Adeno-associated virus vector capable of specific gene delivery to the lung

Abstract

The present invention provides a mutant adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant has an amino acid substitution at a specific position 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 the nucleic acid encoding the mutant AAV1 capsid protein enhances transgene expression in the lungs and bronchi when delivered in an aerosol state through the bronchi, making it useful for the prevention or treatment of respiratory diseases.

Description

[Technical Field]

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

[0002] To effectively carry out gene therapy, the development of gene transfer technology that delivers therapeutic genes to desired target cells and ensures high expression efficiency is a top priority.

[0003] Among these gene transfer technologies, AAV is a non-pathogenic virus that has no side effects on infected cells and is unlikely to cause 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 it a useful method for introducing genes into a variety of cells and a useful vector for gene therapy.

[0005] AAVs exist in a variety of serotypes, each with distinct host and viral characteristics. Serotype 2 (AAV2) has been extensively studied for some time and is capable of infecting a wide variety of cell types. Serotypes 1 (AAV1), 5 (AAV5), and 6 (AAV6) have more tissue-specific infection mechanisms. AAV1 is known to be highly efficient at transducing genes into muscle, liver, airways, and the central nervous system; AAV5 is known to be highly efficient at transducing genes into the central nervous system, liver, and retina; and AAV6 is known to be highly efficient at transducing genes into the heart, muscle, and liver. While the ability to transduce genes into specific tissues varies depending on the serotype, AAVs are still readily transduced into other tissues. Therefore, the development of new AAV vectors with improved tissue specificity, safety, and efficacy is essential.

[0006] Meanwhile, bronchitis and bronchiectasis are major diseases that occur in the lungs and bronchi. Bronchiectasis in particular can lead to complications such as hemoptysis (a condition in which blood or blood-mixed phlegm is coughed up), persistent breathing difficulties, and respiratory failure, so early diagnosis and treatment are essential. Hemoptysis is a potentially life-threatening complication of bronchiectasis, and bronchial artery embolization or surgical treatment can be considered to control hemoptysis.

[0007] Although attempts have been made to improve the efficiency of gene transfer by modifying the AAV capsid protein, no studies have yet been reported on AAV targeting the lungs and bronchi. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2017 / 2011121 (November 23, 2017) [Patent Document 2] Patent Publication No. 2006-171701 (February 4, 2021) Summary of the Invention [Problem to be solved by the invention]

[0009] In response to this, the present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, have completed the present invention by developing a novel protein variant based on the capsid of adeno-associated virus (AAV) serotype 1 that targets the lungs and bronchi, which has the potential to deliver gene therapy drugs that solve the underlying genetic causes of respiratory diseases such as bronchitis or bronchiectasis.

[0010] Therefore, an object of the present invention is to provide a mutant of AAV1 capsid protein in order to improve the efficiency of gene transfer into target tissues or cells by recombinant AAV and / or the efficiency of expression of genetic information.

[0011] Another object of the present invention is to provide a nucleic acid encoding the mutant AAV1 capsid protein.

[0012] Another object of the present invention is to provide a recombinant AAV1 vector comprising a nucleic acid encoding the mutant AAV1 capsid protein.

[0013] A further object of the present invention is to provide a pharmaceutical composition comprising the recombinant AAV1 vector.

[0014] A further object of the present invention is to provide a transfectant comprising the recombinant AAV1 vector. [Means for solving the problem]

[0015] The present invention will now be described in more detail.

[0016] The present invention relates to mutants of the adeno-associated virus serotype 1 (AAV1) capsid protein.

[0017] As used herein, the term "adeno-associated virus" or "AAV" refers to all adeno-associated viruses used in gene therapy, including derivatives, viral subtypes, and naturally occurring and recombinant forms thereof. Various AAV serotypes can 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 naturally occurring terminal repeats (TRs), Rep proteins, and capsid subunits, are publicly available. These 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).

[0018] As used herein, the term "serotype" refers to a subdivision of AAV that can be identified by serological or DNA sequence analysis methods and can be distinguished by its antigenic characteristics.

[0019] The term "capsid" used in the present invention 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 capsid proteins (VP1, VP2, and VP3). The wild-type AAV1 capsid protein has the amino acid sequence shown in SEQ ID NO: 1.

[0020] In one embodiment, the present invention provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant has a substitution of amino acids at any one or more of positions 326, 452, and 456 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein.

[0021] In one embodiment, the present invention provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, which has a mutant AAV1 capsid protein in which the threonine at position 326 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein is substituted with alanine and the glutamine at position 452 is substituted with proline.

[0022] In one embodiment, the present invention relates to a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, which has a mutant AAV1 capsid protein in which alanine at position 456 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein is substituted with threonine.

[0023] In one embodiment, the AAV1 capsid protein variant according to the present invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2 or 3.

[0024] In the examples of the present application, the mutant of the AAV1 capsid protein shown in SEQ ID NO: 2 was designated #3-32, and the mutant of the AAV1 capsid protein shown in SEQ ID NO: 3 was designated #3-65.

[0025] The term "wild type" used in the present invention refers to the type most commonly seen in wild populations of a species. In contrast to mutant types, wild type refers to a phenotype or individual that is considered to be basic. Wild type is also known as normal type. Meanwhile, in this specification, mutant refers to a protein, virus, cell, individual, etc. in which a mutated gene is manifested as a phenotypic change. In addition, in this specification, "mutant" may also refer to the mutated gene itself.

[0026] 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 a mutant of the AAV1 capsid protein. The nucleic acid of the present invention is produced 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 exist in the form of DNA, but may also exist in the form of RNA or a chimera of DNA and RNA. The nucleic acid of the present invention also includes complementary nucleic acids (e.g., cDNA). The nucleic acid of the present invention may be single-stranded or double-stranded, but is preferably double-stranded.

[0027] The present invention relates to a nucleic acid encoding a mutant of the AAV1 capsid protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 3, and although not limited thereto, an example thereof is a nucleic acid having the base sequence shown in SEQ ID NO: 4 or 5.

[0028] The nucleic acids of the present invention can also be operably linked to appropriate control sequences. 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. Control sequences may be native to the AAV from which the capsid protein is derived or may be foreign, and may be natural or synthetic sequences. Recombinant DNA capable of expressing AAV1 capsid protein mutants containing the nucleic acids of the present invention is also included in the present invention.

[0029] The recombinant DNA is useful for delivering the nucleic acid of the present invention to cells in vitro, ex vivo, and in vivo, and for conferring the ability of the cells to express mutant AAV1 capsid proteins. Furthermore, cells into which the nucleic acid of the present invention has been delivered are also useful for producing recombinant AAV particles. The recombinant DNA can be used, in particular, to deliver or introduce the nucleic acid of the present invention into eukaryotic cells, preferably animal cells, more preferably mammalian cells.

[0030] In the present invention, recombinant DNA can be produced by incorporating the nucleic acid of the present invention into DNA used as a vector, such as a plasmid, phage, transposon, cosmid, episomal DNA, or viral genome.

[0031] For example, a packaging plasmid can 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 can further include any nucleic acid sequence, such as a nucleic acid (rep gene) encoding a replicase (Rep) protein. Preferably, the rep gene can include Rep from AAV2.

[0032] Recombinant DNA containing the nucleic acid of the present invention can also be prepared by substituting at least one base in the PLA2 domain-encoding 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 examples include packaging plasmids carrying the cap gene, preferably packaging plasmids carrying the cap gene and the rep gene. As an example, in the present invention, recombinant AAV1 vectors p #3-32 and p #3-65, shown in SEQ ID NO: 6 or 7, were constructed, which are packaging plasmids carrying the nucleic acid (cap gene) encoding the AAV1 capsid protein mutant of the present invention and the rep gene.

[0033] The method for introducing base substitutions into nucleic acids can be carried out by well-known methods and is not particularly limited. For example, the method can be achieved by using commercially available reagents, such as the Mutagenesis Basal Kit (TAKARA BIO INC.), and performing PCR according to the instructions attached to the kit.

[0034] Therefore, the present invention provides a recombinant AAV1 vector comprising a nucleic acid encoding the AAV1 capsid protein mutant.

[0035] 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.

[0036] 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, whether in vitro or in vivo, including human cells of any of a number of tissue types, such as brain, heart, lung, skeletal muscle, liver, kidney, spleen, or pancreas. The term "AAV vector" is sometimes used to refer to an AAV-type viral particle (or virion) that contains at least a nucleic acid molecule encoding a protein of interest.

[0037] As used herein, "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle comprising at least one AAV capsid protein (from all capsid proteins of wild-type AAV) and a polynucleotide rAAV vector encapsulated within the capsid. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., 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, production of rAAV particles necessarily includes production of rAAV, since such vectors are contained within the rAAV particles.

[0038] "Packaging" refers to the series of cellular events that result in the assembly and import of AAV particles into capsids.

[0039] 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."

[0040] 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 available to the industry, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. While adenovirus type 5 of subgroup C is the most commonly used, adenoviruses include many different subgroups. Numerous adenoviruses of human, nonhuman mammalian, and avian origin are publicly available and available from depositories such as the American College of Cardiology (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 College of Cardiology (ATCC).

[0041] "Helper virus functions" refer to functions encoded in the helper virus genome that permit AAV replication and packaging (along with other requirements for replication and packaging 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 polynucleotide sequences encoding the essential functions to producer cells in trans. For example, a plasmid or other expression vector containing nucleotide sequences encoding one or more adenoviral proteins is transfected into producer cells along with the rAAV vector.

[0042] 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).

[0043] 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.

[0044] The tropism of an AAV capsid for a particular cell or tissue type can be determined by measuring the ability of AAV vector particles, including AAV1 capsid proteins, to infect or transduce a particular cell or tissue type using standard assays well known in the art, such as those disclosed in the Examples herein.

[0045] That is, "tropism" refers to the ability of an AAV vector or virion to infect one or more particular cell types, but can also include whether the vector functions to transduce cells into one or more particular cell types. That is, tropism refers to the preferential transfer of the AAV vector or virion to a particular cell or tissue type, and / or the preferential interaction with a cell surface that facilitates entry into the particular cell or tissue type, optionally and preferably the expression (e.g., transcription and optionally translation) of sequences carried by the AAV vector or virion in the cell, e.g., expression of a heterologous nucleotide sequence in the case of a recombinant virus.

[0046] As used herein, the term "transduction" refers to the ability of an AAV vector or virion to infect one or more specific cell types. That is, transduction refers to the introduction of an AAV vector or virion into a cell and the delivery of 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 may be correlated.

[0047] The AAVs described herein contain amino acid modifications in one or more capsid proteins that confer new or enhanced tissue tropism properties. The AAV1 mutants described herein target the lungs and bronchi.

[0048] As used herein, the term "pneumotropic" refers to tropism for the lungs and bronchi.

[0049] In some embodiments, the lung 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 lung tropism of the wild-type AAV capsid protein lacking the peptide.

[0050] The present invention also provides a pharmaceutical composition comprising the recombinant AAV1 vector, which may further comprise a pharmaceutically acceptable carrier.

[0051] The term "pharmaceutically acceptable carrier" includes any substance that, when combined with the active ingredient of the composition, allows the ingredient to retain its biological activity without eliciting an adverse physiological response, such as an unintended immune response. Pharmaceutically acceptable carriers include water, phosphate buffered saline, emulsions such as oil-water emulsions, and wetting agents. Compositions containing such carriers are formulated by well-known conventional methods, such as those 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. Ansel et al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., 3rd ed. American Pharmaceutical Assoc.

[0052] In one embodiment, the pharmaceutical composition according to the present invention may be a pharmaceutical composition for preventing or treating respiratory diseases, specifically, bronchitis or bronchiectasis.

[0053] As used herein, the term "treatment" refers to any type of intervention or process performed on a subject or 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 diseased or disease-free subjects (e.g., prophylactically).

[0054] In one embodiment, the present invention also includes a method for preventing or treating a respiratory disease, specifically, bronchitis or bronchiectasis, comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject.

[0055] 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 administration routes for the antibodies described herein include airway, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, intravitreal, or other parenteral routes, for example, by injection or infusion. The phrase "parenteral administration," as used herein, generally refers to administration modes other than enteral and topical administration by injection, including, but not limited to, airway, intravenous, intraperitoneal, intramuscular, intra-arterial, intraspinal, intralymphatic, intralesional, intrathecal, intraorbital, intracardiac, subcutaneous, transtracheal, subcutaneous, subcuticular, intravitreal, intra-articular, subthecal, subarachnoid, intraspinal, and thoracic epidural injection and infusion, as well as in vivo electroporation. In a specific example, animal studies using pigs demonstrated that the AAV vector of the present invention can achieve lung-specific gene transfer when administered in aerosol form to the airway.

[0056] 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., a respiratory disorder). 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 hypertension as disclosed herein). Thus, a "therapeutically effective amount" is an amount that provides a reduction in the risk, latency, likelihood, or occurrence of a disease or disorder, or partial relief, alleviation, or reduction in any one indicator (e.g., a respiratory disorder), or a reduction in any one clinical symptom of a disease or disorder.

[0057] 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.

[0058] The present invention also provides a drug delivery system comprising the recombinant AAV1 vector. The composition may further comprise a known pharmaceutically acceptable carrier for use in the above-described applications.

[0059] The AAV1 vector of the present invention exhibits tropism for the lungs and bronchi and has the ability to express genes specifically in the lungs, making it suitable for use as a drug delivery vehicle to the lungs and bronchi. Specifically, it can be used as an AAV1 vaccine against infectious agents that infect the respiratory tract. Existing intravenously administered vaccines have the disadvantage of spreading throughout the body and reducing the likelihood of antibody production in the lungs or airways. However, gene delivery using the vector of the present invention increases the efficiency of gene expression near the airways, where infectious agents first come into contact, and thus enhances mucosal immunity compared to existing vaccines. [Effects of the Invention]

[0060] The present invention relates to a recombinant AAV1 capsid mutant that has the ability to specifically target the lungs and bronchi and highly efficiently introduce genes. A recombinant viral vector containing a nucleic acid encoding a mutant AAV1 capsid protein is useful for expressing introduced genes in lung target cells when transmitted in an aerosol state through the bronchi, thereby enabling the prevention or treatment of respiratory diseases. [Brief explanation of the drawings]

[0061] [Figure 1] FIG. 1 shows a comparison of the packaging efficiency of wild-type AAV1 and the recombinant AAV1 vectors of the present invention (mutants #3-32 and #3-65) through genomic evaluation using quantitative PCR analysis. [Figure 2] FIG. 1 shows an analysis of the improvement in transduction efficiency into HEK293T cells as a percentage of GFP-expressing cells among all cultured cells. [Figure 3] To confirm the lung specificity of the recombinant AAV1 vectors (#3-32(a) and #3-65(b) mutants), whole-mount β-galactosidase staining of lungs excised from 8-week-old C57BL / 6 male mice was performed to confirm LacZ expression. This figure shows the results of efficient local delivery into the lungs and bronchi. [Figure 4] To confirm lung and bronchial specificity, LacZ expression in the lungs and bronchi by recombinant AAV1 vectors (mutants #3-32(a) and #3-65(b)) was confirmed by eosin / LacZ staining. [Figure 5a] FIG. 1 shows the cleavage maps of AAV1 vectors #3-32 and #3-65 mutants. [Figure 5b] FIG. 1 shows the cleavage maps of AAV1 vectors #3-32 and #3-65 mutants. [Figure 5c] FIG. 1 shows a cleavage map of the pHelper plasmid. [Figure 5d] FIG. 1 shows a cleavage map of the pCMV GFP plasmid. [Figure 5e] FIG. 1 shows a cleavage map of the pCMV LacZ plasmid. [Figure 6] FIG. 1 shows the results of injecting a recombinant AAV1 vector (#3-65 mutant) through the airways of pigs to confirm the lung specificity of the vector. DETAILED DESCRIPTION OF THE INVENTION

[0062] 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 examples shown below.

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

[0064] Eight-week-old C57BL / 6 male mice were anesthetized with isoflurane and a 1-cm skin incision was made over the airway. A 1x1011vg / 100µl AAV library pool in PBS was loaded into a PenWu microaerosolizer (BioJane, Shanghai, China) (1.25mm length of intratracheal portion, 700µm outer diameter, 430µm inner diameter). The AAV library pool was then injected intratracheally while monitoring needle passage into the airway.

[0065] One week later, the mice were perfused with 0.9% saline through the heart, and the lungs were removed. After homogenization, DNA was extracted from the whole lung using a DNA mini kit (Qiagen). The AAV cap gene-specific forward primer 5'-GCGGAAGCTTCGATCAACTACG-3' (SEQ ID NO: 8) and reverse primer 5'-CGCAGAGACCAAAGTTCAACTGA-3' (SEQ ID NO: 9) were used to amplify the cap gene of AAV mutants exhibiting lung tropism. This was used to create a lung-tropic AAV library pool, which was then intratracheally instilled in the same manner as above. One week later, the mice were perfused and removed. The lungs were then chopped for 30 seconds into small pieces and subjected to single-cell dissociation using collagenase II. DNase I was added to minimize cell loss by preventing cell clustering due to chromosomal DNA released from dead cells. After incubation at 37°C for 4–6 hours, single-cell total lung population was isolated by pipetting. Red blood cell lysis was performed at room temperature in the dark, and the cells were then filtered through a 70 μm pore cell strainer and transferred to FACS buffer. To select AAV variants exhibiting local tropism within lung tissue, 10 μg of α-sma-APC antibody was added and incubated at 4°C. APC (anti-alpha smooth muscle actin antibody)-positive cells were then sorted to identify bronchus-associated cells. After cell lysis and DNA extraction, the bronchus-tropic cap gene was amplified using the AAV cap gene-specific primers.This was used to create a second AAV library, and intratracheal injection and sorting were performed in the same manner. After three rounds of in vivo selection, the final amplified cap gene was subcloned into the pSub2 plasmid via HindIII / NotI restriction and ligation, with HindIII and NotI sequences at both ends. It was then electroporated into DH10β cells, purified (Qiagen Plasmid Maxi Kit), and stored as a lung-tropic plasmid pool. (pSub2 is a plasmid based on pSub201 (ATCC) constructed in the David Schaffer Lab at UC Berkeley, designed to allow for cap gene subcloning using HindIII and NotI. References 1. Narendra Maheshri et al., Nature Biotechnology, 2006; 2. James T. Koerber, Nature Protocols, 2006).

[0066] 2) Construction of recombinant AAV1 vectors (AAV1 #3-32 and #3-65 mutants) (1) Construction of packaging plasmid mutants From the angiotropic plasmid pool, multiple lung-tropic cap genes were subcloned into pXX2 (UC Berkeley, David Schaffer Lab) containing HindIII and NotI sites for cap gene insertion using HindIII / NotI restriction, completing the construction of multiple lung-specific plasmid variants for reporter gene insertion.

[0067] (2) Plasmid transfection into AAV293 cells Construction of AAV1 #3-32 and #3-65 mutants 17 μg of variant plasmid, 17 μg of ITR flanked reporter gene (pCMV-GFP or pCMV-LacZ or pCMV-FGF12-IRES-GFP), and 17 μg of pHelper were formed into a calcium-phosphate complex and transfected into AAV293 cells. After about 48 hours, only the cell pellet was collected and AAV inside the cells was extracted by freezing-thawing. Then, cell debris was removed by centrifugation, and 10 U / mL of benzonase was incubated at 37 °C for 30 minutes to remove nucleic acids released from the virus-producing cells.

[0068] The cleavage maps of the produced AAV1 #3-32 and #3-65 variants are shown in FIGS. 5a and 5b, and the entire nucleotide sequences of the AAV1 #3-32 and #3-65 variants are as follows, respectively.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0069] <AAV1 #3-65 variant> Production of wild-type AAV1 AAV293 cells were transfected with 17 μg of a packaging plasmid containing the AAV2 rep gene and the AAV1 cap gene, 17 μg of an ITR-flanked reporter gene (pCMV-GFP, pCMV-LacZ, or pCMV-FGF12-IRES-GFP), and 17 μg of pHelper in a calcium-phosphate complex. Approximately 48 hours later, cell pellets were collected and intracellular AAVs were 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 10 U / mL benzonase at 37°C for 30 minutes.

[0070] 3) Purification of AAV1 #3-32 and #3-65 mutants 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) with 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 into PBS buffer containing 0.01% Tween 20 using an Amicon Ultra-15 Centrifugal Filter (MWCO 100,000).

[0071] 4) Titer measurement of AAV1 #3-32 and #3-65 mutants The titers of wild-type AAV1 and AAV1 #3-32 and #3-65 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: 10 and 5-GGCGGAGTTGTTACGACATTTTGG-3: SEQ ID NO: 11) and performing qPCR together with the respective standards.

[0072] The packaging efficiency of wild-type AAV1 and recombinant AAV1 vectors (#3-32 and #3-65 mutants) was compared using genomic titers and is shown in Figure 1. This indicates that the #3-32 and #3-65 mutants have improved packaging efficiency compared to wild-type AAV1, suggesting that these mutants may be superior individuals in evolutionary terms.

[0073] Example 2: Confirmation of infection with recombinant AAV1 mutants 1) In vitro experiments To analyze the efficiency and location of gene transfer, AAV1 wild type, #3-32, and #3-65 mutants carrying CMV-GFP as a reporter gene were packaged and infected into HEK293T (2 x 104 cells / 20 μL).

[0074] Each virus carried CMV-FGF12-IRES-GFP. When GFP was carried, the percentage of GFP-expressing cells among the total cultured cells was analyzed by flow cytometry 48 hours after infection of HEK293T cells (MOI 10,000) to confirm infectivity. The results are shown in Figure 2.

[0075] Figure 2 shows the improvement in HEK293T transduction efficiency as analyzed by the percentage of GFP-expressing cells among the total cultured cells.

[0076] 2) In vivo experiments The CMV-LacZ-carrying AAV1 wild type and the #3-32 and #3-65 mutants were packaged and then intrabronchially instilled into 8-week-old C57BL / 6 male mice in the form of an aerosol (1x1011 vg / 100µl).

[0077] To confirm the lung specificity of the recombinant AAV1 vectors (#3-32 and #3-65 mutants), we performed whole-mount β-galactosidase staining of lungs excised from 8-week-old C57BL / 6 male mice 1 week after injection to confirm LacZ expression. As shown in Figures 3a and 3b, this demonstrated local delivery specifically to the lungs and bronchi.

[0078] To confirm the lung specificity of the recombinant AAV1 vectors (#3-32 and #3-65 mutants), we removed the lungs from 8-week-old C57BL / 6 male mice 1 week after injection and examined the expression of LacZ in the lungs and bronchi by eosin / LacZ staining. As shown in Figures 4a and 4b, the vectors were specifically delivered to the lungs and bronchi.

[0079] In addition, wtAAV1 (1.25x1011vg / kg) and #3-65 (5.38x1011vg / kg) vectors carrying LacZ were dispersed in PBS + 0.01% Tween 20 and injected into the airways of pigs in 1ml liquid form. Two weeks later, the pigs were sacrificed, fixed in 4% PFA, and then stained with X-gal. While wtAAV1 showed no gene expression in lung sections, #3-65 showed local LacZ expression in the airways and airway areas within the lung tissue (Figure 6).

Claims

1. A mutant of the adeno-associated virus serotype 1 (AAV1) capsid protein, The mutant is an AAV1 capsid protein mutant in which amino acids at any one or more of positions 326, 452, and 456 in the amino acid sequence of the wild-type AAV1 capsid protein shown in SEQ ID NO: 1 have been substituted.

2. The mutant is an AAV1 capsid protein mutant described in claim 1, in which the threonine at position 326 in the amino acid sequence of the wild-type AAV1 capsid protein shown in SEQ ID NO: 1 is replaced with alanine and the glutamine at position 452 is replaced with proline.

3. A mutant of the AAV1 capsid protein described in claim 1, wherein the alanine at position 456 in the amino acid sequence of the wild-type AAV1 capsid protein shown in SEQ ID NO: 1 is replaced with threonine.

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

5. A nucleic acid encoding a mutant of the AAV1 capsid protein of claim 3.

6. The nucleic acid according to claim 4, having the base sequence shown in SEQ ID NO:

4.

7. The nucleic acid according to claim 5, having the base sequence shown in SEQ ID NO:

5.

8. A recombinant AAV1 vector comprising a nucleic acid encoding a mutant of the AAV1 capsid protein of claim 4 or claim 5.

9. The recombinant AAV vector of claim 8, wherein the AAV1 vector has an improved transduction profile for the lungs and bronchi compared to AAV1 wild-type viral vectors.

10. A pharmaceutical composition comprising the recombinant AAV1 vector of claim 8.

11. 11. The pharmaceutical composition of claim 10, wherein the composition additionally comprises a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 10, wherein the composition is for the prevention or treatment of pneumonia, bronchitis, or bronchiectasis.

13. A gene transfer vector comprising the recombinant AAV1 vector of claim 8.

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