Adeno-associated virus capsid protein mutants

Abstract

The present invention provides a mutant of adeno-associated virus serotype 1 (AAV1) capsid protein, the mutant being an AAV1 capsid protein mutant in which serine at position 430 is replaced with cysteine ​​and isoleucine at position 647 is replaced with valine compared to the amino acid sequence of the wild-type AAV capsid protein, a nucleic acid encoding the mutant AAV1 capsid protein, a recombinant AAV1 vector comprising the nucleic acid encoding the mutant AAV1 capsid protein, and a pharmaceutical composition comprising the vector. In particular, a recombinant viral vector comprising a nucleic acid encoding a mutant AAV1 capsid protein is useful for preventing or treating pulmonary arterial hypertension by improving expression of a transgene in pulmonary vascular target cells when delivered in an aerosol state through the bronchi.

Description

[Technical Field]

[0001] The present invention relates to mutant adeno-associated virus (AAV) capsid proteins, and in particular, recombinant viral vectors containing mutant AAV1 capsid proteins are useful for expressing transgenes in pulmonary vascular target cells when delivered in aerosol form through the bronchi. [Background technology]

[0002] To effectively carry out gene therapy, it is of utmost importance to develop gene transfer techniques that allow therapeutic genes to be delivered to desired target cells with high expression efficiency.

[0003] Among these gene transfer technologies, AAV is a non-pathogenic virus that has no side effects on infected cells and has a low probability of causing mutations in the genetic information of target cells, making it safer than other gene therapy technologies.

[0004] AAV (Adeno-Associated Virus) is a non-enveloped, single-stranded DNA virus that can infect both dividing and non-dividing cells. AAV can replicate only in the presence of a helper virus and is non-pathogenic to humans. These characteristics make AAV a useful method for introducing genes into various cells and a useful vector for gene therapy.

[0005] AAVs exist in various serotypes, and their host and viral characteristics vary depending on the serotype. Serotype 2 (AAV2) is a serotype that has been widely studied for a long time and can infect a variety of cell types. Serotypes 1 (AAV1), 5 (AAV5), and 6 (AAV6) have higher tissue infection specificity. AAV1 is known to be highly efficient at gene transfer to muscle, liver, airways, and the central nervous system; AAV5 is known to be highly efficient at gene transfer to the central nervous system, liver, and retina; and AAV6 is known to be highly efficient at gene transfer to heart, muscle, and liver. Although gene transfer characteristics to specific tissues differ depending on the serotype, they are still easily transduced to other tissues. Therefore, the development of new AAV vectors that can improve tissue specificity, safety, and efficacy is essential.

[0006] On the other hand, pulmonary arterial hypertension is a disease in which the pulmonary arterioles narrow for no particular reason, causing increased pressure in the pulmonary arteries and impaired right ventricular function. Because the main symptoms are common everyday symptoms such as shortness of breath and dizziness, it is often overlooked or mistaken for other diseases. For this reason, it often takes a long time for patients to receive a diagnosis.

[0007] In fact, according to a survey by the Centers for Disease Control and Prevention, it took an average of 1.5 years to accurately diagnose pulmonary arterial hypertension, and accurate diagnosis required cardiac catheterization, which required inserting wires into the body, in addition to being expensive.

[0008] When blood does not flow smoothly from the heart to the lungs, it can lead to breathing difficulties, heart failure, and in severe cases, death. Despite continuous advances in medical technology, the five-year survival rate for pulmonary arterial hypertension is only about half. As the prognosis is very poor, appropriate early diagnosis and treatment are important.

[0009] Attempts have been made to improve gene transfer efficiency by modifying the AAV capsid protein, but no studies have yet been reported on adeno-associated viruses targeting the pulmonary vasculature. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2017-201121 (November 23, 2017) [Patent Document 2] Japanese Patent Publication No. 2021-0010372 (February 4, 2021) Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, the present inventors have made intensive efforts to solve the above problems, and as a result, have completed the present invention by developing a protein variant based on the capsid of a new adeno-associated virus (AAV) serotype 1 that targets the pulmonary blood vessels and has the potential to deliver a gene therapy agent that solves the underlying genetic cause of pulmonary arterial hypertension. [Means for solving the problem]

[0012] Therefore, an object of the present invention is to provide mutants of AAV1 capsid proteins to improve the efficiency of gene transfer into target cells and / or the efficiency of genetic information expression by recombinant AAV.

[0013] Another object of the present invention is to provide a nucleic acid encoding a mutant of the AAV1 capsid protein.

[0014] Another object of the present invention is to provide a recombinant AAV1 vector comprising a nucleic acid encoding the mutant AAV1 capsid protein.

[0015] Yet another object of the present invention is to provide a pharmaceutical composition comprising the recombinant AAV1 vector.

[0016] It is yet another object of the present invention to provide a method for preventing or treating pulmonary hypertension, which comprises administering a therapeutically effective amount of the pharmaceutical composition to a subject. [Effects of the Invention]

[0017] The present invention relates to a recombinant AAV1 capsid mutant capable of highly efficient gene transfer by specifically targeting the pulmonary vasculature. A recombinant viral vector containing a nucleic acid encoding a mutant AAV1 capsid protein is useful for expressing transgenes in pulmonary vascular target cells when delivered in an aerosol state through the bronchi, enabling the prevention or treatment of pulmonary vascular diseases. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows the 3D structure of the recombinant AAV1 vector (#2-3 mutant). [Figure 2a] Figure 2a shows the cleavage map of the #2-3 mutant. [Figure 2b] Figure 2b shows the pHelper plasmid cleavage map. [Figure 2c] FIG. 2c shows the pCMV GFP plasmid cleavage map. [Figure 2d] Figure 2d shows the pCMV LacZ plasmid cleavage map. [Figure 3] FIG. 3 shows the complete base sequence of the recombinant AAV1 vector (#2-3 mutant) according to the present invention, represented by SEQ ID NO: 5. [Figure 4] Figure 4 shows the complete base sequence of the wild-type AAV1 vector. [Figure 5] FIG. 5 shows a comparison of the packaging efficiency and genomic titer of wild-type AAV1 and the recombinant AAV1 vector of the present invention (#2-3 mutant) through quantitative PCR analysis. [Figure 6]FIG. 6 shows the improvement in transduction efficiency of human pulmonary artery smooth muscle cells (HPASMCs) analyzed by the percentage of cells expressing GFP among all cultured cells. [Figure 7] Figure 7 shows the results of efficient local transduction in lung tissue by whole-mount β-galactosidase staining of lungs excised from 8-week-old C57BL / 6 male mice to confirm the vascular specificity of the recombinant AAV1 vector (#2-3 mutant) in lung tissue. [Figure 8] Figure 8 shows the quantification of pulmonary vascular smooth muscle cell targeting of recombinant AAV1 vectors (#2-3 mutants). [Figure 9] Figure 9 shows the Brownian motion of the recombinant AAV1 vector (#2-3 mutant) in 1x PBS (pH 7.4). [Figure 10] Figure 10 shows the targeting and distribution of recombinant AAV1 vectors (#2-3 mutants) to vascular smooth muscle cells after intratracheal injection. [Figure 11] Figure 11 shows the LacZ expression of recombinant AAV1 vector (#2-3 mutant) in blood vessels with a diameter of 100 μm or more, confirmed by x-gal staining [V: vessel, B: bronchus]. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in more detail below.

[0020] The present invention relates to mutants of the adeno-associated virus serotype 1 (AAV1) capsid protein.

[0021] As used herein, the term "adeno-associated virus" or "AAV" refers to all adeno-associated viruses used in gene therapy, including their derivatives, viral subtypes, naturally occurring, and recombinant forms. Various serotypes of AAV may be used as recombinant gene transfer viruses to transduce numerous different cell types. The genomic sequences of various AAV serotypes, as well as the sequences of the natural terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank accession numbers NC_002077 (AAV-1) and AF063497 (AAV-1).

[0022] As used herein, the term "serotype" refers to a subdivision of AAV that can be identified by serological or DNA sequence analysis and can be distinguished by its antigenic characteristics.

[0023] The term "capsid" as used herein refers to a protein encoded by the cap gene present in the viral genome, which constitutes the outer coat of the virus. The wild-type AAV genome or cap gene encodes three types of capsid proteins (VP1, VP2, and VP3). As used herein, all of these are included in the capsid protein. The wild-type AAV1 capsid protein comprises the amino acid sequence set forth in SEQ ID NO:1.

[0024] In one embodiment, the present invention provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the serine at position 430 is substituted with a cysteine ​​and the isoleucine at position 647 is substituted with a valine compared to the amino acid sequence of the wild-type AAV1 capsid protein.

[0025] In one embodiment, the AAV1 capsid protein mutant according to the present invention comprises or consists of the amino acid sequence (VP1) set forth in SEQ ID NO: 3. VP2 corresponds to the amino acid sequence from T138 to the end of SEQ ID NO: 3, and VP3 corresponds to the amino acid sequence from M203 to the end of SEQ ID NO: 3.

[0026] In the examples of this application, the mutant AAV1 capsid protein according to the present invention was designated #2-3.

[0027] The term "wild type" as used herein refers to the type that is most commonly found in wild populations of a species. With respect to mutant types, wild type refers to a phenotype that is considered to be basic or an individual thereof. Wild type is also known as "normal type." Furthermore, as used herein, "mutant" refers to a protein, virus, cell, individual, etc. in which a mutated gene is manifested as a phenotypic change. Furthermore, as used herein, "mutant" can also refer to the mutated gene itself.

[0028] In one embodiment, the present invention includes a nucleic acid encoding a mutant of the AAV1 capsid protein. The nucleic acid of the present invention encodes the mutant of the AAV1 capsid protein. The nucleic acid of the present invention is prepared by substituting at least one base with another base in the base sequence of a nucleic acid (cap gene) encoding the AAV1 capsid protein. The nucleic acid of the present invention may be in the form of DNA, but may also be in the form of RNA in some cases, or may be a chimera of DNA and RNA. The nucleic acid of the present invention also includes a complementary nucleic acid (e.g., cDNA). The nucleic acid of the present invention may be single-stranded or double-stranded, but is preferably double-stranded.

[0029] The present invention is not particularly limited to nucleic acids encoding mutants of AAV1 capsid proteins, but in one embodiment, the nucleic acid may be a nucleic acid having the nucleotide sequence represented by SEQ ID NO:4.

[0030] The nucleic acids of the present invention may be operably linked to an appropriate control sequence. Control sequences include promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, internal ribosome entry sites (IRES), enhancers, etc. Promoter sequences include inducible promoter sequences and constitutive promoter sequences. The control sequence may be native to the AAV from which the capsid protein is derived, or may be foreign, a natural sequence, or a synthetic sequence. Recombinant DNA capable of expressing AAV1 capsid protein mutants containing the nucleic acids of the present invention is also included in the present invention.

[0031] The recombinant DNA is useful for transferring the nucleic acid of the present invention to cells in vitro, ex vivo, and in vivo, thereby conferring to the cells the ability to express mutant AAV1 capsid proteins. Furthermore, cells transfected with the nucleic acid of the present invention are also useful for producing recombinant AAV particles. The recombinant DNA may be used to transfer or introduce the nucleic acid of the present invention into eukaryotic cells, preferably animal cells, and more preferably mammalian cells.

[0032] In the present invention, recombinant DNA may be produced by carrying the nucleic acid of the present invention in DNA used as a vector, such as a plasmid, phage, transposon, cosmid, episomal DNA, or viral genome.

[0033] For example, a packaging plasmid may be prepared by incorporating a nucleic acid (cap gene) encoding a mutant AAV1 capsid protein of the present invention into a plasmid. The packaging plasmid may further include any nucleic acid sequence, such as a nucleic acid (rep gene) encoding a replicase (Rep) protein. Preferably, the rep gene may include Rep from AAV2.

[0034] Recombinant DNA containing the nucleic acid of the present invention can also be prepared by substituting at least one base in the PLA2 domain coding region of the nucleic acid sequence of the cap gene carried by a known packaging plasmid with another base. The packaging plasmid is not particularly limited, but includes packaging plasmids carrying the cap gene, preferably packaging plasmids carrying the cap gene and the rep gene. As an example, in the present invention, a recombinant AAV1 vector p#2-3 represented by SEQ ID NO: 5 was prepared, which is a packaging plasmid carrying the nucleic acid (cap gene) encoding a mutant AAV1 capsid protein of the present invention and the rep gene.

[0035] The method for introducing base substitutions into nucleic acids can be carried out by known methods and is not particularly limited, but can be achieved by using commercially available reagents, for example, Mutagenesis Basal Kit (TAKARA BIO INC.), and performing PCR according to the instructions included with the kit.

[0036] Thus, the present invention includes novel AAV1#2-3 mutants for gene therapy applications, which are recombinant AAV1 vectors comprising nucleic acids encoding the AAV1 capsid protein mutants.

[0037] The recombinant AAV vector of the present invention is useful for gene transfer into target cells, and the gene transferred by the recombinant AAV vector of the present invention is highly expressed in the target cells.

[0038] As used herein, the term "AAV vector" refers to any vector that contains or is derived from components of an adeno-associated virus (AAV) and is suitable for infecting mammalian cells, including human cells of any of many tissue types, such as the brain, heart, lung, skeletal muscle, liver, kidney, spleen, or pancreas, whether in vitro or in vivo. The term "AAV vector" may also be used to refer to an AAV-type viral particle (or virion) that contains at least a nucleic acid molecule encoding a protein of interest.

[0039] As used herein, the term "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein (from all capsid proteins of wild-type AAV) and a polynucleotide rAAV vector encapsulated within the capsid. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, for example, 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 rAAV particles necessarily includes the production of rAAV, since such a vector is contained within the rAAV particle.

[0040] "Packaging" refers to a series of intracellular events that result in the assembly and intracapsid import of AAV particles.

[0041] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and import proteins within the capsid of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0042] A "helper virus" for AAV refers to a virus that enables AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. A variety of such helper viruses for AAV are known in the art, including adenoviruses, rupesviruses, and foxviruses such as vaccinia. While subgroup C type 5 adenoviruses are most commonly used, adenoviruses include many different subgroups. Numerous adenoviruses derived from humans, non-human mammals, and birds are known and available from depositories such as the American Type Culture Collection (ATCC). Herpes family viruses, including herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV), are also available from depositories such as the American Type Culture Collection (ATCC).

[0043] "Helper virus function(s)" refers to the function(s) encoded in the helper virus genome that permit AAV replication and packaging (along with other requirements for replication and packaging as described herein). As described herein, "helper virus functions" may be provided in a variety of ways, including by providing a helper virus or, for example, by providing a polynucleotide sequence encoding the essential function(s) to a producer cell in trans. For example, a plasmid or other expression vector containing a nucleotide sequence encoding at least one adenovirus protein is transfected into a producer cell along with the rAAV vector.

[0044] In one embodiment, the AAV1 vectors of the present invention have an improved transduction profile for target tissues compared to AAV1 vectors containing wild-type capsid proteins, i.e., the AAV1 vectors of the present invention have distinct tissue targeting capabilities (e.g., tissue tropism).

[0045] As used herein, the term "tropism" refers to the specificity of AAV capsid proteins present in AAV viral particles to infect or transduce particular types of cells or tissues.

[0046] The tropism of an AAV capsid for a particular type of cell or tissue may be determined by measuring the ability of AAV vector particles containing AAV1 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 Examples herein.

[0047] That is, "tropism" refers to the ability of an AAV vector or virion to infect at least one particular cell type, but can also include whether the vector functions to transduce cells into at least one particular cell type; i.e., tropism refers to the preferential introduction of the AAV vector or virion into a particular cell or tissue type(s) and / or preferential interaction with the cell surface that facilitates entry into a particular cell or tissue type, optionally and preferably followed by expression (e.g., transcription and optionally translation) of sequences carried by the AAV vector or virion within the cell, e.g., in the case of a recombinant virus, expression of a heterologous nucleotide sequence(s).

[0048] As used herein, the term "transduction" refers to the ability of an AAV vector or virion to infect at least one specific cell type; i.e., transduction refers to introducing an AAV vector or virion into a cell and transferring the genetic material contained within the AAV vector or virion to the cell for expression from the vector genome. In some, but not all, cases, transduction and tropism are correlated.

[0049] The AAVs described herein contain amino acid modifications in at least one capsid protein that confer new or enhanced tissue tropism properties. The AAV1 modifications according to the present invention target the pulmonary vasculature (e.g., the pulmonary artery).

[0050] As used herein, the term "tropism for lung tissue or pulmonary blood vessels" means tropism for lung tissue or pulmonary blood vessels.

[0051] In one embodiment, the pulmonary vascular tropism of the peptide-modified hybrid AAV capsid protein is increased by at least 5%, 10%, 20%, 30%, 40%, 50% or more compared to the pulmonary vascular tropism of the wild-type AAV capsid protein without the peptide.

[0052] The present invention also includes a pharmaceutical composition comprising the recombinant AAV1 vector, which may further comprise a pharmaceutically acceptable carrier.

[0053] The term "pharmaceutically acceptable carrier" includes any substance that, when combined with an active ingredient of a composition, allows the ingredient to have biological activity without eliciting an undesired physiological response, such as an immune response. Pharmaceutically acceptable carriers include water, phosphate buffered saline, emulsions such as oil / water emulsions, and wetting agents. Compositions containing such carriers may be formulated by well-known conventional methods, as described in Remington's Pharmaceutical Sciences, current Ed., Mack Publishing Co., Easton Pa. 18042, USA; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Cansel et al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., 3rd ed., American Pharmaceutical Assoc.

[0054] In one embodiment, the pharmaceutical composition according to the present invention may be a pharmaceutical composition for preventing or treating pulmonary arterial hypertension.

[0055] 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, for the purpose of attempting to reverse, alleviate, ameliorate, inhibit, slow, or prevent the progression, development, severity, or recurrence of disease-related syndromes, complications, symptoms, or biochemical manifestations. Treatment may be performed on subjects with disease or subjects without disease (e.g., for prophylaxis).

[0056] In one embodiment, the present invention also includes a method for preventing or treating pulmonary arterial hypertension, comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject.

[0057] As used herein, "administration" refers to the physical introduction of a therapeutic agent or a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, intravitreal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the term "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, subthecal, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation.

[0058] As used herein, the term "therapeutically effective amount" refers to an amount of a drug alone or in combination with other therapeutic agents that is effective to "treat" a disease or disorder in a subject or to reduce the risk, latency, 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 having or at risk of having a disease or disorder (e.g., pulmonary arterial hypertension as disclosed herein). Hereby, a "therapeutically effective amount" is an amount that reduces the risk, latency, likelihood, or occurrence of a disease or disorder, or provides some relief, alleviation, or reduction in at least one indicator (e.g., pulmonary arterial hypertension), or reduces at least one clinical symptom of a disease or disorder.

[0059] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc. [Example]

[0060] The present invention will be described in more detail below through examples according to the present invention, but the scope of the present invention is not limited to the following examples.

[0061] [Example]

[0062] Example 1: Selection of pulmonary vascular tropic AAV1 capsid protein mutants using an AAV library

[0063] 1) Selection of AAV1 capsid protein mutants Plasmid pools were generated by random point mutagenesis of the cap genes of wild-type AAV variants (AAV1, AAV2, AAV4, AAV6, AAV8, and AAV9) using error-prone PCR, and random 7mer / 9mer insertions were performed using a 3-fold protrusion of each serotype. 7-70 ng of the AAV plasmid library, 25 μg of pBluescript, and 25 μg of pHelper were combined in calcium-phosphate complexes and transfected into AAV293 cells for AAV packaging. An AAV library pool containing the cap gene information of each variant was generated.

[0064] Eight-week-old C57BL / 6 male mice were anesthetized with isoflurane, and a 1-cm incision was made in the skin over the airway. 1 × 10% PBS was added to a PenWu microaerosolizer (BioJane, Shanghai, China) (1.25" length of intratracheal portion, 700 μm of outer diameter, 430 μm of inner diameter). 11 A vg / 100 μl AAV library pool was loaded, and the needle was confirmed to pass through the airway, followed by intratracheal injection.

[0065] One week later, the lungs were excised and perfused with 0.9% saline through the heart. After homogenization, DNA was extracted from the whole lung using a DNA mini kit (Qiagen). The cap gene of an AAV mutant exhibiting whole-lung tropism was amplified using the AAV cap gene-specific forward primer 5'-GCGGAAGCTTCGATCAACTACG-3' (SEQ ID NO: 7) and reverse primer 5'-CGCAGAGACCAAAGTTCAACTGA-3' (SEQ ID NO: 8). This was prepared using a lung-tropic AAV library pool, and intratracheal injection (1 x 10) was performed as described above. 11One week later, the lungs were perfused and removed, chopped for 30 seconds, and then subjected to single cell dissociation using collagenase II. DNase I was added to prevent cell clustering due to chromosomal DNA released from dead cells and minimize cell loss. After incubation at 37°C for 4-6 hours, the total lung population was isolated in the form of single cells by pipetting. Red blood cell lysis was then performed at room temperature in the dark, followed by filtration through a 70μm pore cell strainer and transfer to FACS buffer. To select AAV mutants exhibiting angiotropism, 10μg of APC (anti-alpha smooth muscle actin antibody) was added and the mixture was incubated at 4°C. APC-positive cells were then sorted to select vascular-related cells. After cell lysis and DNA extraction, the cap gene of the AAV mutant transfected into the cells was amplified using primers (5'-GCGGAAGCTTCGATCAACTACG-3': SEQ ID NO: 9) and (5'-CGCAGAGACCAAAGTTCAACTGA-3': SEQ ID NO: 10).The amplified cap gene, containing HindIII and NotI sequences at both ends, was subcloned into the pSub2 plasmid via HindIII / NotI restriction and ligation. It was then electroporated into DH10β cells, purified, and stored as an angiotropic plasmid pool using the Qiagen Plasmid Maxi Kit. (pSub2 is a plasmid constructed by David Schaffer La., UC Berkeley, based on pSub201 (ATCC), and was engineered to allow cap gene subcloning using HindIII and NotI. References 1. Narendra Maheshri et al., Nature Biotechnology, 2006; 2. James T. Koerber, Nature Protocols, 2006.) Among the various mutants selected through three rounds of in vivo selection, mutant #2-3 was finally selected as an AAV gene delivery vector for the prevention and treatment of pulmonary arterial hypertension. It was confirmed to contain the AAV1-based point mutations S430C and I647V (Figure 1).

[0066] 2) Construction of recombinant AAV1 vectors (AAV1#2-3 mutants) (1) Construction of packaging plasmid mutants In the angiotropic plasmid pool, multiple angiotropic cap genes were subcloned into pXX2 (UC Berkeley, David Schaffer Lab) containing HindIII and NotI restriction enzymes for cap gene insertion, completing the creation of multiple angiospecific plasmid mutants carrying reporter genes.

[0067] (2) Plasmid transfection into AAV293 cells Generation of AAV1#2-3 mutant AAV293 cells were transfected with 17μg of mutant plasmid, 17μg of an ITR-flanked reporter gene (pCMV-GFP, pCMV-LacZ, or pCMV-FGF12-IRES-GFP), and 17μg of pHelper in a calcium-phosphate complex. After approximately 48 hours, the cell pellets were collected and intracellular AAV was extracted by freezing and thawing. Cell debris was then removed by centrifugation, and nucleic acids released from virus-producing cells were removed by incubation with 10U / mL benzonase at 37°C for 30 minutes.

[0068] The cleavage map of the constructed #2-3 mutant is shown in FIG. 2, and the entire base sequence is the same as that shown in FIG.

[0069] Generation of wild-type AAV1 AAV293 cells were transfected with 17μg of a packaging plasmid containing the AAV2 rep gene and the AAV1 cap gene, 17μg of an ITR-flanked reporter gene (pCMV-GFP, pCMV-LacZ, or pCMV-FGF12-IRES-GFP), and 17μg of pHelper, forming a calcium-phosphate complex. After approximately 48 hours, the cell pellets were collected and intracellular AAV was extracted by freezing and thawing. Cell debris was then removed by centrifugation, and nucleic acids released from virus-producing cells were removed by incubation with 10U / mL benzonase at 37°C for 30 minutes.

[0070] 3) Purification of AAV1#2-3 mutant The AAV solution was ultracentrifuged using an iodixanol gradient. Iodixanol solutions were prepared at 15%, 25%, 40%, and 54% concentrations and loaded sequentially into ultracentrifuge tubes, followed by the AAV solution. After tube sealing, ultracentrifugation was performed using an Optima XE-90 Ultracentrifuge (Beckman Coulter) and a Vti65.2 rotor (42,000 RPM, 18°C, 2 hours). The AAV layer between the 54% and 40% iodixanol concentrations was extracted and buffer exchanged with PBS buffer containing 0.01% Tween 20 using an Amicon® Ultra-15 Centrifugal Filter (MWCO 100,000).

[0071] 4) Measurement of AAV1#2-3 mutant titer The titers of wild-type AAV1 (wtAAV1) and AAV1#2-3 mutants carrying CMV-FGF12-IRES-GFP were quantified by treating Dnase I (5U)-resistant viruses with proteinase K to extract the viral genome, followed by quantitative PCR (qPCR) using CMV primers (5'-ATGGTGATGCGGTTTTGGCAG-3': SEQ ID NO: 11 and 5'-GGCGGAGTTGTTACGACATTTTGG-3': SEQ ID NO: 12) together with the respective standards.

[0072] The packaging efficiency of wild-type AAV1 and the recombinant AAV1 vector (#2-3 mutant) was compared by genomic titer and is shown in Figure 5. This indicates that the #2-3 mutant has improved packaging efficiency compared to wild-type AAV1, potentially allowing the maintenance of evolutionarily superior individuals.

[0073] Example 2: Confirmation of infection of recombinant AAV1 mutants

[0074] 1) In vitro experiments Wild-type AAV1 and the #2-3 mutant carrying CMV-GFP as a reporter gene for gene transfer efficiency and location analysis were packaged and transfected into human pulmonary arterial smooth muscle cells (HPASMCs; 2 × 10 4 cells / 20 μL).

[0075] Each virus carried CMV-FGF12-IRES-GFP. When the virus carried GFP, the percentage of GFP-expressing cells among all cultured cells was analyzed by flow cytometry 48 hours after infection of HPASMC (MOI 10,000) to confirm its infectivity. The results are shown in Figure 6.

[0076] Figure 6 shows the improvement in transduction efficiency of human pulmonary artery smooth muscle cells (HPASMCs) analyzed as the percentage of cells expressing GFP among all cultured cells. It shows that the #2-3 mutant has superior gene transfer efficacy to vascular cells and tropism to human primary cells (human pulmonary artery smooth muscle cells) compared to wild-type AAV1.

[0077] 2) In vivo experiments The CMV-LacZ-carrying AAV1 wild type and #2-3 mutant were packaged and injected into the trachea of ​​8-week-old C57BL / 6 male mice in the form of aerosol (5 × 10 11 vg / 100μl).

[0078] To confirm the specificity of the recombinant AAV1 vector (#2-3 variant) in lung tissue, lungs excised from 8-week-old C57BL / 6 male mice 1 week after injection were stained with whole-mount β-galactosidase to confirm LacZ expression. The results are shown in Figure 3, confirming efficient local delivery to lung tissue.

[0079] To confirm the pulmonary vascular specificity of the recombinant AAV1 vector (#2-3 variant), 8-week-old C57BL / 6 male mice were injected with the vector, and the lungs were removed 1 week after injection and the presence or absence of LacZ expression in the pulmonary vasculature was confirmed by X-gal staining. The results are shown in Figure 7. This confirmed that the vector was efficiently delivered to the pulmonary vasculature.

[0080] Example 3: Confirmation of the pulmonary vascular smooth muscle cell targeting efficiency of recombinant AAV1 mutants

[0081] To evaluate the targeting efficiency of vascular smooth muscle cells in lung tissue, we attempted to quantify the viral genome transferred to multiple cells at the single cell level. The #2-3 mutant carrying LacZ and wtAAV1 were packaged and administered intratracheally (5 × 10) to 8-week-old C57BL / 6 mice. 11 On days 3 or 7 after injection, lungs were excised and subjected to single-cell dissociation followed by overnight incubation at 4°C with an antibody conjugated to α-SMA (α-SMA-Alexa488, Abcam, ab184675) for cell sorting.

[0082] The viral genome ratios transferred into each cell were calculated relative to the initial injection dose, normalized to the wtAAV1 value, and the fold change in the #2-3 mutant relative to wtAAV1 was confirmed (*p-value<0.01).

[0083] As shown in Figure 8, the viral genome ratio in samples sorted using an antibody conjugated to α-sma tended to increase 3.7±0.9-fold and 2.7±0.5-fold in the #2-3 mutant compared to wtAAV1 on Day 3 and Day 7, respectively.

[0084] Example 4: Confirmation of the motility of recombinant AAV1 mutants

[0085] After intratracheal injection of the #2-3 mutant and wtAAV1 in aerosol form, they penetrate various barriers, including the airway wall, extracellular matrix, and vascular wall, and reach vascular smooth muscle cells. To demonstrate that this is possible not only through the tropism of #2-3 but also through the superior mobility of AAV1, we examined Brownian motion in the liquid phase (PBS phase).

[0086] 1)1xPBS, static state When wtAAV1 or the #2-3 mutant was dispersed at a concentration of 1.0E+09 vg / ml in 1x PBS (pH 7.4) and the trajectories were observed in the static state, it was confirmed that #2-3 exhibited a diffusion-like movement compared to wtAAV1.

[0087] MSD (Mean squared displacement) is defined as msd(τ)=<Δr(τ)2>=<[r(t+τ)-r(t)] 2 >The equation (r(t) is the vector position at time t, τ is the lag time between the two positions (the difference in the trajectories obtained for each vector)) is used to measure Log 10 (MSD 1s ) / pixel 2 A comparison of the distances traveled by the #2-3 mutant (3.12±0.17) compared with AAV1 (2.21±0.49) showed a statistically significant increase (*p-value<0.01). This indicates that the distance traveled by the #2-3 mutant was increased compared with that of wtAAV1 in 1x PBS, pH 7.4 (Figure 9).

[0088] Brownian motion refers to the random movement of particles in a liquid or gas, while diffusion refers to the relatively directional movement of particles. Diffusion can be considered a macroscopic expression of Brownian motion, and the form of diffusion can be predicted by calculating the average movement of Brownian motion.

[0089] That is, in contrast to AAV1, which exhibits erratic movement and remains in a fixed position, the #2-3 mutant exhibited relatively linear movement, indicating diffusion-like movement.

[0090] Example 5: Confirmation of pulmonary vascular tropism of recombinant AAV1 mutants

[0091] 1) Confirmation of the distribution of recombinant AAV1 mutants in lung tissue wtAAV1 or the #2-3 mutant was tagged with Alexa594 and administered intratracheally to 8-week-old C57BL / 6 male mice (3 × 10 11 Forty-eight hours after injection, the lungs were excised under anesthesia and perfused through the heart. After fixation with 4% PFA, the lungs were sectioned and colocalization of AAV distribution within arterioles was confirmed using α-sma antibody (Alexa488).

[0092] Observation of AAV distribution 24 h after intratracheal injection revealed that in the #2-3 mutant, AAV vectors tagged with Alexa594 (a fluorescent substance conjugated to the AAV capsid) moved from the bronchiole to the arteriole.

[0093] At 48 h after intratracheal injection, most of the #2-3 vectors migrated from the bronchioles to the arterioles, and the AAV vectors were confirmed to be localized in the vascular wall. This colocalization with α-sma antibody, a marker for intravascular smooth muscle cells, confirmed the blood vessel tropism of the #2-3 vectors. In contrast, at 48 h, wtAAV1 was confirmed to have a large amount of AAV vector in the air sacs around blood vessels, but did not show colocalization with α-sma antibody (Figure 10).

[0094] Specifically, when the virus was exposed to fluorescence and its movement within lung tissue was examined, it was found that wtAAV1 remained around the bronchi or was unable to reach the blood vessels, whereas the #2-3 mutant passed through the bronchi and moved rapidly toward the blood vessels very smoothly, and the #2-3 vector gene was found concentrated in vascular cells.

[0095] 2) Confirmation of pulmonary vascular tropism by LacZ expression LacZ-loaded wtAAV1 or the #2-3 mutant was intratracheally injected into 8-week-old C57BL / 6 male mice (3 × 10 11 Two weeks later, the mice were anesthetized and perfused by cardiac perfusion. The mice were then sectioned and stained with X-gal. Compared to wtAAV1, the #2-3 mutant mice exhibited LacZ expression in the vascular tissue.

[0096] As shown in Figure 11, the #2-3 vector had improved mobility compared to wtAAV1, improving the rate at which it physically reached vascular sites through the airways and extracellular matrix in lung tissue, and also exhibited vascular tropism, concentrating in blood vessels. Therefore, the #2-3 vector, a recombinant AAV1 of the present invention, is expected to be useful as an AAV vector for the treatment and prevention of pulmonary vascular-targeted diseases.

Claims

1. A mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant has an amino acid sequence represented by SEQ ID NO: 3 in which serine at position 430 is substituted with cysteine ​​and isoleucine at position 647 is substituted with valine, compared to the amino acid sequence of the wild-type AAV1 capsid protein represented by SEQ ID NO:

1.

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

3. The nucleic acid according to claim 2, having the base sequence represented by SEQ ID NO:

4.

4. A recombinant AAV1 vector comprising a nucleic acid encoding a mutant of the AAV1 capsid protein of claim 2.

5. The recombinant AAV1 vector of claim 4, wherein the AAV1 vector has an improved transduction profile for target tissues compared to AAV1 wild-type viral vectors.

6. The recombinant AAV1 vector of claim 5, wherein the target tissue is a pulmonary vasculature.

7. The recombinant AAV1 vector of claim 6, wherein the pulmonary blood vessel is a pulmonary artery.

8. A recombinant AAV1 vector described in claim 4, represented by sequence number 5.

9. A pharmaceutical composition comprising the recombinant AAV1 vector of claim 4.

10. 10. The pharmaceutical composition of claim 9, wherein the composition further comprises a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 9 for the prevention or treatment of pulmonary arterial hypertension.

12. 10. The pharmaceutical composition of claim 9, wherein the composition is administered by intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intravitreal, intraarticular, subthecal, subarachnoid, intraspinal, epidural, or intrasternal injection.

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