Compositions and methods for the treatment of neuropathy associated with glucosylceramidase beta deficiency

Abstract

This disclosure relates to compositions and methods for modifying, for example, enhancing the expression of GCase proteins, whether in vitro and / or in vivo. [Solution] Such compositions include the delivery of adeno-associated virus (AAV) particles. The compositions and methods of the present disclosure are useful for the treatment of subjects diagnosed with or suspected of having Parkinson's disease or related conditions resulting from a deficiency in the amount and / or function of the GBA gene product or related to decreased expression or protein levels of the GCase protein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 057,265, filed on 27 July 2020. The contents of the aforementioned application are incorporated herein by reference.

[0002] Sequence List This application is filed together with an electronic sequence listing. The sequence listing is titled 135333-00120_SL.txt, was created on July 23, 2021, and has a size of 6,773,307 bytes. The information in the electronic sequence listing is incorporated herein by reference in its entirety.

[0003] Described herein are compositions and methods relating to polynucleotides, such as those encoding glucosylceramidase beta (GBA) protein and peptides, for use in the treatment of Parkinson's disease (PD) and related disorders, such as Gaucher disease and Lewy body dementia (collectively, "GBA-related disorders"). In some embodiments, the compositions may be delivered by an adeno-associated virus (AAV) vector. In other embodiments, the compositions described herein may be used to treat subjects in need, such as human subjects diagnosed with GBA-related disorders or other conditions resulting from deficiencies in the amount and / or function of GBA protein, or as research tools for studying such diseases or conditions in cellular or animal models. [Background technology]

[0004] Lysosomal acid glucosylceramidase, commonly known as glucosylcerebrosidase, GCase, or D-glucosyl-N-acylsphingosingle cohydrolase, is a lysosomal membrane protein important for glycolipid metabolism. This enzyme is encoded by the glucosylceramidase beta (GBA) gene (Ensembl Gene ID No. ENSG00000177628). This enzyme, along with saposin A and saposin C, catalyzes the hydrolysis of glucosylceramide to ceramide and glucose. See Non-Patent Literature 1 (the entire contents of which are incorporated herein by reference).

[0005] Mutations in GBA are known to cause disease in humans. Homozygous or compound heterozygous GBA mutations cause Gaucher disease ("GD"). See Non-Patent Literature 2 (which is incorporated herein by reference in its entirety). Gaucher disease is one of the most common lysosomal storage disorders, with an estimated standardized birth rate of 0.4–5.8 per 100,000 in the standard population. Heterozygous GBA mutations can cause Parkinson's disease (PD). In fact, GBA mutations occur in 7–10% of all PD patients, making GBA mutations the most important genetic risk factor for PD. PD-GBA patients have reduced levels of the lysosomal enzyme beta-glucocerebrosidase (GCase), resulting in increased accumulation of the sphingoglycolipid glucosylceramide (GluCer). This correlates with worsening α-synuclein aggregation and associated neurological symptoms. Gaucher disease and Parkinson's disease (PD), as well as other lysosomal storage disorders, such as Lewy body dementia and related diseases, sometimes share a common etiology in the GBA gene. See Non-Patent Document 3 (the entire contents of which are incorporated herein by reference). There are limited treatment options for such diseases.

[0006] Therefore, there has long been a need to develop pharmaceutical compositions and methods for treating PD and other GBA-related disorders, as well as for improving GCase protein deficiencies in patients suffering from GBA-related disorders. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Vaccaro, Anna Maria, et al.Journal of Biological Chemistry 272.27(1997):16862-16867 [Non-Patent Document 2] Sardi, S. Pablo, Jesse M. Cedarbaum, and Patrik Brundin. Movement Disorders 33.5(2018):684-696 [Non-Patent Document 3] Sidransky, E. and Lopez, G. Lancet Neurol.2012 November;11(11):986-998 [Overview of the Initiative]

[0008] This disclosure addresses these challenges by providing AAV-based compositions and methods for treating GCase deficiencies in patients. Disclosed herein are compositions and methods relating to AAV-based gene delivery of GCases that improve loss of function and enhance intracellular lipid transport. The compositions and methods are useful for improving lysosomal glycosphingolipid metabolism and for delaying, halting, or reversing neurodegeneration and other symptoms of PD and GBA-related disorders (e.g., Lewy body dementia (DLB), Gaucher disease (GD)) in subjects (e.g., subjects with mutations in the GBA gene). The β-glucocerebrosidase (GBA) protein may also be referred to herein as the GCase protein.

[0009] Accordingly, in one embodiment, the Disclosure provides an isolated nucleic acid, for example a recombinant nucleic acid, comprising a transgene encoding a GBA protein, wherein the nucleotide sequence encoding the GBA protein comprises a nucleotide sequence, for example a codon-optimized nucleotide sequence, that is at least 88% (e.g., at least 89, 90, 92, 95, 96, 97, 98, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1773. In some embodiments, the nucleic acid further encodes an enhancement element, for example an enhancement element described herein.

[0010] In another embodiment, the Disclosure provides isolated nucleic acids, such as recombinant nucleic acids, comprising a transgene encoding a GBA protein and an enhancement element, wherein the encoded enhancement element optionally comprises the amino acid sequence of SEQ ID NO: 1789 or 1758, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to them; or the amino acid sequence of SEQ ID NO: 1794, 1796, or 1798, or SEQ ID NO: 1794 A cell membrane-permeable peptide comprising, optionally, an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conserved substitutions), relative to 1796 or 1798; and / or a lysosomal targeting sequence comprising, optionally, an amino acid sequence of any of sequence numbers 1800, 1802, 1804, 1806, or 1808, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conserved substitutions), relative to 1800, 1802, 1804, 1806, or 1808.

[0011] In another embodiment, the disclosure provides an isolated viral genome, such as a recombinant viral genome, comprising a nucleic acid containing a transgene encoding a GBA protein, and further comprising a nucleotide sequence encoding a miR binding site that modulates, for example, reduces the expression of the encoded GBA protein in cells or tissues of a DRG, liver, hematopoietic system, or a combination thereof. In some embodiments, the encoded miR binding site comprises a miR183 binding site. In some embodiments, the viral genome further encodes an enhancement element, such as an enhancement element described herein.

[0012] In yet another embodiment, the Disclosure provides an isolated, for example, recombinant viral genome comprising a promoter operably ligated to a nucleic acid containing a transgene encoding the GBA protein described herein. In some embodiments, the viral genome comprises nucleotide sequences encoding internal terminal repeat (ITR) sequences (e.g., ITR regions described herein), enhancers (e.g., enhancers described herein), intron regions (e.g., intron regions described herein), Kozak sequences (e.g., Kozak sequences described herein), exon regions (e.g., exon regions described herein), miR binding sites (e.g., miR binding sites described herein), and / or poly(A) signal regions (e.g., poly(A) signal sequences described herein). In some embodiments, the viral genome comprises the nucleotide sequence of SEQ ID NO: 1812 or SEQ ID NO: 1826, or a nucleotide sequence that is at least 95% identical to them. In some embodiments, the viral genome includes one of the nucleotide sequences of SEQ ID NOs. 1759-1771, SEQ ID NOs. 1809-1811, or SEQ ID NOs. 1813-1827, or a nucleotide sequence that is at least 95% identical to them.

[0013] In yet another embodiment, the disclosure provides isolated AAV particles, such as recombinant AAV particles, comprising a capsid protein and a viral genome comprising a promoter (e.g., one of the promoters described herein) operably linked to a transgene encoding a GBA protein as described herein. In some embodiments, the capsid protein comprises an AAV capsid protein. In some embodiments, the capsid protein comprises a VOY101 capsid protein, an AAV9 capsid protein, or a functional variant thereof.

[0014] In yet another embodiment, the Disclosure provides a method for constructing the viral genome described herein. The method comprises providing the viral genome described herein and nucleic acids encoding a skeletal region suitable for replication of the viral genome in cells, for example, bacterial cells (e.g., the skeletal region includes one or both of a bacterial origin of replication and a selection marker), and excising the virus from the skeletal region, for example, by cleaving nucleic acid molecules upstream and downstream of the viral genome.

[0015] In yet another embodiment, the Disclosure provides a method for producing isolated AAV particles, for example, recombinant AAV particles. The method comprises providing a host cell containing a viral genome as described herein, and incubating the host cell under conditions suitable for encapsulating the viral genome into AAV particles, for example, in the VOY101 capsid protein, thereby producing isolated AAV particles.

[0016] In yet another embodiment, the Disclosure provides a method for targeting and delivering exogenous GBA proteins. The method comprises administering an effective amount of AAV particles or a plurality of AAV particles, the AAV particles comprising a viral genome, for example, a viral genome comprising nucleic acids including a transgene encoding a GBA protein as described herein.

[0017] In yet another embodiment, the Disclosure provides a method for treating a subject having, or diagnosed with, a disease, neurological disorder, or neuromuscular disorder associated with GBA expression. The method comprises administering an effective amount of one or more AAV particles as described herein, wherein the AAV particles comprise a viral genome comprising nucleic acids including a transgene encoding the GBA protein as described herein, for example. In some embodiments, the disease or neurodegenerative or neuromuscular disorder associated with GBA expression includes Parkinson's disease (PD) (e.g., PD associated with mutations in the GBA gene), Lewy body dementia (DLB), Gaucher disease (GD), spinal muscular atrophy (SMA), multiple system atrophy (MSA), or multiple sclerosis (MS).

[0018] In some embodiments, the disclosure provides an AAV virus genome comprising at least one terminal inverted repeat (ITR) and a payload region, the payload region encoding one or more GCase proteins, including a GCase peptide. In some embodiments, the AAV virus genome comprises a 5'ITR, a promoter, a payload region comprising a nucleotide sequence encoding a GCase protein, and a 3'ITR. The encoded protein may be human (Homo sapiens) GCase, cynomolgus monkey (Macaca fascicularis) GCase, or rhesus monkey (Macaca mulatta) GCase, synthetic (non-natural) GCase, or derivatives thereof, such as a variant retaining the function of one or more wild-type GCase proteins. In some embodiments, the GCase may be at least partially humanized.

[0019] The GCases of this disclosure may be co-expressed with saposin proteins. In some embodiments, the transgene encoding the GCase includes a nucleotide sequence encoding a saposin protein. In some embodiments, the saposin protein is saposin A (SapA). In some embodiments, the saposin protein is saposin C (SapC).

[0020] The viral genome may be incorporated into the AAV particle, which comprises the viral genome and a capsid. In some embodiments, the capsid comprises the sequence shown in Table 1.

[0021] In some embodiments, the AAV particles described herein may be used in pharmaceutical compositions. These pharmaceutical compositions may be used to treat disorders or conditions associated with reduced GCase expression, activity, or protein levels. In some embodiments, the disorder or condition is a lysosomal lipid storage disorder. In some embodiments, the disorder or condition associated with reduced GCase protein levels is PD (e.g., PD associated with GBA gene mutations), Gaucher disease (e.g., type 1 GD (e.g., non-neuropathic GD), type 2 (e.g., acute neuropathic GD), or type 3 GD), or other GBA-related disorders (e.g., Lewy body dementia (DLB)). In some embodiments, administration of AAV particles may result in enhanced GCase expression in target cells.

[0022] In some embodiments, the Disclosure provides a method for increasing patient GCase enzyme activity using AAV-mediated gene transduction of an optimized GBA transgene cassette. AAV-mediated gene transduction can be optimized to achieve broad CNS distribution, thereby reducing substrate sphingoglycolipid glucosylceramide / GluCer levels and α-synuclein pathology, and delaying or reversing disease onset in patients with GBA-related disorders, including GBA patients with Parkinson's disease (GBA-PD), Gaucher disease (e.g., type 2 or 3 GD), and Lewy body dementia. In some embodiments, the method involves intrastriatal (ISTR) or intracisional (ICM) administration of an AAV vector packaging a GBA gene supplement transgene cassette optimized as described herein to achieve broad cell-autonomous transduction and cross-collection of therapeutic GCase enzymes.

[0023] Those skilled in the art will be able to recognize or confirm numerous equivalents of the specific embodiments of the present invention described herein simply by using routine experiments. Such equivalents are intended to be encompassed by the embodiments listed below.

[0024] Enumerated embodiments 1. Isolated nucleic acids, e.g., recombinant nucleic acids, comprising a transgene encoding a β-glucocerebrosidase (GBA) protein, wherein the nucleotide sequence encoding the GBA protein is at least 88% (e.g., at least 89, 90, 92, 95, 96, 97, 98, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1773, e.g., isolated nucleic acids comprising a codon-optimized nucleotide sequence.

[0025] 2. An isolated nucleic acid according to Embodiment 1, comprising a nucleotide sequence in which the nucleotide sequence encoding the GBA protein is at least 90% identical to SEQ ID NO: 1773.

[0026] 3. An isolated nucleic acid according to Embodiment 1 or 2, comprising a nucleotide sequence in which the nucleotide sequence encoding the GBA protein is at least 95% identical to SEQ ID NO: 1773.

[0027] 4. An isolated nucleic acid according to any one of Embodiments 1 to 3, wherein the nucleotide sequence encoding the GBA protein comprises the nucleotide sequence of Sequence ID No. 1773. 5. An isolated nucleic acid according to any one of embodiments 1 to 4, further comprising an enhancement element.

[0028] 6. Isolated nucleic acids, such as recombinant nucleic acids, comprising a transgene encoding a β-glucocerebrosidase (GBA) protein and an enhancement element, wherein the encoded enhancement element is: (a) Saposin C polypeptide or a functional fragment or variant thereof, optionally comprising the amino acid sequence of SEQ ID NO: 1789 or 1758, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto; (b) Cell membrane permeable peptides comprising, by any choice, the amino acid sequence of SEQ ID NO: 1794, 1796, or 1798, or an amino acid sequence of SEQ ID NO: 1794, 1796, or 1798 having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions); and / or (c) Isolated nucleic acids comprising a lysosomal targeting sequence comprising, by choice, the amino acid sequence of any of sequence numbers 1800, 1802, 1804, 1806, or 1808, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), to sequence numbers 1800, 1802, 1804, 1806, or 1808.

[0029] 7. An isolated viral genome, e.g., a recombinant viral genome, comprising a nucleic acid containing a transgene encoding a β-glucocerebrosidase (GBA) protein, and further comprising a nucleotide sequence encoding a miR binding site that modulates, for example, reduces the expression of the encoded GBA protein in cells or tissues of the DRG, liver, hematopoietic system, or a combination thereof.

[0030] 8. The viral genome according to Embodiment 7, wherein the nucleic acid further encodes an enhancement element. 9. An isolated nucleic acid according to Embodiment 5 or 6, or a viral genome according to Embodiment 8, wherein the encoded enhancement element comprises a saposin C polypeptide or a functional fragment or variant thereof.

[0031] 10.(i) The encoded saposin C polypeptide or its functional fragment or variant contains and / or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 1789 or 1758. (ii) The nucleotide sequence encoding the encoded saposin C polypeptide or a functional fragment or variant thereof includes the nucleotide sequence of SEQ ID NO: 1787 or 1791, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to them, Isolated nucleic acids as described in Embodiments 5-6 or 9, or viral genomes as described in Embodiment 8 or 9.

[0032] 11. (i) The coded enhancement element includes any amino acid sequence of sequence numbers 1750, 1752, 1754, 1756-1758, 1784, or 1785, an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), to sequence numbers 1750, 1752, 1754, 1756-1758, 1784, or 1785, and / or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to them, and / or (ii) The nucleotide sequence encoding the enhancement element includes one of the nucleotide sequences of SEQ ID NOs. 1751, 1753, 1755, 1858, or 1859, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to them, The isolated nucleic acid described in Embodiment 5, or the viral genome described in Embodiment 8.

[0033] 12. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-11, or a viral genome according to Embodiments 8-11, wherein the encoded enhancement element comprises a cell membrane-permeable peptide.

[0034] 13. (i) The cell membrane permeable peptide comprises the amino acid sequence of any of SEQ ID NOs. 1794, 1796, or 1798, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NOs. 1794, 1796, or 1798. (ii) The nucleotide sequence encoding the cell membrane permeable peptide includes the nucleotide sequence of SEQ ID NO: 1793, 1795, or 1797, or a nucleotide sequence that is at least 80% (e.g., 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to them, An isolated nucleic acid according to Embodiment 6 or 12, or a viral genome according to Embodiment 12.

[0035] 14. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-13, or a viral genome according to any one of Embodiments 8-13, wherein the encoded enhancement element includes a lysosomal targeting sequence.

[0036] 15. (i) The encoded lysosome targeting sequence includes the amino acid sequence of any of sequence numbers 1800, 1802, 1804, 1806, or 1808, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to sequence numbers 1800, 1802, 1804, 1806, or 1808, (ii) The nucleotide sequence encoding the lysosome targeting sequence includes the nucleotide sequence of SEQ ID NOs. 1799, 1801, 1803, 1805, or 1807, or a nucleotide sequence having at least one, two, or three but four or fewer modifications, e.g., substitutions (e.g., conservative substitutions) relative to SEQ ID NOs. 1799, 1801, 1803, 1805, or 1807, An isolated nucleic acid as described in Embodiment 6 or 14, or a viral genome as described in either Embodiment 14.

[0037] 16. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-15, wherein the nucleic acid encodes at least 2, 3, 4 or more enhancement elements, or a viral genome according to any one of Embodiments 8-15.

[0038] 17. Nucleic acids encode two enhancement elements, (i) The first enhancement element comprises a lysosome-targeting sequence, which optionally comprises the amino acid sequence of SEQ ID NO: 1802, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 1802. (ii) The second enhancing element comprises a saposin C polypeptide or a functional fragment or variant thereof, which optionally comprises the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 1789. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-16, or a viral genome according to any one of Embodiments 8-16.

[0039] 18. The isolated nucleic acid or viral genome according to Embodiment 17, wherein the nucleic acids encoding the first and second enhancement elements include the nucleotide sequences of SEQ ID NOs. 1801 and 1787, nucleotide sequences that are at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to SEQ ID NOs. 1801 and 1787, or nucleotide sequences having at least one, two, or three but four or fewer modifications, e.g., substitutions (e.g., conservative substitutions) relative to SEQ ID NOs. 1801 and 1787.

[0040] 19. The nucleic acid encodes the first and second enhancement elements, (i) The first enhancing element comprises a cell membrane permeable peptide, which optionally comprises the amino acid sequence of SEQ ID NO: 1798, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 1798. (ii) The second enhancement element includes a lysosome-targeting sequence, which optionally includes the amino acid sequence of SEQ ID NO: 1802, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 1802. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-17, or a viral genome according to any one of Embodiments 8-18.

[0041] 20. The isolated nucleic acid or viral genome according to Embodiment 19, wherein the nucleic acids encoding the first and second enhancement elements include the nucleotide sequences of SEQ ID NOs. 1797 and 1801, nucleotide sequences that are at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to SEQ ID NOs. 1797 and 1801, or nucleotide sequences having at least one, two, or three but four or fewer modifications, e.g., substitutions (e.g., conservative substitutions) relative to SEQ ID NOs. 1797 and 1801.

[0042] 21. The nucleic acid encodes the first enhancement element, the second enhancement element, and the third enhancement element. (i) The first enhancement element comprises a lysosome-targeting sequence, which optionally comprises the amino acid sequence of SEQ ID NO: 1802, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 1802. (ii) The second enhancing element comprises a cell membrane permeable peptide, which optionally comprises the amino acid sequence of SEQ ID NO: 1798, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions) relative to SEQ ID NO: 1798. (iii) The third enhancing element comprises a saposin C polypeptide or a functional fragment or variant thereof, which optionally comprises the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 1789. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-20, or a viral genome according to any one of Embodiments 8-20.

[0043] 22. The isolated nucleic acid or viral genome according to Embodiment 21, wherein the nucleic acids encoding the first enhancement element, the second enhancement element, and the third enhancement element include the nucleotide sequences of SEQ ID NOs. 1801, 1797, and 1787, nucleotide sequences that are at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to SEQ ID NOs. 1801, 1797, and 1787, or nucleotide sequences having at least one, two, or three but four or fewer modifications, e.g., substitutions (e.g., conserved substitutions), compared to SEQ ID NOs. 1801, 1797, and 1787.

[0044] 23. An isolated nucleic acid according to any one of Embodiments 1-6 or 9-22, or a viral genome according to any one of Embodiments 7-22, wherein the nucleic acid further encodes a linker.

[0045] 24. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-22, or a viral genome according to any one of Embodiments 8-22, wherein the encoded enhancement element and the encoded GBA protein are directly linked, for example, without a linker.

[0046] 25. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-23, or a viral genome according to any one of Embodiments 8-23, wherein an encoded enhancement element and an encoded GBA protein are linked via an encoded linker.

[0047] 26.(i) The coded linker includes the amino acid sequence of any of sequence numbers 1854, 1855, 1843, or 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions) to sequence numbers 1854, 1855, 1843, or 1845, (ii) The nucleotide sequence encoding the linker is any of the nucleotide sequences in Table 2, or a nucleotide sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions) relative to the sequences in Table 2. (iii) The nucleotide sequence encoding the linker comprises one of the nucleotide sequences of SEQ ID NOs. 1724, 1726, 1729, or 1730, or a nucleotide sequence having at least one, two, or three but four or fewer modifications, e.g., substitutions (e.g., conservative substitutions) relative to SEQ ID NOs. 1724, 1726, 1729, or 1730, (iv) The coded linker includes a furin cutting site, (v) The coded linker contains the T2A polypeptide, (vi) The coded linker includes (Gly4Ser)n linker (sequence number 1871) (wherein n is 1 to 10, e.g., n is 3, 4, or 5) and / or (vii) The coded linker includes the (Gly4Ser)3 linker (sequence number 1845), An isolated nucleic acid or viral genome as described in Embodiment 23 or 25.

[0048] 27.(i) The coded linker includes the amino acid sequence of SEQ ID NO: 1854 and / or the amino acid sequence of SEQ ID NO: 1855, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, to SEQ ID NO: 1854 and / or SEQ ID NO: 1855, and / or (ii) The nucleotide sequence encoding the linker includes the nucleotide sequence of SEQ ID NO: 1724 and / or the nucleotide sequence of SEQ ID NO: 1726, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications, such as substitutions, relative to SEQ ID NO: 1724 and / or SEQ ID NO: 1726. An isolated nucleic acid or viral genome according to Embodiment 23 or any one of Embodiments 25-26.

[0049] 28.(i) The coded linker includes the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, relative to SEQ ID NO: 1845. (ii) The nucleotide sequence encoding the linker is the nucleotide sequence of SEQ ID NO: 1730, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications, such as substitutions, relative to SEQ ID NO: 1730. An isolated nucleic acid according to Embodiment 23 or any one of Embodiments 25-27, or a viral genome according to any one of Embodiments 23 or 25-26.

[0050] 29. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-28, or a viral genome according to any one of Embodiments 8-28, wherein the encoded GBA protein and the encoded enhancement element are expressed as a single polypeptide.

[0051] 30. An isolated nucleic acid according to any one of Embodiments 5-6 or 9-28, or a viral genome according to any one of Embodiments 8-28, wherein the single polypeptide comprises a cleavage site located between an encoded GBA protein and an encoded enhancement element, and optionally the cleavage site is a T2A and / or a furin cleavage site.

[0052] 31. (i) The nucleotide sequence encoding the enhancement element is located 5' to the nucleotide sequence encoding the GBA protein, and / or (ii) an isolated nucleic acid according to any one of Embodiments 5-6 or 9-30, or a viral genome according to any one of Embodiments 8-30, wherein the nucleotide sequence encoding the enhancement element is located 3' to the nucleotide sequence encoding the GBA protein.

[0053] 32. An isolated nucleic acid according to any one of Embodiments 1 to 6 or 9 to 31, or a viral genome according to any one of Embodiments 7 to 31, wherein the encoded GBA protein comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical to the amino acid sequence of Sequence ID No. 1775.

[0054] 33. An isolated nucleic acid according to any one of Embodiments 6 or 9-32, or a viral genome according to any one of Embodiments 7-32, wherein the nucleotide sequence encoding the GBA protein includes any one of the nucleotide sequences of SEQ ID NOs. 1773, 1777, or 1781, or a nucleotide sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical to them.

[0055] 34. An isolated nucleic acid according to any one of Embodiments 1-6 or 9-33, or a viral genome according to any one of Embodiments 7-33, wherein the nucleotide sequence encoding the GBA protein includes the nucleotide sequence of Sequence ID No. 1773.

[0056] 35. An isolated nucleic acid according to any one of Embodiments 6 or 9-33, or a viral genome according to any one of Embodiments 7-33, wherein the nucleotide sequence encoding the GBA protein includes the nucleotide sequence of Sequence ID No. 1777.

[0057] 36. An isolated nucleic acid according to any one of Embodiments 6 or 9-33, or a viral genome according to any one of Embodiments 7-33, wherein the nucleotide sequence encoding the GBA protein comprises the nucleotide sequence of Sequence ID No. 1781.

[0058] 37. An isolated nucleic acid according to any one of Embodiments 1-6 or 9-36, or a viral genome according to any one of Embodiments 7-36, wherein the nucleotide sequence encoding the GBA protein is codon-optimized.

[0059] 38. An isolated nucleic acid according to any one of Embodiments 1-6 or 9-37, or a viral genome according to any one of Embodiments 7-37, further encoding a signal sequence.

[0060] 39. An isolated nucleic acid or viral genome according to Embodiment 38, wherein the encoded signal sequence comprises the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto.

[0061] 40. An isolated nucleic acid or viral genome according to Embodiment 38 or 39, wherein the encoded signal sequence comprises the amino acid sequence of SEQ ID NO: 1857, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto.

[0062] 41. An isolated nucleic acid or viral genome according to any one of Embodiments 38 to 40, wherein the nucleotide sequence encoding the signal sequence comprises one of the nucleotide sequences of SEQ ID NOs. 1850-1852 or 1856, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to them.

[0063] 42. The nucleotide sequence that codes for the signal sequence is (i) the 5' side relative to the nucleotide sequence encoding the GBA protein; and / or (ii) An isolated nucleic acid or viral genome according to any one of embodiments 38 to 41, located 5' to the encoded enhancement element.

[0064] 43.(i) The nucleotide sequence encoding the signal sequence includes the nucleotide sequence of SEQ ID NO: 1850 or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the nucleotide sequence encoding the GBA protein includes the nucleotide sequence of SEQ ID NO: 1773 or a nucleotide sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, (ii) The nucleotide sequence encoding the signal sequence includes the nucleotide sequence of SEQ ID NO: 1851 or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the nucleotide sequence encoding the GBA protein includes the nucleotide sequence of SEQ ID NO: 1777 or a nucleotide sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, (iii) The nucleotide sequence encoding the signal sequence includes a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1852, and the nucleotide sequence encoding the GBA protein includes a nucleotide sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1781, By arbitrary selection, the nucleotide sequence encoding the signal sequence is located 5' to the nucleotide sequence encoding the GBA protein. An isolated nucleic acid or viral genome as described in any one of embodiments 38 to 42.

[0065] 44. The encoded signal sequence includes an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 1853, and the encoded GBA protein includes an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 1775, By arbitrary selection, the encoded signal sequence is located at the N-terminus of the encoded GBA protein. An isolated nucleic acid or viral genome as described in any one of embodiments 38 to 43.

[0066] 45. (i) The nucleotide sequence encoding the signal sequence includes one of the nucleotide sequences of SEQ ID NOs. 1850 to 1852, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to them, and the nucleotide sequence encoding the enhancement element includes one of the nucleotide sequences of SEQ ID NOs. 1801, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical to it, and optionally, the nucleotide sequence encoding the signal sequence is located 5' to the nucleotide sequence encoding the enhancement element. (ii) The encoded signal sequence includes the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the encoded enhancement element includes the amino acid sequence of SEQ ID NO: 1802, or an amino acid sequence having at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions, relative to SEQ ID NO: 1802, and optionally the encoded signal sequence is located at the N-terminus of the encoded enhancement element. (iii) The nucleotide sequence encoding the signal sequence includes the nucleotide sequence of SEQ ID NO: 1856, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the nucleotide sequence encoding the enhancement element includes the nucleotide sequence of SEQ ID NO: 1859, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the nucleotide sequence encoding the enhancement element includes the nucleotide sequence of SEQ ID NO: 1859, and optionally, the nucleotide sequence encoding the signal sequence is located 5' to the nucleotide sequence encoding the enhancement element. (iv) The encoded signal sequence includes the amino acid sequence of SEQ ID NO: 1857 or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the encoded enhancement element includes the amino acid sequence of SEQ ID NO: 1785 or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and optionally the encoded signal sequence is located at the N-terminus of the encoded enhancement element, (v) The nucleotide sequence encoding the signal sequence includes the nucleotide sequence of SEQ ID NO: 1856, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the nucleotide sequence encoding the enhancement element includes the nucleotide sequence of SEQ ID NO: 1787, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the nucleotide sequence encoding the enhancement element includes the nucleotide sequence of SEQ ID NO: 1787, and optionally, the nucleotide sequence encoding the signal sequence is located 5' to the nucleotide sequence encoding the enhancement element. (vi) The encoded signal sequence includes the amino acid sequence of SEQ ID NO: 1857, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the encoded enhancement element includes the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence having at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions, relative to SEQ ID NO: 1789, and optionally the encoded signal sequence is located at the N-terminus of the encoded enhancement element. (vii) The nucleotide sequence encoding the signal sequence includes the nucleotide sequence of SEQ ID NO: 1856, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the nucleotide sequence encoding the enhancement element includes the nucleotide sequence of SEQ ID NO: 1791, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the nucleotide sequence encoding the enhancement element includes the nucleotide sequence of SEQ ID NO: 1791, and optionally, the nucleotide sequence encoding the signal sequence is located 5' to the nucleotide sequence encoding the enhancement element. (viii) The encoded signal sequence includes the amino acid sequence of SEQ ID NO: 1857, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 97%, 98%, or 99%) identical thereto, and the encoded enhancement element includes the amino acid sequence of SEQ ID NO: 1758, or an amino acid sequence having at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions, relative to SEQ ID NO: 1758, and optionally the encoded signal sequence is located at the N-terminus of the encoded enhancement element. (ix) The nucleotide sequence encoding the signal sequence includes the nucleotide sequence of SEQ ID NO: 1852, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, and the nucleotide sequence encoding the enhancement element includes the nucleotide sequence of SEQ ID NO: 1793, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, and optionally the nucleotide sequence encoding the signal sequence is located 5' to the nucleotide sequence encoding the enhancement element, and / or (x) The encoded signal sequence includes the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, and the encoded enhancement element includes the amino acid sequence of SEQ ID NO: 1794, or an amino acid sequence having at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions, relative to SEQ ID NO: 1794, and optionally the encoded signal sequence is located at the N-terminus of the encoded enhancement element. An isolated nucleic acid or viral genome as described in any one of embodiments 38 to 44.

[0067] 46. ​​An isolated nucleic acid according to any one of Embodiments 1 to 6 or 9 to 45, or a viral genome according to any one of Embodiments 7 to 45, wherein the nucleic acid comprises a nucleotide sequence encoding a signal sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1850 in a 5' to 3' order, and a nucleotide sequence encoding a GBA protein that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1773, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to it.

[0068] 47. Nucleic acids are arranged in the order from 5' to 3'. (i) A nucleotide sequence encoding a signal sequence containing the nucleotide sequence of SEQ ID NO: 1852, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, and a nucleotide sequence encoding a GBA protein containing the nucleotide sequence of SEQ ID NO: 1781, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto; (ii) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1852; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1781; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1799; (iii) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1852; a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1801; and a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1781; (iv) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1852; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1781; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1803; (v) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the nucleotide sequence of90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the nucleotide sequence of, (vi) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1852; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1781; a nucleotide sequence encoding a linker containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1730; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1797; (vii) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1852; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1781; a nucleotide sequence encoding a linker containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1730; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1793; (viii) A nucleotide sequence encoding a first signal sequence containing the nucleotide sequence of SEQ ID NO: 1852 or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), a nucleotide sequence encoding a GBA protein containing the nucleotide sequence of SEQ ID NO: 1781 or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), and a nucleotide sequence encoding a furin cleavage site containing the nucleotide sequence of SEQ ID NO: 1724 or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), A nucleotide sequence encoding a T2A polypeptide, comprising the sequence and a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1726; a nucleotide sequence encoding a second signal sequence, comprising the nucleotide sequence of, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1856; and a nucleotide sequence encoding an enhancement element, comprising the nucleotide sequence of, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1859; (ix) A nucleotide sequence encoding a first signal sequence containing the nucleotide sequence of SEQ ID NO: 1852 or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto; a nucleotide sequence encoding a GBA protein containing the nucleotide sequence of SEQ ID NO: 1781 or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto; a nucleotide sequence encoding a furin cleavage site containing the nucleotide sequence of SEQ ID NO: 1724 or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto A nucleotide sequence encoding a T2A polypeptide, comprising a column and a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1726; a nucleotide sequence encoding a second signal sequence, comprising a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1856; and a nucleotide sequence encoding an enhancement element, comprising a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1787; (x) A nucleotide sequence encoding a first signal sequence containing the nucleotide sequence of SEQ ID NO: 1852 or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), a nucleotide sequence encoding a GBA protein containing the nucleotide sequence of SEQ ID NO: 1781 or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), and a nucleotide sequence encoding a furin cleavage site containing the nucleotide sequence of SEQ ID NO: 1724 or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) A nucleotide sequence encoding a T2A polypeptide, comprising a column and a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1726; a nucleotide sequence encoding a second signal sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1856; and a nucleotide sequence encoding an enhancement element that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1791; (xi) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1852; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1781; a nucleotide sequence encoding a linker containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1730; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1795; (xii) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1852; a nucleotide sequence encoding a reinforcement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1793; a nucleotide sequence encoding a linker containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1730; and a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1781; (xiii) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1852; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1781; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1807; (xiv) A nucleotide sequence encoding a first signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1852; a nucleotide sequence encoding a first enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1801; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1781; and a nucleotide sequence encoding, or at least 85% (e.g., at least 90%, 92%, 95%) of the nucleotide sequence of SEQ ID NO: 1724. A nucleotide sequence encoding a furin cleavage site containing a nucleotide sequence identical to, by 96%, 97%, 98%, or 99%, the nucleotide sequence of SEQ ID NO: 1726, or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), a nucleotide sequence encoding a T2A polypeptide containing a nucleotide sequence identical to, by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), the nucleotide sequence of, by SEQ ID NO: 1856, or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), a nucleotide sequence encoding a second enhancement element containing a nucleotide sequence identical to, by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), the nucleotide sequence of, by SEQ ID NO: 1787; (xv) A nucleotide sequence encoding a first signal sequence containing the nucleotide sequence of SEQ ID NO: 1852, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, and a nucleotide sequence encoding a GBA protein containing the nucleotide sequence of SEQ ID NO: 1781, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto. A sequence comprising a nucleotide sequence encoding a linker containing the nucleotide sequence of sequence number 1730, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, and a nucleotide sequence encoding a first enhancement element containing the nucleotide sequence of sequence number 1797, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, A nucleotide sequence encoding a furin cleavage site, comprising the nucleotide sequence of SEQ ID NO: 1724, or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), and a nucleotide sequence encoding a T2A polypeptide, comprising the nucleotide sequence of SEQ ID NO: 1726, or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), A nucleotide sequence encoding a second signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the nucleotide sequence encoding a second enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the nucleotide sequence of, or85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the nucleotide sequence of, the nucleotide sequence of, the nucleotide sequence of, the nucleotide sequence of, the nucleotide sequence of, the nucleotide sequence of, the nucleotide sequence of, the nucleotide sequence of, the nucleotide sequence of, the nucleotide sequence of, the nucleotide sequence of, (xvi) A nucleotide sequence encoding a first signal sequence containing the nucleotide sequence of SEQ ID NO: 1852, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, and a nucleotide sequence encoding a first enhancement element containing the nucleotide sequence of SEQ ID NO: 1801, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, and SEQ ID NO: 178 A nucleotide sequence encoding a GBA protein containing a nucleotide sequence of 1, or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), a nucleotide sequence encoding a linker containing a nucleotide sequence of SEQ ID NO: 1730, or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%), and a nucleotide sequence of SEQ ID NO: 1797, or the same A nucleotide sequence encoding a first enhancement element containing a nucleotide sequence identical to at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the nucleotide sequence of SEQ ID NO: 1724, or a nucleotide sequence encoding a furin cleavage site containing a nucleotide sequence identical to thereto by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the nucleotide sequence of SEQ ID NO: 1726, or at least 85% (e.g., A nucleotide sequence encoding a T2A polypeptide containing a nucleotide sequence identical to, at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%, the nucleotide sequence of SEQ ID NO: 1856, or a second signal sequence containing a nucleotide sequence identical to, at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) thereto, and a nucleotide sequence encoding, at least 85% (e.g., at least 90%, 92%,A nucleotide sequence encoding a second enhancement element containing a nucleotide sequence that is identical (95%, 96%, 97%, 98%, or 99%); (xvii) A nucleotide sequence encoding a signal sequence containing the nucleotide sequence of SEQ ID NO: 1851, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto, and a nucleotide sequence encoding a GBA protein containing the nucleotide sequence of SEQ ID NO: 1777, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto; (xviii) A nucleotide sequence encoding a first signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1851, a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1777, a nucleotide sequence encoding a furin cleavage site containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1724, a nucleotide sequence encoding a furin cleavage site A nucleotide sequence encoding a T2A polypeptide, comprising a do sequence and a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1726; a nucleotide sequence encoding a second signal sequence, comprising a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1856; and a nucleotide sequence encoding an enhancement element, comprising a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1787; (xix) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1851; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1777; a nucleotide sequence encoding a linker containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1730; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1797; (xx) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1851; a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1801; and a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1777; (xxi) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to (xxi) the nucleotide sequence of SEQ ID NO: 1851, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) thereto; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to (xxi) the nucleotide sequence of SEQ ID NO: 1777, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) thereto; a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to (xxi) the nucleotide sequence of SEQ ID NO: 1805, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) thereto; (xxii) A nucleotide sequence encoding a first signal sequence containing the nucleotide sequence of SEQ ID NO: 1850, or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%); a nucleotide sequence encoding a GBA protein containing the nucleotide sequence of SEQ ID NO: 1773, or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%); a nucleotide sequence encoding a furin cleavage site containing the nucleotide sequence of SEQ ID NO: 1724, or a nucleotide sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%); A nucleotide sequence encoding a T2A polypeptide, comprising the sequence and a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1726; a nucleotide sequence encoding a second signal sequence, comprising the nucleotide sequence of, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1856; and a nucleotide sequence encoding an enhancement element, comprising the nucleotide sequence of, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1787; (xxiii) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1850; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1773; a nucleotide sequence encoding a linker containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1730; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1797; (xxiv) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1850; a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1801; and a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1773; (xxv) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to (xxv) the nucleotide sequence of SEQ ID NO: 1850, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) thereto; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to (xxv) the nucleotide sequence of SEQ ID NO: 1773, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) thereto; a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to (xxv) the nucleotide sequence of SEQ ID NO: 1805, or a nucleotide sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) thereto; (xxvi) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1851; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1777; a nucleotide sequence encoding a linker containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1730; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1793; or (xxvii) A nucleotide sequence encoding a signal sequence containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1850; a nucleotide sequence encoding a GBA protein containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1773; a nucleotide sequence encoding a linker containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1730; and a nucleotide sequence encoding an enhancement element containing a nucleotide sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the nucleotide sequence of SEQ ID NO: 1793; An isolated nucleic acid according to any one of Embodiments 1 to 6 or 9 to 46, or a viral genome according to any one of Embodiments 7 to 46.

[0069] 48. An isolated nucleic acid according to any one of Embodiments 1 to 6 or 9 to 47, or a viral genome according to any one of Embodiments 7 to 47, wherein the nucleic acid encodes a signal sequence comprising an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto to the amino acid sequence of SEQ ID NO: 1853 in 5' to 3' order, and a GBA protein comprising an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto to the amino acid sequence of SEQ ID NO: 1775, or a viral genome according to any one of Embodiments 7 to 47.

[0070] 49. Nucleic acids are arranged in the order from 5' to 3'. (i) A signal sequence comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853; a GBA protein comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775; and an enhancement element comprising an amino acid sequence comprising, or at least 1, 2, or 3, but no more than 4, modifications, e.g., substitutions, to, SEQ ID NO: 1800; (ii) A GBA protein comprising a signal sequence containing an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853, an enhancement element containing an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1802, and an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775; (iii) A signal sequence comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853; a GBA protein comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775; and an enhancement element comprising an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1804; (iv) A signal sequence comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853; a GBA protein comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775; and an enhancement element comprising an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, to the amino acid sequence of SEQ ID NO: 1806; (v) A signal sequence comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853; a GBA protein comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775; a linker comprising an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 1845, or at least one, two, or three but no more than four modifications, such as substitutions; and an enhancement element comprising an amino acid sequence of SEQ ID NO: 1798, or at least one, two, or three but no more than four modifications, such as substitutions; (vi) A signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%); a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%); a linker comprising the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1794, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions; (vii) A first signal sequence comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853, and a GBA protein comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775, and a fuse comprising an amino acid sequence comprising an amino acid sequence of, or at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions, to SEQ ID NO: 1854. A T2A polypeptide comprising a phosphorus cleavage site and the amino acid sequence of SEQ ID NO: 1855, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, relative to SEQ ID NO: 1855; a second signal sequence comprising the nucleotide sequence of SEQ ID NO: 1857, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, relative to SEQ ID NO: 1857; and an enhancement element comprising an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1785, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) thereto; (viii) A first signal sequence comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853, and a GBA protein comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775, and a fuse comprising an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 1854, or an amino acid sequence having at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions. A T2A polypeptide comprising a phosphorus cleavage site and the amino acid sequence of SEQ ID NO: 1855, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, relative to SEQ ID NO: 1855; a second signal sequence comprising the nucleotide sequence of SEQ ID NO: 1857, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, relative to SEQ ID NO: 1857; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto; (ix) A first signal sequence comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853, and a GBA protein comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775, and a Fury protein comprising an amino acid sequence comprising, or at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions, to, SEQ ID NO: 1854. A T2A polypeptide comprising a cleavage site and the amino acid sequence of SEQ ID NO: 1855, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, relative to SEQ ID NO: 1855; a second signal sequence comprising the nucleotide sequence of SEQ ID NO: 1857, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, relative to SEQ ID NO: 1857; and an enhancement element comprising an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1758, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) thereto; (x) A signal sequence containing an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence having at least 1, 2, or 3 but 4 or fewer modifications, such as substitutions, to SEQ ID NO: 1845, and an enhancement element containing an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of, the amino acid sequence of SEQ ID NO: 1796; (xi) A GBA protein comprising a signal sequence containing the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%); an enhancement element containing the amino acid sequence of SEQ ID NO: 1794, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions; a linker containing the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions; and an amino acid sequence containing the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence identical to it by at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%); (xii) A signal sequence comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853; a GBA protein comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775; and an enhancement element comprising an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions, to SEQ ID NO: 1808; (xiii) A first signal sequence comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853; a first enhancement element comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1802; a GBA protein comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775; and a GBA protein comprising an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1854. A furin cleavage site containing at least one, two, or three, but no more than four, modifications, for example, an amino acid sequence with substitutions; the amino acid sequence of SEQ ID NO: 1855, or a T2A polypeptide containing at least one, two, or three, but no more than four, modifications, for example, an amino acid sequence with substitutions, relative to SEQ ID NO: 1855; the nucleotide sequence of SEQ ID NO: 1857, or a second signal sequence containing at least one, two, or three, but no more than four, modifications, for example, an amino acid sequence with substitutions, relative to SEQ ID NO: 1857; and a second enhancement element containing an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 1789; (xiv) A first signal sequence comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1853; a GBA protein comprising an amino acid sequence identical to or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence of SEQ ID NO: 1775; a linker comprising an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 1845, or at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions, of SEQ ID NO: 1845; and a first enhancement element comprising an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 1798, or at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions, of SEQ ID NO: 1798 A ment, a furin cleavage site containing the amino acid sequence of SEQ ID NO: 1854, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1854, e.g., an amino acid sequence with substitutions, a T2A polypeptide containing the amino acid sequence of SEQ ID NO: 1855, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1855, e.g., an amino acid sequence with substitutions, a second signal sequence containing the nucleotide sequence of SEQ ID NO: 1857, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1857, e.g., an amino acid sequence with substitutions, and an enhancement element containing the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto; or (xv) A first signal sequence containing an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence of SEQ ID NO: 1853; a first enhancement element containing an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence of SEQ ID NO: 1802; a GBA protein containing an amino acid sequence identical to, or at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence of SEQ ID NO: 1775; a linker containing an amino acid sequence having at least 1, 2, or 3 but 4 or fewer modifications, e.g., substitutions, to, or the amino acid sequence of SEQ ID NO: 1845; and at least A first enhancement element comprising one, two, or three but four or fewer modifications, e.g., an amino acid sequence having substitutions; the amino acid sequence of SEQ ID NO: 1854, or a furin cleavage site comprising at least one, two, or three but four or fewer modifications, e.g., an amino acid sequence having substitutions; the amino acid sequence of SEQ ID NO: 1855, or a T2A polypeptide comprising at least one, two, or three but four or fewer modifications, e.g., an amino acid sequence having substitutions; the nucleotide sequence of SEQ ID NO: 1857, or a second signal sequence comprising at least one, two, or three but four or fewer modifications, e.g., an amino acid sequence having substitutions; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence that is at least 85% (e.g., at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical thereto; An isolated nucleic acid according to any one of Embodiments 1-6 or 9-48, or a viral genome according to any one of Embodiments 7-48, which encodes a viral genome.

[0071] 50. An isolated viral genome, e.g., a recombinant viral genome, comprising a promoter operably linked to the nucleic acid described in any one of Embodiments 1 to 6 or Embodiments 9 to 49.

[0072] 51. A viral genome according to any one of embodiments 7 to 49, further comprising a promoter operably linked to a nucleic acid containing a transgene encoding a GBA protein. 52. A viral genome according to any one of embodiments 7 to 50, further comprising an enhancer.

[0073] 53. The viral genome according to Embodiment 52, wherein the enhancer comprises a CMVie enhancer. 54. The viral genome according to Embodiment 52 or 53, wherein the enhancer comprises the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence that is at least 95% identical thereto.

[0074] 55. A viral genome according to any one of embodiments 50 to 54, wherein the promoter comprises a tissue-specific promoter or a ubiquitous promoter. 56. The promoter, (i) EF-1a promoter, chicken β-actin (CBA) promoter and / or derivative CAG, CMV pre-initial enhancer and / or promoter, β-glucuronidase (GUSB) promoter, ubiquitin C (UBC) promoter, neuron-specific enolase (NSE), platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, intercellular adhesion molecule 2 (ICAM-2) promoter, synapsin (Syn) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 ( mGluR2 promoter, neurofilament light chain (NFL) or heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2), glial fibrillary acid protein (GFAP) promoter, myelin basic protein (MBP) promoter, cardiovascular promoters (e.g., αMHC, cTnT, and CMV-MLC2k), liver promoters (e.g., hAAT, TBG), skeletal muscle promoters (e.g., desmin, MCK, C512) or fragments thereof, e.g., cleavage or functional variants; and / or (ii) Any nucleotide sequence of sequence numbers 1832, 1833, 1834, 1835, 1836, 1839, or 1840, or any nucleotide sequence that is at least 95% identical to them; A viral genome according to any one of embodiments 50 to 55, including the viral genome described.

[0075] 57. A viral genome according to any one of embodiments 50 to 56, wherein the promoter comprises a CB promoter or a functional variant thereof. 58. The viral genome according to Embodiment 57, wherein the CB promoter or a functional variant thereof comprises the nucleotide sequence of SEQ ID NO: 1834, or a nucleotide sequence that is at least 95% identical thereto.

[0076] 59. A viral genome according to any one of embodiments 50 to 58, wherein the promoter comprises a CMVie enhancer and a CB promoter. 60. The viral genome according to Embodiment 59, wherein the CMVie enhancer comprises the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence that is at least 95% identical thereto, and the CB promoter comprises the nucleotide sequence of SEQ ID NO: 1834, or a nucleotide sequence that is at least 95% identical thereto.

[0077] 61. A viral genome according to any one of embodiments 50 to 61, wherein the promoter comprises the EF-1α promoter or a functional variant thereof. 62. The viral genome according to Embodiment 61, wherein the EF-1α promoter or a functional variant thereof comprises the nucleotide sequence of SEQ ID NO: 1839 or 1840, or a nucleotide sequence that is at least 95% identical thereto.

[0078] 63. The viral genome according to Embodiment 61 or 62, wherein the EF-1α promoter or its functional variant includes an intron, for example, an intron comprising the nucleotide sequence at positions 242-1,180 of SEQ ID NO: 1839, or an intron comprising the nucleotide sequence of SEQ ID NO: 1841, or a nucleotide sequence that is at least 95% identical thereto.

[0079] 64. The viral genome according to any one of embodiments 61 to 63, wherein the EF-1α promoter or its functional variant does not contain an intron, for example, an intron containing the nucleotide sequence at positions 242 to 1,180 of SEQ ID NO: 1839, or an intron containing the nucleotide sequence of SEQ ID NO: 1841, or a nucleotide sequence that is at least 95% identical thereto.

[0080] 65. A viral genome according to any one of embodiments 50 to 64, wherein the promoter comprises the CBA promoter or a functional variant thereof. 66. The viral genome according to Embodiment 65, wherein the CBA promoter or a functional variant thereof comprises the nucleotide sequence of SEQ ID NO: 1836, or a nucleotide sequence that is at least 95% identical thereto.

[0081] 67. A viral genome according to any one of embodiments 50 to 66, wherein the promoter comprises a CMVie enhancer, a CBA promoter or a functional variant thereof, and an intron.

[0082] 68.(i) The CMVie enhancer contains the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence that is at least 95% identical thereto. (ii) The CBA promoter or its functional variant contains the nucleotide sequence of SEQ ID NO: 1836, or a nucleotide sequence that is at least 95% identical thereto, (iii) The viral genome according to Embodiment 67, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 1837, or a nucleotide sequence that is at least 95% identical thereto.

[0083] 69. A viral genome according to any one of embodiments 50 to 68, wherein the promoter includes a CAG promoter region. 70. The promoter includes the CAG promoter region, and the CAG promoter region is (i) CMVie enhancers, CBA promoters or their functional variants, and introns; and / or (ii) The nucleotide sequence of sequence number 1835, or a nucleotide sequence that is at least 95% identical thereto; A viral genome according to any one of embodiments 50 to 69, including the viral genome described.

[0084] 71. A viral genome according to any one of Embodiments 50 to 70, wherein the promoter comprises the CMV promoter or a functional variant thereof. 72. The viral genome according to Embodiment 71, wherein the CMV promoter or a functional variant thereof comprises the nucleotide sequence of SEQ ID NO: 1832, or a nucleotide sequence that is at least 95% identical thereto.

[0085] 73. The viral genome according to any one of Embodiments 50 to 72, wherein the promoter comprises a CMVie enhancer and a CMV promoter or a functional variant thereof, and optionally, the CMVie enhancer comprises the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide sequence that is at least 95% identical thereto, and the CMV promoter or a functional variant thereof comprises the nucleotide sequence of SEQ ID NO: 1832 or a nucleotide sequence that is at least 95% identical thereto.

[0086] 74. A viral genome according to any one of embodiments 50 to 73, wherein the promoter includes a CMV promoter region. 75. The CMV promoter region, (i) CMVie enhancers and CMV promoters or their functional variants; (i) The nucleotide sequence of sequence number 1833, or a nucleotide sequence that is at least 95% identical thereto; The viral genome described in Embodiment 74, including the viral genome described in Embodiment 74.

[0087] 76. A viral genome according to any one of embodiments 7 to 76, further comprising terminal inversion repeat (ITR) sequences. 77. The viral genome according to Embodiment 76, wherein the ITR sequence is located 5' to the nucleic acid containing the transgene encoding the GBA protein.

[0088] 78. The viral genome according to Embodiment 75 or 76, wherein the ITR sequence is located 3' to the nucleic acid containing the transgene encoding the GBA protein. 79. A viral genome according to any one of embodiments 7 to 78, comprising an ITR located 5' to the nucleic acid containing the transgene encoding the GBA protein and an ITR located 3' to the nucleic acid containing the transgene encoding the GBA protein.

[0089] 80. The viral genome according to any one of embodiments 76 to 79, wherein the ITR includes the nucleic acid sequence of sequence number 1829, 1830, or 1862, or a nucleotide sequence that is at least 95% identical to thereto.

[0090] 81. A viral genome according to any one of embodiments 76 to 80, wherein the ITR comprises the nucleotide sequence of SEQ ID NO: 1860 and / or SEQ ID NO: 1861, or the nucleotide sequence of SEQ ID NO: 1860 and / or SEQ ID NO: 1861 having at least one, two, or three modifications, but four or fewer modifications.

[0091] 82. The viral genome according to any one of embodiments 76 to 81, wherein the ITR is located 5' to the nucleic acid containing the transgene encoding the GBA protein and comprises the nucleotide sequence of SEQ ID NO: 1860 and / or SEQ ID NO: 1861, or the nucleotide sequence of SEQ ID NO: 1860 or SEQ ID NO: 1861 having at least one, two, or three modifications, but four or fewer modifications.

[0092] 83. The viral genome according to any one of embodiments 76 to 81, wherein the ITR is located 3' to the nucleic acid containing the transgene encoding the GBA protein and comprises the nucleotide sequence of SEQ ID NO: 1860 or SEQ ID NO: 1861, or the nucleotide sequence of SEQ ID NO: 1860 and / or SEQ ID NO: 1861 having at least one, two, or three modifications, but four or fewer modifications.

[0093] 84.(i) The ITR located at the 5' end of the nucleic acid containing the transgene encoding the GBA protein contains the nucleotide sequence of SEQ ID NO: 1829, or a nucleotide sequence that is at least 95% identical thereto, and / or (ii) The viral genome according to any one of Embodiments 76 to 83, wherein the ITR located 3' to the nucleic acid encoding the GBA protein includes the nucleotide sequence of SEQ ID NO: 1830, or a nucleotide sequence that is at least 95% identical thereto.

[0094] 85. A viral genome according to any one of embodiments 7 to 84, further comprising a polyadenylated (poly-A) signaling region. 86. The viral genome according to Embodiment 85, wherein the poly(A) signaling region comprises the nucleotide sequence of SEQ ID NO: 1846, or a nucleotide sequence that is at least 95% identical thereto.

[0095] 87. A viral genome according to any one of embodiments 7 to 86, further comprising an intron region. 88. The viral genome according to Embodiment 87, wherein the introns include beta-globin introns.

[0096] 89. The viral genome according to Embodiment 87 or 88, wherein the intron comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1842.

[0097] 90. A viral genome according to any one of embodiments 7 to 89, further comprising, for example, at least one, two, or three exon regions. 91. A viral genome according to any one of embodiments 7 to 90, further comprising a Kozak sequence.

[0098] 92. A viral genome according to any one of embodiments 50 to 91, further comprising a nucleotide sequence encoding a miR binding site, for example, a miR binding site that modulates, for example, reduces the expression of a GBA protein encoded by the viral genome in a cell or tissue expressing the corresponding miRNA.

[0099] 93. The viral genome according to Embodiments 7-92, wherein the encoded miRNA binding sites are complementary, for example, fully complementary or partially complementary, to miRNAs expressed in cells or tissues of the DRG, liver, hematopoietic system, or a combination thereof.

[0100] 94. A viral genome according to any one of embodiments 50 to 93, wherein the encoded miR binding site modulates, for example, reduces the expression of the encoded GBA protein in cells or tissues of the DRG, liver, hematopoietic system, or a combination thereof.

[0101] 95. A viral genome according to any one of embodiments 7 to 94, comprising at least 1 to 5 copies, for example, at least 1, 2, 3, 4, or 5 copies of encoded miR binding sites.

[0102] 96. A viral genome according to any one of embodiments 7 to 95, comprising at least four copies of encoded miR binding sites, wherein all four copies optionally contain the same miR binding site, or at least one, two, three, or all copies contain different miR binding sites.

[0103] 97.4 The viral genome according to Embodiment 96, wherein the encoded miR binding sites are contiguous (e.g., not separated by spacers) or separated by spacers.

[0104] 98. The viral genome according to embodiment 97, wherein the spacer comprises the nucleotide sequence of SEQ ID NO: 1848, or a nucleotide sequence of SEQ ID NO: 1848 having at least 1, 2, or 3 modifications, but having 4 or fewer modifications.

[0105] 99. The encoded miR binding site comprises a miR183 binding site, a miR122 binding site, miR-142-3p, or a combination thereof, and optionally, (i) the encoded miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), or a nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but having 10 or fewer modifications, (ii) the encoded miR122 binding site comprises the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), or a nucleotide sequence of SEQ ID NO: 1865 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but having 10 or fewer modifications, and / or (iii) the encoded miR-142-3p binding site comprises the nucleotide sequence of SEQ ID NO: 1869, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), or a nucleotide sequence of SEQ ID NO: 1869 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but having 10 or fewer modifications, The viral genome according to any one of embodiments 7 to 98.

[0106] 100. The viral genome according to any one of embodiments 7 to 99, wherein the viral genome comprises an encoded miR183 binding site. 101. The viral genome according to any one of embodiments 7 to 100, wherein the viral genome comprises at least 1 to 5 copies, such as 4 copies, of the miR183 binding site, and optionally, each copy is contiguous (e.g., not separated by a spacer) or each copy is separated by a spacer.

[0107] 102. The viral genome according to embodiment 100 or 101, wherein the encoded miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence that is substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), or a nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications but having 10 or fewer modifications.

[0108] 103. The viral genome comprises (i) a first encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence that is substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), or a nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications but having 10 or fewer modifications, and (ii) a first spacer sequence comprising the nucleotide sequence of SEQ ID NO: 1848, or a nucleotide sequence of SEQ ID NO: 1848 having at least 1, 2, or 3 modifications but having 4 or fewer modifications. (iii) A second encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, (iv) A second spacer sequence comprising the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but four or fewer modifications, (v) A third encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, (vi) A third spacer sequence comprising the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but four or fewer modifications, (vii) A nucleotide sequence of sequence number 1847, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), or a fourth encoded miR183 binding site comprising the nucleotide sequence of sequence number 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications. A viral genome according to any one of embodiments 7 to 102, including the viral genome described.

[0109] A viral genome according to any one of embodiments 7 to 103, comprising a row of miR183 binding sites containing 104.4 copies of miR183 binding sites, wherein each copy of the miR binding site in the row is separated by a spacer.

[0110] 105. The viral genome according to Embodiment 104, wherein the encoded miR183 binding site sequence includes the nucleotide sequence of SEQ ID NO: 1849, or a nucleotide sequence that is at least 95% identical thereto.

[0111] 106. A self-complementary viral genome according to any one of embodiments 7 to 105. 107. A single-stranded viral genome as described in any one of embodiments 7 to 106.

[0112] In order from 108.5' to 3', (i) A 5' adeno-associated (AAV) ITR which optionally includes the nucleotide sequence of sequence number 1829, or a nucleotide sequence that is at least 95% identical thereto, (ii) A CMVie enhancer comprising, optionally selected, the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence that is at least 95% identical thereto, (iii) A CB promoter or a functional variant thereof, which optionally includes the nucleotide sequence of SEQ ID NO: 1834, or a nucleotide sequence that is at least 95% identical thereto, (iv) an intron which optionally includes the nucleotide sequence of sequence number 1842, or a nucleotide sequence that is at least 95% identical thereto, (v) A nucleotide sequence encoding a signal sequence, which optionally includes the nucleotide sequence of sequence number 1850, or a nucleotide sequence that is at least 95% identical thereto, (vi) A transgene encoding the GBA protein, comprising a nucleotide sequence that is at least 88% (e.g., at least 89, 90, 92, 95, 96, 97, 98, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1773, (vii) A poly-A signal region comprising, optionally, the nucleotide sequence of sequence number 1846, or a nucleotide sequence that is at least 95% identical thereto, (viii) A 3'AAV ITR which optionally includes the nucleotide sequence of SEQ ID NO: 1830, or a nucleotide sequence that is at least 95% identical thereto, and Isolated viral genomes, including recombinant viral genomes.

[0113] In order from 109.5' to 3', (i) A 5' adeno-associated (AAV) ITR which optionally includes the nucleotide sequence of sequence number 1829, or a nucleotide sequence that is at least 95% identical thereto, (ii) A CMVie enhancer comprising, optionally selected, the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence that is at least 95% identical thereto, (iii) A CB promoter or a functional variant thereof, which optionally includes the nucleotide sequence of SEQ ID NO: 1834, or a nucleotide sequence that is at least 95% identical thereto, (iv) an intron which optionally includes the nucleotide sequence of sequence number 1842, or a nucleotide sequence that is at least 95% identical thereto, (v) A nucleotide sequence encoding a signal sequence, which optionally includes the nucleotide sequence of sequence number 1850, or a nucleotide sequence that is at least 95% identical thereto, (vi) A transgene encoding the GBA protein, which optionally includes the nucleotide sequence of SEQ ID NO: 1773 or a nucleotide sequence that is at least 88% (e.g., at least 89, 90, 92, 95, 96, 97, 98, or 99%) identical to the nucleotide sequence of SEQ ID NO: 1773, (vii) Encoded miR183 binding sites, which optionally include the nucleotide sequence of SEQ ID NO: 1847, or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but 10 or fewer modifications, (viii) A spacer sequence which optionally includes the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but not more than four modifications, (ix) Encoded miR183 binding sites comprising, optionally, the nucleotide sequence of SEQ ID NO: 1847, or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but 10 or fewer modifications, (x) A spacer sequence which optionally includes the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but no more than four modifications, and a spacer, (xi) an encoded miR183 binding site comprising, optionally, the nucleotide sequence of SEQ ID NO: 1847, or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, (xii) A spacer sequence comprising, optionally selected, the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but not more than four modifications, (xiii) Encoded miR183 binding sites, which optionally include the nucleotide sequence of SEQ ID NO: 1847, or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, (xiv) A polyA signal region comprising, optionally, the nucleotide sequence of sequence number 1846, or a nucleotide sequence that is at least 95% identical thereto, (xv)3'AAV ITR, which optionally includes the nucleotide sequence of SEQ ID NO: 1830, or a nucleotide sequence that is at least 95% identical thereto, and Isolated viral genomes, including recombinant viral genomes.

[0114] 110. A viral genome according to any one of embodiments 50 to 109, comprising the nucleotide sequence of sequence number 1812, or a nucleotide sequence that is at least 95% identical thereto.

[0115] 111. A viral genome according to any one of embodiments 50 to 110, comprising the nucleotide sequence of SEQ ID NO: 1826, or a nucleotide sequence that is at least 95% identical thereto.

[0116] In the order from 112.5' ​​to 3', (i) A 5' adeno-associated (AAV) ITR which optionally includes the nucleotide sequence of sequence number 1829, or a nucleotide sequence that is at least 95% identical thereto, (ii) A CMVie enhancer, optionally comprising the nucleotide sequence of SEQ ID NO: 1831 or a nucleotide sequence that is at least 95% identical thereto, and (iii) A CB promoter or a functional variant thereof, optionally comprising the nucleotide sequence of SEQ ID NO: 1834 or a nucleotide sequence that is at least 95% identical thereto, and (iv) An intron, optionally comprising the nucleotide sequence of SEQ ID NO: 1842 or a nucleotide sequence that is at least 95% identical thereto, and (v) A nucleic acid comprising a transgene encoding a β-glucosylceramidase (GBA) protein according to any one of Embodiments 1 to 6 or Embodiments 9 to 49, and (vi) A polyA signal region, optionally comprising the nucleotide sequence of SEQ ID NO: 1846 or a nucleotide sequence that is at least 95% identical thereto, and (vii) A 3' AAV ITR, optionally comprising the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide sequence that is at least 95% identical thereto comprising an isolated viral genome, such as a recombinant viral genome.

[0117] 113. In the order from 5' to 3', (i) A 5' adeno-associated (AAV) ITR, optionally comprising the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide sequence that is at least 95% identical thereto, and (ii) An EF-1α promoter or a functional variant thereof, optionally comprising the nucleotide sequence of SEQ ID NO: 1839 or SEQ ID NO: 1840 or a nucleotide sequence that is at least 95% identical thereto, and (iii) A nucleic acid comprising a transgene encoding a β-glucocerebrosidase (GBA) protein as described in any one of Embodiments 1 to 6 or Embodiments 9 to 49, (iv) A polyA signal region comprising, optionally, the nucleotide sequence of SEQ ID NO: 1846, or a nucleotide sequence that is at least 95% identical thereto, (v) A 3'AAV ITR which optionally includes the nucleotide sequence of SEQ ID NO: 1830, or a nucleotide sequence that is at least 95% identical thereto, and Isolated viral genomes, including recombinant viral genomes.

[0118] In order from 114.5' to 3', (i) A 5' adeno-associated (AAV) ITR which optionally includes the nucleotide sequence of sequence number 1829, or a nucleotide sequence that is at least 95% identical thereto, (ii) A CMVie enhancer comprising, optionally selected, the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence that is at least 95% identical thereto, (iii) A CMV promoter or a functional variant thereof, which optionally includes the nucleotide sequence of SEQ ID NO: 1832, or a nucleotide sequence that is at least 95% identical thereto, (iv) an intron which optionally includes the nucleotide sequence of sequence number 1842, or a nucleotide sequence that is at least 95% identical thereto, (v) A nucleic acid comprising a transgene encoding a β-glucocerebrosidase (GBA) protein as described in any one of Embodiments 1 to 6 or 9 to 49, (vi) A polyA signal region comprising, optionally, the nucleotide sequence of SEQ ID NO: 1846, or a nucleotide sequence that is at least 95% identical thereto, (vii) A 3'AAV ITR which optionally includes the nucleotide sequence of SEQ ID NO: 1830, or a nucleotide sequence that is at least 95% identical thereto, and Isolated viral genomes, including recombinant viral genomes.

[0119] In the order from 115.5' to 3', (i) A 5' adeno-associated (AAV) ITR which optionally includes the nucleotide sequence of sequence number 1829, or a nucleotide sequence that is at least 95% identical thereto, (ii) A CAG promoter or a functional variant thereof, which optionally includes the nucleotide sequence of SEQ ID NO: 1835, or a nucleotide sequence that is at least 95% identical thereto, (iii) A nucleic acid comprising a transgene encoding a β-glucocerebrosidase (GBA) protein as described in any one of Embodiments 1 to 6 or Embodiments 9 to 49, (iv) A polyA signal region comprising, optionally, the nucleotide sequence of SEQ ID NO: 1846, or a nucleotide sequence that is at least 95% identical thereto, (v) A 3'AAV ITR which optionally includes the nucleotide sequence of SEQ ID NO: 1830, or a nucleotide sequence that is at least 95% identical thereto, and Isolated viral genomes, including recombinant viral genomes.

[0120] 116. A viral genome according to any one of Embodiments 7-107 or 112-115, comprising a nucleotide sequence described in any one of Sequence IDs 1759-1771, 1809-1811, 1813-1827, or 1870, or a nucleotide sequence that is at least 95% identical thereto.

[0121] 117. A viral genome according to any one of Embodiments 7 to 116, further comprising a capsid protein, for example, a nucleic acid encoding a structural protein, wherein the capsid protein comprises a VP1 polypeptide, a VP2 polypeptide, and / or a VP3 polypeptide.

[0122] 118. The viral genome according to Embodiment 117, wherein the VP1 polypeptide, VP2 polypeptide, and / or VP3 polypeptide are encoded by at least one Cap gene.

[0123] 119. A viral genome according to any one of Embodiments 7 to 118, further comprising a nucleic acid encoding a Rep protein, for example, a non-structural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68 protein, a Rep52 protein, and / or a Rep40 protein.

[0124] 120. The viral genome according to Embodiment 119, wherein the Rep78 protein, Rep68 protein, Rep52 protein, and / or Rep40 protein are encoded by at least one Rep gene.

[0125] 121. Isolated GBA protein, e.g., recombinant GBA protein, encoded by any one of the isolated nucleic acids of Embodiments 1-6 or 9-49, or by the viral genome described in any one of Embodiments 7-120.

[0126] 122. (i) Capsid proteins and, (ii) The viral genome described in any one of Embodiments 7 to 120 and Isolated AAV particles, including recombinant AAV particles.

[0127] 123.(i) The capsid protein contains the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. (ii) The capsid protein contains the amino acid sequence of SEQ ID NO: 138, which has at least 1, 2, or 3 modifications, but 30, 20, or 10 or fewer modifications. (iii) The capsid protein contains the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto, (iv) The capsid protein contains the amino acid sequence of SEQ ID NO: 11, which has at least 1, 2, or 3 modifications, but 30, 20, or 10 or fewer modifications. (v) The capsid protein comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto, and / or (vi) The nucleotide sequence encoding the capsid protein includes the nucleotide sequence of SEQ ID NO: 137, or a sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto, AAV particles as described in Embodiment 122.

[0128] 124. Capsid proteins, (i) In numbering according to Sequence ID No. 138, an amino acid substitution at position K449, for example, the substitution of K449R, (ii) An insert containing the amino acid sequence of TLAVPFK (SEQ ID NO: 1262), wherein, optionally, the insert is located immediately after position 588 relative to a reference sequence numbered according to SEQ ID NO: 138. (iii) In numbering according to Sequence ID No. 138, an amino acid other than "A" at position 587 and / or an amino acid other than "Q" at position 588, (iv) Amino acid substitutions of A587D and / or Q588G, numbered according to Sequence ID No. 138 AAV particles according to embodiment 122 or 123, including the AAV particles described in embodiment 122 or 123.

[0129] 125. An AAV particle according to any one of Embodiments 122 to 124, comprising an insert in which the capsid protein (i) has an amino acid substitution of K449R in numbering according to Sequence ID No. 138, and (ii) has an amino acid sequence of TLAVPFK (Sequence ID No. 1262), optionally located immediately after position 588 of Sequence ID No. 138.

[0130] 126. An AAV particle according to any one of Embodiments 122 to 124, wherein the capsid protein comprises (i) an amino acid substitution of K449R, numbered according to Sequence ID No. 138, (ii) an insert comprising the amino acid sequence of TLAVPFK (Sequence ID No. 1262), optionally located immediately after position 588 relative to a reference sequence numbered according to Sequence ID No. 138, and (iii) amino acid substitutions of A587D and Q588G, numbered according to Sequence ID No. 138.

[0131] 127. An AAV particle according to any one of Embodiments 122 to 124, wherein the capsid protein comprises (i) an insert containing the amino acid sequence of TLAVPFK (SEQ ID NO: 1262), optionally located immediately after position 588 with respect to a reference sequence numbered according to SEQ ID NO: 138, and (ii) amino acid substitutions A587D and Q588G, numbered according to SEQ ID NO: 138.

[0132] 128. An AAV particle according to any one of Embodiments 122 to 127, wherein the capsid protein comprises one of the capsid proteins listed in Table 1 or a functional variant thereof.

[0133] 129. An AAV particle according to any one of Embodiments 122 to 128, wherein the capsid protein comprises VOY101, VOY201, AAVPHP.N (PHP.N), AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), PHP.B2, PHP.B3, G2B4, G2B5, AAV9, AAVrh10, or a functional variant thereof.

[0134] 130.(i) The capsid protein contains the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is substantially identical thereto (for example, having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), (ii) The capsid protein has at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 1, but no more than 30, 20, or 10 modifications, such as substitutions, and includes an amino acid sequence containing these modifications. (iii) The capsid protein comprises and / or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) (iv) The nucleotide sequence encoding the capsid protein includes the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence that is substantially identical thereto (for example, having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), AAV particles according to any one of embodiments 122 to 129.

[0135] 131. Capsid proteins, (i) VP1 polypeptide, VP2 polypeptide, VP3 polypeptide, or a combination thereof; (ii) an amino acid sequence corresponding to positions 138-743 of SEQ ID NO: 1, e.g., VP2, or a sequence having at least 80% (e.g., at least about 85, 90, 92, 95, 96, 97, 98, or 99%) sequence identity thereto; (iii) an amino acid sequence corresponding to positions 203-743 of SEQ ID NO: 1, e.g., VP3, or a sequence having at least 80% (e.g., at least about 85, 90, 92, 95, 96, 97, 98, or 99%) sequence identity thereto; and / or (iv) The amino acid sequence corresponding to positions 1-743 of SEQ ID NO: 1, e.g., VP1, or a sequence having at least 80% (e.g., at least about 85, 90, 92, 95, 96, 97, 98, or 99%) sequence identity thereto. AAV particles according to any one of embodiments 122 to 130, including the AAV particles described.

[0136] 132. The nucleotide sequence that codes for the capsid protein is (i) CTG start codon; and / or (ii) Nucleotide sequences of sequence number 137 that include 3 to 20 mutations, e.g., substitutions, e.g., mutations 3 to 15, mutations 3 to 10, mutations 3 to 5, mutations 5 to 20, mutations 5 to 15, mutations 5 to 10, mutations 10 to 20, mutations 10 to 15, mutations 15 to 20, mutations 3, mutations 5, mutations 10, mutations 12, mutations 15, mutations 18, or mutations 20. AAV particles according to any one of embodiments 122 to 131, including the AAV particles described.

[0137] 133. A vector comprising an isolated nucleic acid according to any one of Embodiments 1-6 or 9-49, or a viral genome according to any one of Embodiments 7-120. 134. A cell comprising a viral genome as described in any one of Embodiments 7 to 120, a viral particle as described in any one of Embodiments 122 to 132, or a vector as described in Embodiment 133.

[0138] 135. The cell according to Embodiment 134, which is a mammalian cell, e.g., HEK293 cell, an insect cell, e.g., Sf9 cell, or a bacterial cell. 136. A nucleic acid comprising a viral genome as described in any one of Embodiments 7 to 120, and a skeletal region suitable for replication of the viral genome in a cell, for example, a bacterial cell (e.g., the skeletal region includes one or both of a bacterial origin of replication and a selection marker).

[0139] 137. The nucleic acid according to Embodiment 136, wherein the viral genome comprises a nucleotide sequence described in any one of SEQ ID NOs: 1799-1082, 1752-1759, 1803-1821, or 1824-1830.

[0140] 138. A method for constructing a viral genome, (i) To provide a nucleic acid molecule containing the viral genome of Embodiment 136 or 137, or a nucleic acid encoding the viral genome described in any one of Embodiments 7 to 120, (ii) Excision of the viral genome from the skeletal region, for example, by cleaving nucleic acid molecules upstream and downstream of the viral genome. Methods that include...

[0141] 139. A method for producing isolated AAV particles, for example, recombinant AAV particles, (i) To provide a host cell containing a nucleic acid encoding a viral genome according to any one of Embodiments 7 to 120 or a viral genome according to Embodiment 136 or 137, (ii) Incubating host cells under conditions suitable for encapsulating the viral genome in a capsid protein, such as the VOY101 capsid protein. A method comprising, thereby producing isolated AAV particles.

[0142] 140. The method according to Embodiment 139, further comprising introducing a first nucleic acid molecule containing a viral genome into a host cell prior to step (i). 141. The method according to Embodiment 139 or 140, wherein the host cell contains a second nucleic acid encoding a capsid protein, for example, the VOY101 capsid protein.

[0143] 142. The method according to Embodiment 141, further comprising introducing a second nucleic acid into cells. 143. The method according to Embodiment 141 or 142, wherein the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule.

[0144] 144. The method according to any one of Embodiments 139 to 143, wherein the host cell comprises mammalian cells, e.g., HEK293 cells, insect cells, e.g., Sf9 cells, or bacterial cells.

[0145] 145. A pharmaceutical composition comprising AAV particles according to any one of Embodiments 122 to 132, or AAV particles containing a viral genome according to any one of Embodiments 7 to 120, and a pharmaceutically acceptable excipient.

[0146] 146. A method for delivering exogenous GBA protein to a target, comprising administering an effective amount of the pharmaceutical composition described in Embodiment 145, the AAV particle described in any one of Embodiments 122 to 132, the AAV particle containing the viral genome described in any one of Embodiments 7 to 120, or the AAV particle containing the viral genome containing the nucleic acid described in any one of Embodiments 1 to 6 or 9 to 49, thereby delivering the exogenous GBA protein to the target.

[0147] 147. The method according to Embodiment 146, wherein the subject has, has been diagnosed with, or is at risk of having, a disease related to GBA expression, e.g., abnormal or reduced GBA expression, e.g., GBA gene, GBA mRNA, and / or GBA protein expression.

[0148] 148. The method of Embodiment 146 or 147, wherein the subject has, has been diagnosed with, or is at risk of having, a neurodegenerative or neuromuscular disorder. 149. A method for treating a subject having or diagnosed with a disease related to GBA expression, comprising administering an effective amount of a pharmaceutical composition according to Embodiment 145, an AAV particle according to any one of Embodiments 122 to 132, an AAV particle containing a viral genome according to any one of Embodiments 7 to 120, or an AAV particle containing a viral genome containing nucleic acid according to any one of Embodiments 1 to 6 or 9 to 49, thereby treating the disease related to GBA expression in the subject.

[0149] 150. A method for treating a subject having or diagnosed with a neurodegenerative or neuromuscular disorder, comprising administering an effective amount of a pharmaceutical composition according to Embodiment 145, an AAV particle according to any one of Embodiments 122 to 132, an AAV particle containing a viral genome according to any one of Embodiments 7 to 120, or an AAV particle containing a viral genome containing nucleic acid according to any one of Embodiments 1 to 6 or 9 to 49, thereby treating the neurodegenerative or neuromuscular disorder of the subject.

[0150] 151. The method according to any one of Embodiments 147 to 150, wherein the disease or neurodegenerative or neuromuscular disorder associated with the expression of GBA includes Parkinson's disease (PD), Lewy body dementia (DLB), Gaucher disease (GD), spinal muscular atrophy (SMA), multiple system atrophy (MSA), or multiple sclerosis (MS).

[0151] 152. A method for treating a subject having or diagnosed with Parkinson's disease (PD) (e.g., PD associated with a mutation in the GBA gene), comprising administering an effective amount of a pharmaceutical composition according to Embodiment 145, an AAV particle according to any one of Embodiments 122 to 132, an AAV particle containing a viral genome according to any one of Embodiments 7 to 120, or an AAV particle containing a viral genome containing nucleic acid according to any one of Embodiments 1 to 6 or 9 to 49, wherein the subject's PD is treated.

[0152] 153. The method according to Embodiment 151 or 152, wherein PD is related to a mutation in the GBA gene. 154. The method according to any one of Embodiments 151 to 153, wherein the PD is early-onset PD (e.g., before age 50) or juvenile PD (e.g., before age 20).

[0153] 155. The method according to any one of Embodiments 151 to 154, wherein the PD is tremor-dominant, postural instability and gait difficulty PD (PIGD), or sporadic PD (e.g., PD not associated with mutation).

[0154] 156. A method for treating a subject having or diagnosed with Gaucher disease (GD), comprising administering an effective amount of a pharmaceutical composition according to Embodiment 145, an AAV particle according to any one of Embodiments 122 to 132, an AAV particle containing a viral genome according to any one of Embodiments 7 to 120, or an AAV particle containing a viral genome containing nucleic acid according to any one of Embodiments 1 to 6 or 9 to 49, wherein the GD of the subject is treated.

[0155] 157. The method according to Embodiment 151 or 156, wherein GD is neuropathic GD (e.g., affecting cells or tissues of the CNS, e.g., brain and / or spinal cord), non-neuropathic GD (e.g., not affecting cells or tissues of the CNS), or a combination thereof.

[0156] 158. The method according to any one of Embodiments 151 or 156-157, wherein GD is a type I GD (GD1), a type II GD (GD2), or a type III GD (GD3). 159. The method according to Embodiment 158, wherein GD1 is a non-neuropathic GD.

[0157] 160. The method according to Embodiment 158, wherein GD2 is neuropathic GD. 161. The method according to any one of Embodiments 146 to 160, wherein, when measured by an assay, for example, the assay described in Example 7, the subject has a reduced level of GCase activity compared to a reference level.

[0158] 162. The method according to Embodiment 161, wherein the reference level includes the level of GCase activity in a subject that does not have a disease, neuromuscular and / or neurodegenerative disorder associated with GBA expression. 163. The method according to any one of embodiments 149 to 162, wherein treatment includes preventing the progression of a disease in the subject.

[0159] 164. The method according to any one of embodiments 149 to 163, wherein treatment results in improvement of at least one symptom of a disease, neurodegenerative disorder, and / or neuromuscular disorder associated with GBA expression in a subject.

[0160] 165. The method according to Embodiment 164, wherein symptoms of diseases, neurodegenerative disorders, and / or neuromuscular disorders associated with GBA expression include decreased GCase activity, accumulation of glucocerebrosides and other glycolipids in immune cells (e.g., macrophages), accumulation of synuclein aggregates (e.g., Lewy bodies), developmental delay, progressive encephalopathy, progressive dementia, ataxia, myoclonus, oculomotor disorders, bulbar palsy, general weakness, tremors of the limbs, depression, hallucinations, cognitive decline, or a combination thereof.

[0161] 166. The method according to any one of embodiments 146 to 165, wherein the subject is a human. 167. The method according to any one of embodiments 146 to 166, wherein the subject is young, for example, between 6 and 20 years of age.

[0162] 168. The method according to any one of embodiments 146 to 167, wherein the subject is an adult, for example, over 20 years of age. 169. The method according to any one of Embodiments 146 to 168, wherein the subject has mutations in the GBA gene, GBA mRNA, and / or GBA protein.

[0163] 170. The method according to any one of Embodiments 146 to 169, wherein AAV particles are administered to a subject by intravenous, intracerebral, intrathalamic (ITH), intramuscular, intrathecal, intraventricular, intraparenchymal, focused ultrasound (FUS), for example, in combination with intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS in combination with intravenous administration, or intracisional injection (ICM).

[0164] 171. The method according to any one of Embodiments 146 to 170, wherein AAV particles are administered by a dual dose of ITH and ICM. 172. The method according to any one of Embodiments 146 to 170, wherein AAV particles are administered by intravenous injection, and optionally, the intravenous injection is combined with focused ultrasound (FUS), for example, intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS combined with intravenous administration.

[0165] 173. The method according to any one of Embodiments 146 to 172, wherein AAV particles are administered to cells, tissues, or regions of the CNS, e.g., regions of the brain or spinal cord, e.g., parenchyma, cerebral cortex, substantia nigra, caudate nucleus-cerebellum, striatum, corpus callosum, cerebellum, brainstem, caudate nucleus-putamen, thalamus, superior colliculus, spinal cord, or a combination thereof.

[0166] 174. The method according to any one of Embodiments 146 to 173, wherein AAV particles are administered to peripheral cells, tissues, or regions, such as cells or tissues of the lungs, cells or tissues of the heart, cells or tissues of the spleen, cells or tissues of the liver, or a combination thereof.

[0167] 175. The method according to any one of Embodiments 146 to 174, wherein AAV particles are administered into cerebrospinal fluid, serum, or a combination thereof. 176. The method according to any one of embodiments 146 to 175, wherein the AAV particles are administered to at least two tissues or regions of the CNS, for example, bilateral administration.

[0168] 177. The method according to any one of Embodiments 146 to 176, further comprising performing a blood test, performing an imaging test, taking a CNS biopsy sample, taking a tissue biopsy (e.g., a lung, liver, or spleen biopsy), taking a blood or serum sample, or taking an aqueous cerebrospinal fluid biopsy.

[0169] 178. The method according to any one of Embodiments 146 to 177, further comprising evaluating, for example, the level of GBA expression, e.g., GBA gene, GBA mRNA, and / or GBA protein expression in a subject, e.g., in cells, tissues, or body fluids of the subject, wherein optionally the level of GBA protein is measured by an assay described herein, e.g., ELISA, Western blot, or immunohistochemical assay.

[0170] 179. The method according to Embodiment 178, wherein the level of GBA expression is measured before, during, or after treatment with AAV particles. 180. The method according to Embodiment 178 or 179, wherein the cells or tissues are cells or tissues of the central nervous system (e.g., parenchyma) or peripheral cells or tissues (e.g., liver, heart, and / or spleen).

[0171] 181. The method according to any one of Embodiments 146 to 180, wherein the administration results in an increased level of GBA protein expression in the cells or tissue of a subject compared to a reference level, e.g., an untreated subject, e.g., a subject not administered AAV particles.

[0172] 182. The method according to any one of Embodiments 146 to 181, further comprising evaluating, for example, measuring, the level of GCase activity in a subject, for example, in the cells or tissue of the subject, wherein optionally, the level of GCase activity is measured by an assay described herein, for example, the assay described in Example 7.

[0173] 183. Administration, (i) Levels of GCase activity in target cells, tissues (e.g., cells or tissues of the CNS, e.g., cerebral cortex, striatum, thalamus, cerebellum, and / or brainstem) and / or body fluids (e.g., CSF and / or serum), which, optionally, are increased by at least 3, 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, or 5.5 times compared to a reference level, e.g., an untreated subject, e.g., a subject not administered AAV particles. (ii) the level of viral genome (VG) per cell in the target CNS tissue (e.g., cerebral cortex, striatum, thalamus, cerebellum, brainstem, and / or spinal cord), wherein, optionally, the VG level is increased by more than 50 VG per cell compared to peripheral tissue, and is at least 1 / 4 to 1 / 10 lower than the level in CNS tissue, e.g., measured by the assay described herein; and / or (iii) Levels of GBA mRNA expression in cells or tissues (e.g., cells or tissues of the CNS, e.g., cerebral cortex, thalamus, and / or brainstem) that are optionally increased by at least 100 to 1300 times, e.g., 100, 200, 500, 600, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 times compared to a reference level, e.g., an untreated subject (e.g., one not administered AAV particles), or endogenous GBA mRNA levels, e.g., measured by the assay described herein; The method according to any one of embodiments 146 to 182, which results in an increase of at least one, two, or all of the above.

[0174] 184. The method according to any one of Embodiments 146 to 183, further comprising the administration of an additional therapeutic agent and / or treatment suitable for the treatment or prevention of diseases, neurodegenerative disorders, and / or neuromuscular disorders associated with GBA expression.

[0175] 185. The method according to Embodiment 184, wherein additional therapeutic agents include enzyme replacement therapy (ERT) (e.g., imiglucerase, veraglucerase alfa, or taliglucerase alfa); substrate synthesis inhibitory therapy (SRT) (e.g., eliglustat or miglustat); blood transfusion; levodopa, carbidopa, safinamide; dopamine agonists (e.g., pramipexole, rotigotine, or ropinirole); anticholinergic agents (e.g., benzatropine or trihexyphenidyl); cholinesterase inhibitors (e.g., rivastigmine, donepezil, or galantamine); N-methyl-d-aspartate (NMDA) receptor antagonists (e.g., memantine); or a combination thereof.

[0176] 186. An isolated nucleic acid according to any one of Embodiments 1 to 6 or 9 to 49, a viral genome according to any one of Embodiments 7 to 120, AAV particles according to any one of Embodiments 122 to 132, or a pharmaceutical composition according to Embodiment 145, for use in the manufacture of a pharmaceutical product.

[0177] 187. An isolated nucleic acid according to any one of Embodiments 1-6 or 9-49, a viral genome according to any one of Embodiments 7-120, AAV particles according to any one of Embodiments 122-132, or a pharmaceutical composition according to Embodiment 145, for use in the treatment of diseases related to GBA expression, neuromuscular and / or neurodegenerative disorders.

[0178] 188. Use in the manufacture of a drug for use in the treatment of diseases related to GBA expression, neuromuscular and / or neurodegenerative disorders, an effective amount of AAV particles containing a genome according to any one of Embodiments 7 to 120, AAV particles containing a genome containing nucleic acid according to any one of Embodiments 1 to 6 or 9 to 49, AAV particles according to any one of Embodiments 122 to 132, or a pharmaceutical composition according to Embodiment 145.

[0179] 189. An adeno-associated virus (AAV) vector genome containing a sequence selected from one of sequence numbers 1759-1771. 190. An AAV particle comprising the AAV vector genome described in claim 189 and a capsid selected from the group consisting of those listed in Table 1.

[0180] 191. The AAV particle according to claim 190, wherein the capsid comprises the AAV2 serotype. 192. A pharmaceutical composition comprising the AAV particles of claim 190 or claim 191. 193. A method for treating a disorder of the nervous system or neuromuscular system, comprising administering the pharmaceutical composition described in claim 192 to a subject.

[0181] 194. The method according to claim 193, wherein the disorder of the nervous system or neuromuscular system is Parkinson's disease, Gaucher disease, or Lewy body dementia, or a related disorder. 195. The method according to claim 194, wherein the disorder of the nervous system or neuromuscular system is a disorder associated with a decrease in GCase protein levels.

[0182] Details of various aspects or embodiments of this disclosure are described below. Other features, purposes, and advantages of this disclosure will become apparent from this description and the claims. In the description, singular forms include plural forms unless explicitly stated in the context. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of this disclosure. In case of any conflict, this specification shall prevail. [Brief explanation of the drawing]

[0183] [Figure 1A] This report presents LC-MS / MS results quantifying the level of the GBA substrate glucosylsphingosine (GlcSph) in cell lysates from fibroblasts derived from Gaucher disease patients (GD1 patient GM04394, GD1 patient GM00852, and GD2 patient GM00877) and healthy control fibroblasts (CLT GM05758, CTL GM02937, and CTL GM08402). Data are shown as actin-normalized GlcSph. [Figure 1B] This report presents LC-MS / MS results quantifying the level of the GBA substrate glucosylsphingosine (GlcSph) in cell lysates from fibroblasts derived from Gaucher disease patients (GD1 patient GM04394, GD1 patient GM00852, and GD2 patient GM00877) and healthy control fibroblasts (CLT GM05758, CTL GM02937, and CTL GM08402). Data are shown as GlcSph normalized to the lysosomal protein Lamp1. [Figure 1C] This shows GBA protein levels detected in lysates of Gaucher patient-derived fibroblasts (GD1 and GD2) by LC-MS / MS, compared to healthy control fibroblasts (HC). Data are expressed as GBA protein (ng) concentration relative to total protein (mg). [Figure 2A] The X-axis, from left to right, shows GCase activity (RFU / mL normalized to mg protein) in GD-II GM00877 fibroblast pellets 7 days after transduction at a MOI of 103.5 with AAV2 virus particles containing the viral genome constructs GBA_VG1 (SEQ ID NO: 1759), GBA_VG9 (SEQ ID NO: 1767), GBA_VG10 (SEQ ID NO: 1768), GBA_VG11 (SEQ ID NO: 1769), GBA_VG6 (SEQ ID NO: 1764), GBA_VG7 (SEQ ID NO: 1765), GBA_VG12 (SEQ ID NO: 1770), GBA_VG3 (SEQ ID NO: 1761), GBA_VG4 (SEQ ID NO: 1762), GBA_VG5 (SEQ ID NO: 1763), and GBA_VG13 (SEQ ID NO: 1771). The dotted line indicates the baseline level (vehicle treatment). [Figure 2B]The X-axis, from left to right, shows the GCase activity (RFU / mL normalized to mg protein) in acclimatization medium on day 7 after transduction at a MOI of 103.5 for AAV2 virus particles containing the viral genome constructs GBA_VG1 (SEQ ID NO: 1759), GBA_VG9 (SEQ ID NO: 1767), GBA_VG10 (SEQ ID NO: 1768), GBA_VG11 (SEQ ID NO: 1769), GBA_VG6 (SEQ ID NO: 1764), GBA_VG7 (SEQ ID NO: 1765), GBA_VG12 (SEQ ID NO: 1770), GBA_VG3 (SEQ ID NO: 1761), GBA_VG4 (SEQ ID NO: 1762), GBA_VG5 (SEQ ID NO: 1763), and GBA_VG13 (SEQ ID NO: 1771). The dotted line indicates the baseline level (vehicle treatment). [Figure 3] The levels of the GBA substrate glucosylsphingosine (GlcSph) in cell lysates (ng / mg Lamp1) recovered from fibroblasts (GM00877) of GD-II patients 7 days after transduction by an AAV2 vector containing a control without AAV or a viral genome shown on the X-axis (from left to right: GBA_VG1 (SEQ ID NO: 1759), GBA_VG9 (SEQ ID NO: 1767), GBA_VG6 (SEQ ID NO: 1764), GBA_VG7 (SEQ ID NO: 1765), GBA_VG3 (SEQ ID NO: 1761), GBA_VG4 (SEQ ID NO: 1761), and GBA_VG5 (SEQ ID NO: 1763)). [Figure 4A] The X-axis shows the viral genomes (from left to right: GBA_VG1 (SEQ ID NO: 1759), GBA_VG14 (SEQ ID NO: 1809), GBA_VG15 (SEQ ID NO: 1810), GBA_VG16 (SEQ ID NO: 1811), GBA_VG17 (SEQ ID NO: 1812), GBA_VG18 (SEQ ID NO: 1813), GBA_VG19 (SEQ ID NO: 1814), and GBA_VG20 (SEQ ID NO: 1815)) in an AAV2 vector, and the X-axis shows the viral genomes (from left to right: GBA_VG1 (SEQ ID NO: 1819), GBA_VG17 (SEQ ID NO: 1812), GBA_VG18 (SEQ ID NO: 1813), GBA_VG19 (SEQ ID NO: 1814), and GBA_VG20 (SEQ ID NO: 1815)) and the GCase activity measured as RFU / mL normalized to mg protein in fibroblasts of GD-II patients (GD-II GM00877) 7 days after transduction at MOIs of 102.5 (first bar), 103 (second bar), 103.5, and 104 (third bar). [Figure 4B]The X-axis shows the levels of the GBA substrate glucosylsphingosine (GlcSph, ng / mg Lamp1) in cell lysates of fibroblasts derived from GD-II patients 7 days after transduction of an AAV2 vector containing the viral genomes shown on the X-axis (from left to right: GBA_VG1 (SEQ ID NO: 1759), GBA_VG14 (SEQ ID NO: 1809), GBA_VG15 (SEQ ID NO: 1810), GBA_VG16 (SEQ ID NO: 1811), GBA_VG17 (SEQ ID NO: 1812), GBA_VG18 (SEQ ID NO: 1813), GBA_VG19 (SEQ ID NO: 1814), and GBA_VG20 (SEQ ID NO: 1815)) at MOIs of 102.5 (first bar), 103 (second bar), 103.5, and 104 (third bar). [Figure 5] The GC content and distribution of the first codon-optimized nucleotide sequence encoding the GBA protein of SEQ ID NO: 1773, the second codon-optimized nucleotide sequence encoding the GBA protein of SEQ ID NO: 1781, and the wild-type nucleotide sequence encoding the GBA protein of SEQ ID NO: 1777 are shown. [Figure 6A] This study compares the activity of GBA proteins expressed by AAV2 vectorized viral genome constructs: GBA_VG1 (SEQ ID NO: 1759), GBA_VG17 (SEQ ID NO: 1812), and GBA_VG21 (SEQ ID NO: 1816). The X-axis shows GCase activity (RFU / mL), normalized to mg protein, in fibroblasts from GD-II patients treated with AAV2 viral particles containing the viral genome constructs (GBA_VG1 (SEQ ID NO: 1759), GBA_VG17 (SEQ ID NO: 1812), and GBA_VG21 (SEQ ID NO: 1816)) at an MOI of 104.5, compared to a control without AAV. [Figure 6B]This study compares the activity of GBA proteins expressed by AAV2 vectorized viral genome constructs: GBA_VG1 (SEQ ID NO: 1759), GBA_VG17 (SEQ ID NO: 1812), and GBA_VG21 (SEQ ID NO: 1816). The X-axis shows glucosylsphingosine (GlcSph) (ng / mL Lamp1) in fibroblasts from GD-II patients treated at 10⁶ MOI with AAV2 viral particles containing the viral genome constructs (from left to right: GBA_VG1 (SEQ ID NO: 1759), GBA_VG17 (SEQ ID NO: 1812), and GBA_VG21 (SEQ ID NO: 1816)) or in cell lysates of AAV-free control. [Figure 7] This shows the GCase activity (RFU / mL) per mg of protein in rat embryonic dorsal root ganglion (DRG) neurons transduced with an AAV2 vector containing GBA_VG33 (SEQ ID NO: 1828) or an AAV2 vector containing GBA_VG17 (SEQ ID NO: 1812) at an MOI of 103.5 or 104.5, compared to a control without AAV. [Figure 8] This shows the in vivo distribution (VG / cell) versus GCase activity (RFU / mL, multiple of endogenous GCase activity, normalized to mg protein) in the cerebral cortex, striatum, thalamus, brainstem, cerebellum, and liver of wild-type mice one month after IV injection of VOY101.GBA_VG17 (SEQ ID NO: 1812) at 2e13vg / kg. [Modes for carrying out the invention]

[0184] Overview This specification describes, in particular, compositions comprising isolated viral particles for the delivery of proteins, such as GBA proteins, such as vectorized delivery, such as recombinant viral particles, such as AAV particles, as well as methods for preparing and using the same. Adeno-associated viruses (AAVs) are small, non-enveloped, icosahedral capsid viruses of the Parvoviridae family, characterized by a single-stranded DNA viral genome. The Parvoviridae family consists of two subfamilies: the Parvovirinae, which infects vertebrates, and the Densovirinae, which infects invertebrates. The Parvoviridae family includes, but is not limited to, the Dependvirus genus, which contains AAVs that can replicate in vertebrate hosts, including humans, primates, cattle, dogs, horses, and sheep.

[0185] Parvoviruses and other members of the Parvoviridae family are outlined in Kenneth I. Berns, Parvoviridae: The Viruses and Their Replication, Chapter 69 in Fields Virology (3rd Ed. 1996), the entire contents of which are incorporated herein by reference.

[0186] AAV has proven useful as a biological tool due to its relatively simple structure, ability to infect a wide range of cells (including quiescent and dividing cells) without being integrated into the host genome or replicated, and a relatively benign immunogenicity profile. The viral genome can be engineered to contain at least the components necessary to assemble a functional recombinant virus or viral particle that carries a desired payload or is engineered to express or deliver a desired payload to a specific tissue. The viral genome can be modified to contain at least the components necessary to assemble a functional recombinant virus or viral particle that carries or is engineered to express or deliver a desired nucleic acid construct or payload, such as a transgene, polynucleotide encoding a polypeptide, such as a GBA protein, such as GCase, GCase and PSAP, GCase and SapA, or GCase and SapC, GCase and a cell membrane permeable peptide (e.g., ApoEII peptide, TAT peptide, or ApoB peptide), or GCase and a lysosomal targeting sequence (LTS), or is engineered to do so, and can be delivered to target cells, tissues, or organisms. In some embodiments, the target cells are CNS cells. In some embodiments, the target tissue is CNS tissue. The target CNS tissue may be brain tissue. In some embodiments, the brain target includes the caudate nucleus, putamen, thalamus, superior colliculus, cerebral cortex, and corpus callosum.

[0187] Gene therapy offers an alternative approach to PD and related diseases that share a common single-gene etiology, such as Gaucher disease, Lewy body dementia, and related disorders. AAVs are frequently used in gene therapy approaches due to their numerous advantageous features. While we do not wish to be constrained by theory, in some embodiments, GBA proteins (e.g., GCase and related proteins) can be administered and / or delivered using expression vectors, e.g., adeno-associated virus vectors (AAVs) or AAV particles, e.g., AAV particles described herein, to achieve sustained high concentrations, which is thought to enable longer-lasting effects, lower dose therapy, broader biodistribution, and / or more consistent GBA protein levels compared to non-AAV therapies.

[0188] As illustrated in the following examples of this specification, the compositions and methods described herein offer improved features compared to conventional enzyme supplementation approaches, including (i) increased GCase activity in target cells, tissues (e.g., cells or tissues of the CNS, e.g., cerebral cortex, striatum, thalamus, cerebellum, and / or brainstem), and / or body fluids (e.g., CSF and / or serum), (ii) increased in vivo distribution throughout the CNS (e.g., cerebral cortex, striatum, thalamus, cerebellum, brainstem, and / or spinal cord) and periphery (e.g., liver), and / or (iii) increased payload expression, e.g., GBA mRNA expression, in multiple brain regions (e.g., cerebral cortex, thalamus, and brainstem) and periphery (e.g., liver). In some embodiments, the AAV virus genome encoding the GBA protein described herein, comprising an optimized nucleotide sequence encoding the GBA protein (e.g., SEQ ID NO: 1773), results in high biodistribution in the CNS; increased GCase activity in the CNS, peripheral tissues, and / or body fluids; and successful transcription and expression of the transgene. The compositions and methods described herein can be used to treat neuropathic (affecting the CNS) and non-neuropathic (affecting areas other than the CNS) Gaucher diseases (e.g., type 1 GD, type 2 GD, or type 3 GD), PD associated with GBA gene mutations, and disorders associated with deficiency of the GBA protein and / or GCase activity, such as Lewy body dementia (DLB).

[0189] I. Composition Adeno-associated virus (AAV) vector AAV has a genome approximately 5,000 nucleotides long and contains two open reading frames that encode replication proteins (Rep) and capsid structural proteins (Cap). The open reading frames are flanked by two terminal inverted repeat (ITR) sequences that function as origins for replication in the viral genome. The wild-type AAV viral genome contains nucleotide sequences for two open reading frames: one for four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40 encoded by the Rep gene) and the other for three capsid or structural proteins (VP1, VP2, VP3 encoded by the capsid gene or Cap gene). The Rep proteins are important for replication and packaging, while the capsid proteins are assembled to form the AAV protein shell or AAV capsid. Alternative splicing, along with alternative start codons and promoters, generates four different Rep proteins from a single open reading frame and three capsid proteins from a single open reading frame. As a non-limiting example, depending on the AAV serotype, in the case of AAV9 / hu.14 (SEQ ID NO: 123 of US7,906,111; the entire content of which is incorporated herein by reference), VP1 refers to amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. As another non-limiting example, VP1 refers to amino acids 1-743, numbered according to SEQ ID NO: 1, VP2 refers to amino acids 138-743, numbered according to SEQ ID NO: 1, and VP3 refers to amino acids 203-743, numbered according to SEQ ID NO: 1. In other words, VP1 is the full-length capsid sequence, and VP2 and VP3 are shorter components of the full-length sequence. As a result, while the sequence changes in the VP3 region are also changes in VP1 and VP2, the percentage difference compared to the parent sequence is largest because VP3 is the shortest of the three sequences. Although we are describing this in terms of amino acid sequences, the nucleic acid sequences encoding these proteins can be described similarly.In summary, three capsid proteins are assembled to form the AAV capsid protein. While we do not wish to be bound by theory, the AAV capsid protein typically contains VP1:VP2:VP3 in a molar ratio of 1:1:10. As used herein, "AAV serotype" is primarily defined by the AAV capsid. In some cases, the ITR is also specifically described by the AAV serotype (e.g., AAV2 / 9).

[0190] AAV vectors typically require a co-helper (e.g., adenovirus) to perform proliferative infection within a cell. In the absence of such helper function, AAV virions essentially enter the host cell but are not integrated into the cell's genome.

[0191] AAV vectors are being studied for gene therapy delivery due to several unique characteristics. Non-limiting examples of these characteristics include: (i) the ability to infect both dividing and non-dividing cells; (ii) a broad host range of infection, including human cells; (iii) wild-type AAV is not associated with any disease and has not been shown to replicate in infected cells; (iv) there is no cell-mediated immune response to the vector; and (v) it does not integrate into host chromosomes, thus reducing the possibility of long-term genetic alteration. Furthermore, infection with AAV vectors has minimal impact on altering cellular gene expression patterns (Stilwell and Samulski et al., Biotechniques, 2003, 34, 148; this is incorporated herein by reference in its entirety).

[0192] Typically, AAV vectors for GCase protein delivery may be replication-deficient recombinant viral vectors because they lack sequences encoding functional Rep and Cap proteins within the viral genome. In some cases, the deficient AAV vector may lack most or all coding sequences and may essentially contain only one or two AAV ITR sequences and payload sequences. In certain embodiments, the viral genome encodes a GCase protein. In some embodiments, the viral genome encodes a GCase protein and a SapA protein. In some embodiments, the viral genome encodes a GCase protein and a SapC protein. For example, the viral genome may encode human GCase, human GCase+SapA, or human GCase+SapC protein(s).

[0193] In some embodiments, the viral genome may include one or more lysosomal targeting sequences (LTS). In some embodiments, the viral genome may contain one or more cell membrane-permeable peptide sequences (CPPs).

[0194] In some embodiments, the viral genome may include one or more lysosome-targeting sequences and one or more cell membrane-permeable sequences. In some embodiments, the AAV particles of this disclosure can be introduced into mammalian cells.

[0195] AAV vectors can be modified to enhance delivery efficiency. Such modified AAV vectors of the present disclosure can be efficiently packaged and successfully infect target cells with high frequency and minimal toxicity.

[0196] In other embodiments, the AAV particles of this disclosure may be used to deliver GCase proteins to specific tissues of the central nervous system (see, for example, U.S. Patent No. 6,180,613; the entirety of which is incorporated herein by reference).

[0197] As used herein, the terms “AAV vector” or “AAV particle” include a capsid and a viral genome containing a payload. As used herein, “payload” or “payload region” means one or more polynucleotides or polynucleotide regions encoded by or within the viral genome, or the expression products of such polynucleotides or polynucleotide regions, e.g., transgenes, polypeptides or multipolypeptides, e.g., polynucleotides encoding GCase proteins.

[0198] It should be understood that the compositions described herein may have additional conservation or non-essential amino acid substitutions that do not substantially affect their function. AAV Cerotype The AAV particles of this disclosure may include or be derived from any natural or recombinant AAV serotype. According to this disclosure, AAV particles may utilize or be based on a serotype, or include: VOY101, VOY201, AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B -EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, A AVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-E GT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B -TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3(G2A3), AAVG2B4(G2B4), AAVG2B5(G2B5), PHP.S, AAV1, AAV2, AAV2G9, AAV3, AAV3a, A AV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-1 5、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AA V223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / rh.6、AAV3.1 / rh.61 .9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10 AV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、A AV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAV3、AAV5、AAV2 rh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh. R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AA Vhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23、AAVhu.2 AVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41 、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu. .48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AA Vhu.67、AAVhu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.1 8、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh. rh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.8R、AAVrh.8R. A586R mutation、AAVrh8R R533A mutation、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhEr1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAVhEr1.36、AAVhEr r2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhER1.23、AAVhEr3.1、AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-L K04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK16、AAV-LK17、AAV-LK18 AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAV-PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV-b、AAV SM10-2、AAV シャッフル100-1、AAV シャッフル100-3、AAV シャッフル100-7、AAV シャッフル10-2、AAV シャッフル10-6、AAV シャッフル10-8、AAV シャッフル100-2. SM10-1, AAV SM10-8, AAV SM100-3, AAV SM100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, True type AAV (ttAAV), UPENN AAV10, Japanese AAV10 serotype, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、A AV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV It may contain a peptide selected from any of CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 and their variants.

[0199] In some embodiments, the AAV serotype is a sequence described in U.S. Patent Publication No. US20030138772 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV1 (sequences 6 and 64 of US20030138772), AAV2 (sequences 7 and 70 of US20030138772), AAV3 (sequences 8 and 8 of US20030138772) 71), AAV4 (sequence number 63 of US20030138772), AAV5 (sequence number 114 of US20030138772), AAV6 (sequence number 65 of US20030138772), AAV7 (sequence numbers 1-3 of US20030138772), AAV8 (sequence numbers 4 and 95 of US20030138772), AAV9 (sequence numbers 5 and 100 of US20030138772), AAV10 (US20 AAV11 (Sequence ID 117 of US20030138772), AAV12 (Sequence ID 119 of US20030138772), AAVrh10 (Amino acids 1-738 of Sequence ID 81 of US20030138772), AAV16.3 (Sequence ID 10 of US20030138772), AAV29.3 / bb.1 (Sequence ID 11 of US20030138772), AAV 29.4 (Sequence ID 12 of US20030138772), AAV29.5 / bb.2 (Sequence ID 13 of US20030138772), AAV1.3 (Sequence ID 14 of US20030138772), AAV13.3 (Sequence ID 15 of US20030138772), AAV24.1 (Sequence ID 16 of US20030138772), AAV27.3 (Sequence ID 17 of US20030138772), AAV7.2 (sequence number 18 of US20030138772), AAVC1 (sequence number 19 of US20030138772), AAVC3 (sequence number 20 of US20030138772), AAVC5 (sequence number 21 of US20030138772), AAVF1 (sequence number 22 of US20030138772), AAVF3 (sequence number 23 of US20030138772), AAVF5 (sequence number 24 of US20030138772), AAVH6 (sequence number 25 of US20030138772), AAVH2 (sequence number 26 of US20030138772), AAV42-8 (sequence number 27 of US20030138772), AAV42-15 (sequence number 28 of US20030138772), AAV42-5b (sequence number 29 of US20030138772), AAV42-1b (sequence number 30 of US20030138772), AAV42-13 (sequence number 31 of US20030138772), AAV42-3a (sequence number 32 of US20030138772), AAV42-4 (sequence number 33 of US20030138772), AAV42-5a (sequence number 34 of US20030138772), AA V42-10 (sequence number 35 of US20030138772), AAV42-3b (sequence number 36 of US20030138772), AAV42-11 (sequence number 37 of US20030138772), AAV42-6b (sequence number 38 of US20030138772), AAV43-1 (sequence number 39 of US20030138772), AAV43-5 (sequence number 40 of US20030138772), AAV43-12 (sequence number 41 of US20030138772), AAV43-20 (sequence number 42 of US20030138772), AAV4 3-21 (Sequence ID 43 of US20030138772), AAV43-23 (Sequence ID 44 of US20030138772), AAV43-25 (Sequence ID 45 of US20030138772), AAV44.1 (Sequence ID 46 of US20030138772), AAV44.5 (Sequence ID 47 of US20030138772), AAV223.1 (Sequence ID 48 of US20030138772), AAV223.2 (Sequence ID 49 of US20030138772), AAV223.4 (Sequence ID 50 of US20030138772), AAV223.5 (Sequence ID 51 of US20030138772), AAV223.6 (Sequence ID 52 of US20030138772), AAV223.7 (Sequence ID 53 of US20030138772), AAVA3.4 (Sequence ID 54 of US20030138772), AAVA3.5 (Sequence ID 55 of US20030138772), AAVA3.7 (US2003013 It may be or may contain the following variants: 8772 (sequence number 56), AAVA3.3 (sequence number 57 of US20030138772), AAV42.12 (sequence number 58 of US20030138772), AAV44.2 (sequence number 59 of US20030138772), AAV42-2 (sequence number 9 of US20030138772), or a variant thereof.

[0200] In some embodiments, the AAV serotype is a sequence described in U.S. Patent Publication No. US20150159173 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV2 (sequences 7 and 23 of US20150159173), rh20 (sequence 1 of US20150159173), rh32 / 33 (sequence 2 of US20150159173), rh39 (sequences 3, 20 and 36 of US20150159173), rh46 (sequences 4 and 22 of US20150159173). rh73 (sequence number 5 in US20150159173), rh74 (sequence number 6 in US20150159173), AAV6.1 (sequence number 29 in US20150159173), rh.8 (sequence number 41 in US20150159173), rh.48.1 (sequence number 44 in US20150159173), hu.44 (sequence number 45 in US20150159173), hu.29 (sequence number 42 in US20150159173), hu.48 (sequence number 38 in US20150159173), rh54 (sequence number 4 in US20150159173) 9), AAV2 (sequence number 7 of US20150159173), cy.5 (sequence numbers 8 and 24 of US20150159173), rh.10 (sequence numbers 9 and 25 of US20150159173), rh.13 (sequence numbers 10 and 26 of US20150159173), AAV1 (sequence numbers 11 and 27 of US20150159173), AAV3 (sequence numbers 12 and 28 of US20150159173), AAV6 (sequence numbers 13 and 29 of US20150159173), AAV7 (sequence numbers 14 and 30 of US20150159173), A AV8 (sequences 15 and 31 of US20150159173), hu.13 (sequences 16 and 32 of US20150159173), hu.26 (sequences 17 and 33 of US20150159173), hu.37 (sequences 18 and 34 of US20150159173), hu.53 (sequences 19 and 35 of US20150159173), rh.43 (sequences 21 and 37 of US20150159173), rh2 (sequence 39 of US20150159173), rh.37 (sequence 40 of US20150159173), rh.64 (sequence number 43 in US20150159173), rh.48 (sequence number 44 in US20150159173), ch.5 (sequence number 46 in US20150159173), rh.67 (sequence number 47 in US20150159173), rh.58 (sequence number 48 in US20150159173), or their variants, for example, but not limited to, Cy5R1, Cy5R2, Cy It may be, or may have, 5R3, Cy5R4, rh.13R, rh.37R2, rh.2R, rh.8R, rh.48.1, rh.48.2, rh.48.1.2, hu.44R1, hu.44R2, hu.44R3, hu.29R, ch.5R1, rh64R1, rh64R2, AAV6.2, AAV6.1, AAV6.12, hu.48R1, hu.48R2, and hu.48R3.

[0201] In some embodiments, the AAV serotype may be or have sequences described in U.S. Patent No. 7198951 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV9 (sequences 1-3 of U.S. 7198951), AAV2 (sequence 4 of U.S. 7198951), AAV1 (sequence 5 of U.S. 7198951), AAV3 (sequence 6 of U.S. 7198951), and AAV8 (sequence 7 of U.S. 7198951).

[0202] In some embodiments, the AAV cellotype is N Pulicherla et Variations of the AAV9 sequence described by al. (Molecular Therapy 19(6):1070-1078(2011); the entire work is incorporated herein by reference) may or may have such variations, including, but are not limited to, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, and AAV9.84.

[0203] In some embodiments, the AAV serotype may be or have the sequences described in U.S. Patent No. 6156303 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV3B (SEQ ID NOs. 1 and 10 of U.S. 6156303), AAV6 (SEQ ID NOs. 2, 7 and 11 of U.S. 6156303), AAV2 (SEQ ID NOs. 3 and 8 of U.S. 6156303), AAV3A (SEQ ID NOs. 4 and 9 of U.S. 6156303), or derivatives thereof.

[0204] In some embodiments, the AAV serotype may be or have the sequences described in U.S. Patent Publication No. US20140359799 (the contents of which are incorporated herein by reference in their entirety), for example, AAV8 (sequence number 1 of US20140359799), AAVDJ (sequence numbers 2 and 3 of US20140359799), or variants thereof.

[0205] In some embodiments, the serotype may be AAVDJ or a variant thereof, for example, AAVDJ8 (or AAV-DJ8) as described by Grimm et al. (Journal of Virology 82(12):5887-5911 (2008); the entire text of which is incorporated herein by reference). The amino acid sequence of AAVDJ8 may contain two or more mutations to remove the heparin-binding domain (HBD). As a non-limiting example, the AAV-DJ sequence described as Sequence ID No. 1 in U.S. Patent No. 7,588,772 (the entire text of which is incorporated herein by reference) may contain two mutations: (1) R587Q (in which amino acid 587, arginine (R; Arg), is changed to glutamine (Q; Gln)) and (2) R590T (in which amino acid 590, arginine (R; Arg), is changed to threonine (T; Thr)). Another non-limiting example may include three mutations: (1) K406R (lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg)), (2) R587Q (arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln)), and (3) R590T (arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr)).

[0206] In some embodiments, the AAV serotype may be, or may have, an AAV4 sequence described in International Publication No. WO1998011244 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV4 (Sequences 1-20 of WO1998011244).

[0207] In some embodiments, the AAV serotype may be, or may have, a mutation in the AAV2 sequence that generates AAV2G9, as described in International Publication No. WO2014144229 (which is incorporated herein by reference in its entirety).

[0208] In some embodiments, the AAV serotype is a sequence described in International Publication No. WO2005033321 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV3-3 (sequence number 217 of WO2005033321), AAV1 (sequence numbers 219 and 202 of WO2005033321), AAV106.1 / hu.37 (sequence number 10 of WO2005033321), AAV114.3 / hu.40 (sequence number 11 of WO2005033321), AAV127.2 / hu.41 (WO20 AAV128.3 / hu.44 (Sequence ID 81 of WO2005033321), AAV130.4 / hu.48 (Sequence ID 78 of WO2005033321), AAV145.1 / hu.53 (Sequence IDs 176 and 177 of WO2005033321), AAV145.6 / hu.56 (Sequence IDs 168 and 192 of WO2005033321), AAV16.12 / hu.11 (Sequence IDs 153 and 57 of WO2005033321), AAV16.8 / hu.10 (Sequence ID 1 of WO2005033321) AAV161.10 / hu.60 (sequence number 170 of WO2005033321), AAV161.6 / hu.61 (sequence number 174 of WO2005033321), AAV1-7 / rh.48 (sequence number 32 of WO2005033321), AAV1-8 / rh.49 (sequence numbers 103 and 25 of WO2005033321), AAV2 (sequence numbers 211 and 221 of WO2005033321), AAV2-15 / rh.62 (sequence numbers 33 and 114 of WO2005033321), AAV2-3 / rh.61 (WO20050 AAV2-4 / rh.50 (Sequence codes 23 and 108 of WO2005033321), AAV2-5 / rh.51 (Sequence codes 104 and 22 of WO2005033321), AAV3.1 / hu.6 (Sequence codes 5 and 84 of WO2005033321), AAV3.1 / hu.9 (Sequence codes 155 and 58 of WO2005033321), AAV3-11 / rh.53 (Sequence codes 186 and 176 of WO2005033321), AAV3-3 (Sequence code 200 of WO2005033321), AAV33.12 / hu.17 (Sequence ID 4 of WO2005033321), AAV33.4 / hu.15 (Sequence ID 50 of WO2005033321), AAV33.8 / hu.16 (Sequence ID 51 of WO2005033321), AAV3-9 / rh.52 (Sequence IDs 96 and 18 of WO2005033321), AAV4-19 / rh.55 (Sequence ID 117 of WO2005033321), AAV4-4 (Sequence IDs 201 and 218 of WO2005033321), AAV4-9 / rh.54 (Sequence ID 116 of WO2005033321), AAV5 (WO20050 AAV52.1 / hu.20 (sequence number 63 of WO2005033321), AAV52 / hu.19 (sequence number 133 of WO2005033321), AAV5-22 / rh.58 (sequence number 27 of WO2005033321), AAV5-3 / rh.57 (sequence number 105 of WO2005033321), AAV5-3 / rh.57 (sequence number 26 of WO2005033321), AAV58.2 / hu.25 (sequence number 49 of WO2005033321), AAV6 (sequence number of WO2005033321) Numbers 203 and 220), AAV7 (Sequence numbers 222 and 213 of WO2005033321), AAV7.3 / hu.7 (Sequence number 55 of WO2005033321), AAV8 (Sequence numbers 223 and 214 of WO2005033321), AAVH-1 / hu.1 (Sequence number 46 of WO2005033321), AAVH-5 / hu.3 (Sequence number 44 of WO2005033321), AAVhu.1 (Sequence number 144 of WO2005033321), AAVhu.10 (Sequence number 156 of WO2005033321), AAVhu.11 (WO2 AAVhu.12 (Sequence ID 153 of 005033321), AAVhu.13 (Sequence ID 129 of WO2005033321), AAVhu.14 / AAV9 (Sequence IDs 123 and 3 of WO2005033321), AAVhu.15 (Sequence ID 147 of WO2005033321), AAVhu.16 (Sequence ID 148 of WO2005033321), AAVhu.17 (Sequence ID 83 of WO2005033321), AAVhu.18 (Sequence ID 149 of WO2005033321), AAVhu.19 (Sequence ID 133 of WO2005033321), AAVhu.2 (Sequence ID 143 of WO2005033321), AAVhu.20 (Sequence ID 134 of WO2005033321), AAVhu.21 (Sequence ID 135 of WO2005033321), AAVhu.22 (Sequence ID 138 of WO2005033321), AAVhu.23.2 (Sequence ID 137 of WO2005033321), AAVhu.24 (Sequence ID 136 of WO2005033321), AAVhu.25 (Sequence ID 146 of WO2005033321), AAV hu.27 (sequence number 140 of WO2005033321), AAVhu.29 (sequence number 132 of WO2005033321), AAVhu.3 (sequence number 145 of WO2005033321), AAVhu.31 (sequence number 121 of WO2005033321), AAVhu.32 (sequence number 122 of WO2005033321), AAVhu.34 (sequence number 125 of WO2005033321), AAVhu.35 (sequence number 164 of WO2005033321), AAVhu.37 (sequence number 88 of WO2005033321), AAVh u.39 (sequence number 102 of WO2005033321), AAVhu.4 (sequence number 141 of WO2005033321), AAVhu.40 (sequence number 87 of WO2005033321), AAVhu.41 (sequence number 91 of WO2005033321), AAVhu.42 (sequence number 85 of WO2005033321), AAVhu.43 (sequence number 160 of WO2005033321), AAVhu.44 (sequence number 144 of WO2005033321), AAVhu.45 (sequence number 127 of WO2005033321), AAVhu.4 6 (Sequence ID 159 of WO2005033321), AAVhu.47 (Sequence ID 128 of WO2005033321), AAVhu.48 (Sequence ID 157 of WO2005033321), AAVhu.49 (Sequence ID 189 of WO2005033321), AAVhu.51 (Sequence ID 190 of WO2005033321), AAVhu.52 (Sequence ID 191 of WO2005033321), AAVhu.53 (Sequence ID 186 of WO2005033321), AAVhu.54 (Sequence ID 188 of WO2005033321), AAVhu.55 (Sequence ID 187 of WO2005033321), AAVhu.56 (Sequence ID 192 of WO2005033321), AAVhu.57 (Sequence ID 193 of WO2005033321), AAVhu.58 (Sequence ID 194 of WO2005033321), AAVhu.6 (Sequence ID 84 of WO2005033321), AAVhu.60 (Sequence ID 184 of WO2005033321), AAVhu.61 (Sequence ID 185 of WO2005033321), AAVhu.63 (Sequence ID 195 of WO2005033321), AAVhu.64 (W AAVhu.66 (Sequence ID 196 of WO2005033321), AAVhu.67 (Sequence ID 198 of WO2005033321), AAVhu.7 (Sequence ID 150 of WO2005033321), AAVhu.8 (Sequence ID 12 of WO2005033321), AAVhu.9 (Sequence ID 155 of WO2005033321), AAVLG-10 / rh.40 (Sequence ID 14 of WO2005033321), AAVLG-4 / rh.38 (Sequence ID 86 of WO2005033321), AAVLG-4 / rh.38 (Sequence ID 7 of WO2005033321), AAVN721-8 / rh.43 (Sequence ID 163 of WO2005033321), AAVN721-8 / rh.43 (Sequence ID 43 of WO2005033321), AAVpi.1 (Sequence ID 28 of WO2005033321), AAVpi.2 (Sequence ID 30 of WO2005033321), AAVpi.3 (Sequence ID 29 of WO2005033321), AAVrh.38 (Sequence ID 86 of WO2005033321), AAVrh.40 (Sequence ID 92 of WO2005033321), A AVrh.43 (Sequence ID 163 of WO2005033321), AAVrh.44 (Sequence ID 34 of WO2005033321), AAVrh.45 (Sequence ID 41 of WO2005033321), AAVrh.47 (Sequence ID 38 of WO2005033321), AAVrh.48 (Sequence ID 115 of WO2005033321), AAVrh.49 (Sequence ID 103 of WO2005033321), AAVrh.50 (Sequence ID 108 of WO2005033321), AAVrh.51 (Sequence ID 104 of WO2005033321), AAVrh.52 (Sequence ID 96 of WO2005033321), AAVrh.53 (Sequence ID 97 of WO2005033321), AAVrh.55 (Sequence ID 37 of WO2005033321), AAVrh.56 (Sequence ID 152 of WO2005033321), AAVrh.57 (Sequence ID 105 of WO2005033321), AAVrh.58 (Sequence ID 106 of WO2005033321), AAVrh .59 (Sequence ID 42 of WO2005033321), AAVrh.60 (Sequence ID 31 of WO2005033321), AAVrh.61 (Sequence ID 107 of WO2005033321), AAVrh.62 (Sequence ID 114 of WO2005033321), AAVrh.64 (Sequence ID 99 of WO2005033321), AAVrh.65 (Sequence ID 35 of WO2005033321), AAVrh. 68 (sequence number 16 of WO2005033321), AAVrh.69 (sequence number 39 of WO2005033321), AAVrh.70 (sequence number 20 of WO2005033321), AAVrh.72 (sequence number 9 of WO2005033321), or their variants, for example, but not limited to, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVcy.6, AAVrh.12, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.25 / 4215, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.14 may or may have them. Non-exclusive examples of variants include sequence numbers 13, 15, 17, 19, 24, 36, 40, 45, 47, 48, 51-54, 60-62, 64-77, 79, 80, 82, 89, 90, 93-95, 98, 100, 101, 109-113, 118-120, 124, 126, 131, 139, 142, 151, 154, 158, 161, 162, 165-183, 202, 204-212, 215, 219, and 224-236 of WO2005033321 (the entire content of which is incorporated herein by reference).

[0209] In some embodiments, the AAV serotype may be, or may have, a sequence described in International Publication No. WO2015168666 (the entirety of which is incorporated herein by reference), for example, but not limited to, AAVrh8R (sequence number 9 of WO2015168666), AAVrh8R A586R variant (sequence number 10 of WO2015168666), AAVrh8R R533A variant (sequence number 11 of WO2015168666), or a variant thereof.

[0210] In some embodiments, the AAV serotype is a sequence described in U.S. Patent No. 9233131 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAVhE1.1 (sequence number 44 of U.S. 9233131), AAVhEr1.5 (sequence number 45 of U.S. 9233131), AAVhER1.14 (sequence number 46 of U.S. 9233131), AAVhEr1.8 (sequence number 47 of U.S. 9233131), AAVhEr1.16 (sequence number 48 of U.S. 9233131), AAVhEr1.18 (sequence number 49 of U.S. 9233131), AAVhEr1.35 (sequence number 50 of U.S. 9233131), AAVhEr1.7 (sequence number 5 of U.S. 9233131) 1) AAVhEr1.36 (Sequence ID 52 of US9233131), AAVhEr2.29 (Sequence ID 53 of US9233131), AAVhEr2.4 (Sequence ID 54 of US9233131), AAVhEr2.16 (Sequence ID 55 of US9233131), AAVhEr2.30 (Sequence ID 56 of US9233131), AAVhEr2.31 (U It may be or may have a variant thereof, such as S9233131 (sequence number 58), AAVhEr2.36 (sequence number 57 of US9233131), AAVhER1.23 (sequence number 53 of US9233131), AAVhEr3.1 (sequence number 59 of US9233131), AAV2.5T (sequence number 42 of US9233131), or a variant thereof.

[0211] In some embodiments, the AAV serotype is a sequence described in U.S. Patent Publication No. US20150376607 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV-PAEC (Sequence ID 1 of US20150376607), AAV-LK01 (Sequence ID 2 of US20150376607), AAV-LK02 (Sequence ID 3 of US20150376607), AAV-LK03 (Sequence ID 4 of US20150376607), AAV-LK04 (Sequence ID 4 of US20150376607) AAV-LK05 (sequence number 6 of US20150376607), AAV-LK06 (sequence number 7 of US20150376607), AAV-LK07 (sequence number 8 of US20150376607), AAV-LK08 (sequence number 9 of US20150376607), AAV-LK09 (sequence number 10 of US20150376607), AAV-LK10 (sequence number 11 of US20150376607), AAV-LK11 (sequence number 12 of US20150376607), AAV-LK12 (sequence number 6 of US20150376607) AAV-LK13 (US20150376607, SEQ ID NO: 13), AAV-LK14 (US20150376607, SEQ ID NO: 15), AAV-LK15 (US20150376607, SEQ ID NO: 16), AAV-LK16 (US20150376607, SEQ ID NO: 17), AAV-LK17 (US20150376607, SEQ ID NO: 18), AAV-LK18 (US20150376607, SEQ ID NO: 19), AAV-LK19 (US20150376607, SEQ ID NO: 20), AAV-PAEC2 (US20 It may be or may have a variant thereof, such as AAV-PAEC4 (sequence number 22 of US20150376607), AAV-PAEC6 (sequence number 23 of US20150376607), AAV-PAEC7 (sequence number 24 of US20150376607), AAV-PAEC8 (sequence number 25 of US20150376607), AAV-PAEC11 (sequence number 26 of US20150376607), AAV-PAEC12 (sequence number 27 of US20150376607).

[0212] In some embodiments, the AAV serotype may be, or may have, a sequence described in U.S. Patent No. 9163261 (the entirety of which is incorporated herein by reference), for example, AAV-2-pre-miRNA-101 (Sequence ID 1 of U.S. 9163261), or a variant thereof.

[0213] In some embodiments, the AAV serotype may be or have a variant thereof of the sequences described in U.S. Patent Publication No. US20150376240 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV-8h (SEQ ID NO: 6 in US20150376240), AAV-8b (SEQ ID NO: 5 in US20150376240), AAV-h (SEQ ID NO: 2 in US20150376240), AAV-b (SEQ ID NO: 1 in US20150376240).

[0214] In some embodiments, the AAV cellotype is an array described in U.S. Patent Publication No. US20160017295 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV SM10-2 (Sequence ID 22 of US20160017295), AAV Shuffle 100-1 (Sequence ID 23 of US20160017295), AAV Shuffle 100-3 (Sequence ID 24 of US20160017295), AAV Shuffle 100-7 (Sequence ID 25 of US20160017295), AAV Shuffle 10-2 (Sequence ID 34 of US20160017295), AAV Shuffle 10-6 (Sequence ID 35 of US20160017295), AAV Shuffle 10-8 (Sequence ID 36 of US20160017295), AAV Shuffle 100-2 (Sequence ID 37 of US20160017295), AAV SM10-1 (sequence number 38 in US20160017295), AAV SM10-8 (sequence number 39 in US20160017295), AAV SM100-3 (sequence number 40 in US20160017295), AAV SM100-10 (sequence number 41 in US20160017295), or variants thereof may or may have them.

[0215] In some embodiments, the AAV serotype may be or have the sequences described in U.S. Patent Publication No. US20150238550 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, BNP61 AAV (Sequence ID 1 of US20150238550), BNP62 AAV (Sequence ID 3 of US20150238550), BNP63 AAV (Sequence ID 4 of US20150238550), or variants thereof.

[0216] In some embodiments, the AAV serotype is a sequence described in U.S. Patent Publication No. US20150315612 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAVrh.50 (sequence number 108 of US20150315612), AAVrh.43 (sequence number 163 of US20150315612), AAVrh.62 (US2015 AAVrh.48 (sequence number 114 of US20150315612), AAVhu.19 (sequence number 133 of US20150315612), AAVhu.11 (sequence number 153 of US20150315612), AAVhu.53 (sequence number 186 of US20150315612), AAV4-8 / rh.64 (sequence number 15 of US20150315612) AAVLG-9 / hu.39 (sequence number 24 in US20150315612), AAV54.5 / hu.23 (sequence number 60 in US20150315612), AAV54.2 / hu.22 (sequence number 67 in US20150315612), AAV54.7 / hu.24 (sequence number 66 in US20150315612), AAV54.1 / hu.21 (sequence number 6 in US20150315612) 5) AAV54.4R / hu.27 (sequence number 64 in US20150315612), AAV46.2 / hu.28 (sequence number 68 in US20150315612), AAV46.6 / hu.29 (sequence number 69 in US20150315612), AAV128.1 / hu.43 (sequence number 80 in US20150315612), or may have a variant thereof.

[0217] In some embodiments, the AAV serotype may be or have a variant thereof of the sequences described in International Publication No. WO2015121501 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, truetype AAV (ttAAV) (sequence number 2 of WO2015121501), "UPenn AAV10" (sequence number 8 of WO2015121501), "Japan AAV10" (sequence number 9 of WO2015121501).

[0218] According to this disclosure, the selection or use of AAV capsid serotypes may originate from various species. In some embodiments, AAV may be tri-AAV (AAAV). The AAAV serotype may be or have the sequences described in U.S. Patent No. 9,238,800 (the entirety of which is incorporated herein by reference), for example, AAAV (Sequence IDs 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. 9,238,800), or variants thereof.

[0219] In some embodiments, AAV may be bovine AAV (BAAV). The BAAV serotype may be, or have, a sequence described in U.S. Patent No. 9,193,769 (the entirety of which is incorporated herein by reference), for example, BAAV (sequences 1 and 6 of U.S. 9,193,769), or a variant thereof. The BAAV serotype may be, or have, a sequence described in U.S. Patent No. 7,427,396 (the entirety of which is incorporated herein by reference), for example, BAAV (sequences 5 and 6 of U.S. 7,427,396), or a variant thereof.

[0220] In some embodiments, AAV may be goat AAV. The goat AAV serotype may be or may have the sequence described in U.S. Patent No. 7427396 (the entirety of which is incorporated herein by reference), for example, goat AAV (Sequence ID 3 of U.S. 7427396), or a variant thereof.

[0221] In other embodiments, the AAV may be operated as a hybrid AAV from two or more parent serotypes. In some embodiments, the AAV may be an AAV2G9 containing sequences derived from AAV2 and AAV9. The AAV2G9 AAV serotype may be or may have sequences described in U.S. Patent Publication No. US20160017005, the contents of which are incorporated herein by reference in their entirety.

[0222] In some embodiments, AAV may be a serotype generated by an AAV9 capsid library containing mutations in amino acids 390-627 (VP1 numbering), as described in Pulicherla et al. (Molecular Therapy 19(6):1070-1078(2011); the entire content of which is incorporated herein by reference). The serotypes and corresponding nucleotide and amino acid substitutions are AAV9.1 (G1594C;D532H), AAV6.2 (T1418A and T1436X;V473D and I479K), AAV9.3 (T1238A;F413Y), AAV9.4 (T1250C and A1617T;F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T;Q412R, T548A, A587V), AAV9 .6(T1231A;F411I), AAV9.9(G1203A, G1785T;W595C), AAV9.10(A1500G, T1676C;M559T), AAV9.11(A1425T, A1702C, A 1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16(A1775T;Q592L), AAV9.24(T1507C, T1521G;W503R), AAV9.26(A1337G, A1769C;Y446C, Q590P), AAV9.33(A16 67C;D556A), AAV9.34(A1534G, C1794T;N512D), AAV9.35(A1289T, T1450A, C1494T, A1515T, C1794A, G1816A;Q430L, Y 484N, N98K, V606I), AAV9.40(A1694T, E565V), AAV9.41(A1348T, T1362C; T450S), AAV9.44(A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R, A5 55V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T4 92I, H527N), AAV.59(T1336C;Y446H), AAV9.61(A1493T;N498I), AAV9.64(C1531A, A1617T;L511I), AAV9.65(C 1335T, T1530C, C1568A;A523D), AAV9.68(C1510A;P504T), AAV9.80(G1441A;G481R), AAV9.83(C1402A, A1500T ;P468T, E500D), AAV9.87(T1464C, T1468C;S490P), AAV9.90(A1196T;Y399F), AAV9.91(T1316G, A1583T, C1782 This could include, but is not limited to, G, T1806C;L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T;S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T;M559L), and AAV9.95 (T1605A;F535L).

[0223] In some embodiments, the AAV serotype is a sequence described in International Publication No. WO2016049230 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAVF1 / HSC1 (sequences 2 and 20 of WO2016049230), AAVF2 / HSC2 (sequences 3 and 21 of WO2016049230), AAVF3 / HSC3 (W (Sequence codes 5 and 22 of O2016049230), AAVF4 / HSC4 (Sequence codes 6 and 23 of WO2016049230), AAVF5 / HSC5 (Sequence codes 11 and 25 of WO2016049230), AAVF6 / HSC6 (Sequence codes 7 and 24 of WO2016049230), AAVF7 / HSC7 (Sequence codes 8 and 27 of WO2016049230), AAVF8 / HSC8 (WO20 (Sequence codes 9 and 28 of 16049230), AAVF9 / HSC9 (Sequence codes 10 and 29 of WO2016049230), AAVF11 / HSC11 (Sequence codes 4 and 26 of WO2016049230), AAVF12 / HSC12 (Sequence codes 12 and 30 of WO2016049230), AAVF13 / HSC13 (Sequence codes 14 and 31 of WO2016049230), AAVF14 / HSC 14 (SEQ ID NOs. 15 and 32 of WO2016049230), AAVF15 / HSC15 (SEQ ID NOs. 16 and 33 of WO2016049230), AAVF16 / HSC16 (SEQ ID NOs. 17 and 34 of WO2016049230), AAVF17 / HSC17 (SEQ ID NOs. 13 and 35 of WO2016049230), or variants or derivatives thereof, or may contain such.

[0224] In some embodiments, the AAV serotype is a sequence described in U.S. Patent No. 8734809 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV CBr-E1 (sequences 13 and 87 of U.S. 8734809), AAV CBr-E2 (sequences 14 and 88 of U.S. 8734809), AAV CBr-E3 (sequences 15 and 89 of U.S. 8734809), AAV CBr-E4 (sequences 16 and 90 of U.S. 8734809), AAV CBr-E5 (sequences 17 and 91 of U.S. 8734809), AAV CBr-E5 (sequences 18 and 92 of U.S. 8734809), AAV CBr-E6 (sequences 19 and 93 of U.S. 8734809), AAV CBr-E7 (sequences 20 and 94 of U.S. 8734809), AAV CBr-E5 (sequences 18 and 926 (sequences 19 and 93 of U.S. 8734809), AAV CBr-E7 (sequences 20 and 94 of U.S. 8734809), AAV CBr-E5 (sequences 18 and 92 of U.S. 873 CBr-E8 (SEQ ID NOs. 21 and 95 of US8734809), AAV CLv-D1 (SEQ ID NOs. 22 and 96 of US8734809), AAV CLv-D2 (SEQ ID NOs. 23 and 97 of US8734809), AAV CLv-D3 (SEQ ID NOs. 24 and 98 of US8734809), AAV CLv-D4 (SEQ ID NOs. 25 and 99 of US8734809), AAV CLv-D5 (SEQ ID NOs. 26 and 100 of US8734809), AAV CLv-D6 (SEQ ID NOs. 27 and 101 of US8734809), AAV CLv-D7 (SEQ ID NOs. 28 and 102 of US8734809), AAV CLv-D8 (SEQ ID NOs. 29 and 103 of US8734809), AAV CLv-E1 (SEQ ID NOs. 13 and 87 of US8734809), AAV CLv-R1 (SEQ ID NOs. 30 and 104 of US8734809), AAV CLv-R2 (SEQ ID NOs. 31 and 105 of US8734809), AAV CLv-R3 (SEQ ID NOs. 32 and 106 of US8734809), AAV CLv-R4 (SEQ ID NOs. 33 and 107 of US8734809), AAV CLv-R5 (SEQ ID NOs. 34 and 108 of US8734809), AAV CLv-R6 (SEQ ID NOs. 35 and 109 of US8734809), AAV CLv-R7 (SEQ ID NOs. 36 and 110 of US8734809), AAV CLv-R8 (SEQ ID NOs. X and X of US8734809), AAV CLv-R9 (SEQ ID NOs. X and X of US8734809), AAV CLg-F1 (SEQ ID NOs. 39 and 113 of US8734809), AAV CLg-F2 (SEQ ID NOs. 40 and 114 of US8734809), AAV CLg-F3 (SEQ ID NOs. 41 and 115 of US8734809), AAV CLg-F4 (SEQ ID NOs. 42 and 116 of US8734809), AAV CLg-F5 (SEQ ID NOs. 43 and 117 of US8734809), AAV CLg-F6 (SEQ ID NOs. 43 and 117 of US8734809), AAV CLg-F7 (SEQ ID NOs. 44 and 118 of US8734809), AAV CLg-F8 (SEQ ID NOs. 43 and 117 of US8734809), AAV CSp-1 (SEQ ID NOs. 45 and 119 of US8734809), AAV CSp-10 (SEQ ID NOs. 46 and 120 of US8734809), AAV CSp-11 (SEQ ID NOs. 47 and 121 of US8734809), AAV CSp-2 (SEQ ID NOs. 48 and 122 of US8734809), AAV CSp-3 (SEQ ID NOs. 49 and 123 of US8734809), AAV CSp-4 (SEQ ID NOs. 50 and 124 of US8734809), AAV CSp-6 (Sequence IDs 51 and 125 of US8734809), AAV CSp-7 (Sequence IDs 52 and 126 of US8734809), AAVCSp-8 (SEQ ID NOs. 53 and 127 of US8734809), AAV CSp-9 (SEQ ID NOs. 54 and 128 of US8734809), AAV CHt-2 (SEQ ID NOs. 55 and 129 of US8734809), AAV CHt-3 (SEQ ID NOs. 56 and 130 of US8734809), AAV CKd-1 (SEQ ID NOs. 57 and 131 of US8734809), AAV CKd-10 (SEQ ID NOs. 58 and 132 of US8734809), AAV CKd-2 (SEQ ID NOs. 59 and 133 of US8734809), AAV CKd-3 (SEQ ID NOs. 60 and 134 of US8734809), AAV CKd-4 (SEQ ID NOs. 61 and 135 of US8734809), AAV CKd-6 (SEQ ID NOs. 62 and 136 of US8734809), AAV CKd-7 (SEQ ID NOs. 63 and 137 of US8734809), AAV CKd-8 (SEQ ID NOs. 64 and 138 of US8734809), AAV CLv-1 (SEQ ID NOs. 35 and 139 of US8734809), AAV CLv-12 (SEQ ID NOs. 66 and 140 of US8734809), AAV CLv-13 (SEQ ID NOs. 67 and 141 of US8734809), AAV CLv-2 (SEQ ID NOs. 68 and 142 of US8734809), AAV CLv-3 (SEQ ID NOs. 69 and 143 of US8734809), AAV CLv-4 (SEQ ID NOs. 70 and 144 of US8734809), AAV CLv-6 (SEQ ID NOs. 71 and 145 of US8734809), AAV CLv-8 (SEQ ID NOs. 72 and 146 of US8734809), AAV CKd-B1 (SEQ ID NOs. 73 and 147 of US8734809), AAV CKd-B2 (SEQ ID NOs. 74 and 148 of US8734809), AAV CKd-B3 (SEQ ID NOs. 75 and 149 of US8734809), AAV CKd-B4 (SEQ ID NOs. 76 and 150 of US8734809), AAV CKd-B5 (SEQ ID NOs. 77 and 151 of US8734809), AAV CKd-B6 (SEQ ID NOs. 78 and 152 of US8734809), AAV CKd-B7 (SEQ ID NOs. 79 and 153 of US8734809), AAV CKd-B8 (SEQ ID NOs. 80 and 154 of US8734809), AAV CKd-H1 (SEQ ID NOs. 81 and 155 of US8734809), AAV CKd-H2 (SEQ ID NOs. 82 and 156 of US8734809), AAV CKd-H3 (SEQ ID NOs. 83 and 157 of US8734809), AAV CKd-H4 (SEQ ID NOs. 84 and 158 of US8734809), AAV CKd-H5 (SEQ ID NOs. 85 and 159 of US8734809), AAV CKd-H6 (SEQ ID NOs. 77 and 151 of US8734809), AAV CHt-1 (SEQ ID NOs. 86 and 160 of US8734809), AAV CLv1-1 (SEQ ID NO: 171 of US8734809), AAV CLv1-2 (SEQ ID NO: 172 of US8734809), AAV CLv1-3 (SEQ ID NO: 173 of US8734809), AAV CLv1-4 (SEQ ID NO: 174 of US8734809), AAV Clv1-7 (SEQ ID NO: 175 of US8734809), AAV Clv1-8 (SEQ ID NO: 176 of US8734809), AAV Clv1-9 (SEQ ID NO: 177 of US8734809), AAV Clv1-10 (SEQ ID NO: 178 of US8734809), AAV.VR-355 (SEQ ID NO: 181 of US8734809), AAV.hu.48R3 (SEQ ID NO: 183 of US8734809), or their variants or derivatives may be or may contain them.

[0225] In some embodiments, the AAV serotype is a sequence described in International Publication No. WO2016065001 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV CHt-P2 (sequences 1 and 51 of WO2016065001), AAV CHt-P5 (sequences 2 and 52 of WO2016065001), AAV CHt-P9 (sequences 3 and 53 of WO2016065001), AAV CBr-7.1 (sequences 4 and 54 of WO2016065001), AAV CBr-7.2 (sequences 5 and 55 of WO2016065001), AAV CBr-7.3 (sequences 6 and 56 of WO2016065001), AAV CBr-7.4 (Sequence codes 7 and 57 of WO2016065001), AAV CBr-7.5 (Sequence codes 8 and 58 of WO2016065001), AAV CBr-7.7 (Sequence codes 9 and 59 of WO2016065001), AAV CBr-7.8 (Sequence codes 10 and 60 of WO2016065001), AAV CBr-7.10 (Sequence codes 11 and 61 of WO2016065001), AAV CKd-N3 (Sequence codes 12 and 62 of WO2016065001), AAV CKd-N4 (Sequence codes 13 and 63 of WO2016065001), AAV CKd-N9 (Sequence codes 14 and 64 of WO2016065001), AAV CLv-L4 (Sequence IDs 15 and 65 of WO2016065001), AAV CLv-L5 (Sequence codes 16 and 66 of WO2016065001), AAV CLv-L6 (Sequence codes 17 and 67 of WO2016065001), AAV CLv-K1 (Sequence codes 18 and 68 of WO2016065001), AAV CLv-K3 (Sequence codes 19 and 69 of WO2016065001), AAV CLv-K6 (Sequence codes 20 and 70 of WO2016065001), AAV CLv-M1 (Sequence codes 21 and 71 of WO2016065001), AAV CLv-M11 (Sequence codes 22 and 72 of WO2016065001), AAV CLv-M2 (Sequence codes 23 and 73 of WO2016065001), AAV CLv-M5 (Sequence codes 24 and 74 of WO2016065001), AAV CLv-M6 (Sequence codes 25 and 75 of WO2016065001), AAV CLv-M7 (Sequence codes 26 and 76 of WO2016065001), AAV CLv-M8 (Sequence codes 27 and 77 of WO2016065001), AAV CLv-M9 (Sequence codes 28 and 78 of WO2016065001), AAV CHt-P1 (Sequence codes 29 and 79 of WO2016065001), AAV CHt-P6 (Sequence codes 30 and 80 of WO2016065001), AAV CHt-P8 (Sequence codes 31 and 81 of WO2016065001), AAV CHt-6.1 (Sequence codes 32 and 82 of WO2016065001), AAV CHt-6.10 (Sequence codes 33 and 83 of WO2016065001), AAV CHt-6.5 (Sequence codes 34 and 84 of WO2016065001), AAV CHt-6.6 (Sequence codes 35 and 85 of WO2016065001), AAV CHt-6.7 (Sequence codes 36 and 86 of WO2016065001), AAV CHt-6.8 (Sequence codes 37 and 87 of WO2016065001), AAV CSp-8.10 (Sequence codes 38 and 88 of WO2016065001), AAV CSp-8.2 (Sequence codes 39 and 89 of WO2016065001), AAV CSp-8.4 (Sequence codes 40 and 90 of WO2016065001), AAV CSp-8.5 (Sequence codes 41 and 91 of WO2016065001), AAV CSp-8.6 (Sequence codes 42 and 92 of WO2016065001), AAV CSp-8.7 (Sequence codes 43 and 93 of WO2016065001), AAV CSp-8.8 (Sequence codes 44 and 94 of WO2016065001), AAV It may be or may contain CSp-8.9 (SEQ ID NOs. 45 and 95 of WO2016065001), AAV CBr-B7.3 (SEQ ID NOs. 46 and 96 of WO2016065001), AAV CBr-B7.4 (SEQ ID NOs. 47 and 97 of WO2016065001), AAV3B (SEQ ID NOs. 48 and 98 of WO2016065001), AAV4 (SEQ ID NOs. 49 and 99 of WO2016065001), AAV5 (SEQ ID NOs. 50 and 100 of WO2016065001), or variants or derivatives thereof.

[0226] In some embodiments, the AAV particles may have a serotype selected from any of those found in Table 1. In some embodiments, the AAV capsid may comprise any sequence, fragment, or variant thereof from Table 1.

[0227] In some embodiments, the AAV capsid may be encoded by the sequences, fragments, or variants listed in Table 1. In any DNA and RNA sequence mentioned and / or described herein, single-letter notations have the following meanings: A is adenine; C is cytosine; G is guanine; T is thymine; U is uracil; W is a weak base such as adenine or thymine; S is a strong nucleotide such as cytosine and guanine; M is an aminonucleotide such as adenine and cytosine; K is a ketonucleotide such as guanine and thymine; R is purine adenine and guanine; Y is pyrimidine cytosine and thymine; B is any base other than A (e.g., cytosine, guanine, and thymine); D is any base other than C (e.g., adenine, guanine, and thymine); H is any base other than G (e.g., adenine, cytosine, and thymine); V is any base other than T (e.g., adenine, cytosine, and guanine); N is any nucleotide (not a gap); and Z is zero.

[0228] In any of the amino acid sequences mentioned and / or described herein, single-letter notations have the following meanings: G (Gly) is glycine; A (Ala) is alanine; L (Leu) is leucine; M (Met) is methionine; F (Phe) is phenylalanine; W (Trp) is tryptophan; K (Lys) is lysine; Q (Gln) is glutamine; E (Glu) is glutamic acid; S (Ser) is serine; P (Pro) is proline; V (Val) is valine; I (Ile) is Isoleucine; C (Cys) is cysteine; Y (Tyr) is tyrosine; H (His) is histidine; R (Arg) is arginine; N (Asn) is asparagine; D (Asp) is aspartic acid; T (Thr) is threonine; B (Asx) is aspartic acid or asparagine; J (Xle) is leucine or isoleucine; O (Pyl) is pyrrolicin; U (Sec) is selenocysteine; X (Xaa) is any amino acid; and Z (Glx) is glutamine or glutamic acid.

[0229] [Table 1-1]

[0230] Table 1-2

[0231] Table 1-3

[0232] Table 1-4

[0233] Table 1-5

[0234] Table 1-6

[0235] Table 1-7

[0236] Table 1-8

[0237] Table 1-9

[0238] Table 1-10

[0239] Table 1-11

[0240] Table 1-12

[0241] Table 1-13

[0242] Table 1-14

[0243] Table 1-15

[0244] Table 1-16

[0245] Table 1-17

[0246] Table 1-18

[0247] Table 1-19

[0248] Table 1-20

[0249] Table 1-21

[0250] Table 1-22

[0251] Table 1-23

[0252] Table 1-24

[0253] Table 1-25

[0254] Table 1-26

[0255] Table 1-27

[0256] Table 1-28

[0257] Table 1-29

[0258] Table 1-30

[0259] Table 1-31

[0260] Table 1-32

[0261] Table 1-33

[0262] Table 1-34

[0263] Table 1-35

[0264] Table 1-36

[0265] In some embodiments, the AAV serotype is a sequence described in International Patent Publication WO2015038958 (the contents of which are incorporated herein by reference in their entirety), for example, but not limited to, AAV9 (sequences 2 and 11 of WO2015038958 or sequence 137 and 138 herein, respectively), PHP.B (sequences 8 and 9 of WO2015038958, or sequence 5 and 6 herein). These may be, or may have, G2B-13 (SEQ ID NO: 12 in WO2015038958, SEQ ID NO: 7 as specified herein), G2B-26 (SEQ ID NO: 13 in WO2015038958, SEQ ID NO: 5 as specified herein), TH1.1-32 (SEQ ID NO: 14 in WO2015038958, SEQ ID NO: 8 as specified herein), TH1.1-35 (SEQ ID NO: 15 in WO2015038958, SEQ ID NO: 9 as specified herein), or variants thereof. Furthermore, any of the targeted peptides or amino acid inserts described in WO2015038958 may be inserted into any parent AAV serotype, for example, but not limited to, AAV9 (DNA sequence SEQ ID NO: 137 and amino acid sequence SEQ ID NO: 138). In another embodiment, the amino acid insert is inserted between amino acids 588-589 of the parent AAV sequence. The amino acid insert may be, but is not limited to, any of the following amino acid sequences: TLAVPFK (SEQ ID NO: 1262 in this specification), KFPVALT (SEQ ID NO: 1263), LAVPFK (SEQ ID NO: 1264), AVPFK (SEQ ID NO: 1265), VPFK (SEQ ID NO: 1266), TLAVPF (SEQ ID NO: 1267), TLAVP (SEQ ID NO: 1268), TLAV (SEQ ID NO: 1269), SVSKPFL (SEQ ID NO: 1270), FTLTTPK (SEQ ID NO: 1271), MNATKNV (SEQ ID NO: 1272), QSSQTPR (SEQ ID NO: 1273), ILGTGTS (SEQ ID NO: 1274), TRTNPEA (SEQ ID NO: 1275), NGGTSSS (SEQ ID NO: 1276), or YTLSQGW (SEQ ID NO: 1277).Non-limiting examples of nucleotide sequences that may encode an amino acid insert include SEQ ID NOs: 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, or 1287.

[0266] In some embodiments, the AAV serotype may be, or have, a variant thereof, of a sequence described in International Patent Publication WO2017100671 (the contents of which are incorporated herein by reference in their entirety), for example, AAV9 (SEQ ID NO: 45 in WO2017100671, SEQ ID NO: 11 herein), PHP.N (SEQ ID NO: 46 in WO2017100671, SEQ ID NO: 4 herein), PHP.S (SEQ ID NO: 47 in WO2017100671, SEQ ID NO: 10 herein), or a variant thereof. Furthermore, any of the targeted peptides or amino acid inserts described in WO2017100671 may be inserted into any parent AAV serotype, for example, AAV9, for example, but not limited to AAV9. In some embodiments, the amino acid insert is inserted between amino acids 586-592 of the parent AAV (e.g., AAV9). In another embodiment, the amino acid insert is inserted between amino acids 588-589 of the parent AAV sequence. Amino acid inserts are not limited to the following amino acid sequences: AQTLAVPFKAQ (SEQ ID NO: 1288), AQSVSKPFLAQ (SEQ ID NO: 1289), AQFTLTTPKAQ (SEQ ID NO: 1290), DGTLAVPFKAQ (SEQ ID NO: 1291), ESTLAVPFKAQ (SEQ ID NO: 1292), GGTLAVPFKAQ (SEQ ID NO: 1293), AQTLATPFKAQ (SEQ ID NO: 1294), ATTLATPFKAQ (SEQ ID NO: 1295), DGTLATPFKAQ (SEQ ID NO: 1296), GGTLATPFKAQ (SEQ ID NO: 1297), SGSLAVPFKAQ (SEQ ID NO: 1298). 298), AQTLAQPFKAQ (SEQ ID NO: 1299), AQTLQQPFKAQ (SEQ ID NO: 1300), AQTLSNPFKAQ (SEQ ID NO: 1301), AQTLAVPFSNP (SEQ ID NO: 1302), QGTLAVPFKAQ (SEQ ID NO: 1303), NQTLAVPFKAQ (SEQ ID NO: 1304), EGSLAVPFKAQ (SEQ ID NO: 1305), SGNLAVPFKAQ (SEQ ID NO: 1306), EGTLAVPFKAQ (SEQ ID NO: 1307), DSTLAVPFKAQ (SEQ ID NO: 1308), AVTLAVPFKAQ (SEQ ID NO: 1309), AQTLSTPFKAQ (SEQ ID NO: 1310),AQTLPQPFKAQ (SEQ ID NO: 1311), AQTLSQPFKAQ (SEQ ID NO: 1312), AQTLQLPFKAQ (SEQ ID NO: 1313), AQTLTMPFKAQ (SEQ ID NO: 1314), AQTLTTPFKAQ (SEQ ID NO: 1315), AQYTLSQGWAQ (SEQ ID NO: 1316), AQMNATKNVAQ (SEQ ID NO: 1317), AQVSGGHHSAQ (SEQ ID NO: 1318), AQTLTAPFKAQ (SEQ ID NO: 1319), AQTLSKPFKAQ (SEQ ID NO: 1320), QAVRTSL (SEQ ID NO: 1320) 1) YTLSQGW (SEQ ID NO: 1277), LAKERLS (SEQ ID NO: 1322), TLAVPFK (SEQ ID NO: 1262), SVSKPFL (SEQ ID NO: 1270), FTLTTPK (SEQ ID NO: 1271), MNSTKNV (SEQ ID NO: 1323), VSGGHHS (SEQ ID NO: 1324), SAQTLAVPFKAQAQ (SEQ ID NO: 1325), SXXXLAVPFKAQAQ (wherein X may be any amino acid; SEQ ID NO: 1326), SAQXXXVPFKAQAQ (wherein X may be any amino acid; SEQ ID NO: 13 27) SAQTLXXXFKAQAQ (wherein X may be any amino acid; SEQ ID NO: 1328), SAQTLAVXXXAQAQ (wherein X may be any amino acid; SEQ ID NO: 1329), SAQTLAVPFXXXAQ (wherein X may be any amino acid; SEQ ID NO: 1330), TNHQSAQ (SEQ ID NO: 1331), AQAQTGW (SEQ ID NO: 1332), DGTLATPFK (SEQ ID NO: 1333), DGTLATPFKXX (wherein X may be any amino acid; SEQ ID NO: 1334), LAVPFK It may be any of the following: AQ (SEQ ID NO: 1335), VPFKAQ (SEQ ID NO: 1336), FKAQ (SEQ ID NO: 1337), AQTLAV (SEQ ID NO: 1338), AQTLAVPF (SEQ ID NO: 1339), QAVR (SEQ ID NO: 1340), AVRT (SEQ ID NO: 1341), VRTS (SEQ ID NO: 1342), RTSL (SEQ ID NO: 1343), QAVRT (SEQ ID NO: 1344), AVRTS (SEQ ID NO: 1345), VRTSL (SEQ ID NO: 1346), QAVRTS (SEQ ID NO: 1347), or AVRTSL (SEQ ID NO: 1348). Non-limiting examples of nucleotide sequences that can encode an amino acid insert include: SEQ ID NO: 1349, SEQ ID NO: 1350,Examples include SEQ ID NOs: 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358 (wherein N may be A, C, T, or G), 1359 (wherein N may be A, C, T, or G), 1360 (wherein N may be A, C, T, or G), 1361 (wherein N may be A, C, T, or G), 1362 (wherein N may be A, C, T, or G), 1279, 1280, 1281, 1287, or 1363.

[0267] In some embodiments, the AAV serotype is the sequence described in U.S. Patent No. 9624274 (the entirety of which is incorporated herein by reference), for example, but not limited to, AAV1 (sequence number 181 of U.S. 9624274), AAV6 (sequence number 182 of U.S. 9624274), AAV2 (sequence number 183 of U.S. 9624274), AAV3b (sequence number 184 of U.S. 9624274), AAV7 (sequence number 185 of U.S. 9624274), AAV8 (sequence number 186 of U.S. 9624274), AAV10 (sequence number 187 of U.S. 9624274), AAV4 (sequence number 188 of U.S. 9624274), AAV11 (sequence number 189 in US9624274), bAAV (sequence number 190 in US9624274), AAV5 (sequence number 191 in US9624274), GPV (sequence number 192 in US9624274; sequence number 879 in this specification), B19 (sequence number 193 in US9624274; sequence number 880 in this specification), MVM (sequence number 194 in US9624274; sequence number 881 in this specification), FPV (sequence number 195 in US9624274; sequence number 882 in this specification), CPV (sequence number 196 in US9624274; sequence number 883 in this specification) or variants thereof may or may have them. Furthermore, any of the structural protein inserts described in US9624274 may be inserted into any parent AAV serotype, for example, I-453 and I-587 of AAV2 (Sequence ID 183 of US9624274), but not limited to these. The amino acid inserts are not limited to the following amino acid sequences: VNLTWSRASG (SEQ ID NO: 1364), EFCINHRGYWVCGD (SEQ ID NO: 1365), EDGQVMDVDLS (SEQ ID NO: 1366), EKQRNGTLT (SEQ ID NO: 1367), TYQCRVTHPHLPRALMR (SEQ ID NO: 1368), RHSTTQPRKTKGSG (SEQ ID NO: 1369), DSNPRGVSAYLSR (SEQ ID NO: 1370), TITCLWDLAPSK (SEQ ID NO: 1371), KTKGSGFFVF (SEQ ID NO: 1372), THPHLPRALMRS (SEQ ID NO: 1373), GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 1374),LPRALMRS (SEQ ID NO: 1375), INHRGYWV (SEQ ID NO: 1376), CDAGSVRTNAPD (SEQ ID NO: 1377), AKAVSNLTESRSESLQS (SEQ ID NO: 1378), SLTGDEFKKVLET (SEQ ID NO: 1379), REAVAYRFEED (SEQ ID NO: 1380), INPEIITLDG (SEQ ID NO: 1381), DISVTGAPVITATYL (SEQ ID NO: 1382), DISVTGAPVITA (SEQ ID NO: 1383), PKTVSNLTESSSESVQS (SEQ ID NO: 1384), SLMGDEFKAVLET (SEQ ID NO: 1385), QHSVAYTFEED (SEQ ID NO: 1386), INPEIITRDG (SEQ ID NO: 1387), DISLTGDPVITASYL (SEQ ID NO: 1388), DISLTGDPVITA (SEQ ID NO: 1389), DQSIDFEIDSA (SEQ ID NO: 1390), KNVSEDLPLPTFSP TLLGDS (SEQ ID NO: 1391), KNVSEDLPLPT (SEQ ID NO: 1392), CDSGRVRTDAPD (SEQ ID NO: 1393), FPEHLLVDFLQSLS (SEQ ID NO: 1394), DAEFRHDSG (SEQ ID NO: 1395), HYAAAQWDFGNTMCQL (SEQ ID NO: 1396), YAAQWDFGNTMCQ (SEQ ID NO: 1397), RSQKEGLHYT (SEQ ID NO: 1398), SSRTPSDKPVAHW It may be any of the following: ANPQAE (sequence number 1399), SRTPSDKPVAHWANP (sequence number 1400), SSRTPSDKP (sequence number 1401), NADGNVDYHMNSVP (sequence number 1402), DGNVDYHMNSV (sequence number 1403), RSFKEFLQSSLRALRQ (sequence number 1404); FKEFLQSSLRA (sequence number 1405), or QMWAPQWGPD (sequence number 1406).

[0268] In some embodiments, the AAV serotype may be, or may have, an AAV capsid protein containing, for example, one or more amino acid modifications at amino acid positions 585-590 of the natural AAV2 capsid protein, as described in U.S. Patent No. 9475845 (the entirety of which is incorporated herein by reference). Furthermore, the modifications may include, but are not limited to, amino acid sequences RGNRQA (SEQ ID NO: 1407), SSSTDP (SEQ ID NO: 1408), SSNTAP (SEQ ID NO: 1409), SNSNLP (SEQ ID NO: 1410 as used herein), SSTTAP (SEQ ID NO: 1411), AANTAA (SEQ ID NO: 1412), QQNTAP (SEQ ID NO: 1413), SAQAQA (SEQ ID NO: 1414), QANTGP (SEQ ID NO: 1415), NATTAP (SEQ ID NO: 1416), SSTAGP (SEQ ID NO: 1417) This can result in QQNTAA (SEQ ID NO: 1418), PSTAGP (SEQ ID NO: 1419), NQNTAP (SEQ ID NO: 1420), QAANAP (SEQ ID NO: 1421), SIVGLP (SEQ ID NO: 1422), AASTAA (SEQ ID NO: 1423), SQNTTA (SEQ ID NO: 1424), QQDTAP (SEQ ID NO: 1425), QTNTGP (SEQ ID NO: 1426), QTNGAP (SEQ ID NO: 1427), QQNAAP (SEQ ID NO: 1428), or AANTQA (SEQ ID NO: 1429). In some embodiments, the amino acid modification is a substitution at amino acid positions 262-265 of the native AAV2 capsid protein or at the corresponding position of the capsid protein of another AAV containing the targeted sequence. The target sequences are not limited to, but include amino acid sequences such as: NGRAHA (SEQ ID NO: 1430), QPEHSST (SEQ ID NO: 1431), VNTANST (SEQ ID NO: 1432), HGPMQKS (SEQ ID NO: 1433), PHKPPLA (SEQ ID NO: 1434), IKNNEMW (SEQ ID NO: 1435), RNLDTPM (SEQ ID NO: 1436), VDSHRQS (SEQ ID NO: 1437), YDSKTKT (SEQ ID NO: 1438), SQLPHQK (SEQ ID NO: 1439), STMQQNT (SEQ ID NO: 1440), TERYMTQ (SEQ ID NO: 1441), DASLSTS (SEQ ID NO: 1442), DLPNKKT (SEQ ID NO: 1443), DLTAARL (SEQ ID NO: 1444),EPHQFNY (SEQ ID NO: 1445), EPQSNHT (SEQ ID NO: 1446), MSSWPSQ (SEQ ID NO: 1447), NPKHNAT (SEQ ID NO: 1448), PDGMRTT (SEQ ID NO: 1449), PNNNKTT (SEQ ID NO: 1450), QSTTHDS (SEQ ID NO: 1451), TGSKQKQ (SEQ ID NO: 1452), SLKHQAL (SEQ ID NO: 1453), SPIDGEQ (SEQ ID NO: 1454), WIFPWIQL (SEQ ID NO: 1455), CDCRGDCFC (SEQ ID NO: 1456), CNGRC (SEQ ID NO: 1457), CPRECES (SEQ ID NO: 1455) 58), CTTHWGFTLC (SEQ ID NO: 1459), CGRRAGGSC (SEQ ID NO: 1460), CKGGRAKDC (SEQ ID NO: 1461), CVPELGHEC (SEQ ID NO: 1462), CRRETAWAK (SEQ ID NO: 1463), VSWFSHRYSPFAVS (SEQ ID NO: 1464), GYRDGYAGPILYN (SEQ ID NO: 1465), XXXYXXX (SEQ ID NO: 1466), YXNW (SEQ ID NO: 1467), RPLPPLP (SEQ ID NO: 1468), APPLPPR (SEQ ID NO: 1469), DVFYPYPYASGS (SEQ ID NO: 1470), MYWY PY (SEQ ID NO: 1471), DITWDQLWDLMK ​​(SEQ ID NO: 1472), CWDDXWLC (SEQ ID NO: 1473), EWCEYLGGYLRCYA (SEQ ID NO: 1474), YXCXXGPXTWXCXP (SEQ ID NO: 1475), IEGPTLRQWLAARA (SEQ ID NO: 1476), LWXXX (SEQ ID NO: 1477), XFXXYLW (SEQ ID NO: 1478), SSIISHFRWGLCD (SEQ ID NO: 1479), MSRPACPPNDKYE (SEQ ID NO: 1480), CLRSGRGC (SEQ ID NO: 1481), CHWMFSPWC (SEQ ID NO: 1482 ), WXXF (sequence number 1483), CSSRLDAC (sequence number 1484), CLPVASC (sequence number 1485), CGFECVRQCPERC (sequence number 1486), CVALCREACGEGC (sequence number 1487), SWCEPGWCR (sequence number 1488), YSGKWGW (sequence number 1489), GLSGGRS (sequence number 1490), LMLPRAD (sequence number 1491), CSCFRDVCC (sequence number 1492), CRDVVSVIC (sequence number 1493), MARSGL (sequence number 1494), MARAKE (sequence number 1495),MSRTMS (Sequence ID 1496), KCCYSL (Sequence ID 1497), MYWGDSHWLQYWYE (Sequence ID 1498), MQLPLAT (Sequence ID 1499), EWLS (Sequence ID 1500), SNEW (Sequence ID 1501), TNYL (Sequence ID 1502), WDLAWMFRLPVG (Sequence ID 1503), CTVALPGGYVRVC (Sequence ID 1504), CVAYCIEHHCWTC (Sequence ID 1505), CVFAHNYDYLVC (Sequence ID 1506), CVFTSNYAFC (Sequence ID 1507), VHSPNKK (Sequence ID number) (Sequence ID 1508), CRGDGWC (Sequence ID 1509), XRGCDX (Sequence ID 1510), PXXX (Sequence ID 1511), SGKGPRQITAL (Sequence ID 1512), AAAAAAAAAXXXXX (Sequence ID 1513), VYMSPF (Sequence ID 1514), ATWLPPR (Sequence ID 1515), HTMYYHHYQHHL (Sequence ID 1516), SEVGCRAGPLQWLCEKYFG (Sequence ID 1517), CGLLPVGRPDRNVWRWLC (Sequence ID 1518), CKGQCDRFKGLPWEC (Sequence ID 1519), S GRSA (SEQ ID NO: 1520), WGFP (SEQ ID NO: 1521), AEPMPPHSLNFSQYLWYT (SEQ ID NO: 1522), WAYXSP (SEQ ID NO: 1523), IELLQAR (SEQ ID NO: 1524), AYTKCSRQWRTCMTTH (SEQ ID NO: 1525), PQNSKIPGPTFLDPH (SEQ ID NO: 1526), ​​SMEPALPDWWWKMFK (SEQ ID NO: 1527), ANTPCGPYTHDCPVKR (SEQ ID NO: 1528), TACHQHVRMVRP (SEQ ID NO: 1529), VPWMEPAYQRFL (SEQ ID NO: 1530), DP RATPGS (SEQ ID NO: 1531), FRPNRAQDYNTN (SEQ ID NO: 1532), CTKNSYLMC (SEQ ID NO: 1533), CXXTXXXGXGC (SEQ ID NO: 1534), CPIEDRPMC (SEQ ID NO: 1535), HEWSYLAPYPWF (SEQ ID NO: 1536), MCPKHPLGC (SEQ ID NO: 1537), RMWPSSTVNLSAGRR (SEQ ID NO: 1538), SAKTAVSQRVWLPSHRGGEP (SEQ ID NO: 1539), KSREHVNNSACPSKRITAAL (SEQ ID NO: 1540), EGFR (SEQ ID NO: 1541),It may be any of the following: AGLGVR (SEQ ID NO: 1542), GTRQGHTMRLGVSDG (SEQ ID NO: 1543), IAGLATPGWSHWLAL (SEQ ID NO: 1544), SMSIARL (SEQ ID NO: 1545), HTFEPGV (SEQ ID NO: 1546), NTSLKRISNKRIRRK (SEQ ID NO: 1547), LRIKRKRRKRKKTRK (SEQ ID NO: 1548), GGG, GFS, LWS, EGG, LLV, LSP, LBS, AGG, GRR, GGH, and GTV.

[0269] In some embodiments, the AAV serotype may be, or may have, a site-directed mutant capsid protein of a sequence described in U.S. Patent Publication No. US20160369298 (the entirety of which is incorporated herein by reference), for example, AAV2 (SEQ ID NO: 97 in US20160369298; SEQ ID NO: 1549 herein), or a variant thereof, where the specific site is at least one site selected from R447, G453, S578, N587, N587+1, S662 or a fragment thereof of VP1.

[0270] Furthermore, any of the variant sequences listed in US20160369298, but not limited to, the following sequences: SDSGASN (SEQ ID NO: 1550), SPSGASN (SEQ ID NO: 1551), SHSGASN (SEQ ID NO: 1552), SRSGASN (SEQ ID NO: 1553), SKSGASN (SEQ ID NO: 1554), SNSGASN (SEQ ID NO: 1555), SGSGASN (SEQ ID NO: 1556), SASGASN (SEQ ID NO: 1557), SESGTSN (SEQ ID NO: 1558), STTGGSN (SEQ ID NO: 1559), SSAGSTN (SEQ ID NO: 1560), NNDSQA (SEQ ID NO: 1561), NNRNQA (SEQ ID NO: 1562), NNNKQA (SEQ ID NO: 1563), NAKRQA (SEQ ID NO: 1564), NDEHQA (SEQ ID NO: 1565), NTSQKA (SEQ ID NO: 1566), YYLSRTNTPSGTDTQSRLVFSQAGA (SEQ ID NO: 1567), YYLSRTNTDSGTETQSGLDFSQAGA (SEQ ID NO: 1568), YYLSRTNTESGTPTQSALEFSQAGA (SEQ ID NO: 1569), YYLSRTNTHSGTHTQSPLHFSQAGA (SEQ ID NO: 1570), YYLS RTNTSSGTITISHLIFSQAGA (SEQ ID NO: 1571), YYLSRTNTRSGIMTKSSLMFSQAGA (SEQ ID NO: 1572), YYLSRTNTKSGRKTLSNLSFSQAGA (SEQ ID NO: 1573), YYLSRTNDGSGPVTPSKLRFSQRGA (SEQ ID NO: 1574), YYLSRTNAASGHATHSDLKFSQPGA (SEQ ID NO: 1575), YYLSRTNGQAGSLTMSELGFSQVGA (SEQ ID NO: 1576), YYLSRTNSTGGNQTTSQLLFSQLSA (SEQ ID NO: 1577), YF LSRTNNNTGLNTNSTLNFSQGRA (SEQ ID NO: 1578), SKTGADNNNSEYSWTG (SEQ ID NO: 1579), SKTDADNNNSEYSWTG (SEQ ID NO: 1580), SKTEADNNNSEYSWTG (SEQ ID NO: 1581), SKTPADNNNSEYSWTG (SEQ ID NO: 1582), SKTHADNNNSEYSWTG (SEQ ID NO: 1583), SKTQADNNNSEYSWTG (SEQ ID NO: 1584), SKTIADNNNSEYSWTG (SEQ ID NO: 1585), SKTMADNNNSEYSWTG (SEQ ID NO: 1586),SKTRADNNNSEYSWTG (SEQ ID NO: 1587), SKTNADNNNSEYSWTG (SEQ ID NO: 1588), SKTVGRNNNSEYSWTG (SEQ ID NO: 1589), SKTADRNNNSEYSWTG (SEQ ID NO: 1590), SKKLSQNNNSKYSWQG (SEQ ID NO: 1591), SKPTTGNNNSDYSWPG (SEQ ID NO: 1592), STQKNENNNSNYSWPG (SEQ ID NO: 1593), HKDDEGKF (SEQ ID NO: 1594), HKDDNRKF (SEQ ID NO: 1595), HKDDTNKF (SEQ ID NO: 1596), HED SDKNF (SEQ ID NO: 1597), HRDGADSF (SEQ ID NO: 1598), HGDNKSRF (SEQ ID NO: 1599), KQGSEKTNVDFEEV (SEQ ID NO: 1600), KQGSEKTNVDSEEV (SEQ ID NO: 1601), KQGSEKTNVDVEEV (SEQ ID NO: 1602), KQGSDKTNVDDAGV (SEQ ID NO: 1603), KQGSSKTNVDPREV (SEQ ID NO: 1604), KQGSRKTNVDHKQV (SEQ ID NO: 1605), KQGSKGGNVDTNRV (SEQ ID NO: 1606), KQGSGEANVDNGDV (SEQ ID NO: 16 07), KQDAAADNIDYDHV (SEQ ID NO: 1608), KQSGTRSNAAASSV (SEQ ID NO: 1609), KENTNTNDTELTNV (SEQ ID NO: 1610), QRGNNVAATADVNT (SEQ ID NO: 1611), QRGNNEAATADVNT (SEQ ID NO: 1612), QRGNNPAATADVNT (SEQ ID NO: 1613), QRGNNHAATADVNT (SEQ ID NO: 1614), QEENNIAATPGVNT (SEQ ID NO: 1615), QPPNNMAATHEVNT (SEQ ID NO: 1616), QHHNNSAATTIVNT (SEQ ID NO: 16 17) QTTNNRAAFNMVET (SEQ ID NO: 1618), QKKNNNAASKKVAT (SEQ ID NO: 1619), QGGNNKAADDAVKT (SEQ ID NO: 1620), QAAKGGAADDAVKT (SEQ ID NO: 1621), QDDRAAAANESVDT (SEQ ID NO: 1622), QQQHDDAAYQRVHT (SEQ ID NO: 1623), QSSSSLAAVSTVQT (SEQ ID NO: 1624), QNNQTTAAIRNVTT (SEQ ID NO: 1625), NYNKKSDNVDFT (SEQ ID NO: 1626), NYNKKSENVDFT (SEQ ID NO: 1627),NYNKKSLNVDFT (SEQ ID NO: 1628), NYNKKSPNVDFT (SEQ ID NO: 1629), NYSKKSHCVDFT (SEQ ID NO: 1630), NYRKTIYVDFT (SEQ ID NO: 1631), NYKEKKDVHFT (SEQ ID NO: 1632), NYGHRAIVQFT (SEQ ID NO: 1633), NYANHQFVVCT (SEQ ID NO: 1634), NYDDDPTGVLLT (SEQ ID NO: 1635), NYDDPTGVLLT (SEQ ID NO: 1636), NFEQQNSVEWT (SEQ ID NO: 1637), SQSGASN (SEQ ID NO: 1638), NNGSQA (SEQ ID NO: 1639), YYLSRTNTPSGTTTWSRLQFSQAGA (SEQ ID NO: 1640), SKTSADNNNSEYSWTG (SEQ ID NO: 1641), HKDDEEKF (SEQ ID NO: 1642), KQGSEKTNVDIEEV (SEQ ID NO: 1643), QRGNNQAATADVNT (SEQ ID NO: 1644), NYNKKSVNVDFT (SEQ ID NO: 1645), SQSGASNYNTPSGTTTQSRLQFSTSADNNNSEYSWTGATKYH (SEQ ID NO: 1646), SASGASNFNSEGGSLTQSSLGFSTDGENNN SDFSWTGATKYH (SEQ ID NO: 1647), SQSGASNYNTPSGTTTQSRLQFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 1648), SASGASNYNTPSGTTTQSRLQFSTSADNNNSEFSWPGATTYH (SEQ ID NO: 1649), SQSGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 1650), SASGASNYNTPSGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 1651), SQSGASNYNTPSG TTTQSRLQFSTSADNNNSDFSWTGATKYH (SEQ ID NO: 1652), SGAGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 1653), SGAGASN (SEQ ID NO: 1654), NSEGGSLTQSSLGFS (SEQ ID NO: 1655), TDGENNNSDFS (SEQ ID NO: 1656), SEFSWPGATT (SEQ ID NO: 1657), TSADNNNSDFSWT (SEQ ID NO: 1658), SQSGASNY (SEQ ID NO: 1659), NTPSGTTTQSRLQFS (SEQ ID NO: 1660),TSADNNNSEYSWTGATKYH (SEQ ID NO: 1661), SASGASNF (SEQ ID NO: 1662), TDGENNNSDFSWTGATKYH (SEQ ID NO: 1663), SASGASNY (SEQ ID NO: 1664), TSADNNNSEFSWPGATTYH (SEQ ID NO: 1665), NTPSGSLTQSSLGFS (SEQ ID NO: 1666), TSADNNNSDFSWTGATKYH (SEQ ID NO: 1667), SGAGASNF (SEQ ID NO: 1668), CTCCAGVVSVVSMRSRVCVNSGCAGCTDHCVVSRNSG TCVMSACACAA (SEQ ID NO: 1669), CTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAA (SEQ ID NO: 1670), SAAGASN (SEQ ID NO: 1671), YFLSRTNTESGSTTQSTLRFSQAG (SEQ ID NO: 1672), SKTSADNNNSDFS (SEQ ID NO: 1673), KQGSEKTDVDIDKV (SEQ ID NO: 1674), STAGASN (SEQ ID NO: 1675), YFLSRTNTTSGIETQSTLRFSQAG (SEQ ID NO: 1676), SKTDG ENNNSDFS (SEQ ID NO: 1677), KQGAAADDVEIDGV (SEQ ID NO: 1678), SEAGASN (SEQ ID NO: 1679), YYLSRTNTPSGTTTQSRLQFSQAG (SEQ ID NO: 1680), SKTSADNNNSEYS (SEQ ID NO: 1681), KQGSEKTNVDIEKV (SEQ ID NO: 1682), YFLSRTNDASGSDTKSTLLFSQAG (SEQ ID NO: 1683), STTPSENNNSEYS (SEQ ID NO: 1684), SAAGATN (SEQ ID NO: 1685), YFLSRTNGEAGSATLSEL It may be or contain any of the following: RFSQAG (SEQ ID NO: 1686), HGDDADRF (SEQ ID NO: 1687), KQGAEKSDVEVDRV (SEQ ID NO: 1688), KQDSGGDNIDIDQV (SEQ ID NO: 1689), SDAGASN (SEQ ID NO: 1690), YFLSRTNTEGGHDTQSTLRFSQAG (SEQ ID NO: 1691), KEDGGGSDVAIDEV (SEQ ID NO: 1692), SNAGASN (SEQ ID NO: 1693), and YFLSRTNGEAGSATLSELRFSQPG (SEQ ID NO: 1694). Non-limiting examples of nucleotide sequences that can encode amino acid mutation sites include:The following are examples: SEQ ID NOs: 1695, 1696, 1697, 1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, AGCAGGAGCTCCTTGGCCTCAGCGTGCGAG (SEQ ID NOs: 264 in US20160369298; SEQ ID NOs: 1711 in this specification), 1712, 1713, 1714, 1715, 1716, and 1717.

[0271] In some embodiments, the AAV serotype may include, for example, the ophthalmic cell targeting peptides described in International Patent Publication WO2016134375 (the entirety of which is incorporated herein by reference), for example, SEQ ID NOs. 9 and 10 of WO2016134375, for example. Furthermore, any of the ophthalmic cell targeting peptides or amino acids described in WO2016134375 may be inserted into any parent AAV serotype, for example, for example, AAV2 (SEQ ID NOs. 8 of WO2016134375; SEQ ID NOs. 1718 herein), for example, for example, AAV9 (SEQ ID NOs. 11 of WO2016134375; SEQ ID NOs. 1719 herein). In some embodiments, modifications such as insertions are made in the AAV2 protein at P34-A35, T138-A139, A139-P140, G453-T454, N587-R588, and / or R588-Q589. In certain embodiments, insertions are made in AAV9 at D384, G385, 1560, T561, N562, E563, E564, E565, N704, and / or Y705. The ophthalmic cell-targeting peptide may be, but is not limited to, one of the following amino acid sequences: GSTPPPM (SEQ ID NO: 1 in WO2016134375; SEQ ID NO: 1720 herein) or GETRAPL (SEQ ID NO: 4 in WO2016134375; SEQ ID NO: 1721 herein).

[0272] In some embodiments, AAV serotypes may be modified as described in U.S. Patent Publication US20170145405 (the contents of which are incorporated herein by reference in their entirety). AAV serotypes may include modified AAV2 (e.g., modified in Y444F, Y500F, Y730F and / or S662V), modified AAV3 (e.g., modified in Y705F, Y731F and / or T492V), and modified AAV6 (e.g., modified in S663V and / or T492V).

[0273] In some embodiments, AAV serotypes may be modified as described in International Publication WO2017083722 (the contents of which are incorporated herein by reference in their entirety). AAV serotypes may include AAV1 (Y705+731F+T492V), AAV2 (Y444+500+730F+T491V), AAV3 (Y705+731F), AAV5, AAV5 (Y436+693+719F), AAV6 (VP3 variant Y705F / Y731F / T492V), AAV8 (Y733F), AAV9, AAV9 (VP3 variant Y731F), and AAV10 (Y733F).

[0274] In some embodiments, the AAV serotype may include an engineered epitope comprising the amino acid SPAKFA (SEQ ID NO: 24 in WO2017015102; SEQ ID NO: 1722 herein) or NKDKLN (SEQ ID NO: 2 in WO2017015102; SEQ ID NO: 1723 herein), as described in International Patent Publication WO2017015102 (the contents of which are incorporated herein by reference in their entirety). The epitope may be inserted into the region of amino acids 665-670 and / or the region of residues 664-668 of AAV3B (SEQ ID NO: 3), based on the numbering of the VP1 capsid of AAV8.

[0275] In some embodiments, the AAV serotype is one or more of the amino acid residues 262-268, 370-379, 451-459, 472-473, 493-500, 528-534, 547-552, 588-597, 709-710, 716-722 of AAV1 ( For example, an AAV variant may contain a capsid protein containing substitutions in any combination of 2, 3, 4, 5, 6, or 7 amino acids, or substitutions in equivalent amino acid residues of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, bovine AAV, or avian AAV. The amino acid substitutions may be any of the amino acid sequences listed in WO2017058892, but are not limited to these.In some embodiments, AAV has amino acid substitutions in residues 256L, 258K, 259Q, 261S, 263A, 264S, 265T, 266G, 272H, 385S, 386Q, S472R, V473D, N500E547S, 709A, 710N, 716D, 717N, 718N, 720L, A456T, Q457T, N458Q, K459S, T492S, K493A, S586R, S587G, S588N, T589R and / or 722T of AAV1 (SEQ ID NO: 1 of WO2017058892). In any combination, AAV5 (Sequence ID 5 of WO2017058892) 244N, 246Q, 248R, 249E, 250I, 251K, 252S, 253G, 254S, 255V, 256D, 263Y, 377E, 378N, 453L, 456R, 532Q, 533P, 535N, 536P, 537G, 538T, 539T, 540A, 541T, 542Y, 543L, 546N, 653V, 654P, 656S, 697Q, 698F, 704D, 705S, 706T, 707G, 708E, 709Y and / Or any combination of amino acid substitutions at 710R, for AAV5 (Sequence ID 5 of WO2017058892): 248R, 316V, 317Q, 318D, 319S, 443N, 530N, 531S, 532Q, 533P, 534A, 535N, 540A, 541T, 542Y, 543L, 545G, 546N, 697Q, 704D, 706T, 708E, 709Y and / or any combination of amino acid substitutions at 710R, for AAV6 (Sequence ID 6 of WO2017058892): 264S, 266G, 2 It may include any combination of amino acid substitutions at 69N, 272H, 457Q, 588S and / or 589I in AAV8 (SEQ ID NO: 8 of WO2017058892), any combination of amino acid substitutions at 457T, 459N, 496G, 499N, 500N, 589Q, 590N and / or 592A in AAV9 (SEQ ID NO: 9 of WO2017058892), and any combination of amino acid substitutions at 451I, 452N, 453G, 454S, 455G, 456Q, 457N and / or 458Q in AAV9 (SEQ ID NO: 9 of WO2017058892).

[0276] In some embodiments, AAV may include amino acid sequences at positions 155, 156, and 157 of VP1 or positions 17, 18, 19, and 20 of VP2, as described in International Publication No. WO2017066764 (the contents of which are incorporated herein by reference in their entirety). The amino acid sequences may be, but are not limited to, NSS, SXS, SSY, NXS, NSY, SXY, and NXY, where N, X, and Y are, but are not limited to, independently non-serine or non-threonine amino acids, and AAV may be, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some embodiments, the AAV may include a deletion of at least one amino acid at position 156, 157, or 158 of VP1 or position 19, 20, or 21 of VP2, and the AAV may be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0277] In some embodiments, AAV may be a serotype produced by Cre recombination-based AAV targeted evolution (CREATE) as described by Deverman et al. (Nature Biotechnology 34(2):204-209 (2016)) (the entire content of which is incorporated herein by reference). In some embodiments, the AAV serotype thus produced has improved CNS transduction and / or neuronal and astrocyte tropism compared to other AAV serotypes. As non-limiting examples, AAV serotypes may include, but are not limited to, peptides such as PHP.B, PHP.B2, PHP.B3, PHP.A, PHP.S, G2A12, G2A15, G2A3, G2B4, and G2B5. In some embodiments, these AAV serotypes may be AAV9 (SEQ ID NO: 11 or 138) derivatives containing a 7-amino acid insert between amino acids 588-589. Non-limiting examples of these seven amino acid inserts include TLAVPFK (PHP.B; SEQ ID NO: 1262), SVSKPFL (PHP.B2; SEQ ID NO: 1270), FTLTTPK (PHP.B3; SEQ ID NO: 1271), YTLSQGW (PHP.A; SEQ ID NO: 1277), QAVRTSL (PHP.S; SEQ ID NO: 1321), LAKERLS (G2A3; SEQ ID NO: 1322), MNSTKNV (G2B4; SEQ ID NO: 1323), and / or VSGGHHS (G2B5; SEQ ID NO: 1324).

[0278] In some embodiments, the AAV serotype may be as described by Jackson et al (Frontiers in Molecular Neuroscience 9:154 (2016)) (the entire text of which is incorporated herein by reference). In some embodiments, the AAV serotype is PHP.B or AAV9. In some embodiments, when paired with a synapsin promoter, the AAV serotype enhances neuronal transduction compared to when a more ubiquitous promoter (e.g., CBA or CMV) is used.

[0279] In some embodiments, the AAV serotype is a serotype comprising the AAVPHP.N(PHP.N) peptide or a variant thereof. In some embodiments, the AAV serotype is a serotype comprising the AAVPHP.B(PHP.B) peptide or a variant thereof. In some embodiments, the AAV serotype is a serotype comprising the AAVPHP.A(PHP.A) peptide or a variant thereof. In some embodiments, the AAV serotype is a serotype comprising the PHP.S peptide or a variant thereof. In some embodiments, the AAV serotype is a serotype comprising the PHP.B2 peptide or a variant thereof. In some embodiments, the AAV serotype is a serotype comprising the PHP.B3 peptide or a variant thereof. In some embodiments, the AAV serotype is a serotype comprising the G2B4 peptide or a variant thereof. In some embodiments, the AAV serotype is a serotype comprising the G2B5 peptide or a variant thereof. In some embodiments, the AAV serotype is VOY101 or a variant thereof. In some embodiments, the AAV cellotype is VOY201 or a variant thereof.

[0280] In some embodiments, the AAV serotype of the AAV particles, e.g., the AAV particles for vectorized delivery of the GBA protein described herein, is AAV9 or a variant thereof. In some embodiments, the AAV particles, e.g., the recombinant AAV particles described herein, comprise an AAV9 capsid protein. In some embodiments, the AAV9 capsid protein comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the nucleic acid sequence encoding the AAV9 capsid protein comprises the nucleotide sequence of SEQ ID NO: 137. In some embodiments, the AAV9 capsid protein comprises an amino acid sequence that is at least 70%, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identical to SEQ ID NO: 138. In some embodiments, the nucleic acid sequence encoding the AAV9 capsid protein includes a nucleotide sequence that is identical to SEQ ID NO: 137 by at least 70%, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99%.

[0281] In some embodiments, the capsid protein comprises an amino acid sequence that is substantially identical to or to the amino acid sequence of SEQ ID NO: 11 (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the capsid protein comprises an amino acid sequence that has at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 11, but no more than 30, 20, or 10 modifications, such as substitutions, wherein position 449 is K-free, for example, R.

[0282] In some embodiments, the capsid protein comprises an amino acid sequence that is substantially identical to or from the amino acid sequence of SEQ ID NO: 1 (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the capsid protein comprises an amino acid sequence that has at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 1, but no more than 30, 20, or 10 modifications, such as substitutions. In some embodiments, the capsid protein comprises an amino acid sequence encoded by a nucleotide sequence that is substantially identical to or from the nucleotide sequence of SEQ ID NO: 2 (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the nucleotide sequence encoding the capsid protein includes the nucleotide sequence of SEQ ID NO: 2 or a sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0283] In some embodiments, the capsid protein, for example, the AAV9 capsid protein, includes an amino acid sequence that is substantially identical to or from the amino acid sequence of SEQ ID NO: 138 (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the capsid protein includes an amino acid sequence that has at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 138, but no more than 30, 20, or 10 modifications, such as substitutions. In some embodiments, the capsid protein includes an amino acid sequence encoded by a nucleotide sequence that is substantially identical to or from the nucleotide sequence of SEQ ID NO: 137 (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the nucleotide sequence encoding the capsid protein includes the nucleotide sequence of SEQ ID NO: 137 or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the capsid protein includes a substitution at position K449, numbered according to SEQ ID NO: 138, for example, a substitution of K449R.

[0284] In some embodiments, the capsid protein includes an insert containing the amino acid sequence TLAVPFK (SEQ ID NO: 1262). In some embodiments, the insert is located immediately after position 588 relative to a reference sequence numbered according to SEQ ID NO: 138. In some embodiments, the capsid protein includes the amino acid substitutions A587D and Q588G, numbered according to SEQ ID NO: 138.

[0285] In some embodiments, the capsid protein includes an amino acid substitution of K449R, numbered according to SEQ ID NO: 138, and an insert containing the amino acid sequence of TLAVPFK (SEQ ID NO: 1262), where the insert is located immediately after position 588 relative to the reference sequence numbered according to SEQ ID NO: 138.

[0286] In some embodiments, the capsid protein includes an insert comprising the amino acid substitution K449R, numbered according to SEQ ID NO: 138, the amino acid sequence TLAVPFK (SEQ ID NO: 1262), an insert located immediately after position 588 relative to the reference sequence numbered according to SEQ ID NO: 138, and the amino acid substitutions A587D and Q588G, numbered according to SEQ ID NO: 138.

[0287] In some embodiments, the capsid protein is an insert comprising the amino acid sequence TLAVPFK (SEQ ID NO: 1262), wherein the insert is located immediately after position 588 relative to a reference sequence numbered according to SEQ ID NO: 138, and includes amino acid substitutions A587D and Q588G, numbered according to SEQ ID NO: 138.

[0288] In some embodiments, the AAV serotype of the AAV particles for vectorized delivery of, for example, an antibody molecule described herein (e.g., an anti-beta-amyloid antibody molecule), is AAV9 K449R or a variant thereof. In some embodiments, the AAV particles comprise the AAV9 K449 capsid protein. In some embodiments, the AAV9 K449R capsid protein comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the AAV9 K449R capsid protein comprises an amino acid sequence that is at least 70%, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% identical to SEQ ID NO: 11.

[0289] In some embodiments, AAV particles, for example, AAV capsids of AAV particles for vectorized delivery of GBA proteins as described herein, enable cross-brain barrier passage after intravenous administration. Non-limiting examples of such AAV capsids include AAV9, AAV9 K449R, VOY101, VOY201, or peptide inserts, but are not limited to AAVPHP.N(PHP.N), AAVPHP.B(PHP.B), PHP.S, G2A3, G2B4, G2B5, G2A12, G2A15, PHP.B2, PHP.B3, AAV2.BR1, or AAVPHP.A(PHP.A).

[0290] In some embodiments, the AAV serotype is selected for use in terms of its tropism towards central nervous system cells. In some embodiments, the central nervous system cells are neurons. In other embodiments, the central nervous system cells are astrocytes.

[0291] In some embodiments, AAV serotypes are selected for use in terms of their tropism towards muscle cells. In some embodiments, the start codon for translation of the AAV VP1 capsid protein may be CTG, TTG, or GTG, as described in U.S. Patent No. 8163543 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, the nucleotide sequence encoding the capsid protein, e.g., the VP1 capsid protein, includes 3 to 20 mutations (e.g., substitutions) relative to the nucleotide sequence of SEQ ID NO: 137, e.g., 3 to 15 mutations, 3 to 10 mutations, 3 to 5 mutations, 5 to 20 mutations, 5 to 15 mutations, 5 to 10 mutations, 10 to 20 mutations, 10 to 15 mutations, 15 to 20 mutations, 3 mutations, 5 mutations, 10 mutations, 12 mutations, 15 mutations, 18 mutations, or 20 mutations.

[0292] This disclosure refers to structural capsid proteins (including VP1, VP2, and VP3) encoded by the capsid (Cap) gene. These capsid proteins form the outer protein structural shell (i.e., capsid) of viral vectors such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally contain methionine (Met1) as the first amino acid in the peptide sequence, which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for the first methionine (Met1) residue, or generally any first amino acid (AA1), to be cleaved by proteolytic enzymes such as Met-aminopeptidases after or during polypeptide synthesis. This "Met / AA clipping" process often correlates with the corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Met clipping commonly occurs in VP1 and VP3 capsid proteins, but can also occur in VP2 capsid proteins.

[0293] If Met / AA clipping is incomplete, a mixture of one or more (one, two, or three) VP capsid proteins constituting the viral capsid may be produced, some of which may contain the Met1 / AA1 amino acid (Met+ / AA+), and some may lack the Met1 / AA1 amino acid as a result of Met / AA clipping (Met- / AA-). For further considerations regarding Met / AA clipping of capsid proteins, see Jin, et al. Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017 Oct. 28(5):255-267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 February 19. 327(5968):973-977 (the contents of these works are incorporated herein by reference in their entirety).

[0294] According to this disclosure, references to capsid proteins may, in context, refer to independent capsid proteins, viral capsids consisting of mixtures of capsid proteins, and / or polynucleotide sequences (or fragments thereof) that encode, describe, produce, or result in the capsid proteins of this disclosure. Direct references to “capsid proteins” or “capsid polypeptides” (such as VP1, VP2, or VP2) may also include VP capsid proteins containing the Met1 / AA1 amino acid (Met+ / AA+), and corresponding VP capsid proteins lacking the Met1 / AA1 amino acid (Met- / AA-) as a result of Met / AA clipping.

[0295] Furthermore, according to this disclosure, references to specific “sequence numbers” (whether protein or nucleic acid) that each contain or encode one or more capsid proteins containing the Met1 / AA1 amino acid (Met+ / AA+) will be understood to teach VP capsid proteins lacking the Met1 / AA1 amino acid, since examining the sequence will readily reveal any sequence that simply lacks the first listed amino acid (whether or not it is Met1 / AA1).

[0296] As a non-restrictive example, a reference to a (Met+)VP1 polypeptide sequence that is 736 amino acids long and contains the amino acid "Met1" encoded by an AUG / ATG start codon may also be understood as teaching a (Met-)VP1 polypeptide sequence that is 735 amino acids long and does not contain the "Met1" amino acid from the 736 amino acid Met+ sequence. As a second non-restrictive example, a reference to a (AA1+)VP1 polypeptide sequence that is 736 amino acids long and contains the amino acid "AA1" encoded by an arbitrary NNN start codon may also be understood as teaching a (AA1-)VP1 polypeptide sequence that is 735 amino acids long and does not contain the "AA1" amino acid from the 736 amino acid AA1+ sequence.

[0297] References to viral capsids formed from VP capsid proteins (such as references to specific AAV capsid serotypes) may include VP capsid proteins containing the Met1 / AA1 amino acid (Met+ / AA1+), corresponding VP capsid proteins lacking the Met1 / AA1 amino acid as a result of Met / AA1 clipping (Met- / AA1-), and combinations thereof (Met+ / AA1+ and Met- / AA1-).

[0298] As a non-restrictive example, AAV capsidoserotypes may include VP1(Met+ / AA1+), VP1(Met- / AA1-), or combinations of VP1(Met+ / AA1+) and VP1(Met- / AA1-). AAV capsidoserotypes may also include VP3(Met+ / AA1+), VP3(Met- / AA1-), or combinations of VP3(Met+ / AA1+) and VP3(Met- / AA1-), and any similar combination of VP2(Met+ / AA1) and VP2(Met- / AA1-).

[0299] AAV virus genome In some embodiments, the AAV particles of this disclosure function as an expression vector containing a viral genome encoding a GCase protein. The viral genome may encode a GCase protein and enhancers, such as prosaposin (PSAP) or saposin (Sap) polypeptides or their functional variants (e.g., SapA protein or SapC protein), cell membrane permeable peptides (e.g., ApoEII peptide, TAT peptide, or ApoB peptide), lysosome-targeting sequences (LTS), or combinations thereof. In some embodiments, the expression vector is not limited to AAV and may be an adenovirus, retrovirus, lentivirus, plasmid, vector, or any variant thereof.

[0300] In some embodiments, AAV particles, e.g., AAV particles for vectorized delivery of the GBA protein described herein, include a viral genome, e.g., an AAV viral genome (e.g., a vector genome or AAV vector genome). In some embodiments, the viral genome, e.g., an AAV viral genome, further includes terminal inverted repeat (ITR) regions, enhancers, promoters, intron regions, Kozak sequences, exon regions, nucleic acids encoding a transgene encoding a payload (e.g., the GBA protein described herein) with or without enhancement elements, nucleotide sequences encoding miR binding sites (e.g., miR183 binding sites), poly(A) signaling regions, or combinations thereof.

[0301] Viral genome components: Terminal inversion repeats (ITRs) In some embodiments, the viral genome may include at least one terminal inversion repeat (ITR) region. The AAV particles of this disclosure include a viral genome comprising at least one ITR region and a payload region. In some embodiments, the viral genome includes two ITRs. These two ITRs flank the 5' and 3' ends of the payload region. In some embodiments, the ITRs function as origins of replication, including recognition sites for replication. In some embodiments, the ITRs include sequence regions that may be arranged complementaryly and symmetrically. In some embodiments, the ITRs incorporated into the viral genome described herein may consist of native polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0302] The ITR may originate from the same serotype as the capsid, selected from any of the serotypes listed in Table 1 or its derivatives. The ITR may be of a different serotype than the capsid. In some embodiments, an AAV particle has more than one ITR. As a non-limiting example, an AAV particle has a viral genome containing two ITRs. In some embodiments, the ITRs are of the same serotype as each other. In another embodiment, the ITRs are of different serotypes. As a non-limiting example, zero, one, or both of the ITRs have the same serotype as the capsid. In some embodiments, all ITRs in the viral genome of an AAV particle are AAV2 ITRs.

[0303] Independently, each ITR may be about 100 to about 150 nucleotides long. In some embodiments, the ITR may be nucleotides of 100 to 180 lengths, for example, about 100 to 115, about 100 to 120, about 100 to 130, about 100 to 140, about 100 to 150, about 100 to 160, about 100 to 170, about 100 to 180, about 110 to 120, about 110 to 130, about 110 to 140, about 110 to 150, about 110 to 160, about 110 to 170, about 110 to 180, about 120 to 130, about 120 to The ITR includes nucleotides of approximately 140, 120-150, 120-160, 120-170, 120-180, 130-140, 130-150, 130-160, 130-170, 130-180, 140-150, 140-160, 140-170, 140-180, 150-160, 150-170, 150-180, 160-170, 160-180, or 170-180 lengths. In some embodiments, the ITR includes nucleotides of approximately 120-140 lengths, e.g., nucleotides of approximately 130 lengths. In some embodiments, the ITR is 140-142 nucleotides long, e.g., 141 nucleotides long. In some embodiments, the ITR includes nucleotides of length 1205 to 135, for example, nucleotides of length 130. Non-limiting examples of ITR lengths are 102, 130, 140, 141, 142, and 145 nucleotide lengths, having at least 95% identity to them.

[0304] In some embodiments, each ITR may be 141 nucleotides long. In some embodiments, each ITR may be 130 nucleotides long. In some embodiments, the AAV particle contains two ITRs, one ITR being 141 nucleotides long and the other ITR being 130 nucleotides long.

[0305] In some embodiments, the ITR includes one nucleotide sequence of SEQ ID NO: 1829, SEQ ID NO: 1830, or SEQ ID NO: 1862, or a nucleotide sequence that is substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical) to any of the aforementioned sequences. In some embodiments, the ITR includes one nucleotide sequence of SEQ ID NO: 1860, SEQ ID NO: 1861, SEQ ID NO: 1863, or SEQ ID NO: 1864, or a nucleotide sequence having one, two, or three but four or fewer modifications, such as substitutions, to SEQ ID NO: 1860, SEQ ID NO: 1861, SEQ ID NO: 1863, or SEQ ID NO: 1864.

[0306] Viral genome components: promoters and expression enhancers In some embodiments, the payload region of the viral genome includes at least one element that enhances the target specificity and expression of the transgene. See, for example, Powell et al., Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015 (the entire work of which is incorporated herein by reference). Non-exclusive examples of elements that enhance the target specificity and expression of the transgene include promoters, endogenous miRNAs, post-transcriptional regulators (PREs), polyadenylation (PolyA) signal sequences, upstream enhancers (USEs), CMV enhancers, and introns.

[0307] In some embodiments, polypeptide expression in target cells may be driven by a specific promoter, but not limited to species-specific, inducible, tissue-specific, or cell cycle-specific promoter (Parr et al., Nat. Med. 3:1145-9 (1997); the entirety of which is incorporated herein by reference).

[0308] In some embodiments, the viral genome includes one that is sufficient for the expression of a payload encoded by the transgene (e.g., a GBA protein) in, for example, a target cell. In some embodiments, the promoter is considered efficient if it drives the expression of a polypeptide(s) encoded in the payload region of the viral genome of the AAV particle.

[0309] In some embodiments, the promoter is one that is considered efficient in driving expression in the target cells or tissues. In some embodiments, the promoter drives the expression of GCase, GCase and SapA, or GCase and SapC proteins (or more) in a target tissue for a specified period of time. The promoter-driven expression can occur at intervals of 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, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 2 This can be 3 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or a period exceeding 10 years. The onset period can be 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years.

[0310] In some embodiments, the promoter promotes the expression of polypeptides (e.g., GCase polypeptide, GCase polypeptide and prosaposin (PSAP) polypeptide, GCase polypeptide and SapA polypeptide, GCase polypeptide and SapC polypeptide, GCase polypeptide and cell membrane permeable peptide (e.g., ApoEII peptide, TAT peptide, and / or ApoB peptide), or GCase polypeptide and lysosome-targeting peptide) for at least 1, 2, 3, 4, or 5 months. , 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 ​​years, 49 years, 50 years, 55 years, 60 years, 65 years, or beyond 65 years.

[0311] The promoter may be natural or non-natural. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters. In some embodiments, the promoter may be a human promoter. In some embodiments, the promoter may be a truncated type.

[0312] In some embodiments, the viral genome includes one or more promoters, e.g., multiple cell and / or tissue promoters, e.g., ubiquitous promoters, which in some embodiments drive or promote expression in most mammalian tissues. Examples of promoters that drive or promote expression in some embodiments include, but are not limited to, human elongation factor 1α-subunit (EF1α), cytomegalovirus (CMV) pre-early enhancer and / or promoter, chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (GUSB), and ubiquitin C (UBC). Tissue-specific expression elements can be used to restrict expression to specific cell types, e.g., but are not limited to, CNS-specific promoters, B-cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or promoters specific to various nervous system cell or tissue types, e.g., those that can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0313] In some embodiments, the viral genome includes a nervous system-specific promoter, e.g., a promoter that brings about the expression of a payload in neurons, astrocytes, and / or oligodendrocytes. Non-limiting examples of neuron tissue-specific expression elements include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), synapsin 1 (Syn1), methyl-CpG-binding protein 2 (MeCP2), Ca 2+Examples include the promoters of calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), β-globin minigene nβ2, preproenkephalin (PPE), enkephalin (Enk), and excitatory amino acid transporter 2 (EAAT2). Non-limiting examples of tissue-specific expression elements in astrocytes include the glial fibrillary acid protein (GFAP) and EAAT2 promoters. Non-limiting examples of tissue-specific expression elements in oligodendrocytes include the myelin basic protein (MBP) promoter. Prion promoters represent further tissue-specific promoters useful for driving protein expression in CNS tissues (see Loftus, Stacie K., et al. Human molecular genetics 11.24(2002):3107-3114; the entire disclosure is invoked by reference).

[0314] In some embodiments, the promoter may be less than 1kb. The promoters are 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 5 Nucleotide lengths may be 20, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, or greater than 800. The promoter may have a nucleotide length of 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800.

[0315] In some embodiments, the promoter may be the same or different starting promoter or parent promoter, for example, a combination of two or more components of CMV and CBA, without limitation. Each component may be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450, 460, 470, 4 Nucleotide lengths may be 80, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, or greater than 800. Each component may have a nucleotide length of 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800. In some embodiments, the promoter is a combination of a 382-nucleotide CMV enhancer sequence and a 260-nucleotide CBA promoter sequence.

[0316] In some embodiments, the viral genome includes a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives such as CAG, CB6, and CBh), EF-1α, PGK, UBC, GUSB (hGBp), and UCOE (promoter for HNRPA2B1-CBX3). In some embodiments, the viral genome includes the EF-1α promoter or an EF-1α promoter variant.

[0317] In some embodiments, the promoter is a ubiquitous promoter as described in Yu et al. (Molecular Pain 2011, 7:63), Soderblom et al. (E. Neuro 2015), Gill et al. (Gene Therapy 2001, Vol. 8, 1539-1546), and Husain et al. (Gene Therapy 2009) (the contents of each of these are incorporated by reference in their entirety).

[0318] In some embodiments, the promoter is not cell-specific. In some embodiments, the promoter is a ubiquitin c (UBC) promoter. The UBC promoter may have a size of 300–350 nucleotides. As a non-limiting example, the UBC promoter is 332 nucleotides. In some embodiments, the promoter is a β-glucuronidase (GUSB) promoter. The GUSB promoter may have a size of 350–400 nucleotides. As a non-limiting example, the GUSB promoter is 378 nucleotides. In some embodiments, the promoter is a neurofilament light chain (NFL) promoter. The NFL promoter may have a size of 600–700 nucleotides. As a non-limiting example, the NFL promoter is 650 nucleotides. In some embodiments, the promoter is a neurofilament heavy chain (NFH) promoter. The NFH promoter may have a size of 900–950 nucleotides. As a non-limiting example, the NFH promoter is 920 nucleotides. In some embodiments, the promoter is a scn8a promoter. The scn8a promoter may have a size of 450–500 nucleotides. As a non-restrictive example, the scn8a promoter is 470 nucleotides long.

[0319] In some embodiments, the promoter is a phosphoglycerate kinase 1 (PGK) promoter. In some embodiments, the promoter is the chicken β-actin (CBA) promoter or a functional variant thereof.

[0320] In some embodiments, the promoter is a CB6 promoter or a functional variant thereof. In some embodiments, the promoter is a CB promoter or a functional variant thereof. In some embodiments, the promoter is a minimal CB promoter or a functional variant thereof.

[0321] In some embodiments, the promoter is a CBA promoter or a functional variant thereof. In some embodiments, the promoter is a minimal CBA promoter or a functional variant thereof.

[0322] In some embodiments, the promoter is a cytomegalovirus (CMV) promoter or a functional variant thereof. In some embodiments, the promoter is a CAG promoter or a functional variant thereof.

[0323] In some embodiments, the promoter is the EF1α promoter or a functional variant thereof. In some embodiments, the promoter is a GFAP promoter (e.g., as described in Zhang, Min, et al. Journal of neuroscience research 86.13(2008):2848-2856; the entire disclosure is incorporated by reference) that drives the expression of a GCase polypeptide, or a GCase polypeptide and an enhancing element (e.g., GCase and SapA, or GCase and SapC protein expression) in astrocytes.

[0324] In some embodiments, the promoter is a synapsin promoter or a functional variant thereof. In some embodiments, the promoter is an RNA pol III promoter. In a non-limiting example, the RNA pol III promoter is U6. In a non-limiting example, the RNA pol III promoter is H1.

[0325] In some embodiments, the viral genome includes two promoters. In non-limiting examples, the promoters are the EF1α promoter and the CMV promoter. In some embodiments, the viral genome includes an enhancer element, a promoter, and / or a 5'UTR intron. The enhancer element, also referred to herein as the “enhancer,” may be a CMV enhancer, but is not limited to that which; the promoter may be a CMV, CBA, UBC, GUSB, NSE, synapsin, MeCP2, or GFAP promoter, but is not limited to that which; and the 5'UTR / intron may be SV40 or CBA-MVM, but is not limited to that which. As a non-limiting example, enhancers, promoters, and / or introns used in combination may include: (1) CMV enhancer, CMV promoter, SV40 5'UTR intron; (2) CMV enhancer, CBA promoter, SV40 5'UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM 5'UTR intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter; (7) Synapsin promoter; (8) MeCP2 promoter; and (9) GFAP promoter.

[0326] In some embodiments, the viral genome includes an enhancer. In some embodiments, the enhancer includes a CMVie enhancer. In some embodiments, the viral genome includes a CMVie enhancer and a CB promoter. In some embodiments, the viral genome includes a CMVie enhancer and a CMV promoter (e.g., a CMV promoter region). In some embodiments, the viral genome includes a CMVie enhancer, a CBA promoter or a functional variant thereof, and an intron (e.g., a CAG promoter).

[0327] In some embodiments, the viral genome includes an engineered promoter. In other embodiments, the viral genome includes a promoter derived from a naturally expressed protein.

[0328] In some embodiments, the CBA promoter is used for a viral genome of an AAV particle described herein, for example, a GCase protein, or a GCase protein and an enhancement element (e.g., GCase and SapA protein, GCase and SapC protein, or GCase protein and a cell membrane-permeable peptide or variant thereof). In some embodiments, the CBA promoter is engineered to optimally express a GCase polypeptide or a GCase polypeptide and an enhancement element described herein (e.g., a prosaposin or saposin protein or variant thereof; a cell membrane-permeable peptide or variant thereof; or a lysosome-targeting signal).

[0329] Viral genome components: Introns In some embodiments, the vector genome comprises at least one intron or a fragment or derivative thereof. In some embodiments, at least one intron can enhance the expression of GCase proteins and / or enhancement elements described herein (e.g., prosaposin proteins or SapC proteins or variants thereof; cell membrane permeable peptides (e.g., ApoEII peptide, TAT peptide, or ApoB peptide) or variants thereof; and / or lysosomal targeting signals) (see, for example, Powell et al., Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015, which is incorporated herein by reference in its entirety). Non-restrictive examples of introns include MVM (67-97 bp), F.IX truncated intron 1 (300 bp), β-globin SD / immunoglobulin heavy chain splice acceptor (250 bp), adenovirus splice donor / immunoglobulin splice acceptor (500 bp), SV40 late splice donor / splice acceptor (19S / 16S) (180 bp), and hybrid adenovirus splice donor / IgG splice acceptor (230 bp).

[0330] In some embodiments, introns may be 100 to 500 nucleotides long. Introns may have lengths of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nucleotides. Introns may have lengths of 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-500 nucleotides.

[0331] In some embodiments, the AAV vector may be an SV40 intron or a fragment or variant thereof. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a CBA promoter. In some embodiments, the promoter may be an H1 promoter.

[0332] In some embodiments, the AAV vector may contain a beta-globin intron or a fragment or variant thereof. In some embodiments, the intron contains one or more human beta-globin sequences (e.g., fragments / variants thereof). In some embodiments, the promoter may be a CB promoter. In some embodiments, the promoter includes a CMV promoter. In some embodiments, the promoter includes a minimal CBA promoter.

[0333] In some embodiments, the encoded protein(s) may be located downstream of an intron in the expression vector, such as an SV40 intron, a betaglobin intron, or other introns known in the art. Furthermore, the encoded GBA protein may also be located upstream of a polyadenylation sequence in the expression vector. In some embodiments, the encoded protein may be located in the expression vector within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or beyond 30 nucleotides, downstream of a promoter containing an intron (e.g., 3' to the promoter containing the intron) and / or upstream of a polyadenylation sequence (e.g., 5' to the polyadenylation sequence). In some embodiments, the encoded GBA protein may be located in the expression vector within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30, or 25-30 nucleotides downstream of the intron (e.g., 3' side of the intron) and / or upstream of the polyadenylation sequence (e.g., 5' side of the polyadenylation sequence). In some embodiments, the encoded protein may be located in the expression vector within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% of nucleotides downstream of the intron (e.g., 3' side of the intron) and / or upstream of the polyadenylated sequence (e.g., 5' side of the polyadenylated sequence), or beyond 25% of nucleotides.In some embodiments, the encoded protein may be located in the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% of the sequence downstream of the intron (e.g., 3' side of the intron) and / or upstream of the polyadenylation sequence (e.g., 5' side of the polyadenylation sequence) in the expression vector.

[0334] In certain embodiments, the intron sequence is not an enhancer sequence. In some embodiments, the intron sequence is not a sub-component of the promoter sequence. In some embodiments, the intron sequence is a sub-component of the promoter sequence.

[0335] Viral genome components: Untranslated regions (UTRs) In some embodiments, the wild-type untranslated region (UTR) of a gene is transcribed but not translated. Generally, the 5' UTR begins at the transcription start site and ends at the start codon, while the 3' UTR begins immediately after the stop codon and continues to the transcription termination signal.

[0336] Stability and protein production can be enhanced by manipulating the UTR with features typically found in genes that are abundantly expressed in specific target organs. As a non-limiting example, the 5'UTR of mRNA normally expressed in the liver (e.g., albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) can be used in the viral genome of the AAV particles of this disclosure to enhance expression in hepatocyte lines or the liver.

[0337] In some embodiments, the viral genome encoding the transgene described herein (e.g., a transgene encoding the GBA protein) includes a Kozak sequence. While we do not wish to be bound by theory, the wild-type 5' untranslated region (UTR) contains features that play a role in translation initiation. The Kozak sequence is commonly known to be involved in the process by which ribosomes initiate translation of numerous genes, and is typically contained within the 5'UTR. The Kozak sequence has the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), followed by another "G".

[0338] In some embodiments, the 5'UTR of the viral genome contains a Kozak sequence. In some embodiments, the 5'UTR of the viral genome does not contain the Kozak sequence.

[0339] While we do not wish to be bound by theory, it is known that wild-type 3'UTRs have adenosine and uridine stretches embedded within the 3'UTR. These AU-rich signatures are particularly common in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al, 1995; the entire content of which is incorporated herein by reference): Class I AREs, e.g., c-Myc and MyoD, contain several copies of the AUUUA motif dispersed within the U-rich region; Class II AREs, e.g., GM-CSF and TNF-α, have two or more overlapping UUAUUUA(U / A)(U / A) notamers; Class III AREs, e.g., c-Jun and myogenin, are less well-defined. These U-rich regions do not contain the AUUUA motif. Most proteins that bind to AREs are known to destabilize messengers, but members of the ELAV family, particularly HuR, have been demonstrated to increase mRNA stability. HuR binds to all three classes of AREs. By artificially introducing a HuR-specific binding site to the 3'UTR of a nucleic acid molecule, HuR binding is achieved, thereby stabilizing the message in vivo.

[0340] The stability of polynucleotides can be modulated by introducing, removing, or modifying 3'UTR AU-rich elements (AREs). When manipulating specific polynucleotides, such as the payload region of a viral genome, introducing one or more copies of AREs can reduce the stability of the polynucleotide, thereby decreasing translation and consequently reducing protein production. Similarly, to increase intracellular stability, AREs can be identified, removed, or mutated, thereby increasing translation and consequently protein production.

[0341] In some embodiments, the 3'UTR of the viral genome may contain an oligo(dT) sequence for template addition of the poly(A) tail. Any UTR derived from any gene known in the art can be incorporated into the viral genome of an AAV particle. These UTRs, or portions thereof, may be positioned in the same orientation as the selected gene, or their orientation or position may be altered. In some embodiments, the UTRs used in the viral genome of an AAV particle may be inverted, shortened, lengthened, or constructed using one or more other 5'UTRs or 3'UTRs known in the art. As used herein, the term “modified” means, in the case of a UTR, that the UTR has been altered in any way compared to a reference sequence. For example, a 3' or 5' UTR may be modified from a wild-type or native UTR by altering its orientation or position, as taught above, or by including additional nucleotides, deleting nucleotides, exchanging or rearranging nucleotides.

[0342] In some embodiments, the viral genome of the AAV particle contains at least one artificial UTR that is not a variant of the wild-type UTR. In some embodiments, the viral genome of the AAV particle includes UTRs selected from a family of transcripts whose proteins share a common function, structure, features, or properties.

[0343] Viral genome components: miR binding sites Tissue or cell-specific expression of the AAV virus particles of the present invention can be enhanced by introducing tissue or cell-specific regulatory sequences, such as promoters, enhancers, microRNA binding sites, or detargeting sites. While we do not wish to be bound by theory, it is thought that encoded miR binding sites can regulate, for example, prevent, suppress, or otherwise inhibit the expression of target genes on the viral genome of the present invention based on the expression of corresponding endogenous microRNAs (miRNAs) or corresponding regulatory exogenous miRNAs in tissues or cells, such as non-target cells or tissues. In some embodiments, miR binding sites regulate, for example, reduce the expression of payloads encoded by the viral genome of the AAV particles described herein in cells or tissues where the corresponding mRNA is expressed. In some embodiments, miR binding sites regulate, for example, reduce the expression of encoded GBA proteins in cells or tissues of DRGs, liver, hematopoietic systems, or combinations thereof.

[0344] In some embodiments, the viral genome of the AAV particles described herein includes a nucleotide sequence encoding a microRNA binding site, for example, a detargeting site. In some embodiments, the viral genome of the AAV particles described herein includes a miR binding site, a microRNA binding site sequence (miR BS), or a nucleotide sequence encoding its inverse complementary strand.

[0345] In some embodiments, the miR binding site sequence or the nucleotide sequence encoding the miR binding site is located in the 3'-UTR region of the viral genome (e.g., 3' side relative to the nucleic acid sequence encoding the payload), e.g., before the polyA sequence, in the 5'-UTR region of the viral genome (e.g., 5' side relative to the nucleic acid sequence encoding the payload), or both.

[0346] In some embodiments, the encoded miR binding site sequence contains at least 1 to 5 copies, e.g., at least 1 to 3, 2 to 4, 3 to 5, 1, 2, 3, 4, 5 or more copies of miR binding sites (miR BS). In some embodiments, the encoded miR binding site sequence contains 4 copies of miR binding sites. In some embodiments, all copies are identical, e.g., containing the same miR binding site. In some embodiments, the miR binding sites in the encoded miR binding site sequence are contiguous and not separated by spacers. In some embodiments, the miR binding sites in the encoded miR binding site sequence are separated by spacers, e.g., non-coding sequences. In some embodiments, the spacers are about 1 to 6 nucleotides long or about 5 to 10 nucleotides long, e.g., about 7 to 8 nucleotides long. In some embodiments, the spacers are about 8 nucleotides long. In some embodiments, the spacer sequence contains one or more of (i)GGAT, (ii)CACGTG, (iii)GCATGC, or one or more repeats of (i) to (iii). In some embodiments, the spacer comprises the nucleotide sequence of SEQ ID NO: 1848, or the nucleotide sequence of SEQ ID NO: 1848 having at least one, two, or three modifications, but four or fewer modifications.

[0347] In some embodiments, the encoded miR binding site sequence contains at least 1 to 5 copies, e.g., at least 1 to 3, 2 to 4, 3 to 5, 1, 2, 3, 4, 5 or more copies of miR binding sites (miR BS). In some embodiments, at least 1, 2, 3, 4, 5 or all copies are different, e.g., containing different miR binding sites. In some embodiments, the miR binding sites in the encoded miR binding site sequence are contiguous and not separated by spacers. In some embodiments, the miR binding sites in the encoded miR binding site sequence are separated by spacers, e.g., non-coding sequences. In some embodiments, the spacers are about 1 to 6 nucleotides long or about 5 to 10 nucleotides long, e.g., about 7 to 8 nucleotides long or about 8 nucleotides long. In some embodiments, the spacer sequence contains one or more of (i)GGAT, (ii)CACGTG, (iii)GCATGC, or one or more repeats of (i) to (iii). In some embodiments, the spacer comprises the nucleotide sequence of SEQ ID NO: 1848, or the nucleotide sequence of SEQ ID NO: 1848 having at least one, two, or three modifications, but four or fewer modifications.

[0348] In some embodiments, the encoded miR binding site is substantially identical to the host cell's miR (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical). In some embodiments, the encoded miR binding site contains at least 1, 2, 3, 4, or 5 mismatches or 6, 7, 8, 9, or 10 or fewer mismatches relative to the host cell's miR. In some embodiments, the mismatched nucleotides are contiguous. In some embodiments, the mismatched nucleotides are not contiguous. In some embodiments, the mismatched nucleotides are located outside the seed region binding sequence of the miR binding site, for example, on one or both sides of the miR binding site. In some embodiments, the encoded miR binding site is 100% identical to the host cell's miR.

[0349] In some embodiments, the nucleotide sequence encoding the miR binding site is substantially complementary to the host cell's miR (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% complementary). In some embodiments, the sequence complementary to the nucleotide sequence encoding the miR binding site contains at least 1, 2, 3, 4, or 5 mismatches or 6, 7, 8, 9, or 10 or fewer mismatches with respect to the corresponding host cell's miR. In some embodiments, the mismatched nucleotides are contiguous. In some embodiments, the mismatched nucleotides are not contiguous. In some embodiments, the mismatched nucleotides are located outside the seed region binding sequence of the miR binding site, e.g., on one or both sides of the miR binding site. In some embodiments, the encoded miR binding site is 100% complementary to the host cell's miR.

[0350] In some embodiments, the encoded miR binding site or sequence of encoded miR binding sites is about 10 to about 125 nucleotides long, e.g., about 10 to 50 nucleotides, 10 to 100 nucleotides, 50 to 100 nucleotides, 50 to 125 nucleotides, or 100 to 125 nucleotides. In some embodiments, the encoded miR binding site or sequence of encoded miR binding sites is about 7 to about 28 nucleotides long, e.g., about 8 to 28 nucleotides, 7 to 28 nucleotides, 8 to 18 nucleotides, 12 to 28 nucleotides, 20 to 26 nucleotides, 22 nucleotides, 24 nucleotides, or 26 nucleotides, and optionally includes at least one contiguous region (e.g., 7 or 8 nucleotides) that is complementary (e.g., fully complementary or partially complementary) to the seed sequence of the miRNA (e.g., miR122, miR142, miR183).

[0351] In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to a miR expressed in the liver or hepatocytes, such as miR122. In some embodiments, the encoded miR binding site or encoded miR binding site sequence includes a miR122 binding site sequence. In some embodiments, the encoded miR122 binding site includes the nucleotide sequence ACAAACACCATTGTCACACTCCA (SEQ ID NO: 1865), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1865, or having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, for example, which can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome includes at least 3, 4, or 5 copies of encoded miR122 binding sites, for example, a sequence of encoded miR122 binding sites, which optionally include the nucleotide sequence ACAAACACCATTGTCACACTCCACACAAACACCATTGTCACACTCCACACAAACACCATTGTCACACTCCA (SEQ ID NO: 1866), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1866, or having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, for example, which can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, at least two coded miR122 binding sites are connected directly, for example, without spacers.In other embodiments, at least two encoded miR122 binding sites are separated by spacers, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides long, located between two or more consecutive encoded miR122 binding site sequences. In embodiments, the spacers are about 1 to 6 nucleotides long or about 5 to 10 nucleotides long, for example, about 7 to 8 nucleotides long or about 8 nucleotides long. In some embodiments, the spacer sequence includes one or more of (i)GGAT, (ii)CACGTG, (iii)GCATGC, or one or more repeats of (i) to (iii). In some embodiments, the spacer includes the nucleotide sequence of SEQ ID NO: 1848, or the nucleotide sequence of SEQ ID NO: 1848 having at least 1, 2, or 3 modifications, but 4 or fewer modifications.

[0352] In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to the miR expressed in the hematopoietic system, including immune cells (e.g., antigen-presenting cells or APCs, including dendritic cells (DCs), macrophages, and B lymphocytes). In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to the miR expressed in the hematopoietic system and includes, for example, a nucleotide sequence disclosed in US2018 / 0066279 (the entirety of which is incorporated herein by reference).

[0353] In some embodiments, the encoded miR binding site or sequence of encoded miR binding sites includes a miR-142-3p binding site sequence. In some embodiments, the encoded miR-142-3p binding site includes a nucleotide sequence of TCCATAAAGTAGGAAACACTACA (SEQ ID NO: 1869) or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1842, or a nucleotide sequence having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, for example, which can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome includes at least 3, 4, or 5 copies of encoded miR-142-3p binding sites, for example, a sequence of encoded miR-142-3p binding sites. In some embodiments, at least 3, 4, or 5 copies (e.g., 4 copies) of the encoded miR-142-3p binding site are contiguous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides long or about 5 to 10 nucleotides long, e.g., about 7 to 8 nucleotides long or about 8 nucleotides long. In some embodiments, the spacer sequence includes one or more of (i)GGAT, (ii)CACGTG, (iii)GCATGC, or one or more repeats of (i) to (iii). In some embodiments, the spacer includes the nucleotide sequence of SEQ ID NO: 1848, or the nucleotide sequence of SEQ ID NO: 1848 having at least 1, 2, or 3 modifications, but 4 or fewer modifications.

[0354] In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to the miR expressed in DRG (dorsal root ganglion) neurons (e.g., miR183, miR182, and / or miR96 binding sites). In some embodiments, the encoded miR binding site is complementary (e.g., fully complementary or partially complementary) to the miR expressed in DRG neurons. In some embodiments, the encoded miR binding site includes, for example, nucleotide sequences disclosed in WO2020 / 132455 (the entirety of which is incorporated herein by reference).

[0355] In some embodiments, the encoded miR binding site or sequence of encoded miR binding sites includes a miR183 binding site sequence. In some embodiments, the encoded miR183 binding site is

[0356] [ka]

[0357] The nucleotide sequence (SEQ ID NO: 1847), or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1847, or having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, such that the modifications can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, a sequence complementary to the seed sequence (e.g., fully complementary or partially complementary) corresponds to a double-underlined encoded miR-183 binding site sequence. In some embodiments, the viral genome contains at least 3, 4, or 5 copies (e.g., 4 copies) of encoded miR183 binding sites, e.g., encoded miR183 binding sites. In some embodiments, the viral genome includes at least four copies of encoded miR183 binding sites, for example, the encoded miR183 binding sites include four copies of miR183 binding sites. In some embodiments, at least three, four, or five copies (e.g., four copies) of encoded miR183 binding sites are contiguous (e.g., not separated by spacers) or separated by spacers. In some embodiments, the spacers are about 1 to 6 nucleotides long or about 5 to 10 nucleotides long, for example, about 7 to 8 nucleotides long or about 8 nucleotides long. In some embodiments, the spacers include the nucleotide sequence of SEQ ID NO: 1848, or the nucleotide sequence of SEQ ID NO: 1848 having at least one, two, or three modifications, but four or fewer modifications. In some embodiments, the encoded miR183 binding site sequence includes the nucleotide sequence of SEQ ID NO: 1849, or a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1849, or having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications.

[0358] In some embodiments, the encoded miR binding site or encoded miR binding site sequence includes a miR182 binding site sequence. In some embodiments, the encoded miR182 binding site includes the nucleotide sequence AGTGTGAGTTCTACCATTGCCAAA (SEQ ID NO: 1867), having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1867, or a nucleotide sequence having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, for example, such modifications can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome includes at least 3, 4, or 5 copies of encoded miR182 binding sites, e.g., an encoded miR182 binding site sequence. In some embodiments, at least 3, 4, or 5 copies (e.g., 4 copies) of encoded miR182 binding sites are contiguous (e.g., not separated by spacers) or separated by spacers. In some embodiments, the spacers are about 1 to 6 nucleotides long or about 5 to 10 nucleotides long, e.g., about 7 to 8 nucleotides long or about 8 nucleotides long. In some embodiments, the spacers include the nucleotide sequence of SEQ ID NO: 1848, or the nucleotide sequence of SEQ ID NO: 1848 having at least 1, 2, or 3 modifications, but 4 or fewer modifications.

[0359] In some embodiments, the encoded miR binding site or sequence of encoded miR binding sites includes a miR96 binding site sequence. In some embodiments, the encoded miR96 binding site includes the nucleotide sequence AGCAAAAATGTGCTAGTGCCAAA (SEQ ID NO: 1868), having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1868, or a sequence having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, for example, which can result in a mismatch between the encoded miR binding site and the corresponding miRNA. In some embodiments, the viral genome includes at least 3, 4, or 5 copies of encoded miR96 binding sites, e.g., a sequence of encoded miR96 binding sites. In some embodiments, at least 3, 4, or 5 copies (e.g., 4 copies) of encoded miR96 binding sites are contiguous (e.g., not separated by spacers) or separated by spacers. In some embodiments, the spacers are about 1 to 6 nucleotides long or about 5 to 10 nucleotides long, e.g., about 7 to 8 nucleotides long or about 8 nucleotides long. In some embodiments, the spacers include the nucleotide sequence of SEQ ID NO: 1848, or the nucleotide sequence of SEQ ID NO: 1848 having at least 1, 2, or 3 modifications, but 4 or fewer modifications.

[0360] In some embodiments, the encoded miR binding site sequence includes miR122 binding sites, miR142 binding sites, miR183 binding sites, miR182 binding sites, miR96 binding sites, or combinations thereof. In some embodiments, the encoded miR binding site sequence includes at least 3, 4, or 5 copies of miR122, miR142, miR183, miR182, miR96 binding sites, or combinations thereof. In some embodiments, at least two of the encoded miR binding sites are connected directly, for example, without a spacer. In other embodiments, at least two of the encoded miR binding sites are separated by a spacer located between two or more consecutive encoded miR binding site sequences, for example, a spacer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In embodiments, the spacer is about 5–10 nucleotides in length, for example, about 7–8 nucleotides or about 8 nucleotides. In some embodiments, the spacer sequence includes one or more of (i)GGAT, (ii)CACGTG, and (iii)GCATGC, or one or more repeats of (i) to (iii). In some embodiments, the spacer includes the nucleotide sequence of SEQ ID NO: 1848, or the nucleotide sequence of SEQ ID NO: 1848 having at least one, two, or three modifications, but four or fewer modifications.

[0361] In some embodiments, the encoded miR binding site sequence includes at least 3 to 5 copies (e.g., 4 copies) of at least 2, 3, 4, 5, or all combinations of miR122, miR142, miR183, miR182, and miR96 binding sites, where each miR binding site in the sequence is either contiguous (e.g., not separated by a spacer) or separated by a spacer. In some embodiments, the spacer is about 1 to 6 nucleotides long or about 5 to 10 nucleotides long, e.g., about 7 to 8 nucleotides long or about 8 nucleotides long. In some embodiments, the spacer sequence includes one or more of (i) GGAT, (ii) CACGTG, (iii) GCATGC, or one or more repeats of (i) to (iii). In some embodiments, the spacer includes the nucleotide sequence of SEQ ID NO: 1848, or the nucleotide sequence of SEQ ID NO: 1848 having at least 1, 2, or 3 modifications, but 4 or fewer modifications.

[0362] Viral genome components: Polyadenylated sequences In some embodiments, the viral genome of the AAV particle of this disclosure includes at least one polyadenylated (poly-A) sequence. The viral genome of the AAV particle may include a polyadenylated sequence between the 3' end of the payload coding sequence and the 5' end of the 3'UTR. In some embodiments, the poly-A signaling region is located 3' to the payload, for example, a nucleic acid containing a transgene encoding the GBA protein described herein.

[0363] In some embodiments, the poly-A signal region includes nucleotides of about 100 to 600 length, for example, about 100 to 500 nucleotides, about 100 to 400 nucleotides, about 100 to 300 nucleotides, about 100 to 200 nucleotides, about 200 to 600 nucleotides, about 200 to 500 nucleotides, about 200 to 400 nucleotides, about 200 to 300 nucleotides, about 300 to 600 nucleotides, about 300 to 500 nucleotides, about 300 to 400 nucleotides, about 400 to 600 nucleotides, or about 500 to 600 nucleotides. In some embodiments, the poly-A signal region includes nucleotides of about 100 to 150 length, for example, about 127 nucleotides. In some embodiments, the poly-A signal region includes nucleotides of about 450 to 500 length, for example, about 477 nucleotides. In some embodiments, the polyA signal region contains nucleotides of about 520 to about 560 in length, for example, about 552 nucleotides. In some embodiments, the polyA signal region contains nucleotides of about 127 in length.

[0364] In some embodiments, the viral genome includes a human growth hormone (hGH) polyA sequence. In some embodiments, the viral genome includes the above hGH polyA sequence and a payload region encoding a GCase protein, or a GCase and enhancement element (e.g., prosaposin, SapA, or SapC protein, or variants thereof; cell membrane permeable peptides (e.g., ApoEII peptide, TAT peptide, or ApoB peptide); or lysosomal targeting peptides), for example, a payload region encoding a sequence or fragment or variant thereof as described in Tables 3 and 4.

[0365] Viral genome components: Filler sequences In some embodiments, the viral genome includes one or more filler sequences. The filler sequences may be wild-type sequences or manipulated sequences. The filler sequences may be variants of wild-type sequences. In some embodiments, the filler sequences are derivatives of human albumin.

[0366] In some embodiments, the viral genome includes one or more filler sequences such that the length of the viral genome is optimally sized for packaging. In some embodiments, the viral genome includes at least one filler sequence such that the length of the viral genome is approximately 2.3 kb. In some embodiments, the viral genome includes at least one filler sequence such that the length of the viral genome is approximately 4.6 kb.

[0367] In some embodiments, the viral genome is a single-stranded (ss) viral genome, approximately 0.1kb to 3.8kb, for example, but not limited to, 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1kb, 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb. It includes one or more filler sequences having lengths of 1.7kb, 1.8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, or 3.8kb, independently or combined. In some embodiments, the total length of the filler sequences in the vector genome is 3.1kb. In some embodiments, the total length of the filler sequences in the vector genome is 2.7kb. In some embodiments, the total length of the filler sequences in the vector genome is 0.8kb. In some embodiments, the total length of the filler sequences in the vector genome is 0.4kb. In some embodiments, the length of each filler sequence in the vector genome is 0.8kb. In some embodiments, the length of each filler sequence in the vector genome is 0.4 kb.

[0368] In some embodiments, the viral genome is a self-complementary (sc) viral genome and includes one or more filler sequences having lengths of approximately 0.1kb to 1.5kb, for example, but not limited to, 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1kb, 1.1kb, 1.2kb, 1.3kb, 1.4kb, or 1.5kb, independently or collectively. In some embodiments, the total length of the filler sequences in the vector genome is 0.8kb. In some embodiments, the total length of the filler sequences in the vector genome is 0.4kb. In some embodiments, the length of each filler sequence in the vector genome is 0.8kb. In some embodiments, the length of each filler sequence in the vector genome is 0.4kb.

[0369] In some embodiments, the viral genome includes filler sequences in any portion. The viral genome may contain filler sequences in amounts of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0370] In some embodiments, the viral genome is a single-stranded (ss) viral genome and includes one or more filler sequences such that the length of the viral genome is approximately 4.6 kb. In some embodiments, the viral genome includes at least one filler sequence, which is located 3' to the 5' side of the 5' ITR sequence. In some embodiments, the viral genome includes at least one filler sequence, which is located 5' to the promoter sequence. In some embodiments, the viral genome includes at least one filler sequence, which is located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome includes at least one filler sequence, which is located 5' to the 3' ITR sequence. In some embodiments, the viral genome includes at least one filler sequence, which is located between two intron sequences. In some embodiments, the viral genome includes at least one filler sequence, which is located within an intron sequence. In some embodiments, the viral genome includes two filler sequences, the first filler sequence located 3' to the 5' ITR sequence and the second filler sequence located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome includes two filler sequences, the first filler sequence located 5' to the promoter sequence and the second filler sequence located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome includes two filler sequences, the first filler sequence located 3' to the 5' ITR sequence and the second filler sequence located 5' to the 5' ITR sequence.

[0371] In some embodiments, the viral genome is a self-complementary (sc) viral genome and includes one or more filler sequences such that the length of the viral genome is approximately 2.3 kb. In some embodiments, the viral genome includes at least one filler sequence, which is located 3' to the 5' side of the 5' ITR sequence. In some embodiments, the viral genome includes at least one filler sequence, which is located 5' to the promoter sequence. In some embodiments, the viral genome includes at least one filler sequence, which is located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome includes at least one filler sequence, which is located 5' to the 3' ITR sequence. In some embodiments, the viral genome includes at least one filler sequence, which is located between two intron sequences. As a non-limiting example, the viral genome includes at least one filler sequence, which is located within an intron sequence. In some embodiments, the viral genome includes two filler sequences, the first filler sequence located 3' to the 5' ITR sequence and the second filler sequence located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome includes two filler sequences, the first filler sequence located 5' to the promoter sequence and the second filler sequence located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome includes two filler sequences, the first filler sequence located 3' to the 5' ITR sequence and the second filler sequence located 5' to the 5' ITR sequence.

[0372] In some embodiments, the viral genome may contain one or more filler sequences between one or more regions of the viral genome. In some embodiments, filler regions may be located before, but are not limited to, payload regions, terminal inverted repeats (ITRs), promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and / or exon regions. In some embodiments, but are not limited to, filler regions may be located after, but are not limited to, payload regions, terminal inverted repeats (ITRs), promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and / or exon regions. In some embodiments, but are not limited to, filler regions may be located before and after, but are not limited to, payload regions, terminal inverted repeats (ITRs), promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and / or exon regions.

[0373] In some embodiments, the viral genome may include one or more filler sequences that branch off at least one region of the viral genome. The branched region of the viral genome may include regions that are 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% on the 5' side of the filler sequence. In some embodiments, the filler sequence can branch off at least one region, so that 10% of the region is located on the 5' side of the filler sequence and 90% of the region is located on the 3' side of the filler sequence. In some embodiments, the filler sequence can branch off at least one region, so that 20% of the region is located on the 5' side of the filler sequence and 80% of the region is located on the 3' side of the filler sequence. In some embodiments, the filler array can branch at least one region, so that 30% of the region is located on the 5' side of the filler array and 70% of the region is located on the 3' side of the filler array. In some embodiments, the filler array can branch at least one region, so that 40% of the region is located on the 5' side of the filler array and 60% of the region is located on the 3' side of the filler array. In some embodiments, the filler array can branch at least one region, so that 50% of the region is located on the 5' side of the filler array and 50% of the region is located on the 3' side of the filler array. In some embodiments, the filler array can branch at least one region, so that 60% of the region is located on the 5' side of the filler array and 40% of the region is located on the 3' side of the filler array. In some embodiments, the filler array can branch at least one region, so that 70% of the region is located on the 5' side of the filler array and 30% of the region is located on the 3' side of the filler array. In some embodiments, the filler array can be branched into at least one region, so that 80% of the region is located on the 5' side of the filler array and 20% of the region is located on the 3' side of the filler array.In some embodiments, the filler array can be branched into at least one region, so that 90% of the region is located on the 5' side of the filler array and 10% of the region is located on the 3' side of the filler array.

[0374] In some embodiments, the viral genome includes a filler sequence after the 5'ITR. In some embodiments, the viral genome includes a filler sequence after the promoter region. In some embodiments, the viral genome includes a filler sequence after the payload region. In some embodiments, the viral genome includes a filler sequence after the intron region. In some embodiments, the viral genome includes a filler sequence after the enhancer region. In some embodiments, the viral genome includes a filler sequence after the polyadenylation signal sequence region. In some embodiments, the viral genome includes a filler sequence after the exon region.

[0375] In some embodiments, the viral genome includes a filler sequence before the promoter region. In some embodiments, the viral genome includes a filler sequence before the payload region. In some embodiments, the viral genome includes a filler sequence before the intron region. In some embodiments, the viral genome includes a filler sequence before the enhancer region. In some embodiments, the viral genome includes a filler sequence before the polyadenylation signal sequence region. In some embodiments, the viral genome includes a filler sequence before the exon region.

[0376] In some embodiments, the viral genome includes a filler sequence prior to the 3'ITR. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the 5'ITR and the promoter region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the 5'ITR and the payload region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the 5'ITR and the intron region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the 5'ITR and the enhancer region. In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the 5'ITR and the polyadenylation signal sequence region.

[0377] In some embodiments, the filler sequence may be located between two regions, for example, but not limited to, the 5'ITR and the exon region. In some embodiments, the filler sequence may be located between two regions, for example, a promoter region and a payload region. In some embodiments, the filler sequence may be located between two regions, for example, a promoter region and an intron region. In some embodiments, the filler sequence may be located between two regions, for example, a promoter region and an enhancer region. In some embodiments, the filler sequence may be located between two regions, for example, a promoter region and a polyadenylation signal sequence region. In some embodiments, the filler sequence may be located between two regions, for example, a promoter region and an exon region. In some embodiments, the filler sequence may be located between two regions, for example, a promoter region and an exon region.

[0378] In some embodiments, the filler sequence may be located between two regions, for example, a payload region and an intron region. In some embodiments, the filler sequence may be located between two regions, for example, a payload region and an enhancer region. In some embodiments, the filler sequence may be located between two regions, for example, a payload region and a polyadenylation signal sequence region. In some embodiments, the filler sequence may be located between two regions, for example, a payload region and an exon reg...

Claims

1. Isolated nucleic acid, for example, recombinant nucleic acid, comprising a transgene encoding a β-glucocerebrosidase (GBA) protein, wherein the isolated nucleic acid comprises a nucleotide sequence in which the nucleotide sequence encoding the GBA protein is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1773.

2. The isolated nucleic acid according to claim 1, wherein the nucleotide sequence encoding the GBA protein includes the nucleotide sequence of SEQ ID NO: 1773, or a nucleotide sequence that is at least 95% identical to SEQ ID NO: 1773.

3. The isolated nucleic acid according to claim 1 or 2, further comprising a nucleotide sequence encoding a miR binding site that reduces the expression of the GBA protein encoded by the nucleic acid in cells or tissues expressing the corresponding miRNA, wherein the encoded miRNA binding site is optionally complementary, for example, fully complementary or partially complementary, to the miRNA expressed in cells or tissues of DRG, liver, hematopoietic system, or a combination thereof.

4. An isolated viral genome, such as a recombinant viral genome, comprising a nucleic acid containing a transgene encoding a GBA protein, and further comprising a nucleotide sequence encoding a miR binding site that modulates, for example, reduces the expression of the encoded GBA protein in cells or tissues of the DRG, liver, hematopoietic system, or a combination thereof.

5. The coded miR binding site includes the miR183 binding site, the miR122 binding site, the miR-142-3p, or a combination thereof, and can be optionally selected. (i) The coded miR183 binding site includes the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence having at least 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto, or a nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but 10 or fewer modifications. (ii) The coded miR122 binding site comprises the nucleotide sequence of SEQ ID NO: 1865, or a nucleotide sequence having at least 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto, or a nucleotide sequence of SEQ ID NO: 1865 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but 10 or fewer modifications, and / or (iii) The isolated nucleic acid according to claim 3, or the viral genome according to claim 4, wherein the encoded miR-142-3p binding site comprises the nucleotide sequence of SEQ ID NO: 1869, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto, or a nucleotide sequence of SEQ ID NO: 1869 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but 10 or fewer modifications.

6. The nucleic acid further encodes an enhancement element, and the encoded enhancement element is (a) Prosaposin polypeptides, saposin C polypeptides, or functional fragments or variants thereof, comprising, by arbitrary selection, the amino acid sequences of SEQ ID NOs. 1789, 1758, 1750, 1752, 1754, 1756-1758, 1784, or 1785, amino acid sequences having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), or amino acid sequences that are at least 85% identical to those thereof, prosaposin polypeptides, saposin C polypeptides, or functional fragments or variants thereof. (b) A cell membrane-permeable peptide comprising, by any choice, the amino acid sequence of any of SEQ ID NOs: 1794, 1796, or 1798, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), to SEQ ID NOs: 1794, 1796, or 1798; or (c) Lysosomal targeting sequences comprising, by any choice, the amino acid sequence of any of sequence numbers 1800, 1802, 1804, 1806, or 1808, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), to sequence numbers 1800, 1802, 1804, 1806, or 1808, e.g., substitutions (e.g., conservative substitutions) An isolated nucleic acid according to any one of claims 1 to 3, comprising one, two, or all of the above, or a viral genome according to claim 4 or 5.

7. An isolated nucleic acid, for example, a recombinant nucleic acid, comprising a transgene encoding a GBA protein and an enhancement element, wherein the encoded enhancement element is (a) Saposin C polypeptide or a functional fragment or variant thereof, optionally comprising the amino acid sequence of Sequence ID No. 1789 or 1758, or an amino acid sequence that is at least 85% identical thereto; (b) A cell membrane-permeable peptide comprising, by any choice, the amino acid sequence of any of SEQ ID NOs: 1794, 1796, or 1798, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), to SEQ ID NOs: 1794, 1796, or 1798; or (c) Lysosomal targeting sequences comprising, by any choice, the amino acid sequence of any of sequence numbers 1800, 1802, 1804, 1806, or 1808, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions), to sequence numbers 1800, 1802, 1804, 1806, or 1808, e.g., substitutions (e.g., conservative substitutions) The isolated nucleic acid comprising one, two, or all of the above.

8. (i) The coded enhancement element includes the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence having at least one, two, or three but no more than four modifications, for example, substitutions (for example, conservative substitutions), relative to SEQ ID NO: 1789. (ii) The nucleotide sequence encoding the enhancement element includes the nucleotide sequence of Sequence ID No. 1787, or a nucleotide sequence that is at least 85% identical thereto, (ii) The coded enhancement element includes the amino acid sequence of SEQ ID NO: 1802, or an amino acid sequence having at least one, two, or three but no more than four modifications, for example, substitutions (for example, conservative substitutions) relative to SEQ ID NO: 1802, (iii) The nucleotide sequence encoding the enhancement element includes the nucleotide sequence of SEQ ID NO: 1801, or a nucleotide sequence having at least one, two, or three modifications, but no more than four modifications, such as substitutions (e.g., conservative substitutions), relative to SEQ ID NO: 1801. (iv) The coded enhancement element includes the amino acid sequence of SEQ ID NO: 1794, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions) relative to SEQ ID NO: 1794, or (v) The nucleotide sequence encoding the enhancement element is the nucleotide sequence of SEQ ID NO: 1793, or a nucleotide sequence having at least one, two, or three modifications, but four or fewer, such as substitutions (e.g., conservative substitutions) relative to SEQ ID NO: 1793. An isolated nucleic acid according to claim 6 or 7, or a viral genome according to claim 6.

9. An isolated nucleic acid according to claim 7 or 8, wherein the nucleotide sequence encoding the GBA protein comprises one of the nucleotide sequences of SEQ ID NOs: 1773, 1777, or 1781, or a nucleotide sequence that is at least 90% (e.g., at least 92%, 95%, 97%, 98%, or 99%) identical to them, or a viral genome according to any one of claims 4 to 6 or 8.

10. An isolated nucleic acid according to any one of claims 6 to 9, or a viral genome according to any one of claims 6 or 8 to 9, wherein the encoded enhancement element and the encoded GBA protein are linked directly (e.g., without a linker) or via an encoded linker.

11. (i) The coded linker includes the amino acid sequence of any of sequence numbers 1854, 1855, 1843, or 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, e.g., substitutions (e.g., conservative substitutions) to sequence numbers 1854, 1855, 1843, or 1845, (ii) The nucleotide sequence encoding the linker includes any of the nucleotide sequences in Table 2, or a nucleotide sequence having at least one, two, or three but four or fewer modifications, for example, substitutions (for example, conservative substitutions) relative to the sequences in Table 2. (iii) The nucleotide sequence encoding the linker includes any one nucleotide sequence of SEQ ID NOs. 1724, 1726, 1729, or 1730, or a nucleotide sequence having at least one, two, or three but four or fewer modifications, for example, substitutions (for example, conservative substitutions) relative to SEQ ID NOs. 1724, 1726, 1729, or 1730, (iv) The coded linker includes a furin cutting portion, (v) The coded linker comprises a T2A polypeptide, (vi) The coded linker includes a (Gly4Ser)n linker (wherein n is 1 to 10, for example n is 3, 4, or 5), and / or (vii) The coded linker includes three (Gly4Ser) linkers, The isolated nucleic acid or viral genome according to claim 10.

12. (i) The nucleotide sequence encoding the enhancement element is located 5' to the nucleotide sequence encoding the GBA protein, and / or (ii) The nucleotide sequence encoding the enhancement element is located 3' to the nucleotide sequence encoding the GBA protein, An isolated nucleic acid according to any one of claims 6 to 11, or a viral genome according to any one of claims 6 or 8 to 11.

13. Further code the signal sequence, and by choice, (i) The coded signal sequence includes the amino acid sequence of SEQ ID NO: 1853 or 1857, or an amino acid sequence that is at least 85% identical to them, and / or (ii) The nucleotide sequence encoding the signal sequence is located 5' to the nucleotide sequence encoding the GBA protein and / or 5' to the encoded enhancement element, An isolated nucleic acid according to any one of claims 1 to 3 or 5 to 12, or a viral genome according to any one of claims 4 to 6 or 8 to 12.

14. (i) The nucleic acid comprises, in a 5' to 3' order, a nucleotide sequence encoding a signal sequence containing the nucleotide sequence of SEQ ID NO: 1850 or a nucleotide sequence that is at least 85% identical thereto, and a nucleotide sequence encoding a GBA protein containing the nucleotide sequence of SEQ ID NO: 1773 or a nucleotide sequence that is at least 85% identical thereto, or (ii) The nucleic acid encodes a signal sequence containing the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto, in the order from 5' to 3', and a GBA protein containing the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto. An isolated nucleic acid according to any one of claims 1 to 3 or 5 to 13, or a viral genome according to any one of claims 4 to 6 or 8 to 13.

15. The nucleic acid is arranged in the order from 5' to 3', (i) A signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1800, or an amino acid sequence having at least one, two, or three, but no more than four, modifications, such as substitutions, to SEQ ID NO: 1800; (ii) A GBA protein comprising a signal sequence containing the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; an enhancement element containing the amino acid sequence of SEQ ID NO: 1802, or an amino acid sequence that is at least 85% identical thereto; and the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; (iii) A signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1804, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1804; (iv) A signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1806, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1806; (v) A signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; a linker comprising the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence comprising at least one, two, or three, but no more than four, modifications, such as substitutions, to SEQ ID NO: 1845; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1798, or an amino acid sequence comprising at least one, two, or three, but no more than four, modifications, such as substitutions, to SEQ ID NO: 1798; (vi) A signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; a linker comprising the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1845; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1794, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1794; (vii) A first signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; a furin cleavage site comprising the amino acid sequence of SEQ ID NO: 1854, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1854, for example, an amino acid sequence with substitutions; a T2A polypeptide comprising the amino acid sequence of SEQ ID NO: 1855, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1855, for example, an amino acid sequence with substitutions; a second signal sequence comprising the nucleotide sequence of SEQ ID NO: 1857, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1857, for example, an amino acid sequence with substitutions; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1785, or an amino acid sequence that is at least 85% identical thereto; (viiii) A first signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; a furin cleavage site comprising the amino acid sequence of SEQ ID NO: 1854, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1854, for example, an amino acid sequence with substitutions; a T2A polypeptide comprising the amino acid sequence of SEQ ID NO: 1855, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1855, for example, an amino acid sequence with substitutions; a second signal sequence comprising the nucleotide sequence of SEQ ID NO: 1857, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1857, for example, an amino acid sequence with substitutions; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence that is at least 85% identical thereto; (ix) A first signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; a furin cleavage site comprising the amino acid sequence of SEQ ID NO: 1854, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1854, for example, an amino acid sequence with substitutions; a T2A polypeptide comprising the amino acid sequence of SEQ ID NO: 1855, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1855, for example, an amino acid sequence with substitutions; a second signal sequence comprising the nucleotide sequence of SEQ ID NO: 1857, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1857, for example, an amino acid sequence with substitutions; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1758, or an amino acid sequence that is at least 85% identical thereto; (x) A signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; a linker comprising the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1845; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1796, or an amino acid sequence that is at least 85% identical thereto; (xi) A GBA protein comprising: a signal sequence containing the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; an enhancement element containing the amino acid sequence of SEQ ID NO: 1794, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1794; a linker containing the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1845; and an amino acid sequence containing the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; (xi) A signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1808, or an amino acid sequence having at least one, two, or three, but no more than four, modifications, such as substitutions, to SEQ ID NO: 1808; (xiii) A first signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a first enhancement element comprising the amino acid sequence of SEQ ID NO: 1802, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; and the amino acid sequence of SEQ ID NO: 1854, or at least one, two, or three, but four or fewer, modifications, such as substitutions, of SEQ ID NO: 1854. A furin cleavage site containing a sequence, a T2A polypeptide containing the amino acid sequence of SEQ ID NO: 1855, or an amino acid sequence having at least one, two, or three but no more than four modifications, for example, substitutions, relative to SEQ ID NO: 1855, a second signal sequence containing the nucleotide sequence of SEQ ID NO: 1857, or at least one, two, or three but no more than four modifications, for example, substitutions, relative to SEQ ID NO: 1857, and a second enhancement element containing the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence that is at least 85% identical thereto; (xiv) A first signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; a linker comprising the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1845; a first enhancement element comprising the amino acid sequence of SEQ ID NO: 1798, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1798; and the amino acid sequence of SEQ ID NO: 1854 or an enhancement element comprising at least one, two, or three but no more than four modifications to SEQ ID NO: 1854, for example, a furin cleavage site containing an amino acid sequence with substitutions, the amino acid sequence of SEQ ID NO: 1855, or a T2A polypeptide containing at least one, two, or three but no more than four modifications to SEQ ID NO: 1855, for example, an amino acid sequence with substitutions, the nucleotide sequence of SEQ ID NO: 1857, or a second signal sequence containing at least one, two, or three but no more than four modifications to SEQ ID NO: 1857, for example, an amino acid sequence with substitutions, and the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence that is at least 85% identical thereto; (xv) A first signal sequence comprising the amino acid sequence of SEQ ID NO: 1853, or an amino acid sequence that is at least 85% identical thereto; a first enhancement element comprising the amino acid sequence of SEQ ID NO: 1802, or an amino acid sequence that is at least 85% identical thereto; a GBA protein comprising the amino acid sequence of SEQ ID NO: 1775, or an amino acid sequence that is at least 85% identical thereto; a linker comprising the amino acid sequence of SEQ ID NO: 1845, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions, to SEQ ID NO: 1845; and an amino acid sequence of SEQ ID NO: 1798, or an amino acid sequence having at least one, two, or three but no more than four modifications, such as substitutions. A first enhancement element comprising: a furin cleavage site comprising the amino acid sequence of SEQ ID NO: 1854, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1854, for example, an amino acid sequence with substitutions; a T2A polypeptide comprising the amino acid sequence of SEQ ID NO: 1855, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1855, for example, an amino acid sequence with substitutions; a second signal sequence comprising the nucleotide sequence of SEQ ID NO: 1857, or at least one, two, or three but no more than four modifications to SEQ ID NO: 1857, for example, an amino acid sequence with substitutions; and an enhancement element comprising the amino acid sequence of SEQ ID NO: 1789, or an amino acid sequence that is at least 85% identical thereto. An isolated nucleic acid according to any one of claims 7 to 13, or a viral genome according to any one of claims 4 to 6 or 8 to 13, which encodes a viral genome.

16. An isolated viral genome, for example, a recombinant viral genome, comprising a promoter operably linked to the nucleic acid according to any one of claims 1 to 3 or 7 to 15.

17. The viral genome according to claims 4 to 6 or 7 to 15, further comprising a promoter operably linked to the nucleic acid containing the transgene encoding the GBA protein, wherein the promoter comprises a tissue-specific promoter or a ubiquitous promoter.

18. The aforementioned promoter, (i) Chicken β-actin (CBA) promoter and / or derivative CAG, EF-1a promoter, CMV pre-initial enhancer and / or promoter, β-glucuronidase (GUSB) promoter, ubiquitin C (UBC) promoter, neuron-specific enolase (NSE), platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, intercellular adhesion molecule 2 (ICAM-2) promoter, synapsin (Syn) promoter, methyl-CpG-binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 ( mGluR2 promoter, neurofilament light chain (NFL) or heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2), glial fibrillary acidic protein (GFAP) promoter, myelin basic protein (MBP) promoter, cardiovascular promoters (e.g., αMHC, cTnT, and CMV-MLC2k), liver promoters (e.g., hAAT, TBG), skeletal muscle promoters (e.g., desmin, MCK, C512) or fragments thereof, e.g., cleavage or functional variants; and / or (ii) Any nucleotide sequence of sequence numbers 1832, 1833, 1834, 1835, 1836, 1839, or 1840, or a nucleotide sequence that is at least 95% identical to them. The viral genome according to claim 16 or 17, comprising:

19. The promoter or its functional variant, (i) the nucleotide sequence of Sequence ID No. 1834, or a nucleotide sequence that is at least 95% identical thereto; or (ii) The nucleotide sequence of sequence number 1839 or 1840, or a nucleotide sequence that is at least 95% identical thereto. A viral genome according to any one of claims 16 to 18, including the viral genome described in any one of claims 16 to 18.

20. A viral genome according to any one of claims 4 to 6 or 8 to 19, further comprising an enhancer optionally comprising the nucleotide sequence of sequence number 1831, or a nucleotide sequence that is at least 95% identical thereto.

21. The viral genome according to claims 4 to 6 or 8 to 20, comprising an enhancer containing the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence that is at least 95% identical thereto, and a promoter containing the nucleotide sequence of SEQ ID NO: 1834, or a nucleotide sequence that is at least 95% identical thereto.

22. (i) Terminal inverted repeat (ITR) sequences, which are optionally located 5' to the transgene encoding the GBA protein and / or 3' to the transgene encoding the GBA protein; (ii) Polyadenylated (Poly-A) signal region; (iii) Intron region; (iv) Exon regions, e.g., at least one, two, or three exon regions; (v) Kozak array The viral genome according to claims 4 to 6 or 8 to 20, further comprising:

23. (i) The ITR contains the nucleotide sequence of SEQ ID NO: 1829 or 1830, or a nucleotide sequence that is at least 95% identical to them, (ii) The ITR located 5' to the nucleic acid containing the transgene encoding the GBA protein contains the nucleotide sequence of SEQ ID NO: 1829 or a nucleotide sequence that is at least 95% identical thereto, and / or the ITR located 3' to the nucleic acid containing the transgene encoding the GBA protein contains the nucleotide sequence of SEQ ID NO: 1830 or a nucleotide sequence that is at least 95% identical thereto, The viral genome according to claim 22.

24. (i) The polyadenylated (polyA) signal region includes the nucleotide sequence of SEQ ID NO: 1846, or a nucleotide sequence that is at least 95% identical thereto, (ii) The intron includes a beta-globin intron and / or (iii) The intron includes the nucleotide sequence of SEQ ID NO: 1842, or a nucleotide sequence that is at least 95% identical thereto. The viral genome according to claim 22 or 23.

25. (i) at least one to five copies, for example, at least one, two, three, four, or five copies of the coded miR binding site; (ii) at least four copies of coded miR junctions, wherein all four copies optionally contain the same miR junction, or at least one, two, three, or all copies contain different miR junctions, wherein the four copies of coded miR junctions are contiguous, for example, not separated by spacers, or separated by spacers. An isolated nucleic acid according to any one of claims 3, 5-6 or 8-24, or a viral genome according to any one of claims 4, 6 or 8-24, comprising:

26. The aforementioned viral genome, (i) A first encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence having at least 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto, or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, (ii) A first spacer sequence comprising the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but four or fewer modifications, (iii) A second coded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence having at least 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto, or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, (iv) A second spacer sequence comprising the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but four or fewer modifications, (v) A third encoded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence having at least 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto, or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, (vi) A third spacer sequence comprising the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but four or fewer modifications, (vii) A fourth coded miR183 binding site comprising the nucleotide sequence of SEQ ID NO: 1847, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto, or the nucleotide sequence of SEQ ID NO: 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications. An isolated nucleic acid according to any one of claims 3, 5-6 or 8-25, or a viral genome according to any one of claims 4, 6 or 8-25, comprising:

27. In the order from 5' to 3', (i) A 5' adeno-associated (AAV) ITR comprising, optionally, the nucleotide sequence of SEQ ID NO: 1829, or a nucleotide sequence that is at least 95% identical thereto, (ii) A CMVie enhancer comprising, optionally, the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence that is at least 95% identical thereto, (iii) A CB promoter or a functional variant thereof, which optionally includes the nucleotide sequence of SEQ ID NO: 1834, or a nucleotide sequence that is at least 95% identical thereto, (iv) an intron which optionally includes the nucleotide sequence of sequence number 1842, or a nucleotide sequence that is at least 95% identical thereto, (v) A nucleotide sequence encoding a signal sequence, which optionally includes the nucleotide sequence of sequence number 1850, or a nucleotide sequence that is at least 95% identical thereto, (vi) A transgene encoding the GBA protein, comprising the nucleotide sequence of SEQ ID NO: 1773 or a nucleotide sequence that is at least 88% identical to the nucleotide sequence of SEQ ID NO: 1773, (vii) A poly-A signal region comprising, optionally, the nucleotide sequence of sequence number 1846, or a nucleotide sequence that is at least 95% identical thereto, (viiii) A 3'AAV ITR comprising, optionally, the nucleotide sequence of SEQ ID NO: 1830, or a nucleotide sequence that is at least 95% identical thereto, and Isolated viral genomes, including recombinant viral genomes.

28. In the order from 5' to 3', (i) A 5' adeno-associated (AAV) ITR comprising, optionally, the nucleotide sequence of SEQ ID NO: 1829, or a nucleotide sequence that is at least 95% identical thereto, (ii) A CMVie enhancer comprising, optionally, the nucleotide sequence of SEQ ID NO: 1831, or a nucleotide sequence that is at least 95% identical thereto, (iii) A CB promoter or a functional variant thereof, which optionally includes the nucleotide sequence of SEQ ID NO: 1834, or a nucleotide sequence that is at least 95% identical thereto, (iv) an intron which optionally includes the nucleotide sequence of sequence number 1842, or a nucleotide sequence that is at least 95% identical thereto, (v) A nucleotide sequence encoding a signal sequence, which optionally includes the nucleotide sequence of sequence number 1850, or a nucleotide sequence that is at least 95% identical thereto, (vi) A transgene encoding the GBA protein, wherein, by optional selection, the transgene encoding the GBA protein includes the nucleotide sequence of SEQ ID NO: 1773 or a nucleotide sequence that is at least 88% identical to the nucleotide sequence of SEQ ID NO: 1773, (vii) Encoded miR183 binding site, which optionally includes the nucleotide sequence of sequence number 1847, or the nucleotide sequence of sequence number 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but 10 or fewer modifications, and the encoded miR183 binding site, (viiii) A spacer sequence comprising, optionally, the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but four or fewer modifications, and the spacer sequence, (ix) an encoded miR183 binding site comprising, optionally selected, the nucleotide sequence of sequence number 1847, or the nucleotide sequence of sequence number 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but 10 or fewer modifications, the encoded miR183 binding site, (x) A spacer sequence comprising, optionally, the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but four or fewer modifications, and the spacer sequence, (xi) an encoded miR183 binding site comprising, optionally selected, the nucleotide sequence of sequence number 1847, or the nucleotide sequence of sequence number 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but 10 or fewer modifications, the encoded miR183 binding site, (xi) A spacer sequence comprising, optionally, the nucleotide sequence of sequence number 1848, or the nucleotide sequence of sequence number 1848 having at least one, two, or three modifications, but four or fewer modifications, and the spacer sequence, (xiiii) an encoded miR183 binding site comprising, optionally selected, the nucleotide sequence of sequence number 1847, or the nucleotide sequence of sequence number 1847 having at least 1, 2, 3, 4, 5, 6, or 7 modifications, but 10 or fewer modifications, the encoded miR183 binding site, (xiv) A polyA signal region comprising, optionally, the nucleotide sequence of sequence number 1846, or a nucleotide sequence that is at least 95% identical thereto, (xv)3'AAV ITR, which optionally includes the nucleotide sequence of SEQ ID NO: 1830, or a nucleotide sequence that is at least 95% identical thereto, and Isolated viral genomes, including recombinant viral genomes.

29. A viral genome according to any one of claims 4 to 6 or 8 to 28, comprising the nucleotide sequences of SEQ ID NOs: 1812, 1829, 1759-1771, 1809-1811, 1813-1827, or 1870, or nucleotide sequences that are at least 95% identical thereto.

30. (i) It is single-stranded, (ii) further comprising a capsid protein, for example, a nucleic acid encoding a structural protein, wherein the capsid protein comprises a VP1 polypeptide, a VP2 polypeptide, and / or a VP3 polypeptide, wherein optionally the VP1 polypeptide, the VP2 polypeptide, and / or the VP3 polypeptide are encoded by at least one Cap gene; and / or (iii) Further comprising a nucleic acid encoding a Rep protein, for example, a non-structural protein, wherein the Rep protein includes Rep78 protein, Rep68, Rep52 protein, and / or Rep40 protein, and optionally the Rep78 protein, the Rep68 protein, the Rep52 protein, and / or the Rep40 protein are encoded by at least one Rep gene. The viral genome according to any one of claims 4 to 6 or 8 to 29.

31. Isolated GBA protein, for example, recombinant GBA protein, encoded by an isolated nucleic acid according to any one of claims 1 to 3 or 5 to 15, or by a viral genome according to any one of claims 4 to 6 or 8 to 30.

32. (i) Capsid protein and, (ii) The viral genome according to any one of claims 4 to 6 or 8 to 30 Isolated AAV particles, including recombinant AAV particles.

33. (i) The capsid protein comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 90% sequence identity thereto. (ii) The capsid protein comprises the amino acid sequence of SEQ ID NO: 138, which has at least one, two, or three modifications, but 30, 20, or 10 or fewer modifications. (iii) The capsid protein comprises the amino acid sequence of Sequence ID No. 11, or an amino acid sequence having at least 90% sequence identity thereto. (iv) The capsid protein comprises the amino acid sequence of SEQ ID NO: 11, which has at least one, two, or three modifications, but 30, 20, or 10 or fewer modifications. (v) The capsid protein comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence having at least 90% sequence identity thereto, and / or (vi) The nucleotide sequence encoding the capsid protein includes the nucleotide sequence of Sequence ID No. 137, or a sequence having at least 90% sequence identity thereto. AAV particle according to claim 32.

34. The aforementioned capsid protein, (i) In numbering according to Sequence ID No. 138, an amino acid substitution at position K449, for example, a substitution of K449R, (ii) An insert containing the amino acid sequence of TLAVPFK (SEQ ID NO: 1262), wherein, optionally, the insert is located immediately after position 588 relative to a reference sequence numbered according to SEQ ID NO: 138, (iii) Numbering according to Sequence ID No. 138, an amino acid other than "A" at position 587 and / or an amino acid other than "Q" at position 588, (iv) Amino acid substitutions A587D and / or Q588G, numbered according to Sequence ID No. 138 AAV particles according to claim 32 or 33, comprising:

35. (i) The capsid protein comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto, (ii) The capsid protein has at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 1, but includes 30, 20, or 10 or fewer modifications, such as substitutions, (iii) The capsid protein comprises and / or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto. (iv) The nucleotide sequence encoding the capsid protein includes the nucleotide sequence of Sequence ID No. 2, or a nucleotide sequence having at least 90%, 92%, 95%, 97%, 98%, or 99% sequence identity thereto. AAV particle according to any one of claims 32 to 34.

36. A vector comprising a viral genome according to any one of claims 4 to 6 or 8 to 30, or a nucleic acid according to any one of claims 1 to 3 or 5 to 15.

37. A cell comprising a viral genome according to any one of claims 4 to 6 or 8 to 30, a viral particle according to any one of claims 32 to 35, or a vector according to claim 36, wherein the cell is optionally a mammalian cell, for example, a HEK293 cell, an insect cell, for example, an Sf9 cell, or a bacterial cell.

38. A method for producing isolated AAV particles, for example, recombinant AAV particles, (i) To provide a host cell containing a viral genome as described in any one of claims 4 to 6 or 8 to 30, (ii) Incubating the host cells under conditions suitable for encapsulating the viral genome in a capsid protein, for example, the VOY101 capsid protein, The method for producing the isolated AAV particles.

39. A pharmaceutical composition comprising AAV particles according to any one of claims 32 to 35, or AAV particles containing a viral genome according to any one of claims 4 to 6 or 8 to 30, and a pharmaceutically acceptable excipient.

40. A method for delivering an exogenous GBA protein to a target, comprising administering an effective amount of the pharmaceutical composition according to claim 39, AAV particles according to any one of claims 32 to 35, AAV particles containing a viral genome according to any one of claims 4 to 6 or 8 to 30, or AAV particles containing a viral genome containing nucleic acid according to any one of claims 1 to 3 or 5 to 15, thereby delivering the exogenous GBA protein to the target.

41. The aforementioned subject is, (i) GBA expression, e.g., abnormal or reduced GBA expression, e.g., diseases related to the expression of the GBA gene, GBA mRNA, and / or GBA protein; or (ii) The method of claim 40, wherein the person has, has been diagnosed with, or is at risk of having, a neurodegenerative or neuromuscular disorder.

42. A method for treating a subject having or diagnosed with a disease related to GBA expression, comprising administering an effective amount of the pharmaceutical composition according to claim 39, AAV particles according to any one of claims 32 to 35, AAV particles containing a viral genome according to any one of claims 4 to 6 or 8 to 30, or AAV particles containing a viral genome containing nucleic acid according to any one of claims 1 to 3 or 5 to 15, wherein the disease related to GBA expression in the subject is treated.

43. A method for treating a subject having or diagnosed with a neurodegenerative or neuromuscular disorder, comprising administering an effective amount of the pharmaceutical composition according to claim 39, AAV particles according to any one of claims 32 to 35, AAV particles containing a viral genome according to any one of claims 4 to 6 or 8 to 30, or AAV particles containing a viral genome containing nucleic acid according to any one of claims 1 to 3 or 5 to 15, thereby treating the neurodegenerative or neuromuscular disorder of the subject.

44. The method according to any one of claims 41 to 43, wherein the disease associated with the expression of GBA or the neurodegenerative or neuromuscular disorder includes Parkinson's disease (PD), Lewy body dementia (DLB), Gaucher disease (GD), spinal muscular atrophy (SMA), multiple system atrophy (MSA), or multiple sclerosis (MS).

45. The aforementioned PD is (i) Related to mutations in the GBA gene, (ii) Early-onset PD (e.g., before age 50) or early-onset PD (e.g., before age 20), (iii) Tremor-dominant type, postural instability, difficulty walking, PD (PIGD), or (iv) Sporadic PD (e.g., PD not associated with mutation) The method according to claim 44.

46. The aforementioned GD is, (i) neuropathic GD (e.g., affecting cells or tissues of the CNS, e.g., cells or tissues of the brain and / or spinal cord), non-neuropathic GD (e.g., not affecting cells or tissues of the CNS), or a combination thereof; or (ii) Type I GD (GD1), Type II GD (GD2), or Type III GD (GD3), wherein GD1 is non-neuropathic GD and GD2 is neuropathic GD. The method according to claim 44.

47. The aforementioned subject is, (i) Having mutations in the GBA gene, GBA mRNA, and / or GBA protein, and / or (ii) The subject is human, and by arbitrary selection, the subject is either young (e.g., between 6 and 20 years old) or adult (e.g., over 20 years old), The method according to any one of claims 40 to 46.

48. The method according to any one of claims 40 to 47, wherein the AAV particles are administered to the target by intravenous, intracerebral, intrathalamic (ITH) administration, intramuscular, intrathecal, intraventricular, intraparenchymal, focused ultrasound (FUS), for example, in combination with intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS in combination with intravenous administration, intracisional injection (ICM), or dual administration of ITH and ICM.

49. The method according to any one of claims 40 to 48, wherein the AAV particles are administered intravenously, and optionally, the intravenous administration is by focused ultrasound (FUS), for example, in combination with intravenous administration of microbubbles (FUS-MB), or by MRI-guided FUS in combination with intravenous administration.

50. The aforementioned administration, (i) The level of GCase activity in the cells, tissues (e.g., cells or tissues of the CNS, e.g., cerebral cortex, striatum, thalamus, cerebellum, and / or brainstem), and / or body fluids (e.g., CSF and / or serum), which, optionally, is increased by at least 3, 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, or 5.5 times compared to a reference level, e.g., an untreated subject, e.g., a subject not administered the AAV particles. (ii) The level of viral genome (VG) per cell in the target CNS tissue (e.g., cerebral cortex, striatum, thalamus, cerebellum, brainstem, and / or spinal cord), wherein the VG level is increased by more than 50 VG per cell compared to peripheral tissue and is at least 1 / 4 to 1 / 10 lower than the level in the CNS tissue, for example, measured by the assay described herein; and / or (iii) Levels of GBA mRNA expression in cells or tissues (e.g., cells or tissues of the CNS, e.g., cerebral cortex, thalamus, and / or brainstem), which are optionally increased by at least 100 to 1300 times, e.g., 100, 200, 500, 600, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 times, compared to a reference level, e.g., an untreated subject (e.g., one not administered with the AAV particles), or an endogenous GBA mRNA level, e.g., measured by the assay described herein. The method according to any one of claims 40 to 49, which results in an increase of at least one, two, or all of the above.

51. The method according to any one of claims 40 to 50, further comprising the administration of additional therapeutic agents and / or treatments suitable for the treatment or prevention of diseases associated with GBA expression, neurodegenerative disorders, and / or neuromuscular disorders, wherein the additional therapeutic agents optionally include enzyme replacement therapy (ERT) (e.g., imiglucerase, veraglucerase alfa, or taliglucerase alfa); substrate synthesis inhibitory therapy (SRT) (e.g., eliglustat or miglustat); blood transfusion; levodopa, carbidopa, safinamide; dopamine agonists (e.g., pramipexole, rotigotine, or ropinirole); anticholinergic agents (e.g., benzatropine or trihexyphenidyl); cholinesterase inhibitors (e.g., rivastigmine, donepezil, or galantamine); N-methyl-d-aspartate (NMDA) receptor antagonists (e.g., memantine); or a combination thereof.

52. An isolated nucleic acid according to any one of claims 1 to 3 or 5 to 15, a viral genome according to any one of claims 4 to 6 or 8 to 30, AAV particles according to any one of claims 32 to 35, or a pharmaceutical composition according to claim 39, for use in the manufacture of a pharmaceutical product.

53. An isolated nucleic acid according to any one of claims 1 to 3 or 5 to 15, a viral genome according to any one of claims 4 to 6 or 8 to 30, AAV particles according to any one of claims 32 to 35, or a pharmaceutical composition according to claim 39, for use in the treatment of diseases related to GBA expression, neuromuscular and / or neurodegenerative disorders.

54. Use of an effective amount of AAV particles containing the genome described in any one of claims 4 to 6 or 8 to 30, AAV particles containing the nucleic acid described in any one of claims 1 to 3 or 5 to 15, AAV particles described in any one of claims 32 to 35, or the pharmaceutical composition described in claim 39, in the manufacture of a drug for use in the treatment of diseases related to GBA expression, neuromuscular and / or neurodegenerative disorders.

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