Adeno-associated virus variants capable of brain astrocyte-specific gene transfer and applications thereof

Abstract

The present disclosure relates to adeno-associated virus variants capable of brain astrocyte-specific gene transfer. The administration of a recombinant viral vector comprising nucleic acid encoding a mutant of an AAV capsid protein according to the present disclosure allows a therapeutic gene to be transferred to and expressed in specific brain astrocytes, and thus prevention or treatment of brain diseases is remarkably effective.

Description

Adeno-associated virus variants capable of brain astrocyte-specific gene transfer and their applications

[0001] This application claims priority to Republic of Korea Application No. 10-2023-0175840, filed December 6, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates to an adeno-associated virus variant capable of gene transfer specifically to brain astrocytes.

[0003] Adeno-associated virus (AAV) is a promising gene delivery vector associated with gene therapy. AAV exists in various serotypes, and gene transfer properties differ depending on the amino acid sequence within the hypervariable region of the capsid protein. AAV is non-pathogenic, has low immunogenicity, and can stably express transgenes from episomes. However, existing AAVs have diverse tropisms, are limited in their ability to bypass biological barriers, and may be less effective due to neutralizing antibodies in the human body.

[0004] Brain diseases are a major global health problem, with various conditions, including Alzheimer's, Parkinson's, and brain tumors, causing functional and structural damage to the brain. Gene therapy is increasingly gaining attention as a treatment for these conditions. Gene therapy has the potential to fundamentally address the underlying causes of disease by introducing or suppressing specific genes.

[0005] Hypoxic-ischemic brain injury (HIBI) is a serious brain disease that can occur in people of all ages, including newborns, adults, and the elderly. It occurs when oxygen and blood supply to brain tissue is inadequate, damaging nerve cells and supporting cells. In newborns, complications during birth can lead to hypoxic-ischemic brain injury, which can lead to permanent neurological impairment, making early treatment essential.

[0006] Hypoxic-ischemic brain injury involves a complex pathological process, and astrocytes play a crucial role in the survival and recovery of neurons. Astrocytes play a pivotal role in various aspects, including regulating the inflammatory response at the site of injury, protecting neurons, and promoting nerve regeneration. Brain astrocytes play a crucial role in maintaining nervous system homeostasis, regulating neurotransmitters, and regulating the brain's immune response. However, in hypoxic-ischemic brain injury, astrocyte function can be impaired or pathologically altered, potentially exacerbating brain damage.

[0007] Targeting brain astrocytes to restore or regulate their function is considered an important approach in the treatment of hypoxic-ischemic brain injury. Existing pharmacological treatments primarily focus on protecting damaged neurons or suppressing inflammatory responses, but they are limited in effectively targeting specific cell types, particularly brain astrocytes.

[0008] Gene therapy using AAV is emerging as a promising alternative for treating hypoxic-ischemic brain injury. However, existing AAV vectors have low cell-selective delivery efficiency, leading to off-targeting and resulting adverse effects. Therefore, there is a pressing need for technologies that can improve the treatment efficacy and reduce side effects for brain diseases, including hypoxic-ischemic brain injury, by developing novel adeno-associated virus variants capable of specifically delivering genes to astrocytes.

[0009] Therefore, the problem that the present disclosure seeks to solve is to provide an adeno-associated virus variant capable of specifically delivering genes to brain astrocytes.

[0010] To solve the above problem, the inventors of the present disclosure have conducted extensive research efforts and discovered a mutant of adeno-associated virus capsid protein capable of specifically delivering genes to brain astrocytes.

[0011] The present disclosure relates to a mutant of an adeno-associated virus capsid protein capable of delivering genes specifically to brain astrocytes.

[0012] 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. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeat (TR), Rep protein, and capsid subunit, are known in the art. For example, the sequence of AAV1 can be found in GenBank Accession Nos. NC_002077 (AAV-1) and AF063497 (AAV-1).

[0013] 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 properties.

[0014] The term "capsid" as used herein 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 VP1 protein comprises the amino acid sequence represented by SEQ ID NO: 1.

[0015] The present disclosure provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant has an amino acid substitution at one or more of positions 35, 449, and 451 in the amino acid sequence of SEQ ID NO: 1 of a wild-type AAV1 capsid protein. In one aspect of the present disclosure, the mutant comprises one or more of a substitution of asparagine (N) at position 35 with aspartic acid (D), a substitution of threonine (T) at position 449 with serine (S), and a substitution of asparagine (N) at position 451 with lysine (K) in the amino acid sequence of SEQ ID NO: 1 of a wild-type AAV1 capsid protein. In one aspect of the present disclosure, the mutant may comprise all of the N35D, T449S, and N451K substitutions. In the present disclosure, the mutant of the AAV1 capsid protein is designated as AAV1#11-6, which comprises or consists of the amino acid sequence of AAV1#11-6 capsid VP1 represented by SEQ ID NO: 4.

[0016] In addition, the present disclosure provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant has an amino acid substitution at one or more of positions 237 and 582 in the amino acid sequence of SEQ ID NO: 1 of a wild-type AAV1 capsid protein. In one aspect of the present disclosure, the mutant is provided as a mutant of an AAV1 capsid protein comprising at least one substitution of an aspartic acid (D) at position 237 to an asparagine (N) and a valine (V) at position 582 to an aspartic acid (D) in the amino acid sequence of SEQ ID NO: 1 of a wild-type AAV1 capsid protein. In one aspect of the present disclosure, the mutant may comprise both the D237N and V582D substitutions. In the present disclosure, the mutant of the AAV1 capsid protein is designated as AAV1#11-10, which comprises or consists of the amino acid sequence of AAV1#11-10 capsid VP1 represented by SEQ ID NO: 7.

[0017] In addition, the present disclosure provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant comprises at least one substitution selected from the group consisting of a substitution of asparagine (N) at position 35 with aspartic acid (D) (N35D), a substitution of threonine (T) at position 449 with serine (S) (T449S), a substitution of asparagine (N) at position 451 with lysine (K) (N451K), a substitution of aspartic acid (D) at position 237 with asparagine (N) (D237N), and a substitution of valine (V) at position 582 with aspartic acid (D) (V582D) in the amino acid sequence of SEQ ID NO: 1 of a wild-type AAV1 capsid protein. A person skilled in the art will readily understand that, given the superior tropism of the AAV1#11-6 and AAV1#11-10 capsid variants toward astrocytes, another AAV1 capsid protein mutant comprising one or more of these amino acid substitution mutations will also exhibit equivalent tropism.

[0018] In one aspect of the present disclosure, the present disclosure provides capsid protein mutants of other serotypes (e.g., AAV2 to AAV9) that have identical mutations at positions corresponding to the amino acid mutations of the AAV1 capsid protein mutants. The capsid protein mutants of the other serotypes may also have superior tropism for brain astrocytes by including mutations corresponding to the AAV1 capsid protein mutants.

[0019] The present disclosure also provides a nucleic acid encoding a mutant of the AAV1 capsid protein. The nucleic acid of the present disclosure encodes the mutant of the AAV1 capsid protein. The nucleic acid of the present disclosure may be 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 disclosure also includes a complementary nucleic acid (e.g., cDNA). The nucleic acid of the present disclosure may be single-stranded or double-stranded, but is preferably double-stranded.

[0020] The present disclosure provides a nucleic acid encoding a mutant of an AAV capsid protein comprising an amino acid sequence represented by any one of SEQ ID NOs: 4 to 9. The present disclosure also provides a nucleic acid having a base sequence represented by any one of SEQ ID NOs: 13 to 18.

[0021] The nucleic acid of the present disclosure can be operably linked to a suitable control sequence. The control sequence includes a promoter sequence, a polyadenylation signal, a transcription termination sequence, an upstream regulatory domain, internal ribosome entry sites, an enhancer, and the like. The promoter sequence includes an inducible promoter sequence and / or a constitutive promoter sequence. The control sequence may be native to the AAV from which the capsid protein is derived or may be foreign, and may be a natural or synthetic sequence. Recombinant DNA capable of expressing a mutant of an AAV capsid protein comprising the nucleic acid of the present disclosure is also encompassed by the present disclosure.

[0022] The recombinant DNA described above is useful for delivering the nucleic acid of the present disclosure to cells in vitro, ex vivo, and in vivo, and for conferring upon such cells the ability to express mutants of AAV capsid proteins. Furthermore, cells delivered with the nucleic acid of the present disclosure are also useful for producing recombinant AAV particles. The recombinant DNA can be used to deliver or introduce the nucleic acid of the present disclosure into eukaryotic cells, preferably animal cells, and more preferably mammalian cells.

[0023] In the present disclosure, recombinant DNA can be produced by retaining the nucleic acid of the present disclosure in DNA used as a vector. For example, plasmids, phages, transposons, cosmids, episomal DNA, viral genomes, etc. can be used.

[0024] For example, a packaging plasmid can be constructed by harboring a nucleic acid (cap gene) encoding a mutant of the AAV capsid protein of the present disclosure in a plasmid. The packaging plasmid can further comprise any nucleic acid sequence, such as a nucleic acid encoding a replicase (Rep) protein (rep gene).

[0025] 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.

[0026] The present disclosure provides a recombinant AAV1 vector comprising a nucleic acid encoding the AAV capsid protein variant. The recombinant AAV vector is useful for gene introduction into target cells, preferably brain astrocytes. The gene introduced by the recombinant AAV vector of the present disclosure is strongly expressed in the target cells, preferably brain astrocytes.

[0027] The term 'AAV vector' as used herein refers to any vector comprising or derived from components of an adeno-associated virus and suitable for infecting mammalian cells, including human cells of any of many tissue types, such as brain, 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.

[0028] As used herein, the term "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.

[0029] In one aspect of the present disclosure, 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. Various such helper viruses for AAV are known in the art, including adenoviruses, rhesus viruses, and poxviruses such as vaccinia. While adenovirus type 5 of subgroup C is most commonly used, adenoviruses encompass many different subgroups. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the ATCC.

[0030] Helper virus function refers to a function encoded in the helper virus genome that allows AAV replication and packaging (along with other requirements for replication and packaging described herein). As described herein, helper virus function can be provided in a variety of ways, including by providing the helper virus or, for example, by transfecting a producer cell with a polynucleotide sequence encoding the essential function. 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.

[0031] As used herein, the term "tropism" refers to the specificity of an AAV capsid protein present in an AAV viral particle for infecting or transducing a particular type of cell or tissue. The tropism of an AAV capsid for a particular type of cell or tissue can be determined by measuring the ability of an AAV vector particle, including AAV capsid proteins, to infect or transduce a particular type of cell or tissue using standard assays well known in the art, such as those disclosed in the embodiments of the present disclosure.

[0032] In one aspect of the present disclosure, the mutant AAV capsid protein further increases the brain astrocyte tropism of the wild type AAV capsid protein by at least 5%, 10%, 20%, 30%, 40%, 50% or more.

[0033] The present disclosure also provides a pharmaceutical composition comprising the recombinant AAV vector. The composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes any substance that, 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.

[0034] In one aspect of the present disclosure, the pharmaceutical composition may be used for the prevention or treatment of a brain-related disease. The brain-related disease may include, but is not limited to, hypoxic-ischemic brain damage, Alzheimer's disease, Parkinson's disease, brain tumors, multiple sclerosis, cerebral palsy, glioblastoma, metastatic brain cancer, encephalitis, autoimmune neurological disorders, or Alexander's disease. Preferably, the brain-related disease may be hypoxic-ischemic brain damage.

[0035] As used herein, "treatment" refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, delaying, or preventing the progression, development, severity, or recurrence of a disease-related syndrome, complication, symptom, or biochemical sign. Treatment may be performed on a subject with a disease or on a subject without a disease (e.g., for prophylaxis).

[0036] Additionally, the present disclosure provides a method for preventing or treating a brain-related disease, specifically hypoxic-ischemic brain damage, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition.

[0037] As used herein, the term "administration" refers to the physical introduction of a therapeutic agent or a composition comprising a therapeutic agent into a subject using any of various methods and delivery systems known to those skilled in the art. Preferred routes of administration in the present disclosure include intracerebral, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, tracheal, intravitreal, or other parenteral routes of administration, for example, by injection or infusion. Parenteral administration 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, intracerebral, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intravitreal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, tracheal, or intrasternal injection and infusion. Preferably, the administration in the present disclosure may be intracerebral intralesional administration.

[0038] As used herein, the term "therapeutically effective amount" 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 brain-related disease). The therapeutically effective amount includes an amount of a drug or therapeutic agent that provides some improvement or benefit to a subject having or at risk of having a disease or disorder (e.g., hypoxic-ischemic brain injury). 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 brain-related disease), and / or reduces at least one clinical symptom of the disease or disorder.

[0039] In one aspect of the present disclosure, the pharmaceutical composition can be delivered in a volume of about 0.1 mL to about 10 mL, depending on the size of the area to be treated, the viral titer used, the route of administration, and the method of administration. The effective concentration of AAV in the pharmaceutical composition is about 10 per milliliter. 7 10 inland 14 The range of vector genomes (vg / mL) may be within the range. To reduce risks such as toxicity, it is preferable to use the lowest effective concentration of virus. The dosage or administration volume may be selected by the attending physician, taking into account the physical condition of the subject to be treated, preferably a human, the subject's age, the specific disorder, and the degree of development of the disorder in the case of progressive disease.

[0040] In the present disclosure, the term "subject" includes any human or non-human animal. The non-human animal includes all vertebrates, such as mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0041] Additionally, the present disclosure encompasses a drug delivery system comprising the recombinant AAV vector. The composition may further comprise a known pharmaceutically acceptable carrier for use in the above-described applications.

[0042] The present disclosure relates to a recombinant AAV capsid variant capable of specifically targeting brain astrocytes and delivering genes with high efficiency.

[0043] By administering a recombinant viral vector comprising a nucleic acid encoding a mutant of the AAV capsid protein of the present disclosure, a therapeutic gene can be specifically delivered and expressed in brain astrocytes, thereby exhibiting a remarkably excellent effect in preventing or treating brain diseases.

[0044] Figure 1 is a diagram showing 3D modeling of AAV variants. The locations of the mutations on the surface are indicated in red.

[0045] Figure 2 shows the distribution of GFP gene expression of AAV variants delivered to a hypoxic-ischemic brain damage animal model in Experimental Example 2.

[0046] Figure 3 shows the count number and co-expression ratio for each marker after staining cell nuclei using DAPI (blue) and using GFAP (red) as an astrocyte-specific marker in Experimental Example 2.

[0047] Figure 4 shows the results of immunohistochemical staining analysis after injection of the NeuroD1-loaded AAV variant in Experimental Example 3.

[0048] Figure 5 shows the results of analyzing the expression time of GFAP / GFP or NeuN / GFP after injection of NeuroD1-loaded AAV variants in Experimental Example 4.

[0049] Figure 6 shows the body weight change and mortality rate after injection of the NeuroD1-loaded AAV variant in Experimental Example 5.

[0050] Figure 7 shows the DNA copy number and blood test results for each tissue after injection of the NeuroD1-loaded AAV variant in Experimental Example 6.

[0051] Figure 8 shows the amino acid sequence and DNA sequence of AAV1 capsid VP1.

[0052] Figure 9 shows the amino acid sequence and DNA sequence of AAV1 capsid VP2.

[0053] Figure 10 shows the amino acid sequence and DNA sequence of AAV1 capsid VP3.

[0054] Figure 11 shows the amino acid sequence and DNA sequence of AAV1#11-6 capsid VP1.

[0055] Figure 12 shows the amino acid sequence and DNA sequence of AAV1#11-6 capsid VP2.

[0056] Figure 13 shows the amino acid sequence and DNA sequence of AAV1#11-6 capsid VP3.

[0057] Figure 14 shows the amino acid sequence and DNA sequence of AAV1#11-10 capsid VP1.

[0058] Figure 15 shows the amino acid sequence and DNA sequence of AAV1#11-10 capsid VP2.

[0059] Figure 16 shows the amino acid sequence and DNA sequence of AAV1#11-10 capsid VP3.

[0060] Hereinafter, the present invention will be described in detail, using examples and the like, to aid understanding. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0061]

[0062] <AAV 캡시드 단백질 변이체의 선별>

[0063] AAV capsid protein variants with astrocyte tropism were selected through in vivo directed evolution. Specifically, a library pool was generated by inducing random point mutations in the cap genes of wild-type AAV variants (AAV1, AAV2, AAV4, AAV6, AAV8, and AAV9) using error-prone PCR, and these were injected into an animal model of hypoxic-ischemic brain injury. After 2 weeks, each lesion site was isolated, and only astrocytes were sorted from the lesion. Viral genes were recovered from the extracted astrocytes, and each gene clone was generated. Using these gene clones, recombinant AAV variants carrying a GFP fluorescent gene were generated. Thereafter, the above recombinant AAV variants were injected into an animal model of hypoxic-ischemic brain damage, and in this disclosure, two variants capable of inducing high gene expression efficiency in astrocytes in the hypoxic-ischemic brain damage lesion area were selected (AAV1#11-6, AAV1#11-10). Figure 1 shows the 3D modeling results of the two variants.

[0064] The sequence numbers of the amino acid sequences of each mutant capsid are shown in Table 1 below.

[0065] Amino acid sequence of capsidSequence number AAV1 Capsid VP1 SEQ ID NO: 1 AAV1 Capsid VP2 SEQ ID NO: 2 AAV1 Capsid VP3 SEQ ID NO: 3 AAV1#11-6 Capsid VP1 SEQ ID NO: 4 AAV1#11-6 Capsid VP2 SEQ ID NO: 5 AAV1#11-6 Capsid VP3 SEQ ID NO: 6 AAV1#11-10 Capsid VP1 SEQ ID NO: 7 AAV1#11-10 Capsid VP2 SEQ ID NO: 8 AAV1#11-10 Capsid VP3 SEQ ID NO: 9

[0066] Additionally, the sequence numbers of the DNA sequences of each mutant capsid are shown in Table 2 below.

[0067] DNA sequence of capsidSequence number AAV1 Capsid VP1 SEQ ID NO: 10 AAV1 Capsid VP2 SEQ ID NO: 11 AAV1 Capsid VP3 SEQ ID NO: 12 AAV1#11-6 Capsid VP1 SEQ ID NO: 13 AAV1#11-6 Capsid VP2 SEQ ID NO: 14 AAV1#11-6 Capsid VP3 SEQ ID NO: 15 AAV1#11-10 Capsid VP1 SEQ ID NO: 16 AAV1#11-10 Capsid VP2 SEQ ID NO: 17 AAV1#11-10 Capsid VP3 SEQ ID NO: 18

[0068]

[0069] Experimental Example 1: Animal Model of Hypoxic-Ischemic Brain Damage

[0070] One-week-old ICR mice were used as animal models in the experimental animal laboratory of the Yonsei Life Research Institute, Yonsei University College of Medicine, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The carotid artery leading to the right brain of the animal model was ligated to block blood flow, and then brain damage was induced by exposure to a hypoxic environment (8% O2 and 90% N2). Viruses were injected into the lesion site in the acute phase (3 days after spinal cord injury). The date of sacrifice was adjusted according to the timing of gene expression of the injected virus, and perfusion was performed to remove blood. Depending on the subsequent experiment, brain tissue was isolated immediately or obtained after fixation with 4% PFA.

[0071]

[0072] Experimental Example 2: Comparison of immunohistochemical (IHC) results of AAV1#11-6, AAV1#11-10, and wild-type AAV1.

[0073] AAV1#11-6, AAV1#11-10 and wild type AAV1 (1x10) were injected into the lesion site in the acute phase after hypoxic-ischemic brain injury. 8vg / μL; 6 μL) was injected. The injected virus was loaded with the GFP gene, and the presence or absence of GFP gene expression could be confirmed through immunohistochemical staining. On the 7th day after AAV injection, the tissue was isolated, and the distribution of AAV expression around the lesion site was analyzed by immunochemical staining using GFP and GFAP (Fig. 2). In addition, using GFAP (red), an astrocyte-specific marker, and DAPI (blue), a cell nucleus staining marker, the count number and co-expression ratio for each marker are shown in Fig. 3. Looking at the results of the above experiment, it was confirmed that AAV1-based AAV1#11-6 and AAV1#11-10 showed higher gene transfer efficiency in the lesion site than wild-type AAV1.

[0074]

[0075] Experimental Example 3: Analysis of the AAV variant's astroglial infection efficiency.

[0076] Among the AAV variants selected in an animal model of hypoxic-ischemic brain injury, we examined the gene expression distribution of AAV1#11-10, one of the variants with a high GFP expression rate in the animal model, and the parent vector AAV1. Three days after induction of the brain injury model, two types of variants carrying CMV-NeuroD1-T2A-GFP and the parent vector AAV1 were injected into astrocytes. One week after injection, the expression distribution of the variants was analyzed using immunohistochemical staining. The expression of astrocyte markers (GFAP), microglia markers (IBA1), oligodendrocyte markers (MBP), and neuronal markers (NeuN) was confirmed in GFP-positive cells. The results of immunohistochemical staining analysis after injection of NeuroD1-loaded AAV variants are shown in Figure 4.

[0077] In the case of the AAV1#11-10 mutant, the ratios of GFAP were 57.1%, IBA1 1.6%, MBP 5.5%, and NeuN 3.8%. On the other hand, in the case of AAV1, the ratios of GFAP were 35.8%, IBA1 2.6%, MBP 8.9%, and NeuN 2.4%. Among the GFP-positive cells, the cells with the highest proportion were astrocytes, and the increased infection of the AAV1#11-10 mutant into astrocytes was confirmed with a significant statistical difference (p<0.05) compared to the parent vector, AAV1.

[0078]

[0079] Experimental Example 4: Analysis of direct cross-differentiation induction of AAV variants

[0080] To confirm the efficiency of neuronal differentiation of astrocytes by NeuroD1 after gene expression, CMV-NeuroD1-T2A-GFP-loaded AAV1#11-10 and AAV1 were transplanted in the acute phase after hypoxic-ischemic brain injury. 7 and 30 days after transplantation, the expression of astrocytes (GFAP+) or neurons (NeuN+) in AAV-infected cells (GFP+) according to the post-transplantation period was analyzed using immunohistochemical staining to directly analyze the transdifferentiation efficiency. Figure 5 shows the results of analyzing GFAP / GFP or NeuN / GFP by expression time after injection of NeuroD1-loaded AAV variants.

[0081] We confirmed that astrocytes differentiate into neurons by decreasing the proportion of astrocytes and increasing the proportion of neurons among AAV-infected cells. In the case of the AAV1#11-10 mutant, GFAP decreased from 57.1% (day 7) to 18.6% (day 30), and NeuN increased from 3.8% (day 7) to 26.7% (day 30). Meanwhile, in the case of AAV1, GFAP decreased from 35.8% (day 7) to 14.3% (day 30), and NeuN increased from 2.4% (day 7) to 13.6% (day 30).

[0082]

[0083] Experimental Example 5: In vivo safety analysis of AAV variants

[0084] After acute hypoxic-ischemic brain injury, CMV-NeuroD1-T2A-GFP-loaded AAV1, AAV1#11-10 variants, or saline (vehicle group) were transplanted, respectively, and the in vivo safety of the virus transplantation was observed by measuring the mortality rate and body weight of the animals. Figure 6 shows the body weight change and mortality rate of the animal model after injection of the NeuroD1-loaded AAV variants. The mortality rate after virus transplantation was the highest at 25% in the vehicle group, whereas all animals with the AAV1#11-10 variant survived. No statistically significant difference was found in the body weight change of the animal model between each group after transplantation.

[0085]

[0086] Experimental Example 6: Off-target and toxicity testing of AAV variants

[0087] As in Experimental Example 6 above, tissues (brain, heart, lung, liver, kidney, and gonad) from each group were isolated 4 weeks after each virus transplant. After DNA extraction from each isolated tissue, copy number analysis of the transferred gene and blood tests were performed. Figure 7 shows the DNA copy number and blood test results for each tissue after injection of the NeuroD1-loaded AAV variant. The brain showed the highest copy number, followed by the liver and heart, and there was no statistically significant difference compared to the parent vector. Blood tests confirmed that the AST level, which indicates hepatotoxicity, was statistically significantly increased in the vehicle group compared to the groups injected with AAV.

Claims

1. A mutant of adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant is a mutant of the AAV1 capsid protein in which an amino acid present at one or more of positions 237 and 582 in the amino acid sequence of the wild-type AAV1 capsid protein (SEQ ID NO: 1) is substituted.

2. In the first paragraph, the mutant is a mutant of an AAV1 capsid protein comprising at least one substitution of aspartic acid (D) at position 237 to asparagine (N) and a substitution of valine (V) at position 582 to aspartic acid (D) in the amino acid sequence of a wild-type AAV1 capsid protein (SEQ ID NO: 1).

3. In the second paragraph, the mutant is an AAV1 capsid protein mutant including both a substitution of aspartic acid (D) at position 237 with asparagine (N) and a substitution of valine (V) at position 582 with aspartic acid (D) in the amino acid sequence of a wild-type AAV1 capsid protein (SEQ ID NO: 1).

4. A nucleic acid encoding a mutant of the AAV1 capsid protein of paragraph 1.

5. In paragraph 4, the nucleic acid is a nucleic acid having a base sequence represented by sequence number:

16.

6. A recombinant AAV1 vector comprising the nucleic acid of claim 4 or 5.

7. In paragraph 6, the recombinant AAV1 vector is a recombinant AAV1 vector having superior tropism for brain astrocytes compared to an AAV1 wild-type viral vector.

8. A pharmaceutical composition for preventing or treating brain disease comprising the recombinant AAV1 vector of clause 6.

9. A pharmaceutical composition for preventing or treating a brain disease in clause 8, wherein the brain disease is hypoxic ischemic brain damage.

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