BDNF gene therapy

Abstract

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

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of the earlier filing date of U.S. Provisional Patent No. 63 / 379,114, filed on October 11, 2022, under 35 U.S.C. § 119(e), and the said provisional patent is hereby incorporated by reference in its entirety.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic sequence listing (SeqList - 162027 - 53276.xml; size: 216,128 bytes; and creation date: October 10, 2023) are hereby incorporated by reference in their entirety into this application. Technical field

[0005] This disclosure generally relates to compositions and methods of gene therapy for treating metabolic disorders and diseases. Background art

[0006] The most common cause of obesity is excessive food intake, combined with limited energy expenditure and / or lack of physical exercise. Obesity increases the likelihood of developing various diseases, such as diabetes, hypertension, atherosclerosis, coronary artery disease, sleep apnea, gout, rheumatism, and arthritis. In addition, the risk of death is directly related to obesity. For example, a body mass index exceeding 40 results in a reduction in average life expectancy of more than a decade.

[0007] Current drug treatment modalities include appetite suppressants that target receptor classes (e.g., CB1, 5 - HT2c, and NPY); modulators of appetite circuits in the hypothalamus and the molecular actions of ghrelin; and nutrient absorption inhibitors that target lipases. However, none of the current modalities can effectively treat obesity without causing adverse reactions, some of which can be very severe. The incidence of bariatric surgery has increased sharply, reflecting how difficult and refractory severe obesity is to alternative, less invasive strategies. However, bariatric surgery is generally effective, but it is also associated with significant morbidity. Drug 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 compliance with the treatment regimen and may cause inflammation at the injection site. In addition, these therapies may take up to a year for the drug to exert its full efficacy (e.g., 15% - 20% weight loss, which is lower than bariatric surgery).

[0008] Accordingly, there is an urgent need for safer and more effective methods for treating obesity. SUMMARY OF THE INVENTION

[0009] The present disclosure addresses the above needs in several aspects. In one aspect, the present disclosure provides an expression construct for treating 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.

[0010] 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.

[0011] In some embodiments, the promoter sequence comprises a constitutive promoter.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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).

[0016] In some embodiments, the expression construct further comprises a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element comprises the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0017] In some embodiments, the post-transcriptional regulatory element comprises a nucleic acid sequence having at least 90% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 58 - 61 or comprises a nucleic acid sequence selected from SEQ ID NOs: 58 - 61.

[0018] In some embodiments, the post-transcriptional regulatory element comprises a nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 58 or 59 or comprises the nucleic acid sequence of SEQ ID NO: 58 or 59.

[0019] 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 with the nucleic acid sequence of SEQ ID NO: 62 or comprises the nucleic acid sequence of SEQ ID NO: 62 or 66 - 69.

[0020] In some embodiments, the expression construct further comprises a microRNA (miR) binding sequence (miRBS) 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 with any one of (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) SEQ ID NO: 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 NO: 63 and 82.

[0021] In some embodiments, the expression construct further comprises a terminator downstream of the post-transcriptional regulatory element. In some embodiments, the expression construct comprises:

[0022] (a) a promoter sequence comprising SEQ ID NO: 1;

[0023] (b) a nucleic acid sequence encoding BDNF comprising SEQ ID NO: 52;

[0024] (c) a post-transcriptional regulatory element comprising SEQ ID NO: 58;

[0025] (d) a polyadenylation signal comprising SEQ ID NO: 62; and

[0026] (e) a microRNA binding sequence comprising SEQ ID NO:63;

[0027] In some embodiments, the expression construct comprises:

[0028] (a) a promoter sequence comprising SEQ ID NO:1;

[0029] (b) a nucleic acid sequence encoding BDNF comprising SEQ ID NO:53;

[0030] (c) a post-transcriptional regulatory element comprising SEQ ID NO:59;

[0031] (d) a polyadenylation signal comprising SEQ ID NO:62; and

[0032] (e) a microRNA binding sequence comprising (i) SEQ ID NO:64 or (ii) SEQ ID NO:63 and 82.

[0033] In another aspect, the present disclosure also provides a vector comprising the expression construct as described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the viral vector comprises the expression construct as 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 with 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 with a nucleic acid sequence selected from SEQ ID NOs: 11-50 or having at least 90% sequence identity with 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 with SEQ ID NO:11 or SEQ ID NO:22, or the nucleic acid sequence comprises SEQ ID NO:11 or SEQ ID NO:22.

[0034] The present disclosure provides a pharmaceutical composition comprising the vector as described herein and a pharmaceutically acceptable carrier.

[0035] The present disclosure provides a method for treating or preventing a metabolic disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject in need thereof a vector or pharmaceutical composition described herein. In one embodiment, the metabolic disorder is obesity.

[0036] The present disclosure provides 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 DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A 、 1B and 1C are a set of graphs showing that BDNF expression from an optimized constitutive promoter is higher than that from tissue-specific or the original CAG promoter. Figure 1A FACS-based promoter screening of optimized tissue-specific and constitutive promoters operably linked to a fluorescent protein reporter (mClover3) identified preferred promoter candidates for driving BDNF expression. Figure 1B Shows extracellular BDNF expression in transiently transfected mouse N2A (left) and human Be2M17 cells (right). Figure 1C Shows extracellular BDNF expression in transiently transfected mouse N2A cells. OSU: reference construct, see Example 2.

[0038] Figure 2A 、 2B 、2C and 2D show that the BDNF expression constructs disclosed herein are more effective in vitro than the reference construct. Figure 2A Shows extracellular BDNF expression in transiently transfected mouse N2A (left) and human SH-SYFY cells (right). Figure 2B Shows BDNF expression in transiently transfected human neural progenitor cells. Figure 2C Shows the results of six independent experiments depicting that BDNF expressed by the expression construct BDNF-1 in N2A cells is 4-fold more than that of the reference construct OSU. Figure 2D Shows that codon optimization of the BDNF gene does not impair BDNF signaling as indicated by the induction of phospho-ERK1 / 2 in primary neurons after 30 minutes of treatment. C.O.1 = codon-optimized gene sequence #1 (BDNFco CDS); C.O.2 = codon-optimized gene sequence #2 (BDNFco2CDS); C.O.3 = codon-optimized gene sequence #3 (BDNFco3 CDS); C.O.2 = codon-optimized gene sequence #4 (BDNFco4 CDS).

[0039] Figure 3A 、3B And 3C show excellent BDNF expression from selected expression constructs in transduced primary neurons. Figure 3A Show that primary mouse cortical neurons were transduced with AAV2 / 1 at a multiplicity of infection (MOI) of 10,000 or 50,000. Media were collected at 4 and 7 days post-infection (d.p.i.) and prepared for Western blotting. DNA was isolated from neuronal lysates at 7 days post-infection. Figure 3B Show that neuronal transduction was equivalent between groups as quantified by qPCR of viral genomes using probes for transgene or ITR (inverted terminal repeat) sequences. Figure 3C Show that transduction in vitro with constructs BDNF1 and BDNF12 resulted in significantly higher BDNF expression in primary cortical neurons.

[0040] Figure 4A 、 4B 、4C and 4D show that overexpression of BDNF from selected expression constructs resulted in weight loss in a diet-induced obesity mouse model. Figure 4A Show that AAV2 / 1 (2E9 GC / hemisphere) was bilaterally injected into the hypothalamus of 8-week-old wild-type C57Bl6 male mice and weighed weekly. After 7 days, animals were placed on a 45% high-fat or matched control diet. After 21 days, the hypothalamus was dissected for protein analysis. Figure 4B Show Western blots depicting high expression of BDNF expression constructs in vivo. Figure 4C Show the expression of pro- and mBDNF in the hypothalamus as quantified by ELISA. Figure 4D Show the weight changes after AAV2 / 1 delivery in a diet-induced obesity mouse model. ***p < 0.001; ****p < 0.0001. One-way ANOVA with repeated measures. Endpoints from top to bottom: HFD, AAV1-eGFP; control diet; AAV1-OSU; AAV-BDNF-12; AAV-BDNF-1. DETAILED DESCRIPTION

[0041] 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.

[0042] BDNF EXPRESSION CONSTRUCTS

[0043] In one aspect, the present disclosure provides an expression construct for treating metabolic disorders and diseases, including obesity.

[0044] 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" means that a first molecule is linked to a second molecule, wherein the arrangement of the molecules is 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, if a promoter regulates the transcription of a target transcribable polynucleotide molecule in a cell, the promoter is operably linked to the transcribable polynucleotide molecule. Additionally, if two portions of a transcriptional regulatory element are joined together such that the transcriptional activation functionality of one portion is not adversely affected by the presence of the other portion, the two portions are operably linked to each other. The two transcriptional regulatory elements may be operably linked to each other by a linker nucleic acid (e.g., an inserted non-coding nucleic acid), or may be operably linked to each other in the absence of inserted nucleotides.

[0045] BDNF is a key molecular regulator of metabolic function and body weight. Single gene mutations in brain-derived neurotrophic factor BDNF or its receptor NTRK2 result in human obesity. BDNF signaling is also downstream of the leptin-melanocortin pathway, which controls appetite and body weight. The expression constructs and vectors described herein may encode BDNF variants. As used herein, a variant is a protein that contains one or more alterations (including, but not limited to, amino acid additions, substitutions, insertions, deletions, or post-translational modifications) when compared to the parental protein, wherein 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 parental 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 the signaling cascade.

[0046] In some embodiments, the nucleic acid sequence encoding BDNF is the coding sequence from the wild-type BDNF gene. In some embodiments, the nucleic acid sequence encoding BDNF is the coding sequence from the 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 that comprises 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).

[0047] SEQ ID NO:81:

[0048] MTILFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGTLE

[0049] SVNGPKAGSRGLTSLADTFEHVIEELLDEDQKVRPNEENNKDADL

[0050] YTSRVMLSSQVPLEPPLLFLLEEYKNYLDAANMSMRVRRHSDPA

[0051] RRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQL

[0052] KQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTM

[0053] DSKKRIGWRFIRIDTSCVCTLTIKRGR

[0054] 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 with 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] A number of expression control sequences (e.g., native, constitutive, inducible, and / or tissue-specific) are known in the art and can be used to drive the expression of the BDNF transgene, depending on the type of expression desired. For eukaryotic cells, expression control sequences typically include a promoter, an enhancer, and a polyadenylation sequence that may include splice donor and acceptor sites. The polyadenylation sequence is typically inserted downstream of the sequence encoding BDNF and upstream of the 3' ITR sequence.

[0062] Another regulatory component of rAAV that can be used in the methods disclosed herein is an internal ribosome entry site (IRES). The IRES sequence can be used to produce more than one polypeptide from a single gene transcript. The IRES (or other suitable sequence) is used to produce a protein containing more than one polypeptide chain, or to express two different proteins from the same cell or within the same cell. In some embodiments, the IRES is located 3' to the sequence encoding BDNF in the rAAV vector.

[0063] As used herein, the term "promoter" or "regulatory sequence" refers to a nucleic acid sequence required for the expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence can be a core promoter sequence, and in other cases, the sequence can also include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter or regulatory sequence can be, for example, a promoter or regulatory sequence that expresses the 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 essentially only when an inducer corresponding to the promoter is present in the cell. The term "enhancer" as used herein refers to a cis-acting regulatory sequence (e.g., 50 - 1,500 base pairs) that binds one or more proteins (e.g., activator proteins or transcription factors) to increase the transcriptional activation of a nucleic acid sequence. The enhancer can be located upstream of the start site of the gene it regulates or downstream of the start site of the gene by up to 1,000,000 base pairs. The enhancer can be located within an intron region or an exon region of an unrelated gene.

[0064] 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 sequence of SEQ ID NO: 1. In some embodiments, the promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 1.

[0065] In some embodiments, the promoter sequence comprises a constitutive promoter sequence. A "constitutive promoter" refers to a promoter that is capable of promoting continuous transcription of a coding sequence or gene under its control and / or is operably linked thereto.

[0066] 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).

[0067] 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.

[0068] 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 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 a nucleic acid sequence of SEQ ID NO: 62 or 66 - 69. In some embodiments, the expression construct comprises a nucleic acid sequence of SEQ ID NO: 62 or 66 - 69.

[0070] In some embodiments, the expression construct further comprises a microRNA (miR) binding sequence (miRBS or mirBS) located in the 3' UTR. In an embodiment, the miRBS is located between the nucleic acid sequence encoding BDNF and the post-transcriptional regulatory element. In an embodiment, the miRBS comprises a binding sequence for a microRNA selected from miR-142, miR-185, miR-1, miR-122 or a combination thereof. In an embodiment, 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.

[0071] 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 with (i) SEQ ID NO:63, (ii) SEQ ID NO:64 or (iii) SEQ ID NO:63 and 82.

[0072] 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.

[0073] 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%, 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:82. In some embodiments, the microRNA binding sequence comprises SEQ ID NO:63 and 82.

[0074] 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 contains a cloning site (nucleotides 202-209 of SEQ ID NO:64, underlined in Table 7). One of ordinary skill in the art can delete or modify this cloning site (e.g., by substituting one or more nucleotides of nucleotides 202-209 of SEQ ID NO:64 with different nucleotides) without eliminating the functionality of the microRNA binding sequence.

[0075] 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 containing one or more inverted terminal repeats (ITRs). In some embodiments, the ITR sequence is derived from AAV serotype 2 (AAV2).

[0076] 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.

[0077] 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.

[0078] 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 a nucleic acid sequence selected from SEQ ID NOs: 11-50. In some embodiments, the expression construct comprises a nucleic acid sequence selected from SEQ ID NOs: 11-50. 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 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.

[0079] 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.

[0080] 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.

[0081] vector

[0082] In one aspect, there is provided a recombinant vector and its use for introducing a transgene or an expression construct into a cell.

[0083] In some embodiments, the recombinant vector comprises a recombinant DNA construct, the recombinant DNA construct comprising additional DNA elements, the additional DNA elements including DNA fragments that provide for replication of the DNA in a host cell and expression of the target gene at an appropriate level in a target cell. One of ordinary skill in the art is aware of the selection of expression control sequences (promoters, enhancers, etc.) based on the ability to promote expression of the target gene in a target cell.

[0084] As used herein, "vector" refers to a vehicle that contains a polynucleotide to be delivered to a host cell in vitro or in vivo. Non-limiting examples of vectors include recombinant plasmids, yeast artificial chromosomes (YACs), minichromosomes, DNA microcircles or viruses (including virus-derived sequences). A vector may also refer to a viral particle that contains a nucleic acid to be delivered to a host cell in vitro or in vivo. In some embodiments, a vector refers to a viral particle that contains a recombinant viral genome, wherein the viral genome contains one or more ITRs and a transgene.

[0085] In some embodiments, the recombinant vector is a viral vector or a combination of multiple viral vectors.

[0086] In one aspect, there is provided a vector comprising any expression construct disclosed herein.

[0087] In some embodiments, the vector comprises an expression construct that comprises a promoter sequence operably linked to a nucleic acid sequence encoding brain-derived neurotrophic factor (BDNF) or a variant / fragment thereof.

[0088] In some embodiments, the vector comprises a nucleic acid sequence encoding BDNF, wherein the BDNF coding sequence is derived from the wild-type BDNF gene. In some embodiments, the vector comprises a nucleic acid sequence encoding BDNF, wherein the BDNF coding sequence is derived from the 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.

[0089] In some embodiments, the vector comprises a BDNF-encoding 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 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 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.

[0090] In some embodiments, the vector comprises a promoter sequence that 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 nucleic acid 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 sequence of SEQ ID NO:1. In some embodiments, the promoter sequence comprises the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the promoter sequence comprises a constitutive promoter.

[0091] In some embodiments, the vector comprises a nucleic acid containing a post-transcriptional regulatory element. In some embodiments, the vector comprises a nucleic acid containing 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 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.

[0092] In some embodiments, the vector comprises a nucleic acid containing 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 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.

[0093] In some embodiments, the vector comprises a microRNA binding sequence located 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 any one of (i) SEQ ID NO:63, (ii) SEQ ID NO:64, or (iii) SEQ ID NO: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 NO:63 and 82. 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% identity 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 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 the nucleic acid sequence of SEQ ID NO:63, and (ii) 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 the nucleic acid sequence of SEQ ID NO:82. In some embodiments, the microRNA binding sequence comprises SEQ ID NO:63 and 82. 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% identity 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.

[0094] 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 containing one or more inverted terminal repeats (ITRs). In some embodiments, the ITR sequence is 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.

[0095] 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.

[0096] In some embodiments, the vector comprises a nucleic acid that 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 any one of the sequences of SEQ ID NOs: 11, 18-22, 27-30, and 42-45.

[0097] In some embodiments, the vector comprises a nucleic acid sequence selected from SEQ ID NOs: 11-50. In some embodiments, the expression construct comprises a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30, and 42-45.

[0098] 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.

[0099] 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.

[0100] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0101] (a) A promoter sequence comprising the C14_L21 promoter, C26_L21 promoter, C42b_L21 promoter, C46_L21 promoter, B36 promoter, B46 promoter, B46 promoter, B47 promoter or B48 promoter;

[0102] (b) A nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to the promoter sequence;

[0103] (c) A post-transcriptional regulatory element;

[0104] (d) A polyadenylation signal; and / or

[0105] (e) One or more ITRs. In some embodiments, the vector set comprises two ITR sequences.

[0106] In some embodiments, a vector is provided that comprises a nucleic acid comprising one or more of the following:

[0107] (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;

[0108] (b) A nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to the promoter, and wherein 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;

[0109] (c) A post-transcriptional regulatory element, said 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 NO: 58 - 61;

[0110] (d) A polyadenylation signal, said 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 a nucleic acid sequence of SEQ ID NO: 62 or 66 - 69; and / or

[0111] (e) One or more ITRs (e.g., two ITR sequences).

[0112] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0113] (a) A promoter sequence, said 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 of SEQ ID NO: 1;

[0114] (b) A nucleic acid sequence encoding BDNF, wherein said nucleic acid sequence encoding BDNF is operably linked to the promoter, and wherein said 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 of SEQ ID NO: 52;

[0115] (c) A post-transcriptional regulatory element, said 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 of SEQ ID NO: 58;

[0116] (d) A polyadenylation signal, said 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 a nucleic acid sequence of SEQ ID NO: 62;

[0117] (e) A microRNA binding sequence, said 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

[0118] (f) One or more ITRs (e.g., two ITR sequences).

[0119] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0120] (a) A promoter sequence, said 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;

[0121] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0122] (c) A post-transcriptional regulatory element, said 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;

[0123] (d) A polyadenylation signal, said 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;

[0124] (e) A microRNA binding sequence, said 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) the nucleic acid sequences of SEQ ID NO: 63 and 82; and

[0125] (f) One or more ITRs (e.g., two ITR sequences).

[0126] In some embodiments, the vector comprises a nucleic acid that comprises one or more of the following:

[0127] (a) A promoter sequence, said 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;

[0128] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0129] (c) A post-transcriptional regulatory element, said 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;

[0130] (d) A polyadenylation signal, said 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

[0131] (e) One or more ITRs (e.g., two ITR sequences).

[0132] In some embodiments, the vector comprises a nucleic acid that comprises one or more of the following:

[0133] (a) A promoter sequence, said 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;

[0134] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0135] (c) A post-transcriptional regulatory element, said 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;

[0136] (d) A polyadenylation signal, said 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

[0137] (e) One or more ITRs (e.g., two ITR sequences).

[0138] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0139] (a) A promoter sequence, said 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;

[0140] (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;

[0141] (c) A post-transcriptional regulatory element, the 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;

[0142] (d) A polyadenylation signal, the 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

[0143] (e) One or more ITRs (e.g., two ITR sequences).

[0144] In some embodiments, the vector comprises a nucleic acid that comprises one or more of the following:

[0145] (a) A promoter sequence, the 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;

[0146] (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;

[0147] (c) A post-transcriptional regulatory element, said 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;

[0148] (d) A polyadenylation signal, said 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

[0149] (e) One or more ITRs (e.g., two ITR sequences).

[0150] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0151] (a) A promoter sequence, said 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;

[0152] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0153] (c) A post-transcriptional regulatory element, said 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;

[0154] (d) A polyadenylation signal, said 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;

[0155] (e) A microRNA binding sequence, said microRNA binding 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 (i) SEQ ID NO: 64 or (ii) the sequences of SEQ ID NO: 63 and 82; and

[0156] (f) One or more ITRs (e.g., two ITR sequences).

[0157] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0158] (a) A promoter sequence, said 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;

[0159] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0160] (c) A post-transcriptional regulatory element, said 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;

[0161] (d) A polyadenylation signal, said 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;

[0162] (e) A microRNA binding sequence, the microRNA binding 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 (i) SEQ ID NO: 64 or (ii) the sequences of SEQ ID NOs: 63 and 82; and

[0163] (f) One or more ITRs (e.g., two ITR sequences).

[0164] In some embodiments, the vector comprises a nucleic acid, the nucleic acid comprising one or more of the following:

[0165] (a) A promoter sequence, the 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;

[0166] (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 the promoter;

[0167] (c) A post-transcriptional regulatory element, the 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;

[0168] (d) A polyadenylation signal, the 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;

[0169] (e) A microRNA binding sequence, said microRNA binding 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 (i) SEQ ID NO: 64 or (ii) the sequences of SEQ ID NO: 63 and 82; and

[0170] (f) One or more ITRs (e.g., two ITR sequences).

[0171] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0172] (a) A promoter sequence, said 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;

[0173] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0174] (c) A post-transcriptional regulatory element, said 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;

[0175] (d) A polyadenylation signal, said 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;

[0176] (e) A microRNA binding sequence, the microRNA binding 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 (i) SEQ ID NO: 64 or (ii) the sequences of SEQ ID NOs: 63 and 82; and

[0177] (f) One or more ITRs (e.g., two ITR sequences).

[0178] In some embodiments, the vector comprises a nucleic acid, the nucleic acid comprising one or more of the following:

[0179] (a) A promoter sequence, the 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;

[0180] (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 the promoter;

[0181] (c) A post-transcriptional regulatory element, the 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

[0182] (d) A polyadenylation sequence, the 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.

[0183] In some embodiments, the vector comprises a nucleic acid, the nucleic acid comprising one or more of the following:

[0184] (a) A promoter sequence, said 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;

[0185] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0186] (c) A post-transcriptional regulatory element, said 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

[0187] (d) A polyadenylation sequence, said 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.

[0188] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0189] (a) A promoter sequence, said 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;

[0190] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0191] (c) A post-transcriptional regulatory element, said 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

[0192] (d) A polyadenylation sequence, said 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.

[0193] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0194] (a) A promoter sequence, said 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;

[0195] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0196] (c) A post-transcriptional regulatory element, said 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

[0197] (d) A polyadenylation sequence, said 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.

[0198] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0199] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0200] (b) A nucleic acid sequence encoding BDNF, wherein said nucleic acid sequence encoding BDNF is operably linked to the promoter, and wherein said nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO:52;

[0201] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:58;

[0202] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0203] (e) A microRNA binding sequence, said microRNA binding sequence comprising the nucleic acid sequence of SEQ ID NO:63; and

[0204] (f) One or more ITRs (e.g., two ITR sequences).

[0205] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0206] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0207] (b) A nucleic acid sequence encoding BDNF protein, wherein said nucleic acid sequence encoding BDNF protein comprises the nucleic acid sequence of SEQ ID NO:53, and wherein said nucleic acid sequence encoding BDNF protein is operably linked to the promoter;

[0208] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:59;

[0209] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0210] (e) A microRNA binding sequence, said microRNA binding sequence comprising (i) SEQ ID NO:64 or (ii) SEQ ID NO:63 and 82; and

[0211] (f) One or more ITRs (e.g., two ITR sequences).

[0212] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0213] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0214] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO:57, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0215] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:60;

[0216] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62; and

[0217] (e) One or more ITRs (e.g., two ITR sequences).

[0218] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0219] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0220] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO:54, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0221] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:60;

[0222] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62; and

[0223] (e) One or more ITRs (e.g., two ITR sequences).

[0224] In some embodiments, the viral genome comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0225] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0226] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO:53, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0227] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60;

[0228] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and

[0229] (e) One or more ITRs (e.g., two ITR sequences).

[0230] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0231] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 1;

[0232] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 55, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0233] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 60;

[0234] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and

[0235] (e) One or more ITRs (e.g., two ITR sequences).

[0236] In some embodiments, the vector comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0237] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 2;

[0238] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO: 53, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0239] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO: 59;

[0240] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO: 62; and

[0241] (e) One or more ITRs (e.g., two ITR sequences).

[0242] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0243] (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO:3;

[0244] (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 wherein the nucleic acid sequence encoding the BDNF protein is operably linked to the promoter;

[0245] (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:59;

[0246] (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0247] (e) a microRNA binding sequence comprising (i) SEQ ID NO:64 or (ii) SEQ ID NO:63 and 82; and

[0248] (f) one or more ITRs (e.g., two ITR sequences).

[0249] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0250] (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO:4;

[0251] (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 wherein the nucleic acid sequence encoding the BDNF protein is operably linked to the promoter;

[0252] (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:59;

[0253] (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0254] (e) a microRNA binding sequence comprising (i) SEQ ID NO:64 or (ii) SEQ ID NO:63 and 82; and

[0255] (f) one or more ITRs (e.g., two ITR sequences).

[0256] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0257] (a) A promoter sequence comprising the nucleic acid sequence of SEQ ID NO:5;

[0258] (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 wherein the nucleic acid sequence encoding the BDNF protein is operably linked to the promoter;

[0259] (c) A post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:59;

[0260] (d) A polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0261] (e) A microRNA binding sequence comprising (i) SEQ ID NO:64 or (ii) SEQ ID NO:63 and 82; and

[0262] (f) One or more ITRs (e.g., two ITR sequences).

[0263] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0264] (a) A promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0265] (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 wherein the nucleic acid sequence encoding the BDNF protein is operably linked to the promoter;

[0266] (c) A post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:61; and

[0267] (d) A polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:66.

[0268] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0269] (a) A promoter sequence comprising the nucleic acid sequence of SEQ ID NO:9;

[0270] (b) A nucleic acid sequence encoding the BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises 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;

[0271] (e) A post-transcriptional regulatory element, the post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:61; and

[0272] (c) A polyadenylation sequence, the polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:66.

[0273] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0274] (a) A promoter sequence, the promoter sequence comprising the nucleic acid sequence of SEQ ID NO:8;

[0275] (b) A nucleic acid sequence encoding the BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises 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;

[0276] (f) A post-transcriptional regulatory element, the post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:61; and

[0277] (c) A polyadenylation sequence, the polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:66.

[0278] In some embodiments, the vector comprises a nucleic acid comprising one or more of the following:

[0279] (a) A promoter sequence, the promoter sequence comprising the nucleic acid sequence of SEQ ID NO:10;

[0280] (b) A nucleic acid sequence encoding the BDNF protein, wherein the nucleic acid sequence encoding the BDNF protein comprises 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;

[0281] (g) A post-transcriptional regulatory element, the post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:61; and

[0282] (c) A polyadenylation sequence, the polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:66.

[0283] Viral vector

[0284] Viral vectors for expressing a target gene in a target cell, tissue or organism are known in the art and include, for example, AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, poxviral vectors, baculoviral vectors, herpes simplex virus vectors, vaccinia virus vectors or synthetic viral vectors (e.g., chimeric viruses, mosaic viruses or pseudotyped viruses, and / or viruses containing foreign proteins, synthetic polymers, nanoparticles or small molecules).

[0285] AAV vector

[0286] As used herein, the terms "adeno-associated virus (AAV) vector", "AAV gene therapy vector" and "gene therapy vector" refer to vectors having a functional or partially functional ITR sequence and a transgene. As used herein, the term "ITR" refers to an inverted terminal repeat sequence (ITR).

[0287] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires a helper virus to facilitate efficient replication. The 4.7 kb genome of AAV is characterized by two inverted terminal repeat sequences (ITRs) and two open reading frames that encode the Rep protein and the Cap protein, respectively. The Rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD and 40 kD. The functions of these proteins are mainly to regulate AAV replication and to rescue and integrate AAV into the host cell chromosome. 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 viral particle capsid. More than 80% of the total protein in the AAV viral particle consists of VP3. The rep and cap open reading frames at the 5' and 3' ends are flanked by inverted terminal repeat sequences (ITRs) approximately 145 bp in length. The two ITRs are the only cis-elements required for AAV replication, rescue, packaging and integration of the AAV genome. The entire rep and cap domains can be excised and replaced with a therapeutic or reporter gene transgene.

[0288] Recombinant adeno-associated virus "rAAV" vectors include any vector derived from any adeno-associated virus serotype. The rAAV vector may have one or more of the AAV wild-type genes completely or partially deleted, in some embodiments the Rep and / or Cap genes, but retains functional flanking ITR sequences.

[0289] In some embodiments, the viral vector is an rAAV viral particle that contains an rAAV genome and one or more capsid proteins. In some embodiments, the rAAV genome contains the expression construct disclosed herein.

[0290] In some embodiments, the viral vectors disclosed herein comprise a nucleic acid that includes AAV 5’ ITR and 3’ ITR located 5' and 3' respectively of a 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, such as 5' to 3' or head-to-tail or in another alternative configuration. In other embodiments, it may be desirable for the nucleic acid to contain multiple copies of the ITR or to have 5’ ITR (or conversely, 3’ ITR) located both 5' and 3' of the sequence encoding BDNF. The ITR sequences can be located immediately upstream and / or downstream of the heterologous molecule, or intervening sequences can be present. The ITR need not be a wild-type nucleotide sequence and can be altered (e.g., by insertion, deletion, or substitution of nucleotides) so long as the sequence provides for functional rescue, replication, and packaging. The ITR can be selected from AAV2 or from other AAV serotypes as described herein.

[0291] In some embodiments, the viral vector is an AAV vector, such as AAV1 (i.e., an AAV that contains AAV1 ITR and AAV1 capsid proteins), AAV2 (i.e., an AAV that contains AAV2 ITR and AAV2 capsid proteins), AAV3 (i.e., an AAV that contains AAV3 ITR and AAV3 capsid proteins), AAV4 (i.e., an AAV that contains AAV4 ITR and AAV4 capsid proteins), AAV5 (i.e., an AAV that contains AAV5 ITR and AAV5 capsid proteins), AAV6 (i.e., an AAV that contains AAV6 ITR and AAV6 capsid proteins), AAV7 (i.e., an AAV that contains AAV7 ITR and AAV7 capsid proteins), AAV8 (i.e., an AAV that contains AAV8 ITR and AAV8 capsid proteins), AAV9 (i.e., an AAV that contains AAV9 ITR and AAV9 capsid proteins), AAVrh74 (i.e., an AAV that contains AAVrh74 ITR and AAVrh74 capsid proteins), AAVrh.8 (i.e., an AAV that contains AAVrh.8 ITR and AAVrh.8 capsid proteins), or AAVrh.10 (i.e., an AAV that contains AAVrh.10 ITR and AAVrh.10 capsid proteins).

[0292] In some embodiments, the viral vector is a pseudotyped AAV vector that contains 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 that contains AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., an AAV that contains AAV2 ITRs and AAV10 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 1 (i.e., an AAV that contains AAV2 ITRs and AAV1 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 7m8 (i.e., an AAV that contains AAV2 ITRs and AAV7m8 capsid proteins).

[0293] 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 chimeric capsid protein that contains one or more of the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In some embodiments, the vector contains a genome derived from AAV1. In some embodiments, the vector contains a genome derived from AAV2.

[0294] In one aspect, a viral genome is provided, the viral genome comprising a nucleic acid, the nucleic acid comprising (a) a promoter sequence operably linked to a nucleic acid sequence encoding BDNF. In some embodiments, 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 and 70 - 79, or comprises 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 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 sequence of SEQ ID NO:1. In some embodiments, the promoter sequence comprises the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the promoter sequence comprises a constitutive promoter.

[0295] In some embodiments, the viral genome comprises a nucleic acid containing a post-transcriptional regulatory element. In some embodiments, the vector comprises a nucleic acid containing 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.

[0296] In some embodiments, the viral genome comprises a nucleic acid containing 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.

[0297] In some embodiments, the viral genome comprises a microRNA binding sequence 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 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of (i) SEQ ID NO:63, (ii) SEQ ID NO:64, or (iii) SEQ ID NO:63 and 82. 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% identity 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 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 the nucleic acid sequence of SEQ ID NO:63, and (ii) 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 the nucleic acid sequence of SEQ ID NO:82. In some embodiments, the microRNA binding sequence comprises SEQ ID NO:63 and 82. 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% identity 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.

[0298] In some embodiments, the microRNA binding sequence comprises (i) SEQ ID NO:63, (ii) SEQ ID NO:64, or (iii) SEQ ID NO:63 and 82.

[0299] In some embodiments, the viral genome comprises a terminator downstream of the post-transcriptional regulatory element.

[0300] In some embodiments, the viral genome comprises a nucleic acid containing one or more inverted terminal repeats (ITRs). In some embodiments, the ITR sequence is derived from AAV serotype 1 (AAV1). 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.

[0301] 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 NO:11 - 50.

[0302] 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 one of the sequences of SEQ ID NO:11, 18 - 22, 27 - 30, and 42 - 45.

[0303] In some embodiments, the viral genome comprises a nucleic acid sequence selected from SEQ ID NO:11 - 50. In some embodiments, the expression construct comprises a nucleic acid sequence selected from SEQ ID NO:11, 18 - 22, 27 - 30, and 42 - 45.

[0304] 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.

[0305] 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.

[0306] In some embodiments, the viral genome comprises a nucleic acid comprising one or more of the following:

[0307] (a) a promoter sequence comprising the C14_L21 promoter, the C26_L21 promoter, the C42b_L21 promoter, the C46_L21 promoter, the B36 promoter, the B46 promoter, the B46 promoter, the B47 promoter, or the B48 promoter;

[0308] (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to the promoter sequence;

[0309] (c) WPRE;

[0310] (d) a polyadenylation signal; and / or

[0311] (e) one or more ITRs. In some embodiments, the viral genome comprises two ITR sequences.

[0312] In some embodiments, the viral genome comprises a nucleic acid comprising one or more of the following:

[0313] (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;

[0314] (b) a nucleic acid sequence encoding BDNF, wherein the nucleic acid sequence encoding BDNF is operably linked to the promoter, and wherein 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;

[0315] (c) A post-transcriptional regulatory element, said 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 NO: 58 - 61;

[0316] (d) A polyadenylation signal, said 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 a nucleic acid sequence of SEQ ID NO: 62 or 66 - 69; and

[0317] (e) One or more ITRs (e.g., two ITR sequences).

[0318] In some embodiments, the viral genome comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0319] (a) A promoter sequence, said 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 of SEQ ID NO: 1;

[0320] (b) A nucleic acid sequence encoding BDNF, wherein said nucleic acid sequence encoding BDNF is operably linked to the promoter, and wherein said 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 of SEQ ID NO: 52;

[0321] (c) A post-transcriptional regulatory element, said 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 of SEQ ID NO: 58;

[0322] (d) A polyadenylation signal, said 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 a nucleic acid sequence of SEQ ID NO: 62;

[0323] (e) A microRNA binding sequence, said 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

[0324] (f) One or more ITRs (e.g., two ITR sequences).

[0325] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0326] (a) A promoter sequence, said 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;

[0327] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0328] (c) A post-transcriptional regulatory element, said 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;

[0329] (d) A polyadenylation signal, said 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;

[0330] (e) A microRNA binding sequence, the 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) the nucleic acid sequences of SEQ ID NO: 63 and 82; and

[0331] (f) One or more ITRs (e.g., two ITR sequences).

[0332] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0333] (a) A promoter sequence, the 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;

[0334] (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 the promoter;

[0335] (c) A post-transcriptional regulatory element, the 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;

[0336] (d) A polyadenylation signal, the 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

[0337] (e) One or more ITRs (e.g., two ITR sequences).

[0338] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0339] (a) A promoter sequence, said 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;

[0340] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to a promoter;

[0341] (c) A post-transcriptional regulatory element, said 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;

[0342] (d) A polyadenylation signal, said 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

[0343] (e) One or more ITRs (e.g., two ITR sequences).

[0344] In some embodiments, the viral genome comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0345] (a) A promoter sequence, said 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;

[0346] (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;

[0347] (c) A post-transcriptional regulatory element, the 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;

[0348] (d) A polyadenylation signal, the 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).

[0349] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0350] (a) A promoter sequence, the 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;

[0351] (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;

[0352] (c) A post-transcriptional regulatory element, the 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;

[0353] (d) A polyadenylation signal, the 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

[0354] (e) One or more ITRs (e.g., two ITR sequences).

[0355] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0356] (a) A promoter sequence, the 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;

[0357] (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 the promoter;

[0358] (c) A post-transcriptional regulatory element, the 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;

[0359] (d) A polyadenylation signal, the 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;

[0360] (e) A microRNA binding sequence, said microRNA binding 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 (i) SEQ ID NO: 64 or (ii) the sequences of SEQ ID NO: 63 and 82; and

[0361] (f) One or more ITRs (e.g., two ITR sequences).

[0362] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0363] (a) A promoter sequence, said 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;

[0364] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to a promoter;

[0365] (c) A post-transcriptional regulatory element, said 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;

[0366] (d) A polyadenylation signal, said 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;

[0367] (e) A microRNA sequence, the microRNA 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 (i) SEQ ID NO: 64 or (ii) SEQ ID NO: 63 and 82; and

[0368] (f) One or more ITRs (e.g., two ITR sequences).

[0369] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0370] (a) A promoter sequence, the 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;

[0371] (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 the promoter;

[0372] (c) A post-transcriptional regulatory element, the 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;

[0373] (d) A polyadenylation signal, the 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;

[0374] (e) A microRNA sequence, wherein the microRNA 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 (i) SEQ ID NO: 64 or (ii) SEQ ID NO: 63 and 82; and

[0375] (f) One or more ITRs (e.g., two ITR sequences).

[0376] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0377] (a) A promoter sequence, wherein 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 sequence of SEQ ID NO: 5;

[0378] (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 the promoter;

[0379] (c) A post-transcriptional regulatory element, wherein 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: 59;

[0380] (d) A polyadenylation signal, wherein the polyadenylation signal 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: 62;

[0381] (e) A microRNA binding sequence, said microRNA binding 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 (i) SEQ ID NO: 64 or (ii) the sequences of SEQ ID NO: 63 and 82; and

[0382] (f) One or more ITRs (e.g., two ITR sequences).

[0383] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0384] (a) A promoter sequence, said 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;

[0385] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0386] (e) A post-transcriptional regulatory element, said 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

[0387] (c) A polyadenylation sequence, said 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.

[0388] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0389] (a) A promoter sequence, wherein 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 sequence of SEQ ID NO:9;

[0390] (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 the promoter;

[0391] (e) A post-transcriptional regulatory element, wherein 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:61; and

[0392] (c) A polyadenylation sequence, wherein the polyadenylation 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 sequence of SEQ ID NO:66.

[0393] In some embodiments, the viral genome comprises a nucleic acid that comprises one or more of the following:

[0394] (a) A promoter sequence, wherein 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 sequence of SEQ ID NO:8;

[0395] (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 the promoter;

[0396] (c) A post-transcriptional regulatory element, said 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

[0397] (d) A polyadenylation sequence, said 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.

[0398] In some embodiments, the viral genome comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0399] (a) A promoter sequence, said 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;

[0400] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a 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 said nucleic acid sequence encoding a BDNF protein is operably linked to a promoter;

[0401] (e) A post-transcriptional regulatory element, said 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

[0402] (c) A polyadenylation sequence, said 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.

[0403] In some embodiments, the viral genome comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0404] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0405] (b) A nucleic acid sequence encoding BDNF, wherein said nucleic acid sequence encoding BDNF is operably linked to the promoter, and wherein said nucleic acid sequence encoding BDNF comprises the nucleic acid sequence of SEQ ID NO:52;

[0406] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:58;

[0407] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0408] (e) A microRNA binding sequence, said microRNA binding sequence comprising the nucleic acid sequence of SEQ ID NO:63; and

[0409] (f) One or more ITRs (e.g., two ITR sequences).

[0410] In some embodiments, the viral genome comprises nucleic acid, said nucleic acid comprising one or more of the following:

[0411] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0412] (b) A nucleic acid sequence encoding BDNF protein, wherein said nucleic acid sequence encoding BDNF protein comprises the nucleic acid sequence of SEQ ID NO:53, and wherein said nucleic acid sequence encoding BDNF protein is operably linked to the promoter;

[0413] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:59;

[0414] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0415] (e) A microRNA binding sequence, said microRNA binding sequence comprising (i) SEQ ID NO:64 or (ii) SEQ ID NO:63 and 82; and

[0416] (f) One or more ITRs (e.g., two ITR sequences).

[0417] In some embodiments, the viral genome comprises nucleic acid, said nucleic acid comprising one or more of the following:

[0418] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0419] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO:57, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0420] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:60;

[0421] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62; and

[0422] (e) One or more ITRs (e.g., two ITR sequences).

[0423] In some embodiments, the viral genome comprises a nucleic acid comprising one or more of the following:

[0424] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0425] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO:54, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0426] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:60;

[0427] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62; and

[0428] (e) One or more ITRs (e.g., two ITR sequences).

[0429] In some embodiments, the viral genome comprises a nucleic acid comprising one or more of the following:

[0430] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0431] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO:53, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0432] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:60;

[0433] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62; and

[0434] (e) One or more ITRs (e.g., two ITR sequences).

[0435] In some embodiments, the viral genome comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0436] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0437] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO:55, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0438] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:60;

[0439] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62; and

[0440] (e) One or more ITRs (e.g., two ITR sequences).

[0441] In some embodiments, the viral genome comprises a nucleic acid, said nucleic acid comprising one or more of the following:

[0442] (a) A promoter sequence, said promoter sequence comprising the nucleic acid sequence of SEQ ID NO:2;

[0443] (b) A nucleic acid sequence encoding a BDNF protein, wherein said nucleic acid sequence encoding a BDNF protein comprises the nucleic acid sequence of SEQ ID NO:53, and wherein said nucleic acid sequence encoding a BDNF protein is operably linked to the promoter;

[0444] (c) A post-transcriptional regulatory element, said post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:59;

[0445] (d) A polyadenylation signal, said polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62; and

[0446] (e) One or more ITRs (e.g., two ITR sequences).

[0447] In some embodiments, the viral genome comprises a nucleic acid comprising one or more of the following:

[0448] (a) A promoter sequence comprising the nucleic acid sequence of SEQ ID NO:3;

[0449] (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 wherein the nucleic acid sequence encoding the BDNF protein is operably linked to the promoter;

[0450] (c) A post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:59;

[0451] (d) A polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0452] (e) A microRNA binding sequence comprising (i) SEQ ID NO:64 or (ii) SEQ ID NO:63 and 82; and

[0453] (f) One or more ITRs (e.g., two ITR sequences).

[0454] In some embodiments, the viral genome comprises a nucleic acid comprising one or more of the following:

[0455] (a) A promoter sequence comprising the nucleic acid sequence of SEQ ID NO:4;

[0456] (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 wherein the nucleic acid sequence encoding the BDNF protein is operably linked to the promoter;

[0457] (c) A post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:59;

[0458] (d) A polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0459] (e) A microRNA binding sequence comprising (i) SEQ ID NO:64 or (ii) SEQ ID NO:63 and 82; and

[0460] (f) One or more ITRs (e.g., two ITR sequences).

[0461] In some embodiments, the viral genome comprises a nucleic acid comprising one or more of the following:

[0462] (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO:5;

[0463] (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 wherein the nucleic acid sequence encoding the BDNF protein is operably linked to the promoter;

[0464] (c) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:59;

[0465] (d) a polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO:62;

[0466] (e) a microRNA binding sequence comprising (i) SEQ ID NO:64 or (ii) SEQ ID NO:63 and 82; and

[0467] (f) one or more ITRs (e.g., two ITR sequences).

[0468] In some embodiments, the viral genome comprises a nucleic acid comprising one or more of the following:

[0469] (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO:1;

[0470] (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 wherein the nucleic acid sequence encoding the BDNF protein is operably linked to the promoter;

[0471] (h) a post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:61; and

[0472] (c) a polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:66.

[0473] In some embodiments, the viral genome comprises a nucleic acid comprising one or more of the following:

[0474] (a) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO:9;

[0475] (b) A nucleic acid sequence encoding BDNF protein, wherein the nucleic acid sequence encoding BDNF protein comprises the nucleic acid sequence of SEQ ID NO:56, and wherein the nucleic acid sequence encoding BDNF protein is operably linked to a promoter;

[0476] (i) A post-transcriptional regulatory element, the post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:61; and

[0477] (c) A polyadenylation sequence, the polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:66.

[0478] In some embodiments, the viral genome comprises a nucleic acid, the nucleic acid comprising one or more of the following:

[0479] (a) A promoter sequence, the promoter sequence comprising the nucleic acid sequence of SEQ ID NO:8;

[0480] (b) A nucleic acid sequence encoding BDNF protein, wherein the nucleic acid sequence encoding BDNF protein comprises the nucleic acid sequence of SEQ ID NO:56, and wherein the nucleic acid sequence encoding BDNF protein is operably linked to a promoter;

[0481] (j) A post-transcriptional regulatory element, the post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:61; and

[0482] (c) A polyadenylation sequence, the polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:66.

[0483] In some embodiments, the viral genome comprises a nucleic acid, the nucleic acid comprising one or more of the following:

[0484] (a) A promoter sequence, the promoter sequence comprising the nucleic acid sequence of SEQ ID NO:10;

[0485] (b) A nucleic acid sequence encoding BDNF protein, wherein the nucleic acid sequence encoding BDNF protein comprises the nucleic acid sequence of SEQ ID NO:56, and wherein the nucleic acid sequence encoding BDNF protein is operably linked to a promoter;

[0486] (k) A post-transcriptional regulatory element, the post-transcriptional regulatory element comprising the nucleic acid sequence of SEQ ID NO:61; and

[0487] (c) A polyadenylation sequence, the polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:66.

[0488] Other viral vectors

[0489] 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 deletions in the E1 and E3 regions. A transcription cassette can be inserted into the E1 region, thereby generating a recombinant E1 / E3 deletion AV vector. Adenovirus vectors also include helper-dependent high-capacity adenovirus vectors (also referred to as high-capacity "gutless" or "gutted" vectors), which do not contain viral coding sequences. These vectors contain cis-acting elements required for viral DNA replication and packaging, mainly the inverted terminal repeats (ITRs) and the packaging signal (CY). These helper-dependent AV vector genomes have the potential to carry foreign DNA ranging from a few 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 such as those in the CNS. Lentiviral vectors are derived from the human immunodeficiency virus and, like the virus, integrate into the host genome, thereby providing the potential for very long-term gene expression. Cationic lipids, polymers, or both can also be used as carriers to introduce polynucleotides including plasmids, YACs, minichromosomes, and microcircles carrying a target gene containing an expression construct into cells or organisms by non-viral vector systems. Conjugated poly-L-lysine (PLL) polymers and polyethyleneimine (PEI) polymer systems can also be used to deliver vectors to cells. Other methods for delivering vectors to cells for cell cultures and organisms include hydrodynamic injection and electroporation, as well as the use of ultrasound. For a review of viral and non-viral delivery systems for gene delivery, see Nayerossadat, N. et al. (Adv Biomed Res. 2012; 1:27), which is incorporated herein by reference.

[0490] rAAV virion production

[0491] The rAAV viral particles disclosed herein can be constructed and produced using the materials and methods described herein and materials and methods known to those of skill in the art. Such engineering methods for constructing any embodiment of the present disclosure are known to those skilled in nucleic acid manipulation techniques 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. In addition, methods suitable for generating rAAV constructs in an adenovirus capsid have been described in U.S. Patent Nos. 5,856,152 and 5,871,982. Briefly, to package the rAAV genome into rAAV viral particles, a host cell is used that contains the sequences necessary to express AAV rep and AAV cap or functional fragments thereof, as well as the helper genes required for AAV production. The AAV rep and cap sequences are obtained from AAV sources as identified herein. The AAV rep and cap sequences can be introduced into the host cell in any manner known to those of skill in the art, including but not limited to transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated microprojectiles, viral infection, and protoplast fusion. In some embodiments, the rep and cap sequences can be transfected into the host cell by one or more nucleic acid molecules and stably exist in the cell as episomes. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cell. Another embodiment has rep and cap sequences that are transiently expressed in the host cell. For example, a useful nucleic acid molecule for such transfection contains, from 5' to 3', a promoter, an optional spacer region inserted between the promoter and the start site of the rep gene sequence, the AAV rep gene sequence, and the AAV cap gene sequence. As used herein, the term "from 5' to 3'" refers only to the order of specific genetic elements in a nucleic acid sequence. In some embodiments, the specific genetic elements are linked to each other by a linker nucleic acid (e.g., an inserted non-coding nucleic acid). In some embodiments, the specific genetic elements are linked to each other in the absence of inserted nucleotides. In some embodiments, some of the specific genetic elements are linked to each other by a linker nucleic acid, while other genetic elements are linked to each other in the absence of inserted nucleotides.

[0492] The rep and cap sequences, along with their expression control sequences, can be provided on a single vector, or each sequence can 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 can be provided on a vector containing other DNA sequences to be introduced into the host cell. In some embodiments, the promoter used in such constructs can be any suitable constitutive, inducible, or native promoter known to those skilled in the art. The molecule providing the rep and cap proteins can be in any form that transfers these components into the host cell. Desirably, such a molecule is in the form of a plasmid, which can contain other non-viral sequences, such as the sequences of marker genes. Such a molecule does not contain AAV ITR and generally does not contain the AAV packaging sequence. To avoid the occurrence of homologous recombination, other viral sequences, especially adenovirus sequences, are avoided in such plasmids. The plasmid is desirably constructed so that it can be stably transfected into cells.

[0493] Although the molecule providing the rep and cap can be transiently transfected into the host cell, it is preferred to stably transform the host cell with the sequences necessary for expressing functional rep / cap proteins in the host cell, for example, as an episome or by integration into the chromosome of the host cell. Depending on the promoter controlling the expression of the host cell for such stable transfection, the rep / cap proteins can be transiently expressed (e.g., by using an inducible promoter).

[0494] The methods for constructing the embodiments of the present disclosure are conventional genetic engineering or recombinant engineering techniques, such as those described in the above references. For example, according to the manufacturer's instructions, the calcium phosphate method (Clontech) or Effectene TM reagent (Qiagen, Valencia, Calif.) can be used to generate rAAV using a triple transfection method. See also Herzog et al., 1999, Nature Medic., 5(1):56 - 63. For the methods used in the following examples, a plasmid with a transgene (a helper plasmid containing rep and cap), and a plasmid providing the adenovirus helper functions of E2A, E4 Orf6, and VA are employed. Although the present disclosure provides illustrative examples of specific constructs, using the information provided herein, those skilled in the art can select and design other suitable constructs using the selection of spacers, promoters, and other elements (including at least one translation start point and termination signal, and optionally adding a polyadenylation site).

[0495] Then, rAAV viral particles are produced by culturing host cells containing an rAAV virus as described herein, said host cells containing an rAAV genome to be packaged into the rAAV viral particles, an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing their expression. Suitable viral helper genes, such as adenovirus E2A, E4Orf6, and VA, and other possible helper genes, can be provided to the culture in a variety of ways known in the art. In some embodiments, suitable viral helper genes are provided on separate plasmids. Thereafter, recombinant AAV viral particles directing the expression of the BDNF transgene are isolated from the cells or cell culture in the absence of contaminating helper virus or wild-type AAV.

[0496] Expression of the BDNF transgene can be measured in a manner known in the art. For example, target cells can be infected in vitro, and the copy number of the transgene in the cells can be monitored by Southern blotting or quantitative polymerase chain reaction (PCR). RNA expression levels can be monitored by Northern blotting or quantitative reverse transcriptase (RT)-PCR; and protein expression levels can be monitored by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or by the specific methods detailed in the examples below.

[0497] Pharmaceutical Compositions

[0498] Provided herein are pharmaceutical compositions comprising any of the vectors or viral particles disclosed herein and a pharmaceutically acceptable carrier.

[0499] In some embodiments, contamination by rAAV containing a gene encoding BDNF is evaluated by conventional methods and then formulated into a pharmaceutical composition suitable for storage and / or administration to a patient.

[0500] 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, such as HEPES, to maintain the pH at an appropriate physiological level.

[0501] The carriers of the present disclosure can be formulated into pharmaceutical compositions. In addition to the carriers, these compositions may also contain pharmaceutically and / or physiologically acceptable excipients, carriers, buffers, stabilizers, antioxidants, preservatives, or other additives well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials can be determined by those skilled in the art according to the route of administration. Pharmaceutical compositions are generally in liquid form. Liquid pharmaceutical compositions usually contain a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oils, or synthetic oils. Additional carriers are provided in International Patent Publication No. WO 00 / 15822, which is incorporated herein by reference. It may include physiological saline solution, magnesium chloride, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. In some cases, surfactants such as Pluronic acid (PF68) at 0.001% may be used. In some cases, Ringer's injection solution, lactated Ringer's injection solution, or Hartmann's solution is used. As needed, preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included.

[0502] Pharmaceutical compositions can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Injectable preparations can be presented in unit dosage forms, for example, in ampoules or in multi-dose containers, and are added with preservatives. The agent can be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient can be in the form of a powder that is reconstituted with a suitable vehicle (e.g., sterile pyrogen-free water) before use. The agent can also be formulated as a rectal composition, such as (for example) a suppository containing a conventional suppository base (such as cocoa butter or other glycerides) or a retention enema.

[0503] "Parenteral" administration of the composition includes, for example, subcutaneous (s.c.) injection, intravenous (i.v.) injection, intramuscular (i.m.) injection, or intrasternal injection, or infusion techniques.

[0504] For delayed release, the carrier can be included in a pharmaceutical composition formulated for slow release, such as in microcapsules formed from biocompatible polymers or in a liposome carrier system according to methods known in the art.

[0505] If the carrier is to be stored for a long time, it can be frozen in the presence of glycerol.

[0506] Method of Use

[0507] A method for treating or preventing a metabolic disorder in a subject in need thereof is provided herein. In some embodiments, the method includes administering to the subject a therapeutically effective amount of the carrier or pharmaceutical composition as described herein. In some embodiments, the metabolic disorder is obesity.

[0508] Methods for reducing or eliminating symptoms of a metabolism-related disorder in a subject in need thereof are also within the scope of the present disclosure. In some embodiments, the method comprises administering to the subject a carrier or pharmaceutical composition as described herein. In certain embodiments, the symptoms of the metabolism-related disorder are one or more of obesity, insulin sensitivity, syndrome X, and diabetes.

[0509] "Metabolic disorder" refers to a disorder, disease, and affliction caused or characterized by abnormal weight gain, energy use or expenditure, altered response 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 deficiencies or excesses in metabolic pathways that result in metabolic imbalances of carbohydrates, lipids, proteins, and / or nucleic acids. Examples of metabolic disorders include, but are not limited to, metabolic syndrome, insulin deficiency or insulin resistance-related disorders, diabetes (such as, for example, type 2 diabetes), glucose intolerance, dyslipidemia, atherosclerosis, hypertension, preeclampsia, cardiomyopathy, stroke, non-alcoholic fatty liver disease (NAFLD), hyperglycemia, hepatic steatosis of different etiologies, dyslipidemia, dysfunction of the immune system associated with overweight and obesity, cardiovascular disease, hypercholesterolemia, elevated triglycerides, asthma, sleep apnea, osteoarthritis, neurodegeneration, gallbladder disease, syndrome X, inflammatory disorders and immune disorders, atherogenic dyslipidemia, and cancer.

[0510] "Obesity" refers to a subject affliction in which the subject has a body mass index (BMI) greater than or equal to 30. BMI is typically calculated by dividing the body weight of a human subject in kilograms (or pounds) by the square of the height in meters (or feet).

[0511] 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 farm animals. Mammals include, but are not limited to, human or non-human mammals such as cows, horses, dogs, sheep, or cats, etc. An individual and a patient are also subjects herein.

[0512] As used herein, the terms "treat", "treated", "treating", or "treatment" refer to a therapeutic treatment in which the object is to slow down (reduce) an undesired physiological disorder, condition, or disease, or to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to: alleviation of symptoms; reduction in the extent of a disorder, condition, or disease; stabilization (i.e., no worsening) of the state of a disorder, condition, or disease; delay in the onset of a disorder, condition, or disease or slowing of the progression of a disorder, condition, or disease; amelioration of one or more symptoms of the state of a disorder, condition, or disease; and remission (whether partial or total) or enhancement or improvement of a disorder, condition, or disease. Treatment includes eliciting a clinically significant response without an excessive level of side effects. Treatment also includes an extension of survival as compared to the expected survival in the absence of treatment. The terms "prevent", "prevention", etc. refer to acting before the onset of an overt disease or condition to prevent the development of the disease or condition or to minimize the extent of the disease or condition, or to slow its progression.

[0513] In another aspect, the present disclosure further provides a method of increasing the level or activity of BDNF in a subject in need thereof. The method comprises administering to the subject a vector or pharmaceutical composition as described herein.

[0514] The level or activity of BDNF can be measured by assaying or estimating the protein level or mRNA level. Methods for assaying or estimating the protein level or mRNA level are well known in the art. For example, the protein level (e.g., protein expression level) of BDNF can be assayed by SDS-PAGE, Western blotting, or immunoassay (e.g., immunoblot assay, immunoprecipitation assay). The mRNA level can be assayed by RT-PCR.

[0515] The level of BDNF can be measured by assaying the protein itself (by Western blotting, ELISA, RIA, and other techniques known to those skilled in the art), by assaying the mRNA encoding these proteins (such as quantitative PCR, Northern blotting, ribonuclease protection assay, RNA dot blotting, and other techniques known to those skilled in the art), or by assaying the activity of the regulatory elements of the gene of BDNF. For example, the activity of the regulatory elements can be evaluated by a reporter gene construct comprising a DNA fragment from a promoter, enhancer, and / or intron element coupled to the cDNA encoding a reporter gene (such as luciferase, β-galactosidase, green fluorescent protein, or other easily assayable reporter genes). These reporter gene constructs can be stably or transiently transfected into cells.

[0516] Routes and methods of administration

[0517] In some embodiments, the vectors or pharmaceutical compositions disclosed herein are administered to a subject by intratumoral, intravenous, subcutaneous, intraosseous, oral, transdermal, intracerebral, intraparenchymal, sustained release, controlled release, delayed release, as a suppository or sublingually.

[0518] In some embodiments, the vector or pharmaceutical composition is administered by stereotactic microinjection.

[0519] The dose of the disclosed vectors can be determined according to various parameters, particularly according to the age, weight and condition of the patient to be treated, the specific disorder, and the degree of progression of the disease (if progressive), the route of administration, and the desired regimen. A physician will be able to determine the route of administration and dose required for any particular patient.

[0520] An effective amount of rAAV carrying a nucleic acid sequence encoding BDNF under the control of a promoter sequence is desirably between about 1×10 9 and about 2×10 12 (e.g., about 5x10 9 and about 2x10 11 ) rAAV genome particles or between about 1×10 10 and about 2×10 11 genome particles. Genome particles are defined herein as AAV capsids containing single-stranded DNA molecules, which can be quantified by sequence-specific methods such as real-time PCR. In some embodiments, a volume between about 150 and about 800 μl (e.g., about 100 to about 500 μl, such as about 100 to about 200 μl) of about 1×10 9 and about 2×10 12 (e.g., about 5x10 9 and about 2x10 11 ) rAAV genome particles can be provided. Other doses within these ranges can be selected by the attending physician.

[0521] In some embodiments, the dose can be provided as one or more doses for bilateral administration into the hypothalamus. In some embodiments, multiple "booster" doses of the pharmaceutical compositions disclosed herein may be required. For example, depending on the duration of the transgene in the target cells, booster doses can be delivered at 6-month intervals or annually after the first administration. Other similar tests can be used to determine the status of the treated subject over time. The attending physician can select the appropriate tests.

[0522] Articles and kits

[0523] Also provided are cartridges or articles for use in the methods described herein. In some aspects, the cartridge contains the compositions described herein (e.g., compositions for delivering a BDNF-encoding transgene) in a suitable package. Suitable packages for the compositions described herein are known in the art and include, for example, vials (such as sealed vials), vessels, ampoules, bottles, jars, flexible packages (such as sealed Mylar or plastic bags), etc. These articles can be further sterilized and / or sealed.

[0524] Also provided are cartridges containing the compositions described herein. These cartridges can further contain instructions regarding methods of using the compositions (such as the uses described herein). The cartridges described herein can also include other materials that are desirable from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing the administration of the composition or performing any method as described herein. For example, in some embodiments, the cartridge contains rAAV for expressing a BDNF-encoding transgene in target cells, a pharmaceutically acceptable carrier suitable for injection, and one or more of the following: buffers, diluents, filters, needles, syringes, and a package insert with instructions for performing the injection.

[0525] Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein are performed in any suitable order. With respect to any method provided, the steps of the method can occur simultaneously or sequentially. When the steps of the method occur sequentially, unless otherwise indicated, the steps can occur in any order.

[0526] In cases where the method includes combinations of steps, each combination or sub-combination of steps is covered by the scope of the present disclosure unless otherwise stated herein.

[0527] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure. The publications disclosed herein are provided only for their disclosure prior to the filing date of the present disclosure. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Additionally, the provided publication dates may differ from the actual publication dates that may need to be independently confirmed.

[0528] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or variations according to the present invention will occur to those skilled in the art, and these are all included within the spirit and scope of the present application and the scope of the appended claims.

[0529] Examples

[0530] Example 1

[0531] Cell culture

[0532] The Neuro2A (N2A), BE2-M17, and SH-SY5Y immortalized cell lines are maintained at 37 °C in 5% CO2. Cells are transiently transfected with plasmids using TurboFect transfection reagent. Two days later, the culture medium and cell lysates are collected for protein analysis. After 30 passages, a fresh cell aliquot is thawed and passaged twice before being used for subsequent experiments.

[0533] Primary neuron culture

[0534] Pregnant female mice at embryonic day 16 (E16) are anesthetized with CO2 and sacrificed by cervical dislocation. Under a dissection hood, 24 - 26 embryos are collected per experiment through an incision in the maternal abdomen, removed from the amniotic sac, and decapitated in ice-cold Hank's balanced salt solution (HBSS). Using fine scissors and forceps, the brain is dissected rapidly, and the cortex is cleared of meninges and separated under a dissecting microscope. The cortex is collected in ice-cold HBSS and kept on ice until all embryos are dissected. In a tissue culture hood, the HBSS is removed, and the cortical tissue is 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 is dissociated by continuous trituration with a 25-ml serological pipette, followed by trituration with 10- and 5-ml serological pipettes. The cell suspension is washed once with DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin and passed through a 40-μm cell strainer, then counted on a hemocytometer. The single cells are seeded at a density of 400,000 cells per well in poly-d-lysine (0.1 mg ml -1 )-coated wells of a 24-well plate. The cells are grown in Neurobasal medium supplemented with B-27 TM supplement, N-2 supplement, and 0.5 mM L-glutamine and maintained at 37 °C in 5% CO2. Half of the medium is changed every 3 - 4 days to replenish the culture.

[0535] Primary neuron transduction

[0536] On day 1 in vitro, AAV2 / 1 particles are added to the neuronal medium at multiplicities of infection (MOI) of 10,000 and 50,000. The medium is changed 72 hours later according to the standard neuronal protocol.

[0537] BDNF activity assay

[0538] The medium from transfected N2A cells was collected after 2 days and spun at 500 rpg to remove any cell debris. The BDNF levels in this cleared medium were evaluated by ELISA. Medium containing a standardized amount of BDNF was added to the primary cultures and incubated for 15 - 30 minutes, after which the lysates were collected and prepared for Western blotting.

[0539] Western blot

[0540] The cells were gently washed with PBS and then ice-cold lysis buffer (CST) containing DNase and protease / phosphatase inhibitors was added. The cells were lysed on the plate with agitation on ice for 15 minutes and then collected. The 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. The proteins were then normalized to a standard concentration using 4x LDS, 10x DTT, and lysis buffer. The samples were denatured at 85 °C for 10 minutes.

[0541] 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 total ERK1 / 2 and phospho-ERK1 / 2 analysis, 10 μg of protein samples were separated on a 4% - 12% Bolt Bis-tris precast denaturing gel (Invitrogen, USA) using MOPS buffer. The gels were then transferred to nitrocellulose membranes. The membranes were probed overnight at 4 °C with primary antibodies (total ERK1 / 2, phospho-ERK1 / 2, BDNF, Gapdh, β-actin) diluted in Licor TBS blocking buffer. The membranes were then washed and probed for one hour at room temperature with fluorescently conjugated anti-mouse or anti-rabbit IgG secondary antibodies. The membranes were imaged on the LICOR Odyssey system. Protein amounts were normalized using β-actin or Gapdh.

[0542] ELISA

[0543] Tissue samples were dissolved in SDS-free CSTRIPA buffer containing DNase and protease / phosphatase inhibitor mixture. The samples were sonicated twice and then spun to remove cell debris. The samples were then used for pro-BDNF and mature BDNF ELISA (Biosensis) according to the manufacturer's protocol.

[0544] Animal

[0545] Male C57BL / 6 mice at 7 weeks of age were purchased from The Charles River Laboratories. Mice were housed five animals per cage at a constant temperature (23 °C) on a 12-hour light / dark cycle (light from 0700 to 1900 h) with free access to food and water. All studies were reviewed by the Institutional Animal Care and Use Committee (IACUC). Animals were randomly grouped according to body weight before surgery. At the end of the study, the hypothalamus was rapidly dissected and snap-frozen in liquid nitrogen.

[0546] Stereotaxic surgery

[0547] To overexpress BDNF in the adult hypothalamus, adeno-associated virus (0.5 μl, 2e9 GC per hemisphere, serotype 1) was bilaterally injected into 8-week-old male C57Bl / 6 mice. Animals were anesthetized with isoflurane and fixed to a Kopf stereotaxic frame using ear bars and incisor bars. The skull was exposed through a midline incision in the scalp, and two small holes were drilled in the skull with a dental drill above the injection site (1.2 mm posterior to bregma, 0.5 mm lateral to the midline, 5.85 mm dorsal to bregma). The rAAV vector was bilaterally injected (2e9 genome particles per site) into the hypothalamus at a rate of 0.1 ml / min using a 5-μl Hamilton Neuro syringe connected to a Micro4 microinjection pump controller (World Precision Instruments). At the end of the infusion, the syringe was slowly withdrawn from the brain, and Vetbond was applied to close the scalp. The mice were placed in clean cages and carefully monitored until they recovered from anesthesia. Mice were fed a normal chow diet (NCD, 11% fat, 28% protein, 61% carbohydrate, caloric density 3.4 kcal / g, study diet; also referred to as the "control diet" in Figure 4D ). On day 7 after rAAV injection, mice in the appropriate groups were placed on a high-fat diet (HFD, 45% fat, caloric density 4.73 kcal g−1, study diet) for the duration of the study (21 days after injection).

[0548] Example 2

[0549] Several BDNF gene therapy vectors for the treatment of obesity have been developed. For an overview of the expression constructs, see Table 1-8. Different expression constructs were designed by varying various cis-regulatory components. As shown herein, in direct comparisons in primary mouse cortical neurons as well as in various immortalized neuronal cell lines, the BDNF expression constructs achieved significantly higher expression compared to previously disclosed constructs. In addition, delivery of the AAV-mediated BDNF expression vector to the hypothalamus of mice was more effective than the disclosed CAG-BDNF construct in preventing weight gain induced by a high-fat diet. By designing gene therapy vectors that allow for high-level BDNF expression, therapeutic efficacy at lower viral vector doses and potentially lower immune responses and reduced safety risks can be achieved. Other advantages of the disclosed gene therapy vectors include: a single administration provides long-term treatment without the need to comply with an injection regimen; it limits peripheral side effects as it is administered locally to the brain; and it directly targets the mechanism area. Collectively, these results demonstrate that the disclosed expression constructs provide a means for a potent and effective gene therapy for treating rare obesity populations as well as more common forms of obesity caused by genetic or environmental factors.

[0550] BDNF expression from the constitutive promoter is higher than from the tissue-specific or native CAG promoter

[0551] Promoters are an integral part of gene therapy that affect the level, timing, persistence, and cell type specificity of transgene expression. Therefore, a library of tissue-specific and constitutive promoters that are stronger than commonly used reference promoters (NSE, Syn, CAmkIIa) in mouse N2A cells was developed. These promoters are equal to or greater than CMV, and some are even stronger than CAG. A subset of each class of these promoters was selected and applied to BDNF to increase 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 tested promoters (Figure 1). This promoter was then carried forward into subsequent experiments and multiple rounds of expression construct optimization.

[0552] The BDNF expression constructs disclosed herein are more efficient in vitro than the reference constructs

[0553] Four different codon optimization algorithms were applied to the BDNF transgene. Relative to the reference construct CAG-BDNF HA-WPRE-bGH (also referred to herein as the OSU construct), which evaluated how codon optimization affects BDNF expression, has been described in Cao et al., Molecular therapy of obesity and diabetes by a physiological autoregulatory approach, Nat Med. April 2009; 15(4):447-54 and U.S. Patent No. 9,265,843. The codon optimization method includes the following combinations: (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 important genes expressed in the putamen or other subregions of the brain, (2) removing CpG motifs, and (3) removing unwanted motifs (e.g., cryptic splicing sites, TLR9 activation motifs) and (4) introducing required motifs (e.g., TLR9 inhibitory motifs) where possible. Although some species-dependent effects were observed (N2A data vs. SH-SY5Y), increased protein expression was consistently observed in the case of codon optimization #1 and #3. See Table 5 for the sequence. Thus, the codon-optimized genes BDNFco and BDNFco3 were used for further development ( Figure 2A ).

[0554] The expression constructs BDNF-1 (containing BDNFco) and BDNF-12 (containing BDNFco3) showed significantly higher expression than the reference construct in both N2A cells ( Figure 2B ) and human neural progenitor cells (NPCs) ( Figure 2C ). The results showed that the codon-optimized sequences had the same signaling activity (ERK1 / 2 phosphorylation) and thus had the same functionality as the wild-type BDNF sequence in the reference construct ( Figure 2D ).

[0555] Next, BDNF-1 and BDNF-12 were tested in primary mouse cortical neurons using AAV2 / 1-mediated transduction ( Figure 3A ). Equal transduction was achieved with a cross-construct (WPRE and ITR2 qPCR) ( Figure 3B ). Consistent with the previous data from transiently transfected cells, as indicated by Western blotting, the expression constructs BDNF-1 and BDNF-12 had significantly higher BDNF expression compared to the reference construct ( Figure 3C ).

[0556] To test the expression and efficacy of the disclosed constructs in vivo, an obesity model induced by a high-fat diet (HFD) in wild-type adult mice was used. The virus was bilaterally injected into the hypothalamus of each mouse. The animals were placed on a high-fat diet 7 days after surgery. Body weight was recorded weekly, and tissues were collected 21 days after surgery ( Figure 4A ).

[0557] Compared with the endogenous levels in the control diet group, the levels of proBDNF, which is more than 1000-fold higher in the expression construct BDNF1 expression (648.1 ± 33.5 vs. 0.6 ± 0.1 ng / mg total protein), and the levels of mature BDNF (mBDNF), which is more than 200-fold higher (260.6 ± 20.7 vs. 1.3 ± 0.3 ng / mg total protein) ( Figure 4C ). Compared with the endogenous levels in the control diet group, the levels of proBDNF, which is more than 400-fold higher in the expression construct BDNF12 expression (648.1 ± 33.5 vs. 0.6 ± 0.1 ng / mg total protein), and the levels of mBDNF, which is more than 84-fold higher (260.6 ± 20.7 vs. 1.3 ± 0.3 ng / mg total protein) ( Figure 4C ). Compared with the OSU reference construct, the expression constructs BDNF-1 and BDNF-12 expressed 19.2-fold and 11.3-fold more mBDNF protein, respectively, in the mouse hypothalamus ( Figure 4B ).

[0558] BDNF overexpression led to a concentration-dependent decrease in body weight in wild-type animals. As Figure 4D shown, the weight gain caused by 45% HFD in animals treated with the OSU reference construct (0.9 ± 0.3 g) was lower than that of the control group of wild-type mice (HFD: 4.9 ± 0.4 g; AAV2 / 1-eGFP + HFD: 4.5 ± 0.3 g). Compared with the control group and the OSU reference group, mice treated with the expression constructs and BDNF-12 not only prevented the weight gain caused by HFD but also showed a significant weight loss despite being on HFD (BDNF-1: -4.3 ± 0.4 g; BDNF-12: -2.9 ± 0.9 g; Figure 4D ).

[0559] Although the foregoing written description of the invention enables one of ordinary skill in the art to make and use those currently considered to be its best mode, one of ordinary skill in the art will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiments, methods, and examples herein.

[0560] Table 1. Sequence elements of representative constructs. *This construct contains two copies of the BDNF coding sequence (SEQ ID NO:57) separated by an IRES sequence. Seq: Sequence.

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567] Table 2. Sequence elements (5' to 3') of the selected representative constructs, with SEQ ID NO.

[0568]

[0569]

[0570]

[0571]

[0572] Table 3. Overview of the promoter sequences. For the sequences, see Table 6.

[0573] SEQ ID NO Name 1 C14_L21 2 B36 3 B46 4 B47 5 B48 6 CAG(OSU) 7 C42b 8 C42b_L21 9 C26_L21 10 C46_L21

[0574] Table 4. Overview of the expression construct sequences. For the sequences, see Table 8.

[0575]

[0576]

[0577] Table 5. Overview of the additional sequences. ITR = inverted terminal repeat sequence. PTRE = post-transcriptional regulatory element. polyA = polyadenylation sequence. IRES = internal ribosome entry site. For the sequences, see Table 7.

[0578]

[0579] Table 6. Promoter sequences.

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586] Table 7. Additional sequences.

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600] Table 8. Construct sequences.

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

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[0634]

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[0641]

[0642]

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[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

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[0653]

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[0671]

[0672]

Claims

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

2. The expression construct according to claim 1, wherein the promoter sequence (i) comprises a nucleic acid sequence having at least 90% sequence identity with 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. The expression construct according to 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 according to claim 1, wherein the promoter sequence comprises a constitutive promoter.

5. The expression construct according to any one of the preceding claims, wherein the nucleic acid sequence encoding the BDNF is a wild-type BDNF gene.

6. The expression construct according to any one of the preceding claims, wherein the nucleic acid sequence encoding the BDNF is a human BDNF gene.

7. The expression construct according to any one of the preceding claims, wherein the nucleic acid sequence encoding the BDNF is a codon-optimized sequence.

8. The expression construct according to 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 with 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. The expression construct according to any one of the preceding claims, wherein the nucleic acid sequence encoding the BDNF comprises (i) a nucleic acid sequence having at least 90% sequence identity with 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. The expression construct according to any one of the preceding claims, wherein the expression construct further comprises a post-transcriptional regulatory element.

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

12. The expression construct according to claim 10, wherein the post-transcriptional regulatory element (i) comprises a nucleic acid sequence having at least 90% sequence identity with 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. The expression construct according to claim 10, wherein the post-transcriptional regulatory element (i) comprises a nucleic acid sequence having at least 90% sequence identity with 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. The expression construct according to any one of the preceding claims, wherein the expression construct further comprises a polyadenylation signal.

15. The expression construct according to claim 14, wherein the polyadenylation signal (i) comprises a nucleic acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 62 or 66 - 69 or (ii) comprises any one of SEQ ID NO: 62 or 66 - 69.

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

62.

17. The expression construct according to any one of the preceding claims, wherein the expression construct further comprises a microRNA binding sequence located between the nucleic acid sequence encoding the BDNF and the post - transcriptional regulatory element.

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

19. The expression construct according to claim 1, wherein 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.

20. The expression construct according to claim 1, wherein 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 SEQ ID NO:

64.

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

22. The vector according to claim 21, wherein the vector is a viral vector.

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

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

25. The viral vector according to 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. The viral vector according to claim 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, 18-22, 27-30 and 42-45 or (ii) comprises a nucleic acid sequence selected from SEQ ID NOs: 11, 18-22, 27-30 and 42-45.

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

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

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

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

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

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

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

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

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