Adeno-associated virus vector capable of smooth muscle cell-specific gene delivery

Abstract

The present disclosure relates to an adeno-associated virus vector capable of smooth muscle cell-specific gene delivery. With significantly enhanced tropism for smooth muscle cells, the adeno-associated virus variants of the present disclosure can be advantageously applied as a gene therapeutic agent for treating smooth muscle-related diseases.

Description

Adeno-associated virus vector capable of smooth muscle cell-specific gene transfer

[0001] This application claims the benefit of priority to Republic of Korea Application No. 10-2024-0015880, filed February 1, 2024, Republic of Korea Application No. 10-2024-0081460, filed June 21, 2024, and Republic of Korea Application No. 10-2024-0178930, filed December 4, 2024, all of which are hereby incorporated by reference in their entirety.

[0002] The present disclosure relates to an adeno-associated virus vector capable of smooth muscle cell-specific gene transfer.

[0003] Gene therapy, an innovative treatment method that delivers genes into a patient's cells for the treatment of specific diseases, has attracted significant attention in recent years. Successful gene therapy requires an efficient and safe gene delivery system capable of selectively delivering genes to target cells. Adeno-associated virus (AAV) vectors are widely studied as one such delivery system. AAV vectors possess characteristics such as nonpathogenicity, stable gene expression, and the potential for selective delivery to various cells and tissues, making them an important tool in the field of gene therapy.

[0004] Smooth muscle cells (SMCs) are found in various organs, including blood vessels, bronchi, and the digestive tract. Diseases associated with SMCs include arteriosclerosis, hypertension, asthma, irritable bowel syndrome (IBS), and pulmonary hypertension. Atherosclerosis is a disease characterized by the accumulation of cholesterol and inflammatory cells in the blood vessel walls, narrowing the vessel. The proliferation and migration of SMCs play a crucial pathological role. Hypertension is primarily caused by dysfunction of smooth muscle cells, which regulate vascular contraction and relaxation. Asthma is a disease characterized by excessive contraction of airway smooth muscle cells, narrowing the airways and causing breathing difficulties. Irritable bowel syndrome is a disease characterized by symptoms such as abdominal pain, constipation, and diarrhea due to dysregulation of intestinal smooth muscle cell activity. Most of these diseases are closely linked to abnormal activity or dysfunction of SMCs.

[0005] Therefore, gene delivery technologies targeting smooth muscle cells could play a crucial role in treating these diseases. However, most AAV vectors developed to date lack specificity for smooth muscle cells, leading to the problem of gene delivery to non-target cells and tissues. This increases the likelihood of undesirable side effects and can reduce therapeutic efficacy.

[0006] To enable smooth muscle cell-specific gene delivery, strategies are being studied that involve modifying the capsid protein of AAV vectors or utilizing specific promoters. Modifying the capsid protein enhances binding to receptors on the surface of smooth muscle cells, thereby enhancing intracellular uptake efficiency. Furthermore, utilizing specific promoters is designed to selectively express genes only in target cells. These approaches can simultaneously enhance the safety and efficacy of gene therapy by improving specificity for smooth muscle cells.

[0007] Therefore, the challenge that the present disclosure seeks to address is to overcome the limitations of existing technologies and maximize gene therapy efficiency by providing a novel AAV vector that enables smooth muscle cell-specific gene delivery.

[0008] In order to solve the above problem, the inventors of the present disclosure have conducted research efforts and discovered that by improving the capsid protein of adeno-associated virus (preferably by point mutation, peptide insertion mutation, or both), the tropism for smooth muscle cells is significantly increased, thereby completing the present invention.

[0009] The present disclosure provides an adeno-associated virus mutant, preferably an adeno-associated virus capsid protein mutant, capable of specifically targeting smooth muscle cells. The capsid protein of the adeno-associated virus mutant may have at least one amino acid substituted with a different amino acid compared to the wild-type capsid protein, and may further include a peptide insertion sequence in the capsid protein.

[0010] 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 subunits, 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), AF063497 (AAV-1).

[0011] In one aspect of the present disclosure, the adeno-associated virus mutant may be a mutant derived from serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh74, but is not limited thereto. The term serotype refers to a subdivision of AAV that can be identified by serological or DNA sequencing methods and distinguished by its antigenic characteristics. The adeno-associated virus mutant may be a mutant derived from wild-type AAV1.

[0012] In one aspect of the present disclosure, the term "capsid" refers to a protein encoded by the cap gene present in the genome of a virus, 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).

[0013] In one aspect of the present disclosure, the mutant of the adeno-associated virus capsid protein may be a mutant of the AAV1 capsid protein, and any one or more amino acids selected from the group consisting of the 14th, 21st, 23rd, 24th, 31st, 32nd, 35th, 36th, 37th, 38th, 39th, 42nd, 137th, 310th, 415th, 430th, 492nd, 516th, 583rd, 647th, 660th, and 688th amino acids may be substituted with another amino acid compared to the wild-type AAV1 capsid protein (SEQ ID NO: 1). In one aspect of the present disclosure, when the mutant of the adeno-associated virus capsid protein is a mutant of the AAV1 capsid protein, the 14th amino acid substitution compared to the wild-type AAV1 capsid protein Asparagine (N) may be substituted with threonine (T) (N14T), the 21st amino acid substitution may be substituted with glutamic acid (E) (E21Q), the 23rd amino acid substitution may be substituted with tryptophan (W) (W23R), the 24th amino acid substitution may be substituted with lysine (K) (D24K), the 31st amino acid substitution may be substituted with lysine (K) (K31T), the 32nd amino acid substitution may be substituted with proline (P) (P32T), the 35th amino acid substitution may be substituted with arginine (R) (N35R), and the 36th amino acid substitution may be substituted with glutamine (Q) The 37th amino acid substitution may be a substitution of glutamine (Q) with arginine (R) (Q37R), the 38th amino acid substitution may be a substitution of lysine (K) with histidine (H) (K38H), and the 39th amino acid substitution may be a substitution of glutamine (Q) with lysine (K) (Q39K).The 42nd amino acid substitution may be a substitution of glycine (G) with serine (S) (G42S), the 137th amino acid substitution may be a substitution of lysine (K) with glutamic acid (E) (K137E), the 310th amino acid substitution may be a substitution of lysine (K) with arginine (R) (K310R), the 415th amino acid substitution may be a substitution of threonine (T) with isoleucine (I) (T415I), the 430th amino acid substitution may be a substitution of serine (S) with arginine (R), cysteine ​​(C), or glutamic acid (E) (S430R, S430C, or S430E), the 492nd amino acid substitution may be a substitution of threonine (T) with alanine (A) (T492A), and the 516th amino acid substitution may be a substitution of serine (S). Threonine (T) may be substituted (S516T), the 583rd amino acid substitution may be a substitution of asparagine (N) with aspartic acid (D) (N583D), the 647th amino acid substitution may be a substitution of isoleucine (I) with alanine (A), valine (V), or tyrosine (Y) (I647A, I647V, or I647Y), the 660th amino acid substitution may be a substitution of alanine (A) with valine (V) (A660V), and the 688th amino acid substitution may be a substitution of glutamine (Q) with arginine (R) (Q688R).

[0014] In one aspect of the present disclosure, the mutant of the adeno-associated virus capsid protein can comprise S430C and I647V substitutions compared to a wild-type AAV1 capsid protein, the amino acid sequence of which is represented by SEQ ID NO: 2. Furthermore, the mutant of the adeno-associated virus capsid protein can comprise S516T, N583D and A660V substitutions compared to a wild-type AAV1 capsid protein, the amino acid sequence of which is represented by SEQ ID NO: 3. Furthermore, the mutant of the adeno-associated virus capsid protein can comprise K310R substitution compared to a wild-type AAV1 capsid protein, the amino acid sequence of which is represented by SEQ ID NO: 4. Furthermore, the mutant of the adeno-associated virus capsid protein can comprise W23R and T415I substitutions compared to a wild-type AAV1 capsid protein, the amino acid sequence of which is represented by SEQ ID NO: 5. In addition, the mutant of the adeno-associated virus capsid protein may include N14T, E21Q, D24K, K31T, P32T, N35R, Q36E, Q37R, K38H, Q39K and G42S substitutions compared to the wild-type AAV1 capsid protein, and the amino acid sequence of such a mutant is represented by SEQ ID NO: 6. In addition, the mutant of the adeno-associated virus capsid protein may include Q688R substitution compared to the wild-type AAV1 capsid protein, and the amino acid sequence of such a mutant is represented by SEQ ID NO: 7. In addition, the mutant of the adeno-associated virus capsid protein may include T492A substitution compared to the wild-type AAV1 capsid protein, and the amino acid sequence of such a mutant is represented by SEQ ID NO: 8.Additionally, the mutant of the adeno-associated virus capsid protein may include a K137E substitution compared to the wild-type AAV1 capsid protein, and the amino acid sequence of such a mutant is represented by SEQ ID NO: 9.

[0015] In one aspect of the present disclosure, the 430th amino acid substitution may be, in addition to serine being substituted with cysteine, a substitution of serine with arginine (R) or glutamic acid (E) (S430R, S430E), and the amino acid sequences of these mutants are represented by SEQ ID NOs: 10 or 11, respectively. In addition, the 647th amino acid substitution may be, in addition to isoleucine being substituted with valine, a substitution of isoleucine with alanine (A) or tyrosine (Y) (I647A, I647Y), and the amino acid sequences of these mutants are represented by SEQ ID NOs: 12 or 13, respectively.

[0016] In one aspect of the present disclosure, the adeno-associated virus capsid protein mutant may be a mutant of an AAV capsid protein of a serotype other than AAV1, such as AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh74. In such cases, amino acid substitutions may occur at amino acid positions of the other serotype corresponding to each substitution position of the wild-type AAV1.

[0017] In one aspect of the present disclosure, the peptide insertion sequence included in the capsid protein of the adeno-associated virus mutant may be a peptide insertion sequence composed of X1X2X3X4X5X6X7 (chemical formula I). ​​By including the specific peptide insertion sequence in the capsid protein of the adeno-associated virus mutant together with amino acid substitutions at specific positions of the adeno-associated virus capsid protein mutant, the tropism of the adeno-associated virus mutant for the surface receptor of smooth muscle cells can be significantly increased.

[0018] In one aspect of the present disclosure, the peptide insertion sequence consisting of X1X2X3X4X5X6X7 (chemical formula I) can be inserted into a loop region, preferably a GH loop region, of a wild-type adeno-associated virus, preferably a wild-type AAV1. Preferably, the peptide insertion sequence can be inserted between two adjacent amino acids among amino acids 580 to 600 of the wild-type AAV1, and more preferably between amino acids 588 to 589. Alternatively, the peptide insertion sequence can be inserted into an amino acid position of a capsid protein of another serotype corresponding to the insertion site of the wild-type AAV1.

[0019] In one aspect of the present disclosure, the X1 amino acid can be any one selected from the group consisting of S, L, A, P, N, V, D, R, E, Q, and T. The X2 amino acid can be any one selected from the group consisting of S, N, G, R, T, A, P, E, V, Q, and K. The X3 amino acid can be any one selected from the group consisting of T, E, S, N, V, P, R, A, G, K, and D. The X4 amino acid can be any one selected from the group consisting of S, T, P, K, D, Q, Y, A, V, L, and N. The X5 amino acid can be any one selected from the group consisting of P, S, T, D, G, A, N, L, K, R, and I. The X6 amino acid can be any one selected from the group consisting of L, Q, T, V, N, G, P, S, A, K, and D. The above X7 amino acid may be any one selected from the group consisting of S, T, P, G, Q, K, A, R, V, H, and E.

[0020] In one aspect of the present disclosure, the formula I may comprise at least one or more of the amino acids designated for X1 to X7, or may comprise all of them.

[0021] In one aspect of the present disclosure, the formula I is (a) wherein X1 is any one selected from the group consisting of A, R, N, D, L, P, and S, wherein X7 is any one selected from the group consisting of A, Q, G, P, S, T, and V, and wherein X2, X3, X4, X5, and X6 are independently any amino acid;

[0022] (b) wherein X5 is any one selected from the group consisting of A, N, G, I, P, S, and T, wherein X6 is any one selected from the group consisting of R, N, Q, G, L, T, and V, and wherein X1, X2, X3, X4, and X7 are independently any amino acid; or

[0023] (c) wherein X2 is any one selected from the group consisting of R, N, Q, G, P, S, and T, wherein X3 is any one selected from the group consisting of N, D, E, P, S, T, and V, and wherein X1, X4, X5, X6, and X7 can independently be any amino acid.

[0024] In one aspect of the present disclosure, the insertion sequence may be any one of the insertion sequences described in Table 1 below. Preferably, the insertion sequence may be any one selected from the group consisting of SGVSILG (SEQ ID NO: 151), SPNSAGV (SEQ ID NO: 152), and SQDHGRS (SEQ ID NO: 264).

[0025] In one aspect of the present disclosure, the capsid amino acid sequence of an adeno-associated virus mutant having SPNSAGV as a peptide insertion sequence and no point mutations is represented by SEQ ID NO: 14. In addition, the capsid amino acid sequence of an adeno-associated virus mutant having SGVSILG as a peptide insertion sequence and no point mutations is represented by SEQ ID NO: 15. In addition, the capsid amino acid sequence of an adeno-associated virus mutant having SPNSAGV as a peptide insertion sequence and S430C and I647V point mutations is represented by SEQ ID NO: 16. In addition, the capsid amino acid sequence of an adeno-associated virus mutant having SGVSILG as a peptide insertion sequence and S430C and I647V point mutations is represented by SEQ ID NO: 17. Mutants comprising a combination of the above peptide insertion sequences and point mutations can synergistically and significantly increase tropism for smooth muscle.

[0026] In one aspect of the present disclosure, the cell to which the mutant of the adeno-associated virus capsid protein can exhibit tropism may be a smooth muscle cell, and preferably a human smooth muscle cell. The human smooth muscle cell may be a smooth muscle cell present in the digestive system (esophagus, stomach, small intestine, large intestine, rectum, and anus), respiratory system (lungs, trachea, bronchi, bronchioles), urinary system (kidneys, ureters, bladder, urethra), reproductive system (vas deferens, epididymis, prostate, uterus, fallopian tubes, vagina), vascular system (arteries, veins), lymphatic vessels, eye (iris muscle, ciliary muscle), skin (around hair roots), and others (bile duct, pancreatic duct, etc.), but is not limited thereto.

[0027] The term "tropism" may refer 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. Tropism refers to 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 into one or more specific cell types. That is, tropism refers to the preferential entry of an AAV vector or virion into a particular cell or tissue type and / or preferential interaction with a cell surface that facilitates entry into a particular cell or tissue type, followed by expression (transcription and translation) of sequences carried by the AAV vector or virion in the cell, e.g., expression of heterologous nucleotide sequences in the case of a recombinant virus.

[0028] In addition, the present disclosure provides a nucleic acid encoding a mutant of the adeno-associated virus capsid protein. The nucleic acid can encode a mutant of the AAV1 capsid protein. The base sequence corresponding to the amino acid sequence of SEQ ID NOs: 1 to 17 is represented as SEQ ID NOs: 18 to 34. The nucleic acid in the present disclosure 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 disclosure may exist in the form of DNA, but may also 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.

[0029] In one aspect of the present disclosure, the nucleic acid may be operably linked to a suitable 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, and may be a natural or synthetic sequence.

[0030] The present disclosure also provides a recombinant adeno-associated virus vector comprising the nucleic acid. The recombinant AAV vector is useful for gene introduction into target cells, and the gene introduced by the recombinant AAV vector can be strongly expressed in the target cells. The recombinant AAV vector is useful for delivering the nucleic acid of the present disclosure to cells in vitro, ex vivo, and in vivo, and for conferring the ability to express mutants of the AAV1 capsid protein to the cells. Furthermore, the 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. In the present disclosure, the 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. For example, a packaging plasmid can be produced by harboring a nucleic acid (cap gene) encoding a mutant of the AAV1 capsid protein of the present disclosure 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 add Rep derived from AAV2. A recombinant AAV vector comprising the nucleic acid of the present disclosure 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 in a known packaging plasmid with another base. The packaging plasmid is not particularly limited, but examples thereof include a packaging plasmid loaded with a cap gene, and preferably a packaging plasmid loaded with both a cap gene and a rep gene.

[0031] In one aspect of the present disclosure, an AAV virus refers to a viral particle comprised of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector. 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. Therefore, the production of an rAAV particle necessarily involves the production of rAAV, since the vector is contained within the rAAV particle. The term packaging refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. The AAV rep and cap genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of adeno-associated viruses. AAV rep and cap are referred to herein as AAV packaging genes.

[0032] Helper viruses for AAV refer to viruses that enable 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. 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), which are also available from depositories such as the ATCC. Helper virus function refers to a function encoded in the helper virus genome that allows AAV replication and packaging. As described herein, helper virus function can be provided in various ways, including by providing a helper virus or, for example, by transfecting a producer cell with a polynucleotide sequence encoding an essential function. For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenovirus proteins can be transfected into a producer cell together with the recombinant AAV vector.

[0033] In addition, the present disclosure provides a pharmaceutical composition for preventing or treating a smooth muscle cell-related disease comprising the recombinant adeno-associated virus vector. The smooth muscle cell-related disease includes, but is not limited to, vascular smooth muscle-related diseases (e.g., hypertension, aneurysm, atherosclerosis), leiomyoma, leiomyosarcoma, smooth muscle relaxation disorders (e.g., achalasia, bladder outlet obstruction), smooth muscle hypercontraction (e.g., bronchospasm, intestinal spasm, uterine hypercontraction), congenital smooth muscle abnormalities (e.g., congenital megacolon, pulmonary hypertension), smooth muscle infections and inflammatory diseases (e.g., endometritis), and other metabolic and hereditary smooth muscle diseases (e.g., congenital leiomyomatosis, mitochondrial diseases).

[0034] As used herein, the term "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 a subject without a disease (e.g., for prophylaxis).

[0035] The present disclosure also provides a method for preventing or treating a smooth muscle-related disease, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition. The administration refers to physically introducing the therapeutic agent or a composition comprising the therapeutic agent into the subject using any of various methods and delivery systems known to those skilled in the art. Preferred routes of administration include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, intravitreal, or other parenteral routes of administration, for example, by injection or infusion. The 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, 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.

[0036] In one aspect of the present disclosure, a 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., pulmonary arterial hypertension). 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 developing a disease or disorder. 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 or at least one clinical symptom of the disease or disorder.

[0037] In one aspect of the present disclosure, the 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.

[0038] The adeno-associated virus mutant of the present disclosure has remarkably excellent tropism for smooth muscle cells and can be usefully used as a gene therapy agent for smooth muscle-related diseases.

[0039] Figure 1 shows the results of imaging the lung tissue tropism of mutants selected three times in mouse lung smooth muscle cells using X-gal staining.

[0040] Figure 2 shows the results of immunohistochemical imaging of the migration and transduction abilities of wild-type AAV1, mutants AAV1.S430C, AAV1.I647V, and AAV1.p2CV into pulmonary artery smooth muscle cells.

[0041] Figure 3 shows the results of monitoring the movement trajectories of wild-type AAV1, mutants AAV1.S430C, AAV1.I647V, and AAV1.p2CV in pH7.4 PBS.

[0042] Figure 4 shows the results of measuring the MSD values ​​of moving trajectories of wild-type AAV1, mutants AAV1.S430C, AAV1.I647V, and AAV1.p2CV in pH 7.4 PBS according to the length of measurement time.

[0043] Figure 5 shows the results showing the moving trajectories and MSD values ​​in pH 7.4 PBS for mutants AAV1.I647A, AAV1.I647V, AAV1.I647Y, AAV1.S430R, AAV1.S430C, and AAV1.S430E.

[0044] Figure 6 shows the results of delivering the green fluorescent protein (GFP) gene to human pulmonary artery smooth muscle cells (hPASMC) in vitro using wild-type AAV1, mutants AAV1.S430C, AAV1.I647V, and AAV1.p2CV, and measuring the percentage of GFP-expressing cells and average brightness through fluorescence imaging.

[0045] Figure 7 shows the results of delivering the human FGF12 (hFGF12) gene to a pulmonary arterial hypertension mouse model in vivo via airway aerosol administration using wild-type AAV1 and mutant AAV1.p2CV, and measuring the amount of hFGF12 mRNA expression in pulmonary smooth muscle cells (PSMC) via RT-PCR.

[0046] Figure 8 shows the results of delivering the hFGF12 gene to human pulmonary artery smooth muscle cells (hPASMC) in vitro using AAV1-based adeno-associated virus mutants in Experimental Example 14 and measuring the hFGF12 mRNA expression level through cDNA qPCR.

[0047] Figures 9 and 10 show the results of delivering the human FGF12 (hFGF12) gene to human pulmonary artery smooth muscle cells (hPASMC) in vitro using AAV1-based adeno-associated virus mutants in Experimental Example 14, and measuring the mRNA expression levels of ACTA and TAGLN genes expressed through hFGF12 through cDNA qPCR.

[0048] Figures 11 and 12 show the results of an in vivo experiment on the preventive effect of pulmonary arterial hypertension (PAH) using AAV1-based adeno-associated virus mutants in Experimental Example 15.

[0049] 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.

[0050]

[0051] Experimental Example 1: Production of an AAV1-based point mutant adeno-associated virus library.

[0052] Random mutations were introduced into the wild-type AAV1 cap gene using error-prone PCR based on wild-type AAV1. During error-prone PCR, various concentrations of manganese(II) chloride (Sigma Aldrich, St. Louis, MO; final concentrations of 0.05 mM, 0.15 mM, and 0.3 mM in the total PCR mixture) 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. Taq polymerase (Thermo Fisher Scientific, Waltham, MA) was used to extend each mutated cap gene. Each error-prone PCR product was cloned downstream of rep2, which is bordered by inverted terminal repeats (ITRs), to generate a plasmid library containing AAV capsid genes with random point mutations.

[0053] The combination of the above AAV1-based point 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 h after transfection, HEK293T cells were harvested, lysed through three freeze-thaw cycles, and the lysate was treated with benzonase chelator (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).

[0054]

[0055] Experimental Example 2: Screening of lung smooth muscle cell-targeting variants from an AAV1-based point mutant adeno-associated virus library.

[0056] The recombinant AAV1 variant library prepared in Experimental Example 1 was aerosolized into the trachea of ​​8-week-old male C57BL6 mice using a PenWu microaerosolizer (BioJane, Shanghai, China) (1.25" length of intratracheal portion, 700 μm outer diameter, 430 μm inner diameter). Two weeks later, the mice were necropsied. Smooth muscle cells were isolated from lung tissue obtained at the necropsy using flow cytometry. In the flow cytometry, smooth muscle cells were labeled and isolated using APC-conjugated α-sma antibody (Abcam, Cambridge, UK). The genes of AAV1 variants were recovered from the isolated lung smooth muscle cells using Qiagen 51306.

[0057] The recovered capsid DNA was amplified and inserted into a plasmid, transfected into E. coli, and cloned to obtain a selected library. An AAV mutant library was prepared using the library, and the process of aerosol administration to the mouse airway and selection was repeated three times in total. Mutation information of AAV1-based mutants discovered through Sanger sequencing of capsid DNA recovered from multiple single colonies obtained after transfecting E. coli during the library selection repetition process is shown in Table 1. Among the discovered mutants, a mutant in which serine at position 430 of the wild-type AAV1 capsid amino acid sequence was substituted with cysteine ​​(S430C) and isoleucine at position 647 was substituted with valine (I647V) was designated AAV1.p2CV. In addition, a mutant in which serine at position 516 was substituted with threonine (S516T), asparagine at position 583 was substituted with aspartic acid (N583D), and alanine at position 660 was substituted with valine (A660V) was named AAVp5. In addition, a mutant in which lysine (K) at position 310 was substituted with arginine (R) (K310R) was named AAVp3.1. In addition, a mutant in which tryptophan (W) at position 23 was substituted with arginine (R) (W23R), and threonine (T) at position 415 was substituted with isoleucine (I) (T415I) was named AAVp3.2.Additionally, a mutant in which the 14th asparagine was replaced with threonine (N14T), the 21st glutamic acid was replaced with glutamine (E21Q), the 24th aspartic acid was replaced with lysine (D24K), the 31st lysine was replaced with threonine (K31T), the 32nd proline was replaced with threonine (P32T), the 35th asparagine was replaced with arginine (N35R), the 36th glycine was replaced with glutamic acid (Q36E), the 37th glutamine was replaced with arginine (Q37R), the 38th lysine was replaced with histidine (K38H), the 39th glutamine was replaced with lysine (Q39K), and the 42nd glycine was replaced with serine (G42S) was named AAVp3.44. Additionally, a mutant in which glutamine at position 688 was substituted with arginine (Q688R) was designated AAVp3.55. Additionally, a mutant in which threonine at position 492 was substituted with alanine (T492A) was designated AAVp3.55. Additionally, a mutant in which lysine at position 137 was substituted with glutamic acid (K137E) was designated AAVp3.97.

[0058] The DNA recovered during the above process was amplified in quantity through PCR, and the capsid gene of the library selected from pulmonary smooth muscle cells was read using Pacbio-SMRT (Pacific Bioscience) next-generation sequencing (NGS) equipment. Mutation information was collected from the NGS results, and mutants containing the point mutations present in Table 1 are shown in Table 2.

[0059] In the above process, there were mutants that were found to be overlapping in the Sanger sequencing results and NGS results, and these were p2CV, AAVp5, AAVp3.97, AAVp3.56, AAVp3.55, AAVp3.2, and AAVp3.1. In addition, the mutants that contained the point mutations of the mutants discovered in Table 1 were indicated by adding "partial_" to the mutant characteristics in Table 2, and a wide variety of combinations of mutations other than those in Table 1 were discovered in the lung smooth muscle cell NGS results. This shows that mutants formed by combinations of each point mutation present in mutants with improved lung smooth muscle cell tropism can improve lung smooth muscle cell tropism.

[0060] Point mutation sequence number variant name S430C, I647V2AAV1.p2CVS516T, N583D, A660V3AAVp5K310R4AAVp3.1W23R, T415I5AAVp3.2N14T, E21Q, D24K, K31T, P32T, N35R, Q36E, Q37R, K38H, Q39K, G42S6AAVp3.44Q688R7AAVp3.55T492A8AAVp3.56K137E9AAVp3.97

[0061] 점 돌연변이변이체 특성S430C, I647VAAV1.p2CVK310RAAVp3.1W23R, T415IAAVp3.2Q688RAAVp3.55T492AAAVp3.56K137EAAVp3.97S516T, N583D, A660VAAVp5I647Vpartial_AAV.p2CVY414C, I647Vpartial_AAV.p2CVT242S, D556G, I647Vpartial_AAV.p2CVS430Cpartial_AAV.p2CVL53I, I647Vpartial_AAV.p2CVK123R, I647Vpartial_AAV.p2CVD9G, D556G, I647Vpartial_AAV.p2CVK31Q, D154G, K310R, V582G, M599Vpartial_AAVp3.1K310R, S663Tpartial_AAVp3.1K310R, A598Vpartial_AAVp3.1W23Rpartial_AAVp3.2W23R, F173Lpartial_AAVp3.2N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105Kpartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42Spartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136V, Q148H, Q151V, I159T, T162A, K168R, S179A, E180D, E190Q, T194A, A196S, A197G, V198L, P200T, T201N, S205T, G207Spartial_AAVp3.44N14Tpartial_AAVp3.44Q37Rpartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136V, Q148H, Q151V, I159T, T162A, K168R, S179A, E180D, E190Q, T194A, A196S, A197G, V198L, P200T, T201N, S205T, G207S, A224Spartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67Epartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136V, Q148H, Q151V, I159T, T162A, K168Rpartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92Kpartial_AAVp3.44K31T, M203Kpartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136Vpartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136V, Q148H, Q151V, I159T, T162A, K168R, S179A, E180D, E190Q, T194A, A196S, A197G, V198L, P200T, T201N, S205T, G207S, A224S, L235Mpartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136V, Q148H, Q151V, I159T, T162A, K168R, S179A, E180D, E190Q, T194A, A196S, A197G, V198L, P200T, T201Npartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136V, Q148H, Q151V, I159T, T162A, K168R, S179A, E180Dpartial_AAVp3.44N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, G135P, A136V, H272Tpartial_AAVp3.44N14T, E21Qpartial_AAVp3.44K31Tpartial_AAVp3.44P32T, S385Gpartial_AAVp3.44N14T, T138A, S205Tpartial_AAVp3.44N14T, L129F, N383D, V558Ipartial_AAVp3.44N14T, L129F, N383Dpartial_AAVp3.44N14T, L129Fpartial_AAVp3.44N14T, D24T, F56Gpartial_AAVp3.44K31T, M203K, S663Tpartial_AAVp3.44G42Spartial_AAVp3.44E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, Y273Hpartial_AAVp3.44D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136V, Q148H, Q151V, I159T, T162A, K168R, S179A, E180D, E190Q, T194A, A196S, A197G, V198L, P200T, T201Npartial_AAVp3.44D24A, K31T, K84Q, T589A, I683Vpartial_AAVp3.44G42S, T492Apartial_AAVp3.44, partial_AAVp3.55G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136V, Q148H, Q151V, I159T, T162A, K168R, S179A, E180D, E190Q, T194A, A196S, A197G, V198L, P200T, T201N, S205T, G207S, A224S, L235M, T492Apartial_AAVp3.44, partial_AAVp3.56N14T, E21Q, Q688Rpartial_AAVp3.44, partial_AAVp3.56K31Q, M599V, Q688Rpartial_AAVp3.55I159V, Q688Rpartial_AAVp3.55D24A, Q688Rpartial_AAVp3.55T326A, Q688Rpartial_AAVp3.55Q688R, N718Dpartial_AAVp3.55Q386H, Q688Rpartial_AAVp3.55P571H, Q688Rpartial_AAVp3.55P32S, P191L, P571H, Q688Rpartial_AAVp3.55N458S, Q688Rpartial_AAVp3.55M211V, K641M, Q688Rpartial_AAVp3.55K641M, Q688Rpartial_AAVp3.55G189R, Q688Rpartial_AAVp3.55G163S, M211V, K641M, Q688Rpartial_AAVp3.55A196V, Q688Rpartial_AAVp3.55T176A, T492Apartial_AAVp3.56S205A, T492Apartial_AAVp3.56N172K, T492Apartial_AAVp3.56D24A, T492Apartial_AAVp3.56T492A, V654Ipartial_AAVp3.56T492A, T589Apartial_AAVp3.56T492A, S588Npartial_AAVp3.56T492A, S547Gpartial_AAVp3.56T492A, R694Hpartial_AAVp3.56T492A, N717Tpartial_AAVp3.56T492A, N496Spartial_AAVp3.56T492A, K533Rpartial_AAVp3.56T492A, I727Vpartial_AAVp3.56T492A, G544Vpartial_AAVp3.56T492A, F638L, G639W, L640T, K641Q, N642E, P644A, P645S, Q646S, I647D, L648P, I649H, K650Qpartial_AAVp3.56T492A, A709Tpartial_AAVp3.56S385G, T492A, N710Spartial_AAVp3.56S385G, T492Apartial_AAVp3.56S109T, T492Apartial_AAVp3.56Q164R, T492Apartial_AAVp3.56P420S, T492Apartial_AAVp3.56N35Y, T492Apartial_AAVp3.56N302K, T492Apartial_AAVp3.56N214S, T492Apartial_AAVp3.<h2 style=";text-align:left;direction:ltr">56L256I, T492Apartial_AAVp3.56L129F, T492Apartial_AAVp3.56K77E, T492Apartial_AAVp3.56K38R, E418D, T492A, N657Tpartial_AAVp3.56K33R, V125I, V132A, T492A, S703Ypartial_AAVp3.56K169R, T492Apartial_AAVp3.56K123R, T492Apartial_AAVp3.56G174C, T492A, T589Apartial_AAVp3.56G135A, T492Apartial_AAVp3.56G112C, T411A, T492Apartial_AAVp3.56F399S, T492Apartial_AAVp3.56D24A, L129F, T492Apartial_AAVp3.56K137E, K169R, N451Spartial_AAVp3.97N14T, E21Q, D24K, A29P, K31P, A34P, N35A, Q36E, Q37R, K38H, Q39K, G42S, A67E, Q81R, K84D, A85S, R92K, Q105K, G135P, A136V, Q148H, Q151V, I159T, T162A, K168R, S179A, E180D, E190Q, T194A, A196S, A197G, V198L, P200T, T201N, S205T, G207S, A224S, L235M, P250Spartial_AAVp3.44N583Dpartial_AAVp5A660Vpartial_AAVp5S516T, N583Dpartial_AAVp5A660V, D712Gpartial_AAVp5S516Tpartial_AAVp5N583D, A660Vpartial_AAVp5S499G, E575G, M599I, A660Vpartial_AAVp5S181L, A660Vpartial_AAVp5P475L, A660V, D712Gpartial_AAVp5K77E, S516T, N583D, A660Vpartial_AAVp5G594R, A660V, D712Gpartial_AAVp5G539D, A660V, D712Gpartial_AAVp5G356D, S516T, N583Dpartial_AAVp5D24A, N583D, S680Npartial_AAVp5A71V, A660V, D712Gpartial_AAVp5A3V, D24A, K84Q, N583Dpartial_AAVp5.

[0062]

[0063] Experimental Example 3: Production of adeno-associated virus mutants containing AAV1-based point mutations.

[0064] The capsid gene recovered in the process of Experimental Example 2 above and confirmed through Sanger sequencing was subcloned into pXX2 (UC Berkeley, David Schaffer Lab) containing HindIII and NotI, thereby completing the construction of a lung smooth muscle cell-specific plasmid mutant for loading the CMV-GFP gene and CMV-LacZ gene.

[0065] HEK293T cells were transfected with 17 μg of adeno-associated virus mutant plasmids containing the constructed AAV1-based point mutations, 17 μg of ITR-flanked reporter genes (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 the AAV inside the cells was extracted by freezing-thawing. Afterwards, cell debris was removed by centrifugation, and nucleic acids from virus-producing cells were removed by incubating with 10 U / mL of benzonase at 37 °C for 30 min to secure an AAV solution. AAV solution was 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 vector was 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).

[0066]

[0067] Experimental Example 4: Histological Analysis for Observing Lung Tissue Specificity of Adeno-Associated Virus Mutants Containing AAV1-Based Point Mutations

[0068] Each of the adeno-associated virus mutants AAVp5, AAVp3.1, AAVp3.2, AAVp2CV, AAVp3.44, AAVp3.55, AAVp3.56, and AAVp3.97 loaded with CMV-LacZ produced through the above Experimental Example 3 was aerosolized into the trachea of ​​8-week-old male C57BL6 mice using a PenWu microaerosolizer (BioJane, Shanghai, China) (1.25" length of intratracheal portion, 700 μm outer diameter, 430 μm inner diameter). Three weeks later, lung tissue was extracted and the expression of LacZ was confirmed through X-gal staining.

[0069] As a result, AAVp5 was able to confirm high LacZ gene expression throughout the lung tissue, and the remaining mutants were able to successfully deliver the LacZ gene to the lung airways or bronchi (Fig. 1).

[0070]

[0071] Experimental Example 5: Confirmation of Pulmonary Artery Smooth Muscle Cell Transduction Ability of Individual Point Mutations in AAV1.p2CV

[0072] AAV1.p2CV has a high spreading capacity and is known to deliver payload genes well to pulmonary artery smooth muscle cells through airway aerosol administration (see Domestic Application No. 10-2022-0120442). Therefore, the effects of the wild-type AAV1 point mutations S430C and I647V of AAV1.p2CV on pulmonary artery smooth muscle cell tropism were confirmed. The mutant in which serine at position 430 in the AAV1 amino acid sequence was substituted with cysteine ​​(S430C) was named AAV1.S430C, and the mutant in which isoleucine at position 647 was substituted with valine (I647V) was named AAV1.I647V, and the corresponding mutants were prepared in the same manner as in Experimental Example 3.

[0073] Wild-type AAV1 and mutants AAV1.S430C, AAV1.I647V, and AAV1.p2CV were administered intratracheally to male C57BL / 6 mice in the same manner as in Experimental Example 2. On days 1 and 2, the mice were necropsied, and the AAV movement into pulmonary artery smooth muscle cells was imaged using immunohistochemistry. For immunohistochemistry, AAV was stained with Alexa594 (Thermo Fisher Scientific) in lung tissue sections obtained at necropsy, and pulmonary smooth muscle cells were stained with Alexa488-conjugated anti-α-SMA primary antibody (Abcam; 1:100), and cell nuclei were stained with DAPI.

[0074] On day 1, wild-type AAV1 showed a very small amount of infection around the pulmonary artery (V) in the image, AAV1.S430C reached the bronchial (B) smooth muscle cells, AAV1.I647V moved beyond the bronchial smooth muscle cells, and AAV1.p2CV reached the area around the pulmonary artery. On day 2, wild-type AAV1 was observed around the pulmonary artery but its signal did not overlap with the pulmonary artery smooth muscle cells, AAV1.S430C overlapped with the pulmonary artery smooth muscle cells but showed a pattern of being widely spread around the pulmonary artery, AAV1.I647V showed a lot of signal overlapping with the pulmonary artery smooth muscle cells and a small amount was expressed in the vicinity of the pulmonary artery, and AAV1.p2CV completely overlapped the signal with the pulmonary artery smooth muscle cells (Fig. 2). Accordingly, it was confirmed that the combination of the S430C mutation and the I647V mutation produced a synergistic effect, further enhancing the tropism toward pulmonary artery smooth muscle cells.

[0075]

[0076] Experimental Example 6: Confirmation of the spreading capacity of individual point mutations and their combinations in AAV1.p2CV.

[0077] The trajectories of wild-type AAV1, mutants AAV1.S430C, AAV1.I647V, and AAV1.p2CV used in Experimental Example 5 above in pH 7.4 PBS were monitored for 1 second using NanoSights (Malvern Panalytical). As shown in Fig. 3, compared to wild-type AAV1, mutants AAV1.S430C, AAV1.I647V, and AAV1.p2CV moved a longer distance in the same amount of time. The change in Mean Squared Distance (MSD) according to the length of the observation time was such that, while the wild-type AAV1 remained at 0 even as the observation time increased, the AAV1.S430C increased slightly, and the AAV1.I647V increased rapidly and then remained constant, but the AAV1.p2CV containing both mutations simultaneously showed a remarkably steep increase (Fig. 4). This shows that when individual point mutations are combined, the synergistic effect can significantly enhance the diffusion capacity.

[0078]

[0079] Experimental Example 7: Confirmation of changes in diffusion capacity according to individual point mutations in AAV1.p2CV.

[0080] The changes in diffusion capacity were observed by substituting the 430th and 647th amino acids, which are two point mutation sites of AAV1.p2CV, with different types. The mutant in which serine at position 430 of the AAV1 amino acid sequence was substituted with arginine (S430R) was named AAV1.S430R, the mutant in which serine at position 430 was substituted with glutamic acid (S430E) was named AAV1.S430E, the mutant in which isoleucine at position 647 was substituted with alanine (I647A) was named AAV1.I647A, and the mutant in which isoleucine at position 647 was substituted with tyrosine (I647Y) was named AAV1.I64Y.

[0081] As a result of monitoring the moving trajectories for 1 second for mutants AAV1.I647A, AAV1.I647V, AAV1.I647Y, AAV1.S430R, AAV1.S430C, and AAV1.S430E in the same manner as in Experimental Example 6 (Fig. 5), AAV1.I647A moved farther than AAV1.I647V, and AAV1.I647Y moved less. In addition, AAV1.S430R moved less than AAV1.S430C, and AAV1.S430E moved farther than AAV1.S430E. The MSD values ​​calculated in the experiment are shown on the right side of Fig. 5.

[0082] The molecular weight of tyrosine is 181.191 Da, that of valine is 117.148 Da, and that of alanine is 89.094 Da, so it was found that the mutant with a substitution at the 647th amino acid was affected by the molecular weight. In addition, compared to the negative charge of the pH 7.4 VP1 protein which is -15.0, when the 430th serine is substituted with arginine, the negative charge becomes -13.6, and when it is substituted with glutamic acid, the negative charge becomes -15.6. Therefore, it can be seen that when the 430th amino acid of wild-type AAV1 is substituted, the diffusion ability of the mutant is proportional to the size of the negative charge.

[0083]

[0084] Experimental Example 8: Confirmation of human pulmonary artery smooth muscle cell (hPSMC) transduction ability of individual point mutations of AAV1.p2CV.

[0085] In Experimental Example 3 above, wild-type AAV1, mutants AAV1.S430C, AAV1.I647V, and AAV1.p2CV were loaded with CMV-GFP and delivered to human pulmonary artery smooth muscle cells (hPASMC) in vitro. The percentage of GFP-expressing cells and the average intensity of the GFP signal were measured, and the results are shown in Fig. 6. As a result of comparing the GFP expression rates of the four AAV-based viral vectors, the values ​​of AAV1.S430C and AAV1.I647V were slightly reduced compared to wild-type AAV1, but AAV1.p2CV, which has two point mutations at the same time, showed an expression rate equivalent to that of AAV1. In terms of intensity, the values ​​were slightly reduced in AAV1.I647V compared to wild-type AAV1, but increased in AAV1.S430C and AAV1.p2CV. Thus, it can be seen that the combination of S430C and I647V mutations has a synergistic effect on gene transferability and expression level.

[0086]

[0087] Experimental Example 9: Production of a pulmonary arterial hypertension mouse model

[0088] Creation of the pulmonary arterial hypertension mouse model and animal experiments were performed according to a protocol approved by the KAIST IACUC (No. KA2024-012-v1) and complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

[0089] To minimize the potential influence of hormonal and gender differences on functional and histological outcomes, 10-week-old adult male C57BL / 6 mice were used. Mice were maintained on a 12-h light / dark cycle and had unrestricted access to food. Mice were housed in hypoxic cages with oxygen concentrations of 8.5% of atmospheric pressure for 3 weeks, during which time they were administered Sugen 5416 (cat. no.: S8442, SIGMA Aldrich) at a dose of 20 mg / kg weekly.

[0090]

[0091] Experimental Example 10: Preparation of AAV1 and AAV1.p2CV-based 7mer peptide-inserted adeno-associated virus libraries.

[0092] The AAV1 capsid gene and the capsid gene of AAV1.p2CV obtained in Experimental Example 2 were subcloned into the pSub2 (UC Berkeley, David Schaffer Lab) plasmid to obtain plasmids pSub2-AAV1 and pSub2-AAV1.p2CV for inserting 7-mer peptides. To prevent the generation of a stop codon, 21 synthetic DNA duplexes consisting of seven NNK codons followed by two random DNA sequences (NN) followed by guanine or thymine were inserted between amino acids at positions 588 and 589 in the capsid amino acid sequence of wild-type AAV1 in the plasmid (between nucleotides at positions 1764 and 1765 on the DNA basis), thereby constructing an AAV1 and AAV1.p2CV-based 7-mer peptide insertion plasmid library.

[0093] The constructed AAV1 and AAV1.p2CV-based 7mer peptide insertion plasmids and pHelper, pBluescript plasmids were complexed with PEIpro (Polyplus, cat no.: 101000033) and transfected into HEK293T cells. After approximately 72 hours, only the cell pellet was collected, and AAV within the cells was extracted by freezing and thawing. Cell debris was removed by centrifugation, and nucleic acids from virus-producing cells were removed by incubating with 10 U / mL of benzonase at 37°C for 30 minutes to secure an AAV solution. The AAV solution was purified using iodixanol density gradient ultracentrifugation (OptiPrep, Alere Technologies AS, Oslo, Norway) with a Vti65.2 rotor (Beckman Coulter; 42,000 rpm). The purified AAV vector was buffer-exchanged into 1 1 / 2 PBS solution containing 0.01% (v / v) Tween 20 (Sigma-Aldrich) using an Amicon tube (Merck Millipore, Billerica, MA; molecular weight cutoff: 100 kDa).

[0094]

[0095] Experimental Example 11: Screening of pulmonary smooth muscle cell-targeting variants in a pulmonary arterial hypertension mouse model from an AAV1 and AAV1.p2CV-based 7mer peptide-inserted adeno-associated virus library.

[0096] AAV1 and AAV1.p2CV-based 7mer peptide insertion variant libraries were administered in the form of aerosols to 10-week-old male C57BL6 mice through a PenWu microaerosolizer (BioJane, Shanghai, China) (1.25" length of intratracheal portion, 700 μm outer diameter, 430 μm inner diameter). After about 1 day of recovery, pulmonary hypertension was induced according to Experimental Example 9 above. After the completion of the 3-week pulmonary artery induction process, perfusion was performed immediately, and major organs (lung, liver, kidney, and heart) were removed. Pulmonary smooth muscle (PSMC) cells labeled with APC-conjugated α-sma antibody (Abcam, Cambridge, UK) were isolated from the removed lung tissues through flow cytometry, and the remaining lung cells were designated as negative. The expression of AAV1 and AAV1.p2CV-based variants in PSMC and negative, liver, kidney, and heart cells The gene surrounding the 7mer peptide insertion was recovered using Qiagen 51306. The amount of DNA was then amplified by PCR, and the inserted gene was read using an Illumina MiSeq (Illumina, Inc.) next-generation sequencing (NGS) device. In addition, mutation information on the 7mer peptide inserted into the wild-type AAV1 could be collected through this for score calculation. The inserted DNA read through NGS was converted into amino acid sequence and expressed as the inserted 7mer peptide, and the number of NGS copies for each peptide was obtained by tissue. The score was defined as the value obtained by dividing the ratio of each 7mer peptide sequence to the total NGS copies for each tissue by the ratio of the corresponding 7mer peptide sequence to the NGS copies of the recombinant mutant library.

[0097] The cell- and tissue-specific specificity scores for PSMC, negative, liver, kidney, and heart of the 7mer peptide insertion variants discovered through NGS in the above manner were calculated. Variants with higher lung smooth muscle cell scores than wild-type AAV1 were selected, and each insertion sequence is shown in Table 3 below. In addition, the specificity scores for PSMC, negative, liver cells, kidney cells, or heart cells of AAV1 capsid mutants containing each insertion sequence are also shown. The NGS copy ratio of the corresponding insertion peptide in the mutant library is defined as VG_freq and its value is shown.

[0098] 삽입 서열서열번호VG_freqPSMCnegativeliverkidneyheartGRPQNRA355.58422E-07118.18510023.687820IILSLRA365.47301E-07175.04940044.310120HLSRCGP375.58422E-07139.04940043.427680ARRLRLL385.58422E-07172.27620055.271590SSPSLLS395.58422E-07193.05920078.959410RPLSRHR405.58422E-0779.760860047.375650STSKGSV415.47301E-07279.280642.9131418.5922380.563860SGPVCVC421.11684E-06109.436417.37199069.089490LGRASGC435.58422E-07131.560938.27645055.271590PTLLTPT445.58422E-0761.727650094.75130ILSVKLP455.58422E-0768.690700106.59520RSALLRD465.58422E-07451.7571230.4075071.063470GRKRART475.58422E-07160.459767.659350161.86686.334668TRMRNGT485.47301E-07235.6937126.9262056.39470AATARST495.58422E-07379.9726222.42340146.07496.334668ASLLSKL505.58422E-07138.7867103.3464067.11550GSRAKTR515.47301E-07131.111598.88679084.592050FVLLRAV525.58422E-07136.2328106.0606000SPPLLPC535.47301E-07288.1315242.136208.0563860SVMRRPR545.47301E-0789.568972.250320108.76120SPMTLAT555.58422E-07248.803747.54440841.74350PLGPAKR565.58422E-07122.2212135.881407.8959410TDSSTLD575.47301E-07178.5868210.8928040.281930VLTQSVV585.47301E-0781.2113291.423640128.90220LGQSNVL595.58422E-07158.7044195.209921.2409478.9594112.66934HKLRCLP605.47301E-07119.089145.53150157.099512.92678PKVLGGA615.47301E-0781.21132103.49370140.98680FLLVVVS625.47301E-0760.0741383.96658016.112770TKQTKKT635.47301E-07159.0939268.673556.8904552.366516.463388APSRRTV645.58422E-0798.85102204.3471015.7918812.66934AASECAG655.58422E-0790.49727162.74820356.3878621.8844SITSTSP665.47301E-07112.2185274.270904.0281930FPVARTT675.58422E-07171.4109151.356401368.71712.66934GRARGSD685.47301E-0768.75919242.55250116.81760ASSPALS695.53974E-0644.88196166.1575.877852134.480362.48529LLPSLKG705.58422E-0738.1615204.7790138.1796.334668SLRQSDD715.58422E-0746.5589100973.178614111.13PTPAKPP725.47301E-07126.43190000SAQRRVV731.11684E-0695.16410011.843910FTMPSVG745.47301E-07209.22380032.225540LTPVRSC751.66415E-0629.4572600011.30251RPSAALH765.58E-071.97E+020.00E+000.00E+001.18E+020.00E+00FDSFAVR775.58422E-07281.35090059.219560SAGTISC785.47E-071.96E+028.77E+010.00E+009.26E+010.00E+00LIGNLTA795.47301E-07164.50530040.281936.463388ASGKTPM805.47E-071.90E+022.54E+022.79E+011.57E+024.08E+01TGAAFSV815.47E-071.90E+020.00E+000.00E+002.42E+010.00E+00EPAVRHR821.11684E-0678.670535.741468015.791883.167334RPRTTYR835.47301E-0760.688880020.140960PAPESTE845.47E-071.73E+020.00E+002.71E+004.03E+010.00E+00MDPKSVR851.10572E-06140.49429.235114051.8398215.99595WSVATSS861.65302E-0681.40450036.009830VVMNRPA875.47301E-07134.2495.858134060.422890PKPATKK885.47E-071.57E+020.00E+000.00E+004.03E+000.00E+00ETSERKV895.47301E-0786.778090044.310120VFLHVAP905.58422E-07520.987863.156150185.55466.334668PDVNARS915.58422E-07166.84340098.6992763.19298NRPQLLN925.47301E-07192.26411.8657880120.84580FPGHCGY935.47E-071.51E+020.00E+000.00E+005.64E+011.27E+02NKKQKKP945.47301E-0745.299220032.225540LMTVPPT955.47301E-07485.66978.108450165.1559421.6996GGGILRS965.47301E-07109.12990096.676630GGAIAHN975.58422E-0753.42610051.323620PWSLVVL985.47301E-07127.80385.8581340124.8746.463388TAQQSER995.47301E-07477.6965123.142068.47928696.9481HPKKDGL1005.58E-071.23E+020.00E+000.00E+002.37E+010.00E+00HVANRLC1015.58422E-07294.874884.117020165.81480LLTRAMC1025.58422E-0734.890510039.479710PGYLCGL1035.58422E-0776.748340090.803320LSLLWPP1045.58E-071.17E+020.00E+000.00E+003.95E+001.88E+02FGAHRFL1055.58422E-0785.5856500106.59520PRALLCT1065.58422E-0727.258210031.5837742.17989ADSRAMD1075.58422E-07365.3542129.83280185.554669.52765FCRVVVG1085.47301E-07106.798511.716270140.98680GTEVPRR1095.58422E-0737.071170055.271590MPSVVHR1105.58422E-07451.7862148.204338.26613225.03431149.056PTTVMTP1115.47E-071.11E+020.00E+000.00E+008.06E+017.81E+02IRILLHR1125.47301E-07221.6908124.973565.01766278.44980RHSTLAT1135.58422E-07139.666493.77731063.167530LSTKRQS1145.47301E-0756.7366800145.01490TRHPLPL1155.58422E-0788.3166166.98379015.791880HPSTNQP1165.47301E-07259.2707205.03470104.7330VSVCVPC1175.47301E-07264.9012212.6999052.366510SSRHIRR1185.47E-079.30E+010.00E+004.09E+013.63E+010.00E+00RAGVRCD1195.58422E-07149.1063120.5708035.531740RKIHPSP1205.47301E-07137.1909111.3045024.169160VESRPRN1215.58422E-07299.1214243.055542.4818859.219560WPQTPAG1225.58422E-07115.124697.26419011.843910KRASTLP1235.47301E-0733.37452035.217996.676630SGSSEHG1245.58422E-0790.38686566.4915123.909415.7918812.66934DSGLGRR1255.58422E-07155.917130.139937.17165114.49110FLIVASV1265.47301E-07225.8429195.9078080.563866.463388QLALVSR1275.47301E-07102.348582.094690128.90226.463388VIRVSMS1285.58422E-07404.2371380.24240288.20190AKRWGTN1295.47301E-07130.165141.7999088.620240RGGKGPK1301.11E-068.20E+010.00E+000.00E+003.39E+010.00E+00SWGAGDS1311.10572E-0694.14081112.118115.64445034.09882HVQWGTN1325.47301E-07165.4178208.89360108.761212.92678RRHRTRT1335.58422E-07554.8693151.861602468.80763.19298SMGSGPV1345.58422E-07110.3089148.1191059.2195623.17588FRLKVGF1355.47E-076.79E+018.82E+010.00E+004.03E+010.00E+00SDLISLG1361.66415E-0687.5630697.1842112.47335282.345958.01561CGSAKLT1375.47301E-07134.1364199.1765036.253740QTTEPST1381.10572E-0684.9029894.72050222.6046583.4281YGVSSSR1395.47301E-07298.5246483.29180100.704812.92678LADPRSV1405.58422E-07291.7621477.98410110.54320PDFLGVP1415.47301E-07201.2513342.76140100.70480SLGAWRG1425.47E-075.87E+010.00E+000.00E+001.05E+020.00E+00VYVQVPR1435.58422E-07127.8166227.7449035.531740LPVTKED1445.58422E-0772.95615137.1473055.271590SAFKPDL1455.58422E-07197.7166317.09820688.511842.17989LDQKRQP1465.58422E-07123.0633177.0880579.20220TPTDGTR1475.58422E-0766.7394133.4901043.427680QVVVPPS1485.47301E-07301.7791635.8641080.563866.463388PAVHHAS1492.04E-055.55E+011.07E+028.55E+001.26E+027.20E+02HKGLNKY1505.47E-074.89E+010.00E+000.00E+003.22E+010.00E+00SGVSILG1519.97375E-0648.635496.009412.973157286.22041414.075SPNSAGV1521.11017E-0536.37383.270332.93303891.8542132.14369PSPTRTH1535.47E-074.45E+010.00E+000.00E+000.00E+000.00E+00SIARRIV1545.47301E-07131.3264336.5960157.09956.463388PPDLVPN1555.58422E-07141.1445369.6906075.011440SSKIVPS1566.6677E-0635.1278197.13164052.317715.37251TQLVRKT1575.58422E-0753.4261158.8473027.635790EAVTLKR1585.58422E-07120.768404.1274055.271590FSVGNGA1591.10572E-0629.85079100.519726.818225.9815176.398381RTTPPWL1605.58422E-0734.89051129.3276035.531740NPLVPVP1615.47301E-07182.0177731.77340116.81760QSKETVR1625.58422E-0737.07117167.3029082.907380PSLSLYL1635.58422E-0757.78741265.1515063.167530VWVKKKP1645.58422E-07101.5674532.8826047.37565320.4442HSGHVCV1655.47301E-0747.83681263.0762056.39470DTPTPTT1665.47301E-0725.58713150.3588000ARTQWLP1675.47301E-0724.47465210.8928068.479280LRGDLLN1681.11684E-0638.07292244.327403.94797149.5191ERTTRKV1695.58422E-07142.8373988.69530122.387112.66934PTPDRTA1705.47301E-0740.04942395.7393040.281930ARPKPNV1715.47301E-07342.69830000RISSQST1725.58E-074.12E+021.06E+020.00E+007.50E+010.00E+00SAVHHAS1735.58E-073.20E+021.91E+000.00E+005.63E+021.03E+02FIVCARV1745.58422E-0723.987230000SEARSSR1751.10572E-06121.3447001.9938390GRSVFSG1765.58E-072.33E+021.86E+020.00E+004.74E+010.00E+00NDTPPPR1775.58422E-07169.51610019.739850FSCRKRA1785.47301E-0733.37452004.0281930AQRQLAR1795.47301E-07176.030024.169160AWQPVGA1805.47301E-07336.71930048.3383175.35391SRLENQS1815.47301E-07223.32020040.2819312.92678YAVVPLY1825.47E-072.10E+021.41E+021.90E+012.42E+014.30E+01CTKISTS1835.58422E-0740.2129200019.004PRRDSQV1841.11684E-0691.421980017.765870DRISTDA1855.47301E-07152.51680036.253740LSKYRAW1865.58E-071.92E+022.16E+020.00E+004.34E+010.00E+00SNPTELT1875.58E-071.92E+020.00E+000.00E+004.34E+010.00E+00AFFPVPL1885.58422E-0742.522810011.843910ITRFKEC1895.47301E-07259.66630084.592050QKKTKTQ1905.58E-071.77E+021.13E+020.00E+000.00E+006.33E+00GPDAARK1915.58E-071.72E+020.00E+000.00E+003.95E+000.00E+00KKKKKKK1921.09E-061.69E+022.31E+020.00E+003.02E+013.23E+00TQQTVPE1931.11E-061.59E+022.61E+010.00E+007.98E+013.20E+00KWTQVSP1945.47301E-07163.973511.71627056.39476.463388VCLPWTS1955.47E-071.57E+021.37E+020.00E+004.43E+010.00E+00NPPAREG1965.58422E-07141.244610.97179059.219560EVSTPNN1975.58422E-07196.88470098.6992716.84121LFGRAGY1985.47E-071.57E+023.91E+000.00E+001.21E+020.00E+00MKLYRLS1991.0946E-0623.362160012.084580TEQKNHQ2005.58E-071.57E+023.02E+020.00E+001.97E+010.00E+00SKWTAWA2015.58422E-0752.3357702.65511827.635790GFQWLPT2025.58422E-07150.0006032.7995482.907380TKDNAPD2035.58422E-07285.941634.448810114.49116.334668SFMSTNL2045.47301E-07267.22444.77892048.338310QRKPAHE2055.47E-071.56E+024.25E+020.00E+007.65E+010.00E+00PRYWPSL2065.47301E-0753.399230040.281930EGTPNIN2071.10572E-06141.030125.85832013.95687142.716QKPQNTK2085.47E-071.42E+020.00E+000.00E+002.82E+010.00E+00RPVDRFR2095.58422E-0745.79380039.479710SFVRGYC2105.47301E-07112.14610096.676636.463388TDVPMDE2115.47E-071.37E+027.68E+010.00E+003.63E+010.00E+00KGMPEPR2125.58422E-07123.5845375.124931.86141764.34650TRSLESM2135.47E-071.19E+025.22E+010.00E+001.21E+010.00E+00SCLGADL2145.58422E-07347.243987.7742947.79212339.52550TCHSRSV2155.47301E-0794.561130032.22554320.4922LGVRVMH2165.58E-071.15E+021.04E+020.00E+003.95E+010.00E+00RPFSHPT2175.58E-071.11E+021.32E+020.00E+003.55E+010.00E+00DVSFRVL2185.47301E-07212.293299.588270124.8740SSCCLAR2195.47301E-0723.362160040.281930PPKSTFY2201.09E-061.06E+021.18E+020.00E+008.06E+000.00E+00AWVVDLA2215.58422E-07103.8919636.36360236.87820ICTVHRV2225.58E-079.92E+010.00E+000.00E+009.08E+010.00E+00FWSFLSR2235.58422E-0727.258210059.219560FPWVSWL2245.47301E-07415.6078263.1288072.507470LLRGIGP2255.47E-079.90E+010.00E+000.00E+001.17E+021.94E+01TETLRNP2265.58422E-07269.8324162.674995.53308440.5288639.033GKRIAEA2275.58422E-07112.733485.945650130.2830KGGADLP2285.58E-079.27E+012.30E+020.00E+001.42E+021.27E+01EDVPRCC2295.58422E-07224.4348160.91950102.6472788.5948LIVHFLY2305.58E-078.80E+017.46E+010.00E+007.50E+010.00E+00SESSTQT2315.58422E-0726.167890082.907380NTFHNPR2325.47301E-0765.6365514.9263122.31068104.733451.653VIDCRSV2335.58422E-07286.5587285.2664015.791880WDSSLFP2345.58422E-07238.5757236.62650189.502612.66934QVMPGMA2351.11684E-0640.1795544.80146035.531740RGPVRYH2361.11E-068.12E+012.77E+010.00E+006.58E+011.17E+01GTVRSNQ2372.74763E-0677.58754316.56840125.8733224.9281LTSFDLG2388.28E-067.59E+019.08E+012.51E+001.10E+026.32E+01MSRLDLN2395.47E-077.57E+010.00E+000.00E+005.24E+010.00E+00PKALMIT2402.21E-067.35E+010.00E+000.00E+008.00E+015.85E+00RSPCEGR2415.58E-076.91E+010.00E+003.64E+011.03E+026.33E+00ISPGLFC2425.47301E-07142.7044199.17650128.90220EANPLTS2435.58422E-07151.5557216.262031.5837716.84121LCSRVFS2445.47301E-07101.2063167.9332072.507476.463388LGCMCAA2455.58422E-07338.0626542.15060600.833216.84121SQVNLGS2465.58422E-0771.8512700414.94980LCHSPTS2475.47301E-07229.2633408.11660153.0713503.3601AIPQTMQ2485.58E-075.85E+010.00E+000.00E+000.00E+000.00E+00NAHVLHS2495.47301E-07132.3026246.28410145.014912.92678NHTVRRR2505.58422E-07149.986287.0734015.791880LNLRHHT2515.58E-075.67E+019.51E+010.00E+008.69E+010.00E+00VTTREAQ2525.47301E-0750.06178259.3446040.281930FVTREYR2535.58E-074.71E+011.01E+020.00E+005.92E+010.00E+00KNKKKKA2545.47301E-0762.2991149.8776036.253740VLLWELG2555.58E-074.66E+010.00E+000.00E+000.00E+000.00E+00AMASLMA2563.89783E-0662.16542137.6415.14128214.966338.6236VFLPGFS2575.47301E-07157.7963403.596748.7632584.592050VRQYCVI2585.58E-074.03E+010.00E+000.00E+000.00E+000.00E+00PPGRIMP2595.47E-074.00E+010.00E+000.00E+001.21E+010.00E+00PRVDIPT2605.58E-073.84E+010.00E+000.00E+001.18E+010.00E+00VKGDNNP2617.75118E-0656.32146137.28846.739993327.8728241.8231RPDGLKL2625.58422E-07111.2135337.1616067.11556.334668SNTSALQ2631.09E-063.34E+014.19E+010.00E+000.00E+001.03E+02SQDHGRS2642.26506E-0537.92321106.43693.192118138.4919952.4114VLRPEER2651.09E-063.04E+010.00E+000.00E+000.00E+000.00E+00VMESVRS2665.58422E-0768.6907393.1557063.167530RFPGAPP2675.47301E-0796.34162591.2933084.592050KKTTKKK2685.58E-072.51E+011.91E+000.00E+005.53E+016.33E+00IHWAGVL2695.47301E-0764.52407618.8356036.253740GTCCSTR2705.47301E-0755.62419561.3256092.6484440.83024WLSSKTI2715.47E-073.75E+023.91E+000.00E+009.67E+013.23E+01YKCTPPS2725.58422E-0796.866340000HVMPQRW2735.47E-073.42E+023.84E+020.00E+008.06E+011.72E+01LSYWLVL2745.58422E-0725.077560000QDNLSGS2751.6419E-0623.474220000PIELFGN2765.47E-072.14E+023.91E+003.90E+018.86E+010.00E+00RCPNNTV2771.11684E-0677.5802005.9219560LSRFSHF2785.58E-072.12E+021.76E+022.12E+017.90E+010.00E+00PQTPSCR2795.47301E-0756.00927008.0563860RLFYLAC2805.47301E-0724.47465004.0281930KLAIVHI2815.47E-072.06E+022.84E+020.00E+001.29E+020.00E+00PQPTWLW2825.47E-071.99E+023.11E+027.04E+012.30E+020.00E+00QTRNRWT2835.58422E-0734.89051007.8959410AICFCCC2845.47301E-07196.68430068.479280TETQFTY2855.58E-071.83E+020.00E+000.00E+006.71E+010.00E+00EGKAMYS2865.58E-071.72E+020.00E+000.00E+006.32E+010.00E+00EPRDMKT2875.47E-071.70E+020.00E+000.00E+007.99E+023.66E+01NYWESSR2881.10572E-0648.92765032.2091719.938390KSGMAKQ2895.47E-071.52E+020.00E+000.00E+002.82E+010.00E+00NLPREFG2905.58E-071.51E+023.18E+020.00E+002.42E+021.27E+01NEAHHTP2915.58E-071.49E+020.00E+000.00E+007.90E+000.00E+00TWPCTSY2925.47E-071.44E+023.32E+010.00E+001.61E+010.00E+00GENCLLL2935.58422E-07158.62310103.8652122.38710MTMTTQK2945.58E-071.41E+027.31E+010.00E+002.37E+010.00E+00NEKSAAQ2955.58E-071.31E+022.95E+010.00E+001.58E+010.00E+00NETSKQA2963.88671E-0669.9833620.347626.8068922.4172236.339WKGRTMM2975.58E-071.19E+020.00E+000.00E+001.46E+020.00E+00EEKLTIN2985.58422E-07211.876300106.5952819.807VNRYLFG2995.58422E-07292.4939102.57370102.6472824.2863KPHPKTE3005.47E-079.79E+012.44E+021.12E+017.65E+013.44E+01NGTIISD3011.12E-069.57E+010.00E+000.00E+000.00E+000.00E+00QPGLHWR3025.58E-079.41E+010.00E+000.00E+009.48E+010.00E+00YLSGVFV3035.58422E-07121.456180.544950207.02250DLAKEQN3045.58E-078.72E+011.19E+020.00E+009.48E+010.00E+00WFSWCVL3055.47301E-0756.736681.8657880181.268717.18343WNGQSQS3065.58422E-07208.7346194.783998.39861102.64720KSCEGML3075.47301E-07511.2326513.09180120.84580KQRPTWR3085.47301E-07121.3629124.9735028.197350FVYLSYF3095.47E-078.56E+010.00E+000.00E+009.26E+010.00E+00ALQCELI3105.47E-078.45E+010.00E+001.12E+013.19E+020.00E+00VCLVHSQ3115.47301E-0783.43629242.13620181.26870LCRAYEP3121.12E-068.16E+010.00E+000.00E+001.97E+010.00E+00LAGHIIK3135.58422E-0773.05201228.5789023.6878225.33867CGWVFAC3145.58E-077.09E+015.38E+010.00E+006.71E+010.00E+00CWNQADD3155.58422E-0792.67793125.0344067.115535.84522AILMENP3165.47E-076.60E+011.44E+020.00E+001.21E+020.00E+00QMRNLAT3175.58422E-07206.8791377.54990138.1790CPAFAWA3185.58422E-07163.1137296.64250144.84136.334668WRPYVMP3195.47301E-0757.84916106.3499080.563860KNVDAAT3201.11684E-06109.1163215.827039.479718.420607ASFTGCF3211.12E-064.66E+019.78E+010.00E+006.32E+010.00E+00LREYDTT3225.47E-074.56E+010.00E+000.00E+003.64E+021.29E+01NHANKKS3235.58422E-0747.79729129.8328082.907380AFITRSF3245.58E-073.71E+010.00E+000.00E+001.18E+010.00E+00QNSEKRM3251.11684E-0634.34535103.317605.9219560FFFFVVC3265.58422E-0729.4388700395.20990ENKKKKH3275.58E-073.05E+010.00E+000.00E+001.58E+010.00E+00IVYGFSC3285.47E-074.52E+026.09E+020.00E+002.38E+020.00E+00FPWWPGQ3295.47E-073.74E+021.08E+020.00E+003.63E+013.44E+01EQDSSAL3301.6419E-0654.132430000LLYPCEM3315.58E-072.89E+024.21E+010.00E+009.87E+010.00E+00KNVPWLR3325.47E-072.64E+025.47E+010.00E+001.57E+022.36E+01CGYTFLR3331.12E-062.46E+020.00E+000.00E+001.97E+010.00E+00KVDPVIG3341.65302E-0639.881450000IYIDTTG3355.47301E-07189.2288008.0563860TMTEPIQ3365.58E-072.14E+025.10E+024.01E+013.55E+010.00E+00FCKFRLE3375.47E-072.13E+022.34E+020.00E+006.04E+011.72E+01SHIDGPE3385.47E-071.96E+022.38E+020.00E+007.65E+014.93E+02FVLCEWE3395.47E-071.48E+020.00E+000.00E+002.01E+010.00E+00RFHPIAH3405.58E-071.31E+020.00E+000.00E+000.00E+000.00E+00VLNSDCF3415.47E-071.11E+020.00E+000.00E+000.00E+000.00E+00LSDAFFK3425.58E-071.07E+020.00E+000.00E+003.95E+000.00E+00NEGMPFC3435.58422E-07244.4569208.4639063.167530RWVGQSK3445.47E-078.97E+010.00E+000.00E+000.00E+000.00E+00YQQDNVH3455.58E-078.71E+011.13E+020.00E+002.01E+023.80E+01CMIEDVD3465.47E-076.28E+010.00E+000.00E+001.21E+010.00E+00RGGFQIF3475.47E-075.69E+010.00E+000.00E+002.82E+010.00E+00QQETLYP3483.86447E-0629.8356288.496136.056137112.992719.49087RMITIEP3491.09E-063.17E+011.27E+018.37E+005.44E+013.23E+00QKHSMLE35 05.58422E-0728.34854128.2261015.7918825.33867IQYMSHQ3515.47E-072.43E+020 .00E+000.00E+008.46E+010.00E+00TMLEDMI3525.47E-072.23E+024.69E+020.00E+0 03.42E+021.29E+01RQEKQNK3535.58E-071.87E+020.00E+000.00E+006.71E+011.27E +01YMETWDF3545.47E-071.38E+020.00E+000.00E+000.00E+000.00E+00CCACYQA3551 .09E-069.86E+010.00E+000.00E+004.23E+010.00E+00YRFCAQI3565.47E-077.01E+0 10.00E+000.00E+002.82E+011.72E+01YQCEHQA3575.58E-072.40E+024.42E+020.00E +003.16E+010.00E+00>AAV11.47E-032.33E+013.35E+016.62E+021.15E+022.84E+02.

[0099]

[0100] Experimental Example 12: Production of AAV1-based adeno-associated virus mutants

[0101] The DNA sequence corresponding to the 7mer peptide selected in the same manner as in Experimental Example 10 was inserted into the recombinant AAV plasmid vector pAAV2 / 1 (Adgene), which consists of the wild-type AAV1 capsid gene in the rep gene of wild-type AAV2. In addition, pAAV2 / 1.p2CV was prepared by substituting the AAV1.p2CV capsid DNA in the wild-type AAV1 capsid gene position of pAAV2 / 1 in the pSub2-AAV1.p2CV obtained in Experimental Example 10, and the DNA sequence corresponding to the 7mer peptide selected in the same manner as in Experimental Example 10 was inserted. The recombinant mutant in which the SPNSAGV sequence was inserted between amino acids at positions 588 and 589 in the capsid amino acid sequence of wild-type AAV1 in the above manner was named AAV1.SP, and the corresponding plasmid was named pAAV2 / 1.SP. In addition, the recombinant mutant having the SGVSILG sequence inserted was named AAV1.SG, and the corresponding plasmid was named pAAV2 / 1.SG. In addition, the recombinant mutant having the SPNSAGV sequence inserted in addition to the AAV1.p2CV, like the AAV1.SP, was named AAV1.p2CV.SP, and the corresponding plasmid was named pAAV2 / 1.p2CV.SP. In addition, the recombinant mutant having the SGVSILG sequence inserted in addition to the AAV1.p2CV, like the AAV1.SG, was named AAV1.p2CV.SG, and the corresponding plasmid was named pAAV2 / 1.p2CV.SG.

[0102] To load the human FGF12 (hFGF12) gene, a pulmonary arterial hypertension therapeutic gene, into the AAV1-based adeno-associated virus mutant manufactured by the above method, plasmids pAAV2 / 1.p2CV, pAAV2 / 1.SP, pAAV2 / 1.p2CV.SP, pAAV2 / 1.SG, or pAAV2 / 1.p2CV.SG and ITR flanked hFGF12 gene (pCMV-hFGF12) and pHelper were complexed using PEIpro (Polyplus, cat no.: 101000033) and transfected into HEK293T cells. After approximately 72 hours, only the cell pellet was collected, and AAV inside the cells was extracted by freezing-thawing. Afterwards, cell debris was removed through centrifugation, and nucleic acids from virus-producing cells were removed by incubating with 10 U / mL of benzonase at 37°C for 30 minutes to secure an AAV solution. The AAV solution was 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 1 / 2 PBS solution containing 0.01% (v / v) Tween 20 (Sigma-Aldrich) using Amicon tubes (Merck Millipore, Billerica, MA; molecular weight cutoff: 100 kDa) to obtain recombinant mutants AAV1.p2CV, AAV1.SP, AAV1.p2CV.SP, AAV1.SG, and AAV1.p2CV.SG carrying the therapeutic gene hFGF12.

[0103]

[0104] Experimental Example 13: Comparison of transduction and expression abilities of wild-type AAV1 and AAV1.p2CV in lung cells of a pulmonary hypertension mouse model.

[0105] In Experimental Example 12, the hFGF12 gene was loaded onto wild-type AAV1 and mutant AAV1.p2CV, and pulmonary airway aerosol administration was performed in the same manner as in Experimental Example 11 in a mouse model in which pulmonary arterial hypertension was induced according to Experimental Example 9. Two weeks after administration, an autopsy was performed, and mRNA was isolated from the lung tissue obtained. The results of reverse transcription polymerase chain reaction (RT-PCR) for the hFGF12 gene delivered via AAV were analyzed using ImageJ, and the graph is shown in Fig. 7. As a result, it can be seen that the mutant AAV1.p2CV has a higher hFGF12 mRNA expression level in lung tissue than the wild-type AAV1.

[0106]

[0107] Experimental Example 14: Transduction and Expression Experiments of AAV Mutants in Human Pulmonary Artery Smooth Muscle Cells (hPASMCs)

[0108] In vitro hFGF12 gene transfer and mRNA expression experiments were performed on human pulmonary artery smooth muscle cells using recombinant AAV mutants AAV1.p2CV, AAV1.SP, AAV1.p2CV.SP, AAV1.SG, and AAV1.p2CV.SG loaded with the therapeutic gene hFGF12 prepared in Experimental Example 12 above. In the experiment, human pulmonary artery smooth muscle cells (hPASMC, American Type Culture Collection (ATCC); 2x10 4After infecting hPASMCs (20 μl / cells) with recombinant AAV mutants loaded with hFGF12 at an MOI of 100,000, mRNA was isolated from hPASMCs using the RNeasy Plus kit (Qiagen) 3 days later, and complementary DNA (cDNA) for the mRNA was obtained. hFGF12 and ACTA and TAGLN genes expressed by it were extracted from the obtained cDNA. The cDNA titer of each gene was measured by qPCR, and the qPCR results for cDNA of GAPDH mRNA expression were normalized. The experimental results for each gene are shown in Figures 8 to 10.

[0109] As shown in the experimental results in Figures 8 to 10, it was confirmed that the point mutation based on AAV1 (preferably the point mutation of the p2CV mutant) and the 7mer peptide (preferably SPNSAGV or SGVSILG) insertion mutation between the 588th and 589th amino acid positions had a synergistic effect, significantly improving the specificity for human pulmonary artery smooth muscle cells. Meanwhile, the p2CV mutant had excellent diffusion ability and could deliver genes to pulmonary artery smooth muscle cells via airway aerosol administration (see Domestic Application No. 10-2022-0120442), but according to Experimental Example 8, the targeting specificity for human pulmonary artery smooth muscle cells was slightly higher than that of the wild-type AAV1. However, when the 7mer peptide was inserted into the wild-type AAV1, it showed a higher hFGF12 expression level than p2CV. Furthermore, when the 7mer peptide insertion mutation was combined with the p2CV point mutation, the specificity for human pulmonary artery smooth muscle cells was unexpectedly and significantly improved compared to when inserted into wild-type AAV1. These experimental results support the synergistic effect of the combination. Furthermore, according to Experimental Example 13, AAV1.p2CV showed a higher hFGF12 expression level than wild-type AAV1 in a pulmonary arterial hypertension mouse model. Considering the synergistic effect of the combination, it can be seen that the combination of the point mutations disclosed in Table 1 and the 7mer peptide insertion mutation disclosed in Table 3 can significantly enhance the targeting specificity for pulmonary smooth muscle cells.

[0110]

[0111] Experimental Example 15: Experimental Effect on the Prevention of Pulmonary Arterial Hypertension

[0112] The recombinant AAV1 and recombinant mutants AAV1.p2CV, AAV1.p2CV.SP, and AAV1.p2CV.SG carrying the therapeutic gene hFGF12, manufactured in the manner of Experimental Example 12, were used to test the in vivo preventive effect on pulmonary arterial hypertension (PAH) in the mouse model manufactured in Experimental Example 9. Each AAV vector of wild-type recombinant AAV1, AAV1.p2CV, AAV1.p2CV.SP, and AAV1.p2CV.SG carrying hFGF12 was aerosolized into the trachea of ​​mice using a PenWu microaerosolizer (BioJane, Shanghai, China) (1.25" length of intratracheal portion, 700 μm outer diameter, 430 μm inner diameter), and pulmonary arterial hypertension was induced in a Su5416+hypoxic environment.

[0113] Figure 11 shows the results of measuring right ventricular systolic pressure (RVSP), and Figure 12 shows the results of measuring pulmonary vascular thickness (intima-media thickness). In addition, Normal represents normal mice, and SHAM represents PAH mice that underwent only airway securing surgery without virus injection (t-test for Normal: *>0.05, **>0.01, ***>0.001; t-test for SHAM: 쪌 >0.05, 쪌쪌 >0.01, 쪌쪌쪌 >0.001). AAV1.p2CV is a p2CV mutant containing two point mutations, S430C and I647V, in wild-type AAV1, and mutants combined with SPNSAGV or SGVSILG peptide insertions were designated AAV1.p2CV.SP or AAV1.p2CV.SG, respectively.

[0114] As shown in the results in Figures 11 and 12, it was confirmed that when a point mutation based on wild-type AAV1 or a 7mer insertion mutation was combined with it, the blood vessel thickness value was maintained at a value close to normal, and the effect of preventing pulmonary arterial hypertension was significantly improved.

Claims

1. A mutant of the AAV1 (adeno-associated virus serotype 1) capsid protein, Compared to the amino acid sequence of the wild-type AAV1 capsid protein represented by sequence number 1, Asparagine (N) at position 14 is replaced with threonine (T) (N14T), Glutamic acid (E) at position 21 is replaced with glutamine (Q) (E21Q), Tryptophan (W) at position 23 is replaced with arginine (R) (W23R), Aspartic acid (D) at position 24 is replaced with lysine (K) (D24K), Lysine (K) at position 31 is substituted with threonine (T) (K31T), Proline (P) at position 32 is replaced with threonine (T) (P32T), Asparagine (N) at position 35 is replaced with arginine (R) (N35R), Glutamine (Q) at position 36 is replaced with glutamic acid (E) (Q36E), Glutamine (Q) at position 37 is replaced with arginine (R) (Q37R), Lysine (K) at position 38 is replaced with histidine (H) (K38H), Glutamine (Q) at position 39 is replaced with lysine (K) (Q39K), Glycine (G) at position 42 is replaced with serine (S) (G42S) Lysine (K) at position 137 is substituted with glutamic acid (E) (K137E) Lysine (K) at position 310 is replaced with arginine (R) (K310R), Threonine (T) at position 415 is substituted with isoleucine (I) (T415I) Serine (S) at position 430 is replaced with arginine (R) (S430R), Substitution of serine (S) at position 430 with cysteine (C) (S430C) Substitution of serine (S) at position 430 with glutamic acid (E) (S430E) Threonine (T) at position 492 is replaced with alanine (A) (T492A) Substitution of serine (S) at position 516 with threonine (T) (S516T) Asparagine (N) at position 583 is replaced with aspartic acid (D) (N583D) Isoleucine (I) at position 647 is replaced with alanine (A) (I647A) Isoleucine (I) at position 647 is replaced with valine (V) (I647V) Isoleucine (I) at position 647 is substituted with tyrosine (Y) (I647Y) Alanine (A) at position 660 is substituted with valine (V) (A660V), and Contains at least one amino acid substitution selected from the group consisting of a substitution of glutamine (Q) at position 688 with arginine (R) (Q688R), A mutant of AAV1 capsid protein containing an insertion sequence of X1X2X3X4X5X6X7 (chemical formula I).

2. In the first paragraph, the mutant of the AAV1 capsid protein is compared to the wild type AAV1 capsid protein. Contains S430C and I647V substitutions; or Contains S516T, N583D and A660V substitutions; Including K310R substitution; Contains W23R and T415I substitutions; or Contains N14T, E21Q, D24K, K31T, P32T, N35R, Q36E, Q37R, K38H, Q39K and G42S substitutions; Contains a Q688R substitution; or Contains a T492A substitution; or Contains a K137E substitution; or Including S430R substitution; Including S430E substitution; Contains an I647A substitution; or A mutant of AAV1 capsid protein containing the I647Y substitution.

3. In the first paragraph, the insertion sequence is a mutant of the AAV1 capsid protein inserted between the 588th and 589th amino acids of the amino acid sequence of wild-type AAV1 (SEQ ID NO: 1).

4. In paragraph 1, the X1 amino acid is any one selected from the group consisting of S, L, A, P, N, V, D, R, E, Q, and T, or The above X2 amino acid is any one selected from the group consisting of S, N, G, R, T, A, P, E, V, Q, and K, The above X3 amino acid is any one selected from the group consisting of T, E, S, N, V, P, R, A, G, K, and D, or The above X4 amino acid is any one selected from the group consisting of S, T, P, K, D, Q, Y, A, V, L, and N, The above X5 amino acid is any one selected from the group consisting of P, S, T, D, G, A, N, L, K, R, and I, or The above X6 amino acid is any one selected from the group consisting of L, Q, T, V, N, G, P, S, A, K, and D, or A mutant of the AAV1 capsid protein wherein the X7 amino acid is any one selected from the group consisting of S, T, P, G, Q, K, A, R, V, H, and E.

5. In the first paragraph, the insertion sequence is a mutant of an AAV1 capsid protein selected from the group consisting of SGVSILG, SPNSAGV, and SQDHGRS.

6. In the first paragraph, the mutant of the AAV1 capsid protein is a mutant of the AAV1 capsid protein having a tropism for smooth muscle cells.

7. In the first paragraph, the mutant of the AAV1 capsid protein comprises S430C and I647V substitutions compared to the wild-type AAV1 capsid protein, The above insertion sequence is a mutant of the AAV1 capsid protein, SPNSAGV.

8. In the first paragraph, the mutant of the AAV1 capsid protein comprises S430C and I647V substitutions compared to the wild-type AAV1 capsid protein, The above insertion sequence is a mutant of the AAV1 capsid protein SGVSILG.

9. A nucleic acid having a base sequence represented by SEQ ID NO: 25, encoding a mutant of the AAV1 capsid protein in paragraph 7.

10. A nucleic acid having a base sequence represented by SEQ ID NO: 26, encoding a mutant of the AAV1 capsid protein in claim 8.

11. A recombinant AAV1 vector comprising a nucleic acid encoding a mutant of the AAV1 capsid protein of claim 9.

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

13. A recombinant AAV vector according to claim 11 or 12, wherein the recombinant AAV1 vector has an improved transduction profile for smooth muscle cells compared to an AAV1 wild-type viral vector.

14. A pharmaceutical composition for preventing or treating a smooth muscle-related disease comprising the recombinant AAV1 vector of claim 11 or 12.

15. A pharmaceutical composition according to claim 14, wherein the smooth muscle-related disease is pulmonary hypertension.

16. A mutant of AAV1 (adeno-associated virus serotype 1) capsid protein, Compared to the amino acid sequence of the wild-type AAV1 capsid protein represented by sequence number 1, Asparagine (N) at position 14 is replaced with threonine (T) (N14T), Glutamic acid (E) at position 21 is replaced with glutamine (Q) (E21Q), Tryptophan (W) at position 23 is replaced with arginine (R) (W23R), Aspartic acid (D) at position 24 is replaced with lysine (K) (D24K), Lysine (K) at position 31 is substituted with threonine (T) (K31T), Proline (P) at position 32 is replaced with threonine (T) (P32T), Asparagine (N) at position 35 is replaced with arginine (R) (N35R), Glutamine (Q) at position 36 is replaced with glutamic acid (E) (Q36E), Glutamine (Q) at position 37 is replaced with arginine (R) (Q37R), Lysine (K) at position 38 is replaced with histidine (H) (K38H), Glutamine (Q) at position 39 is replaced with lysine (K) (Q39K), Glycine (G) at position 42 is replaced with serine (S) (G42S) Lysine (K) at position 137 is substituted with glutamic acid (E) (K137E) Lysine (K) at position 310 is replaced with arginine (R) (K310R), Threonine (T) at position 415 is substituted with isoleucine (I) (T415I) Serine (S) at position 430 is replaced with arginine (R) (S430R), Substitution of serine (S) at position 430 with cysteine (C) (S430C) Substitution of serine (S) at position 430 with glutamic acid (E) (S430E) Threonine (T) at position 492 is replaced with alanine (A) (T492A) Substitution of serine (S) at position 516 with threonine (T) (S516T) Asparagine (N) at position 583 is replaced with aspartic acid (D) (N583D) Isoleucine (I) at position 647 is replaced with alanine (A) (I647A) Isoleucine (I) at position 647 is replaced with valine (V) (I647V) Isoleucine (I) at position 647 is substituted with tyrosine (Y) (I647Y) Alanine (A) at position 660 is substituted with valine (V) (A660V), and A mutant of AAV1 capsid protein comprising at least one amino acid substitution selected from the group consisting of a substitution of glutamine (Q) at position 688 with arginine (R) (Q688R).

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