Adeno-associated virus deformed complex platform with improved gene expression and reduced genotoxicity.

Abstract

We disclose an adeno-associated virus (AAV) complex platform containing an asymmetrically deformed reverse repeat (ITR). The AAV complex offers advantages such as improved productivity and transgene expression efficiency, as well as reduced genotoxicity, due to the presence of a deformed asymmetric ITR among its two ITRs. We also disclose compositions and gene therapy methods containing the adeno-associated virus complex.

Description

[Technical Field]

[0001] This invention relates to an adeno-associated virus deformation complex platform and its applications. [Background technology]

[0002] Adeno-associated virus (AAV) is a single-stranded DNA virus with a genome size of approximately 4.6 kbp and is a helper-dependent human parvovirus. The genome consists of ITRs at both ends and two ORFs (open reading frames), rep and cap. The N-terminus of the genome codes for the rep gene, which is involved in viral replication and viral gene expression, while the C-terminus codes for the cap gene, which encodes the viral capsid protein. The ITR is involved in the replication of the AAV genome and the packaging of AAV particles.

[0003] On the other hand, AAV gene carriers are non-pathogenic human virus-derived carriers, are safe, do not induce cellular immune responses, and have a broad host range. Furthermore, AAV gene carriers transmit genes to both non-dividing and dividing cells, and are particularly characterized by the long-lasting expression of genes transmitted by AAV gene carriers in vivo.

[0004] However, the aforementioned AAV has a problem in that, due to the inverse repeat sequence (ITR), up to approximately 4.4 kb of the protein coding sequence is capsidized, resulting in a decrease in DNA packaging capacity. Furthermore, when genes inserted in the 3'→5' direction and genes inserted in the 5'→3' direction are expressed on the dielectric of the host cell, there is a competitive relationship between the two strands, which leads to a decrease in the expression rate of the transmitted gene. In addition, there is a possibility of inducing cancer during the process of the transmitted gene being inserted into the dielectric of the host cell.

[0005] Therefore, as a gene carrier, it is necessary to develop an AAV complex in which the ITR is modified within the AAV properties, improving DNA packaging ability, reducing the probability of insertion into the chromosomes of infected cells, while improving productivity and expression efficiency. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] One specific example described herein relates to an adeno-associated virus (AAV) complex comprising a polynucleotide sequence that encodes a transgene between a first inverted terminal repeat (ITR) and a second ITR, wherein in either the first or second ITR, all or part of a stem-loop structure consisting of a rep-binding element (RBE), RBE', A, A', B, B', C, C', and D regions is modified. The adeno-associated virus complex may further include an operablely linked promoter, a polynucleotide sequence that encodes a transgene, and a polyadenylated sequence between the first and second ITRs. The AAV is an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In the adeno-associated virus complex, the first ITR is undeformed, and the second ITR is deformed. The deformation of the stem-loop structure is at least one of insertion, deletion, or substitution. In the adeno-associated virus complex, either the first ITR or the second ITR is deformed so as not to form a stem-loop structure. In the adeno-associated virus complex, either the first ITR or the second ITR is deformed in whole or in part in a stem-loop structure consisting of the RBE, RBE', A, A', B, B', C, C', and D regions. In the adeno-associated virus complex, one of the first ITR and the second ITR contains a trs (terminal resolution site) sequence and an RBE sequence, with all C, C', B', B, RBE', A', and D sequences deleted from after the RBE. The promoter is a tissue-specific promoter. In the adeno-associated virus complex, the transgene is a therapeutic gene.In the adeno-associated virus complex, the transgene may be GFP, Luciferase, TP53, RPE65, TPP1, or FVIII.

[0007] Other specific examples relate to gene therapy methods that include the step of administering an effective dose of the adeno-associated virus complex described herein to an individual in need.

[0008] Further specific examples relate to compositions comprising adeno-associated virus complexes as described herein. These compositions may further comprise a pharmaceutically acceptable carrier. These compositions may be used for therapeutic gene delivery for gene therapy. [Means for solving the problem]

[0009] The specific examples shown in the attached drawings are described below in detail, where the same reference number refers to the same element. In this regard, these specific examples can take many forms and should not be construed as being limited to the descriptions presented herein. Therefore, these specific examples are described solely by reference to the drawings, which are for the purpose of illustrating the described examples. The term "and / or" as used herein includes all any combination of at least one of the related listed items. When an expression such as "at least one of ~" precedes an element list, it modifies the entire list of elements, not the individual elements of the list.

[0010] One specific example concerns adeno-associated virus complexes containing modified reverse repeat sequences.

[0011] Another specific example relates to cells transformed with the adeno-associated virus complex.

[0012] Further specific examples relate to a method of transgene transfer that includes the step of administering an effective amount of the adeno-associated virus complex.

[0013] Still other specific examples relate to a gene therapy method comprising the step of administering an effective amount of the adeno-associated virus complex.

[0014] Still other specific examples relate to a method of treating a disease in an individual, comprising the step of administering an effective amount of an adeno-associated virus complex according to one aspect to an individual who needs it.

[0015] Still other specific examples relate to a composition comprising the adeno-associated virus complex.

[0016] Still other specific examples relate to the use of the adeno-associated virus complex for gene therapy.

[0017] Additional specific examples are described in some of the following explanations, some will become clear from the explanations, or may be learned by implementing the presented specific examples of the present invention.

[0018] In one specific example, an adeno-associated virus (AAV) complex containing a modified inverted terminal repeat (ITR) is disclosed.

[0019] As used herein, the term "adeno-associated virus (AAV)" refers to a single-stranded DNA virus with a genome size of approximately 4.6 kbp and is a helper vector-dependent human parvovirus. The genome consists of inverted terminal repeats (ITRs) at both ends and two open reading frames (ORFs), rep and cap. The N-terminus of the genome encodes the rep gene involved in viral replication and the expression of viral genes, and the C-terminus encodes the cap gene that encrypts the viral capsid protein. The ITRs are involved in the replication of the AAV genome and the packaging of AAV particles. The ITRs contain RBE (rep-binding element), RBE’, A, A’, B, B’, C, C’, and D regions and form a stem-loop (hairpin) structure. The structure of the AAV ITR is widely known in the literature such as Goncalves, M.A. Virology Journal, 2(1):43 (2005), etc., and the said literature is incorporated herein by reference.

[0020] The sequence of the ITR can be based on the ITR sequence of a virus belonging to the genus Dependovirus of the family Parvoviridae.

[0021] The sequence of the ITR can be based on the ITR sequence of AAV. The ITR sequence of AAV is known.

[0022] Examples of AAV include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, etc., and in addition, other AAV serotypes currently known or discovered in the future may also be included. AAV can include known AAV derivatives. AAV can include modified or artificial AAV.

[0023] Therefore, the sequence of the ITR can be based on an ITR sequence of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The sequence of the ITR can be based on an ITR sequence of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. The first ITR and the second ITR can be based on ITR sequences of the same or different AAV serotypes.

[0024] Alternatively, another type of virus belonging to the genus Dependovirus of the family Parvoviridae may be used instead of the aforementioned AAV.

[0025] In this specification, the term "AAV complex" may be used interchangeably with "AAV vector," "AAV carrier," "recombinant AAV," and "recombinant AAV vector."

[0026] In one specific example, the AAV complex includes a heterogeneous polynucleotide sequence between the first ITR and the second ITR.

[0027] The heterogeneous polynucleotide sequence may be operably positioned between the first ITR and the second ITR.

[0028] In one specific example, the genome of the AAV complex includes a first ITR (5'-ITR), a polynucleotide sequence encoding the transgene, and a second ITR (3'-ITR) in the 5' to 3' direction.

[0029] In a particular example, the AAV complex may include a polynucleotide sequence that encodes a transgene between the first ITR and the second ITR, and in either the first ITR or the second ITR, all or part of a stem-loop structure consisting of RBE, RBE', A, A', B, B', C, C', and D regions may be modified.

[0030] In a specific example, the AAV complex may include a promoter operably linked between the first ITR and the second ITR, a polynucleotide sequence encoding a transgene, and a polyadenylated sequence.

[0031] In a specific example, the genome of the AAV complex includes a first ITR (5'-ITR), a promoter, a polynucleotide sequence encoding the transgene, a polyadenylated sequence, and a second ITR (3'-ITR) in the 5'-3' direction.

[0032] In certain additional examples, the AAV complex comprises a promoter operably linked between a first ITR and a second ITR, a polynucleotide sequence encoding a transgene, and a polyadenylation sequence, wherein in either the first ITR or the second ITR, all or part of a stem-loop structure consisting of RBE, RBE', A, A', B, B', C, C', and D regions is modified.

[0033] In certain specific examples, the AAV complex may include asymmetrically deformed ITRs. In one specific example, the AAV complex has a deformed first ITR and / or second ITR. In another specific example, the AAV complex has a deformed first ITR and an undeformed second ITR. In yet another specific example, the AAV complex has an undeformed first ITR and a deformed second ITR. In yet another specific example, the AAV complex has an undeformed 5'-ITR on the (+) strand of the transgene and a deformed 3'-ITR on the (+) strand. In yet another specific example, the AAV complex has a deformed 5'-ITR on the (-) strand of the transgene and an undeformed 3'-ITR on the (-) strand. In other words, a deformed second ITR means that the 3'-ITR on the (+) strand and / or the 5'-ITR on the (-) strand of the transgene are deformed.

[0034] The AAV complex described herein, by including an asymmetrically modified ITR, exhibits improved productivity, increased transgene expression rates, and reduced genotoxicity, making it suitable for use as an AAV transduction platform for transmitting diverse genes into target cells. Furthermore, the AAV complex described herein can be used as a target transduction medium for short-term, highly efficient transgene expression while suppressing prolonged transgene expression in host cells.

[0035] Of the first and second ITRs, the non-modified ITRs are wild-type ITRs. Of the first and second ITRs, the non-modified ITRs may be AAV wild-type ITRs.

[0036] In certain specific examples, the non-deformed ITR among the first and second ITRs may be a functional derivative possessing substantially the same functional properties as the wild-type ITR (e.g., AAV wild-type ITR).

[0037] In this specification, the term "functional derivative" means a derivative having substantially the same functional properties. The derivative means a similar compound obtained by chemically altering part of the structure of a compound. The derivative means a compound in which a hydrogen atom or a specific group of atoms is substituted by another atom or group of atoms. Methods for producing derivatives of a compound while maintaining substantially the same functional properties are known in the art.

[0038] In a specific example, in either the first ITR or the second ITR, all or part of the stem-loop structure consisting of regions RBE, RBE', A, A', B, B', C, C', and D may be deformed.

[0039] In one specific example, the deformation of the stem-loop structure (hairpin structure) may be selected from insertion, deletion, and replacement.

[0040] In one specific example, the deformation of the stem-loop structure (hairpin structure) includes deformation that includes a single stem and a single loop. For example, the deformed ITR may include the removal of the B-B' arm so that the C-C' arm remains, or the removal of the C-C' arm so that the B-B' arm remains.

[0041] In one specific example, the deformation of the stem-loop structure (hairpin structure) includes deformation to have a single stem instead of two loops. For example, the deformed ITR may include the absence of B-B' and C-C' arms.

[0042] In one specific example, the deformed ITR may include a deletion of the C' region such that the severed C loop and B-B' arm remain. Similarly, the deformed ITR may include a deletion of the B region such that the severed B loop and C-C' arm remain.

[0043] In one specific example, the modified ITR may include base pair deletions in at least one of the C, C', B, or B' portions such that complementary base pairing occurs between the C and B' portions and between the C' and B portions, generating a single arm.

[0044] In one specific example, the modified ITR may include one, two, three, four, five, or six nucleotide modifications (e.g., deletion, substitution, or addition) in at least one region selected between A' and C, C and C', C' and B, B and B', and B' and A.

[0045] In one specific example, the deformation of the stem-loop structure (hairpin structure) may include deformation of the structural elements. Specifically, deformation of the structural elements may include changes in the height of the stem and / or changes in the number of nucleotides in the loop. For example, the height of the stem may be approximately 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range of nucleotides within that number. In yet another example, the loop may have approximately 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range of nucleotides within that number.

[0046] In other specific examples, the interval between two elements (e.g., RBE and hairpin, not limited to) can be altered (e.g., increased or decreased) to change the functional interaction with the larger Rep protein. For example, the interval can be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides or more, or any range of nucleotides within the aforementioned number.

[0047] In one specific example, either the first ITR or the second ITR is deformed so as not to form a stem-loop structure (hairpin structure). In another specific example, the first ITR is deformed so as not to form a stem-loop structure. In yet another specific example, the second ITR is deformed so as not to form a stem-loop structure. In yet another specific example, the first ITR may not be deformed, and the second ITR may be deformed so as not to form a stem-loop structure.

[0048] The expression "deformed so as not to form a stem-loop sequence (hairpin structure)" means that the ITR's structure is deformed by the alteration of its sequence so that it does not form a stem-loop structure (hairpin structure) but exists as an open-end or free-end. In one embodiment, the AAV complex can suppress the formation of circular dimers and circular concatemers in infected cells and inhibit the integration of AAV into the host genome observed in AAV, by deforming either the first ITR or the second ITR so as not to form a stem-loop structure (hairpin structure). Furthermore, this deformation can increase the productivity of the AAV complex and the expression rate of the RUNX3 gene.

[0049] In one specific example, either the first ITR or the second ITR is deformed into a blunt end. In another specific example, either the first ITR or the second ITR is deformed into a sticky end. In yet another specific example, the second ITR is deformed into a blunt end or a sticky end. In yet another specific example, the second ITR is deformed into a blunt end. The deformation to a blunt end or a sticky end can be performed by a person of ordinary skill using methods known to the art.

[0050] In one specific example, all or part of the stem-loop structure consisting of the RBE, RBE', A, A', B, B', C, C', and D regions is deleted in either the first ITR or the second ITR. In another specific example, part of the stem-loop structure consisting of the RBE, RBE', A, A', B, B', C, C', and D regions is deleted in either the first ITR or the second ITR. In yet another specific example, either the first ITR or the second ITR contains the trs (terminal resolution site) sequence and the RBE sequence, and all of the C, C', B', B, RBE', A', and D sequences are deleted from RBE onward. In other specific examples, the first ITR remains unchanged, while the second ITR includes the trs sequence and the RBE sequence, with all of the C, C', B', B, RBE', A', and D sequences deleted from RBE onward.

[0051] In other specific examples, either the first ITR or the second ITR contains, is required to contain, or may contain, any one nucleotide sequence or its complementary sequence from sequence numbers 1 through 9. In other specific examples, the first ITR remains unchanged, and the second ITR contains, is required to contain, or may contain, any one nucleotide sequence or its complementary sequence from sequence numbers 1 through 9.

[0052] The sequence of sequence number 1 can be based on the AAV2 ITR sequence.

[0053] The sequence of sequence number 2 can be based on the AAV1 ITR sequence.

[0054] The sequence of sequence number 3 can be based on the AAV3 ITR sequence.

[0055] The sequence of sequence number 4 can be based on the AAV4 ITR sequence.

[0056] The sequence of sequence number 5 can be based on the AAV6 ITR sequence.

[0057] The sequence of sequence number 6 can be based on the AAV7 ITR sequence.

[0058] The sequence of sequence number 7 can be based on the AAV5 ITR sequence.

[0059] The sequence of sequence number 8 can be based on the AAV8 ITR sequence.

[0060] The sequence of sequence number 9 can be based on the AAV9 ITR sequence.

[0061] In specific examples, the sequences of sequence numbers 1 to 9 are obtained by partially deleting the AAV ITR sequence. The sequences of sequence numbers 1 to 9 include the trs sequence and the RBE sequence within the AAV ITR sequence. The sequences of sequence numbers 1 to 9 are obtained by completely deleting the C, C', B', B, RBE', A', and D sequences from RBE onwards.

[0062] Generally, RNA polymerases produce mRNA with a sequence complementary to the target gene in the promoter region. This process is called "transcription," and it proceeds in the 5' to 3' direction. On the other hand, when inserting a target gene into an AAV complex, the gene is inserted into the double helix of the AAV complex DNA in the 5' to 3' direction and the 3' to 5' direction, respectively. Therefore, while the transcription process of the target gene is progressing, the AAV complex DNA double helix causes transcription of the target gene in both directions, leading to interference and a decrease in the expression efficiency of the target gene. However, in one embodiment of the AAV complex, by modifying the stem-loop structure, specifically by deleting all or part of the stem-loop structure, interference from the double helix structure of the AAV complex DNA during the transcription of the target gene can be avoided, thereby improving the expression efficiency of the target gene.

[0063] For example, in an AAV complex containing asymmetrically deformed ITRs, the first ITR may remain undeformed, while the second ITR may be deformed in a way that prevents the formation of a stem-loop structure. As a result, the 5'-ITR on the (-) strand of the transgene does not form a hairpin structure, and transcription of the transgene proceeds complementary to that strand in the 5'-3' direction. On the other hand, the 3'-ITR on the (+) strand of the transgene does not form a hairpin structure, and transcription of the transgene does not proceed on that strand. In other words, transcription of the transmission gene proceeds only in the 5'-3' direction, and the competitive factor in the 3'-5' direction is removed, thus increasing the efficiency of gene expression.

[0064] In one embodiment, an AAV complex (pAAV-GC ITR vector) was produced in which an asymmetrically deformed ITR was created by partially modifying the hairpin structure of the second ITR among the wild-type AAV ITRs contained in the vector, using an AAV vector containing diverse transgenes. The produced AAV complex was compared with an AAV complex in which the hairpin structure was not deformed (pAAV-WT ITR vector) and an AAV complex in which both ends of the hairpin structure were deformed to be symmetrical (pAAV-BC del.ITR vector), and the productivity, transgene expression rate, and genotoxicity of each were confirmed. As a result, it was confirmed that the AAV complex containing the asymmetrically deformed ITR (pAAV-GC ITR vector) showed a significant increase in viral productivity and transgene expression rate, and very low genotoxicity, compared with the AAV complex in which the ITR was not deformed (pAAV-WT ITR vector) and the AAV complex containing the symmetrically deformed ITR (pAAV-BC del.ITR vector).

[0065] In this specification, the term "promoter" refers to a site that regulates the transcription of a gene. The promoter is operablely ligated to the coding sequence of a transgene. The promoter is either a tissue-specific promoter or an inducible promoter. The tissue-specific promoter is not limited to any type of promoter that induces the specific expression of a gene in a specific type of cell or tissue in vivo. The tissue-specific promoter can be appropriately selected to be specific to the target tissue so that the transgene is expressed according to the type of transgene. Non-restrictive examples of the aforementioned tissue-specific promoters include liver-specific TBG (thyroxin binding globulin) promoters, insulin promoters, glucagon promoters, somatostatin promoters, PPY (pancreatic polypeptide) promoters, Syn (synapsin-1) promoters, creatine kinase (MCK) promoters, mammalian desmin (DES) promoters, A-MHC (α-myosin heavy chain) promoters, cTnT (cardiac troponin T) promoters, and SPC (Surfactant Protein C) promoters.

[0066] The aforementioned heterogeneous polynucleotides may encode a transgene.

[0067] In this specification, the term "transgene" refers to a gene that has been transmitted from one organism to another, either naturally or through various genetic engineering techniques. The term "transgene" may be used interchangeably with "transfer gene" and "target gene."

[0068] The aforementioned transgene is not limited to a specific type, as long as it is the target gene to be introduced into the host cell. In specific examples, the AAV complex can be used as an AAV transmission platform for transmitting diverse transgenes, as it offers the advantages of improved AAV productivity, improved transgene expression rates, and reduced genotoxicity, regardless of the type of transgene.

[0069] In one specific example, the introduced gene is a therapeutic gene. When the introduced gene is a therapeutic gene, the AAV complex according to one embodiment can be used as a gene therapy agent. Therefore, the AAV complex can be an AAV vector for gene therapy.

[0070] In one specific example, the transgene may be, but is not limited to, GFP, Luciferase, TP53, RPE65, TPP1, or FVIII. When the transgene is TP53, the AAV complex for transmitting the transgene can be used as a gene therapy agent for anti-cancer treatment. When the transgene is RPE65, the AAV complex for transmitting the transgene can be used as a gene therapy agent for inherited retinal disease (IRD). When the transgene is TPP1 (CKN2), the AAV complex for transmitting the transgene can be used as a gene therapy agent for Batten disease. When the transgene is FVIII, the AAV complex for transmitting the transgene can be used as a gene therapy agent for hemophilia.

[0071] The aforementioned introduced gene may be of human or animal origin.

[0072] In one specific example, the AAV complex may further include post-transcriptional regulatory elements. The AAV complex may include a first ITR in the 5'-to-3' direction, a promoter sequence, a polynucleotide sequence encoding the transgene, a post-transcriptional regulatory element, a polyadenylated sequence, and a second ITR.

[0073] The aforementioned post-transcriptional regulatory element may include WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

[0074] In one specific example, the AAV complex may further include a gene junction between the promoter and the polynucleotide sequence encoding the transgene.

[0075] In this specification, the term "gene junction" refers to an undefined sequence between the promoter end and the start of the target gene sequence. Specifically, a promoter is a site where transcription machinery complexes bind to regulate a gene, and the boundary between the known promoter sequence end boundary and the gene start site whose expression is to be regulated is generally ambiguous. Therefore, optimization of the junction is necessary to establish a successful promoter-gene expression relationship. The sequence of the gene junction can be appropriately selected by conventional methods by those skilled in the art.

[0076] The AAV complex may be manipulated to encode a sorting marker or reporter that provides selection or confirmation of contaminated cells. The sorting marker or reporter is known to the art. Non-limiting examples of sorting markers include genes that provide resistance to ampicillin, streptovidine, kanamycin, hygromycin, etc. Non-limiting examples of reporters include luciferase, green fluorescent protein (GFP), etc.

[0077] We also disclose cells transformed with adeno-associated virus complexes.

[0078] The specific components of the adeno-associated virus complex are as described above.

[0079] In this specification, the term "transformation" refers to the change in the genetic properties of an organism due to the introduction of DNA from an external source. Transformation is the phenomenon in which DNA, a type of nucleic acid extracted from cells of one strain of an organism, is introduced into living cells of another strain, causing the DNA to enter those cells and altering their genetic traits. In other words, "transformation" means making it possible to introduce genes into host cells and express them within those host cells.

[0080] The methods for introducing the AAV complex described herein into cell lines and transforming them include, but are not limited to, methods known in the art, such as transient transfection using lipofectamine, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, and electroporation. Preferably, transformation can be performed using the lipofectamine 2000 reagent.

[0081] In other specific examples, a method for transmitting a transgene to an individual is disclosed, which includes the step of administering an effective amount of an adeno-associated virus complex according to one embodiment to an individual in need.

[0082] Further specific examples include gene therapy methods that involve administering an effective dose of adeno-associated virus complex to an individual in need.

[0083] Further specific examples include methods for treating a disease in an individual, which involve administering an effective dose of adeno-associated virus complexes to the individual in need.

[0084] In the method described above, the specific contents of the adeno-associated virus complex are as stated above.

[0085] In the methods described above, the AAV complex is administered to the individual either on its own or the AAV complex is formulated into a dosage form that can be administered to the individual and then administered to the individual. In one specific example, the AAV complex may be administered to the individual in the form of a composition containing the AAV complex. For example, the AAV complex may be administered to the individual in a dosage form of a composition containing the AAV complex and a pharmaceutically acceptable carrier.

[0086] The individual is one that requires the expression of a transgene transmitted by the AAV complex. The individual is one that suffers from or is likely to suffer from a disease to which gene therapy is applicable. The individual is one that suffers from or is likely to suffer from a disease that is treatable by the expression of a transgene transmitted by the AAV complex. The individual may be a mammal, such as a human, cattle, horse, pig, dog, sheep, goat, or cat.

[0087] In this specification, the term "gene therapy" means a treatment that utilizes genes to treat or prevent a disease. AAV vectors for delivering therapeutic genes into cells can be used as gene therapy agents. Diseases to which gene therapy is readily applicable include, but are not limited to, diseases caused by the deletion of a single gene. Non-limiting examples of diseases to which gene therapy is applicable include cancer, cardiovascular diseases, genetic disorders such as inherited retinal dystrophy (IRD), Batten disease, haemophilia, cystic fibrosis, muscular dystophy, thalassemia, and sickle cell anemia, neurological disorders, infectious diseases (such as acquired immunodeficiency), and joint diseases.

[0088] The cancer may be one in which tumor suppressor genes are inactivated. The cancer may be, for example, a KRAS-mutated cancer. The cancer may be, for example, a KRAS-mutated solid tumor. The cancer may be, for example, a KRAS-mutated lung cancer. In certain specific cases, if the activity of the tumor suppressor genes is restored, cancer cells are eliminated and normal cells are present, making treatment of KRAS-mutated lung cancer possible. The tumor suppressor genes may be, for example, sPD-1, VHL, MMAC1, DCC, p53, NF1, WT1, RB, BRAC1, BRAC2, and RUNX3 genes.

[0089] In one specific example, the lung cancer is either non-small cell lung cancer or small cell lung cancer. Non-small cell lung cancer includes, for example, squamous cell carcinoma, large cell carcinoma, and lung adenocarcinoma.

[0090] Therefore, in specific cases, the AAV complex can be used in gene therapy because it can activate a specific gene in a disease caused by reduced activity of that gene, thereby preventing or treating the disease.

[0091] In this specification, the term "prevention" means all actions that suppress or delay the onset of a disease by administering the AAV complex. The term "treatment" means all actions that improve or beneficially modify the symptoms of a disease by administering the AAV complex.

[0092] In a particular example, the method may further include the step of administering a second active ingredient to the individual. The second active ingredient is an active ingredient for the prevention or treatment of a disease to be treated. The active ingredient may be administered simultaneously with, separately from, or sequentially with the AAV complex.

[0093] In certain specific examples, the AAV complex can be formulated as an injection suitable for administration by any appropriate route, such as intravenously, intraarterially, subcutaneously, intradermally, intraperitoneally, intramuscularly, intraarticularly, or intrathecally, and can be administered to an individual. The AAV complex can be administered systemically or locally, either alone or in combination with other pharmaceutically active compounds.

[0094] The desirable dosage of the AAV complex varies depending on the patient's condition and body weight, the degree of the disease, the drug form, the administration route, and the duration, but can be appropriately selected by those skilled in the art. In one specific example, the dosage of the AAV complex is about 1.0×10 6 vg / kg to about 1.0×10 16 vg / kg, about 1.0×10 8 vg / kg to about 1.0×10 16 vg / kg, about 1.0×10 10 vg / kg to about 1.0×10 16 vg / kg, about 1.0×10 10 vg / kg to about 1.0×10 14 vg / kg, about 1.0×1十 12 vg / kg to about 1.0×10 14 vg / kg, for example, about 1.0×10 12 vg / kg, about 1.0×10 13 vg / kg, about 1.0×10 14 vg / kg. In certain specific examples, the dosage of the AAV complex is about 1.0×10 13 vg / kg. The administration can be once a day, multiple times a day, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

[0095] As used herein, the term "about" can be used to include a range of ±10% of the specified numerical value.

[0096] Other aspects relate to compositions containing adeno-associated virus complexes.

[0097] Another aspect relates to the use of the adeno-associated virus complex for gene therapy.

[0098] The specific details of the adenovirus complex and gene therapy are as described above.

[0099] In a particular example, the composition is a gene therapy composition. The composition is a therapeutic gene delivery composition for gene therapy. The composition is a pharmaceutical composition. The composition may further comprise a pharmaceutically acceptable carrier. The carrier comprises excipients, diluents, or adjuvants. As the carrier, a carrier suitable for delivering the AAV complex into a living organism can be used. Specifically, the carrier may be selected to be suitable for formulation into a parenteral dosage form (e.g., an injectable preparation). For example, the carrier may be selected to be suitable for formulation into an intravenous injection preparation. The carrier may be an aqueous solution, such as water or a buffered saline solution.

[0100] In certain specific examples, the composition may be prepared in any dosage form by conventional methods. The composition may be formulated into a form suitable for delivering an AAV vector to an individual. The composition may be formulated in an aqueous solution, for example, in water or buffered saline solution. The composition may be formulated, for example, as a parenteral dosage form (e.g., an injection, e.g., bolus injection or continuous infusion). In one specific example, the pharmaceutical composition may be formulated into an injectable form suitable for administration by any suitable route, such as intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, or intravertebral cavity. In certain specific examples, the composition may be formulated to be administered by intravenous or subcutaneous injection. The composition may also be manufactured in systemic or topical dosage forms. The composition may be provided as a unit dose form, such as an ampoule, pre-filled syringe, small injectable container, or multi-dose container with preservatives added.

[0101] In specific examples, when the composition is used for gene therapy for a specific disease, it may further contain a second therapeutic agent that has a preventive or therapeutic effect on that disease. The pharmaceutical composition may be a single composition or individual compositions.

[0102] In certain specific examples, the composition may contain the AAV complex in an effective amount. The term “effective amount” means an amount sufficient to produce the desired preventive or therapeutic effect when administered to an individual requiring prevention or treatment of a disease. The effective amount can be appropriately selected by those skilled in the art depending on the cell or individual. The effective amount may be determined depending on the severity of the disease, the patient’s age, weight, health, sex, the patient’s sensitivity to the drug, the time of administration, the route of administration, the elimination ratio, the duration of treatment, factors including drugs compounded or used in combination with the composition used, and other factors well known in the medical field. In one specific example, the composition may contain about 1.0 × 10 6 vg / kg or approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 8 vg / kg or approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 10 vg / kg or approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 10 vg / kg or approximately 1.0 × 10 14 vg / kg, approximately 1.0×10 12 vg / kg or approximately 1.0 × 10 14 vg / kg, for example, approximately 1.0 × 10⁻⁶ 12 vg / kg, approximately 1.0×10 13 vg / kg, approximately 1.0×10 14 The AAV complex may contain a dose of vg / kg. In a specific example, the pharmaceutical composition may contain approximately 1.0 × 10⁻⁶ 13 The administration may include an AAV complex in a dose of vg / kg. The administration may be once daily, multiple times daily, or once a week, once every two weeks, once every three weeks, or once every four weeks or once a year. [Effects of the Invention]

[0103] One embodiment of the AAV complex, having an asymmetric ITR in which one of the two ITRs is deformed, offers advantages such as increased productivity and transgene expression efficiency, and reduced genotoxicity, and can therefore be used as an AAV transduction platform.

[0104] Specifically, one embodiment of the AAV complex has the following advantages:

[0105] 1. Deletion of any one of the 1.2 ITRs partially omits the self-renewal step in the host cell (or packaging cell), increasing the efficiency of packaging to viral particles within the same production period, thus increasing self-renewal efficiency and improving the productivity of the AAV complex compared to the existing AAV complex.

[0106] 2. The expression rate (expression level) of the transgene is higher compared to typical wild-type (WT) AAV.

[0107] 3. The dielectric of WT AAV forms a concatemer structure essential for the recombination of the transgene within infected cells. Specifically, it has been reported that the transgene is inserted into the host cell's chromosome by forming a concatemer structure, which is a multimer in which multiple identical dielectrics are linked together. On the other hand, one embodiment of the AAV complex has an asymmetric deformed ITR, and therefore does not form a concatemer structure, thus suppressing the insertion of the transgene into the host chromosome (i.e., recombination). Therefore, it has the advantage of reducing genotoxicity.

[0108] 4. An AAV complex having an asymmetrically deformed ITR according to one embodiment is an AAV transmission platform with improved productivity and transgene expression rates and reduced genotoxicity, and can be used to transmit a variety of genes into target cells. [Brief explanation of the drawing]

[0109] Specific examples, features, and advantages of the present invention will become even clearer from the following description, along with the accompanying drawings.

[0110] [Figure 1] This shows a cleavage map of an adeno-associated virus (AAV) vector according to one embodiment.

[0111] [Figure 2] The structure of an AAV vector according to one embodiment is shown.

[0112] [Figure 3A] This is a schematic genome diagram of the AAV vector containing the asymmetrically deformed ITR from Example 1.

[0113] [Figure 3B] This is a schematic genome diagram of the AAV vector containing an ITR that is not symmetrically deformed, as in Comparative Example 1.

[0114] [Figure 3C] This is a schematic genome diagram of the AAV vector containing the symmetrically deformed ITR of Comparative Example 2.

[0115] [Figure 4] These are Western blot results confirming the AAV productivity of Example 1 (GC), Comparative Example 1 (WT), and Comparative Example 2 (BC-del) for GFP gene expression.

[0116] [Figure 5] These are the qPCR viral genome quantification results confirming AAV productivity for GFP gene expression in Example 1 (pAAV-GC), Comparative Example 1 (pAAV-WT), and Comparative Example 2 (pAAV-BC del).

[0117] [Figure 6]This is a qPCR viral genome quantification result confirming the AAV productivity of pAAV-GC vectors, pAAV-WT vectors, and pAAV-BC del vectors for the expression of various genes (GFP, Luciferase, TP53, RPE65, TPP1, or FVIII).

[0118] [Figure 7] This is a Western blot analysis of the expression of the transgene GFP in the AAV complex, based on the ITR type (ITR WT, ITR BC del, or ITR GC).

[0119] [Figure 8] This shows the relative expression rate (%) of transgenes in the AAV complex according to the ITR type (pAAV-GC, pAAV-BC del, or pAAV-WT).

[0120] [Figure 9A] The results of fluorescence microscopy imaging of GFP expression at passage numbers P3 and P10 are shown.

[0121] [Figure 9B] Table 4, which quantifies the percentage of GFP-expressing cells, is shown in a diagram.

[0122] [Figure 10] Table 5, which quantifies the relative gene expression levels in P7, is shown in a diagrammatic form.

[0123] [Figure 11] The PCR amplification results confirming the presence or absence of concatemer formation of the transgene in host cells are shown. [Modes for carrying out the invention]

[0124] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided only to further facilitate understanding of the present invention and do not limit the scope of the present invention. [Examples]

[0125] Examples

[0126] Example 1. Production of an adeno-associated virus complex for GFP gene expression containing an asymmetrically deformed ITR.

[0127] 1-1. Production of recombinant adeno-associated virus vectors into which the GFP gene has been introduced

[0128] A recombinant adeno-associated virus (AAV) vector into which the GFP gene was introduced was manufactured. Specifically, the CBA-GFP (Chicken Beta-Actin promoter-GFP) gene, cloned into the CS4-GFP vector (Chungbuk National University Institute of Oncology), was PCR amplified using the primers shown in Table 1 below to clone it into the AAV2 vector. These primers were prepared by synthesizing the restriction enzymes NdeI and HindIII. After treating the amplified CBA-GFP DNA with the restriction enzymes NdeI and HindIII, it was cloned into the NdeI-HindIII position of the MCS (Multi Cloning Site) of a wild-type adeno-associated virus (AAV) 2 empty vector using T4 DNA ligase to obtain the AAV2-CBA-GFP plasmid. The ampicillin resistance gene was removed from the AAV2-CBA-GFP plasmid using the BspHI restriction enzyme sites located at both ends of the ampicillin resistance gene. A kanamycin resistance gene was inserted into the site where the ampicillin resistance gene had been removed. The kanamycin resistance gene was recombined using the PCR primers shown in Table 1 below.

[0129] [Table 1]

[0130] 1-2. Deformation of ITR structure

[0131] Site-directed mutagenesis was induced to deform a portion of the hairpin structure of the second ITR, one of the AAV2 wild-type reverse repeat sequences (ITRs) contained in the vector prepared in Example 1-1. Specifically, using the 5'-phosphorylation primers shown in Table 2 below, all or one or more of the C, C', B', B, RBE', A', and D sequences were deleted from after the RBE of the 5'-direction ITR of the (-) strand of the GFP gene in the AAV2 wild-type ITR contained in the vector. For example, all of the C, C', B', B, RBE', A', and D sequences were deleted from after the RBE of the 5'-direction ITR of the (-) strand of the GFP gene. As a result, the second ITR was deformed so that it did not form a hairpin structure. Consequently, an adenovirus complex for GFP gene expression was obtained containing an asymmetrically deformed ITR, in which the first ITR was not deformed and the second ITR was deformed.

[0132] As in Example 1, the AAV vector containing the asymmetrically deformed ITR was named pAAV-GC ITR (abbreviated as pAAV-GC).

[0133] Figure 1 shows a crack map of an AAV vector according to one embodiment.

[0134] Figure 2 shows the structure of an AAV vector according to one embodiment.

[0135] Figure 3A is a schematic genome diagram of the AAV vector containing the asymmetrically deformed ITR from Example 1.

[0136] [Table 2]

[0137] Example 2. Preparation of an AAV vector for Luciferase gene expression containing an asymmetrically deformed ITR.

[0138] An AAV complex for Luciferase gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the Luciferase gene (2,387 bp, GenBank accession no. M15077.1) was introduced instead of the GFP gene.

[0139] Example 3. Preparation of an AAV vector for TP53 gene expression containing an asymmetrically deformed ITR.

[0140] An AAV complex for TP53 gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the TP53 gene (2,512 bp, GenBank accession no. NM_000546.6) was introduced instead of the GFP gene.

[0141] Example 4. Preparation of an AAV vector for RPE65 gene expression containing an asymmetrically deformed ITR.

[0142] An AAV complex for RPE65 gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the RPE65 gene (2,605 bp, GenBank accession no. NM_000329.3) was introduced instead of the GFP gene.

[0143] Example 5. Preparation of an AAV vector for TPP1 gene expression containing an asymmetrically deformed ITR.

[0144] An AAV complex for TPP1(CLN2) gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the TPP1(CLN2) gene (1,693 bp, GenBank accession no. NM_000391.4) was introduced instead of the GFP gene.

[0145] Example 6. Preparation of an AAV vector for FVIII gene expression containing an asymmetrically deformed ITR.

[0146] An AAV complex for FVIII gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the FVIII gene (673 bp, GenBank accession no. NM_000132.4) was introduced instead of the GFP gene.

[0147] Comparative Example

[0148] Comparative Example 1. Preparation of an AAV complex for GFP gene expression containing an undeformed, symmetrical ITR.

[0149] An AAV composite containing an undeformed, symmetrical ITR was fabricated using the same method as in Example 1-1, except that the hairpin structure of the ITR was not deformed.

[0150] An AAV vector containing a symmetrical ITR that is not deformed, as in Comparative Example 1, was named pAAV-WT ITR (abbreviated as pAAV-WT).

[0151] Figure 3B is a schematic genome diagram of the unmodified, symmetrical ITR-containing AAV vector of Comparative Example 1.

[0152] Comparative Example 2. Production of an AAV complex for GFP gene expression containing a symmetrically deformed ITR.

[0153] An AAV complex for GFP gene expression was produced using the same method as in Example 1-2, except that the C-C'-B'-RBE sequence was deleted from both ends of the ITR of the AAV complex produced in Example 1-1.

[0154] As shown in Comparative Example 2, an AAV vector containing a symmetrically deformed ITR was named pAAV-BC del.ITR (abbreviated as pAAV-BC del).

[0155] Figure 3C is a schematic genome diagram of the AAV vector containing the symmetrically deformed ITR of Comparative Example 2.

[0156] Comparative Example 3. Production of an AAV complex for Luciferase gene expression containing an undeformed, symmetrical ITR.

[0157] An AAV complex for Luciferase gene expression containing an unmodified, symmetrical ITR was prepared using the same method as in Comparative Example 1, except that the Luciferase gene was introduced instead of the GFP gene.

[0158] Comparative Example 4. Production of an AAV complex for Luciferase gene expression containing a symmetrically deformed ITR.

[0159] An AAV complex for Luciferase gene expression containing a symmetrically modified ITR was prepared using the same method as in Comparative Example 2, except that the Luciferase gene was introduced instead of the GFP gene.

[0160] Comparative Example 5. Preparation of an AAV complex for TP53 gene expression containing an undeformed, symmetrical ITR.

[0161] An AAV complex for TP53 gene expression containing an unmodified, symmetrical ITR was prepared using the same method as in Comparative Example 1, except that the TP53 gene was introduced instead of the GFP gene.

[0162] Comparative Example 6. Preparation of an AAV complex for TP53 gene expression containing a symmetrically deformed ITR.

[0163] An AAV complex for TP53 gene expression containing a symmetrically modified ITR was prepared using the same method as in Comparative Example 2, except that the TP53 gene was introduced instead of the GFP gene.

[0164] Comparative Example 7. Preparation of an AAV complex for RPE65 gene expression containing an undeformed, symmetrical ITR.

[0165] An AAV complex for RPE65 gene expression containing an unmodified, symmetrical ITR was prepared using the same method as in Comparative Example 1, except that the RPE65 gene was introduced instead of the GFP gene.

[0166] Comparative Example 8. Preparation of an AAV complex for RPE65 gene expression containing a symmetrically deformed ITR.

[0167] An AAV complex for RPE65 gene expression containing a symmetrically modified ITR was prepared using the same method as in Comparative Example 2, except that the RPE65 gene was introduced instead of the GFP gene.

[0168] Comparative Example 9. Preparation of an AAV complex for TPP1 gene expression containing an undeformed, symmetrical ITR.

[0169] An AAV complex for TPP1 gene expression containing an unmodified, symmetrical ITR was prepared using the same method as in Comparative Example 1, except that the TPP1 gene was introduced instead of the GFP gene.

[0170] Comparative Example 10. Preparation of an AAV complex for TPP1 gene expression containing a symmetrically deformed ITR.

[0171] An AAV complex for TPP1 gene expression containing a symmetrically modified ITR was prepared using the same method as in Comparative Example 2, except that the TPP1 gene was introduced instead of the GFP gene.

[0172] Comparative Example 11. Preparation of an AAV complex for FVIII gene expression containing an undeformed, symmetrical ITR.

[0173] An AAV complex for FVIII gene expression containing a non-modified ITR was prepared using the same method as in Comparative Example 1, except that the FVIII gene was introduced instead of the GFP gene.

[0174] Comparative Example 12. Preparation of an AAV complex for FVIII gene expression containing a symmetrically deformed ITR.

[0175] An AAV complex for FVIII gene expression containing a symmetrically modified ITR was prepared using the same method as in Comparative Example 2, except that the FVIII gene was introduced instead of the GFP gene.

[0176] Experimental example

[0177] Experimental Example 1. Confirmation of the productivity of the AAV complex.

[0178] The productivity of recombinant AAV complexes was confirmed using a specific example.

[0179] Specifically, the AAV complexes prepared in Examples 1 to 6 and Comparative Examples 1 to 12 were used to plasma-infect 293T cells, which are human embryonic kidney (HEK) cells. The molar ratio of the HEK293 cell line:REP / CAP plasmid (Agilent Corporation):AAV complex was 1:1:1, and the standard AAV production method was followed. The AAV produced from the HEK293 lysate was obtained by ultracentrifugation, and the amounts of viral protein (VP) and viral genome were quantified by Western blot (WB) and qPCR, respectively. qPCR results #1, 2, and 3 represent biological replicates.

[0180] Figure 4 shows the Western blot results confirming AAV productivity for GFP gene expression, Example 1 (GC), Comparative Example 1 (WT), and Comparative Example 2 (BC-del).

[0181] Figure 5 shows the qPCR viral genome quantification results confirming AAV productivity for GFP gene expression, Example 1 (pAAV-GC), Comparative Example 1 (pAAV-WT), and Comparative Example 2 (pAAV-BC del).

[0182] Figure 6 shows the qPCR viral genome quantification results confirming AAV productivity for various gene expression vectors (GFP, Luciferase, TP53, RPE65, TPP1, or FVIII) using pAAV-GC vectors, pAAV-WT vectors, and pAAV-BCdel vectors.

[0183] As a result, as shown in Figures 4 to 6, the pAAV-GC vector demonstrated significantly higher AAV productivity compared to the pAAV-WT vector and the pAAV-BCdel vector, even when various genes were introduced.

[0184] Therefore, it was found that pAAV-GC vectors containing asymmetrically modified ITRs partially omit the self-renewal step in host cells, increasing the packaging efficiency to viral particles within the same production period, thus increasing self-renewal efficiency and improving the productivity of pAAV-GC vectors compared to existing AAV transduction devices.

[0185] Experimental Example 2. Confirmation of gene expression rate of the AAV complex.

[0186] 2-1. Confirmation of GFP gene expression rate

[0187] The target gene expression rate of recombinant AAV complexes was confirmed using a specific example. Specifically, HEK293, a cell line derived from a normal fetal kidney, was infected with the same amount of AAV complex (Example 1, Comparative Example 1, or Comparative Example 2). After 72 hours, the GFP signal expressed by the cells was imaged using an inverted fluorescence microscope. Subsequently, each cell was disrupted, the protein was purified, loaded onto an SDS page gel, and the GFP gene expression rate was confirmed by Western blotting. Western blot band intensity was quantified using ImageJ software. The detection value of the GFP protein in the pAAV-GC vector-treated sample with the strongest band intensity was set to 100%, and the expression rates of each gene were quantified as relative values. Biological replication was performed twice for each group.

[0188] Figure 7 shows the results of Western blotting to confirm the expression of the transgene GFP in the AAV complex according to the ITR type (ITR WT, ITR BC del, or ITR GC).

[0189] Table 3 shows the results of quantifying the relative expression rate (%) of the transgene GFP according to ITR type in the AAV complex.

[0190] [Table 3]

[0191] As a result, as shown in Figure 7 and Table 3, the expression rate of the GFP gene was significantly higher when using the pAAV-GC vector (Example 1) compared to the pAAV-WT vector (Comparative Example 1) and the pAAV-BC del vector (Comparative Example 2).

[0192] 2-2. Confirmation of the expression rates of diverse genes

[0193] Using the same method as in Experimental Example 2-1, experiments were conducted to confirm the expression rates of various transgenes, GFP, Luciferase, TP53, RPE65, TPP1, and FVIII, using Examples 1 to 6 and Comparative Examples 1 to 12.

[0194] For the GFP and Luciferase genes, the same cell line as in Experimental Example 2-1 was used. For the TP53, TPP1, RPE65, and FVIII genes, HEK293 cells were used in which each gene was knocked out using CRISPR / Cas9 (Synthego, USA) to eliminate the influence of endogenous genes unique to the target cells.

[0195] After confirming that no endogenous gene expression occurred in the cell line, the cells were cultured in a 6-well cell culture plate. 1.0 × 10 5 1.0 × 10 per cell 8The concentration of the virus solution was quantified and applied to cells so that individual virus particles were processed. Two days after AAV treatment, proteins expressed from each introduced gene were detected by Western blot (WB). The antibodies used were as follows: GFP (Invitrogen A-11122), Luciferase (Invitrogen PA1-179), TP53 (MA5-14067), TPP1 (PA5-102819), RPE65 (MA1-16578), and FVIII (PA5-104451). WB band intensity was quantified using ImageJ software. The result band of pAAV-GC was set to 100%, and pAAV-WT and pAAV-BCdel were quantified as relative values. Biological replication was performed three times for each group.

[0196] Table 4 shows the quantified relative expression rates (%) of transgenes in the AAV complex based on ITR type (pAAV-GC, pAAV-BC del, or pAAV-WT).

[0197] Figure 8 shows the relative expression rates (%) of transgenes in the AAV complex according to ITR type (pAAV-GC, pAAV-BC del, or pAAV-WT).

[0198] [Table 4]

[0199] As a result, as shown in Table 4 and Figure 8, the expression rates of all transgenes were approximately 2 to 10 times higher when using pAAV-GC vectors containing asymmetrically deformed ITRs compared to pAAV-WT vectors containing non-deformed symmetric ITRs and symmetrically deformed pAAV-BC del vectors.

[0200] Therefore, it was found that pAAV-GC vectors containing asymmetrically modified ITRs can be used to transmit diverse transgenes into target cells as a transduction platform with significantly increased transgene expression efficiency.

[0201] Experimental Example 3. Confirmation of the genotoxicity of the AAV complex.

[0202] 3-1. Confirmation of whether the transgene GFP is inserted into the host chromosome.

[0203] An experiment was conducted to confirm that, when using a recombinant AAV complex with a specific example, the transgene is not recombined into the chromosome of the target cell.

[0204] Specifically, H460 cells were infected with pAAV-GC (Example 1), pAAV-WT (Comparative Example 1), and pAAV-BC del (Comparative Example 2), each containing the GFP gene. Cells expressing GFP were isolated, and the same number of cells were seeded into culture plates. The cells were subcultured by diluting them by 1 / 3 every 3 days, and the expression rate of the GFP gene was tracked to confirm whether or not the GFP gene was genetically transmitted to the offspring.

[0205] Figure 9A shows the results of fluorescence microscopy imaging of GFP expression at passage numbers P3 and P10.

[0206] Table 5 shows the percentage of GFP-expressing cells at subculturing stages P1 to P10.

[0207] [Table 5]

[0208] Figure 9B is a diagrammatic representation of Table 4, which quantifies the percentage of GFP-expressing cells.

[0209] As a result, as shown in Figures 9A and 9B and Table 5, after P7, the expression of the transgene GFP was observed very rarely in cells infected with the pAAV-GC vector (Example 1). On the other hand, in cells infected with the pAAV-WT vector (Comparative Example 1) or the pAAV-BC del vector (Comparative Example 2), the transgene GFP was observed continuously.

[0210] Therefore, it was found that the AAV complex of Example 1 exhibited significantly reduced genotoxicity in gene transmission to offspring compared to the AAV complexes of Comparative Examples 1 and 2.

[0211] 3-2. Confirmation of insertion of diverse transgenes into host chromosomes

[0212] Using the same method as in Experimental Example 3-1, experiments were conducted using Examples 1 to 6 and Comparative Examples 1 to 12 to confirm the insertion of various transgenes, GFP, Luciferase, TP53, RPE65, TPP1, and FVIII, into host chromosomes.

[0213] For the GFP and Luciferase genes, the same cell line as in Experimental Example 3-1 was used. For the TP53, TPP1, RPE65, and FVIII genes, HEK293 cells were used in which each gene was knocked out using CRISPR / Cas9 (Synthego, USA) to eliminate the influence of endogenous genes unique to the target cells.

[0214] After confirming that endogenous gene expression did not occur in the cell line, proteins expressed from each gene introduced by AAV were detected by Western blotting. The antibodies used were as follows: GFP (Invitrogen A-11122), Luciferase (Invitrogen PA1-179), TP53 (MA5-14067), TPP1 (PA5-102819), RPE65 (MA1-16578), and FVIII (PA5-104451). WB band intensity was quantified using ImageJ software. The result band of pAAV-GC was set to 100%, and pAAV-WT and pAAV-BCdel were quantified as relative values.

[0215] Table 6 shows the results (%) of relative gene expression levels at passage number P7.

[0216] [Table 6]

[0217] Figure 10 shows a diagrammatic representation of Table 5, which quantifies the relative gene expression levels in P7.

[0218] As a result, as shown in Table 6 and Figure 10, transgene expression was observed very rarely in cells infected with the pAAV-GC vector in P7. On the other hand, it was confirmed that transgene expression was continuously observed in cells infected with the pAAV-WT vector or the pAAV-BC del vector.

[0219] Therefore, we found that pAAV-GC vectors containing asymmetrically deformed ITRs exhibited significantly reduced genotoxicity in the transmission of introduced genes to offspring compared to pAAV-WT vectors containing non-deformed symmetric ITRs and symmetrically deformed pAAV-BC del vectors.

[0220] 3-3. Confirmation of concatemer formation in target cells of the transgene.

[0221] When a transgene is transmitted to a host cell using an AAV vector, we conducted an experiment to confirm whether or not concatemers, which are found when a transgene is inserted into the host chromosome, are formed.

[0222] Specifically, in Experimental Example 3-1, DNA was extracted from each GFP-expressing cell in P7. The extracted DNA was amplified by PCR, and electrophoresis was performed to confirm the residual form of the DNA that was transmitted to the host cell.

[0223] Figure 11 shows the PCR amplification results confirming the presence or absence of concatemer formation of the transgene in host cells.

[0224] As a result, as shown in Figure 11, it was confirmed that in the case of the pAAV-GC vector (Example 1) lacking the hairpin structure of one ITR, concatemers, which are well known as a characteristic of AAV vectors, could not be formed. On the other hand, in the case of the pAAV-WT vector (Comparative Example 1) and the pAAV-BC del vector (Comparative Example 2), in which both ITRs had apin structures, the transgene was observed as polymers in various forms, such as monomers, dimers, and concatemers.

[0225] Therefore, we observed that when using the pAAV-GC vector, the transgene does not form a concatemer structure in the host cell, and insertion into the host cell's chromosome is suppressed.

[0226] In summary, it was found that the pAAV-GC vector containing an asymmetrically modified ITR is suitable as a target signaling medium for expressing a transgene efficiently for a short period while suppressing the long-term expression of the transgene in the host cell.

[0227] The foregoing description of the present invention is illustrative, and a person with ordinary skill in the art to which the invention pertains will understand that embodiments and specific examples can be easily modified without altering the technical idea or essential features of the invention. Therefore, the embodiments should be understood to be illustrative and not limiting in all respects.

[0228] The specific examples described herein should be considered descriptive and not restrictive. Descriptions of features or embodiments within each specific example should generally be considered applicable to other similar features or embodiments of other specific examples. While one or more specific examples have been described with reference to the drawings, those skilled in the art will understand that various modifications to form and detail are possible without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. An adeno-associated virus (AAV) complex comprising a polynucleotide sequence that encodes a transgene between a first inverted terminal repeat (ITR) and a second ITR, An adeno-associated virus complex in which all or part of the stem-loop structure consisting of the RBE (rep-binding element), RBE', A, A', B, B', C, C', and D region is deformed in either the first ITR or the second ITR.

2. The adeno-associated virus complex according to claim 1, further comprising a promoter operably linked between a first ITR and a second ITR, a polynucleotide sequence encoding a transgene, and a polyadenylation sequence.

3. The adeno-associated virus complex according to claim 1, wherein the AAV is an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

4. The adeno-associated virus complex according to claim 1, wherein the first ITR is not deformed and the second ITR is deformed.

5. The adeno-associated virus complex according to claim 1, wherein the deformation of the stem-loop structure is at least one of insertion, deletion, or substitution.

6. The adeno-associated virus complex according to claim 1, wherein either the first ITR or the second ITR is deformed so as not to form a stem-loop structure.

7. The adeno-associated virus complex according to claim 1, wherein in either the first ITR or the second ITR, all or part of a stem-loop structure consisting of the RBE, RBE', A, A', B, B', C, C', and D regions is deleted.

8. The adeno-associated virus complex according to claim 1, wherein either the first ITR or the second ITR includes a trs (terminal resolution site) sequence and an RBE sequence, and all C, C', B', B, RBE', A', and D sequences are deleted from after the RBE.

9. The adeno-associated virus complex according to claim 2, wherein the promoter is a tissue-specific promoter.

10. The adeno-associated virus complex according to claim 1, wherein the introduced gene is a therapeutic gene.

11. The adeno-associated virus complex according to claim 1, wherein the introduced gene is GFP, Luciferase, TP53, RPE65, TPP1, or FVIII.

12. A gene therapy method comprising the step of administering an effective amount of the adeno-associated virus complex described in claim 1 to an individual in need thereof.

13. A composition comprising the adeno-associated virus complex described in claim 1.

14. The composition according to claim 13, further comprising a pharmaceutically acceptable carrier.

15. The composition according to claim 13, for use in the delivery of therapeutic genes for gene therapy.

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