid nanoparticles.Attorney Docket No. 14640.0112-00304

[0164] In some embodiments, AAV particles comprising a viral genome 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

[0165] 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 GBA1, and / or alters the release profile of GBA1. 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.

[0166] 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, 21stEdition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; the relevant 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

[0167] In some embodiments, AAV formulations may comprise at least one excipient which is an inactive ingredient. 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).Attorney Docket No. 14640.0112-00304

[0168] Formulations of AAV particles may include cations or anions. In some embodiments, the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+, or combinations thereof. In one embodiment, 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 relevant contents of each of which are herein incorporated by reference in their entirety).

[0169] In some embodiments, the formulation comprises an AAV particle or pharmaceutical composition comprising an AAV particle, wherein the AAV particle comprises an AAV capsid comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the AAV particle comprises a TTM-043 capsid and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 16.

[0170] In some embodiments, the formulation comprises an AAV particle or pharmaceutical composition comprising an AAV particle, wherein the AAV particle comprises an AAV capsid comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the AAV particle comprises a TTM-043 capsid and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 17.V. Uses and Applications

[0171] The compositions of the disclosure may be administered to a subject or used in the manufacture of a medicament for treating a GB Al -related disorder or at least one symptom thereof in a subject. Such a GB Al -related disorder may be a neuromuscular or a neurological disorder or condition. In some embodiments, the GB Al -related 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., GD type 1, GD type 2, or GD type 3), Dementia with Lewy Bodies (DLB), Lewy Body Dementia (LBD), Spinal muscular atrophy (SMA), Multiple System Atrophy (MSA), Multiple sclerosis (MS), Alzheimer’s Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Pure Autonomic Failure, Neurodegeneration with brain iron accumulation, type 1 (NBIA 1), or Hallervorden-Spatz Syndrome.

[0172] In some embodiments, the subject has or has been diagnosed with having a GBA1- related disorder, In some embodiments, the GBAl-related disorder is PD (e.g., a PD associated with a GBA1 mutation), PDD, GD (e.g., GD type 1, GD type 2, or GD type 3), DLB, LBD, SMA, MSA, MS, AD, ALS, Pure Autonomic Failure, NBIA 1, or Hallervorden- Spatz Syndrome.Attorney Docket No. 14640.0112-00304

[0173] 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, or at least one symptom thereof. 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, or at least one symptom thereof.

[0174] In some embodiments, the disclosure provides a method for treating Parkinson’s Disease (PD), e.g., a PD with one or more mutations in a GBA1 gene, or at least one symptom thereof. In certain embodiments, the Parkinson’s Disease is associated with one or more mutations in a GBA1 gene. In some embodiments, the subject has or has been diagnosed with having PD.

[0175] In some embodiments, the disclosure provides a method for treating Gaucher Disease (GD) (e.g., GD type 1, GD type 2, or GD type 3) or at least one symptom thereof. 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 subject has or has been diagnosed with having GD (e.g., GD type 1, 2, or 3).

[0176] In some embodiments, the disclosure provides a method for treating Lewy Body Dementia (LBD) or at least one symptom thereof. In some embodiments, the subject has or has been diagnosed with having LBD. In some embodiments, the disclosure provides a method for treating Dementia with Lewy Bodies (DLB) or at least one symptom thereof. In some embodiments, the subject has or has been diagnosed with having DLB.

[0177] In some embodiments, the subject has one or more mutations in the GBA1 gene.

[0178] In some embodiments, the subject has lower GCase activity as compared to GCase activity in an individual who does not have a GB Al -related disorder. In some embodiments, the level of GCase activity is measured by a 4-MUG assay or a SensoLyte Blue Glucocerebrosidase assay.

[0179] In some embodiments, administration of the AAV particle or pharmaceutical composition results in an increase in GCase activity in a cell, tissue, (e.g., a cell or tissue of the CNS), and / or fluid (e.g., CSF and / or serum) of the subject relative to baseline or relative to GCase activity in an individual with a GB Al -related disorder who has not been administered the AAV particle or pharmaceutical composition. In some embodiments, GCase activity in the subject after administration is increased by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold relative to baseline.Attorney Docket No. 14640.0112-00304

[0180] In some embodiments, administration of the AAV particle or pharmaceutical composition results in an increase in the number or level of viral genomes (VG) per cell level in a CNS tissue of the subject relative to the number or level of VG per cell in a peripheral tissue of the subject. In some embodiments, the subject’s VG per cell level in the CNS tissue is increased by greater than 50 VG per cell relative to the subject’s VG per cell level in a peripheral tissue.

[0181] In some embodiments, administration of the AAV particle or pharmaceutical composition results in an increase in GBA1 mRNA expression in a cell or tissue (e.g., a cell or tissue of the CNS) of the subject relative to baseline or relative to GCase activity in an individual with a GB Al -related disorder who has not been administered the AAV particle or pharmaceutical composition. In some embodiments, GBA1 mRNA expression in the subject after administration is increased by at least 100-1300-fold as compared to baseline.

[0182] In some embodiments, administering an AAV particle or pharmaceutical composition described herein results in prevention of progression of a GB Al -related disorder or at least one symptom of the disorder in the subject. In some embodiments, administering an AAV particle or pharmaceutical composition described herein results in amelioration of at least one symptom of the disorder and / or a change in one or more biomarkers of the disorder.Exemplary non-limiting biomarkers comprise 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), a level of neurofilament light chain, or a combination thereof. Exemplary non-limiting symptoms comprise 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.

[0183] In some embodiments, administration of the AAV particles comprising viral genomes (e.g., recombinant viral genomes) that encode GBA1 protein may protect central nervous system (CNS) pathways from degeneration or stabilize the condition of a subject suffering from degeneration in a CNS pathway.

[0184] In some embodiments, the delivery 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 fllipin staining and quantification). In some embodiments, the delivery of the AAV particles improves at least one symptom of a GBAl-related disorder, including, for example, cognitive, muscular, physical, and sensory symptoms of GBAl- related disorders. In some embodiments, the delivery of the AAV particles stabilizes at leastAttorney Docket No. 14640.0112-00304 one symptom of a GB Al -related disorder, including, for example, cognitive, muscular, physical, and sensory symptoms of GB Al -related disorders.

[0185] In some embodiments, a method of delivery or treatment comprising administering an AAV particle or pharmaceutical composition disclosed herein by intravenous administration.

[0186] 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.

[0187] 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.

[0188] In some embodiments, the delivery of the AAV particles may halt or slow the disease progression of GBAl-related disorders by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% using a known analysis method and comparator group for GBAl- related disorders. As a non-limiting example, the delivery 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 fllipin staining and quantification).

[0189] In some embodiments, delivery of an AAV particle described herein increases the amount of GBA1 protein in a tissue by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 97%, at least 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.Attorney Docket No. 14640.0112-00304

[0190] In some embodiments, the AAV particles and AAV viral 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 GB Al -related disorder or cells or tissues harboring at least one mutation in a GBA1 gene, the AAV particles and AAV viral genomes described herein restore GBA activity to normal levels, as defined by GBA activity levels in subjects, tissues, and cells not afflicted with a GB Al -related disorder or not harboring a GBA1 gene mutation. In some embodiments, the AAV particles and AAV viral genomes described herein effectively reduce a-synuclein levels in subjects having a GB Al -related disorder or cells or tissues harboring at least one mutation in a GBA1 gene. In some embodiments, the AAV particles and AAV viral genomes described herein effectively prevent a-synuclein mediated pathology.

[0191] In some embodiments, delivery of an AAV particle described herein increases Gcase expression by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% in a subject relative to baseline. In some embodiments, delivery of an AAV particle described herein increases Gcase activity by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% in a subject relative to baseline. In some embodiments, delivery of an AAV particle described herein increases Gcase expression and / or activity by at least 30% in the cortex relative to baseline. In some embodiments, delivery of an AAV particle described herein increases Gcase expression and / or activity by at least 30% in the putamen relative to baseline. In some embodiments, delivery of an AAV particle described herein increases Gcase expression and / or activity by at least 30% in the substantia nigra relative to baseline.Therapeutic applications

[0192] The present disclosure provides methods for treating at least one symptom of a GBAl-related disorder such as but not limited to PD (e.g., a PD associated with a GBA1 mutation), PDD, GD (e g., GD type 1, GD type 2, or GD type 3), DLB, LBD, SMA, MSA, MS, AD, ALS, Pure Autonomic Failure, NBIA 1, Hallervorden-Spatz Syndrome, 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.

[0193] 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,Attorney Docket No. 14640.0112-00304 disease progression may be measured by tests or diagnostic tool(s) known to those skilled in the art. As another non-limiting example, disease progression may be measured by change in the pathological features of the brain, CSF, or other tissues or fluids of the subject.

[0194] In some embodiments, treatment comprising administration of an AAV particle or pharmaceutical composition disclosed herein may result in prevention of progression of a GB Al -related disorder. In some embodiments, treatment may result in prevention of progression of at least one symptom of a GB Al -related disorder. In some embodiments, the treatment may result in stabilizing the condition of a subject who has, has been diagnosed with having, or is at risk of having a GB Al -related disorder. In some embodiments, the treatment may result in stabilizing the condition of at least one symptom of a GB Al -related disorder. In some embodiments, the treatment may result in amelioration (e.g., reducing the severity of) at least one symptom of a GB Al -related disorder. In some embodiments, the treatment reverses or partially reverses at least one symptom of a GB Al -related disorder. In some embodiments, the treatment may prevent or delay the onset of one or more symptoms of a GB Al -related disorder. In some embodiments, the treatment improves at least one symptom of a GB Al -related disorder. In some embodiments, the GB Al -related disorder is PD (e.g., a PD associated with a GBA1 mutation), PDD, GD (e.g., GD type 1, GD type 2, or GD type 3), DLB, or LBD.Exemplary Compositions for Delivery or Treatment Methods or Uses

[0195] In some embodiments, the present disclosure provides a method of delivering GBA1 to a subject comprising administering to the subject (e.g., via IV administration) an AAV particle comprising an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 (e.g., the AAV capsid is TTM-043) and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the present disclosure provides a method of treating (e.g., ameliorating, stabilizing the condition of, or preventing progression of) at least one symptom of a GBA1- related disorder (e.g., PD, PDD, GD, LBD, or DLB) in a subject, the method comprising administering to the subject (e.g., via IV administration) an AAV particle comprising an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 (e.g., the AAV capsid is TTM-043) and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 16.

[0196] In some embodiments, the present disclosure provides a method of delivering GBA1 to a subject comprising administering to the subject (e.g., via IV administration) an AAV particle comprising an AAV capsid variant comprising the amino acid sequence of SEQ IDAttorney Docket No. 14640.0112-00304NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 (e.g., the AAV capsid is TTM-043) and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the present disclosure provides a method of treating (e.g., ameliorating, stabilizing the condition of, or preventing progression of) at least one symptom of a GBA1- related disorder (e.g., PD, PDD, GD, LBD, or DLB) in a subject, the method comprising administering to the subject (e.g., via IV administration) an AAV particle comprising an AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22 (e.g., the AAV capsid is TTM-043) and a viral genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 17.VI. Delivery of AAV ParticlesDelivery to Cells

[0197] 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.Delivery to Subjects

[0198] 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.

[0199] In some embodiments, the AAV particles may be delivered to bypass anatomical blockages (e.g., the blood brain barrier).

[0200] 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.

[0201] In some embodiments, the AAV particles may be delivered using intrathecal infusion.

[0202] In some embodiments, a subject may be administered the AAV particles described herein using a bolus infusion.

[0203] 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 ofAttorney Docket No. 14640.0112-00304 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.

[0204] In some embodiments, the AAV particles may be delivered to a subject via a single route of administration.

[0205] In some embodiments, the AAV particles may be delivered to a subject via a multisite route of administration. For example, a subject may be administered the AAV particles at 2, 3, 4, 5, or more than 5 sites.

[0206] 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.

[0207] In some embodiments, if continuous delivery (continuous infusion) of the AAV particles is used, the continuous infusion may be for 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or more than 24 hours.

[0208] 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.

[0209] 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.

[0210] 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 intracerebroventri cul ar admini strati on .

[0211] In some embodiments, an AAV particle described herein is administered intravenously.

[0212] 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.

[0213] 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 XYZAttorney Docket No. 14640.0112-00304 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.

[0214] 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.

[0215] 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 cortex, striatum, thalamus, cerebellum, brainstem, and / or spinal cord) as compared to a baseline level in the subject.

[0216] 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 cortex, striatum, thalamus, cerebellum, brainstem, and / or spinal cord) by transducing cells in these CNS regions. Transduction may also be referred to as the number of cells that are positive for GBA1 protein.

[0217] In some embodiments, delivery of AAV particles comprising a viral genome encoding GBA1 as described herein to neurons in the brain (e.g., the cortex, striatum, thalamus, cerebellum, and / or brainstem) 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).

[0218] 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.

[0219] 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.AdministrationAttorney Docket No. 14640.0112-00304

[0220] 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).

[0221] 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, prophylactically 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.

[0222] 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. In some embodiments, a single unit dose is provided as a discrete dosage form (e.g., a tablet, capsule, patch, loaded syringe, vial, efc.). It may be administered as a single unit dose. The viral particles may be formulated in buffer only or in a formulation described herein.

[0223] 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.

[0224] 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.Attorney Docket No. 14640.0112-00304Combinations

[0225] 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.

[0226] The therapeutic agents may be approved by the US Food and Drug Administration or may be in clinical trial or at the preclinical research stage. The therapeutic agents may utilize any therapeutic modality known in the art, with non-limiting examples including gene silencing or interference (e.g.., miRNA, siRNA, RNAi, shRNA), gene editing (e.g.., TALEN, CRISPR / Cas9 systems, zinc finger nucleases), and gene, protein or enzyme replacement.

[0227] 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 agent and / or therapy.

[0228] In some embodiments, the at least one additional agent and / or therapy comprises an agent and / or therapy for treating the GB Al -related disorder. In some embodiments, the at least one additional agent and / or therapy for treating the GB Al -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.

[0229] 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 beAttorney Docket No. 14640.0112-00304 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 hydroxychloroquine. In some embodiments, the corticosteroid comprises prednisone, prednisolone, methylprednisolone, and / or dexamethasone.Measurement of Expression

[0230] 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).

[0231] In certain embodiments, the GBA1 protein is detectable by an ELISA.

[0232] In certain embodiments, the GBA1 protein is detectable by an immunohistochemistry assay.

[0233] In certain embodiments, the GBA1 protein is detectable by Western blot.

[0234] 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 (e.g., the cortex, striatum, thalamus, cerebellum, brainstem, and / or spinal cord). 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 DevicesKitsAttorney Docket No. 14640.0112-00304

[0235] In some aspects, the present disclosure provides a variety of kits for conveniently and / or effectively carrying out methods of the 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.

[0236] Any of the vectors, constructs, or GBA1 sequences proteins 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.

[0237] 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.

[0238] 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.Attorney Docket No. 14640.0112-00304

[0239] In some embodiments, kits may include instructions for employing kit components as well as the use of any other reagent not included in the kit. Instructions may include variations that may be implemented.Devices

[0240] 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.

[0241] 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.

[0242] 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.

[0243] Methods 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

[0244] 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.

[0245] 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 present invention pertains.

[0246] The articles “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise 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 the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includesAttorney Docket No. 14640.0112-00304 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.

[0247] The term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps.

[0248] 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.

[0249] 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.

[0250] 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 insert 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. 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, 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.

[0251] Administering'. As used herein, the term “administering” refers to providing a pharmaceutical agent or composition to a subject.

[0252] 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.Attorney Docket No. 14640.0112-00304

[0253] 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.

[0254] 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.

[0255] 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 insertion and / or substitution (i.e., replacement). In some embodiments, the AAV capsid variant described herein has the ability to encapsulate (i.e., encapsidate) 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.

[0256] Cis-Elements: As used herein, cis-elements or the synonymous term “cis-regulatory elements” refer to regions of non-coding DNA which regulate the transcription of nearby genes. The Latin prefix “cis” translates to “on this side.” Cis-elements are found in the vicinity of the gene, or genes, they regulate. Examples of cis-elements include a Kozak sequence, SV40 introns, or a portion of the backbone.

[0257] CNS structures: As used herein, “CNS structures” refers to structures of the central nervous system and sub-structures thereof. Non-limiting examples of structures in the spinal cord may include, ventral horn, dorsal horn, white matter, and nervous system pathways or nuclei within. Non-limiting examples of structures in the brain include, forebrain, midbrain, hindbrain, diencephalon, telencephalon, myelencephalon, metencephalon, mesencephalon, prosencephalon, rhombencephalon, cortices, frontal lobe, parietal lobe, temporal lobe, occipital lobe, cerebrum, thalamus, hypothalamus, tectum, tegmentum, cerebellum, pons,Attorney Docket No. 14640.0112-00304 medulla, amygdala, hippocampus, basal ganglia, corpus callosum, pituitary gland, putamen, striatum, ventricles and sub-structures thereof.

[0258] CNS Cells: As used herein, “CNS cells” refers to cells of the central nervous system and sub-structures thereof. Non-limiting examples of CNS cells include, neurons and subtypes 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, large aspiny neurons, GABAergic neurons and / or glutamatergic neurons.

[0259] 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.

[0260] 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 590 of SEQ ID NO: 20 refers to the amino acid at the 590thposition from the N-terminus in SEQ ID NO: 20 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 to the designated sequence. For instance, an amino acid corresponding to position 590 of SEQ ID NO: 20 may also be referred to as an amino acid at position 590, numbered according to SEQ ID NO: 20, relative to a reference sequence of SEQ ID NO: 20, or as numbered according to a sequence corresponding to SEQ ID NO: 20.

[0261] 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 a disease, disorder, and / or condition.

[0262] Excipient'. As used herein, the term “excipient” refers to an inactive substance that serves as the vehicle or medium for an active pharmaceutical agent or other active substance.

[0263] Formulation'. As used herein, a “formulation” includes at least one active ingredient (e.g., an AAV particle) and at least one inactive ingredient (e.g., a pharmaceutically acceptable excipient).Attorney Docket No. 14640.0112-00304

[0264] 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.

[0265] 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).

[0266] 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.

[0267] 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.

[0268] Humanized'. As used herein, the term “humanized” refers to a non-human sequence of a polynucleotide or a polypeptide which has been altered to increase its similarity to a corresponding human sequence.

[0269] Identity-. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g, between oligonucleotide molecules (e.g, DNA molecules and / or RNA molecules) and / or between 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 two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, theAttorney Docket No. 14640.0112-00304 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; the relevant contents each of which are incorporated herein by reference in their 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 between 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); the relevant contents of which are incorporated herein by reference in their 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 identity may be determined using BLAST, Clustal Omega, or EMBOSS Needle. In some embodiments, sequence identity is determined using local alignment. In some embodiments, sequence identity is determined using global alignment.

[0270] 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.

[0271] 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.

[0272] 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 ofAttorney Docket No. 14640.0112-00304 component with 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.

[0273] miRNA binding site: As used herein, a “miRNA binding site” or “miR binding site” refers to an 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.

[0274] 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.

[0275] 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).

[0276] 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.

[0277] 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. Within a sequence, an amino acid position is counted from the / ' / -terminus.

[0278] Preventing'. As used herein, the term “preventing” refers to partially or completely delaying onset of a disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features,Attorney Docket No. 14640.0112-00304 or manifestations of a particular disease, disorder, and / or condition; partially or completely delaying progression from a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. The term “prevention” or “preventing” of a disease, disorder and / or condition may be considered a subset within the meaning with the term “treatment” or treating” of the disease, disorder and / or condition.

[0279] Recombinant: As used herein, the term “recombinant” refers to a biomolecule (e.g., nucleic acid or protein), cell, or organism - including but not limited to a nucleic acid, viral genome, and / or AAV particle disclosed herein - that is engineered from and / or contains genetic material from different sources (e.g., from at least two different sources). “Recombinant” may also be used in reference to technologies or laboratory techniques that combine different sources of genetic material.

[0280] Region: As used herein, the term “region” refers to a sequence portion, domain, zone, or general area. Regions may comprise terminal regions. When referring to a protein or protein module, a region may comprise a linear sequence of amino acids within the protein or protein module or may comprise a three-dimensional area. 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, a “terminal region” refers to regions located at the ends or termini of a given agent. In 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 (i.e., the start of a protein). C-termini refer to the end of a protein comprising an amino acid with a free carboxyl group. N- and / or C- terminal regions may comprise the N- and / or C-termini as well as neighboring amino acids. When referring to polynucleotides, terminal regions may comprise 5’ and / or 3’ termini. 5’ and / or 3 ’-terminal regions may comprise the 5’ and / or 3 / termini as well as neighboring nucleotides.

[0281] 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).

[0282] 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 sub-species level.Attorney Docket No. 14640.0112-00304

[0283] Signal Sequence: As used herein, the phrase “signal sequence” refers to a sequence which can direct the transport or localization.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] Split Dose: 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.

[0288] 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 / or the subject or patient may be susceptible to, suspected of having, or have a GBA1- related disorder.

[0289] 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.Attorney Docket No. 14640.0112-00304

[0290] 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.

[0291] 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 lifestyles 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.

[0292] Target Cell: As used herein, a “target cell” refers to one or more cells of interest. The target cell 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 subject. In the context of delivery of an AAV particle, a target cell may refer to one or more cells to which the AAV particle is delivered (e.g., preferentially delivered) and / or one or more cells that the AAV particle transduces. In the context of expression of a GB Al -encoding sequence or the effects of a GB Al -encoding sequence, a target cell may refer to one or more cells in which expression of GBA1 is desired or achieved and / or one or more cells in which an increase in GBA1 (e.g., a gene, mRNA, or protein expression and / or level) is desired or achieved. A target cell may be comprised in a “target tissue.”

[0293] Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject, elicits a desired biological and / or pharmacological effect.

[0294] 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 a disease, disorder, and / or condition, to treat, improve symptoms of, delayAttorney Docket No. 14640.0112-00304 progression of symptoms, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition.

[0295] Total Daily Dose: 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.

[0296] Treating'. As used herein, the term “treating” refers to partially or completely alleviating (e.g., alleviating at least one symptom), ameliorating, improving, relieving, delaying onset of, inhibiting or slowing progression of (e.g., stabilizing), 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 (e.g., a subject exhibiting at least one symptom) for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0297] Unit Dose: 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.

[0298] 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.

[0299] 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.

[0300] Viral genome'. As used herein, a “viral genome,” “vector genome,” or “VG” is a polynucleotide comprising at least one nucleotide sequence encoding GBA1. A viral genome comprises at least one copy of a GB Al -encoding sequence. A viral genome may be a recombinant viral genome.Attorney Docket No. 14640.0112-00304EXAMPLESExample 1. Individual Capsid Characterization of TTM-043 in NHPs

[0301] This example describes the transduction level, tropism, ability to cross the blood brain barrier, and overall spatial distribution in the central nervous system (CNS) and peripheral tissues of the AAV capsid variant TTM-043 relative to AAV9 following intravenous administration in cynomolgus macaques (Macaca fascicularis).Example 1A.

[0302] AAV particles were generated with the TTM-043 capsid variant or the AAV9 capsid control which comprised a self-complementary viral genome encoding a histone H2b protein driven by a ubiquitous CAG promoter. The AAV particles were administered to a first group of male cynomolgus macaques (Macaca fascicularis,' 4-6 kg body weight; over 2 years old) intravenously in a single solution, at a total dose per group of 2el3 VG / kg or a dose per capsid of 4el2 VG / kg. 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 / 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.

[0303] As shown in Table 8, TTM-043 demonstrated increased CNS transduction and / or biodistribution in several regions of the brain (greater than 30% of cells transduced observed in several regions for multiple capsid variants) and spinal cord of the cynomolgus macaques after intravenous administration at a relatively low dose of 4el2 vg / kg. More specifically, TTM-043 was capable of transducing up to 30% of cells in the caudate, putamen, and cortex. TTM-043 also showed improved delivery to the spinal cord relative to AAV9 at the dose of 4el2 vg / kg.

[0304] Cell-typing was also performed in the putamen, substantia nigra, and temporal cortex of the brain to measure the percent of neurons (NeuN+ cells) and astrocytes (Sox9+ cells) that were transduced by the AAV particles comprising the TTM-043 capsid variant or the AAV9 controls (Table 9). TTM-043 was capable of transducing up to 31.5% of neurons and 62.6% of astrocytes in the putamen; 13.4% of neurons and 63.4% of astrocytes in the temporal cortex; and 33.7% of neurons and 28.7% of astrocytes in the substantia nigra (Table 9). Co-localization of TTM-043 with motor neurons (ChAT+ cells) was also observed in the spinal cord by immunofluorescence microscopy (Table 9). Across the lumbar, cervical, and thoracic spinal cord, TTM-043 was capable of transducing 67.5% of motor neurons (Table 9).Attorney Docket No. 14640.0112-00304

[0305] In the peripheral tissues, the TTM-043 capsid variant exhibited robust liver detargeting relative to AAV9 (Table 10).Table 8. 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 macaquesTable 9. Quantification of neurons (%NeuN positive cells), motor neurons (%chAT positive cell) and / or astrocytes (% Sox9 cells) transduced with TTM-043 in the putamen, temporal cortex, substantia nigra, and spinal cordAttorney Docket No. 14640.0112-00304Table 10. 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

[0306] Taken together, these data demonstrate that TTM-043 is an enhanced CNS-tropic capsid in cynomolgus macaques that is capable of crossing the blood brain barrier following intravenous injection, even at a low dose of 4el2 vg / kg. TTM-043 was also capable of transducing both neurons and astrocytes in several brain tissues as well as motor neurons in the spinal cord. Further, TTM-043 demonstrated robust liver de-targeting relative to AAV9. Example IB.

[0307] AAV particles were generated with the TTM-043 capsid variant which each comprised a self-complementary viral genome encoding a histone H2b protein with an HA tag driven by a ubiquitous CAG promoter. The AAV particles comprising the TTM-043 capsid variant were administered to cynomolgus macaques (Macaca fascicularis) (n=3 male monkeys; 7.4-11 years of age) intravenously at a dose of 3el3 VG / kg. The in-life period was 28 days and then various CNS and peripheral tissues were collected for measuring transgene mRNA (expression) by RT-qPCR, viral DNA (biodistribution) by ddPCR, andAttorney Docket No. 14640.0112-00304 immunohistochemistry (IHC) / chromogenic and immunofluorescent quantification of percent positive cells in various tissues (cellular tropism).

[0308] As shown in Table 11, substantial and widespread transduction of TTM-043 was observed in the brain and spinal cord of NHPs following intravenous administration of the AAV particles comprising the TTM-043 capsid variant. More specifically, TTM-043 demonstrated superior viral genome biodistribution in a variety of CNS tissues and regions, broader expression in the CNS as shown by both transgene mRNA expression and IHC (Table 11 and Table 12), as well as a highly neurotropic and astrocytic tropism in the brain and the spinal cord (Table 12 and Table 13). TTM-043 when administered intravenously at a dose of 3el3 vg / kg was capable of transducing up to 7-66% of neurons (HA and NeuN positive cells) and 72-96% of astrocytes (HA and Sox9 positive cells) in multiple brain regions; up to 87% of Purkinje Neurons in the cerebellum; up to 93-100% of motor neurons (HA and ChAT positive cells) in the spinal cord; and up to 82-98% of astrocytes (HA and Sox9 positive cells) in the spinal cord (Table 12). TTM-043 was also able to transduce 84% of the dopaminergic neurons in the substantia nigra, as indicated by cells that were positive for both tyrosine hydroxylase (TH) and HA (payload tag). The TTM-043 capsid variant was well tolerated in the NHPs.Table 11: Quantification of viral genome copies per diploid genome (vg / dg) (biodistribution) by ddPCR; transgene mRNA expression by RT-qPCR (mRNA = transgene mRNA expression relative to a housekeeping gene (mTBP)); and the percentage of cells transduced with the TTM-043 capsid variant measured by colocalized staining of HA (payload tag) and DAB in the brain and spinal cord of NHPs (each value represents the average from 3 NHPs; for regions of the DRG, sensory neuron data are shown)Attorney Docket No. 14640.0112-00304Table 12: Quantification of the percentage of cells transduced with the TTM-043 capsid variant measured by co-localized staining of HA (payload tag) and either NeuN (neurons) or Sox9 (astrocytes) in the brain of NHPs (each value represents the average from 3 NHPs)Table 13: Quantification of the percentage of cells transduced with the TTM-043 capsid variant measured by co-localized staining of HA (payload tag) and either ChAT (motor neurons) or Sox9 (astrocytes) in the gray matter of the spinal cord of NHPs (each value represents the average from 3 NHPs)

[0309] The biodistribution and mRNA expression following intravenous administration of AAV particle comprising the TTM-043 capsid variant at a dose of 3el3 vg / kg was also measured in the peripheral tissues of the liver, heart, and muscle (vastus lateralis and gastrocnemius) as provided in Table 14). TTM-043 showed very low biodistribution and mRNA expression in the liver in NHPs (Table 14), and demonstrated substantially reduced liver tropism.Attorney Docket No. 14640.0112-00304Table 14: Quantification of viral genome copies per diploid genome (vg / dg) (biodistribution) by ddPCR, and transgene mRNA expression relative to a housekeeping gene (mTBP) by RT-ddPCR in the peripheral tissues of NHPs (each value represents the average from 3 NHPs)

[0310] Taken together, the individual characterization of the TTM-043 capsid variant further demonstrates and confirms that the TTM-043 is an enhanced CNS tropic capsid in cynomolgus macaques, capable of crossing the blood brain barrier following intravenous injection.Example 2: Individual Characterization of TTM-043 in Mice

[0311] 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 brain, heart, and liver of TTM-043 relative to AAV9 following intravenous injection in mice.

[0312] AAV particles were generated with TTM-043 or AAV9 encapsulating a self- complementary genome encoding a fluorescent reporter construct, ZsGreen-HA, driven by a CAG promoter. The TTM-043 capsid variant and AAV9 control were tested by intravenously administering, by tail vein injection, the AAV particle formulation at 1 e 13 VG / kg to three BALB / c mice. The in-life period was 28 days and then various CNS and peripheral tissues were collected for measuring biodistribution and transgene mRNA expression.

[0313] The brains isolated from mice injected with the AAV particles were assayed to calculate ZsGreen expression and / or transgene DNA. Data were provided as fold over AAV9 (Table 15). TTM-043 demonstrated increased CNS tropism and expression in the brain relative to AAV9 (Table 15). TTM-043 also demonstrated reduced mRNA and DNA expression in the liver by qPCR relative to the AAV9 control (Table 15). Similar results were observed by immunohistochemistry (IHC) staining of the brain (including the cortex, thalamus, and cerebellum), spinal cord (grey matter), and liver for transduction by AAV particles comprising TTM-043. TTM-043 demonstrated decreased staining by IHC in the liver relative to the AAV9 control. TTM-043 also demonstrated reduced staining by IHC in the heart relative to AAV9.Attorney Docket No. 14640.0112-00304Table 15. ZsGreen Expression and Transgene DNA and / or RNA expression for TTM- 043 relative to AAV9 in miceExample 3. Binding and Transcytosis of TTM-043

[0314] This Example investigates the ability of ALPL to transport the AAV capsid variant TTM-043 across the cell membrane.

[0315] The ability of ALPL to transport TTM-043 across the cell membrane (transcytosis) was investigated using a transwell transcytosis assay and Madin-Darby Canine Kidney (MDCK) cells engineered to overexpress ALPL. MDCK cells were used as they demonstrate clear apico-basolateral polarity and well-defined tight junctions.

[0316] AAV particles comprising the TTM-043 capsid variant or an AAV9 control capsid were added to the top of the MDCK cells expressing ALPL from the single clone, and the ability of these particles to move from the top to the bottom chamber was measured by qPCR. The percent of the AAV particles comprising TTM-043 capsid variant or the AAV9 control detected in the bottom chamber relative to the load was then calculated. As shown in FIG. 1, transcytosis was observed for the TTM-043 capsid variants, as evidenced by the increased levels of virus in the bottom chamber, whereas little to no transcytosis was observed for the AAV9 control.

Claims

Attorney Docket No. 14640.0112-00304CLAIMSWhat is claimed is:

1. An adeno-associated virus (AAV) particle comprising a viral genome comprising a P- glucocerebrosidase 1 (GBAl)-encoding sequence and an AAV9 capsid variant, wherein the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 22 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 thereto; wherein the AAV9 capsid variant comprises the amino acid sequence of HDSPHK (SEQ ID NO: 1) present at amino acids 252-257 numbered according to SEQ ID NO: 22 and comprises the amino acid R at position 388, the amino acid T at position 390, the amino acid L at position 392, the amino acid Q at position 393, and the amino acid L at position 394, each numbered according to the amino acid sequence of SEQ ID NO: 22.

2. The AAV particle of claim 1, wherein the AAV9 capsid variant comprises:(i) 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%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 20, wherein the amino acid sequence comprises HDSPHK (SEQ ID NO: 1) present at amino acids 454-459 numbered according to SEQ ID NO: 20 and comprises the amino acid R at position 590, the amino acid T at position 592, the amino acid L at position 594, the amino acid Q at position 595, and the amino acid L at position 596, each numbered according to the amino acid sequence of SEQ ID NO: 20; and / or(ii) 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%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 21, wherein the amino acid sequence comprises HDSPHK (SEQ ID NO: 1) present at amino acids 317-322 numbered according to SEQ ID NO: 21 and comprises the amino acid R at position 453, the amino acid T at position 455, the amino acid L at position 457, the amino acid Q at position 458, and the amino acid L at position 459, each numbered according to the amino acid sequence of SEQ ID NO: 21.Attorney Docket No. 14640.0112-003043. The AAV particle of claim 2, wherein the AAV9 capsid variant comprises:(i) an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 20;(ii) an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 21; and / or(iii) an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 22.

4. The AAV particle of claim 2 or claim 3, wherein the AAV9 capsid variant comprises:(i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 20;(ii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 21; and / or(iii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 22.

5. The AAV particle of any one of claims 1-4, wherein the AAV9 capsid variant comprises:(i) the amino acid sequence of SEQ ID NO: 20;(ii) the amino acid sequence of SEQ ID NO: 21; and / or(iii) the amino acid sequence of SEQ ID NO: 22.

6. The AAV particle of any one of claims 1-5, wherein the viral genome encodes a wildtype GBA1 protein.

7. The AAV particle of any one of claims 1-6, wherein the viral genome encodes a human GBA1 protein, a canine GBA1 protein, or an equine GBA1 protein.

8. The AAV particle of any one of claims 1-7, wherein the viral genome encodes a human GBA1 protein, optionally wherein the human GBA1 protein comprises the amino acid sequence of SEQ ID NO: 3.Attorney Docket No. 14640.0112-003049. The AAV particle of any one of claims 1-8, wherein the GB Al -encoding sequence comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 10.

10. The AAV particle of any one of claims 1-9, wherein the GB Al -encoding sequence comprises a nucleotide sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 10.

11. The AAV particle of any one of claims 1-10, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10.

12. The AAV particle of any one of claims 1-11, wherein the GBAl-encoding sequence further comprises a signal sequence-encoding sequence, wherein the signal sequenceencoding sequence comprises a nucleotide 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%, at least 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 9.

13. The AAV particle of claim 12, wherein the signal sequence-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 9.

14. The AAV particle of any one of claims 1-13, wherein the GBAl-encoding sequence comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 11.

15. The AAV particle of claim 14, wherein the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11.

16. The AAV particle of any one of claims 1-15, wherein the viral genome further comprises a promoter operably linked to the GBAl-encoding sequence.Attorney Docket No. 14640.0112-0030417. The AAV particle of claim 16, wherein the promoter comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 7.

18. The AAV particle of claim 16 or claim 17, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 7.

19. The AAV particle of any one of claims 1-18, wherein the viral genome further comprises an enhancer.

20. The AAV particle of claim 19, wherein the enhancer comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 6.

21. The AAV particle of claim 19 or claim 20, wherein the enhancer comprises the nucleotide sequence of SEQ ID NO: 6.

22. The AAV particle of any one of claims 1-21, wherein the viral genome further comprises an intron.

23. The AAV particle of claim 22, wherein the intron comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 8.

24. The AAV particle of claim 22 or claim 23, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 8.

25. The AAV particle of any one of claims 1-24, wherein the viral genome further comprises a polyadenylation (poly A) region.Attorney Docket No. 14640.0112-0030426. The AAV particle of claim 25, wherein the polyA region comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 14.

27. The AAV particle of claim 25 or claim 26, wherein the polyA region comprises the nucleotide sequence of SEQ ID NO: 14.

28. The AAV particle of any one of claims 1-27, wherein the viral genome further comprises an inverted terminal repeat (ITR).

29. The AAV particle of claim 28, wherein the ITR comprises 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%, at least 99%, or 100% identical) to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 15.

30. The AAV particle of claim 28 or claim 29, wherein the ITR comprises the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 15.

31. The AAV particle of any one of claims 28-30, wherein the viral genome comprises a 5’ ITR and a 3’ ITR, wherein the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 5 and the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 15.

32. The AAV particle of any one of claims 1-31, wherein the viral genome further comprises a nucleotide sequence encoding one or more microRNA183 (miR183) binding sites.

33. The AAV particle of claim 32, wherein the nucleotide sequence encoding the one or more miR183 binding sites encodes four miR183 binding sites.

34. The AAV particle of claim 33, wherein each of the four miR183 binding sites is encoded by a nucleotide sequence that has up to three modifications relative to the nucleotide sequence of SEQ ID NO: 12.Attorney Docket No. 14640.0112-0030435. The AAV particle of claim 33 or 34, wherein each of the four miR183 binding sites is encoded by a nucleotide sequence that comprises the nucleotide sequence of SEQ ID NO: 12.

36. The AAV particle of any one of claims 1-35, 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 a nucleotide sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 13.

37. The AAV particle of claim 36, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13.

38. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(i) a 5’ inverted terminal repeat (ITR);(ii) a promoter;(iii) the GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence that is at least 90% identical (e.g., at least 93% identical) thereto; and(iv) a 3’ ITR.

39. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(i) a 5’ inverted terminal repeat (ITR);(ii) a promoter;(iii) the GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 90% identical (e.g., at least 94% identical) thereto; and(iv) a 3’ ITR.

40. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(i) a 5’ inverted terminal repeat (ITR);Attorney Docket No. 14640.0112-00304(ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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; and(vi) a 3’ ITR.

41. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(i) a 5’ inverted terminal repeat (ITR);(ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotideAttorney Docket No. 14640.0112-00304 sequence of SEQ ID NO: 10 or a nucleotide 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;(vi) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 14 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.

42. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 5 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: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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;(vi) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 14 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; andAttorney Docket No. 14640.0112-00304(vii) a 3’ ITR comprising the nucleotide sequence of SEQ ID NO: 15 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.

43. The AAV particle of claim 42, wherein:(i) the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 5;(ii) the enhancer comprises the nucleotide sequence of SEQ ID NO: 6;(iii) the promoter comprises the nucleotide sequence of SEQ ID NO: 7;(iv) the intron comprises the nucleotide sequence of SEQ ID NO: 8;(v) the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: i i;(vi) the polyA region comprises the nucleotide sequence of SEQ ID NO: 14; and(vii) the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 15.

44. The AAV particle of any one of claims 1-43, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 16 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 any one of claims 1-44, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 16.

46. The AAV particle of any one of claims 1-31 or 38-45, wherein the viral genome consists of the nucleotide sequence of SEQ ID NO: 16.

47. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(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: 10 or a nucleotide sequence that is at least 90% identical (e.g., at least 93% identical) thereto;(iv) a nucleotide sequence encoding at least one microRNA183 (miR183) binding site, e.g., encoding a miR183 binding site series; andAttorney Docket No. 14640.0112-00304(v) a 3’ ITR.

48. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(i) a 5’ inverted terminal repeat (ITR);(ii) a promoter;(iii) the GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 90% identical (e.g., at least 94% identical) thereto;(iv) a nucleotide sequence encoding at least one microRNA183 (miR183) binding site, e.g., encoding a miR183 binding site series; and(v) a 3’ ITR.

49. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(i) a 5’ inverted terminal repeat (ITR);(ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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;(vi) a nucleotide sequence encoding at least one microRNA183 (miR183) binding site, e.g., encoding a miR183 binding site series; andAttorney Docket No. 14640.0112-00304(vii) a 3’ ITR.

50. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(i) a 5’ inverted terminal repeat (ITR);(ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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;(vi) a nucleotide sequence encoding a microRNA183 (miR183) binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13 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.

51. The AAV particle of any one of claims 1-8, wherein the viral genome comprises, in 5’ to 3’ order:(i) a 5’ inverted terminal repeat (ITR) comprising the nucleotide sequence of SEQ ID NO: 5 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;Attorney Docket No. 14640.0112-00304(ii) an enhancer comprising the nucleotide sequence of SEQ ID NO: 6 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: 7 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: 8 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 GB Al -encoding sequence, wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence that is at least 94% identical (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) thereto; or wherein the GB Al -encoding sequence comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide 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;(vi) a nucleotide sequence encoding a microRNA183 (miR183) binding site series, wherein the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13 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;(vii) a polyadenylation (poly A) region comprising the nucleotide sequence of SEQ ID NO: 14 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: 15 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.

52. The AAV particle of claim 51, wherein:(i) the 5’ ITR comprises the nucleotide sequence of SEQ ID NO: 5;(ii) the enhancer comprises the nucleotide sequence of SEQ ID NO: 6;(iii) the promoter comprises the nucleotide sequence of SEQ ID NO: 7;(iv) the intron comprises the nucleotide sequence of SEQ ID NO: 8;(v) the GBAl-encoding sequence comprises the nucleotide sequence of SEQ ID NO: i i;Attorney Docket No. 14640.0112-00304(vi) the nucleotide sequence encoding the miR183 binding site series comprises the nucleotide sequence of SEQ ID NO: 13;(vii) the polyA region comprises the nucleotide sequence of SEQ ID NO: 14; and(viii) the 3’ ITR comprises the nucleotide sequence of SEQ ID NO: 15.

53. The AAV particle of any one of claims 1-52, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 17 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.

54. The AAV particle of any one of claims 1-53, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 17.

55. The AAV particle of any one of claims 1-54, wherein the viral genome consists of the nucleotide sequence of SEQ ID NO: 17.

56. An adeno-associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 11 and an AAV9 capsid variant comprising:(i) the amino acid sequence of SEQ ID NO: 20;(ii) the amino acid sequence of SEQ ID NO: 21; and / or(iii) the amino acid sequence of SEQ ID NO: 22.

57. An adeno-associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 10 and an AAV9 capsid variant comprising:(i) the amino acid sequence of SEQ ID NO: 20;(ii) the amino acid sequence of SEQ ID NO: 21; and / or(iii) the amino acid sequence of SEQ ID NO: 22.

58. An adeno-associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 16 and an AAV9 capsid variant comprising:(i) the amino acid sequence of SEQ ID NO: 20;(ii) the amino acid sequence of SEQ ID NO: 21; and / or(iii) the amino acid sequence of SEQ ID NO: 22.Attorney Docket No. 14640.0112-0030459. An adeno-associated virus (AAV) particle comprising a viral genome comprising the nucleotide sequence of SEQ ID NO: 17 and an AAV9 capsid variant comprising:(i) the amino acid sequence of SEQ ID NO: 20;(ii) the amino acid sequence of SEQ ID NO: 21; and / or(iii) the amino acid sequence of SEQ ID NO: 22.

60. A cell comprising the AAV particle of any one of claims 1-59, 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.

61. A method of making the AAV particle of any one of claims 1-59, the method comprising:(i) providing a cell comprising the viral genome comprising a GB Al -encoding sequence and a nucleic acid encoding the AAV9 capsid variant; and(ii) incubating the cell under conditions suitable to encapsulate the viral genome in the AAV9 capsid variant; thereby making the AAV particle.

62. The method of claim 61, wherein the viral genome comprises(a) the nucleotide sequence of SEQ ID NO: 16 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(b) the nucleotide sequence of SEQ ID NO: 17 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 AAV9 capsid variant comprises:(i) the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 90% identity (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% identity) thereto;(ii) the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 90% identity (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% identity) thereto; and / orAttorney Docket No. 14640.0112-00304(iii) the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 90% identity (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% identity) thereto.

63. The method of claim 61, wherein the viral genome comprises(a) the nucleotide sequence of SEQ ID NO: 16 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(b) the nucleotide sequence of SEQ ID NO: 17 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 AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22.

64. The method of claim 63, wherein the viral genome comprises the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 17 and the AAV9 capsid variant comprises the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22.

65. The method of any one of claims 61-64, further comprising, prior to step (i), introducing into the cell a nucleic acid comprising the viral genome.

66. The method of any one of claims 61-65, further comprising, prior to step (i), introducing into the cell the nucleic acid encoding the AAV9 capsid variant.

67. The method of any one of claims 61-66, wherein the cell comprises a mammalian cell (e.g., an HEK293 cell), an insect cell (e.g., an Sf9 cell), or a bacterial cell.

68. A pharmaceutical composition comprising the AAV particle of any one of claims 1- 59, and a pharmaceutically acceptable excipient.

69. 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 68 or the AAV particle of any one of claims 1-59.Attorney Docket No. 14640.0112-0030470. The method of claim 69, wherein the cell is in a subject.

71. The method of claim 69, wherein the subject has, has been diagnosed with having, or is at risk of having a GB Al -related disorder, optionally wherein the GB Al -related disorder is a GB Al -related neurodegenerative or neuromuscular disorder.

72. The method of claim 70 or claim 71, wherein the subject has, has been diagnosed with having, or is at risk of having 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.

73. A method of treating a subject having or diagnosed with having a GB Al -related disorder, or treating at least one symptom thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 68 or the AAV particle of any one of claims 1-59, optionally wherein the GB Al -related disorder is a GB Al -related neurodegenerative or neuromuscular disorder.

74. The method of claim 73, 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.

75. The method of claim 74, wherein the GBAl-related disorder is PD.

76. The method of claim 74, wherein the GBAl-related disorder is GD (e.g., GD type 1,2, or 3).

77. The method of claim 76, wherein the GD is GD type 1.

78. The method of claim 76, wherein the GD is GD type 2.Attorney Docket No. 14640.0112-0030479. The method of claim 76, wherein the GD is GD type 3.

80. The method of claim 74, wherein the GBAl-related disorder is DLB.

81. The method of claim 74, wherein the GBAl-related disorder is LBD.

82. A method of treating a subject having or diagnosed with having Parkinson’s Disease(PD), or treating at least one symptom thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 68 or the AAV particle of any one of claims 1-59.

83. A method of treating a subject having or diagnosed with having Gaucher Disease (GD) (e.g., GD type 1, 2, or 3), or treating at least one symptom thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 68 or the AAV particle of any one of claims 1-59.

84. The method of claim 83, wherein the GD is GD type 1.

85. The method of claim 83, wherein the GD is GD type 2.

86. The method of claim 83, wherein the GD is GD type 3.

87. A method of treating a subject having or diagnosed with having Lewy Body Dementia(LBD), or treating at least one symptom thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 68 or the AAV particle of any one of claims 1-59.

88. A method of treating a subject having or diagnosed with having Dementia with Lewy Bodies (DLB), or treating at least one symptom thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 68 or the AAV particle of any one of claims 1-59.Attorney Docket No. 14640.0112-0030489. The method of any one of claims 73-88, wherein the subject has one or more mutations in the GBA1 gene.

90. The method of any one of claims 73-89, wherein the subject has lower GCase activity as compared to GCase activity in an individual who does not have a GB Al -related disorder, optionally wherein the level of GCase activity is measured by a 4-MUG assay or a SensoLyte Blue Glucocerebrosidase assay.

91. The method of any one of claims 73-90, wherein the treating results in prevention of progression of the disorder or at least one symptom of the disorder in the subject.

92. The method of any one of claims 73-91, wherein the treating results in amelioration of at least one symptom of the disorder and / or a change in one or more biomarkers of the disorder.

93. The method of claim 92, wherein the one or more biomarkers comprises 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), a level of neurofilament light chain, or a combination thereof.

94. The method of any one of claims 73-93, 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.

95. The method of any one of claims 70-94, wherein the subject is a human.

96. The method of any one of claims 70-95, wherein the AAV particle is delivered to a cell, tissue, or region of the CNS, e.g., of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord), or a combination thereof, of the subject.

97. The method of any one of claims 70-96, wherein the AAV particle or pharmaceutical composition is delivered to the subject via intravenous administration.Attorney Docket No. 14640.0112-0030498. The method of any one of claims 70-97, 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.

99. The method of claim 98, wherein the level of GBA1 protein expression is measured by an enzyme-linked immunosorbent assay (ELISA), a Western blot, or an immunohistochemistry assay.

100. The method of claim 98 or claim 99, wherein evaluating the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and / or GBA1 protein expression) is performed before and after administration of the AAV particle or pharmaceutical composition, optionally wherein the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and / or GBA1 protein expression) before administration is compared to the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and / or GBA1 protein expression) after administration.

101. The method of any one of claims 98-100, comprising evaluating the level of GBA1 expression (e.g., GBA1 gene expression, GBA1 mRNA expression, and / or GBA1 protein expression) in a cell or tissue of the central nervous system (e.g., parenchyma).

102. The method of claim 100 or claim 101, wherein the subject’s level of GBA1 protein expression after administration is increased relative to the subject’s level of GBA1 protein expression before administration.

103. The method of any one of claims 70-102, further comprising evaluating, e.g., measuring, the level of GCase activity in the subject.

104. The method of any one of claims 70-103, wherein the administration of the AAV particle or pharmaceutical composition results in an increase in:(i) GCase activity in a cell, tissue, (e.g., a cell or tissue of the CNS, e.g., putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord)), and / or fluid (e.g., CSFAttorney Docket No. 14640.0112-00304 and / or serum) of the subject relative to baseline or relative to GCase activity in an individual with a GB Al -related disorder who has not been administered the AAV particle or pharmaceutical composition;(ii) the number or level of viral genomes (VG) per cell in a CNS tissue (e.g., of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord)) of the subject relative to the number 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., of the putamen, caudate, cortex (e.g., temporal cortex, motor cortex), substantia nigra, or spinal cord (e.g., lumbar spinal cord, thoracic spinal cord, and / or cervical spinal cord)) of the subject relative to baseline or relative to GCase activity in an individual with a GB Al -related disorder who has not been administered the AAV particle or pharmaceutical composition.

105. The method of any one of claims 73-104, further comprising administering to the subject an additional agent and / or therapy suitable for treatment of the GB Al -related disorder or at least one symptom thereof.

106. The method of claim 105, wherein the 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., quetiapine, clozapine, pramipexole, rotigotine, or ropinirole), 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.

107. The method of any one of claims 70-106, further comprising administering a blood transfusion to the subject.

108. The method of any one of claims 70-107, further comprising administering an immunosuppressant to the subject.

109. The method of claim 108, wherein the immunosuppressant comprises a corticosteroid (e.g., prednisone, prednisolone, methylprednisolone, and / or dexamethasone), rapamycin, mycophenolate mofetil, tacrolimus, rituximab, and / or eculizumab hydroxychloroquine.Attorney Docket No. 14640.0112-00304110. The pharmaceutical composition of claim 68 or the AAV particle of any one of claims 1-59, for use in the treatment of a GB Al -related disorder or at least one symptom thereof in a subject; optionally wherein the GB Al -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 (LBD), 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.

111. The pharmaceutical composition or AAV particle of claim 110, wherein the GBA1- related disorder is GD (e.g., GD type 1, 2, or 3).

112. The pharmaceutical composition or AAV particle of claim 111, wherein the GD is GD type 1.

113. The pharmaceutical composition or AAV particle of claim 111, wherein the GD is GD type 2.

114. The pharmaceutical composition or AAV particle of claim 111, wherein the GD is GD type 3.

115. The pharmaceutical composition or AAV particle of claim 110, wherein the GBA1- related disorder is PD.

116. The pharmaceutical composition or the AAV particle of claim 110, wherein the GB Al -related disorder is LBD.

117. The pharmaceutical composition or the AAV particle of claim 110, wherein the GB Al -related disorder is DLB.

118. Use of the pharmaceutical composition of claim 68, the AAV particle of any one of claims 1-59, or the cell of claim 60 in the manufacture of a medicament for the treatment of a GB Al -related disorder or at least one symptom thereof in a subject; optionally wherein theAttorney Docket No. 14640.0112-00304GB Al -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.

119. The use of claim 118, wherein the GB Al -related disorder is PD.

120. The use of claim 118, wherein the GBAl-related disorder is GD (e.g., GD type 1, 2, or 3).

121. The use of claim 120, wherein the GD is GD type 1.

122. The use of claim 120, wherein the GD is GD type 2.

123. The use of claim 120, wherein the GD is GD type 3.

124. The use of claim 118, wherein the GBAl-related disorder is LBD.

125. The use of claim 118, wherein the GBAl-related disorder is DLB.

126. The AAV particle of any one of claims 1-59 or the pharmaceutical composition of claim 68 for use in a method of treating a disorder of any one of claims 73-109.

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