KR20230091788A - Adeno-associated virus vector capable of lung cell-specific gene delivery - Google Patents

Abstract

The present invention provides a mutant of an adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant is a mutant of the AAV1 capsid protein in which an amino acid at a specific position is substituted in comparison with an amino acid sequence of a wild-type AAV capsid protein, a nucleic acid encoding the mutant of the AAV1 capsid protein, a recombinant AAV1 vector containing the nucleic acid encoding the mutant of the AAV1 capsid protein, and a pharmaceutical composition containing the vector. According to the present invention, the recombinant viral vector containing the nucleic acid encoding the mutant of the AAV1 capsid protein increases transgene expression in lung cells when delivered through intratracheal injection, thereby being useful for preventing or treating lung diseases.

Description

Adeno-associated virus vector capable of lung cell-specific gene delivery}

The present invention relates to mutants of the adeno-associated virus (AAV) capsid protein, and in particular, recombinant viral vectors comprising mutants of the AAV1 capsid protein are useful for gene transfer into lung cells via intratracheal injection.

In order to effectively perform gene therapy, the highest priority is to develop a gene transfer technology that can deliver a therapeutic gene to a desired target cell to obtain high expression efficiency.

Among these gene delivery technologies, AAV is a non-pathogenic virus that has no side effects on infected cells and has a low probability of causing mutations in target cell genetic information, so it is superior in safety compared to other gene therapy technologies.

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. Due to these characteristics, it is a useful method for introducing genes into various cells and is used as a useful vector for gene therapy.

It is known that AAV has various serotypes, and the characteristics of the host or virus are different depending on the serotype. Serotype 2 (AAV2) is a serotype that has been widely studied for a long time and can infect various cells. Serotype 1 (AAV1), serotype 5 (AAV5), and serotype 6 (AAV6) are serotypes with more tissue infection specificity. AAV6 is known to have high gene transduction efficiency into the heart, muscle, liver, etc. Although the characteristics of gene transfer to a specific tissue vary depending on the serotype, it is still easy to transfer to other tissues, so it is essential to develop a new AAV vector that can improve safety and efficiency by improving tissue specificity. .

Although attempts have been made to improve gene transfer efficiency by modifying the AAV capsid protein, studies on adeno-associated viruses that deliver genes specifically and effectively only to lung cells have not yet been reported.

International Publication No. 2017-201121 (2017.11.23.) Japanese Patent Laid-Open No. 2021-0010372 (2021.02.04.)

Therefore, as a result of diligent efforts to solve the above problems, the inventors of the present invention have developed a novel adeno-associated virus (AAV) serotype 1 capsid targeting lung cells with the potential to deliver gene therapy to solve the fundamental genetic cause of lung disease. The present invention was completed by developing a protein variant having a basic skeleton.

Accordingly, an object of the present invention is to provide a mutant of AAV1 capsid protein to improve gene transfer efficiency and/or genetic information expression efficiency by recombinant AAV into a target tissue or cell.

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

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

Another object of the present invention is to provide a pharmaceutical composition comprising the recombinant AAV1 vector.

Another object of the present invention is to provide a gene delivery system comprising the recombinant AAV1 vector.

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

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

As used herein, the term "adeno-associated virus" or "AAV" refers to all adeno-associated viruses for use 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 number of different cell types. The genomic sequences of the various serotypes of AAV as well as sequences of 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. For example, GenBank registration numbers NC_002077 (AAV-1) and AF063497 (AAV-1) may be referred to.

As used herein, the term "serotype" refers to a subdivision of AAV that can be identified by serological or DNA sequencing methods and distinguished by its antigenic properties.

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

In one embodiment, the present invention is a mutant of adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant is at positions 516, 583 and 660 in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein. Provided are mutants of AAV1 capsid protein in which amino acids at one or more positions are substituted.

In one embodiment, the present invention is a mutant of adeno-associated virus serotype 1 (AAV1) capsid protein, wherein the mutant has serine at position 516 replaced with threonine in the amino acid sequence of SEQ ID NO: 1 of the wild-type AAV1 capsid protein. and a mutant of AAV1 capsid protein in which asparagine at position 583 is substituted with aspartic acid and alanine at position 660 is substituted with valine.

In one embodiment, the AAV1 capsid protein mutant according to the present invention comprises or consists of the amino acid sequence represented by SEQ ID NO: 2.

The term wild type used in the present invention refers to a type that is most commonly seen in wild populations among species. For mutant types, wild type refers to the phenotype or individual that is considered to be the underlying. The wild type is also called the normal type as a nickname. Meanwhile, in the present specification, a mutant means a protein, virus, cell, individual, etc. in which a mutated gene appears as a phenotypic change. In addition, in this specification, "mutant" may refer to the gene itself which caused a mutation.

In one embodiment, the present invention includes a nucleic acid encoding a mutant of the AAV1 capsid protein. The nucleic acids of the present invention encode mutants of the AAV1 capsid protein. The nucleic acid of the present invention is produced by replacing at least one base with another base in the base sequence of a nucleic acid encoding AAV1 capsid protein (cap gene). The nucleic acid of the present invention may exist in the form of DNA, but in some cases may be in the form of RNA or a chimera of DNA and RNA. Also, the nucleic acids of the present invention include complementary nucleic acids (eg, cDNA). The nucleic acids of the present invention may be single-stranded or double-stranded, but are preferably double-stranded.

The present invention is not particularly limited as a nucleic acid encoding a mutant of AAV1 capsid protein consisting of the amino acid sequence represented by SEQ ID NO: 2, but in one embodiment, a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 3 is exemplified.

Nucleic acids of the present invention may 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, and the like. Promoter sequences include inducible promoter sequences and constitutive promoter sequences. The control sequence may be native or exogenous to the AAV from which the capsid protein is derived, or may be a natural sequence or a synthetic sequence. Recombinant DNA capable of expressing a mutant of AAV1 capsid protein containing the nucleic acid of the present invention is also included in the present invention.

The recombinant DNA is useful for delivering the nucleic acid of the present invention to cells in vitro, ex vivo and in vivo, and imparting the ability to express a mutant of the AAV1 capsid protein to the cell. In addition, cells to 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 to deliver or introduce the nucleic acid of the present invention into a eukaryotic cell, preferably an animal cell, more preferably a mammalian cell.

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

For example, a packaging plasmid can be constructed by retaining a nucleic acid (cap gene) encoding a mutant of the AAV1 capsid protein of the present invention in a plasmid. The packaging plasmid may further contain any nucleic acid sequence, such as a nucleic acid encoding a replicase (Rep) protein (rep gene). Preferably, the rep gene can add AAV2-derived Rep.

Recombinant DNA containing the nucleic acid of the present invention can also be prepared by replacing at least one base in the PLA2 domain coding region with another base in the nucleic acid sequence of the cap gene loaded in a known packaging plasmid. Although not particularly limited as the above packaging plasmid, a packaging plasmid loaded with a cap gene, preferably a packaging plasmid loaded with a cap gene and a rep gene is exemplified. As an example, in the present invention, a recombinant AAV1 vector represented by SEQ ID NO: 4, which is a packaging plasmid loaded with a nucleic acid (cap gene) encoding a mutant of the AAV1 capsid protein of the present invention and a rep gene, was prepared.

The method of introducing a base substitution into a nucleic acid can be carried out by a known method, and is not particularly limited, but is supplied with a kit using a commercially available reagent, for example, Mutagenesis Basal Kit (TAKARA BIO INC.) This can be achieved by performing PCR according to the instructions of

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

The recombinant AAV vectors of the present invention are useful for gene transfer into target cells. Genes introduced by the recombinant AAV vectors of the present invention are strongly expressed in the target cells.

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

As used herein, "AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to at least one AAV capsid protein (by all capsid proteins of wild-type AAV) and a polynucleotide rAAV vector encapsidated. refers to viral particles composed of If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene 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, as such vectors are contained within rAAV particles.

“Packaging” refers to the series of intracellular events that lead to the assembly and encapsidation of AAV particles.

The AAV "rep" and "cap" genes refer to polynucleotide sequences that encode replication and capsid entry proteins of adeno-associated viruses. AAV rep and cap are referred to herein as AAV "packaging genes".

A “helper virus” for AAV refers to a virus that allows AAV (eg, 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 adenovirus, lepesvirus, and poxviruses such as vaccinia. Adenoviruses include many different subgroups, although adenovirus type 5 of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV) as well as cytomegaloviruses (CMV) and pseudorabies viruses : PRV); They are also available from depositories such as the ATCC.

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

In one embodiment, the AAV1 vector according to the present invention has an improved transduction profile for the target tissue compared to an AAV1 vector comprising a wild-type capsid protein. That is, the AAV1 vector according to the present invention has a clear tissue-targeting ability (eg, tissue tropism).

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

The tropism of an AAV capsid for a particular type of cell or tissue is determined by infecting or transfecting a particular type of cell or tissue, including the AAV1 capsid protein, using standard assays well known in the art, such as those described in the Examples herein. It can be determined by measuring the ability of AAV vector particles to transduce.

That is, "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 occurs after preferential introduction of an AAV vector or virion into a particular cell or tissue type(s) and/or preferential interaction with a cell surface that facilitates entry into a particular cell or tissue type, where appropriate and preferred. Preferably it means the expression (eg transcription and optionally translation) of a sequence carried by an AAV vector or virion in a cell, eg the expression of a heterologous nucleotide sequence(s) in the case of a recombinant virus.

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 means introducing an AAV vector or virion into a cell and transferring the genetic material contained in the AAV vector or virion into the cell to obtain expression from the vector genome. In some, but not all, cases, transduction and enhancement may be correlated.

The AAVs described herein include amino acid modifications in one or more capsid proteins that confer new or enhanced tissue tropism properties. AAV1 variants according to the present invention target lung cells.

As used herein, the term "pulmonary cell tropism" means tropism for lung cells.

In some embodiments, the pulmonary cell tropism of an AAV vector comprising a mutated AAV capsid protein is at least 5%, 10%, 20%, An additional increase of 30%, 40%, 50% or more.

In addition, the present invention includes a pharmaceutical composition comprising the recombinant AAV1 vector. The composition may further include a pharmaceutically acceptable carrier.

The "pharmaceutically acceptable carrier", when combined with the active ingredients of the composition, enables the ingredients to retain biological activity without causing disruptive physiological reactions, such as unintended immune responses, Including any substance. Pharmaceutically acceptable carriers include water, phosphate buffered saline, emulsions such as oil/water emulsions, and wetting agents. Compositions containing these carriers are described in Remington's Pharmaceutical Sciences, current Ed., Mack Publishing Co., Easton Pa. 18042, USA; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., 7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., 3rd ed. Amer. It is formulated by well-known conventional methods such as those used in Pharmaceutical Assoc.

In one embodiment, the pharmaceutical composition according to the present invention may be a pharmaceutical composition for preventing or treating lung diseases, specifically lung cancer, pulmonary fibrosis, or chronic obstructive pulmonary disease.

As used herein, the term "treatment" is performed on a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, or delaying or preventing the progression, development, severity, or recurrence of a disease-related syndrome, complication, symptom, or biochemical sign. Refers to any type of intervention or process or administration of an active agent to a subject.Treatment can be made to a subject with a disease or to a subject without a disease (eg for prophylaxis).

Further, in one embodiment, the present invention includes a method for preventing or treating lung diseases, specifically lung cancer, pulmonary fibrosis, or chronic obstructive pulmonary disease, comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject.

As used herein, "administration" refers to the physical introduction of a therapeutic agent or composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for the antibodies described herein include airway, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, intravitreal, or other parenteral routes of administration, for example by injection or infusion. As used herein, the phrase “parenteral administration” refers to modes of administration other than enteral and topical administration, generally by injection, including, but not limited to, airway, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal , intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, tracheal, subcutaneous, subepidermal, intravitreal, intraarticular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injections and infusions only but not including in vivo electroporation. In a specific embodiment, it was confirmed that the AAV vector of the present invention can deliver genes specifically to lung cells by intratracheal injection in animal experiments using mice.

As used herein, the term "therapeutically effective amount" refers to an agent alone or another drug effective to "treat" a disease or disorder in a subject or to reduce the risk, potential, likelihood, or incidence of a disease or disorder (eg, lung disease). Refers to the amount of a drug in combination with other therapeutic agents. 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 (eg, pulmonary arterial hypertension as disclosed herein). Accordingly, a "therapeutically effective amount" reduces the risk, potential, likelihood, or incidence of a disease or disorder, or provides some relief, relief, and/or reduces at least one indicator (eg, lung disease) and/or An amount that reduces at least one clinical symptom of a disease or disorder.

As used herein, the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, including mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

In addition, the present invention includes a drug delivery system comprising the recombinant AAV1 vector. The composition may further include a known pharmaceutically acceptable carrier for use as described above.

The AAV1 vector of the present invention exhibits tropism to lung cells and has the ability to express genes specifically in lung cells, so it can be used as a delivery vehicle for delivering drugs or genes to lung cells. Specifically, it can be used as an AAV1 vaccine against infectious agents that infect the respiratory tract. Existing vaccines injected through a vein have the disadvantage of spreading throughout the body and reducing the possibility of antibody production in the vicinity of the lungs. It can increase immunity to infectious agents.

The present invention relates to a recombinant AAV1 capsid variant having high-efficiency gene transfer ability by specifically targeting lung cells, and a recombinant viral vector comprising a nucleic acid encoding a mutant of AAV1 capsid protein is delivered to the lungs by intratracheal injection. It is useful for expression of transgenes in cells and is effective in preventing or treating lung diseases.

Figure 1 compares the genomic potency of wild-type AAV1 and the packaging efficiency of the recombinant AAV1 vector of the present invention by quantitative PCR analysis.
Figure 2 shows the results of efficient local delivery into lung cells, in which LacZ expression was confirmed by whole mount β-galactosidase staining in the lungs excised from 8-week-old C57BL/6 male mice, in order to confirm the lung cell specificity of the recombinant AAV1 vector. will be.
Figure 3 confirms LacZ expression in lung cells by the recombinant AAV1 vector through eosin / LacZ staining in order to confirm lung cell specificity.
Figure 4a shows a cleavage map of the recombinant AAV1 vector of the present invention.
Figure 4b shows the pHelper plasmid cleavage map.
Figure 4c shows the pCMV LacZ plasmid cleavage map.

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

[Example]

Example 1: Selection and construction of AAV1 capsid protein variants

1) Selection of AAV1 capsid protein variants

A plasmid pool was created by inducing random point mutations in the cap gene of wild-type AAV variants (AAV1, AAV2, AAV4, AAV6, AAV8, AAV9) using error-prone PCR and inserting random 7mer/9mer into 3-fold protrusion of each serotype. produced. 7-70ng of the AAV plasmid library, 25μg of pBluescript, and 25μg of pHelper were formed in a calcium-phosphate complex and transfected into AAV293 cells to proceed with AAV packaging, and an AAV library pool loaded with the cap gene information of each variant. was produced.

8-week-old C57BL/6 male mice were respiratory anesthetized with isoflurane, and then a 1 cm incision was made in the skin of the airway area. PenWu micro-aerosolizer (BioJane, Shanghai, China) (1.25 length of intratracheal portion, 700μm of outer diameter, 430μm of inner diameter) loaded with 1x10 ¹¹ vg/100 μl of AAV library pool in PBS and passing the needle through the airway , and intratracheal injection was performed.

After one week, 0.9% physiological saline was flowed through the heart to perform perfusion, and the lungs were removed. After homogenization, DNA was extracted from the whole lung using a DNA mini kit (Qiagen), and AAV cap gene-specific forward primer 5'-GCGGAAGCTTCGATCAACTACG-3' (SEQ ID NO: 5), reverse primer ) 5'-CGCAGAGACCAAAGTTCAACTGA-3' (SEQ ID NO: 6) was used to amplify the cap gene of AAV variants showing tropism in the entire lung. The finally amplified cap gene gene, including HindIII and NotI sequences at both ends, was subcloned into the pSub2 plasmid through HindIII/NotI restriction and ligation, electroporated into DH10β, and the plasmid was purified (Qiagen Plasmid Maxi Kit) to lung tissue ( cells) were stored in the form of a plasmid pool. (pSub2 is a plasmid prepared by David Schaffer Lab, UC Berkeley, based on pSub201 (ATCC), and is designed to subclon the cap gene using HindIII and NotI, Reference 1. Narendra Maheshri et al., Nature Biotechnology, 2006; 2. James T Koerber, Nature Protocols, 2006)

2) Construction of recombinant AAV1 vector

① Production of packaging plasmid mutants

In the angiogenic plasmid pool, a number of lung cell-specific cap genes were subcloned into pXX2 (UC Berkeley, David Schaffer Lab) into which HindIII and NotI were introduced for cap gene insertion through HindIII/NotI restriction, resulting in a number of reporter gene loadings. Construction of lung cell specific plasmid mutants was completed.

② Plasmid introduction into AAV293 cells

Construction of AAV1 variants

AAV293 cells were transfected with 17 μg of mutant plasmid, 17 μg of ITR flanked reporter gene (pCMV-GFP or pCMV-LacZ or pCMV-FGF12-IRES-GFP), and 17 μg of pHelper in a calcium-phosphate complex, followed by about 48 hours. After collecting only the cell pellets, AAV inside the cells was extracted through freezing-thawing. Thereafter, cell debris was removed by centrifugation, and nucleic acids from virus-producing cells were removed by incubation with 10 U/mL of benzonase at 37° C. for 30 minutes.

The cleavage map of the prepared AAV1 mutant is shown in FIG. 4a, and the entire base sequence is as follows (SEQ ID NO: 4).

Construction of wild-type AAV1

17 μg of packaging plasmid containing the rep gene of AAV2 and the cap gene of AAV1, 17 μg of ITR flanked reporter gene (pCMV-GFP or pCMV-LacZ or pCMV-FGF12-IRES-GFP), and 17 μg of pHelper form a calcium-phosphate complex. AAV293 cells were transfected, and after about 48 hours, only cell pellets were collected and AAV inside the cells was extracted through freezing-thawing. Thereafter, cell debris was removed by centrifugation, and nucleic acids from virus-producing cells were removed by incubation with 10 U/mL of benzonase at 37° C. for 30 minutes.

3) AAV1 variant purification

The AAV solution was subjected to ultracentrifugation using an iodixanol gradient. Iodixa solutions were prepared in 15%, 25%, 40%, and 54% and sequentially loaded into ultracentrifugation tubes, and the AAV solution was loaded thereon. 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 located in the middle of 54% and 40% iodixanol was extracted, and buffer exchange was performed with a PBS buffer containing 0.01% Tween 20 using an Amicon Ultra-15 Centrifugal Filter (MWCO 100,000).

4) Measurement of AAV1 mutant titer

The titer of wild-type AAV1 and AAV1 mutants carrying CMV-FGF12-IRES-GFP was determined by extracting the viral genome by treating a virus resistant to Dnase I (5U) with proteinase K, and then quantitative PCR (qPCR) for CMV. ) using primers (5-ATGGTGATGCGGTTTTGGCAG-3: SEQ ID NO: 7 and 5-GGCGGAGTTGTTACGACATTTTGG-3: SEQ ID NO: 8), qPCR was performed with each standard for quantification.

The packaging efficiencies of wild-type AAV1 and recombinant AAV1 vectors were compared by genomic titers and shown in FIG. 1 . This means that AAV1 mutants have improved packaging efficiency compared to wild-type AAV1, which means that the organism can be maintained with excellent evolutionary characteristics.

Example 2: Confirmation of Recombinant AAV1 Variant Infection

AAV1 wild-type and AAV1 mutants loaded with CMV-LacZ were individually packaged and injected into 8-week-old C57BL/6 male mice in the form of an intratracheal aerosol (1x10 ¹¹ vg/100 μl).

In order to confirm the lung cell specificity of the recombinant AAV1 vector, LacZ expression was confirmed by whole mount β-galactosidase staining in the lungs extracted 1 week after injection of 8-week-old C57BL/6 male mice, as shown in FIG. It was confirmed that it was specifically delivered locally to lung cells.

In addition, in order to confirm the lung cell specificity of the recombinant AAV1 vector, 8-week-old C57BL/6 male mice were excised 1 week after injection, and LacZ expression in lung cells was confirmed through eosin / LacZ staining. As a result, FIG. As shown in 3, it was confirmed that it was delivered specifically to lung cells.

Claims (10)

As a mutant of the adeno-associated virus serotype 1 (AAV1) capsid protein,
The mutant is an AAV1 capsid protein mutant in which amino acids present at one or more positions among positions 516, 583, and 660 in the amino acid sequence of the wild-type AAV1 capsid protein represented by SEQ ID NO: 1 are substituted.
According to claim 1,
In the mutant, in the amino acid sequence of the wild-type AAV1 capsid protein represented by SEQ ID NO: 1, serine at position 516 is substituted with threonine, asparagine at position 583 is substituted with aspartic acid, and alanine at position 660 is substituted with valine. mutants of the AAV1 capsid protein.
A nucleic acid encoding a mutant of the AAV1 capsid protein of claim 2.
According to claim 3,
The nucleic acid is a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 3.
A recombinant AAV1 vector comprising a nucleic acid encoding a mutant of the AAV1 capsid protein of claim 3 or 4.
According to claim 5,
wherein the AAV1 vector has an improved transduction profile for lung cells when compared to an AAV1 wild-type viral vector.
A pharmaceutical composition comprising the recombinant AAV1 vector according to claim 5.
According to claim 7,
The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
According to claim 7,
The composition is a pharmaceutical composition for the prevention or treatment of lung cancer, pulmonary fibrosis or chronic obstructive pulmonary disease.
A gene delivery system comprising the recombinant AAV1 vector according to claim 5.

Images