BDNF gene therapy

Abstract

The present disclosure provides BDNF expression constructs and vectors containing such constructs for treating metabolic disorders, including but not limited to obesity. The disclosed BDNF gene therapy is more effective than existing BDNF-based gene therapy constructs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Application No. 63 / 379,114, filed October 11, 2022, which is incorporated herein by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (SeqList-162027-53276.xml, size: 216,128 bytes; and creation date: October 10, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE DISCLOSURE The present disclosure relates generally to compositions and methods for gene therapy to treat metabolic disorders and diseases. [Background technology]

[0004] Obesity is most commonly caused by excessive food intake combined with limited energy expenditure and / or lack of exercise.Obesity increases the likelihood of developing various diseases, such as diabetes mellitus, hypertension, atherosclerosis, coronary artery disease, sleep apnea, gout, rheumatism and arthritis.In addition, mortality risk is directly correlated with obesity, and thus, for example, a body mass index of over 40 reduces life expectancy by more than 10 years.

[0005] Current pharmacological treatment modalities include appetite suppressants that target receptor classes (e.g., CB1, 5-HT2c, and NPY); modulators of the hypothalamic appetite circuit and the molecular action of ghrelin; and nutrient absorption inhibitors that target lipase. However, none of the current modalities have been shown to effectively treat obesity without causing adverse effects, some of which can be very severe. The incidence of bariatric surgery has increased dramatically, reflecting how problematic and refractory severe obesity is to alternative, less invasive strategies. However, while bariatric surgery is often effective, it carries its own significant morbidity. Pharmacological therapies such as semaglutide (Wilding JPH, et al. N Engl J Med. 2021 Mar 18;384(11):989-1002) and tirzepatide (Ania M. Jastreboff et al., N Engl J Med 2022;387:205-216) require patient adherence and can cause inflammation at the injection site. Furthermore, these therapies can take up to a year for the drug to have its full effect (e.g., 15–20% weight loss, less than bariatric surgery).

[0006] Therefore, safer and more effective approaches to treating obesity are urgently needed. Summary of the Invention

[0007] The present disclosure addresses the aforementioned needs in several aspects. In one aspect, the present disclosure provides expression constructs for the treatment of metabolic disorders and diseases. In some embodiments, the expression construct comprises a promoter sequence operably linked to a nucleic acid sequence encoding brain-derived neurotrophic factor (BDNF) or a variant thereof.

[0008] In some embodiments, the promoter sequence comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1-10, or comprises a nucleic acid sequence selected from SEQ ID NOs: 1-10. In some embodiments, the promoter sequence comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 1, or comprises the nucleic acid sequence of SEQ ID NO: 1.

[0009] In some embodiments, the promoter sequence comprises a constitutive promoter.

[0010] In some embodiments, the nucleic acid sequence encoding BDNF is a wild-type BDNF gene or coding sequence. In some embodiments, the nucleic acid sequence encoding BDNF is a human BDNF gene or coding sequence. In some embodiments, the nucleic acid sequence encoding BDNF is a codon-optimized coding sequence.

[0011] In some embodiments, the nucleic acid sequence encoding BDNF comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79, or comprises a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79.

[0012] In some embodiments, the nucleic acid sequence encoding BDNF comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:52 or 53, or comprises the nucleic acid sequence of SEQ ID NO:52 or 53.

[0013] In some embodiments, the nucleic acid sequence encoding BDNF encodes a protein comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 81 (human wild-type BDNF).

[0014] In some embodiments, the expression construct further comprises a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0015] In some embodiments, the post-transcriptional regulatory element comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 58-61, or comprises a nucleic acid sequence selected from SEQ ID NOs: 58-61.

[0016] In some embodiments, the post-transcriptional control element comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:58 or 59, or comprises the nucleic acid sequence of SEQ ID NO:58 or 59.

[0017] In some embodiments, the expression construct further comprises a polyadenylation signal. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 62, or comprises the nucleic acid sequence of SEQ ID NO: 62 or 66-69.

[0018] In some embodiments, the expression construct further comprises a microRNA (miR) binding sequence (miRBS) located in the 3'UTR, e.g., between the nucleic acid sequence encoding BDNF and the post-transcriptional regulatory element. In some embodiments, the microRNA binding sequence comprises a nucleic acid sequence having at least 90% sequence identity to any one of (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) SEQ ID NOs: 63 and 82. In some embodiments, the microRNA binding sequence comprises any one of (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) SEQ ID NOs: 63 and 82.

[0019] In some embodiments, the expression construct further comprises a terminator downstream of the post-transcriptional regulatory element. In some embodiments, the expression construct comprises: (a) a promoter sequence comprising SEQ ID NO: 1; (b) a nucleic acid sequence encoding BDNF comprising SEQ ID NO: 52; (c) a post-transcriptional regulatory element comprising SEQ ID NO: 58; (d) a polyadenylation signal comprising SEQ ID NO: 62; and (e) A microRNA binding sequence comprising SEQ ID NO: 63.

[0020] In some embodiments, the expression construct comprises: (a) a promoter sequence comprising SEQ ID NO: 1; (b) a nucleic acid sequence encoding BDNF comprising SEQ ID NO: 53; (c) a post-transcriptional regulatory element comprising SEQ ID NO: 59; (d) a polyadenylation signal comprising SEQ ID NO: 62; and (e) a microRNA binding sequence comprising (i) SEQ ID NO: 64, or (ii) SEQ ID NOs: 63 and 82.

[0021] In another aspect, the disclosure provides a vector comprising an expression construct described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some embodiments, the viral vector comprises an expression construct described herein and one or more inverted terminal repeats (ITRs). In some embodiments, the vector comprises a genome derived from AAV serotype AAV2. In some embodiments, the vector comprises a capsid derived from AAV serotype AAV1. In some embodiments, the vector comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 11-50, or comprises a nucleic acid sequence selected from SEQ ID NOs: 11-50. In some embodiments, the vector comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 11-50, or comprises at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30, and 42-45. In some embodiments, the vector comprises a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30, and 42-45. In some embodiments, the vector comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 22, or the nucleic acid sequence comprises SEQ ID NO: 11 or SEQ ID NO: 22.

[0022] Provided herein is a pharmaceutical composition comprising a vector described herein and a pharmaceutically acceptable carrier.

[0023] Provided herein are methods for treating or preventing a metabolic disorder in a subject in need thereof. In some embodiments, the method comprises administering to a subject in need thereof a vector or pharmaceutical composition described herein. In one embodiment, the metabolic disorder is obesity.

[0024] Provided herein is a method for increasing BDNF levels in a subject in need thereof. The method comprises administering to the subject in need thereof a vector or pharmaceutical composition described herein. In some embodiments, the vector or pharmaceutical composition is administered by stereotaxic microinjection. [Brief explanation of the drawings]

[0025] [Figure 1] (A) A series of diagrams showing higher BDNF expression from an optimized constitutive promoter than from a tissue-specific or original CAG promoter. A FACS-based promoter screen of optimized tissue-specific and constitutive promoters operably linked to a fluorescent protein reporter (mClover3) identified preferred promoter candidates for driving BDNF expression. (B) A series of diagrams showing higher BDNF expression from an optimized constitutive promoter than from a tissue-specific or original CAG promoter. Extracellular BDNF expression by transiently transfected mouse N2A (left) and human Be2M17 cells (right) is shown. OSU: Reference construct; see Example 2. (C) A series of diagrams showing higher BDNF expression from an optimized constitutive promoter than from a tissue-specific or original CAG promoter. OSU: Reference construct; see Example 2. [Figure 2]Figure 1A shows that the BDNF expression construct disclosed herein is more potent than the reference construct in vitro. Figure 1B shows extracellular BDNF expression in transiently transfected mouse N2A (left) and human SH-SYFY cells (right). CO1 = codon-optimized gene sequence #1 (BDNFco CDS), CO2 = codon-optimized gene sequence #2 (BDNFco2 CDS), CO3 = codon-optimized gene sequence #3 (BDNFco3 CDS), CO2 = codon-optimized gene sequence #4 (BDNFco4 CDS). Figure 1B shows that the BDNF expression construct disclosed herein is more potent than the reference construct in vitro. Figure 1C shows BDNF expression in transiently transfected human neural progenitor cells. Figure 1C shows that the BDNF expression construct disclosed herein is more potent than the reference construct in vitro. (A) and (B) show the results of six independent experiments demonstrating that the expression construct BDNF-1 expresses four times more BDNF than the reference construct OSU in N2A cells. (B) shows that the BDNF expression construct disclosed herein is more potent than the reference construct in vitro. (C) shows that codon optimization of the BDNF gene does not impair BDNF signaling, as demonstrated by the induction of phosphorylated ERK1 / 2 in primary neurons after 30 minutes of treatment. [Figure 3]Figure 1A shows superior BDNF expression from selected expression constructs in transduced primary neurons. Primary mouse cortical neurons were transduced with AAV2 / 1 at a multiplicity of infection (MOI) of 10,000 or 50,000. Culture medium was collected at 4 and 7 days post-infection (dpi) and prepared for Western blot analysis. DNA was isolated from neuronal lysates at 7 dpi. Figure 1B shows superior BDNF expression from selected expression constructs in transduced primary neurons. Neuronal transduction was comparable between groups, as quantified by qPCR of the viral genome using probes for either the transgene or the ITR (inverted terminal repeat) sequences. Figure 1C shows superior BDNF expression from selected expression constructs in transduced primary neurons. In vitro transduction of constructs BDNF1 and BDNF12 results in significantly higher BDNF expression in primary cortical neurons. [Figure 4]Figure A shows that overexpression of BDNF from selected expression constructs results in weight loss in a mouse model of diet-induced obesity. Eight-week-old wild-type C57B16 male mice were injected bilaterally into the hypothalamus with AAV2 / 1 (2E9GC / hemisphere) and weighed weekly. Seven days later, animals were fed a 45% high-fat or matched control diet. Twenty-one days later, hypothalamus samples were dissected for protein analysis. Figure B shows that overexpression of BDNF from selected expression constructs results in weight loss in a mouse model of diet-induced obesity. Western blots showing high expression of BDNF expression constructs in vivo are shown. Figure C shows that overexpression of BDNF from selected expression constructs results in weight loss in a mouse model of diet-induced obesity. Expression of pro-BDNF and mBDNF in the hypothalamus, as quantified by ELISA, is shown. Figure D shows that overexpression of BDNF from selected expression constructs results in weight loss in a mouse model of diet-induced obesity. Figure 1 shows weight change after AAV2 / 1 delivery in a diet-induced obese mouse model. ***p<0.001; ****p<0.0001. One-way ANOVA repeated measures. Endpoints from top to bottom: HFD, AAV1-eGFP; control diet; AAV1-OSU; AAV-BDNF-12; AAV-BDNF-1. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present disclosure provides BDNF gene therapy expression constructs and vectors for treating metabolic disorders such as obesity. The disclosed BDNF gene therapy is more effective than existing BDNF-based gene therapy constructs.

[0027] BDNF expression construct In one aspect, the disclosure provides expression constructs for treating metabolic disorders and diseases, including obesity.

[0028] In some embodiments, the expression construct comprises a promoter sequence operably linked to a nucleic acid sequence encoding brain-derived neurotrophic factor (BDNF) or a variant thereof. As used herein, "operably linked" refers to a first molecule bound to a second molecule, arranged such that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single, contiguous molecule, and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule of interest if it regulates the transcription of the transcribable polynucleotide molecule in a cell. Furthermore, two portions of a transcriptional regulatory element are operably linked to each other if the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements may be operably linked to each other via a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to each other without any intervening nucleotides.

[0029] BDNF is an important molecular regulator of metabolic function and body weight. Single gene mutations in brain-derived neurotrophic factor (BDNF) or its receptor (NTRK2) cause obesity in humans. BDNF signaling is also downstream of the leptin-melanocortin pathway and regulates appetite and body weight. The expression constructs and vectors described herein can encode BDNF variants. As used herein, a variant is a protein that contains one or more changes, including but not limited to, amino acid additions, substitutions, insertions, deletions, or post-translational modifications, compared to the parent protein, and the variant retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of the parent protein. In embodiments, the BDNF variants disclosed herein retain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ability of BDNF to activate signaling cascades.

[0030] In some embodiments, the nucleic acid sequence encoding BDNF is a coding sequence from a wild-type BDNF gene. In some embodiments, the nucleic acid sequence encoding BDNF is a coding sequence from a human BDNF (e.g., human wild-type BDNF) gene. In some embodiments, the nucleic acid sequence encoding BDNF is a codon-optimized sequence. In some embodiments, the nucleic acid sequence encoding BDNF encodes a protein comprising a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 81 (human wild-type BDNF).

[0031] SEQ ID NO: 81: MTILFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADTFEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR

[0032] In some embodiments, the nucleic acid sequence encoding BDNF has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79. In some embodiments, the nucleic acid sequence encoding BDNF is encoded by a nucleic acid sequence comprising a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79.

[0033] In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 52.

[0034] In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 53.

[0035] In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 54. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 54.

[0036] In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 55.

[0037] In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO:56.

[0038] In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 57.

[0039] Numerous expression control sequences, such as natural, constitutive, inducible, and / or tissue-specific sequences, are known in the art and can be used to drive the expression of the BDNF transgene, depending on the type of expression desired. In eukaryotic cells, expression control sequences typically include promoter sequences, enhancer sequences, and polyadenylation sequences, which may include splice donor and splice acceptor sites. The polyadenylation sequence is generally inserted downstream of the BDNF-encoding sequence and upstream of the 3'ITR sequence.

[0040] Another regulatory component of rAAV useful in the methods disclosed herein is an internal ribosome entry site (IRES).IRES sequences can be used to produce multiple polypeptides from a single gene transcript.IRES (or other suitable sequences) can be used to produce proteins containing multiple polypeptide chains, or to express two different proteins from or within the same cell.In some embodiments, IRES is located 3' of the sequence encoding BDNF in the rAAV vector.

[0041] As used herein, the term "promoter" or "regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence; in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. A promoter or regulatory sequence may, for example, express a gene product in a tissue-specific manner. An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell. As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50 to 1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase the transcriptional activity of a nucleic acid sequence. Enhancers can be located up to 1,000,000 base pairs upstream or downstream of the start site of the gene they regulate. Enhancers can be located within intronic or exon regions of unrelated genes.

[0042] In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 1-10. In some embodiments, the promoter sequence comprises a sequence selected from SEQ ID NOs: 1-10. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid of SEQ ID NO: 1. In some embodiments, the promoter sequence for a nucleic acid comprises the nucleic acid of SEQ ID NO: 1.

[0043] In some embodiments, the promoter sequence comprises a constitutive promoter sequence. A "constitutive promoter" refers to a promoter capable of promoting the continuous transcription of a coding sequence or gene under its control and / or to which it is operably linked.

[0044] In some embodiments, the expression construct comprises a post-transcriptional regulatory element. In some embodiments, the expression construct comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0045] In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 58-61. In some embodiments, the post-transcriptional regulatory element comprises a nucleic acid sequence selected from SEQ ID NOs: 58-61.

[0046] In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 58 or 59. In some embodiments, the post-transcriptional regulatory element comprises the nucleic acid sequence of SEQ ID NO: 58 or 59.

[0047] In some embodiments, the expression construct comprises a polyadenylation signal. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 62 or 66-69. In some embodiments, the expression construct comprises the nucleic acid sequence of SEQ ID NO: 62 or 66-69.

[0048] In some embodiments, the expression construct further comprises a microRNA (miR) binding sequence (miRBS or mirBS) located in the 3'UTR. In embodiments, the miRBS is located between the nucleic acid sequence encoding BDNF and the post-transcriptional regulatory element. In embodiments, the miRBS comprises binding sequence(s) for a microRNA, wherein the microRNA is selected from miR-142, miR-185, miR-1, miR-122, or a combination thereof. In embodiments, the miRBS comprises one or more (e.g., 1, 2, 3, or 4) binding sites for miR-142, one or more (e.g., 1, 2, 3, or 4) binding sites for miR-185, one or more (e.g., 1, 2, 3, or 4) binding sites for miR-1, and one or more (e.g., 1, 2, 3, or 4) binding sites for miR-122.

[0049] In some embodiments, the microRNA-binding sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) SEQ ID NOs: 63 and 82.

[0050] In some embodiments, the microRNA-binding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the microRNA-binding sequence comprises the nucleic acid sequence of SEQ ID NO: 63.

[0051] In some embodiments, the microRNA-binding sequence comprises (i) a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 63, and (ii) a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the nucleic acid sequence of SEQ ID NO: 82. In some embodiments, the microRNA-binding sequence comprises SEQ ID NOs: 63 and 82.

[0052] In some embodiments, the microRNA-binding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 64. In some embodiments, the microRNA-binding sequence comprises the nucleic acid sequence of SEQ ID NO: 64. SEQ ID NO: 64 includes a cloning site (nucleotides 202-209 of SEQ ID NO: 64, underlined in Table 7). One skilled in the art can delete or modify this cloning site (e.g., by replacing one or more nucleotides of nucleotides 202-209 of SEQ ID NO: 64 with different nucleotides) without abolishing the function of the microRNA-binding sequence.

[0053] In some embodiments, the expression construct further comprises a terminator downstream of the post-transcriptional regulatory element. In some embodiments, the expression construct comprises a nucleic acid comprising one or more inverted terminal repeats (ITRs). In some embodiments, the ITR sequences are derived from AAV serotype 2 (AVV2).

[0054] In some embodiments, the 5' ITR sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the 5' ITR sequence comprises the nucleic acid sequence of SEQ ID NO: 51.

[0055] In some embodiments, the 3' ITR sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 65. In some embodiments, the 3' ITR sequence comprises the nucleic acid sequence of SEQ ID NO: 65.

[0056] In some embodiments, an expression construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 11-50. In some embodiments, an expression construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30, and 42-45. In some embodiments, the expression construct comprises a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30, and 42-45.

[0057] In some embodiments, the expression construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the expression construct comprises the nucleic acid sequence of SEQ ID NO: 11.

[0058] In some embodiments, the expression construct comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the expression construct comprises the nucleic acid sequence of SEQ ID NO: 22.

[0059] vector In one aspect, recombinant vectors and their use for the introduction of transgenes or expression constructs into cells are provided.

[0060] In some embodiments, a recombinant vector comprises a recombinant DNA construct that includes additional DNA elements, including DNA segments that result in replication of the DNA in a host cell and expression of a target gene in the target cell at an appropriate level. Those skilled in the art will understand that expression control sequences (promoters, enhancers, etc.) are selected based on their ability to promote expression of the target gene in the target cell.

[0061] As used herein, "vector" refers to a vehicle containing a polynucleotide that is delivered into a host cell either in vitro or in vivo. Non-limiting examples of vectors include a recombinant plasmid, a yeast artificial chromosome (YAC), a minichromosome, a DNA minicircle, or a virus (containing a sequence derived from a virus). A vector may also refer to a virion containing a nucleic acid that is delivered to a host cell either in vitro or in vivo. In some embodiments, a vector refers to a virion containing a recombinant viral genome, where the viral genome includes one or more ITRs and a transgene.

[0062] In some embodiments, the recombinant vector is a viral vector or a combination of viral vectors.

[0063] In one aspect, a vector is provided that includes any of the expression constructs disclosed herein.

[0064] In some embodiments, the vector comprises an expression construct comprising a promoter sequence operably linked to a nucleic acid sequence encoding brain-derived neurotrophic factor (BDNF) or a variant / fragment thereof.

[0065] In some embodiments, the vector comprises a nucleic acid sequence encoding BDNF, wherein the BDNF coding sequence is derived from a wild-type BDNF gene. In some embodiments, the vector comprises a nucleic acid sequence encoding BDNF, wherein the BDNF coding sequence is derived from a human BDNF (e.g., human wild-type BDNF) gene. In some embodiments, the vector comprises a nucleic acid sequence encoding BDNF, wherein the BDNF coding sequence is a codon-optimized sequence.

[0066] In some embodiments, the vector comprises a BDNF coding sequence, wherein the BDNF is encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79. In some embodiments, the BDNF is encoded by a nucleic acid sequence comprising a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 54. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 54.In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 56. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 57.

[0067] In some embodiments, the vector comprises a promoter sequence comprising a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 1-10. In some embodiments, the promoter sequence comprises a nucleic acid sequence selected from SEQ ID NOs: 1-10. In some embodiments, the promoter sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid of SEQ ID NO: 1. In some embodiments, the promoter sequence for the nucleic acid comprises the nucleic acid of SEQ ID NO: 1. In some embodiments, the promoter sequence comprises a constitutive promoter.

[0068] In some embodiments, the vector comprises a nucleic acid comprising a post-transcriptional regulatory element. In some embodiments, the vector comprises a nucleic acid comprising a WPRE. In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 58-61. In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 58 or 59. In some embodiments, the post-transcriptional regulatory element comprises the nucleic acid sequence of SEQ ID NO: 58 or 59.

[0069] In some embodiments, the vector comprises a nucleic acid comprising a polyadenylation signal. In some embodiments, the polyadenylation signal is derived from the bovine growth hormone (bGH) polyadenylation signal, the human growth hormone (hGH) polyadenylation signal, or the SV40 polyadenylation signal. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62 or 66-69. In some embodiments, the polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 62 or 66-69.

[0070] In some embodiments, the vector comprises a microRNA-binding sequence disposed between the nucleic acid sequence encoding BDNF and the post-transcriptional regulatory element. In some embodiments, the microRNA sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) SEQ ID NOs: 63 and 82. In some embodiments, the microRNA-binding sequence comprises any one of (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) SEQ ID NOs: 63 and 82. In some embodiments, the microRNA-binding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the microRNA-binding sequence comprises the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the microRNA-binding sequence comprises (i) a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 63, and (ii) a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the nucleic acid sequence of SEQ ID NO: 82. In some embodiments, the microRNA-binding sequence comprises SEQ ID NOs: 63 and 82.In some embodiments, the microRNA-binding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 64. In some embodiments, the microRNA-binding sequence comprises the nucleic acid sequence of SEQ ID NO: 64.

[0071] In some embodiments, the vector further comprises a terminator downstream of the post-transcriptional regulatory element. In some embodiments, the vector comprises a nucleic acid comprising one or more inverted terminal repeats (ITRs). In some embodiments, the ITR sequences are derived from AAV. In some embodiments, the 5' ITR sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:51. In some embodiments, the 5' ITR sequence comprises the nucleic acid sequence of SEQ ID NO:51. In some embodiments, the 3' ITR sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:65. In some embodiments, the 3' ITR sequence comprises the nucleic acid sequence of SEQ ID NO:65.

[0072] In some embodiments, the vector comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 11-50.

[0073] In some embodiments, the vector comprises a nucleic acid comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 11, 18-22, 27-30, and 42-45.

[0074] In some embodiments, the vector comprises a nucleic acid sequence selected from SEQ ID NOs: 11 to 50. In some embodiments, the expression construct comprises a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30, and 42-45.

[0075] In some embodiments, the vector comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the expression construct comprises the nucleic acid sequence of SEQ ID NO: 11.

[0076] In some embodiments, the vector comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the expression construct comprises the nucleic acid sequence of SEQ ID NO: 22.

[0077] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence including a C14_L21 promoter, a C26_L21 promoter, a C42b_L21 promoter, a C46_L21 promoter, a B36 promoter, a B46 promoter, a B46 promoter, a B47 promoter, or a B48 promoter; (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to a promoter sequence; (c) posttranscriptional regulatory elements; (d) a polyadenylation signal; and / or (e) One or more ITRs. In some embodiments, the vector comprises two ITR sequences.

[0078] In some embodiments, a vector is provided that includes a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 1-10; (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to a promoter, and the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 58-61; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62 or 66-69; and / or (e) One or more ITRs (e.g., two ITR sequences).

[0079] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to a promoter, and the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 52; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 58; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 63; and (f) one or more ITRs (e.g., two ITR sequences).

[0080] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0081] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 57, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0082] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 54, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0083] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0084] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 55, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0085] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:2; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0086] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 3; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0087] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:4; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0088] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 5; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0089] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 61; and (d) a polyadenylation sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 66.

[0090] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 9; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 61; and (d) a polyadenylation sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 66.

[0091] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:8; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 61; and (d) a polyadenylation sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 66.

[0092] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 10; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 61; and (d) a polyadenylation sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 66.

[0093] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to a promoter, and the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 52; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 58; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising the nucleic acid sequence of SEQ ID NO: 63; and (f) one or more ITRs (e.g., two ITR sequences).

[0094] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising (i) SEQ ID NO: 64, or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0095] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 57, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0096] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 54, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0097] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0098] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 55, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0099] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 2; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0100] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 3; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising (i) SEQ ID NO: 64, or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0101] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 4; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising (i) SEQ ID NO: 64, or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0102] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 5; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising (i) SEQ ID NO: 64, or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0103] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 56, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 61; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 66.

[0104] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 9; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 56, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (e) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 61; (c) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 66.

[0105] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 8; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 56, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (f) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 61; (c) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 66.

[0106] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 56, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (g) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 61; (c) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 66.

[0107] viral vectors Viral vectors for expressing target genes in target cells, tissues, or organisms are known in the art and include, for example, AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, poxvirus vectors, baculovirus vectors, herpes simplex virus vectors, vaccinia virus vectors, or synthetic viral vectors (e.g., chimeric, mosaic, or pseudotyped viruses, and / or viruses containing foreign proteins, synthetic polymers, nanoparticles, or small molecules).

[0108] AAV vectors As used herein, the terms "adeno-associated virus (AAV) vector," "AAV gene therapy vector," and "gene therapy vector" refer to a vector having functional or partially functional ITR sequences and a transgene. As used herein, the term "ITR" refers to an inverted terminal repeat (ITR).

[0109] Adeno-associated viruses (AAVs) are small, single-stranded DNA viruses that require a helper virus to promote efficient replication. The 4.7-kb genome of AAV is characterized by two inverted terminal repeats (ITRs) and two open reading frames encoding the Rep and Cap proteins, respectively. The Rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. These proteins primarily function in AAV replication and rescue, as well as in regulating AAV integration into host cell chromosomes. The Cap reading frame encodes three structural proteins with molecular weights of 85 kD (VP1), 72 kD (VP2), and 61 kD (VP3), which form the virion capsid. VP3 accounts for more than 80% of the total protein in AAV virions. Adjacent to the 5' and 3' ends of the Rep and Cap open reading frames are inverted terminal repeats (ITRs) approximately 145 bp long. The two ITRs are the only cis elements essential for AAV replication, rescue, packaging, and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with therapeutic or reporter transgenes.

[0110] Recombinant adeno-associated virus "rAAV" vectors include any vector derived from any adeno-associated virus serotype. rAAV vectors can have all or part of the AAV wild-type genes deleted, in some embodiments, one or more of the Rep and / or Cap genes, but retain functional flanking ITR sequences.

[0111] In some embodiments, the viral vector is a rAAV virion comprising a rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome comprises an expression construct disclosed herein.

[0112] In some embodiments, the viral vectors disclosed herein comprise a nucleic acid comprising the AAV 5'ITR and 3'ITR located 5' and 3', respectively, to the sequence encoding BDNF. However, in some embodiments, it may be desirable for the nucleic acid to contain 5'ITR and 3'ITR sequences arranged in tandem, e.g., 5'-3', or head-to-tail, or in another alternative configuration. In still other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITRs or to have the 5'ITR (or conversely, the 3'ITR) located both 5' and 3' to the sequence encoding BDNF. The ITR sequences may be located immediately upstream and / or downstream of the heterologous molecule, or intervening sequences may be present. The ITRs need not be wild-type nucleotide sequences and can be modified (e.g., by nucleotide insertion, deletion, or substitution) so long as the sequences provide functional rescue, replication, and packaging. The ITRs can be selected from AAV2 or from other AAV serotypes, as described herein.

[0113] In some embodiments, the viral vector is an AAV vector, e.g., AAV1 (i.e., AAV containing AAV1 ITRs and AAV1 capsid protein), AAV2 (i.e., AAV containing AAV2 ITRs and AAV2 capsid protein), AAV3 (i.e., AAV containing AAV3 ITRs and AAV3 capsid protein), AAV4 (i.e., AAV containing AAV4 ITRs and AAV4 capsid protein), AAV5 (i.e., AAV containing AAV5 ITRs and AAV5 capsid protein), AAV6 (i.e., AAV containing AAV6 ITRs and AAV6 capsid protein), AAV7 (i.e., AAV containing AAV7 ITRs and AAV7 capsid protein), AAV8 (i.e., AAV containing AAV8 ITRs and AAV8 capsid protein), AAV9 (i.e., AAV9 ITRs and AAV9 capsid protein), AAVrh.74 (i.e., an AAV containing AAVrh74 ITRs and AAVrh74 capsid protein), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid protein), or AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid protein).

[0114] In some embodiments, the viral vector is a pseudotyped AAV vector containing ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., an AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., an AAV containing AAV2 ITRs and AAV10 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 1 (i.e., an AAV containing AAV2 ITRs and AAV1 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., an AAV containing AAV2 ITRs and AAV7m8 capsid proteins).

[0115] In some embodiments, the vector is a viral vector. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector contains a recombinant capsid protein, such as a capsid protein containing a chimera of one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In some embodiments, the vector comprises a genome derived from AAV1. In some embodiments, the vector comprises a genome derived from AAV2.

[0116] In one aspect, a viral genome is provided, comprising (a) a nucleic acid comprising a promoter sequence operably linked to a nucleic acid sequence encoding BDNF. In some embodiments, the BDNF is encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to or comprising a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 54. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 54.In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 56. In some embodiments, the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 1-10. In some embodiments, the promoter sequence comprises a sequence selected from SEQ ID NOs: 1-10. In some embodiments, the promoter sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid of SEQ ID NO:1.In some embodiments, the promoter sequence for the nucleic acid comprises the nucleic acid of SEQ ID NO: 1. In some embodiments, the promoter sequence comprises a constitutive promoter.

[0117] In some embodiments, the viral genome comprises a nucleic acid comprising a post-transcriptional regulatory element. In some embodiments, the vector comprises a nucleic acid comprising a WPRE. In some embodiments, the post-transcriptional regulatory element comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 58-61. In some embodiments, the post-transcriptional regulatory element comprises a nucleic acid sequence selected from SEQ ID NOs: 58-61. In some embodiments, the post-transcriptional regulatory element comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 58 or 59. In some embodiments, the post-transcriptional regulatory element comprises the nucleic acid sequence of SEQ ID NO: 58 or 59.

[0118] In some embodiments, the viral genome comprises a nucleic acid comprising a polyadenylation signal. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 62 or 66-69. In some embodiments, the expression construct comprises the nucleic acid sequence of SEQ ID NO: 62 or 66-69. In some embodiments, the polyadenylation signal comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62. In some embodiments, the polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 62.

[0119] In some embodiments, the viral genome comprises a microRNA-binding sequence located in the 3'UTR, for example, between the nucleic acid sequence encoding BDNF and the post-transcriptional regulatory element. In some embodiments, the microRNA-binding sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) any one of SEQ ID NOs: 63 and 82. In some embodiments, the microRNA-binding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the microRNA-binding sequence comprises the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the microRNA-binding sequence comprises (i) a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 63, and (ii) a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the nucleic acid sequence of SEQ ID NO: 82. In some embodiments, the microRNA-binding sequence comprises SEQ ID NOs: 63 and 82.In some embodiments, the microRNA-binding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 64. In some embodiments, the microRNA-binding sequence comprises the nucleic acid sequence of SEQ ID NO: 64.

[0120] In some embodiments, the microRNA binding sequence comprises (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) SEQ ID NOs: 63 and 82.

[0121] In some embodiments, the viral genome further comprises a terminator downstream of the post-transcriptional regulatory element.

[0122] In some embodiments, the viral genome comprises a nucleic acid comprising one or more inverted terminal repeats (ITRs). In some embodiments, the ITR sequences are derived from AAV serotype 1 (AVV1). In some embodiments, the 5' ITR sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:51. In some embodiments, the 5' ITR sequence comprises the nucleic acid sequence of SEQ ID NO:51. In some embodiments, the 3' ITR sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:65. In some embodiments, the 3' ITR sequence comprises the nucleic acid sequence of SEQ ID NO:65.

[0123] In some embodiments, the vector comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 11-50.

[0124] In some embodiments, the viral genome comprises a nucleic acid comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 11, 18-22, 27-30, and 42-45.

[0125] In some embodiments, the viral genome comprises a nucleic acid sequence selected from SEQ ID NOs: 11 to 50. In some embodiments, the expression construct comprises a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30, and 42-45.

[0126] In some embodiments, the viral genome comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the expression construct comprises the nucleic acid sequence of SEQ ID NO: 11.

[0127] In some embodiments, the viral genome comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the expression construct comprises the nucleic acid sequence of SEQ ID NO: 22.

[0128] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence including a C14_L21 promoter, a C26_L21 promoter, a C42b_L21 promoter, a C46_L21 promoter, a B36 promoter, a B46 promoter, a B46 promoter, a B47 promoter, or a B48 promoter; (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to a promoter sequence; (c)WPRE; (d) a polyadenylation signal; and / or (e) One or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0129] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 1-10; (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to a promoter, and the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 58-61; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62 or 66-69; and (e) One or more ITRs (e.g., two ITR sequences).

[0130] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to a promoter, and the nucleic acid sequence encoding BDNF comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 52; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 58; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 63; and (f) one or more ITRs (e.g., two ITR sequences).

[0131] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0132] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 57, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0133] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 54, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0134] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; and one or more ITRs (e.g., two ITR sequences).

[0135] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 55, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0136] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:2; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0137] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 3; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0138] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:4; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0139] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 5; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 53, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to (i) SEQ ID NO: 64 or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0140] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (e) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 61; and (c) a polyadenylation sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 66.

[0141] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 9; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (e) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 61; and (c) a polyadenylation sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 66.

[0142] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO:8; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 61; and (d) a polyadenylation sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 66.

[0143] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 10; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 56, and wherein the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (e) a post-transcriptional regulatory element comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 61; and (c) a polyadenylation sequence comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of SEQ ID NO: 66.

[0144] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to a promoter, and the nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO: 52; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 58; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising the nucleic acid sequence of SEQ ID NO: 63; and (f) one or more ITRs (e.g., two ITR sequences).

[0145] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising (i) SEQ ID NO: 64, or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0146] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 57, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0147] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 54, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0148] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0149] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 55, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0150] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 2; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and (e) One or more ITRs (e.g., two ITR sequences).

[0151] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 3; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising (i) SEQ ID NO: 64, or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0152] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 4; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising (i) SEQ ID NO: 64, or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0153] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 5; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59; (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; (e) a microRNA-binding sequence comprising (i) SEQ ID NO: 64, or (ii) SEQ ID NOs: 63 and 82; and (f) one or more ITRs (e.g., two ITR sequences).

[0154] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 56, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (h) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 61; (c) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 66.

[0155] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 9; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 56, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (i) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 61; (c) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 66.

[0156] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 8; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 56, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (j) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 61; (c) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 66.

[0157] In some embodiments, the viral genome comprises nucleic acid comprising one or more of the following: (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a nucleic acid sequence encoding a BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 56, and the nucleic acid sequence encoding the BDNF protein is operably linked to a promoter; (k) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 61; (c) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 66.

[0158] Other viral vectors Other viral vectors include adenovirus (AV) vectors, such as those based on human adenovirus type 2 and human adenovirus type 5, which have been rendered replication-deficient by deletion of the E1 and E3 regions. A transcription cassette can be inserted into the E1 region to obtain an E1 / E3-deleted recombinant AV vector. Adenovirus vectors include helper-dependent, large-capacity adenovirus vectors (also known as large-capacity, "gutless" or "gutted" vectors) that do not contain viral coding sequences. These vectors contain cis-acting elements necessary for viral DNA replication and packaging, primarily inverted terminal repeats (ITRs) and packaging signals (CYs). These helper-dependent AV vector genomes have the potential to carry foreign DNA from several hundred base pairs to approximately 36 kb. Alternatively, other systems, such as lentiviral vectors, can be used. Lentivirus-based systems can transduce non-dividing as well as dividing cells, making them useful for applications targeting non-dividing cells of the CNS, for example. Lentiviral vectors are derived from the human immunodeficiency virus and, like that virus, integrate into the host genome, providing the potential for very long-term gene expression. Polynucleotides, including plasmids, YACs, minichromosomes, and minicircles, carrying target gene-containing expression constructs can also be introduced into cells or organisms using non-viral vector systems, for example, cationic lipids, polymers, or both as carriers. Conjugated poly-L-lysine (PLL) and polyethyleneimine (PEI) polymer systems can also be used to deliver vectors into cells. Other methods for delivering vectors into cells include hydrodynamic injection, electroporation, and ultrasound, both in cell culture and in organisms. For a review of viral and non-viral delivery systems for gene delivery, see Nayerossadat, N. et al. (Adv Biomed Res. 2012;1:27), incorporated herein by reference.

[0159] rAAV virion production The rAAV virions disclosed herein can be constructed and produced using materials and methods described herein as well as materials and methods known to those of skill in the art. Such engineering methods used to construct any embodiment of the present disclosure are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; and International Patent Publication No. WO 95 / 13598. Additionally, suitable methods for producing rAAV constructs within adenovirus capsids are described in U.S. Patent Nos. 5,856,152 and 5,871,982. Briefly, to package the rAAV genome into rAAV virions, host cells containing sequences necessary for expressing AAV rep and AAV cap or functional fragments thereof, as well as helper genes essential for AAV production, are used. The AAV rep and cap sequences are obtained from AAV sources found herein. The AAV rep and cap sequences can be introduced into host cells by any method known to those skilled in the art, including, but not limited to, transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In some embodiments, the rep and cap sequences can be transfected into host cells via one or more nucleic acid molecules and stably present in the cells as episomes. In other embodiments, the rep and cap sequences are stably integrated into the cell's genome. In other embodiments, the rep and cap sequences are transiently expressed in the host cell.For example, a nucleic acid molecule useful for such transfection comprises, from 5' to 3', a promoter, an optional spacer inserted between the promoter and the start of the rep gene sequence, the AAV rep gene sequence, and the AAV cap gene sequence. As used herein, the term "5' to 3'" simply refers to the order of specific genetic elements in a nucleic acid sequence. In some embodiments, specific genetic elements are linked to each other via linker nucleic acid (e.g., intervening non-coding nucleic acid). In some embodiments, specific genetic elements are linked to each other with no intervening nucleotides. In some embodiments, some of the specific genetic elements are linked to each other via linker nucleic acid, while others are linked to each other with no intervening nucleotides.

[0160] The rep and cap sequences, along with their expression control sequences, may be provided on a single vector, or each sequence may be provided on its own vector. In some embodiments, the rep and cap sequences are provided on the same vector. Alternatively, the rep and cap sequences may be provided on a vector containing other DNA sequences that can be introduced into host cells. In some embodiments, the promoter used in this construct may be any suitable constitutive, inducible, or native promoter known to those of skill in the art. The molecule providing the rep and cap proteins may be in any form that delivers these components to host cells. Desirably, this molecule is in the form of a plasmid, which may contain other non-viral sequences, such as sequences for marker genes. This molecule does not contain AAV ITRs and generally does not contain AAV packaging sequences. To avoid the occurrence of homologous recombination, other viral sequences, particularly adenoviral sequences, are avoided in this plasmid. Desirably, this plasmid is constructed so that it can be stably transfected into cells.

[0161] Although the molecules providing rep and cap can be transiently transfected into host cells, it is preferred that the host cells be stably transformed with the sequences necessary to express functional rep / cap proteins in the host cell, e.g., as an episome or by integration into the host cell chromosome. Depending on the promoter controlling expression in such stably transfected host cells, the rep / cap proteins can be transiently expressed (e.g., through the use of an inducible promoter).

[0162] Methods used to construct embodiments of the present disclosure are conventional genetic or recombinant engineering techniques, as described in the references above. For example, rAAV can be produced using a triple transfection method using either the calcium phosphate method (Clontech) or Effectene™ reagent (Qiagen, Valencia, Calif.), according to the manufacturer's instructions. See Herzog et al., 1999, Nature Medic., 5(1):56-63, for the method used in the following examples, which employs a plasmid carrying the transgene, a helper plasmid containing AAV rep and cap, and a plasmid supplying the adenoviral helper functions of E2A, E4Orf6, and VA. While the present disclosure provides illustrative examples of specific constructs using the information provided herein, those of skill in the art will be able to select and design other suitable constructs using their selection of spacers, promoters, and other elements, including at least one translational start and stop signal, and the optional addition of a polyadenylation site.

[0163] rAAV virions are then produced by culturing host cells containing the rAAV viruses described herein, which contain the rAAV genome, AAV rep sequence, and AAV cap sequence to be packaged into the rAAV virions, under the control of regulatory sequences that direct their expression. Suitable viral helper genes, such as adenovirus E2A, E4Orf6, and VA, among other possible helper genes, can be provided to the culture by various methods known in the art. In some embodiments, suitable viral helper genes are provided on a separate plasmid. Recombinant AAV virions that direct expression of the BDNF transgene are then isolated from the cells or cell culture in the absence of contaminating helper virus or wild-type AAV.

[0164] The expression of BDNF transgene can be measured by methods known in the art. For example, target cells can be infected in vitro, and the copy number of the transgene in cells can be monitored by Southern blotting or quantitative polymerase chain reaction (PCR). RNA expression level can be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and protein expression level can be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or by the specific method detailed in the following examples.

[0165] Pharmaceutical Composition Provided herein are pharmaceutical compositions comprising any of the vectors or viral particles disclosed herein and a pharmaceutically acceptable carrier.

[0166] In some embodiments, the rAAV containing the gene encoding BDNF is assessed for contamination by conventional methods and then formulated into a pharmaceutical composition suitable for storage and / or administration to a patient.

[0167] Formulation of the vectors disclosed herein involves the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly a vehicle or carrier suitable for subretinal injection, such as buffered saline or other buffers, e.g., HEPES, to maintain the pH at an appropriate physiological level.

[0168] The vectors of the present disclosure can be formulated into pharmaceutical compositions. These compositions may contain, in addition to the vector, pharmaceutically and / or physiologically acceptable excipients, carriers, buffers, stabilizers, antioxidants, preservatives, or other additives known to those skilled in the art. Such materials should be non-toxic and not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials can be determined by one of skill in the art according to the route of administration. Pharmaceutical compositions are typically in liquid form. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Additional carriers are provided in International Patent Publication No. WO 00 / 15822, which is incorporated herein by reference. Physiological saline, magnesium chloride, dextrose, or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may also be included. In some cases, surfactants, such as 0.001% pluronic acid (PF68), may be used. In some cases, Ringer's solution, lactated Ringer's solution, or Hartmann's solution is used. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, as required.

[0169] Pharmaceutical compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable preparations can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with added preservatives. The medicaments can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. The medicaments can also be formulated into rectal compositions, such as suppositories or retention enemas, containing, for example, conventional suppository bases, such as cocoa butter or other glycerides.

[0170] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0171] For delayed release, the vectors may be included in pharmaceutical compositions formulated for sustained release, such as microcapsules formed from biocompatible polymers or liposome carrier systems by methods known in the art.

[0172] For long-term storage of vectors, they may be frozen in the presence of glycerol.

[0173] How to use Provided herein are methods for treating or preventing a metabolic disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a vector or pharmaceutical composition described herein. In some embodiments, the metabolic disorder is obesity.

[0174] Also within the scope of the present disclosure is a method for reducing or eliminating a symptom of a metabolic-related disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject a vector or pharmaceutical composition described herein. In certain embodiments, the symptom of the metabolic-related disorder is one or more of obesity, insulin sensitivity, syndrome X, and diabetes.

[0175] "Metabolic disorders" refer to disorders, diseases, and conditions caused by or characterized by abnormal weight gain, energy use or expenditure, altered responses to ingested or endogenous nutrients, energy sources, hormones, or other signaling molecules in the body, or altered metabolism of carbohydrates, lipids, proteins, nucleic acids, or combinations thereof. Metabolic disorders can be associated with either deficiencies or excesses in metabolic pathways that result in imbalances in carbohydrate, lipid, protein, and / or nucleic acid metabolism. Examples of metabolic disorders include, but are not limited to, metabolic syndrome, disorders associated with insulin deficiency or insulin resistance, diabetes mellitus (e.g., type 2 diabetes), impaired glucose tolerance, dyslipidemia, atherosclerosis, hypertension, preeclampsia, cardiac disease, stroke, non-alcoholic fatty liver disease (NAFLD), hyperglycemia, fatty liver of various etiologies, dyslipidemia, immune system dysfunction associated with overweight and obesity, cardiovascular disease, high cholesterol, elevated triglyceride levels, asthma, sleep apnea, osteoarthritis, neurodegeneration, gallbladder disease, syndrome X, inflammatory and immune disorders, atherogenic dyslipidemia, and cancer.

[0176] "Obesity" refers to the condition of a subject in which said subject has a body mass index (BMI) of 30 or greater. BMI is typically calculated by dividing a human subject's weight in kilograms (or pounds) by the square of their height in meters (or feet).

[0177] In some embodiments, the subject is a mammal. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and livestock. Mammals include, but are not limited to, human or non-human mammals, such as, for example, cows, horses, dogs, sheep, or cats. Individuals and patients are also subjects herein.

[0178] As used herein, the terms "treat," "treated," "treating," or "treatment" refer to therapeutic treatment, the purpose of which is to slow (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; a decrease in the severity of the condition, disorder, or disease; stabilization (i.e., not worsening) of the state of the condition, disorder, or disease; a delay in the onset of the condition, disorder, or disease, or a delay in the progression of the condition, disorder, or disease; an improvement in one or more symptoms of the condition, disorder, or disease; and remission (partial or complete), or improvement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to the expected survival if not receiving treatment. The terms "prevent", "prevention" and the like refer to acting before the onset of an obvious disease or disorder to prevent the onset of the disease or disorder, or to minimize the extent of the disease or disorder, or to delay the course of its development.

[0179] In yet another aspect, the present disclosure further provides a method for increasing BDNF levels or activity in a subject in need thereof, the method comprising administering to the subject a vector or pharmaceutical composition described herein.

[0180] The level or activity of BDNF can be measured by determining or estimating protein level or mRNA level.The method of determining or estimating protein level or mRNA level is well known in the art.For example, the protein level (such as protein expression level) of BDNF can be determined by SDS-PAGE, Western blot or immunoassay (such as immunoblotting assay, immunoprecipitation assay).The mRNA level can be determined by RT-PCR.

[0181] Measurement of BDNF levels can be performed by assaying the proteins themselves (by Western blotting, ELISA, RIA, and other techniques known to those of skill in the art), by assaying mRNA encoding these proteins (such as quantitative PCR, Northern blotting, RNase protection assay, RNA dot blotting, and other techniques known to those of skill in the art), or by assaying the activity of regulatory elements of genes for BDNF. For example, the activity of regulatory elements can be assessed using reporter constructs that combine DNA segments of promoter, enhancer, and / or intron elements with cDNA encoding a reporter (such as luciferase, beta-galactosidase, green fluorescent protein, or other easily assayed reporter genes). These reporter constructs can be stably or transiently transfected into cells.

[0182] Route and method of administration In some embodiments, a vector or pharmaceutical composition disclosed herein is administered to a subject intratumorally, intravenously, subcutaneously, intraosseously, orally, transdermally, intracerebrally, intraparenchyma, sustained release, controlled release, delayed release, as a suppository, or sublingually.

[0183] In some embodiments, the vector or pharmaceutical composition is administered by stereotaxic microinjection.

[0184] The dosage of the vectors of the present disclosure can be determined according to various parameters, in particular the age, weight, and condition of the patient to be treated, the specific disorder and, if progressive, the extent to which the disorder has progressed, the route of administration, and the required regimen. A physician can determine the route of administration and dosage required for a particular patient.

[0185] An effective amount of rAAV carrying a nucleic acid sequence encoding BDNF under the control of a promoter sequence is preferably about 1×10 9 ~about 2×10 12 (For example, about 5 × 10 9 ~about 2×10 11 ) rAAV genome particles, or approximately 1 × 10 10 ~about 2×10 11 The genome particle ranges between about 1 x 10 and 1 x 10. A genome particle is defined herein as an AAV capsid containing a single-stranded DNA molecule that can be quantified using a sequence-specific method (such as real-time PCR). In some embodiments, the genome particle ranges between about 1 x 10 and 1 x 10. 9 ~about 2×10 12 (For example, about 5 × 10 9 ~about 2×10 11 ) rAAV genome particles may be provided in a volume of between about 150 and about 800 μl (e.g., between about 100 and about 500 μl, e.g., between about 100 and about 200 μl). Additional dosages within these ranges can be selected by the attending physician.

[0186] In some embodiments, the dose may be provided as one or more doses administered bilaterally into the hypothalamus. In some embodiments, it may be desirable to administer multiple "booster" doses of the pharmaceutical compositions disclosed herein. For example, depending on the duration of the transgene in the target cells, booster doses can be delivered at six-month intervals or annually after the initial administration. Other similar tests can be used to determine the status of the treated subject over time. The selection of the appropriate test can be performed by the attending physician.

[0187] Manufactured Products and Kits Kits or articles of manufacture for use in the methods described herein are also provided. In some embodiments, the kits include a composition described herein (e.g., a composition for delivery of a BDNF-encoding transgene) in suitable packaging. Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and / or sealed.

[0188] Kits containing the compositions described herein are also provided. These kits may further include instructions or instructions for using the compositions, such as the uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts containing instructions for administering the compositions or performing any of the methods described herein. For example, in some embodiments, the kit includes an rAAV for expression of a BDNF-encoding transgene in target cells, a pharmaceutically acceptable carrier suitable for injection, and one or more of a buffer, diluent, filter, needle, syringe, and package insert containing instructions for performing the injection.

[0189] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. For any of the methods provided, the method steps may be performed simultaneously or sequentially. When method steps are performed sequentially, the steps may be performed in any order unless otherwise noted.

[0190] Where a method includes a combination of steps, any combination or subcombination of steps is encompassed within the scope of the present disclosure unless otherwise noted herein.

[0191] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent not inconsistent with this disclosure. The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present disclosure. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0192] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. [Example]

[0193] Example 1 cell culture Neuro2A (N2A), BE2-M17, and SH-SY5Y immortalized cell lines were maintained at 37°C in 5% CO2. Cells were transiently transfected with plasmids using TurboFect Transfection Reagent. After 2 days, medium and cell lysates were collected for protein analysis. After 30 passages, a new aliquot of cells was thawed and passaged twice before use in subsequent experiments.

[0194] Primary neuron culture Embryonic day 16 (E16) mating-day-controlled pregnant female mice were anesthetized with CO2 and sacrificed by cervical dislocation. In a dissection hood, 24–26 embryos per experiment were collected by removing the amniotic membrane through a maternal abdominal incision and decapitated in ice-cold Hank's balanced salt solution (HBSS). Using sharp scissors and forceps, the brains were rapidly dissected, and the cortices were removed from the meninges and isolated under a dissection microscope. The cortices were collected in ice-cold HBSS and kept on ice until all embryos were dissected. In a tissue culture hood, the HBSS was removed, and the cortical tissue was digested with 0.25% trypsin-EDTA at 37°C for 12 minutes, followed by DNase 1 treatment at 37°C for 10 minutes. The tissue was dissociated by successive triturations with a 25 ml serological pipette, followed by triturations with 10 ml and 5 ml serological pipettes. The cell suspension was washed once with DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin, passed through a 40 μM cell strainer, and then counted using a hemocytometer. Single cells were plated at a density of 400,000 cells per well in a 24-well plate containing poly-d-lysine (0.1 mg ml). -1 ) coated wells. Cells were grown in Neurobasal medium supplemented with B-27™ supplement, N-2 supplement, and 0.5 mM L-glutamine and maintained at 37°C in 5% CO2. Cultures were fed with half the medium change every 3-4 days.

[0195] Primary neuron transduction On day 1 in vitro, AAV2 / 1 particles were added to the neuronal culture medium at multiplicities of infection (MOI) of 10,000 and 50,000. The medium was changed after 72 hours according to standard neuronal protocols.

[0196] BDNF activity assay The medium from transfected N2A cells was collected after 2 days and centrifuged at 500 rpm to remove any cellular debris. BDNF levels in this clear medium were assessed by ELISA. Medium containing a standardized amount of BDNF was added to the primary cultures and incubated for 15–30 minutes, after which lysates were collected and prepared for Western blotting.

[0197] Western blotting After gently washing the cells with PBS, ice-cold lysis buffer (CST) containing DNAse and protease / phosphatase inhibitors was added. Cells were lysed on ice for 15 minutes with agitation on the plate and then collected. Samples were vortexed vigorously to ensure complete lysis and then pelleted at maximum speed in a microcentrifuge for 10 minutes. The supernatant was collected, and the protein concentration was determined by BCA assay. Protein was then normalized to a standard concentration using 4x LDS, 10x DTT, and lysis buffer. Samples were denatured at 85°C for 10 minutes.

[0198] For BDNF analysis, 10 μg of protein samples were separated on a 4-12% Tris-Glycine Mini-PROTEAN denaturing gel (BioRad) using Tris-Glycine-SDS buffer. For analysis of total ERK1 / 2 and phosphorylated ERK1 / 2, 10 μg of protein samples were separated on a 4-12% Bolt Bis-tris precast denaturing gel (Invitrogen, USA) using MOPS buffer. The gel was then transferred to a nitrocellulose membrane. The membrane was probed overnight at 4°C with primary antibodies (total ERK1 / 2, phosphorylated ERK1 / 2, BDNF, Gapdh, and beta-actin) diluted in Licor TBS blocking buffer. The membrane was then washed and probed for 1 hour at room temperature with fluorescently conjugated anti-mouse or anti-rabbit IgG secondary antibodies. The membrane was imaged using a Licor Odyssey system. Protein amounts were normalized using beta-actin or Gapdh.

[0199] ELISA Tissue samples were lysed in CST RIPA buffer without SDS and containing DNase and a protease / phosphatase inhibitor cocktail. Samples were sonicated twice and then centrifuged to remove cellular debris. Samples were then used in pro-BDNF and mature BDNF ELISA (Biosensis) according to the manufacturer's protocol.

[0200] animal Seven-week-old male C57BL / 6 mice were purchased from Charles River Laboratories. Mice were housed five animals per cage at a constant temperature (23°C) with a 12-h light-dark cycle (lights on 07:00–19:00) and free access to food and water. All studies were reviewed by the Institutional Animal Care and Use Committee (IACUC). Animals were randomly assigned to groups based on preoperative body weight. At the end of the study, hypothalamus were rapidly dissected and flash-frozen in liquid nitrogen.

[0201] Stereotactic surgery To overexpress BDNF in the adult hypothalamus, adeno-associated virus (0.5 μl, 2e9 GC per hemisphere, serotype 1) was injected bilaterally into 8-week-old male C57Bl / 6 mice. Animals were anesthetized with isoflurane and secured in a Kopf stereotaxic frame via ear and incisor bars. A midline incision was made in the scalp to expose the skull, and two small holes were drilled with a dental drill at the top of the skull for injection sites (1.2 mm posterior to bregma, 0.5 mm lateral to the midline, and 5.85 mm dorsal to bregma). rAAV vectors were injected bilaterally into the hypothalamus (2e9 genome particles per site) at a rate of 0.1 ml per minute using a 5 μl Hamilton Neuro syringe attached to a Micro4 microsyringe pump controller (World Precision Instruments). At the end of the injection, the syringe was slowly withdrawn from the brain, and the scalp was closed with Vetbond. Mice were placed in clean cages and closely monitored until they recovered from anesthesia. They were fed a standard diet (NCD, 11% fat, 28% protein, 61% carbohydrate, caloric density 3.4 kcal / g, Research Diets; also referred to as "control diet" in Figure 4D). Seven days after rAAV injection, mice in the appropriate groups were placed on a high-fat diet (HFD, 45% fat, caloric density 4.73 kcal g, Research Diets) for the duration of the study (21 days post-injection).

[0202] Example 2 We have developed several BDNF gene therapy vectors for the treatment of obesity. See Tables 1–8 for an overview of the expression constructs. Different expression constructs were designed by modifying various cis-regulatory components. As shown herein, the BDNF expression construct achieved significantly higher expression compared to previously published constructs in head-to-head comparisons in primary mouse cortical neurons and various immortalized neuronal cell lines. Furthermore, AAV-mediated delivery of the BDNF expression vector to the hypothalamus of mice was more effective than the published CAG-BDNF construct in preventing weight gain induced by a high-fat diet. By designing gene therapy vectors that enable high levels of BDNF expression, we can achieve therapeutic efficacy with lower viral vector doses and potentially lower immune responses and reduced safety risks. Other advantages of the disclosed gene therapy vectors include a single administration resulting in long-term treatment without the need for adherence to injection regimens; local administration to the brain limits peripheral side effects; and direct targeting of mechanistic regions. Collectively, these results demonstrate that the expression constructs of the present disclosure provide a powerful and effective gene therapy tool for treating rare obese populations due to genetic or environmental factors, as well as more common forms of obesity.

[0203] Higher BDNF expression from the constitutive promoter than from tissue-specific or the original CAG promoter Promoters are essential components of gene therapy, influencing transgene expression levels, timing, persistence, and cell-type specificity. Therefore, we developed a library of tissue-specific and constitutive promoters that were more potent than commonly used reference promoters (NSE, Syn, and CAMkIIa) in mouse N2A cells. These promoters were comparable to or even more potent than CMV promoters, and some were even more potent than CAG promoters. A subset of each class of these promoters was selected and applied to BDNF to increase its expression. In both mouse (N2A) and human cells (BE2M17), the C14 promoter drove some of the highest levels of BDNF expression in vitro among all promoters tested (Figure 1). This promoter was carried over into subsequent rounds of experiments and optimization of the expression construct.

[0204] The BDNF expression constructs disclosed herein are more potent than the reference constructs in vitro.

[0205] Four different codon optimization algorithms were applied to the BDNF transgene. Codon optimization was performed using the reference construct CAG-BDNF described in Cao et al., Molecular therapy of obesity and diabetes by a physiological autoregulatory approach, Nat Med. 2009 Apr;15(4):447-54 and U.S. Pat. No. 9,265,843. HAWe evaluated the effect of codon optimization on BDNF expression compared with the -WPRE-bGH construct (also referred to herein as the OSU construct). The codon optimization method included a combination of (1) removing rare codons with a usage frequency of less than 25% and replacing them with more frequent codons based on the codon usage table of top genes expressed in the putamen or other subregions of the brain, (2) removing CpG motifs, (3) removing undesirable motifs (e.g., cryptic splice sites, TLR9 activation motifs), and (4) introducing desirable motifs (e.g., TLR9 inhibitory motifs) where possible. While some species-dependent effects were observed (N2A data vs. SH-SY5Y), increased protein expression was consistently observed with codon optimization #1 and #3. See Table 5 for sequences. Therefore, the codon-optimized genes BDNFco and BDNFco3 were used for further development (Figure 2A).

[0206] The expression constructs BDNF-1 (containing BDNFco) and BDNF-12 (containing BDNFco3) showed significantly higher expression than the reference constructs in both N2A cells (Figure 2B) and human neural progenitor cells (NPCs) (Figure 2C). The codon-optimized sequences had the same signaling activity (ERK1 / 2 phosphorylation) as the wild-type BDNF sequence found in the reference construct, thus demonstrating their functionality (Figure 2D).

[0207] Next, BDNF-1 and BDNF-12 were tested in primary mouse cortical neurons using AAV2 / 1-mediated transduction (Figure 3A). Equivalent transduction was achieved between the constructs (WPRE and ITR2 qPCR) (Figure 3B). Consistent with previous data from transiently transfected cells, the expression constructs BDNF-1 and BDNF-12 significantly increased BDNF expression compared with the reference construct, as shown by Western blot analysis (Figure 3C).

[0208] To test the in vivo expression and efficacy of the disclosed constructs, a high-fat diet (HFD)-induced obesity model was used in wild-type adult mice. Virus was injected bilaterally into the hypothalamus of each mouse. Starting on day 7 after surgery, animals were placed on a high-fat diet. Body weights were recorded weekly, and tissues were collected on day 21 after surgery (Figure 4A).

[0209] The expression construct BDNF1 expressed over 1000-fold higher levels of BDNF precursor (proBDNF) (648.1 ± 33.5 vs. 0.6 ± 0.1 ng / mg total protein) and 200-fold higher levels of mature BDNF (mBDNF) (260.6 ± 20.7 vs. 1.3 ± 0.3 total protein) than endogenous levels in the control diet group (Fig. 4C). The expression construct BDNF12 expressed over 400-fold higher levels of proBDNF (648.1 ± 33.5 vs. 0.6 ± 0.1 ng / mg total protein) and 84-fold higher levels of mBDNF (260.6 ± 20.7 vs. 1.3 ± 0.3 total protein) than endogenous levels in the control diet group (Fig. 4C). Compared to the OSU reference construct, the expression constructs BDNF-1 and BDNF-12 expressed 19.2- and 11.3-fold more mBDNF protein, respectively, in the mouse hypothalamus (FIG. 4B).

[0210] Overexpression of BDNF induces a concentration-dependent decrease in body weight in wild-type animals. As shown in Figure 4D, animals treated with the OSU reference construct (0.9 ± 0.3 g) showed a 45% HFD-induced weight gain less than the control group of wild-type mice (HFD: 4.9 ± 0.4 g; AAV2 / 1-eGFP+HFD: 4.5 ± 0.3 g). In contrast to the control and OSU reference groups, mice treated with the expression construct and BDNF-12 not only prevented HFD-induced weight gain but also showed significant weight loss despite consuming an HFD (BDNF-1: -4.3 ± 0.4 g, BDNF-12: -2.9 ± 0.9 g, Figure 4D).

[0211] While the foregoing written description of the invention will enable one of ordinary skill in the art to make and use what is presently believed to be the best mode thereof, those skilled in the art will understand and recognize that there are variations, combinations, and equivalents to the specific embodiments, methods, and examples herein.

[0212] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 3] [Table 4-1] [Table 4-2] [Table 5]

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

Table 8-19

Table 8-20

Table 8-21

Table 8-22

Table 8-23

Table 8-24

Table 8-25

Table 8-26

Table 8-27

Table 8-28

Table 8-29

Table 8-30

Table 8-31

Table 8-32

Table 8-33

Table 8-34

Table 8-35

Table 8-36

Table 8-37

Table 8-38

Table 8-39

Table 8-40

Table 8-41

Table 8-42

Table 8-43

Table 8-44

Table 8-45

Table 8-46

Table 8-47

Table 8-48

Table 8-49

Table 8-50

Table 8-51

Table 8-52

Table 8-53

Table 8-54

Table 8-55

Table 8-56

Table 8-57

Table 8-58

Table 8-59

Table 8-60

Table 8-61

Table 8-62

Table 8-63

Table 8-64

Table 8-65

Table 8-66

Table 8-67

Table 8-68

Table 8-69

Table 8-70

Table 8-71

Table 8-72

Table 8-73

Table 8-74

Table 8-75

Table 8-76

Claims

1. An expression construct comprising a promoter sequence operably linked to a nucleic acid sequence encoding brain-derived neurotrophic factor (BDNF) or a variant thereof.

2. 2. The expression construct of claim 1, wherein the promoter sequence (i) comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1-10, or (ii) comprises a nucleic acid sequence selected from SEQ ID NOs: 1-10.

3. 2. The expression construct of claim 1, wherein the promoter sequence (i) comprises a nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 1, or (ii) comprises the nucleic acid sequence of SEQ ID NO:

1.

4. The expression construct of claim 1 , wherein the promoter sequence comprises a constitutive promoter.

5. 10. The expression construct of claim 1, wherein the nucleic acid sequence encoding the BDNF is a wild-type BDNF gene.

6. 10. The expression construct of claim 1, wherein the nucleic acid sequence encoding the BDNF is the human BDNF gene.

7. 10. The expression construct of claim 1, wherein the nucleic acid sequence encoding the BDNF is a codon-optimized sequence.

8. 10. The expression construct of any one of the preceding claims, wherein the nucleic acid sequence encoding the BDNF (i) comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79, or (ii) comprises a nucleic acid sequence selected from SEQ ID NOs: 52-57 or 70-79.

9. 10. The expression construct of any one of the preceding claims, wherein the nucleic acid sequence encoding the BDNF (i) comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 52 or 53, or (ii) comprises the nucleic acid sequence of SEQ ID NO: 52 or 53.

10. 10. An expression construct according to any one of the preceding claims, further comprising a post-transcriptional regulatory element.

11. 11. The expression construct of claim 10, wherein the post-transcriptional regulatory element comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

12. 11. The expression construct of claim 10, wherein the post-transcriptional regulatory element (i) comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 58-61, or (ii) comprises a nucleic acid sequence selected from SEQ ID NOs: 58-61.

13. 11. The expression construct of claim 10, wherein the post-transcriptional control element (i) comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 58 or 59, or (ii) comprises the nucleic acid sequence of SEQ ID NO: 58 or 59.

14. 10. An expression construct according to any one of the preceding claims, further comprising a polyadenylation signal.

15. 15. The expression construct of claim 14, wherein the polyadenylation signal (i) comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 62 or 66-69, or (ii) comprises any one of SEQ ID NOs: 62 or 66-69.

16. 16. The expression construct of claim 15, wherein the polyadenylation signal (i) comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 62, or (ii) comprises SEQ ID NO:

62.

17. 10. The expression construct of any one of the preceding claims, further comprising a microRNA binding sequence located between the nucleic acid sequence encoding the BDNF and the post-transcriptional control element.

18. The microRNA binding sequence is (a) a nucleic acid sequence having at least 90% sequence identity to (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) SEQ ID NOs: 63 and 82; or (b) The expression construct of claim 17, comprising (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) SEQ ID NOs: 63 and 82.

19. 10. The expression construct of claim 1, comprising: (a) a promoter sequence comprising SEQ ID NO: 1; (b) a nucleic acid sequence encoding BDNF comprising SEQ ID NO: 52; (c) a post-transcriptional regulatory element comprising SEQ ID NO: 58; (d) a polyadenylation signal comprising SEQ ID NO: 62; and (e) the expression construct comprising a microRNA binding sequence comprising SEQ ID NO:

63.

20. 10. The expression construct of claim 1, comprising: (a) a promoter sequence comprising SEQ ID NO: 1; (b) a nucleic acid sequence encoding BDNF comprising SEQ ID NO: 53; (c) a post-transcriptional regulatory element comprising SEQ ID NO: 59; (d) a polyadenylation signal comprising SEQ ID NO: 62; and (e) the expression construct comprising a microRNA binding sequence comprising SEQ ID NO:

64.

21. A vector comprising the expression construct of any one of claims 1 to 20.

22. 22. The vector of claim 21, which is a viral vector.

23. 23. The vector of claim 22, wherein the viral vector is an adeno-associated viral (AAV) vector.

24. A viral vector comprising (i) an expression construct according to any one of claims 1 to 20 and (ii) one or more inverted terminal repeats (ITRs).

25. 26. The viral vector of claim 23 or 25, wherein the viral vector (i) comprises a nucleic acid sequence having at least 90% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 11-50, or (ii) comprises a nucleic acid sequence selected from SEQ ID NOs: 11-50.

26. 26. The viral vector of claim 25, comprising: (i) a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30, and 42-45; or (ii) a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30, and 42-45.

27. 27. The viral vector of claim 26, wherein the expression construct comprises (i) a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 11 or 22, or (ii) SEQ ID NO: 11 or 22.

28. 25. The viral vector of claim 23 or 24, wherein the vector comprises a genome derived from AAV serotype AAV2.

29. The viral vector of any one of claims 23 to 28, wherein the vector comprises a capsid derived from AAV serotype AAV1.

30. A pharmaceutical composition comprising the vector according to any one of claims 21 to 30 and a pharmaceutically acceptable carrier.

31. 31. A method for treating a metabolic disorder in a subject in need thereof, the method comprising administering to the subject the vector of any one of claims 21 to 30 or the pharmaceutical composition of claim 30.

32. 32. The method of claim 31, wherein the metabolic disorder is obesity.

33. A method for increasing BDNF levels in a subject in need thereof, the method comprising administering to the subject a vector described in any one of claims 21 to 30 or a pharmaceutical composition described in claim 30.

34. The method of any one of claims 31 to 33, wherein the vector or the pharmaceutical composition is administered by stereotaxic microinjection.

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