Adeno-associated virus vector capable of delivering astrocyte-specific gene

Abstract

The present invention relates to mutants of adeno-associated virus (AAV) capsid proteins. A recombinant viral vector carrying a mutant of the AAV1 capsid protein is useful for the specific expression of introduced genes in astrocytes at spinal cord injury lesion sites.

Description

Adeno-associated virus vector capable of astrocyte-specific gene transfer

[0001] The present invention relates to a mutant of an adeno-associated virus (AAV) capsid protein, and a recombinant viral vector comprising a mutant of an AAV1 capsid protein is useful for expressing a transgene specifically in astrocytes in a spinal cord injury lesion site.

[0002]

[0003] In order to effectively perform gene therapy, the development of gene delivery technology that can deliver therapeutic genes to the desired target cells and achieve high expression efficiency is of utmost priority.

[0004] Among these gene transfer technologies, AAV is a non-pathogenic virus that has no side effects on infected cells and has a low probability of causing mutations in the genetic information of target cells, making it superior to other gene therapy technologies in terms of safety.

[0005] Adeno-Associated Virus (AAV) is a non-enveloped, single-stranded DNA virus that can infect both dividing and non-dividing cells. AAV can replicate only in the presence of a helper virus and is non-pathogenic to humans. This characteristic makes it a useful method for introducing genes into various cells and a useful vector for gene therapy.

[0006] AAV exists in various serotypes, and it is known that the host and virus characteristics differ depending on the serotype. Serotype 2 (AAV2) is a serotype that has been widely studied for a long time and can infect various cells. Serotypes 1 (AAV1), serotype 5 (AAV5), and serotype 6 (AAV6) are serotypes with more tissue specificity. AAV1 is known to have high gene transfer efficiency in muscles, liver, airway, and central nervous system, AAV5 in the central nervous system, liver, and retina, and AAV6 in the heart, muscle, and liver. Although the gene transfer characteristics to specific tissues vary depending on the serotype, it is still easy to transfer to other tissues, so the development of new AAV vectors that can improve tissue specificity, thereby enhancing safety and efficiency, is essential.

[0007] Meanwhile, damage to the spinal cord due to trauma or other causes can lead to paralysis of bodily functions. Spinal cord nerve tissue has a simple structure compared to the brain, but nerve regeneration is extremely difficult. Pathological phenomena following spinal cord injury can be broadly divided into two stages: primary and secondary. Primary injury occurs within minutes of injury, with cells at the site of injury undergoing necrosis. During this period, cell destruction is so rapid that pharmacological treatment is nearly impossible. Secondary injury, which follows the primary injury, progresses slowly over hours to days. Not only do cells at the site of injury degenerate, but also surrounding neurons and oligodendrocytes undergo gradual cell death through apoptosis. This cell death continues to progress, centered around the injury site, eventually expanding the damaged area within the spinal cord. In addition, the axons, which are the pathways for nerve signals, and the myelin sheath that helps the axons function degenerate, eventually forming empty spaces like holes, making it impossible for nerve signals to transmit any further, resulting in permanent loss of function.

[0008] Currently, the only treatment approved by the U.S. Food and Drug Administration (FDA), high-dose methylprednisolone, is administered within 8 hours of injury and is intended to reduce acute inflammation but not promote neurological recovery [Z. Liu et al., High-dose methylprednisolone for acute traumatic spinal cord injury: a meta-analysis. Neurology 93, e841-e850 (2019)]. Therefore, novel treatments to promote functional recovery are needed.

[0009] Pathologically, traumatic spinal cord injury triggers a series of processes including rapid neuronal cell death, forming a glial scar [MD Norenberg, J. Smith, A. Marcillo, The pathology of human spinal cord injury: defining the problems. Journal of neurotrauma 21, 429-440 (2004)]. This scar, composed primarily of reactive astrocytes, initially acts as a protective barrier by isolating damaged neurons from healthy tissue, but impedes neuronal regeneration [JE Burda, MV Sofroniew, Reactive gliosis and the multicellular response to CNS damage and disease. Neuron 81, 229-248 (2014)]. Therefore, direct in vivo reprogramming of these reactive astrocytes to convert them into functional neurons is considered a promising therapeutic strategy.

[0010] However, the specific delivery of astrocyte reprogramming factors to astrocytes is challenging. When delivered to cells other than astrocytes, such as neurons, oligodendrocytes, and microglia, it can disrupt normal gene expression and lead to cell death or functional defects. For example, overexpression of these transcription factors in neurons can disrupt gene expression patterns, leading to abnormal neuronal behavior, loss of function, and even cell death. To ensure safe and effective therapeutic gene delivery or reprogramming to astrocytes surrounding neural injury sites, it is necessary to improve the astrocyte specificity of AAV vectors and minimize conversion in non-target cells.

[0011] Accordingly, although attempts are being made to improve gene transfer efficiency by modifying AAV capsid proteins, research on adeno-associated viruses targeting astrocytes in the spinal cord injury lesion area is limited.

[0012]

[0013] Accordingly, the inventors of the present invention have made extensive efforts to solve the above-mentioned problem and have completed the present invention by developing a protein variant based on the capsid of a novel adeno-associated virus (AAV) serotype 1 that targets spinal cord astrocytes and has the potential to deliver a gene therapy agent that addresses the fundamental genetic cause of spinal cord injury-related diseases.

[0014]

[0015] Accordingly, the purpose of the present invention is to provide a mutant of AAV1 capsid protein for improving the efficiency of gene introduction into target tissues or cells by recombinant AAV and / or improving the efficiency of genetic information expression.

[0016] In addition, another object of the present invention is to provide a nucleic acid encoding a mutant of the AAV1 capsid protein.

[0017] In addition, another object of the present invention is to provide a recombinant AAV1 vector comprising a nucleic acid encoding a mutant of the AAV1 capsid protein.

[0018] In addition, another object of the present invention is to provide a pharmaceutical composition comprising the recombinant AAV1 vector.

[0019] In addition, another object of the present invention is to provide a gene delivery vehicle comprising the recombinant AAV1 vector.

[0020]

[0021] The present invention relates to a recombinant AAV1 capsid variant having a high-efficiency gene transfer ability by specifically targeting spinal cord astrocytes, and a recombinant viral vector comprising a nucleic acid encoding a mutant of an AAV1 capsid protein is useful for the expression of a transgene in spinal cord astrocyte target cells, thereby enabling the prevention or treatment of spinal cord injury-related diseases.

[0022]

[0023] Figure 1a shows the 3D structure of a recombinant AAV1 variant (AAVSN2 variant). This variant is an AAV1-based variant with a total of three point mutations (D595H, D712E, F713Y), and all three sites are exposed on the surface [red site: 595, blue site: 712, pink site: 713].

[0024] Figure 1b shows the 3D structure of a recombinant AAV1 variant (AAVSN3 variant). This variant is an AAV1-based variant with a total of four point mutations (K315R F584L A598V N642H). Among the four sites, the F584L A598V site is exposed on the surface, and the remaining K315R N642H site is an internal structure [red site: 315, blue site: 584, pink site: 598, orange site: 642].

[0025] Figure 2a shows a cleavage map of the pCMV GFP plasmid.

[0026] Figure 2b shows a pHelper plasmid cleavage map.

[0027] Figures 3a, 3b and 3c show the cleavage maps of the wild-type AAV1 vector, AAVSN2 and AAVSN3 mutants, respectively.

[0028] Figure 4 shows a cleavage map of the NeuroD1-P2A-GFP plasmid.

[0029] Figure 5 shows a comparison of the genomic titers of the packaging efficiency of wild-type AAV1 and the recombinant AAV1 variants (AAVSN2, AAVSN3 variants) of the present invention using quantitative PCR analysis.

[0030] Figure 6 shows the ratio of cells expressing GFP when AAVSN3-CMV-GFP was administered to primary mixed glial cells extracted from the spinal cord of 7-week-old Sqrague-Dawley male rats to confirm the astrocyte specificity of the recombinant AAV1 variant (AAVSN3 variant).

[0031] Figure 7a shows the genomic titer of AAVSN2-GFP directly administered to the injured site of the spinal cord to confirm the astrocyte transduction ability of a recombinant AAV1 variant (AAVSN2 variant) in the spinal cord injury site in 7-week-old Sqrague-Dawley male rats 5 days after a left spinal cord injury, and astrocytes were extracted and compared with wild-type AAV1-GFP using quantitative PCR analysis.

[0032] Figure 7b shows an experiment conducted in the same manner as Figure 7a to confirm the astrocyte transduction ability of a recombinant AAV1 variant (AAVSN3 variant) in the spinal cord injury site, and the genomic titer was compared with that of wild-type AAV1-GFP.

[0033] Figure 7c shows the genomic titer measured by quantitative PCR at each location in the spinal cord of rats with left spinal cord injury to confirm the transduction ability of the recombinant AAV1 variant (AAVSN3 variant) over a distance from the spinal cord injury site, and the results were compared with those of wild-type AAV1.

[0034] Figure 8 shows the ratio of astrocytes expressing the GFP gene through wild-type AAV1 vector and recombinant AAV1 variants (AAVSN2 and AAVSN3 variants) in the lesion site of a rat spinal cord injury, as observed by flow cytometry, showing the astrocyte transduction ability of recombinant AAV1 variants in the lesion site.

[0035] Figure 9 shows the amount of GFP expression according to the distance from the injury site by analyzing images in which the GFP gene was expressed through a wild-type AAV1 vector and recombinant AAV1 variants (AAVSN2 and AAVSN3 variants) in the left spinal cord injury site of a rat.

[0036] Figure 10 shows a low-magnification image showing the results of local delivery within the nerve injury area confirmed by immunochemical staining at 2 weeks after injection of a recombinant AAV carrying GFP into the spinal cord injury area to confirm the gene transfer and expression ability of wild-type AAV1 and recombinant AAV1 variants of the present invention (AAVSN2 and AAVSN3 variants) in the spinal cord injury area of ​​a rat.

[0037] Figure 11a shows the gene transfer ability of wild-type AAV1 vector and recombinant AAV1 variant (AAVSN2 variant) in astrocytes near the spinal cord injury site confirmed at high magnification using immunohistochemistry and fluorescence imaging.

[0038] Figure 11b shows the gene transfer ability of wild-type AAV1 vector and recombinant AAV1 variant (AAVSN3 variant) in astrocytes near the spinal cord injury site confirmed at high magnification using immunohistochemistry and fluorescence imaging.

[0039] Figure 12 shows the gene transfer ability of wild-type AAV1 vector and recombinant AAV1 variants (AAVSN2 and AAVSN3 variants) in astrocytes near the lesion site of a left spinal cord injury confirmed at high magnification through immunochemical staining and fluorescence imaging, and the transduction ability to astrocytes was quantified using the ImageJ program.

[0040] Figure 13 shows the gene transfer ability of wild-type AAV1 vectors and recombinant AAV1 variants (AAVSN2 and AAVSN3 variants) in astrocytes and other cells near the lesion site of a left spinal cord injury [GFAP: astrocytes, Tuj1: neurons, Iba1: microglia].

[0041] Figure 14 shows the immune cells in the central nervous system by labeling Iba1 (rabbit anti-Iba1 antibody (1:500 dilution; Wako Pure Chemical Industries)) through immunochemical staining when the wild-type AAV1 vector and recombinant AAV1 variants (AAVSN2 and AAVSN3 variants) carrying the NeuroD1-P2A-GFP gene were administered to the lesion site of a left spinal cord injury.

[0042] Figure 15 shows the results of measuring the viral genome in other major organs other than the spinal cord by qPCR and normalizing it to GAPDH when wild-type AAV1 vector and recombinant AAV1 variants (AAVSN2 and AAVSN3 variants) were administered to the lesion site of a left spinal cord injury.

[0043] Figure 16 shows the recovery of motor ability of rats over time as a function of time in terms of changes in BBB (Basso, Beattie, and Bresnahan) scores when the NeuroD1-P2A-GFP and CMV-GFP genes were delivered to the left spinal cord injury lesion site using a wild-type AAV1 vector and a recombinant AAV1 variant (AAVSN2 variant).

[0044] Figure 17 shows the results comparing the degree of reprogramming of astrocytes into neural cells when NeuroD1-P2A-GFP was delivered to the left spinal cord injury lesion site via a wild-type AAV1 vector and a recombinant AAV1 variant (AAVSN2 variant).

[0045] Figure 18 shows changes in body weight after introduction of therapeutic genes into recombinant AAV1 variants [(a) AAVSN2 variant; (b) AAVSN3 variant].

[0046] Figure 19 shows the results of blood toxicity tests after introduction of therapeutic genes into recombinant AAV1 variants [(a) AAVSN2 variant; (b) AAVSN3 variant].

[0047]

[0048] Hereinafter, the present invention will be described in more detail as follows.

[0049] The present invention relates to a mutant of adeno-associated virus serotype 1 (AAV1) capsid protein.

[0050] The term "adeno-associated virus" or "AAV" as used herein refers to all adeno-associated viruses used in gene therapy, including their derivatives, viral subtypes, and naturally occurring and recombinant forms. Various serotypes of AAV can be used as recombinant gene transfer viruses to transduce a variety of different cell types. The genomic sequences of various AAV serotypes, as well as the sequences of the native terminal repeat (TR), Rep protein, and capsid subunit, are known in the art. These sequences can be found in the literature or in public databases such as GenBank. For example, see GenBank Accession No. NC_002077 (AAV-1) and AF063497 (AAV-1).

[0051] The term "serotype" as used herein means a subdivision of AAV that can be identified by serological or DNA sequencing methods and distinguished by its antigenic characteristics.

[0052] The term "capsid" as used in the present invention refers to a protein encoded by the cap gene present in the viral genome, which constitutes the outer shell of the virus. The wild-type AAV genome or cap gene encodes three types of capsid proteins (VP1, VP2, and VP3). The wild-type AAV1 capsid protein comprises the amino acid sequence represented by SEQ ID NO: 1.

[0053] In one embodiment, the invention provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant comprises a mutant of an AAV1 capsid protein having one or more mutations selected from among D595H, D712E, F713Y, K315R, F584L, A598V, and N642H in the amino acid sequence of SEQ ID NO: 1 of a wild-type AAV1 capsid protein.

[0054] The above D595H mutation is a substitution of aspartic acid at position 595 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with histidine.

[0055] The above D712E mutation is a substitution of aspartic acid at position 712 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with glutamic acid.

[0056] The above F713Y mutation is a substitution of phenylalanine at position 713 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with tyrosine.

[0057] The above K315R mutation is a substitution of lysine at position 315 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with arginine.

[0058] The above F584L mutation is a substitution of phenylalanine at position 584 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with leucine.

[0059] The above A598V mutation is a substitution of alanine at position 598 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with valine.

[0060] The above N642H mutation is a substitution of asparagine at position 642 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with histidine.

[0061] In one embodiment, the present invention can provide a mutant of adeno-associated virus serotype 1 (AAV1) capsid protein having one or more mutations selected from T194A, A196S, A197G, T201N, S205A, L129F, A224S, N223S, H272T, S268T, T265S, A263G, S262N and S264T.

[0062] The above T194A mutation is a substitution of threonine at position 194 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with alanine.

[0063] The above A196S mutation is a substitution of alanine at position 196 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with serine.

[0064] The above A197G mutation is a substitution of alanine at position 197 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with glycine.

[0065] The above T201N mutation is a substitution of threonine at position 201 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with asparagine.

[0066] The above S205A mutation is a substitution of serine at position 205 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with alanine.

[0067] The above L129F mutation is a substitution of leucine at position 129 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with phenylalanine.

[0068] The above A224S mutation is a substitution of alanine at position 224 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with serine.

[0069] The above N223S mutation is a substitution of asparagine at position 223 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with serine.

[0070] The above H272T mutation is a substitution of histidine at position 272 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with threonine.

[0071] The above S268T mutation is a substitution of serine at position 268 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with threonine.

[0072] The above T265S mutation is a substitution of threonine at position 265 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with serine.

[0073] The above A263G mutation is a substitution of alanine at position 263 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with glycine.

[0074] The above S262N mutation is a substitution of serine at position 262 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with asparagine, and / or

[0075] The above S264T mutation is a substitution of serine at position 264 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein with threonine.

[0076] In one embodiment, the invention provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant comprises or consists of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5.

[0077] In one embodiment, the mutant of the AAV1 capsid protein according to the present invention comprises or consists of the amino acid sequence (VP1) set forth in SEQ ID NO: 3 or SEQ ID NO: 5. VP2 corresponds to the amino acid sequence from T138 to the end of SEQ ID NO: 3 or SEQ ID NO: 5, and VP3 corresponds to the amino acid sequence from M203 to the end of SEQ ID NO: 3 or SEQ ID NO: 5.

[0078] The mutant of the AAV1 capsid protein represented by the above SEQ ID NO: 3 was named SN2 and has mutations of D595H, D712E, and F713Y.

[0079] The mutant of the AAV1 capsid protein represented by the above SEQ ID NO: 5 was named SN3 and has mutations of K315R, F584L, A598V, and N642H.

[0080] In one embodiment, the invention provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant has mutations K315R, D595H, D712E, and F713Y.

[0081] In one embodiment, the invention provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant has mutations F584L, A598V, and N642H.

[0082] The term "wild type" as used in the present invention refers to the form most commonly found in wild populations among species. With respect to mutants, the wild type refers to the phenotype considered basic or that individual. The wild type is also called the normal type as an alias. Meanwhile, in this specification, the term "mutant" refers to a protein, virus, cell, individual, etc. in which a mutated gene appears as a phenotypic change. Furthermore, in this specification, the term "mutant" may also refer to the gene itself that has mutated.

[0083] The present invention also includes a nucleic acid encoding a mutant of the AAV1 capsid protein.

[0084] The nucleic acid of the present invention encodes a mutant of the AAV1 capsid protein. The nucleic acid of the present invention is produced by substituting at least one base in the base sequence of a nucleic acid (cap gene) encoding the AAV1 capsid protein with a different base. The nucleic acid of the present invention may exist in the form of DNA, but may also be in the form of RNA or a chimera of DNA and RNA. The nucleic acid of the present invention also includes a complementary nucleic acid (e.g., cDNA). The nucleic acid of the present invention may be single-stranded or double-stranded, but is preferably double-stranded.

[0085] The above wild-type AAV1 cap gene is represented by the base sequence of SEQ ID NO: 2.

[0086] The above D595H mutation can be encoded by substituting GAT at positions 1783 to 1785 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with CAC or CAT.

[0087] The above D712E mutation can be encoded by substituting GAT at positions 2134 to 2136 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with GAA or GAG.

[0088] The above F713Y mutation can be encoded by substituting TTT at positions 2137 to 2139 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with TAT or TAC.

[0089] The above K315R mutation can be encoded by substituting AAA at positions 943 to 945 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with AGA, AGG, CGA, CGC, CGG, or CGT.

[0090] The above F584L mutation can be encoded by substituting TTT at positions 1750 to 1752 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with CTA, CTC, CTG, CTT, TTA, or TTG.

[0091] The above A598V mutation can be encoded by substituting GCT at positions 1792 to 1794 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with GTA, GTC, GTG, or GTT.

[0092] The above N642H mutation can be encoded by substituting AAC at positions 1924 to 1926 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with CAC or CAT.

[0093] The present invention is not particularly limited to a nucleic acid encoding a mutant of an AAV1 capsid protein having an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5, but as an example, a nucleic acid having a base sequence represented by SEQ ID NO: 4 or SEQ ID NO: 6 is exemplified.

[0094] The above T194A mutation can be encoded by substituting ACC at positions 580 to 582 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with GCA, GCC, GCG, or GCT.

[0095] The above A196S mutation can be encoded by substituting GCT at positions 586 to 588 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with AGC, AGT, TCA, TCC, TCG, or TCT.

[0096] The above A197G mutation can be encoded by substituting GCT at positions 599 to 591 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with GGA, GGC, GGG, or GGT.

[0097] The above T201N mutation can be encoded by substituting ACT at positions 601 to 603 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with AAC or AAT.

[0098] The above S205A mutation can be encoded by substituting TCA at positions 613 to 615 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with GCA, GCC, GCG, or GCT.

[0099] The above L129F mutation can be encoded by substituting CTC at positions 385 to 387 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with TTC or TTT.

[0100] The above A224S mutation can be encoded by substituting GCC at positions 670 to 672 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with AGC, AGT, TCA, TCC, TCG, or TCT.

[0101] The above N223S mutation can be encoded by substituting AAT at positions 667 to 669 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with AGC, AGT, TCA, TCC, TCG, or TCT.

[0102] The above H272T mutation can be encoded by substituting CAC at positions 814 to 816 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with ACA, ACC, ACG, or ACT.

[0103] The above S268T mutation can be encoded by substituting AGC at positions 802 to 804 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with ACA, ACC, ACG, or ACT.

[0104] The above T265S mutation can be encoded by substituting ACG at positions 793 to 795 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with AGC, AGT, TCA, TCC, TCG, or TCT.

[0105] The above A263G mutation can be encoded by substituting GCT at positions 787 to 789 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with GGA, GGC, GGG, or GGT.

[0106] The above S262N mutation can be encoded by substituting AGT at positions 784 to 786 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with AAC or AAT.

[0107] The above S264T mutation can be encoded by substituting TCA at positions 790 to 792 in the base sequence of SEQ ID NO: 2 of the wild-type AAV1 cap gene with ACA, ACC, ACG, or ACT.

[0108] The nucleic acid of the present invention may be operably linked to an appropriate control sequence. Control sequences include a promoter sequence, a polyadenylation signal, a transcription termination sequence, an upstream regulatory domain, an internal ribosome entry site (IRES), an enhancer, and the like. Promoter sequences include an inducible promoter sequence and a constitutive promoter sequence. The control sequence may be native to the AAV from which the capsid protein is derived, or may be foreign, a natural sequence, or a synthetic sequence. Recombinant DNA capable of expressing a mutant of an AAV1 capsid protein comprising the nucleic acid of the present invention is also encompassed by the present invention.

[0109] The recombinant DNA described above is useful for delivering the nucleic acid of the present invention to cells in vitro, ex vivo, and in vivo, and for conferring upon said cells the ability to express mutants of the AAV1 capsid protein. Furthermore, cells delivered with the nucleic acid of the present invention are also useful for producing recombinant AAV particles. The recombinant DNA described above can be used to deliver or introduce the nucleic acid of the present invention into eukaryotic cells, preferably animal cells, and more preferably mammalian cells.

[0110] In the present invention, recombinant DNA can be produced by retaining the nucleic acid of the present invention in DNA used as a vector. For example, plasmids, phages, transposons, cosmids, episomal DNA, viral genomes, etc. can be used.

[0111] For example, a packaging plasmid can be constructed by harboring a nucleic acid (cap gene) encoding a mutant of the AAV1 capsid protein of the present invention in a plasmid. The packaging plasmid can additionally contain any nucleic acid sequence, such as a nucleic acid (rep gene) encoding a replicase (Rep) protein. Preferably, the rep gene can be supplemented with Rep derived from AAV2.

[0112] A recombinant DNA comprising the nucleic acid of the present invention can also be produced by substituting at least one base in the PLA2 domain coding region of the nucleic acid sequence of the cap gene loaded on a known packaging plasmid with a different base. The packaging plasmid is not particularly limited, but a packaging plasmid loaded with a cap gene, preferably a packaging plasmid loaded with a cap gene and a rep gene, is exemplified. For example, in the present invention, a recombinant AAV1 vector represented by SEQ ID NO: 7 or SEQ ID NO: 8, which is a packaging plasmid loaded with a nucleic acid (cap gene) encoding a mutant of the AAV1 capsid protein of the present invention and a rep gene, was produced, i.e., pxx_AAVSN2 or pxx_AAVSN3.

[0113] The method of introducing a base substitution into a nucleic acid can be carried out by a known method, and there is no particular limitation, but it can be achieved by using a commercially available reagent, for example, Mutagenesis Basal Kit (TAKARA BIO INC.), and performing PCR according to the instructions included with the kit.

[0114] Accordingly, the present invention provides a recombinant AAV1 vector comprising a nucleic acid encoding the AAV1 capsid protein variant.

[0115] The recombinant AAV vector of the present invention is useful for gene introduction into target cells. The gene introduced by the recombinant AAV vector of the present invention is strongly expressed in the target cells.

[0116] The term "AAV vector" as used herein refers to any vector comprising or derived from components of an adeno-associated virus (AAV) and suitable for infecting mammalian cells, including human cells of any of many tissue types, such as brain, spinal cord, heart, lung, skeletal muscle, liver, kidney, spleen, or pancreas, whether in vitro or in vivo. The term "AAV vector" may be used to refer to an AAV-type viral particle (or virion) comprising at least a nucleic acid molecule encoding a protein of interest.

[0117] As used herein, "AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to a viral particle comprised of at least one AAV capsid protein (any capsid protein of wild-type AAV) and a polynucleotide rAAV vector encapsidated within the capsid. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector." Thus, the production of an rAAV particle necessarily involves the production of rAAV, since such vector is contained within the rAAV particle.

[0118] “Packaging” refers to a series of intracellular events that result in the assembly and incorporation of AAV particles into capsids.

[0119] The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding adeno-associated virus replication and capsid entry proteins. AAV rep and cap are referred to herein as AAV "packaging genes."

[0120] A "helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. A variety of such helper viruses for AAV are known in the art, including adenoviruses, rhesus viruses, and poxviruses such as vaccinia. Adenovirus type 5 of subgroup C is the most commonly used, but adenoviruses include many different subgroups. Numerous adenoviruses from humans, non-human mammals, and birds are known and available from depositories such as the ATCC. Viruses in the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); these are also available from depositories such as the ATCC.

[0121] "Helper virus function(s)" refers to the function(s) encoded in the helper virus genome that allow AAV replication and packaging (along with other requirements for replication and packaging described herein). As described herein, "helper virus functions" can be provided in a variety of ways, including by providing a helper virus or, for example, by transfecting a producer cell with a polynucleotide sequence encoding the essential function(s). For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenovirus proteins is transfected into a producer cell together with the rAAV vector.

[0122] In one embodiment, the AAV1 vector according to the present invention has an improved transduction profile for spinal cord astrocytes compared to an AAV1 vector comprising a wild-type capsid protein. That is, the AAV1 vector according to the present invention has the ability to target spinal cord astrocytes (e.g., astrocyte tropism).

[0123] As used herein, the term “tropism” refers to the specificity of an AAV capsid protein present in an AAV viral particle to infect or transduce a particular type of cell or tissue.

[0124] The tropism of an AAV capsid for a particular type of cell or tissue can be determined by measuring the ability of the AAV vector particle to infect or transduce a particular type of cell or tissue, including AAV1 capsid proteins, using standard assays well known in the art, such as those disclosed in the Examples herein.

[0125] That is, "tropism" means the ability of an AAV vector or virion to infect one or more specific cell types, but may also include whether the vector functions to transduce cells to one or more specific cell types; that is, tropism means preferential entry of the AAV vector or virion into a specific cell or tissue type(s) and / or preferential interaction with the cell surface that facilitates entry into a specific cell or tissue type, followed by expression (e.g., transcription and, if desired, translation) of sequences carried by the AAV vector or virion in the cell, e.g., expression of heterologous nucleotide sequence(s) in the case of a recombinant virus.

[0126] As used herein, the term "transduction" refers to the ability of an AAV vector or virion to infect one or more specific cell types; that is, transduction refers to introducing an AAV vector or virion into a cell and transferring the genetic material contained within the AAV vector or virion into the cell, thereby achieving expression from the vector genome. In some, but not all, cases, transduction and tropism may be correlated.

[0127] The AAVs described herein comprise amino acid modifications in one or more capsid proteins that confer new or enhanced tissue tropism properties. The AAV1 variants according to the present invention target astrocytes in the spinal cord.

[0128] The term "astrocytic tropism in the spinal region" as used herein means tropism for astrocytes in the spinal region.

[0129] In some embodiments, the astrocyte tropism of the peptide-modified hybrid AAV capsid protein in the spinal cord region is further increased by at least 5%, 10%, 20%, 30%, 40%, 50% or more compared to the astrocyte tropism of the wild-type AAV capsid protein without the peptide in the spinal cord region.

[0130] The present invention also encompasses a pharmaceutical composition comprising the recombinant AAV1 vector. The composition may further comprise a pharmaceutically acceptable carrier.

[0131] The term "pharmaceutically acceptable carrier" includes any substance which, when combined with the active ingredient of the composition, allows the ingredients to retain biological activity without causing adverse physiological responses, such as unintended immune responses. Pharmaceutically acceptable carriers include water, phosphate buffered saline, emulsions such as oil / water emulsions, and wetting agents. Compositions containing such carriers are described in Remington's Pharmaceutical Sciences, current Ed., Mack Publishing Co., Easton Pa. 18042, USA; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., 3rd ed. Amer. Pharmaceutical Assoc.

[0132] In one embodiment, the spinal cord injury-related disease may be a traumatic spinal cord injury, an inflammatory spinal cord injury, a vascular spinal cord injury, a tumor spinal cord injury, etc.

[0133] The term “treatment” as used herein refers to any type of intervention or process performed on a subject or the administration of an active agent to a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, or delaying or preventing the progression, development, severity or recurrence of a disease-related syndrome, complication, symptom or biochemical sign. Treatment can be performed on a subject with a disease or on a subject without a disease (e.g., for prophylaxis).

[0134] In addition, in one embodiment, the present invention includes a method for preventing or treating spinal cord injury-related diseases such as traumatic spinal cord injury, inflammatory spinal cord injury, vascular spinal cord injury, and tumor spinal cord injury, comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of the recombinant AAV1 vector.

[0135] As used herein, "administration" refers to the physical introduction of a therapeutic agent or a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, intravitreal, or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" as used herein generally refers to modes of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intravitreal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In a specific embodiment, the AAV vector of the present invention was administered using a micropipette in an animal experiment using rats with a left-sided spinal cord injury, thereby confirming that gene transfer to astrocytes in a spinal cord injury animal model was possible.

[0136] The term "therapeutically effective amount," as used herein, refers to an amount of a drug, alone or in combination with another therapeutic agent, that is effective in "treating" a disease or disorder in a subject or reducing the risk, potential, likelihood, or occurrence of a disease or disorder (e.g., a spinal cord injury-associated disorder). A "therapeutically effective amount" includes an amount of a drug or therapeutic agent that provides some improvement or benefit to a subject who has or is at risk for having a disease or disorder (e.g., a spinal cord injury as disclosed herein). Accordingly, a "therapeutically effective amount" is an amount that reduces the risk, potential, likelihood, or occurrence of a disease or disorder, or provides some relief, alleviation, or reduction in at least one indicator (e.g., a spinal cord injury-associated disorder), and / or reduces at least one clinical symptom of the disease or disorder.

[0137] The term "subject" as used herein includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0138] The present invention also encompasses a gene delivery vehicle comprising the recombinant AAV1 vector. The composition may further comprise a known pharmaceutically acceptable carrier for use in the above-described applications.

[0139] The AAV1 vector of the present invention exhibits a tropism for spinal cord astrocytes and has the ability to express genes specifically in spinal cord astrocytes, and therefore can be used as a vector for delivering genes to spinal cord astrocytes.

[0140]

[0141] Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

[0142]

[0143] [Example]

[0144] Example 1: Screening of AAV1 capsid protein variants using an AAV library.

[0145] 1) Construction of AAV1 mutant plasmid library

[0146] Random mutations were introduced into the wild-type AAV1 cap gene using error-prone PCR, which is commonly used in studies targeting central nervous system diseases. During error-prone PCR, various concentrations of manganese(II) chloride (Sigma-Aldrich, St. Louis, MO; final concentrations in the total PCR mixture were 0.05 mM, 0.15 mM, and 0.3 mM) and dNTP ratios (2 mM ATP, 2 mM GTP, 1 mM TTP, and 1 mM CTP) were used in the cap gene amplification process, resulting in random mutations in each cap gene. Each mutated cap gene was extended using Taq polymerase (Thermo Fisher Scientific, Waltham, MA). The forward primer used was 5'-GCGGAAGCTTCGATCAACTACGC-3' (SEQ ID NO: 9), and the reverse primer used was 5'-GGGCGGCCGCAATTACAGATTACGAGTCAGGTATCTGGTG-3' (SEQ ID NO: 10). Each error-prone PCR product downstream of rep2, bordered by inverted terminal repeats (ITRs), was cloned to generate a plasmid library containing the cap gene with arbitrary mutations.

[0147] 2) Creation of a rat model of left-sided spinal cord injury

[0148] The generation of the left spinal cord injury model and animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Yonsei University College of Medicine (No. 2022-0044) and complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

[0149] To minimize the potential influence of hormonal and gender differences on functional and histological outcomes, adult male Sprague-Dawley rats weighing 240 ± 22 g (7 weeks old) were used. Rats were maintained on a 12-h light / dark cycle and had unrestricted access to food. Rats were anesthetized by intraperitoneal injection of 4.24 mg / kg xylazine (Bayern Korea, Seoul, Korea), 97.5 mg / kg ketamine (Yuhan Co., Seoul, Korea), and 1 mg / kg acepromazine (Samu Median, Seoul, Korea).

[0150] Under anesthesia, an incision was made along the posterior midline, and the parathoracic muscles were removed to expose the thoracic spine. A lumbar discectomy was performed at T9 under aseptic conditions to expose the spinal cord. The spinal cord was contusioned using an Infinite Horizon (IH) impactor device, and a force of 200 kdyn was applied to the dorsal surface of the spinal cord. Virus was injected 7 days after spinal cord injury. The rats were re-anesthetized as described above, the discectomy site was re-exposed, and the virus was injected into the left and right sides of the lesion center using a glass micropipette (diameter: 0.3 mm). The needle was inserted 1.0 mm into the spinal cord from the dorsal surface, and each injection took more than 1 minute. The needle was kept in place for 1 minute after each injection to prevent cell leakage through the injection track.

[0151] All surgical sites were sutured to the inner layer (muscle layer) and stapled to the outer layer (skin). All rats received an intraperitoneal injection of 50 mg / kg cefazolin (Chong Kun Dang Pharmaceutical, Seoul, Korea) to prevent bacterial infection. The bladders of injured rats were manually emptied twice daily until the animals were sacrificed.

[0152] 3) Production of a recombinant AAV1 variant library

[0153] The combination of the above AAV1 mutant plasmid library, Bluescript plasmid (Stratagene, La Jolla, CA), and adenovirus helper plasmid (pHelper; Stratagene, La Jolla, CA) was suspended in 2.5 M CaCl2 solution, and the solution was slowly combined with 2X HEPES buffer solution containing 280 mM NaCl, 1.5 mM Na2HPO4, 50 mM HEPES, pH 7.1. The final mixture was added dropwise to HEK293T cells cultured in 15 cm tissue culture plates for transfection. 72 hours after transfection, HEK293T cells were harvested, lysed through three freeze-thaw cycles, and the lysate was treated with Benzonase II (Sigma-Aldrich; 1 unit / mL) to remove cellular genomic contaminants. Viral vectors were purified using iodixanol density gradient ultracentrifugation (OptiPrep; Alere Technologies AS, Oslo, Norway) using a Vti65.2 rotor (Beckman Coulter; 42,000 rpm). The purified AAV vectors were buffer-exchanged into 1 × PBS solution containing 0.01% (v / v) Tween-20 (Sigma-Aldrich) using Amicon tubes (Merck Millipore, Billerica, MA; molecular weight cutoff: 100 kDa). Finally, the genomic titer of the Dnase-resistant AAV vectors was determined using quantitative PCR (Mini Opticon; Bio-Rad, Hercules, CA).

[0154] 4) Screening of AAV1 capsid protein variants

[0155] The recombinant AAV1 variant library was injected into a 200 kdyn contusion spinal cord injury rat model constructed using the above method on day 5. One week after viral implantation, the spinal cord injury lesion was isolated, and astrocytes were isolated from the lesion using the MACS sorting technique. (MACS sorting is a widely used method for isolating specific cells, using a magnet and antibodies that bind to specific cell surface markers to isolate cells.)

[0156] The viral genome was recovered from isolated astrocytes using a QIAamp DNA mini kit (Qiagen 51306), and the DNA amount was amplified by PCR. The capsid gene of the selected library was read using a Pacbio-SMRT (Pacific Bioscience) next-generation sequencing (NGS) device. The NGS results allowed us to collect mutation information on variants arising from AAV1, and among them, the top eight mutants with more than 100 NGS copy numbers were confirmed to contain mutations at D595H, D712E, and F713Y and mutations at K315R, F584L, A598V, and N642H in the amino acid sequence of the wild-type AAV1 capsid protein indicated by SEQ ID NO: 1 (Table 1). Table 1 below shows the mutants discovered as a result of NGS in a spinal cord injury model.

[0157] Number serotypePoint mutationcopy1>AAV1D595H, D712E, F713Y110702>AAV1K315R, F584L, A598V, N642H53193>AAV1K315R, D595H, D712E, F713Y7924>AAV1F584L, A598V, N642H6415>AAV1D595H2276>AAV1D712E, F713Y1647>AAV1K315R1258>AAV1D712E115

[0158] When the copy numbers of each mutation were added for all mutants obtained from the NGS results and mutations with a high frequency found in astrocytes with a copy number of 100 or more were pooled, D595H, D712E, F713Y, K315R, F584L, A598V, T194A, A196S, A197G, T201N, S205A, L129F, A224S, N223S, H272T, S268T, T265S, A263G, S262N, and S264T were identified in that order in the amino acid sequence of the wild-type AAV1 capsid protein represented by SEQ ID NO: 1, confirming that these mutations increase the specificity for astrocytes in the spinal cord injury lesion area. Table 2 below shows the frequency of discovery for each point mutation in the NGS results.

[0159] mutationcopyD712E13638D595H13562F713Y13486K315R7172A598V6855F584L6828N642H6828T194A248A196S243A 197G235T201N216S205A209L129F187A224S176N223S174H272T131S268T129T265S116A263G112S262N111S264T111

[0160] In addition, the cap gene portion was extracted from the PCR results of the selected mutant library prepared before NGS by treating it with HindIII and NotI restriction enzymes, ligated to the pSub2 backbone plasmid, and then transfected into E. coli. A single colony was randomly selected and subjected to Sanger sequencing. As a result, two mutants containing mutations D595H, D712E, and F713Y and mutations K315R, F584L, A598V, and N642H in the amino acid sequence of the wild-type AAV1 capsid protein indicated by SEQ ID NO: 1 were found with the highest frequency of 4 and 2 times, respectively, and the ranking was confirmed to be consistent with the NGS results, and these were named AAVSN2 and AAVSN3, respectively. From the NGS and Sanger sequencing results, each gene clone of the AAV variants selected from the astrocytes of the highest spinal cord injury lesion site was constructed, and after selecting astrocyte-tropism mutations in the spinal cord injury site from the library pool, each cap gene obtained through HindIII / NotI restriction enzymes was introduced into the AAVSN2 and AAVSN3 gene clones to construct recombinant AAV1 variants carrying the CMV-GFP or NeuroD1-P2A-GFP gene.

[0161] Afterwards, a recombinant AAV1 variant (AAVSN2 variant) was injected into an animal model of left-sided spinal cord injury, and it was confirmed that it could induce high gene expression efficiency in astrocytes in the left-sided spinal cord injury lesion area.

[0162] In addition, we confirmed that the recombinant AAV1 variant (AAVSN3 variant) had a high infection efficiency specifically in astrocytes in the lesion area of ​​the left spinal cord injury.

[0163] 5) Production of recombinant AAV1 vectors (AAV1 SN2 and SN3 variants)

[0164] ① Production of packaging plasmid mutants

[0165] The cap genes of each of the AAVSN2 and AAVSN3 mutants obtained from Sanger sequencing were subcloned into pXX2 (UC Berkeley, David Schaffer Lab) containing HindIII and NotI, thereby completing the construction of astrocyte-specific plasmid mutants for loading the CMV-GFP gene and genes such as CMV-GFP and NeuroD1-P2A-GFP.

[0166] ② Plasmid introduction into astrocytes

[0167] Production of AAV1 SN2 and SN3 variants

[0168] The constructed AAVSN2 or AAVSN3 mutant plasmid 17 smid smid introduced cell-specific reporter gene (pCMV-GFP [Fig. 2a] or pNeuroD1-P2A-GFP [Fig. 4]) 17 roD1-P2A-G [Fig. 2b] 17 r was transfected into HEK293T cells by forming calcium-phosphate complexes, and 72 hours later, HEK293T cells were harvested, lysed through three freeze-thaw cycles, and then centrifuged to remove cell debris, and incubated with 10 U / mL of benzonase at 37°C for 30 minutes to remove nucleic acids from virus-producing cells.

[0169] The cleavage maps of the constructed AAVSN2 and AAVSN3 mutant plasmids are shown in Figs. 3a, 3b, and 3c, and the entire base sequences of the AAV1 SN2 and SN3 mutant plasmids are as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0170] ③ Production of wild-type AAV1

[0171] HEK293T cells were transfected with 17 μg of packaging plasmid containing the rep gene of AAV2 and the cap gene of AAV1, 17 μg of ITR-flanked reporter gene (pCMV-GFP or pCMV-LacZ or pCMV-FGF12-IRES-GFP), and 17 μg of pHelper in the form of calcium-phosphate complexes. After approximately 48 h, only the cell pellet was collected, and AAV inside the cells was extracted by freezing-thawing. Cell debris was then removed by centrifugation, and nucleic acids from virus-producing cells were removed by incubation with 10 U / mL of benzonase at 37°C for 30 minutes.

[0172] 6) Purification of wild-type AAV1 and AAVSN2 and AAVSN3 mutants

[0173] AAV solutions were purified using iodixanol density gradient ultracentrifugation (OptiPrep; Alere Technologies AS, Oslo, Norway) using a Vti65.2 rotor (Beckman Coulter; 42,000 rpm). The purified AAV vectors were buffer-exchanged into 1 × PBS solution containing 0.01% (v / v) Tween-20 (Sigma-Aldrich) using Amicon tubes (Merck Millipore, Billerica, MA; molecular weight cutoff: 100 kDa). Finally, the genomic titer of the Dnase-resistant AAV vectors was determined using quantitative PCR (Mini Opticon; Bio-Rad, Hercules, CA).

[0174] 7) Measurement of titers of wild-type AAV1 and AAVSN2 and AAVSN3 mutants

[0175] Twelve hours after transduction with AAVSN2-CMV-GFP, AAVSN3-CMV-GFP, or AAV1-CMV-GFP, the medium was changed to DMEM supplemented with 1% FBS and 1% PS, and virus harvest was performed 60 hours after transduction. Virus titer was confirmed using qPCR (Fig. 5).

[0176] AAVSN2-GFP showed a statistically significant difference in titer compared to wild-type AAV1-GFP, which has good packaging efficiency. This suggests that the AAVSN2 variant is easier to produce than wild-type AAV1.

[0177] For AAVSN3-GFP, no statistically significant difference was observed compared to wild-type AAV1-GFP, which has good packaging efficiency. This suggests that the AAVSN3 variant is as easy to produce as wild-type AAV1.

[0178]

[0179] Example 2: Confirmation of infection with recombinant AAV1 variant virus

[0180] 1) In vitro experiments

[0181] Since astrocytes could not be isolated from the primary culture obtained from the spinal cord of a 7-week-old male Sqrague-Dawley rat, 2×10 primary mixed glial cells containing astrocytes were prepared. 5After seeding in 24-well cells, AAVSN3-CMV-GFP and AAV1-CMV-GFP were infected with MOIs of 10,000 and 50,000, respectively, 12 hours later. Three days after infection, trypsin was treated, and single cells were labeled with GFP antibody (Alexa Fluor® 488-conjugated anti-GFP antibody (1:500 dilution; Thermo Fisher Scientific)) and GFAP antibody (Alexa Fluor® 647-conjugated anti-GFAP antibody (1:500 dilution; BioLegend)) to tag Alexa 488 and Alexa 647 fluorescence, respectively, and then flow cytometry analysis was performed.

[0182] Through this, astrocytes with strong GFAP signals were isolated, and the ratio of cells expressing the GFP gene by AAV was measured. As a result, there was no statistically significant difference at MOI 10000, but at MOI 50000, it was confirmed that AAVSN3-GFP had a statistically significant higher virus infection efficiency in primary astrocytes than AAV1-GFP (Fig. 6).

[0183] 2) In vivo experiment - confirmation of the virus genome

[0184] In 7-week-old male Sqrague-Dawley rats, AAVSN2 or AAVSN3, wild-type AAV1 (5 × 10) was injected into the central part of the spinal cord lesion 5 days (acute period) after a 200 kdyn contusion spinal cord injury. 8 vg / μL; 6 μL) was injected using a micropipette. (At this time, the injected virus was loaded with the GFP gene, and gene expression can be confirmed through GFP expression.)

[0185] Two weeks after AAV transplantation, perfusion was performed to remove blood, and the spinal cord, including the injured area, was excised and harvested. DNA was recovered from the tissue, and the viral genome was measured by qPCR.

[0186] Each viral genome value was normalized to GAPDH and then analyzed.

[0187] Compared to AAV1-GFP, AAVSN2-GFP showed a statistically significant increase in viral genome within spinal cord tissues including the injury site. That is, AAVSN2 was confirmed to infect more viruses in the spinal cord injury site than wild-type AAV1 (Fig. 7a).

[0188] In addition, for AAV1-GFP, the viral genome within the spinal cord tissue including the injured area did not show a statistically significant difference compared to the model only, but for AAVSN3-GFP, a statistically significant increase was confirmed compared to the model only. In other words, it was confirmed that AAVSN3 infected more viruses in the spinal cord injured area than wild-type AAV1 (Fig. 7b).

[0189] Meanwhile, AAV genome copies per 100 ng of genomic DNA recovered from each tissue were analyzed. No statistically significant differences were observed between the wild-type AAV1 and AAVSN3 groups in the central region centered around the injury site. In spinal cord tissues in areas ② and ③, according to distance from the center, AAVSN3 showed a statistically significant increase compared to wild-type AAV1. This suggests that AAVSN3 can infect the entire spinal cord region, including the injury site, compared to wild-type AAV1 (Fig. 7c).

[0190] 3) In vivo experiment - Confirmation of astrocyte gene transfer ability

[0191] In 7-week-old male Sqrague-Dawley rats, AAVSN2 or AAVSN3, wild-type AAV1 (5 × 10) was injected into the central part of the spinal cord lesion 5 days (acute period) after a 200 kdyn contusion spinal cord injury. 8vg / μL; 6 μL) was injected using a micropipette. (At this time, the injected virus was loaded with the GFP gene, and gene expression could be confirmed through GFP expression.)

[0192] Two weeks after AAV transplantation, perfusion was performed to remove blood, and the spinal cord including the injured area was excised and recovered. Single cells were isolated using a neural tissue isolation kit.

[0193] For AAVSN2, flow cytometry was performed after tagging single cells with GFP antibody and GFAP antibody, respectively, with Alexa 488 and rhodamine fluorescence. Samples that were not administered AAV vectors to the lesion site of rats with left-sided spinal cord injury were defined as Model only, and samples in which GFP antibody or GFAP antibody was not added to these Model only were designated as GFP(-)GFAP(-), and samples in which GFP antibody or GFAP antibody was added to Model only were designated as GFP(-)GFAP(+), and gating on GFAP was performed. GFP(-) was designated as Model only, and the group that received the virus was designated as GFP(+), and gating on GFP was performed.

[0194] The proportion of astrocytes among the GFP expressing cells shown in Fig. 8 is (GFP + astrocytes) / ((GFP + astrocytes) + (GFP + It was calculated as a value of 1. When the ratio of astrocytes among all GFP-expressing cells was graphed, AAVSN2-GFP and AAVSN3-GFP showed a higher ratio than AAV1-GFP, confirming that AAVSN2-GFP and AAVSN3-GFP have a higher gene transfer efficiency specifically to astrocytes than other cells in the spinal cord lesion area compared to AAV1-GFP [Figure 8].

[0195] 4) In vivo experiment - Confirmation of stellate cell gene expression intensity

[0196] In 7-week-old male Sqrague-Dawley rats, AAVSN2 or AAVSN3, wild-type AAV1 (5 × 10) was injected into the central part of the spinal cord lesion 5 days (acute period) after a 200 kdyn contusion spinal cord injury. 8 vg / μL; 6μL) was injected using a micropipette. (At this time, the injected virus was loaded with the GFP gene, and the presence or absence of GFP gene expression could be confirmed through immunohistochemical staining. The mock virus was loaded with the GFP gene.)

[0197] Eight weeks after AAV transplantation, perfusion was performed to remove blood, tissues were fixed with 4% PFA, and sections were prepared using a cryostat. The sectioned samples were analyzed for AAV distribution and gene expression intensity within the central nervous system using immunohistochemistry using GFP.

[0198] Compared to AAV1-GFP, AAVSN2-GFP was confirmed to show gene expression specifically around the damaged area.

[0199] The injured area was set to distance = 0, and the expression intensity was confirmed in the central graph of Figure 9, and it was confirmed that the use of AAVSN2 was more effective than wild-type AAV1 in intensive treatment of the spinal cord injured area.

[0200] Additionally, compared to AAV1-GFP, AAVSN3-GFP showed overall gene expression over a wider area relative to the wound site.

[0201] The damaged area was set to distance = 0, and the expression intensity was confirmed in the graph on the right side of Figure 9, and it was confirmed that the use of AAVSN3 was more effective than wild-type AAV1 in the overall treatment of the spinal cord.

[0202]

[0203] Example 3: Confirmation of gene transfer ability of recombinant AAV1 variants in astrocytes near the spinal cord.

[0204] In 7-week-old male Sqrague-Dawley rats, AAVSN2 or AAVSN3, wild-type AAV1 (5 × 10) was injected into the central part of the spinal cord lesion 5 days (acute period) after a 200 kdyn contusion spinal cord injury. 8 vg / μL; 6μL) was injected using a micropipette. (At this time, the injected virus was loaded with the GFP gene, and gene expression could be confirmed through GFP expression. The mock virus was loaded with the GFP gene.)

[0205] Eight weeks after AAV transplantation, perfusion was performed to remove blood, tissues were fixed with 4% PFA, and sections were prepared using a cryostat. The sectioned samples were analyzed for immune cell distribution within the central nervous system using immunohistochemistry using GFP and GFAP.

[0206] As a result of spinal cord confirmation near the overall spinal cord injury lesion site, it was confirmed that AAV1-based AAVSN2 or AAVSN3 showed high-efficiency gene transfer efficiency in the spinal cord injury site compared to wild-type AAV1 through the induction of a strong GFP signal that was concentrated in the spinal cord injury lesion site [Figure 10].

[0207] In addition, high-magnification examination of the vicinity of the lesion site revealed that in both AAVSN2 and AAVSN3, the GFP signal showed a pattern similar to the GFAP signal, and that the two signals were co-expressed in more areas than in wild-type AAV1. This confirmed that AAVSN2 or AAVSN3 had a higher infection rate for astrocytes than wild-type AAV1 [Figure 11].

[0208] In addition, high-magnification images were analyzed using the imageJ program to measure the ratio of GFAP+ cells among GFP+ cells, and it was confirmed that AAVSN2 and AAVSN3 had higher transduction ability to astrocytes in the lesion area of ​​the left spinal cord injury than wild-type AAV1 [Figure 12].

[0209]

[0210] Example 4: Confirmation of gene transfer ability of recombinant AAV1 variants in astrocytes and other cells near the spinal cord.

[0211] In 7-week-old male Sqrague-Dawley rats, AAVSN2 or AAVSN3, wild-type AAV1 (5 × 10) was injected into the central part of the spinal cord lesion 5 days (acute period) after a 200 kdyn contusion spinal cord injury. 8 vg / μL; 6μL) was injected using a micropipette. (At this time, the injected virus was loaded with the GFP gene, and gene expression could be confirmed through GFP expression. The mock virus was loaded with the GFP gene.)

[0212] Eight weeks after AAV transplantation, perfusion was performed to remove blood, tissues were fixed with 4% PFA, and sections were prepared using a cryosectioner. The sectioned samples were analyzed for the distribution of immune cells in the central nervous system by immunohistochemistry using GFP, GFAP, Tuj1 (rabbit monoclonal anti-Tuj1 antibody (1:2000 dilution; Covance), and Iba1 (rabbit anti-Iba1 antibody (1:500 dilution; Wako Pure Chemical Industries)), respectively. (GFAP: astrocytes, Tuj1: neurons, Iba1: microglia)

[0213] IHC images were analyzed using the imageJ program to quantitatively analyze the percentage of each signal (GFAP, Tuj1, Iba1) among GFP+ cells.

[0214] Compared to AAV1-GFP, AAVSN2-GFP increased GFP expression in astrocytes and decreased GFP expression in neurons. There was no statistically significant difference in microglia. This suggests that AAVSN2 reverses the neuronal tropism of wild-type AAV1 and increases its tropism for astrocytes [Figure 13].

[0215]

[0216] Comparative Example 2: Confirmation of gene transfer ability of recombinant AAV1 variants in microglia near the spinal cord.

[0217] In 7-week-old male Sqrague-Dawley rats, AAVSN2 or AAVSN3, wild-type AAV1 (1 × 10) was injected into the central part of the spinal cord lesion 5 days (acute period) after a 200 kdyn contusion spinal cord injury. 9 vg / μL; 10μL) was injected using a micropipette. (At this time, the injected virus was loaded with the NeuroD1-P2A-GFP gene, and the presence or absence of GFP gene expression could be confirmed through immunohistochemical staining. The mock virus was loaded with the GFP gene.)

[0218] Eight weeks after AAV transplantation, perfusion was performed to remove blood, tissues were fixed with 4% PFA, and sections were prepared using a cryostat. The sectioned samples were analyzed for immune cell distribution within the central nervous system by immunohistochemistry using Iba1 (rabbit anti-Iba1 antibody (1:500 dilution; Wako Pure Chemical Industries)).

[0219] We confirmed that immune cells were statistically significantly reduced in the spinal cord injury lesion area when compared to AAVSN2-NeuroD1 or AAVSN3-NeuroD1 and AAV1-NeuroD1 or Model only. In other words, it was confirmed that gene transfer ability was low in microglia near the spinal cord [Figure 14].

[0220]

[0221] Comparative Example 3: Confirmation of gene transfer ability of recombinant AAV1 variants in tissues other than the spinal cord.

[0222] In 7-week-old male Sqrague-Dawley rats, AAVSN2 or AAVSN3, wild-type AAV1 (5 × 10) was injected into the central part of the spinal cord lesion 5 days (acute period) after a 200 kdyn contusion spinal cord injury. 8 vg / μL; 6μL) was injected using a micropipette. (At this time, the injected virus was loaded with the GFP gene, and gene expression could be confirmed through GFP expression. The mock virus was loaded with the GFP gene.)

[0223] Two weeks after AAV transplantation, perfusion was performed to remove blood and recover liver, spleen, lung, kidney, brain, and heart tissues.

[0224] DNA was recovered from the tissue and viral genome values ​​were measured by qPCR.

[0225] Each viral genome value was normalized to GAPDH and then analyzed.

[0226] There was no statistically significant difference between the AAV1-GFP and AAVSN2-GFP groups in the liver, spleen, lung, kidney, brain, and heart. However, in the case of AAV1-GFP, there was a statistically significant increase compared to the model only group in the liver and lung, but in the case of AAVSN2-GFP, there was no statistically significant difference compared to the model only group in any tissue. In other words, it was confirmed that the off-target issue to other tissues after transplantation was reduced in the case of AAVSN2-GFP compared to AAV1-GFP.

[0227] In the case of AAVSN3-GFP, there was no statistically significant difference in the liver compared to AAV1-GFP, but there was a statistically significant increase in the difference compared to the model only group. In the spleen, lung, kidney, brain, and heart, there was no statistically significant difference compared to AAV1GFP or compared to the model only. In other words, it was confirmed that there was no difference in off-target effect compared to AAV1-GFP, and there was no statistically significant difference compared to the model only in the spleen, lung, kidney, brain, and heart [Figure 15].

[0228]

[0229] Example 4: Confirmation of recovery of spinal cord injury site by recombinant AAV1 variant.

[0230] In 7-week-old male Sqrague-Dawley rats, AAVSN2 and wild-type AAV1 (1 × 10) were injected into the central part of the spinal cord injury 5 days after a 200 kdyn contusion spinal cord injury (acute phase). 9 vg / μL; 10μL) was injected using a micropipette. (At this time, the injected virus is loaded with the NeuroD1-P2A-GFP gene, which can reprogram astrocytes into neurons, thus inducing functional improvement of damaged neural function. By inserting P2A with the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 11), GFP was prevented from functionally affecting the NeuroD1 protein. The presence or absence of gene expression can be confirmed by GFP signal through immunohistochemical staining.)

[0231] After AAV transplantation, a test was conducted to quantify motor ability according to items from 0 to 21 (normal) for the movement of three joints (ankle, knee, and hip) at one-week intervals. After confirming that there were no statistically significant differences among the four groups before injection, the test was conducted for 8 weeks. From the 4th week, statistically significant differences were confirmed in AAVSN2-NeuroD1 (square: left graph) and Model only (diamond: left graph). There were no statistically significant differences between the remaining groups.

[0232] Comparison of AAVSN2-NeuroD1 and AAVSN2-GFP confirmed that the NeuroD1 gene actually induces functional enhancement by inducing reprogramming of astrocytes into neurons.

[0233] Comparison of AAVSN2-NeuroD1 and AAV1-NeuroD1 confirmed that AAVSN2 had a higher efficiency of NeuroD1 gene transfer to astrocytes near the damaged area than AAV1, and that the BBB score was statistically significantly increased in the case of AAVSN2-NeuroD1 compared to Model only [Figure 16].

[0234]

[0235] Example 5: Confirmation of reprogramming after introduction of recombinant AAV1 variant therapeutic gene.

[0236] In 7-week-old male Sqrague-Dawley rats, AAVSN2 and wild-type AAV1 (1 × 10) were injected into the central part of the spinal cord lesion 5 days after a 200 kdyn contusion spinal cord injury (acute phase). 9 vg / μL; 10μL) was injected using a micropipette. (At this time, the injected virus was loaded with the NeuroD1-P2A-GFP gene, and the presence or absence of GFP gene expression could be confirmed through immunohistochemical staining.)

[0237] At 3 and 8 weeks after AAV transplantation, perfusion was performed to remove blood, tissues were fixed in 4% PFA, and sections were prepared using a cryostat. The sectioned samples were analyzed for AAV distribution in the central nervous system by immunohistochemistry using GFP and GFAP or Tuj1 (rabbit monoclonal ant-Tuj1 antibody (1:2000 dilution; Covance)).

[0238] The images of the spinal cord injury lesion site where AAVSN2-NeuroD1 and AAV1-NeuroD1 were transplanted are shown in the upper part of Figure 17.

[0239] At week 3, we confirmed that AAVSN2-NeuroD1 had the highest co-expression of GFAP and GFP signals, and at week 8, we confirmed that AAVSN2-NeuroD1 had the highest co-expression of Tuj1 and GFP signals. As a result of quantitative analysis, AAVSN2-NeuroD1 statistically significantly decreased GFAP+ GFP+ co-expression at weeks 3 and 8, and statistically significantly increased Tuj1+GFP+ co-expression at weeks 3 and 8, confirming that in vivo reprogramming of astrocytes into neurons in the spinal cord injury area was effective. When AAVSN2-NeuroD1 was transplanted, it was confirmed that the reprogrammed neurons formed synaptic connections through synapsin-1 [bottom of Figure 17].

[0240] More specifically, the lower left graph of Figure 17 shows that the number of cells co-expressing GFAP and GFP, i.e., cells in which the ND1 gene was delivered to astrocytes via AAV, decreased over time from week 3 to week 8, whereas the number of neurons co-expressing TUJ1 and GFP, i.e., containing the ND1 delivered via AAV, was very small at week 3 but increased dramatically at week 8. The above two phenomena indicate that astrocytes that received the ND1 gene were reprogrammed into neurons.

[0241] Also, the reason why the signals of synapsin-1 and TUJ1 GFP overlap in the image on the lower right is because the image is from week 8 (after treatment), not week 3. Therefore, it is desirable data that after treatment, TUJ1+GFP+ co-expression increases and synapses are formed, resulting in co-expression of TUJ1+GFP+ signals and synapsin-1.

[0242] This confirmed that the ND1 gene was transferred to astrocytes and reprogrammed them into neurons.

[0243]

[0244] Example 6: Confirmation of body weight changes after introduction of recombinant AAV1 variant therapeutic genes.

[0245] In 7-week-old male Sqrague-Dawley rats, AAVSN2 or AAVSN3, wild-type AAV1 (1 × 10) was injected into the central part of the spinal cord injury 5 days after a 200 kdyn contusion spinal cord injury. 9 vg / μL; 10μL) was injected using a micropipette. (At this time, the injected virus was loaded with the NeuroD1-P2A-GFP gene, and the presence or absence of GFP gene expression could be confirmed through immunohistochemical staining. The mock virus was loaded with the GFP gene.)

[0246] From the creation of the spinal cord injury animal model to the AAV transplantation, body weight was measured at one-week intervals, and it was confirmed that there was no statistically significant difference between all groups [Fig. 18].

[0247]

[0248] Example 7: Blood toxicity test after introduction of recombinant AAV1 variant therapeutic gene

[0249] In 7-week-old male Sqrague-Dawley rats, AAVSN3 or AAVSN2, wild-type AAV1 (1 × 10) was injected into the central part of the spinal cord lesion 5 days after a 200 kdyn contusion spinal cord injury. 9vg / μL; 10μL) was injected using a micropipette. (At this time, the injected virus was loaded with the NeuroD1-P2A-GFP gene, and the presence or absence of GFP gene expression could be confirmed through immunohistochemical staining. The mock virus was loaded with the GFP gene.)

[0250] Eight weeks after AAV transplantation, blood was collected, plasma was separated, and blood toxicity tests for GPT (Glutamioc Pyruvic Transaminase), GOT (Glutamioc Oxaloacetic Transaminase), and BUN (Blood Urea Nitrogen) were performed.

[0251] We confirmed that there was no statistically significant difference among all groups: AAV1-NeuroD1, AAVSN2-NeuroD1 or AAVSN3-NeuroD1, AAVSN3-GFP, Model only, and Intact [Fig. 19].

Claims

1. As a mutant of the adeno-associated virus serotype 1 (AAV1) capsid protein, The above mutant is a mutant of adeno-associated virus serotype 1 (AAV1) capsid protein having one or more mutations selected from among D595H, D712E, F713Y, K315R, F584L, A598V, and N642H in the amino acid sequence of the wild-type AAV1 capsid protein represented by SEQ ID NO:

1.

2. In paragraph 1, The above mutant is a mutant of the AAV1 capsid protein having one or more mutations additionally selected from T194A, A196S, A197G, T201N, S205A, L129F, A224S, N223S, H272T, S268T, T265S, A263G, S262N and S264T.

3. In paragraph 1, The above mutant is a mutant of the AAV1 capsid protein having mutations D595H, D712E and F713Y.

4. In paragraph 1, The above mutant is a mutant of the AAV1 capsid protein having mutations of K315R, F584L, A598V, and N642H.

5. In paragraph 1, The above mutant is a mutant of the AAV1 capsid protein having mutations of K315R, D595H, D712E and F713Y.

6. In paragraph 1, The above mutant is a mutant of the AAV1 capsid protein having mutations of F584L, A598V and N642H.

7. A nucleic acid encoding a mutant of the AAV1 capsid protein of claim 1.

8. In paragraph 6, A nucleic acid having a base sequence represented by SEQ ID NO:

4.

9. In paragraph 7, A nucleic acid having a base sequence represented by SEQ ID NO:

6.

10. A recombinant AAV1 vector comprising a nucleic acid encoding a mutant of the AAV1 capsid protein of claim 1.

11. In paragraph 10, The above AAV1 vector is a recombinant AAV1 vector having an improved transduction profile for astrocytes compared to the AAV1 wild-type viral vector.

12. In paragraph 11, A recombinant AAV1 vector represented by SEQ ID NO:

7.

13. In paragraph 12, A recombinant AAV1 vector represented by SEQ ID NO:

8.

14. A pharmaceutical composition comprising a recombinant AAV1 vector according to claim 10.

15. In paragraph 14, A pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

16. In paragraph 14, A pharmaceutical composition for preventing or treating diseases related to spinal cord injury.

17. In paragraph 14, A pharmaceutical composition wherein the spinal cord injury-related disease is one selected from the group consisting of traumatic spinal cord injury, inflammatory spinal cord injury, vascular spinal cord injury, and tumor spinal cord injury.

18. A gene delivery vehicle comprising a recombinant AAV1 vector according to Article 10.

19. A method for preventing or treating a spinal cord injury-related disease, comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of a recombinant AAV1 vector according to claim 10.

20. In paragraph 19, A method for preventing or treating a spinal cord injury-related disease, wherein the spinal cord injury-related disease is one selected from the group consisting of traumatic spinal cord injury, inflammatory spinal cord injury, vascular spinal cord injury, and tumor spinal cord injury.

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