Recombinant adeno-associated virus vector

Abstract

The present invention provides adeno-associated virus (rAAV) vectors, particularly rAAV vectors comprising genetic constructs comprising genes encoding tyrosine receptor kinase B (TrkB) and brain-derived neurotrophic factor (BDNF). The present invention also extends to pharmaceutical compositions comprising the rAAV vectors and the use of such vectors and compositions in gene therapy methods for preventing or treating a range of optic nerve and cochlear disorders, or for promoting neural regeneration and / or survival. The present invention also provides methods for producing the rAAV vectors.

Description

[Technical Field]

[0001] The present invention relates to recombinant adeno-associated virus (rAAV) vectors, particularly rAAV vectors comprising genetic constructs containing genes encoding tyrosine receptor kinase B (TrkB) and brain-derived neurotrophic factor (BDNF). The invention also extends to pharmaceutical compositions comprising the rAAV vectors, and to the use of such vectors and compositions in gene therapy methods for preventing or treating a range of optic nerve and cochlear disorders, or for promoting neural regeneration and / or survival. The invention also provides methods for producing rAAV vectors. [Background technology]

[0002] Retinal ganglion cells (RGCs) are cells that function as the final pathway for transmitting all visual information processed by the retina to the brain. RGCs are the cells primarily affected in optic neuropathies or optic neuritis, including optic neuropathy (also known as glaucomatous optic neuropathy), hereditary optic neuropathy, ischemic optic neuropathy, and neurodegenerative diseases (Non-Patent Documents 1 and 2). Because the regenerative capacity of RGCs is limited, blindness after optic nerve damage is known to be irreversible (Non-Patent Document 3). Glaucomatous optic neuropathy, the most common optic nerve disorder, is a progressive optic nerve degeneration characterized by RGC axonal damage and concomitant RGC death, leading to visual loss (Non-Patent Documents 4 and 5). Glaucoma, including open-angle glaucoma, normal-tension glaucoma, angle-closure glaucoma, congenital glaucoma, and secondary glaucoma, is the leading cause of irreversible vision loss worldwide. The incidence of glaucoma increases with age. In 2013, the global prevalence of glaucoma among people aged 40 to 80 was estimated at approximately 3.5%, or approximately 64.3 million cases, and was predicted to reach approximately 76 million cases by 2020 and 111.8 million cases by 2040 (Non-Patent Document 6). Currently, with the rapidly increasing elderly population, glaucoma is an urgent social and medical problem.

[0003] Elevated intraocular pressure (IOP) is the most important risk factor for glaucoma (Non-Patent Document 7). Current glaucoma treatment is based on preventing further optic nerve damage by lowering IOP with topically applied drugs (Non-Patent Document 8). The main drugs used to lower IOP are of five types: β-adrenergic receptor antagonists, adrenergic receptor agonists, parasympathomimetics, prostaglandin analogs, and carbonic anhydrase inhibitors. Despite their effectiveness in lowering IOP, these drugs can cause serious side effects in some patients, adversely affecting their quality of life. Furthermore, compliance and adherence to the administration of IOP-lowering eye drops is low, especially in patients who require multiple medications. When IOP reduction is insufficient and further reduction is required, laser trabeculoplasty may be performed; however, many patients are unable to achieve this reduction. Therefore, protecting RGCs and their axons in glaucoma is an important therapeutic approach to be used in addition to conventional IOP-lowering treatments, and is particularly important for patients who do not benefit from conventional treatments (Non-Patent Document 9).

[0004] Brain-derived neurotrophic factor (BDNF), along with nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4 / 5 (NT-4 / 5), is a member of the neurotrophin family of growth factors (Non-Patent Documents 10 and 11). Neurotrophins play an important role in the development, survival, and function of a wide variety of neurons in the peripheral and central nervous systems. Neurotrophins bind to two families of cell surface receptors: p75 neurotrophin receptors (p75NTR) and tropomyosin-related kinase (Trk) receptors. NGF binds primarily to TrkA and BDNF, NT-4 / 5 binds to TrkB, and NT-3 binds primarily to TrkC. BDNF is one of the neurotrophins that can most effectively prevent RGC death after axonal injury (see Non-Patent Documents 12-15). BDNF is usually synthesized as preproBDNF, which contains a signal peptide sequence (Non-Patent Document 16). The signal peptide is then cleaved off, converting preproBDNF to proBDNF. The N-terminal sequence of proBDNF is cleaved intracellularly or extracellularly, resulting in the generation of mature BDNF (mBDNF). While mBDNF activates the TrkB receptor to maintain cell survival, proBDNF is known to preferentially activate the p75NTR receptor to induce cell death (Non-Patent Document 17).

[0005] Animal models of glaucoma have shown a decrease in BDNF or an increase in IOP in the retina after optic nerve crush injury (see Non-Patent Documents 18-20). In animal models of glaucoma, intraocular BDNF supplementation by recombinant protein administration or gene therapy can increase RGC survival compared to untreated controls (Non-Patent Documents 21-24). On the other hand, it has been suggested that the protective effect of BDNF supplementation alone on RGCs is expected to be only transient due to downregulation of TrkB receptors (Non-Patent Document 25). In this context, rAAV vectors have been constructed in which the CAG promoter drives the expression of the TrkB and BDNF genes to maintain the long-term effects of BDNF in the retina. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2017 / 072498 [Patent Document 2] International Publication No. 2018 / 185468 [Non-patent literature]

[0007] [Non-Patent Document 1] Int. J. Mol. Sci.,2020.21(7):2262 [Non-licensed document 2] Hum. Mol. Genet.,2017.26(R2):p.R139-R150 [Non-licensed document 3] Science.2017.356(6342):p.1031-1034

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Non-licensed literature 9

Non-licensed literature 10

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Non-licensed Document 13

Non-licensed Document 14

[0008] However, the present inventors have found that there are serious discrepancies in yield when producing prior art rAAV vectors designed according to the teachings of Patent Document 1 and Non-Patent Document 26. In particular, the present inventors have found a problem in that rAAV vectors containing the TrkB gene and the BDNF gene designed according to the teachings of these documents exhibit genomic fragmentation (or cleavage) of the rAAV genomic DNA during the production process. When the genomic DNA is fragmented, efficient production of the rAAV vector containing the TrkB gene and the BDNF gene is significantly hindered, resulting in reduced rAAV vector production efficiency and reduced yield. Therefore, the present inventors attempted to design and produce an rAAV vector that contains both the TrkB gene and the BDNF gene but does not cleave or fragment the genomic DNA. The present inventors found that an rAAV vector comprising a cytomegalovirus (CMV) promoter operably linked to the TrkB gene and the mature BDNF gene reduces genomic DNA fragmentation, and therefore higher efficiency of the rAAV vector is observed, resulting in better yields. [Means for solving the problem]

[0009] Accordingly, in a first aspect of the present invention there is provided a recombinant adeno-associated virus (rAAV) vector comprising a genetic construct, said genetic construct comprising, in a 5' to 3' direction: cytomegalovirus (CMV) promoter; a first coding sequence encoding tyrosine kinase receptor B (TrkB); A nucleotide sequence encoding a linker for producing TrkB and mature brain-derived neurotrophic factor (mBDNF) as individual proteins; and a second coding sequence encoding mBDNF; in that order, wherein the CMV promoter is operably linked to the first and second coding sequences. Advantageously, an rAAV vector comprising the TrkB gene and the mature BDNF gene and a CMV promoter operably linked to these genes can reduce genomic DNA cleavage or fragmentation during the production process. Therefore, the claimed rAAV vector of the present invention can be produced with high production efficiency. Pharmaceutical compositions containing the rAAV vector can be used for the prevention or treatment of optic nerve disorders and / or retinal degenerative diseases in which retinal ganglion cells degenerate, such as glaucoma and glaucomatous optic neuropathy. The CMV promoter is operably linked to a first coding sequence encoding tyrosine kinase receptor B (TrkB) and a second coding sequence encoding mature brain-derived neurotrophic factor (mBDNF). As used herein, "operably linked" means that the promoter sequence is linked to the first and second coding sequences in such a manner that the proteins encoded by the coding sequences can be expressed in a host cell.

[0010] In one embodiment, the CMV promoter comprises a nucleotide sequence comprising a TATA box sequence derived from a CMV IE promoter and a CMV-derived sequence. One embodiment of a nucleotide sequence encoding a CMV promoter is represented herein below as SEQ ID NO:1.

[0011] [ka] [SEQ ID NO: 1] Thus, in one embodiment, the CMV promoter comprises the nucleotide sequence set forth in SEQ ID NO: 1, or a fragment or variant thereof.

[0012] The genetic construct contained in the rAAV vector of the invention, in one embodiment, comprises a first coding sequence encoding naturally occurring TrkB or a functional variant thereof. It will be appreciated by those skilled in the art that "naturally occurring" TrkB describes the gene as it is found in its natural form, without any non-natural mutations or modifications introduced.

[0013] TrkB functions to activate intracellular signaling molecules downstream of TrkB (e.g., extracellular signal-regulated kinase (ERK)) upon binding with BDNF and neurotrophin-4 / 5 (NT-4 / 5). TrkB function can be assessed using methods known to those skilled in the art, such as ligand binding assays and detecting the activity of intracellular signaling molecules. In some embodiments, the nucleotide sequence encoding TrkB is a nucleotide sequence encoding a mammalian TrkB, and in some embodiments, a nucleotide sequence encoding a human TrkB.

[0014] In one embodiment, TrkB comprises the amino acid sequence represented herein below as SEQ ID NO: 2 (Accession No. NP_001018074.1).

[0015] [ka] TIFF2026504355000004.tif167170[SEQ ID NO:2] Thus, in one embodiment, the first coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 2, or a fragment or variant thereof.

[0016] One embodiment of a nucleotide sequence encoding TrkB is represented herein below as SEQ ID NO:3.

[0017] [ka] Thus, in one embodiment, the first coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 3 or a fragment or variant thereof.

[0018] The genetic construct contained in the rAAV vector of the invention can include a second coding sequence encoding naturally occurring mature BDNF. It will be appreciated by those skilled in the art that "naturally occurring" mBDNF refers to the gene as it is found in its natural form, without any non-natural mutations or modifications introduced into it.

[0019] BDNF is a ligand for TrkB and is known to exist in the forms of preproBDNF, proBDNF, and mBDNF (mature BDNF). Specifically, BDNF is first synthesized as a precursor protein, preproBDNF, which is transported into the rough endoplasmic reticulum and converted to proBDNF via cleavage of the signal peptide. ProBDNF is converted to mBDNF via cleavage of the N-terminal peptide sequence. Both proBDNF and mBDNF are secreted extracellularly, and of these, proBDNF preferentially activates the p75NTR receptor, while mBDNF activates the TrkB receptor. The function of proBDNF or mBDNF can be evaluated using methods known to those skilled in the art, such as receptor binding assays and detection of the activity of intracellular signaling molecules downstream of the receptor. In one embodiment, the nucleotide sequence encoding mBDNF is a nucleotide sequence encoding mammalian mBDNF, and in another embodiment, a nucleotide sequence encoding human mBDNF. Thus, in one embodiment, the nucleotide sequence encoding human mBDNF can be a nucleotide sequence encoding human mBDNF or a functional variant thereof.

[0020] In one embodiment, mBDNF comprises the amino acid sequence represented herein below by SEQ ID NO: 4 (amino acids 129 to 247 of the amino acid sequence of Accession No. NP_001137277.1).

[0021] [ka] [SEQ ID NO: 4] Thus, in one embodiment, the second coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 4, or a fragment or variant thereof.

[0022] One embodiment of a nucleotide sequence encoding mBDNF is represented herein below as SEQ ID NO:5.

[0023] [ka] [SEQ ID NO: 5] Thus, in one embodiment, the second coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 5, or a fragment or variant thereof.

[0024] Because the rAAV vector of the present invention comprises a gene construct encoding mBDNF, in some embodiments, the gene construct further encodes a signal peptide. Thus, in some embodiments, the gene construct comprised in the rAAV vector further comprises a nucleotide sequence encoding a signal peptide. The nucleotide sequence encoding the signal peptide is located 5' to the nucleotide sequence encoding mBDNF. Thus, the gene construct contained in the rAAV vector of the present invention comprises, from 5' to 3', a nucleotide sequence encoding the signal peptide and a nucleotide sequence encoding mBDNF. In one embodiment, the nucleotide sequence encoding the signal peptide is located 3' to the nucleotide sequence encoding the linker. Thus, in some embodiments, the genetic construct contained in the rAAV vector of the invention comprises, from 5' to 3', a cytomegalovirus (CMV) promoter, a nucleotide sequence encoding TrkB, a nucleotide sequence encoding a linker, a nucleotide sequence encoding the signal peptide, and a nucleotide sequence encoding mBDNF.

[0025] The nucleotide sequence encoding the signal peptide used in the present invention is not particularly limited as long as it encodes a signal peptide having the function of promoting the extracellular secretion of mBDNF; for example, see Patent Documents 1 and 2, and Non-Patent Documents 26 and 27. In one embodiment, the nucleotide sequence encoding the signal peptide is a nucleotide sequence encoding a natural amino acid sequence contained in the N-terminus of BDNF protein and having the function of promoting the extracellular secretion of proBDNF and mBDNF. In one embodiment, the nucleotide sequence encoding the signal peptide is an amino acid sequence obtained by modifying the natural amino acid sequence contained in the N-terminus of BDNF protein and encoding an amino acid sequence having the function of promoting the extracellular secretion of proBDNF and mBDNF. In one embodiment, the nucleotide sequence encoding the signal peptide is a nucleotide sequence encoding a natural amino acid sequence included at the N-terminus of the BDNF protein and having the function of promoting extracellular secretion of proBDNF and mBDNF.

[0026] In one embodiment, the signal peptide comprises the amino acid sequence represented herein below as SEQ ID NO: 20 (BDNF signal peptide: SP).

[0027] [ka] [SEQ ID NO: 20] Thus, in one embodiment, the nucleotide sequence encoding the signal peptide encodes the amino acid sequence set forth in SEQ ID NO: 20, or a fragment or variant thereof.

[0028] One embodiment of a nucleotide sequence encoding the signal peptide is represented herein below as SEQ ID NO:21.

[0029] [ka] [SEQ ID NO: 21] Thus, in one embodiment, the signal peptide comprises the nucleotide sequence set forth in SEQ ID NO: 21, or a fragment or variant thereof.

[0030] In one embodiment, the nucleotide sequence encoding the signal peptide is a nucleotide sequence encoding a signal peptide that has been modified from the native amino acid sequence contained at the N-terminus of the BDNF protein and has the function of promoting extracellular secretion of proBDNF and mBDNF. In one embodiment, the signal peptide comprises the amino acid sequence represented herein below as SEQ ID NO: 6 (nv3 signal peptide: mSP).

[0031] [ka] [SEQ ID NO: 6] Thus, in one embodiment, the nucleotide sequence encoding the signal peptide encodes the amino acid sequence set forth in SEQ ID NO: 6, or a fragment or variant thereof.

[0032] One embodiment of a nucleotide sequence encoding the signal peptide is represented herein as SEQ ID NO:7 below.

[0033] [ka] [SEQ ID NO: 7] Thus, in one embodiment, the signal peptide comprises the nucleotide sequence set forth in SEQ ID NO: 7, or a fragment or variant thereof.

[0034] The gene construct further comprises a nucleotide sequence encoding a linker for producing TrkB and mBDNF as individual proteins. The linker is disposed between the nucleotide sequence encoding TrkB and the nucleotide sequence encoding mBDNF. Thus, the gene construct contained in the rAAV vector of the present invention comprises, from 5' to 3', a nucleotide sequence encoding TrkB, a nucleotide sequence encoding a linker for producing TrkB and mBDNF as individual proteins, and a nucleotide sequence encoding mBDNF. Here, the term "linker for producing TrkB and mBDNF as individual proteins" refers to a linker that allows genes encoding the two proteins in succession to be translated into the two proteins by ribosomal skipping in a host cell, or a linker that allows the two proteins to be translated into a single polypeptide and then digested or cleaved in the host cell to release the two proteins as separate proteins. In one embodiment, the linker can be digested or cleaved to produce TrkB and mBDNF as individual proteins. The linker-encoding nucleotide sequence is a nucleotide sequence encoding a virus-derived peptide, specifically a P2A peptide, which is a 2A peptide derived from porcine teschovirus-1.

[0035] In one embodiment, the nucleotide sequence encoding the linker may be a nucleotide sequence encoding a linker comprising a 2A peptide and an additional linker peptide. Thus, in one embodiment, the nucleotide sequence encoding the linker comprises a nucleotide sequence encoding a linker comprising a 2A peptide and an additional linker peptide. The additional linker peptide can be any linker peptide that allows TrkB and mBDNF to be generated as individual proteins, without limitation, and an example thereof is a GSG (glycine-serine-glycine) sequence. Thus, in one embodiment, the nucleotide sequence encoding the linker is a nucleotide sequence encoding a linker consisting of a 2A peptide and GSG added to the N-terminus of the 2A peptide. When the C-terminal amino acid of the polypeptide located at the N-terminus of the additional linker peptide is G, SG (serine-glycine) can be added to the N-terminus of the P2A peptide as an additional linker peptide. Thus, in one embodiment, the nucleotide sequence encoding the linker is a nucleotide sequence encoding a linker consisting of SG and P2A peptides (also referred to as "SG-P2A peptide" in this specification).

[0036] In one embodiment, the linker comprises the amino acid sequence represented herein below as SEQ ID NO:8.

[0037] [ka] [SEQ ID NO: 8] Thus, in one embodiment, the nucleotide sequence encoding the linker encodes the amino acid sequence set forth in SEQ ID NO: 8, or a fragment or variant thereof.

[0038] One embodiment of a nucleotide sequence encoding a linker is represented herein below as SEQ ID NO:9.

[0039] [ka] Thus, in one embodiment, the linker comprises the nucleotide sequence set forth in SEQ ID NO: 9, or a fragment or variant thereof.

[0040] In one embodiment, the genetic construct contained in the rAAV vector of the present invention further comprises a post-transcriptional regulatory element. As used herein, the term "post-transcriptional regulatory element" refers to a non-coding sequence that regulates gene expression through post-transcriptional control. Post-transcriptional regulatory elements that can be used in the present invention include, without limitation, any element that can regulate gene expression through post-transcriptional regulation, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in one embodiment, the genetic construct comprises a nucleotide sequence encoding a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) that enhances the expression of two transgenes, namely, TrkB receptor and mBDNF. In one embodiment, the WPRE coding sequence is located 3' to the transgene coding sequence, and in some embodiments, it is located 3' to the mBDNF coding sequence.

[0041] In one embodiment, the post-transcriptional regulatory element is a WPRE (hereinafter also referred to as WPRE(S)), defined as a nucleotide sequence of 247 bp in length (SEQ ID NO: 10) lacking the β element. One embodiment of a nucleotide sequence encoding a WPRE is represented herein below as SEQ ID NO:10.

[0042] [ka] [SEQ ID NO: 10] Thus, in one embodiment, the WPRE comprises the nucleotide sequence set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0043] In one embodiment, the gene construct contained in the rAAV vector comprises a nucleotide sequence encoding a polyA signal sequence. The term "polyA signal sequence" as used herein refers to a sequence known to those skilled in the art, which is a DNA sequence positioned at the 3' end of a gene and capable of adding a polyadenosine (polyA) tail to the 3' end of mRNA transcribed from the gene. In one embodiment, the polyA signal sequence is a simian virus 40 (SV40) polyA signal sequence, a human β-globin polyA signal sequence, a rabbit β-globin polyA signal sequence, a bovine growth hormone polyA signal sequence, or a human growth hormone polyA signal sequence. In certain embodiments, the polyA signal sequence is an SV40 polyA signal sequence.

[0044] In one embodiment, the polyA signal sequence is positioned 3' to the transgene coding sequence, and in some embodiments, 3' to the WPRE coding sequence. One embodiment of a nucleotide sequence encoding a polyA signal sequence is represented herein as SEQ ID NO:11 below.

[0045] [ka] [SEQ ID NO: 11] Thus, in one embodiment, the polyA signal sequence comprises the nucleotide sequence set forth in SEQ ID NO: 11, or a fragment or variant thereof.

[0046] Thus, in one embodiment, the rAAV vector comprises a genetic construct comprising, in the 5' to 3' direction, a CMV promoter sequence, a first coding sequence encoding TrkB, a nucleotide sequence encoding a linker peptide, a nucleotide sequence encoding a signal peptide, a second coding sequence encoding mBDNF, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in other embodiments, the rAAV vector comprises a genetic construct comprising, in the 5' to 3' direction, a CMV promoter sequence, a first coding sequence encoding TrkB, a nucleotide sequence encoding a linker peptide, a nucleotide sequence encoding a signal peptide, a second coding sequence encoding mBDNF, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a simian virus 40 (SV40) polyA signal sequence. Thus, in other embodiments, the rAAV vector comprises a genetic construct comprising, in the 5' to 3' direction, a CMV promoter sequence, a first coding sequence encoding TrkB, a nucleotide sequence encoding a linker peptide (in one embodiment, a P2A peptide), a nucleotide sequence encoding a signal peptide, a second coding sequence encoding mBDNF, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a simian virus 40 (SV40) polyA signal sequence.

[0047] In one embodiment, the rAAV vector contains left and / or right inverted terminal repeats (ITRs). In one embodiment, each ITR is located at the 5' and / or 3' end of the AAV genome. As used herein, "inverted terminal repeats (ITRs)" refers to sequences known to those skilled in the art that are present at each end of the AAV genomic DNA and form a hairpin loop. AAVs are classified into different serotypes, such as AAV1 and AAV2, based on capsid protein sequences, and AAV genomes of different serotypes contain different ITR sequences. However, an AAV genome containing ITRs from one serotype can be packaged into a capsid from another serotype. Each ITR may be a wild-type sequence or a mutant with ITR functionality. In one embodiment, each ITR is an ITR from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9, etc., or a modified ITR thereof. In one embodiment, each ITR is an AAV2-derived ITR. One embodiment of a nucleotide sequence encoding the 5' ITR is represented herein below as SEQ ID NO:12.

[0048] [ka] One embodiment of a nucleotide sequence encoding the 3' ITR is represented herein below as SEQ ID NO:13.

[0049] [ka] [SEQ ID NO: 13] Thus, in one embodiment, the 5' ITR and 3' ITR comprise the nucleotide sequence set forth in SEQ ID NO: 12 and the complementary sequence to the nucleotide sequence set forth in SEQ ID NO: 12 (the nucleotide sequence set forth in SEQ ID NO: 13), respectively. Thus, in one embodiment, the rAAV vector comprises a genetic construct comprising, from 5' to 3', a 5' ITR, a CMV promoter sequence, a first coding sequence encoding TrkB, a nucleotide sequence encoding a linker peptide (in one embodiment, a P2A peptide), a nucleotide sequence encoding a signal peptide, a second coding sequence encoding mBDNF, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a Simian Virus 40 (SV40) polyA signal sequence, and a 3' ITR.

[0050] The rAAV vectors of the present invention may further contain various expression regulatory elements, including a translation initiation codon, a translation termination codon, a Kozak sequence, a splicing junction, and the like, depending on the promoter, host cell, and the like used (e.g., Goeddel, Gene Expression Technology, Methods in Enzymology, 1990, p. 185, Academic Press, San Diego).

[0051] Gene constructs contained within the rAAV vectors of the invention can be synthesized based on sequence information using standard polynucleotide synthesis methods known in the art. Polynucleotide variants can be generated by introducing mutations into specific sites of a given polynucleotide using methods known to those skilled in the art, such as site-directed mutagenesis. As noted above, those skilled in the art will appreciate the nucleotide sequences of embodiments of the genetic construct contained within the rAAV vector of the first aspect, as well as the amino acid sequence of the encoded transgene. However, for the avoidance of doubt, in one embodiment, the rAAV vector of the invention is an rAAV vector comprising a genetic construct comprising the nucleotide sequence represented herein below in SEQ ID NO:14.

[0052] [ka] TIFF2026504355000019.tif239170Thus, in one embodiment, the rAAV vector according to the first aspect comprises a genetic construct comprising the nucleotide sequence set forth in SEQ ID NO: 14 or a variant or fragment thereof. The gene construct consisting of the nucleotide sequence represented by SEQ ID NO:14 (hereinafter also referred to as "CMV-hTrkB-P2A-mSP-hmBDNF") contains, from 5' to 3', a CMV promoter sequence consisting of the nucleotide sequence represented by SEQ ID NO:1, a nucleotide sequence encoding TrkB consisting of the nucleotide sequence represented by SEQ ID NO:3, a nucleotide sequence encoding the SG-P2A peptide consisting of the nucleotide sequence represented by SEQ ID NO:9, a nucleotide sequence encoding a signal peptide consisting of the nucleotide sequence represented by SEQ ID NO:7, and a nucleotide sequence encoding mBDNF consisting of the nucleotide sequence represented by SEQ ID NO:5, in this order.

[0053] In other embodiments, the rAAV vector of the present invention is an rAAV vector comprising a genetic construct comprising the nucleotide sequence represented herein below by SEQ ID NO: 15.

[0054] [ka] TIFF2026504355000021.tif222170[SEQ ID NO: 15] Thus, in other embodiments, the rAAV vector according to the first aspect comprises a genetic construct comprising the nucleotide sequence set forth in SEQ ID NO: 15, or a variant or fragment thereof. Here, the gene construct consisting of the nucleotide sequence represented by SEQ ID NO: 15 (hereinafter also referred to as "CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA") is a gene construct comprising, in the 5' to 3' direction, a CMV promoter sequence consisting of the nucleotide sequence represented by SEQ ID NO: 1, a nucleotide sequence encoding TrkB consisting of the nucleotide sequence represented by SEQ ID NO: 3, a nucleotide sequence encoding the SG-P2A peptide consisting of the nucleotide sequence represented by SEQ ID NO: 9, a nucleotide sequence encoding a signal peptide consisting of the nucleotide sequence represented by SEQ ID NO: 7, a nucleotide sequence encoding mBDNF consisting of the nucleotide sequence represented by SEQ ID NO: 5, a WPRE consisting of the nucleotide sequence represented by SEQ ID NO: 10, and an SV40 poly(A) signal sequence consisting of the nucleotide sequence represented by SEQ ID NO: 11.

[0055] In other embodiments, the rAAV vector of the present invention is an rAAV vector comprising a genetic construct comprising the nucleotide sequence represented herein below by SEQ ID NO: 16.

[0056] [ka] TIFF2026504355000023.tif251170[SEQ ID NO: 16] Thus, in other embodiments, the rAAV vector according to the first aspect comprises a genetic construct comprising the nucleotide sequence set forth in SEQ ID NO: 16, or a variant or fragment thereof. The gene construct consisting of the nucleotide sequence set forth in SEQ ID NO: 16 (hereinafter also referred to as "ITR-CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA-ITR") is a gene construct comprising AAV2-derived ITRs (the nucleotide sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively) on the 5' and 3' sides of "CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA" (SEQ ID NO: 15).

[0057] In other embodiments, the rAAV vector of the present invention is an rAAV vector comprising a genetic construct comprising the nucleotide sequence represented herein below by SEQ ID NO: 17.

[0058] [ka] TIFF2026504355000025.tif233170[SEQ ID NO: 17] Thus, in other embodiments, the rAAV vector according to the first aspect comprises a genetic construct comprising the nucleotide sequence set forth in SEQ ID NO: 17, or a variant or fragment thereof. The gene construct consisting of the nucleotide sequence set forth in SEQ ID NO: 17 (hereinafter also referred to as "ITR-CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA-ITR(2)") contains "CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA" (SEQ ID NO: 15) with AAV2-derived ITRs (the nucleotide sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively) on the 5' and 3' ends, and the nucleotide sequence between the 5' ITR and the CMV promoter is different from that of SEQ ID NO: 16.

[0059] In the present invention, the AAV serotype is not particularly limited as long as it is capable of expressing TrkB and BDNF in host cells, and AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9, rAAV2.7m8 vector, rAAV2 Max vector, etc. can be used. The rAAV vector of the present invention derived from any of the AAV serotypes mentioned herein is represented by the rAAV1 vector, rAAV2 vector, rAAV2.7m8 vector, rAAV2 Max vector, rAAV3 vector, rAAV4 vector, rAAV5 vector, rAAV8 vector, or rAAV9 vector. The rAAV vector of the present invention may be a modified rAAV vector in which the amino acid sequence of the capsid protein has been modified.

[0060] In one embodiment, the rAAV vector of the present invention is an rAAV2 vector. In one embodiment, the rAAV vector is an rAAV2.7m8 vector. The rAAV2.7m8 vector contains an omentum-specific 7m8 peptide insertion (N587_R588insLALGETTRPA) between amino acids 587 and 588 and has been shown to exhibit improved photoreceptor transduction after intravitreal injection compared to unmodified rAAV2. See WO2012 / 145601 and Reid et al., 2017. Improvement of photoreceptor targeting via intravitreal delivery in mouse and human retina using combinatory rAAV2 capsid mutant vectors. Investigative Ophthalmology & Visual Science, 58(14), pp. 6429-6439.

[0061] One embodiment of the amino acid sequence of the capsid of the rAAV2.7m8 vector is provided herein below as SEQ ID NO: 18.

[0062] [ka] [SEQ ID NO: 18] One embodiment of a nucleotide sequence encoding the Cap gene of the rAAV2.7m8 vector is provided herein as SEQ ID NO:22 below.

[0063] [ka] [SEQ ID NO: 22] In other embodiments, the rAAV vector is an rAAV2 Max vector, which contains five point mutations: Y272F; Y444F; Y500F; Y730F; and T491V (derived from rAAV2 [QuadYF+TV; see WO2008 / 124724, WO2013 / 173512, and WO2015 / 126972]), and a peptide insert, N587_R588insLALGETTRPA (derived from rAAV2.7m8), and has been shown to demonstrate high levels of transduction. See Reid et al., 2017. Improvement of photoreceptor targeting via intravitreal delivery in mouse and human retina using combinatory rAAV2 capsid mutant vectors. Investigative ophthalmology & visual science, 58(14), pp.6429-6439.

[0064] One embodiment of an amino acid sequence encoding the capsid of the rAAV2 Max vector is provided herein as SEQ ID NO: 19 below.

[0065] [ka] [SEQ ID NO: 19] One embodiment of a nucleotide sequence encoding the Cap gene of the rAAV2 Max vector is provided herein as SEQ ID NO:23 below.

[0066] [ka] [SEQ ID NO: 23] The rAAVs described herein can be used to treat optic neuropathy and cochlear disorders, and more generally to promote neuronal regeneration and survival, hi one embodiment, the rAAVs described herein can be used to treat optic neuropathy and / or retinal degenerative diseases associated with degeneration of retinal ganglion cells.

[0067] Thus, in a second aspect, there is provided a recombinant vector according to the first aspect for use as a medicament or in therapy. In a third aspect, there is provided an rAAV vector according to the first aspect for use in the treatment, prevention or amelioration of optic nerve damage or cochlear damage, or for promoting nerve regeneration and / or survival. In one embodiment, there is provided an rAAV vector according to the first aspect for use in the treatment, prevention or amelioration of optic neuropathy and / or a retinal degenerative disease associated with degeneration of retinal ganglion cells. A fourth aspect includes a method for treating, preventing, or ameliorating optic nerve damage or cochlear damage in a subject, or for promoting nerve regeneration and / or survival in a subject, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of an rAAV vector according to the first aspect.

[0068] In one embodiment, there is provided a method for treating, preventing or ameliorating a retinal degenerative disease associated with optic neuropathy and / or degeneration of retinal ganglion cells, comprising administering to a subject in need of such treatment a therapeutically effective amount of an rAAV vector according to the first aspect. In one embodiment, the rAAV vectors of the invention are used in gene therapy techniques, where BDNF encoded by the vector activates TrkB encoded by the vector, thereby promoting the survival of retinal ganglion cells (RGCs) or cochlear cells. As shown in the examples, the rAAV vectors of the present invention can provide a protective effect on the overall thickness of the retinal nerve fiber layer (RNFL) composed of RGC axons, and improve their photo-induced negative response (PhNR), which is related to the function of RGCs and their axons. Thus, in a preferred embodiment, the rAAV vectors of the present invention protect the overall thickness of the RNFL composed of RGC axons. In another preferred embodiment, the rAAV vectors of the present invention improve the PhNR (i.e., increase the PhNR amplitude) related to the function of RGCs and their axons.

[0069] In one embodiment, the rAAV for use according to the third aspect or the method according to the fourth aspect is for preventing or treating glaucoma and glaucomatous optic neuropathies, hereditary optic neuropathies, ischemic optic neuropathies, and neurodegenerative diseases associated with retinal ganglion cell degeneration. Glaucoma and glaucomatous optic neuropathies herein include open-angle glaucoma, normal-tension glaucoma, angle-closure glaucoma, congenital glaucoma, and secondary glaucoma. Hereditary optic neuropathies herein include Leber's hereditary optic neuropathy and dominantly inherited optic atrophy. Neurodegenerative diseases associated with retinal ganglion cell degeneration herein include Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple system atrophy. In some embodiments, the optic neuropathy and / or retinal degenerative disease being treated is glaucoma, hi other embodiments, the optic neuropathy and / or retinal degenerative disease being treated is glaucomatous optic neuropathy. In one embodiment, the cochlear disorder being treated can be hearing loss or deafness. The cochlear cells can be hair cells or spiral ganglion cells, which are neurons that transmit auditory signals from the ear to the brainstem via their axons. The hair cells can be inner ear hair cells or outer ear hair cells.

[0070] In other embodiments, the vectors may be used to promote nerve regeneration and / or survival. In a fifth aspect, there is provided a pharmaceutical composition comprising a recombinant rAAV vector according to the first aspect and a pharmaceutically acceptable vehicle. In a sixth aspect, there is provided a method of preparing a pharmaceutical composition according to the fifth aspect, the method comprising contacting a recombinant rAAV vector according to the first aspect with a pharmaceutically acceptable vehicle.

[0071] The pharmaceutical compositions of the present invention can be prepared by commonly used methods using diluents commonly used in the art, i.e., pharmaceutical diluents, pharmaceutical carriers, etc. Examples of dosage forms of the pharmaceutical compositions include parenteral preparations such as injections and infusions. When formulating, diluents, carriers, excipients, etc. appropriate for the dosage form can be used in a pharmaceutically acceptable manner. The pharmaceutical compositions of the present invention can be prepared as sustained-release formulations. In one embodiment, the pharmaceutical compositions of the present invention are administered as injections. In one embodiment, the pharmaceutical compositions of the present invention can be administered intraocularly, subretinally, intravitreal, or suprachoroidally. When formulating the rAAV vectors of the present invention, diluents, carriers, excipients, etc. appropriate for the dosage form can be used in a pharmaceutically acceptable manner.

[0072] The "subject" (target) in the preventive or therapeutic methods of the present invention refers to a human or non-human animal in need of said prevention or treatment, and in one embodiment, refers to a human in need of said prevention or treatment. Examples of "administration" to a subject include intraocular administration, intravitreal administration, subretinal administration, and suprachoroidal administration.

[0073] The effective amount of the rAAV vector of the present invention can be appropriately optimized taking into consideration the severity of the disease, previous treatments, the general health and age of the subject, the administration method, other diseases, etc. The dose of the rAAV vector of the present invention can also be expressed as the number of copies (vg) of the vector genome administered per eye (vg / eye). vg can also be expressed in genome copies (GC). In one embodiment, the effective amount of the rAAV vector of the present invention is about 1 x 10 6 ~1×10 14 In one embodiment, the effective dose of an rAAV vector of the invention is about 1 x 10 vg / eye. 8 ~1×10 13In other embodiments, the effective dose of an rAAV vector of the invention is about 1 x 10 vg / eye. 10 ~1×10 12 In other embodiments, the effective dose of an rAAV vector of the invention is about 1 x 10 vg / eye. 11 ~1×10 12 vg / eye. The rAAV vectors of the present invention can be used in combination with therapeutic or prophylactic agents for various diseases for which the therapeutic or prophylactic agents are expected to be effective. The combined administration can be performed simultaneously, sequentially in separate operations, or at a desired time interval. The formulations for simultaneous administration can be a combined drug or can be individually formulated separate products.

[0074] The present inventors have also developed a method for producing an rAAV vector according to the first aspect. Accordingly, in a seventh aspect, the present invention further provides a method for producing an rAAV vector according to the first aspect, the method comprising: (i) introducing into an rAAV vector-producing cell a genetic construct, wherein the genetic construct comprises, in a 5' to 3' direction: cytomegalovirus (CMV) promoter; a first coding sequence encoding tyrosine kinase receptor B (TrkB); A nucleotide sequence encoding a linker for producing TrkB and mature brain-derived neurotrophic factor (mBDNF) as individual proteins; and a second coding sequence encoding mature brain-derived neurotrophic factor (mBDNF); in that order, wherein the CMV promoter is operably linked to a first and a second coding sequence; and (ii) culturing the rAAV vector-producing cells to produce the rAAV vector of claim 1; Includes. In one embodiment, a method for producing rAAV includes the steps of introducing a genetic construct into an rAAV vector-producing cell; culturing the rAAV vector-producing cell; and collecting the culture medium from the rAAV vector-producing cell and / or lysing the rAAV vector, and purifying the rAAV vector from the culture medium and / or lysate. A method for producing an rAAV vector may include introducing a gene construct into an rAAV vector-producing cell. The step of introducing a gene construct into an rAAV vector-producing cell may include introducing into the rAAV vector-producing cell, in addition to the gene construct, a plasmid containing a Rep gene and a Cap gene, and a plasmid containing a gene derived from a helper virus that promotes AAV replication (e.g., the adenovirus VA, E2A, and E4 genes). AAV capsid proteins constitute the external, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for virion assembly. For construction of rAAV virions, see, for example, US 5,173,414; US 5,139,941; US ​​5,863,541; US ​​5,869,305; US 6,057,152; and US 6,376,237; as well as Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al. 77:423 (2003). The step of introducing a gene construct into an rAAV vector-producing cell can be carried out using methods known to those skilled in the art. The method for producing an rAAV vector may include a step of recovering the culture medium from rAAV vector-producing cells and / or a lysate of the rAAV vector-producing cells, which can be obtained, for example, by treating the rAAV vector-producing cells with a detergent or ultrasound. The method for producing an rAAV vector may further include a step of purifying the rAAV vector. For example, ion exchange chromatography and / or hydrophobic interaction chromatography, cesium chloride density gradient centrifugation, sucrose gradient centrifugation, iodixanol density gradient centrifugation, ultrafiltration, dialysis, affinity chromatography, polyethylene glycol precipitation, and ammonium sulfate precipitation can be used to purify the rAAV vector from the lysate.

[0075] In an eighth aspect, the present invention provides an rAAV vector producing cell comprising the genetic construct of the rAAV vector of the first aspect. The rAAV vector-producing cells used in the present invention are not particularly limited and can be any cell known in the art and capable of producing rAAV upon introduction of a construct. The rAAV vector-producing cells used in the present invention include various cells, including normal cells commonly used in the technical field of the present invention and artificially established cells. The rAAV vector-producing cells used in the present invention include, for example, animal cells (e.g., CHO cells, HEK293 cells, HeLa cells), insect cells (e.g., Sf9 cells), bacteria (e.g., Escherichia coli), yeast (Saccharomyces species, Pichia species), and the like. In one embodiment, the rAAV vector-producing cells of the present invention are animal cells. In one embodiment, the rAAV vector-producing cells of the present invention are HEK293 cells or cells derived therefrom (e.g., HEK293T cells).

[0076] It will be understood that the present invention extends to any nucleic acid or peptide, or variant, derivative or analogue thereof, that substantially comprises the amino acid or nucleic acid sequence (including variants or fragments thereof) of any of the sequences referred to herein. The terms "substantially an amino acid / nucleotide / peptide sequence," "variant" and "fragment" may refer to a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any one of the sequences referred to herein, such as 40% identity with the sequences identified as SEQ ID NOs: 1-26. Also contemplated are amino acid / polynucleotide / polypeptide sequences that have greater than 65%, in some embodiments greater than 70%, in some embodiments greater than 75%, and in some embodiments greater than 80% sequence identity to any of the sequences referenced herein. In some embodiments, the amino acid / polynucleotide / polypeptide sequence has at least 85%, in some embodiments at least 90%, in some embodiments at least 92%, in some embodiments at least 95%, in some embodiments at least 97%, in some embodiments at least 98%, and in some embodiments at least 99% identity to any of the sequences referenced herein. Those skilled in the art will understand how to calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences.To calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences, the two sequences should first be aligned and then the sequence identity value should be calculated.The identity percentage of two sequences can take different values ​​depending on (i) the method used to align sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, such as local vs. global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and gap penalty, such as function form and constant. After alignment, there are many different ways to calculate the identity ratio between two sequences.For example, the number of identities can be divided by: (i) the length of the shortest sequence; (ii) the length of alignment; (iii) the average length of sequences; (iv) the number of non-gap positions; or (iv) the number of equivalent positions excluding overhangs.In addition, it is understood that identity ratio also depends strongly on length.Therefore, the shorter the pair of sequences, the higher the sequence identity that can be expected to occur by chance.

[0077] It will be appreciated, therefore, that accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is one method for generating a multiple alignment of proteins or DNA according to the present invention. Suitable parameters for ClustalW may be as follows: DNA alignment: gap opening penalty = 15.0, gap extension penalty = 6.66, and matrix = identity; gap opening penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet; DNA and protein alignment: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will recognize that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0078] In some embodiments, calculation of the percent identity between two amino acid / polynucleotide / polypeptide sequences can then be calculated from the alignment, such as (N / T)*100, where N is the number of positions where the sequences share identical residues, and T is the total number of positions being compared, including gaps but excluding overhangs. In some embodiments, overhangs are included in the calculation. Thus, one method for calculating the percent identity between two sequences includes (i) preparing a sequence alignment using, for example, the ClustalW program with appropriate parameter sets, as described above; and (ii) inserting the values ​​of N and T into the following formula: sequence identity=(N / T)*100.

[0079] Alternative methods for identifying similar sequences are known to those of skill in the art. For example, a substantially similar nucleotide sequence is encoded by a sequence that hybridizes to a DNA sequence or its complement under stringent conditions. Stringent conditions mean that the nucleotides hybridize to filter-bound DNA or RNA in 3× sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2× SSC / 0.1% SDS at about 20-65°C. Alternatively, a substantially similar polypeptide may differ from the sequences set forth in, for example, SEQ ID NOs: 3 and 5 by at least one but fewer than 5, 10, 20, 50, or 100 amino acids.

[0080] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be varied or modified to provide functional variants thereof without substantially affecting the sequence of the protein encoded thereby. Suitable nucleotide variants include sequences altered by the substitution of different codons encoding the same amino acid within the sequence, thus producing a silent change. Other suitable variants include all or portions of a sequence comprising a homologous nucleotide sequence, but altered by the substitution of different codons encoding amino acids with side chains of similar biophysical properties to the amino acid being replaced, resulting in a conservative change. For example, small nonpolar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar, hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It is therefore understood which amino acids can be substituted with amino acids with similar biophysical properties, and those skilled in the art will know the nucleotide sequences encoding these amino acids.

[0081] All of the features described in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. For a better understanding of the present invention and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief explanation of the drawings]

[0082] [Figure 1] FIG. 1 shows a schematic map of the gene construct "ITR-CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA-ITR" (SEQ ID NO: 16) contained in the rAAV according to the present invention and represented by "#036" throughout the examples. [Figure 2] Figure 2 shows the results of Western blot analysis (n=2) of the expression of transgene products (hmBDNF, TrkB) and the presence of activated TrkB (phosphorylated TrkB: pTrkB) in HEK293 cells 2 days after transduction with rAAV#036 shown in Figure 1. In the figure, rAAV#036 is abbreviated as "#036," and hmBDNF is abbreviated as "BDNF." [Figure 3] This figure shows the results of ELISA of the expression level of the transgene product (hmBDNF) in mouse retinal tissue 3 weeks after intravitreal administration of rAAV#036 shown in Figure 1 at doses of 3.0 x 10 (3.0e7) vg / 1 μL, 9.0 x 10 (9.0e7) vg / 1 μL, or 2.7 x 10 (2.7e8) vg / 1 μL per eye. Bars in the graph represent the mean ± standard error of the mean for each group (n = 8 or 9). In the figure, rAAV#036 is abbreviated as "#036" and hmBDNF is abbreviated as "BDNF." [Figure 4]This figure shows the results of Western blot analysis (n=3) of the expression of transgene products (hmBDNF, TrkB) and the presence of activated TrkB (pTrkB) in mouse retinal tissue 3 weeks after intravitreal administration of rAAV#036 shown in Figure 1 at a dose of 2.7 × 10 (2.7e8) vg / 1 μL / eye. In the figure, rAAV#036 is abbreviated as "#036," and hmBDNF is abbreviated as "BDNF." [Figure 5]

[0033] Figure 1 shows the results of alkaline agarose gel electrophoresis analysis of the genomic DNA of rAAV#007 (sCAG-hTrkB-P2A-SP-hmBDNF-WPRE(S)-SV40pA), rAAV#008 (CMV-hTrkB-P2A-SP-hmBDNF-WPRE(S)-SV40pA), and rAAV#036 (CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA). The total genome lengths of rAAV#007, rAAV#008, and rAAV#036 are approximately 4.8 kb, 4.6 kb, and 4.6 kb, respectively. [Figure 6]

[0036] Figure 1 shows the productivity of rAAV#007, rAAV#008, and rAAV#036. The vertical axis shows the relative titer (calculated using ITR primers) of the vector genome concentration of rAAV#008 compared to rAAV#007, and of rAAV#036 compared to rAAV#008 in the cell lysate. [Figure 7]

[0036] Figure 1 shows the results of alkaline agarose gel electrophoresis analysis of the genomic DNA of rAAV#036, rAAV2.Max#036, and rAAV2.7m8#036. The total genome length of rAAV#036, rAAV2.Max#036, and rAAV2.7m8#036 is approximately 4.6 kb. [Figure 8]

[0036] Figure 1 shows the productivity of rAAV#036, rAAV2.Max#036, and rAAV2.7m8#036. The vertical axis shows the relative titer of the vector genome concentration of rAAV#036, rAAV2.Max#036, and rAAV2.7m8#036 in cell lysates (calculated using ITR primers). [Figure 9]Figure 1 shows vector copy numbers (copies / µg DNA) measured by real-time PCR in monkey retinal tissue 8 weeks after intravitreal administration of rAAV#036, rAAV2.Max#036, and rAAV2.7m8#036 at a dose of 6.3 x 10 vg / 70 µL per eye. Bars in the graph represent the mean ± standard error of the mean for each group (n = 3). [Figure 10] 1 shows GAPDH-corrected BDNF and TrkB RNA expression levels in monkey retinal tissues 8 weeks after intravitreal administration of rAAV#036, rAAV2.Max#036, and rAAV2.7m8#036 at a dose of 6.3×10 vg / 70 μL / eye, using real-time PCR. Bars in the graph represent the mean ± standard error of the mean for each group (n=3). [Figure 11] 1A-1C show data from non-laser-treated and laser-treated eyes after intravitreal administration of vehicle or rAAV2.7m8#036 at doses of 6.0 x 10 (6.0e10) vg / 70 µL or 3.0 x 10 (3.0e11) vg / 70 µL per eye showing global retinal nerve fiber layer (RNFL) thickness using optical coherence tomography (OCT) circular scans of the optic nerve head. Bars in the graphs represent the mean ± standard error of the mean for each group (n = 3-5). [Figure 12] 1 shows the change in the percent photopic negative response (PhNR) amplitude from pre-administration using suprafoveal focal electroretinograms in laser-untreated and laser-treated eyes after intravitreal administration of vehicle or rAAV2.7m8#036 at a dose of 6.0 x 10 (6.0e10) vg / 70 μL or 3.0 x 10 (3.0e11) vg / 70 μL per eye. Bars in the graph represent the mean ± standard error of the mean for each group (n = 3 or 5). DETAILED DESCRIPTION OF THE INVENTION

[0083] [Example] The present inventors have observed significant discrepancies in yield when producing some of the rAAV vectors described in Patent Document 1 and Non-Patent Document 26. In particular, the present inventors have observed a serious problem: rAAV vectors containing the TrkB gene and the BDNF gene designed according to the teachings of prior art documents exhibit fragmentation or cleavage of the rAAV genomic DNA during the production process. Shortened genomic DNA hinders efficient production of rAAV vectors containing the TrkB gene and the BDNF gene, reducing the production efficiency of the rAAV vector. Therefore, the present inventors have attempted to obtain an rAAV vector containing both the TrkB gene and the BDNF gene with reduced shortening of the genomic DNA. [Example]

[0084] Production of rAAV constructs Using the methods described in Patent Document 1 and Non-Patent Document 26, pAAV-sCAG-hTrkB-P2A-SP-hmBDNF-WPRE(S)-SV40pA (SEQ ID NO: 24) was obtained (this plasmid construct is also referred to as #007). Plasmid construct pAAV-CMV-hTrkB-P2A-SP-hmBDNF-WPRE(S)-SV40pA (SEQ ID NO: 25) containing a CMV promoter (SEQ ID NO: 1) was obtained (this plasmid construct is also referred to as #008). Plasmid construct pAAV-CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA (SEQ ID NO: 26; the signal peptide was modified from that of #008) was obtained (this plasmid construct is also referred to as #036). Plasmid construct #036 contains the polynucleotide "ITR-CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA-ITR" (SEQ ID NO: 16), which contains the polynucleotide "CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA" (SEQ ID NO: 15). The polynucleotide "CMV-hTrkB-P2A-mSP-hmBDNF" (SEQ ID NO: 14) is a region spanning from the CMV promoter to the nucleotide sequence encoding hmBDNF (SEQ ID NO: 15). A map of the polynucleotide "ITR-CMV-hTrkB-P2A-mSP-hmBDNF-WPRE(S)-SV40pA-ITR" (SEQ ID NO: 16) contained in plasmid construct #036 is shown in Figure 1. The rAAV2 vectors were generated using plasmid construct #007 (containing an sCAG promoter), plasmid construct #008 (containing a CMV promoter), and plasmid construct #036. The resulting rAAV2 vectors are designated rAAV#007, rAAV#008, and rAAV#036, respectively. rAAV2.7m8 was produced using plasmid construct #036 and designated rAAV2.7m8#036. rAAV2 Max was produced using plasmid construct #036 and designated rAAV2 Max#036. rAAV2.7m8 comprises a capsid containing the amino acid sequence of SEQ ID NO:18. rAAV2 Max comprises a capsid containing the amino acid sequence of SEQ ID NO:19. [Example]

[0085] Expression of transgene products and activation of TrkB in HEK293 cells transduced with rAAV#036 One day before the rAAV transduction experiment, HEK293 cells were seeded at 1 × 10 cells / well onto collagen I-coated 24-well microplates (Iwaki, product number 4820-010) and cultured statically at 37°C in 5% CO in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Co. LLC, product number D6429) supplemented with 10% fetal bovine serum (FBS, Hyclone, product number SH30070.03) and 1% penicillin-streptomycin (Thermo Fisher Scientific, product number 15070-063). One day after cell seeding, the medium was completely replaced with 425 μL of DMEM containing 1% FBS and 1% penicillin-streptomycin, and 75 μL of rAAV#036 or Dulbecco's phosphate-buffered saline (DPBS, Wako Pure Chemical Industries, Ltd., product number 045-29795) was added dropwise to the cells, followed by static culture at 37°C and 5% CO. For the addition, rAAV#036 was added to DPBS at a final concentration of 2.5 × 10 9 The concentration was adjusted in advance to reach vg / mL. Two days after rAAV addition, the cells were washed with DPBS, and then cell lysis buffer was added. The lysate was collected and stored at -80°C. The cell lysis buffer was adjusted to a final concentration of 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, Thermo Fisher Scientific, product number 15630-080), 135 mM sodium chloride (NaCl, Wako Pure Chemical Industries, Ltd., product number 191-01665), 1% Triton® X-100 (Nacalai Tesque, product number 35501-15), 0.1% Benzonase® Nuclease (Merck Millipore, product number 70664), and 1% Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, product number 78441). The thawed solution was left on ice for 20 minutes, then centrifuged at 15,000 rpm for 5 minutes at 4°C using a Hitachi centrifuge. The supernatant was used for the following tests. The protein concentration of the sample was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, model number 23227) and determined from the absorbance at 562 nm using a microplate reader (SpectraMax Plus 384, Molecular Devices, LLC.). Western blot analysis was performed using equal amounts of protein in the samples to confirm the expression of the transgene products (hmBDNF and TrkB) and the activation of TrkB (phosphorylated TrkB (pTrkB)) in HEK293 cells. Primary antibodies used were rabbit anti-BDNF [EPR1292] antibody (Abcam plc., product number ab108319), rabbit anti-TrkB [80E3] antibody (Cell Signaling Technology, product number 4603S), rabbit anti-phospho-TrkB [Tyr515] polyclonal antibody (Thermo Fisher Scientific, product number PA5-36695), and rabbit anti-β-actin antibody (Cell Signaling Technology, product number 4967S). Secondary antibodies used were ECL™ anti-rabbit IgG and HRP-conjugated F(ab')2 fragment (donkey) (GE Healthcare, product number NA934V). Western blots were detected using Amersham™ ECL™ Prime Western Blotting Detection Reagents (GE Healthcare, model number RPN2232), and images were acquired using a ChemiDoc Touch imaging system (Bio-Rad Laboratories, Inc.). Expression of the transgene products hmBDNF and TrkB, as well as activation of TrkB, was confirmed in cells transfected with rAAV#036 (Figure 2). In the figure, rAAV#036 is abbreviated as "#036," and hmBDNF is abbreviated as "BDNF." [Example]

[0086] Expression of transgene products and activation of TrkB in mouse retinal tissue after intravitreal injection of rAAV#036 Five-week-old male C57BL / 6J mice (Charles River Japan, Inc.) were intravitreally administered with vehicle or rAAV#036, and the expression level of BDNF in the retinal tissue was analyzed 3 weeks after administration. A solution of DPBS supplemented with 0.001% Pluronic™ F-68 (Thermo Fisher Scientific, model number 24040032) was used as the vehicle. rAAV#036 was administered at a dose of 3.0 × 10 per eye. 7 (3.0e7)vg / 1μL, 9.0×10 7 (9.0e7)vg / 1μL, or 2.7×10 8 The drug was administered intravitreally at a dose of (2.7e8)vg / 1μL. A glass pipette (Sankyo Medic) connected to a microinjector FemtoJet® 4i (Eppendorf) was inserted into the vitreous of 5-week-old C57BL / 6J mice under anesthesia, and 1μL was administered per eye. Three weeks after administration, the mice were euthanized by exsanguination under isoflurane anesthesia, and retinal tissue was collected. The collected retinal tissue was frozen on dry ice, and then added with the same cell lysis buffer used in the expression analysis of the introduced gene product in cultured cells in Example 2. The tissue was homogenized using a Biomasher (Nippi Corporation, model number 320103) and stored at -80°C. The thawed solution was left on ice for 20–30 minutes and then centrifuged at 15,000 rpm at 4°C for 10 minutes in a Hitachi centrifuge. The supernatant was used for the following experiments. The protein concentration of the samples was measured using the Pierce™ BCA Protein Assay Kit and determined from the absorbance at 562 nm using a microplate reader. The amount of hmBDNF protein expression was calculated from the absorbance (absorbance at 450 nm minus absorbance at 540 nm) measured using the Human Free BDNF Quantikine™ ELISA Kit (R&D Systems, Inc., catalog no. DBD00) and corrected for total protein concentration (Figure 3). As shown in Figure 3, hmBDNF expression was confirmed in the retinal tissue of mice administered rAAV#036 intravitreally. Furthermore, Western blot analysis was used to evaluate the expression of the transgene products (hmBDNF and TrkB) and the activation of TrkB in the retina following administration of rAAV#036. For this evaluation, a high dose (2.7 × 10 8 A group administered rAAV#036 (vg / 1 μL) and a group administered vehicle were given, and three samples from each group showing values ​​closest to the median in hmBDNF expression analysis using ELISA were selected. The reagents and procedures used for this evaluation were the same as those in Example 2. Expression of hmBDNF and TrkB as transgene products and activation of TrkB (pTrkB) were confirmed in mouse retinal tissue transduced with rAAV#036 (Figure 4). In the figure, rAAV#036 is abbreviated as "#036," and hmBDNF is abbreviated as "BDNF." [Example]

[0087] rAAV genomic DNA analysis After subsequent treatment with DNase I and then proteinase K, the AAV genomic DNA of rAAV#036 was purified by isopropanol precipitation. DNA concentration was measured using a fluorometer (Thermo Fisher Scientific, Qubit® Fluorometer and Qubit® dsDNA HS Assay Kit), and 160 ng of AAV genomic DNA was analyzed by electrophoresis on an alkaline agarose gel containing 50 mM sodium hydroxide (NaOH). The AAV genomic DNA and DNA size markers used for electrophoresis were denatured at 95°C for 5–10 min in the presence of 50 mM NaOH / 0.3% SDS. After electrophoresis, the gel was stained with a single-stranded DNA staining reagent (Biotium, model number 41003, GelRed™), and DNA was detected using a UV transilluminator (Bio-Rad Laboratories, Inc., ChemiDoc MP Imaging System) (Figure 5). For rAAV#007, AAV genomic DNA analysis was performed on rAAV#007 and rAAV#008 in the same manner, except that genomic DNA purification was performed using a DNA purification column (QIAGEN, model number 28104, QIAquick™ PCR Purification Kit). For electrophoresis, 200 ng of genomic DNA was used for rAAV#007 and rAAV#008. For rAAV#036 and rAAV#008 (both of which contain a CMV promoter), as shown in Figure 5, see Patent Document 1 and Non-Patent Document 26 (e.g., rAAV#007). [Example]

[0088] Assessment of rAAV productivity 0.2% Triton X-100 and 200 mM NaCl (both final concentrations) were added to the culture medium of rAAV#007 and rAAV#008 producer cells to obtain cell lysates. The cells were then treated with DNase I and Exo I, followed by protease treatment and purification of AAV genomic DNA using a QIAamp MinElute Virus Spin Kit (QIAGEN, model no. 57704). Real-time PCR was then performed using the AAVpro® Titration Kit for Real Time PCR (Takara Bio, model no. 6233) and the ITR primers provided with the kit. A calibration curve was created using the standard DNA provided with the kit, and the relative titer of the vector genome concentration in the cell lysates was calculated (Figure 6). For rAAV#008 and rAAV#036, AAV genomic DNA was extracted by DNase I / Exo I treatment followed by proteinase K treatment, diluted with water, and then subjected to real-time PCR. Cell lysates were obtained 5 days after transfection in the same manner as above, and the vg contained in each cell lysate was quantified. As shown in Figure 6, for rAAV#008 containing a CMV promoter, see Patent Document 1 and Non-Patent Document 26 (e.g., rAAV#007). Furthermore, rAAV#036 was confirmed to exhibit high productivity, similar to rAAV#008. [Example]

[0089] rAAV2.7m8 vector and rAAV2 Max vector AAV genomic DNA analysis and rAAV productivity evaluation were performed on rAAV2.Max#036 and rAAV2.7m8#036. The genomic integrity of rAAV2.Max#036 and rAAV2.7m8#036 was confirmed (Figure 7). The relative titer contained in each cell lysate was quantified. rAAV2.7m8#036 was confirmed to have improved productivity compared to rAAV#036 and rAAV2.Max#036 (Figure 8). [Example]

[0090] Expression of transgene products in monkey retinal tissue after intravitreal injection of rAAV2.7m8 vector and rAAV2 Max vector Female cynomolgus monkeys (Shin-Nihon Biological Research Institute) were injected with rAAV#036, rAAV2.Max#036, and rAAV2.7m8#036 at 6.3 × 10 per eye. 10 The insulin was administered intravitreally at a dose of 70 μL / vg. Under anesthesia, a 30G MYSHOT® insulin syringe (NIPRO Pharma Vietnam Co., Ltd.) was inserted into the vitreous of each monkey, and 70 μL was administered per eye. Eight weeks after administration, the monkeys were euthanized by exsanguination under anesthesia, and retinal tissue was collected. The retinal tissue samples were frozen and homogenized using biomarkers. DNA and RNA were then isolated using Nucleospin® RNA / Protein (Takara Bio, model number 740933) and Nucleospin® RNA / DNA Buffer Set (Takara Bio, model number 740944). The DNA and RNA concentrations of the samples were measured using a NanoDrop™ 8000 spectrophotometer (Thermo Fisher Scientific). Vector copy number and RNA expression levels were analyzed by real-time PCR using Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, product number 4368708). Vector copy number was calculated using primers designed based on the CMV promoter sequence of SEQ ID NO: 26. RNA primers were designed based on the BDNF and TrkB sequences of SEQ ID NO: 26, respectively. BDNF and TrkB RNA expression levels were normalized by GAPDH. In monkey retina, increased vector copy number was observed with rAAV2.7m8#036 compared to rAAV#036 and rAAV2.Max#036 (Figure 9). Similarly, enhanced BDNF and TrkB RNA expression levels were observed with rAAV2.7m8#036 compared to rAAV#036 and rAAV2.Max#036 in monkey retina (Figure 10). Thus, these data indicate that rAAV vectors according to the present invention, when administered in vivo, demonstrate increased transduction efficiency and can increase BDNF and TrkB RNA expression levels in retinal tissue. [Example]

[0091] rAAV2.7m8#036-associated retinal ganglion cell (RGC) structure and function in a monkey ocular hypertension model Male cynomolgus monkeys (Shin Nippon Biomedical Laboratories, Ltd.) aged 4 to 9 years were used as an experimental glaucoma model. Laser irradiation was performed at a wavelength of 532 nm, uniformly irradiating the trabecular meshwork in a 360° angle, as previously described (Ophthalmic Res., 2017. 58(2): 99-106). As previously reported, intraocular pressure (IOP) increased in laser-treated eyes compared with untreated eyes. Vehicle or rAAV2.7m8#036 laser irradiation resulted in a 6.0 × 10 per eye dose of 6.0 × 10 per eye 19 days after laser irradiation. 10(6.0e10)vg / 70μL or 3.0×10 11 The drug was administered intravitreally at a dose of (3.0e11) vg / 70 μL. A solution of 0.01% Poloxamer 188 (Merck Millipore, product number 137097) in PBS was used as the vehicle. Under anesthesia, monkeys were injected into the vitreous with a 30G MYSHOT® insulin syringe or a BD Ultra-Fine® 8 mm x 30G needle-equipped BD insulin syringe (Becton Dickinson & Co.), and 70 μL was administered per eye. Perioptic nerve head retinal nerve fiber layer (RNFL) thickness and photopic negative response (PhNR) were measured in both eyes of each monkey under anesthesia 16 weeks after laser application. Global RNFL thickness, measured by circular scanning of both optic nerve discs, was measured as previously described (Ophthalmic Res., 2017. 58(2): 99-106). Suprafoveal electroretinograms were measured using a contact lens-type electrode placed on the cornea using a Kowa ER-80 (Kowa Co., Ltd.) and PuREC (PC100-A, Mayo Ltd.) with a light stimulus (duration: 100 ms, stimulus light: 5, background light: 5, stimulus light size: 15°, intensity: 3.082 cds / m², background light: white). PhNR is a slow, negative-going wave reflecting the activity of RGCs and their axons. Decreased PhNR amplitude has been reported in patients with glaucoma (Doc Ophthalmol., 2018. 136(3): 207-211; Invest Ophthalmol Vis Sci., 2008. 49: 2201-2207). PhNR amplitude was measured from the peak of the b-wave to the maximum amplitude at the trough immediately after the i-wave, as reported (Doc Ophthalmol., 2018. 136(3): 207-211). A protective effect of rAAV2.7m8#036 on global RNFL thickness was observed in laser-treated eyes, with global RNFL thickness remaining similar to that in non-laser-treated eyes. In contrast, global RNFL thickness in vehicle-administered laser-treated eyes was reduced compared to non-laser-treated eyes (Figure 11). A protective effect of rAAV2.7m8#036 on the rate of change in PhNR amplitude from pre-administration in laser-treated eyes was also observed. In contrast, the rate of change in PhNR in vehicle-administered laser-treated eyes was reduced compared to non-laser-treated eyes (Figure 12). Thus, these data demonstrate that the rAAV vector according to the present invention exhibits a protective effect on RGC-related structure and function in an experimental monkey model of glaucoma.

[0092] conclusion As demonstrated throughout the Examples, the inventors have discovered that rAAV vectors comprising a cytomegalovirus (CMV) promoter operably linked to a naturally occurring TrkB gene and a naturally occurring mature BDNF gene demonstrate reduced genomic DNA fragmentation / cleavage, which increases the production efficiency of the claimed rAAV vectors of the invention comprising a CMV promoter operably linked to naturally occurring TrkB and mBDNF, increases retinal transduction efficiency, and demonstrates protective effects on RGC-associated structure and function in an experimental monkey model of glaucoma. References 1. Patent Document 1 2. Patent Document 2 3. Non-patent document 26 4. Non-patent document 27

Claims

1. 1. A recombinant adeno-associated virus (rAAV) vector comprising a genetic construct, the genetic construct comprising, in a 5′ to 3′ direction: cytomegalovirus (CMV) promoter; a first coding sequence encoding tyrosine kinase receptor B (TrkB); A nucleotide sequence encoding a linker for producing TrkB and mature brain-derived neurotrophic factor (mBDNF) as individual proteins; and a second coding sequence encoding mBDNF; in that order, wherein the CMV promoter is operably linked to a first and a second coding sequence.

2. 2. The recombinant rAAV vector of claim 1, wherein the CMV promoter comprises the nucleotide sequence set forth in SEQ ID NO: 1 or a fragment or variant thereof.

3. 2. The recombinant rAAV vector of claim 1, wherein the first coding sequence encodes a naturally occurring TrkB or a functional variant thereof.

4. 2. The recombinant rAAV vector of claim 1, wherein the first coding sequence encodes the amino acid sequence set forth in SEQ ID NO:2 or a fragment or variant thereof, and / or the first coding sequence comprises the nucleotide sequence set forth in SEQ ID NO:3 or a fragment or variant thereof.

5. 2. The recombinant rAAV vector of claim 1, wherein the second coding sequence encodes a naturally occurring mBDNF.

6. 2. The recombinant rAAV vector of claim 1, wherein the second coding sequence encodes the amino acid sequence set forth in SEQ ID NO:4 or a fragment or variant thereof, and / or the second coding sequence comprises the nucleotide sequence set forth in SEQ ID NO:5 or a fragment or variant thereof.

7. The recombinant rAAV vector of claim 1, wherein the gene construct further comprises a nucleotide sequence encoding a signal peptide, and optionally the signal peptide is located 5' to the nucleotide sequence encoding mBDNF and / or the nucleotide sequence encoding the signal peptide is located 3' to the nucleotide sequence encoding a linker.

8. 8. The recombinant rAAV vector of claim 7, wherein the nucleotide sequence encoding the signal peptide encodes the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 20, or a fragment or variant thereof, and / or the signal peptide comprises the nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 21, or a fragment or variant thereof.

9. The recombinant rAAV vector of claim 1 , wherein the linker is a P2A peptide.

10. 2. The recombinant rAAV vector of claim 1, wherein the nucleotide sequence encoding the linker encodes the amino acid sequence set forth in SEQ ID NO: 8 or a fragment or variant thereof, and / or the linker comprises the nucleotide sequence set forth in SEQ ID NO: 9 or a fragment or variant thereof.

11. 2. The recombinant rAAV vector of claim 1, wherein the genetic construct further comprises a nucleotide sequence encoding a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), optionally wherein the WPRE comprises the nucleotide sequence set forth in SEQ ID NO: 10, or a fragment or variant thereof.

12. 2. The recombinant rAAV vector of claim 1, wherein the genetic construct further comprises a nucleotide sequence encoding a polyA signal sequence, and optionally the polyA signal sequence comprises the nucleotide sequence set forth in SEQ ID NO: 11 or a fragment or variant thereof.

13. 2. The recombinant rAAV vector of claim 1, comprising the genetic construct comprising, in the 5' to 3' direction, a CMV promoter sequence, a first coding sequence encoding TrkB, a nucleotide sequence encoding a P2A linker peptide, a nucleotide sequence encoding a signal peptide, a second coding sequence encoding mBDNF, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a Simian Virus 40 (SV40) polyA signal sequence.

14. 2. The recombinant rAAV vector of claim 1, wherein the genetic construct comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 14 to 17, or a variant or fragment thereof.

15. The recombinant rAAV vector of claim 1 , wherein the rAAV vector is an rAAV2 vector.

16. The recombinant rAAV vector of claim 1, wherein the rAAV vector is an rAAV2.7m8 vector.

17. 10. The recombinant rAAV vector of claim 1 for use as a medicine or in therapy.

18. The recombinant rAAV vector of claim 1 for use in treating, preventing, or ameliorating optic nerve disorders and / or retinal degenerative diseases accompanied by degeneration of retinal ganglion cells.

19. The recombinant rAAV vector of claim 18, wherein the optic nerve disorder and / or retinal degenerative disease is glaucoma or optic neuropathy.

20. A method for treating, preventing, or ameliorating optic neuropathy and / or retinal degenerative disease in a subject, comprising administering to a subject in need of treatment a therapeutically effective amount of the recombinant rAAV vector described in claim 1.

21. A pharmaceutical composition comprising the recombinant rAAV vector of claim 1 and a pharmaceutically acceptable vehicle.

22. 22. A method for preparing the pharmaceutical composition of claim 21, comprising contacting the recombinant rAAV vector of claim 1 with a pharmaceutically acceptable vehicle.

23. 2. A method for producing the recombinant rAAV vector of claim 1, comprising the steps of: (i) introducing into an rAAV vector-producing cell a genetic construct, wherein the genetic construct comprises, in a 5′ to 3′ direction: cytomegalovirus (CMV) promoter; a first coding sequence encoding tyrosine kinase receptor B (TrkB); A nucleotide sequence encoding a linker for producing TrkB and mature brain-derived neurotrophic factor (mBDNF) as individual proteins; and a second coding sequence encoding mBDNF; in that order, wherein the CMV promoter is operably linked to a first and a second coding sequence; and (ii) culturing the rAAV vector-producing cells to produce the recombinant rAAV vector of claim 1; A method comprising:

24. 2. An rAAV vector producing cell comprising the genetic construct of the recombinant rAAV vector of claim 1.

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