Methods for the prevention or treatment of adeno-associated virus complexes with enhanced RUNX3 gene expression and their KRAS mutations in lung cancer.

Abstract

This invention provides an adeno-associated virus (AVV) complex for RUNX3 gene expression containing an asymmetrically deformed inverse repeat sequence (ITR). The AAV complex has the advantage of improved productivity and gene expression efficiency compared to existing AVV complexes, as one of the two ITRs is a deformed asymmetric ITR, which increases the self-renewal efficiency in the host cell and increases the efficiency of transgene expression.

Description

[Technical Field]

[0001] The present invention relates to adeno-associated virus complexes with enhanced RUNX3 expression and methods for preventing or treating KRAS-mutated lung cancer. [Background technology]

[0002] The KRAS gene is a substance involved in the function of the GTP enzyme protein, which plays a crucial role in the signaling pathways related to cell differentiation, proliferation, and survival. The KRAS protein binds to GTP when transmitting signals at the cellular level, thereby aiding the growth of cancer cells.

[0003] KRAS mutations are relatively common tumor-induced mutations, found in approximately 20% of solid tumors. In particular, these mutations are most frequently found in adenocarcinomas of the pancreas, intestines, and colorectal cancer, as well as lung cancer. Specifically, in patients with lung cancer, pancreatic cancer, and intestinal cancer caused by KRAS mutations, post-treatment prognosis is almost always worse than in patients without KRAS mutations. Furthermore, unlike other non-small cell lung cancers (NSCLCs), all targeted therapies attempted for KRAS-mutated cancers have been discontinued at the clinical stage due to side effects.

[0004] 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.

[0005] However, the aforementioned AAV has a problem in that, due to the inverted terminal repeat (ITR) sequence, up to approximately 4.4 kb of the protein coding sequence is capsidized, resulting in a reduced DNA packaging capacity. Furthermore, for gene therapy agents used in cancer treatment, it is desirable that the introduced gene disappears simultaneously with the death of cancer cells, rather than being expressed over a long period.

[0006] Therefore, as a gene carrier for anti-cancer therapy, it is necessary to develop an AAV complex that is useful for the prevention or treatment of KRAS-mutated solid tumors by modifying the ITR among the AAV properties, thereby improving DNA packaging ability, reducing the probability of insertion into the chromosomes of infected cells, improving productivity and expression efficiency, and selectively killing KRAS-mutated solid tumors. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] In one specific example, an adeno-associated virus (AAV) complex is disclosed, comprising a polynucleotide sequence that encodes the RUNX3 (RUNt-related transcription factor 3) protein 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 include an operablely linked SPC promoter, a polynucleotide sequence that encodes the RUNX3 protein, 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 certain specific examples, the first ITR is not deformed, and the second ITR is deformed. The deformation of the stem-loop structure is an insertion, deletion, or substitution. In the complex, either the first ITR or the second ITR may be deformed so as not to form a stem-loop structure. In the complex, either the first ITR or the second ITR may have all or part of the stem-loop structure consisting of the RBE (rep-binding element), RBE', A, A', B, B', C, C', and D regions deleted. Either the first ITR or the second ITR contains a terminal resolution site (trs) sequence and an RBE sequence, with all C, C', B', B, RBE', A', and D sequences deleted from the RBE onward. In the complex, the first ITR is an AAV wild-type ITR, and the second ITR is composed of, or may be composed of, a nucleotide sequence selected from the group consisting of sequence numbers 1 to 9.In the complex, the first ITR is an AAV wild-type ITR, and the second ITR consists of, or may be, the nucleotide sequence of SEQ ID NO: 1. The adeno-associated virus complex may further include a gene junction containing SEQ ID NO: 10 between the nucleotide sequences that encode the SPC promoter and RUNX3.

[0008] In other specific examples, a method for treating KRAS-mutated lung cancer is disclosed, comprising the step of administering an effective amount of the adeno-associated virus complex to an individual in need. The lung cancer is non-small cell lung cancer or small cell lung cancer, where non-small cell lung cancer may be selected from the group consisting of squamous cell carcinoma, large cell carcinoma, and lung adeno carcinoma.

[0009] Further specific examples disclose a pharmaceutical composition for the prevention or treatment of KRAS-mutated lung cancer, comprising the adeno-associated virus complex. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. [Means for solving the problem]

[0010] The specific examples shown in the attached drawings are described in detail below, and throughout the drawings, the same reference numeral refers to the same element. In this regard, these specific examples have diverse forms and should not be construed as being limited to the descriptions presented herein. Accordingly, these specific examples will be described later with reference to the drawings to illustrate aspects of this description. 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.

[0011] Various specific examples will be presented in part in the following description, some will become clear from the description, or may also be learned by implementing the specific examples presented in the present invention.

[0012] In one specific example, an adeno-associated virus (AAV) complex containing a modified inverted terminal repeat (ITR) is disclosed.

[0013] In another specific example, cells transformed by the adeno-associated virus complex are disclosed.

[0014] In yet another specific example, a method for treating KRAS mutant cancer is disclosed, which includes the step of administering an effective amount of the adeno-associated virus complex.

[0015] In yet another specific example, a pharmaceutical composition for preventing or treating KRAS mutant lung cancer containing the adeno-associated virus complex is disclosed.

[0016] In an additional specific example, an adeno-associated virus (AAV) complex containing a modified inverted terminal repeat (ITR) is disclosed.

[0017] 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, which is a helper vector-dependent human parvovirus. The genome is composed of inverted terminal repeats (ITRs) at both ends and two open reading frames (ORFs), rep and cap. The N-terminal of the genome encodes the rep gene involved in viral replication and the expression of viral genes, and the C-terminal 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 include the RBE (rep-binding element), RBE’, A, A’, B, B’, C, C’, and D regions and form a stem-loop structure (hairpin structure). The structure of the AAV ITR is widely known in the literature such as, for example, Goncalves, M.A. Virology Journal, 2(1):43 (2005), which is incorporated herein by reference.

[0018] In certain specific examples, the sequence of the ITR can be based on the ITR sequence of a virus belonging to the genus Dependovirus of the family Parvoviridae.

[0019] In certain alternative specific examples, the sequence of the ITR can be based on the ITR sequence of AAV. The ITR sequence of AAV is known.

[0020] AAV includes, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and may also include other AAV serotypes that are currently known or may be discovered in the future. AAV may include known AAV derivatives. AAV may include modified or artificial AAVs.

[0021] 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.

[0022] As an alternative, other types of viruses belonging to the genus Dependovirus of the family Parvoviridae could be used instead of AAV.

[0023] In this specification, the term "AAV complex" may be used interchangeably with "AAV vector," "AAV carrier," "recombinant AAV," and "recombinant AAV vector."

[0024] In a specific example, the AAV complex includes a polynucleotide sequence between the first ITR and the second ITR that encodes the RUNX3 (RUNt-related transcription factor 3) protein.

[0025] The polynucleotide sequence that encodes the RUNX3 protein may be operably positioned between the first ITR and the second ITR.

[0026] In a specific example, the genome of the AAV complex includes, in the 5' to 3' direction, a first ITR (5'-ITR), an SPC promoter, a polynucleotide sequence encoding the RUNX3 protein, a polyadenylated sequence, and a second ITR (3'-ITR).

[0027] In a particular example, the AAV complex may include a polynucleotide sequence that encodes the RUNX3 protein 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.

[0028] In one specific example disclosed herein, the AAV complex comprises an operably linked SPC (Surfactant Protein C) promoter, a polynucleotide sequence that encodes the RUNX3 protein, and a polyadenylation sequence between a first ITR and a second ITR.

[0029] In a specific example, the genome of the AAV complex includes, in the 5' to 3' direction, a first ITR (5'-ITR), an SPC promoter, a polynucleotide sequence encoding the RUNX3 protein, a polyadenylated sequence, and a second ITR (3'-ITR).

[0030] In certain other specific examples, the AAV complex comprises an operablely linked SPC promoter, a polynucleotide sequence encoding the RUNX3 protein, and a polyadenylation sequence between a first ITR and a second ITR, wherein one of the first ITR and the second ITR may have a deformed stem-loop structure consisting of all or part of the RBE, RBE', A, A', B, B', C, C', and D regions.

[0031] In certain other 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 target gene 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 target gene 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 target gene are deformed. In one embodiment, the AAV complex, by including an asymmetrically deformed ITR, can increase the productivity of the AAV complex and the expression rate of the RUNX3 gene.

[0032] Of the first and second ITRs, the non-modified ITRs are wild-type ITRs. Of the first and second ITRs, the non-modified ITRs are AAV wild-type ITRs.

[0033] The non-deformed ITR among the first and second ITRs may be functional derivatives that possess substantially the same functional properties as the wild-type ITR (e.g., AAV wild-type ITR).

[0034] 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.

[0035] In either the first ITR or the second ITR, all or part of the stem-loop structure consisting of the RBE (rep-binding element), RBE', A, A', B, B', C, C', and D regions is deformed.

[0036] In one specific example, the deformation of the stem-loop structure (hairpin structure) may be selected from insertion, deletion, and replacement.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In one specific example, the deformation of the stem-loop structure (hairpin structure) may include deformation of the structure of the structural elements. Specifically, deformation of the structure 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.

[0043] In other specific examples, the spacing 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 spacing 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 that number.

[0044] 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.

[0045] The expression "deformed so as not to form a stem-loop structure (hairpin structure)" means that the structure of the ITR is deformed by the sequence change of the ITR 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 suppress the integration of AAV into the host genome observed in the 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.

[0046] 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 either 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.

[0047] 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 may be unmodified, while the second ITR may contain the trs sequence and the RBE sequence, with all of the C, C', B', B, RBE', A', and D sequences deleted from RBE onward.

[0048] 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.

[0049] The sequence of sequence number 1 can be based on the AAV2 ITR sequence.

[0050] The sequence of sequence number 2 can be based on the AAV1 ITR sequence.

[0051] The sequence of sequence number 3 can be based on the AAV3 ITR sequence.

[0052] The sequence of sequence number 4 can be based on the AAV4 ITR sequence.

[0053] The sequence of sequence number 5 can be based on the AAV6 ITR sequence.

[0054] The sequence of sequence number 6 can be based on the AAV7 ITR sequence.

[0055] The sequence of sequence number 7 can be based on the AAV5 ITR sequence.

[0056] The sequence of sequence number 8 can be based on the AAV8 ITR sequence.

[0057] The sequence of sequence number 9 can be based on the AAV9 ITR sequence.

[0058] 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 may be obtained by completely deleting the C, C', B', B, RBE', A', and D sequences from RBE onwards.

[0059] 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.

[0060] 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 RUNX3 gene no longer forms a hairpin structure, and transcription of the RUNX3 gene proceeds complementary to that strand in the 5'-3' direction. On the other hand, in the (+) strand of the RUNX3 gene, the 3'-ITR no longer forms a hairpin structure, and transcription of the RUNX3 gene does not proceed on that strand. In other words, transcription of the transgene 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.

[0061] In one embodiment, an AAV complex (Example 1) was produced in an AAV vector containing the RUNX3 gene, in which the hairpin structure of the second ITR of the wild-type AAV contained in the vector was partially deformed to create an asymmetrically deformed ITR (ITR). The produced AAV complex was compared with an AAV complex in which the hairpin structure was not deformed (Comparative Example 1) and an AAV complex in which both ends of the hairpin structure were deformed to be symmetrical (Comparative Example 2), and the productivity and expression rate of the RUNX3 gene were confirmed for each. As a result, it was confirmed that the AAV complex containing the asymmetrically deformed ITR (Example 1) showed a more than 3-fold increase in viral productivity and a more than 6-fold increase in the expression rate of the RUNX3 gene compared with the AAV complex in which the ITR was not deformed (Comparative Example 1) and the AAV complex containing the symmetrically deformed ITR (Comparative Example 2).

[0062] In this specification, the term "RUNX3 (RUNt-related transcription factor 3) protein" refers to one of the RUNX family genes, which are expressed by the RUNX3 gene and consist of RUNX1, RUNX2, and RUNX3. The RUNX family genes play important roles in normal development and tumorigenesis, and function as transcription regulators of the Smad family, which are subordinate factors that mediate TGF-β and its signal transmission.

[0063] The RUNX3 protein may contain one or more amino acid sequences selected from SEQ ID NOs. 19 and 20. The polynucleotide sequences that encode the RUNX3 protein may be selected from SEQ ID NOs. 21 and 22.

[0064] The aforementioned RUNX3 protein may be of human or animal origin.

[0065] The aforementioned RUNX3 protein can be synthesized by chemical synthesis methods of our industry (WH Freemanand Co., Proteins, Structures and Molecular Principles, 1983) and can be produced by genetic engineering methods of our industry (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, Sambrook et al., Molecular Cloning, A Laboratory Manual, etc.).

[0066] The RUNX3 protein is an amino acid variant having a different sequence from one another due to deletion, insertion, substitution, or combination thereof of amino acid residues, to the extent that it does not affect the function of the protein. Amino acid exchange in proteins that does not alter the overall activity of the molecule is known in the field. In some cases, modification may be carried out by phosphorylation, sulfation, acrylation, glycosylation, methylation, or farnesylation.

[0067] Therefore, in one specific example, the RUNX protein may include peptides and their variants or fragments having substantially identical amino acid sequences to proteins containing any one or more amino acid sequences selected in SEQ ID NO: 19 and SEQ ID NO: 20. The substantially identical proteins may have 80% or more homology, specifically 90% or more, and more specifically 95% or more homology to the RUNX3 protein.

[0068] 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'-3' direction, an SPC promoter sequence, a polynucleotide sequence encoding the RUNX3 protein, post-transcriptional regulatory elements, a polyadenylated sequence, and a second ITR.

[0069] The aforementioned post-transcriptional regulatory element may include WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

[0070] In one specific example, the AAV complex may further include a gene junction between the polynucleotide sequences that encode the SPC promoter and the RUNX3 protein.

[0071] 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.

[0072] As a result, the inventors found an optimized sequence by substituting polynucleotides in various combinations to regulate the expression of the RUNX3 gene by the SPC promoter, and confirmed that polynucleotides in the 22 bp to 28 bp range are suitable. Of the optimized sequences, the promoter DNA and the RUNX3 DNA sequence are prepared by PCR using primers designed with restriction enzymes not included in the vector itself or the DNA sequence to be amplified. Therefore, the inventors ultimately selected a junction that further has restriction enzyme regions that do not cleave the vector, promoter, or gene sequence.

[0073] Therefore, in one specific example, the gene junction may include sequence number 11. Furthermore, the gene junction may determine the presence or absence of RUNX3 gene expression or influence the expression efficiency of the RUNX3 gene depending on its length or structure.

[0074] 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.

[0075] Another embodiment provides cells transformed with an adeno-associated virus complex according to one embodiment.

[0076] The specific components of the adeno-associated virus complex are as described above.

[0077] 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.

[0078] In specific examples, methods for introducing the AAV complex into cell lines and transforming them include, but are not limited to, methods known to 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.

[0079] Another embodiment provides a method for preventing or treating lung cancer, comprising the step of administering an effective amount of an adeno-associated virus complex according to one embodiment to a cell or an organism.

[0080] The specific components of the adeno-associated virus complex are as described above.

[0081] In the above method, the AAV complex may be administered to an individual as is, or the AAV complex may be formulated into a dosage form that can be administered to an individual and then administered to the individual. In one specific example, the AAV complex may be administered to an individual in the form of a composition containing the AAV complex as described below. For example, the AAV complex may be administered to an individual in the form of a composition containing the AAV complex and a pharmaceutically acceptable carrier.

[0082] The individual is one that requires the expression of the RUNX3 gene transmitted by the AAV complex. The individual is one that has or is likely to have a disease to which gene therapy is applicable. The individual is one that has or is likely to have a disease to which treatment is possible by the expression of the RUNX3 gene transmitted by the AAV complex. The individual is a mammal, for example, a human, a cattle, a horse, a pig, a dog, a sheep, a goat, or a cat. The individual may be one that has or is likely to have cancer.

[0083] The aforementioned lung cancer may be KRAS-mutated lung cancer.

[0084] In this specification, the term "KRAS-mutated lung cancer" refers to lung cancer in which the KRAS mutation gene is activated and the tumor suppressor gene is deactivated. If the activity of the tumor suppressor gene is restored, lung cancer cells are eliminated and normal cells are present, making KRAS-mutated lung cancer treatable. The tumor suppressor gene may be, for example, the sPD-1, VHL, MMAC1, DCC, p53, NF1, WT1, RB, BRAC1, BRAC2, or RUNX3 gene.

[0085] In one specific example, the lung cancer is either non-small cell lung cancer or small cell lung cancer. Examples of non-small cell lung cancer include squamous cell carcinoma, large cell carcinoma, and lung adenocarcinoma.

[0086] In one embodiment, the injection of recombinant AAV containing the RUNX3 gene into a non-small cell lung cancer mouse model with activated KRAS mutations was confirmed to promote lung cancer cell death and suppress cancer development. Furthermore, when normal lung epithelial cell lines and non-small cell lung cancer cell lines were infected, there was no change in the cell death rate in normal lung epithelial cells, while RUNX3 and cell death markers were significantly increased in lung cancer cell lines.

[0087] Therefore, one embodiment of the AAV complex can specifically eliminate lung cancer cells by activating the RUNX3 gene in lung cancers that have developed due to reduced RUNX3 protein activity, and thus can be used for the prevention or treatment of KRAS-mutated lung cancer. Furthermore, it can prevent the recurrence of the aforementioned KRAS-mutated lung cancer.

[0088] 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 alter the symptoms of a disease by administering the AAV complex.

[0089] 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 lung cancer. The active ingredient may be administered simultaneously with, separately from, or sequentially with the AAV complex.

[0090] The AAV complex may be administered to an individual in the form of an injectable preparation suitable for administration via any suitable route, such as intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, or intravertebral cavity. The AAV complex may be administered systemically or topically, alone or in combination with other pharmaceutically active compounds.

[0091] 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 ....... 14 vg / kg, about 1.0×10 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 a specific example, the dosage of the AAV complex is about 1.0×10 13 [[ID=2... 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.

[0092] In this specification, the term "about" can be used to include a range of ±10% of the specified numerical value.

[0093] Another aspect provides a pharmaceutical composition for the prevention or treatment of lung cancer comprising an adeno-associated virus complex according to one aspect. Still another aspect provides the use of an adeno-associated virus complex according to one aspect for the manufacture of a medicament for the treatment of lung cancer.

[0094] The specific details of the adenovirus complex and lung cancer are as described above.

[0095] It should be noted that there are some incomplete parts in the original text (such as "about 1.0×10 " without complete numerical values in some lines), and the translation is done as accurately as possible based on the available content.The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier may include excipients, diluents, or adjuvants. As the carrier, a carrier suitable for delivering the AAV complex into a living organism may 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.

[0096] The pharmaceutical composition may be prepared in any dosage form by conventional methods. The composition may be formulated into a form suitable for delivering the A AV 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 a particular specific example, 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.

[0097] In certain specific examples, the pharmaceutical composition may further include one or more anticancer agents. Exemplary anticancer agents may include cetuximab, panitumumab, erlotinib, gefitinib, trastuzumab, T-DM1, Perjeta, lapatinib, paclitaxel, taxol, tamoxifen, cisplatin, or combinations thereof. The pharmaceutical composition may be a single composition or individual compositions. For example, a composition of an antibody or its antigen-binding fragment may be a parenteral dosage form, and an anticancer agent may be an orally administered dosage form.

[0098] The pharmaceutical 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 in need of 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.

[0099] In one specific example, the pharmaceutical composition is approximately 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 13vg / 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]

[0100] One embodiment of the adeno-associated virus complex has the advantage of improved productivity and gene expression efficiency compared to existing AAV complexes, because it has an asymmetric ITR in which one of the two ITRs is deformed, which increases the self-renewal efficiency in the host cell and increases the efficiency of transgene expression. [Brief explanation of the drawing]

[0101] Specific examples, features, and advantages of the present invention will become even clearer from the following description, along with the accompanying drawings.

[0102] [Figure 1] This shows a cleavage map of an adeno-associated virus (AAV) vector according to one embodiment.

[0103] [Figure 2] The structure of an AAV vector according to one embodiment is shown.

[0104] [Figure 3A] This is a schematic genome diagram of the AAV vector containing the asymmetrically deformed ITR of Example 1.

[0105] [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.

[0106] [Figure 3C]This is a schematic genome diagram of the symmetrically deformed ITR-containing AAV vector of Comparative Example 2.

[0107] [Figure 4A] This shows the results of confirming the expression of target genes from AAV complexes according to one embodiment using Western blotting. C1: AAV complex from Comparative Example 1, C2: AAV complex from Comparative Example 2, WT: wild-type AAV complex, RX001: AAV complex from Example 1, Control 293 cell: HEK293 cell line not infected with AAV complex, empty: culture medium with no additives (double negative control), Control positive: purified RUNX3 protein.

[0108] [Figure 4B] This shows the results of quantifying the expression rate of target genes from AAV complexes according to one embodiment. C1: AAV complex from Comparative Example 1, C2: AAV complex from Comparative Example 2, WT: wild-type AAV complex, RX001: AAV complex from Example 1, Control 293 cell: HEK293 cell line not infected with AAV complex, empty: culture medium with no additives (double negative control), Control positive: purified RUNX3 protein.

[0109] [Figure 5A] This shows the results of H&E and TUNEL staining of lung cancer tissue infected according to Example 1.

[0110] [Figure 5B] The results of H&E and TUNEL staining of lung cancer tissue infected by Comparative Example 1 are shown.

[0111] [Figure 6A] These are microscopic images taken 1, 3, and 5 days after infecting KRAS-mutated lung cancer cell lines (H460, Calu6) and normal lung epithelial cells (WI38) with Example 1.

[0112] [Figure 6B] This shows the results of FACS (fluorescence-activated cell sorting) analysis at 0 hours (top), 16 hours (middle), and 32 hours (bottom) after infecting KRAS-mutated lung cancer cell lines (H460, Calu6) and normal lung epithelial cells (WI38) with Example 1.

[0113] [Figure 6C] The results of FACS analysis were obtained at 0 hours (top), 16 hours (middle), and 32 hours (bottom) after infecting the KRAS-mutated lung cancer cell line (Calu6) with Example 1.

[0114] [Figure 6D] This graph quantifies the FACS analysis results after 1 and 3 days following infection of KRAS-mutated lung cancer cell line (H460) and normal lung epithelial cells (WI38) with Example 1.

[0115] [Figure 6E] This shows the results of detecting RUNX3 and cleaved caspase 3 expressed from the KRAS-mutated lung cancer cell line infected in Example 1.

[0116] [Figure 7A] This is the result of examining the internal structure of cancerous tissue after injecting a non-small cell lung cancer xenograft model with Example 1 and raising it for 12 days. AAV2-Control: AAV injection with a genome lacking RUNX3 (AAV-empty), AAV2-SPC-RUNX3: AAV complex injection of Example 1.

[0117] [Figure 7B] The following are the results of measuring the volume of cancerous tissue after injecting non-small cell lung cancer xenograft models with Example 1 and raising them for 12 days. Con1-4: Mice treated with AAV-control (Empty), DI1-4: Mice treated with AAV2-SPC-RUNX3 (RX001), respectively. [Modes for carrying out the invention]

[0118] 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]

[0119] Examples

[0120] Example 1. Production of an adeno-associated virus complex for RUNX3 gene expression containing an asymmetrically deformed ITR.

[0121] 1-1. Production of adeno-associated virus vectors into which the RUNX3 gene and SPC promoter have been introduced.

[0122] Adeno-associated virus (AAV) vectors into which the RUNX3 gene was introduced were manufactured. Specifically, the humanRUNX3 gene (NCBI reference: NM_004350.2, 412nt to 1659nt) was synthesized and then PCR-amplified using the primers shown in Table 1 below. These primers were prepared by synthesizing the restriction enzymes KpnI and HindIII. In addition, an SPC (Surfactant Protein C) promoter (GenBank accession no. AC122268, 148366nt to 149406nt) specifically expressed in lung epithelial cells was PCR-amplified using the primers shown in Table 1 below. These primers were prepared by synthesizing the restriction enzymes NheI and KpnI. Subsequently, the amplified RUNX3 and SPC promoter DNA were treated with the restriction enzyme KpnI, respectively, and then the KpnI portion was ligated using T4 DNA ligase to produce the NheI-SPC-KpnI-RUNX3-HindIII DNA fragment. The NheI-SPC-KpnI-RUNX3-HindIII DNA fragment was then cloned while removing the GFP gene located at the Nhe-HindIII position in the MCS (Multi Cloning Site) of an adeno-associated virus (AAV) 2 GFP vector (Chungbuk National University Oncology Research Institute). To induce the expression of the SPC promoter from the AAV2-GFP vector, the chicken beta actin promoter was removed using the restriction enzymes NdeI and BgIII. Subsequently, in the AAV2-SPC-RUNX3 plasmid from which the chicken beta actin promoter had been removed, the ampicillin resistance gene was removed using the BspH I restriction enzyme portions 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.

[0123] [Table 1]

[0124] 1-2. Deformation of ITR structure

[0125] 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 (rep_binding element) of the 5'-direction ITR of the (-) strand of the RUNX3 gene, one of the AAV2 wild-type ITRs 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 RUNX3 gene. As a result, the second ITR was deformed so that it no longer formed a hairpin structure. As a result, we obtained an adenovirus complex for RUNX3 gene expression containing an asymmetrically deformed ITR, in which the first ITR remained undeformed and the second ITR was deformed.

[0126] Figure 1 shows a cleavage map of an adeno-associated virus vector according to one embodiment.

[0127] Figure 2 shows the structure of an adeno-associated virus vector according to one embodiment.

[0128] Figure 3A is a schematic genome diagram of the adeno-associated virus vector containing the asymmetrically deformed ITR of Example 1.

[0129] [Table 2]

[0130] Comparative Example

[0131] Comparative Example 1. Undeformed, Symmetrical Production of an AAV complex for RUNX3 gene expression containing ITR

[0132] An AAV composite containing a symmetrically undeformed ITR was manufactured using the same method as in Example 1-1, except that the hairpin structure of the ITR was not deformed.

[0133] Figure 3B is a schematic genome diagram of the unmodified, symmetrical ITR-containing AAV vector of Comparative Example 1.

[0134] Comparative Example 2. Preparation of an AAV complex for RUNX3 gene expression containing a symmetrically deformed ITR.

[0135] An AAV complex containing an ITR that was modified to be symmetrical at both ends was manufactured using the same method as in Example 1-2, except that the C-C'-B'-RBE sequence was deleted at both ends of the ITR of the AAV complex manufactured in Example 1-1.

[0136] Figure 3C is a schematic genome diagram of the AAV vector containing the symmetrically deformed ITR of Comparative Example 2.

[0137] Experimental example

[0138] Experimental Example 1. Confirmation of the productivity of the AAV complex.

[0139] The productivity of recombinant AAV complexes according to one embodiment was confirmed. Specifically, the AAV complexes produced in Example 1 and Comparative Examples 1 and 2 were used to transform 293T cells, which are human embryonic kidney (HEK) cells, and the number of viral particles expressed in the cells was measured. Wild-type AAV complexes were used as a negative control group.

[0140] First, 293T cells (Chungbuk National University Tumor Research Institute) were added to DMEM (Welgene, LM001-05) medium supplemented with 10% fetal bovine serum (Welgene, S001-01) and 1X Antibiotic (Welgene, LS203-01) in a 1x10⁶ mixture. 6The individual cells were dispensed into 75T Flasks (SPL, 70075) and incubated for 24 hours. Subsequently, for transformation, a mixture was prepared by adding the virus complexes prepared in Example 1 and Comparative Examples 1 and 2, helper Plasmid (aldevron), and AAV2 rep / capplasmid (aldevron) in a ratio of 1:3:1 (9 μg:27 μg:9 μg). Then, 100 μl of TOMTMT (Welgene, TR004-01) was added, followed by the addition of Transmission Grade Linear Polyethylenimine Hydrochloride (PEI, MW40,000) (Polysciences Inc, 24765-1) in a ratio of 1:2 (45 μl:90 μl) based on the total plasmid amount, and the mixture was left at room temperature for 15 minutes. After changing the cell culture medium, the aforementioned mixture was added, and after 48 or 72 hours, 0.5 M EDTA, pH 8.0 (TransLab, 15-10ED18) equivalent to 1 / 80 of the total volume was added, and the cells were allowed to suspend at room temperature for 10 minutes. The suspended cells were collected in a 50 ml Centrifuge tube (SPL, 50050), and after primary centrifugation at 4°C Centrifuge 2000 g for 10 minutes, secondary centrifugation was performed under the same conditions for 1 minute to completely remove the supernatant. Subsequently, viral DNA was extracted using RT-PCR, and the extracted DNA was quantified using qPCR. Table 3 shows the number of viral particles expressed in 293T cells.

[0141] [Table 3]

[0142] As a result, as shown in Table 3, it was confirmed that Example 1 had a significantly higher number of AAV complex particles compared to the negative control group and Comparative Examples 1 and 2. Specifically, the number of particles in Example 1 was approximately four times higher than that of the negative control group and Comparative Example 2, and approximately three times higher than that of Comparative Example 1.

[0143] In other words, in one embodiment of the AAV complex, productivity can be improved by asymmetrically deforming the ITR.

[0144] Experimental Example 2. Confirmation of gene expression rate of the AAV complex.

[0145] The target gene expression rate of recombinant AAV complexes according to one embodiment was confirmed. Specifically, H460 non-small cell lung cancer cell lines were infected with the same amount of complexes prepared in Example 1 and Comparative Examples 1 and 2. After 48 hours, the cells were lysed, the proteins were purified and loaded onto SDS page gel, and the expression rate of the RUNX3 gene was confirmed by Western blotting. Subsequently, the gene expression rates of Example 1 and Comparative Examples 1 and 2 were calculated by setting the detection rate of the purified RUNX3 protein to 100%. Wild-type AAV complexes were used as a negative control group. In addition, HEK293 cell lines not infected with AAV complexes were used as an additional negative control group. Unlike cancer cells, which do not express RUNX3 well, HEK293 cells are normal cells and not cancer cells, so a large amount of endogenous RUNX3 gene expression was detected.

[0146] Figure 4A shows the results of Western blotting to confirm the expression of target genes in one embodiment of the AAV complex.

[0147] Figure 4B shows the results of quantifying the expression rate of target genes in one embodiment of the AAV complex.

[0148] As a result, as shown in Figures 4A and 4B, the expression rate of the RUNX3 gene in the virus produced by Example 1 was significantly increased by more than twofold compared to the negative control group and Comparative Examples 1 and 2.

[0149] When combined with the results of Experimental Example 1, it can be seen that the AAV complex using one method increased productivity by more than three times and the expression rate of the RUNX3 gene increased by more than two times, resulting in an overall increase in efficiency of more than six times.

[0150] Experimental Example 3. Confirmation of the therapeutic effect of AAV complex on lung cancer.

[0151] 3-1. Confirmation of cell death in KRAS-mutated non-small cell lung cancer

[0152] We confirmed the cell death induction effect of a recombinant AAV complex in one embodiment against KRAS-mutated non-small cell lung cancer. Specifically, RUNX3 lox / lox KRAS wt / LSL Adeno5-CRE 2.5×10¹⁶ C57B6 mice (6 to 8 weeks old) with the genotype exhibit a KRAS mutation through the respiratory system, while simultaneously depleting RUNX3. e7 The mice were infected with particles. After that, the mice were reared for 6 weeks to check for the development of non-small cell lung cancer, and then the AAV complex of Example 1 or Comparative Example 1 was used to infect the respiratory tract of mice in which lung cancer was confirmed. After 2 weeks, the mice were euthanized, and lung tissue was excised to prepare specimens for histopathological examination. The specimens were then stained with H&E (hematoxylin & eosin) and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick-end labelling), and the stained tissues were observed under a microscope.

[0153] Figures 5A and 5B show the results of H&E and TUNEL staining of lung cancer tissue infected by Example 1 and Comparative Example 1, respectively.

[0154] As a result, as shown in Figure 5A, in the case of lung cancer tissue infected with the AAV complex of Example 1, TUNEL staining was observed at a high level at the cancer site, and the spacing between H&E-stained areas was relatively wide. On the other hand, as shown in Figure 5B, in the case of lung cancer tissue infected with the AAV complex of Comparative Example 1, TUNEL staining was hardly observed, and the spacing between H&E-stained areas was relatively narrow.

[0155] In other words, the lung cancer tissue infected by Example 1 shows that cell death occurs at the site of cancer development, no tumor mass is observed, and the original lung structure is preserved. Therefore, the AAV complex according to one embodiment can be usefully used for the prevention or treatment of lung cancer because it promotes the death of lung cancer cells and suppresses cancer development.

[0156] 3-2. Confirmation of cell death in human non-small cell lung cancer

[0157] The cell death induction effect of a recombinant AAV complex in one embodiment of non-small cell lung cancer was confirmed. Specifically, the AAV complex of Example 1 was used to infect non-small cell lung cancer cell lines H460 and Calu6, and a normal lung cell line WI38. Subsequently, each cell line was cultured for 1 to 7 days, and the presence or absence of cell death was checked daily or every 3 days from the start of culture, and then for 3 to 7 days, by flow cytometry (fluorescence-activated cell sorting: FACS). In addition, after extracting proteins from the H460 cell line, the expression of RUNX3 and cleaved caspase 3 was confirmed by Western blotting.

[0158] Figure 6A shows microscopic images taken 1, 3, and 5 days after infecting KRAS-mutated lung cancer cell lines (H460, Calu6) and normal lung epithelial cells (WI38) with Example 1.

[0159] Figure 6B shows the FACS analysis results at 0 hours (top), 16 hours (middle), and 32 hours (bottom) after infecting normal lung epithelial cells (WI38) with Example 1.

[0160] Figure 6C shows the FACS analysis results at 0 hours (top), 16 hours (middle), and 32 hours (bottom) after infecting the KRAS-mutated lung cancer cell line (Calu6) with Example 1.

[0161] Figure 6D is a graph quantifying the FACS analysis results after 1 day and 3 days following infection of KRAS-mutated lung cancer cell line (H460) and normal lung epithelial cells (WI38) with Example 1.

[0162] Figure 6E shows the results of detecting RUNX3 and cleaved caspase 3 expressed from the KRAS-mutated lung cancer cell line infected in Example 1.

[0163] As a result, as shown in Figure 6A, in the case of lung cancer cell lines infected with the complex of Example 1, it was confirmed that most cells had died on day 5 of infection. On the other hand, it was confirmed that no dead cells were observed in normal lung epithelial cells.

[0164] Furthermore, as shown in Figures 6B to 6D, in the case of lung cancer cell line (H460) infected with the complex of Example 1, it was confirmed that the cell death rate increased by more than four times on day 3 of infection compared to day 1 of infection. On the other hand, no change in the cell death rate over time was observed in normal lung epithelial cells (WI38).

[0165] Furthermore, as shown in Figure 6E, in the case of lung cancer cell lines infected with the complex of Example 1, a sustained increase in the expression of RUNX3 and cleaved caspase 3 was observed from the second day after infection.

[0166] In other words, one embodiment of the AAV complex is non-toxic to normal cells and selectively kills only KRAS-mutated lung cancer cell lines, and therefore can be usefully used for the prevention or treatment of KRAS-mutated lung cancer.

[0167] 3-3. Confirmation of cancer tissue necrosis in a xenograft model of non-small cell lung cancer.

[0168] In one embodiment, the necrotic effect of recombinant AAV complexes on cancer tissue was confirmed in a xenograft model of non-small cell lung cancer. Specifically, 2 × 10¹⁶ H460 non-small cell lung cancer cells were transplanted into the dorsal flank of 6-week-old nude mice. 5 Cell / MICE was used for subcutaneous injection to produce a non-small cell lung cancer xenograft model. The above Example 1 was then processed into 2 × 10⁻⁶ units. 7 viral genome / mm 3The solution was then diluted by mixing it with an equal amount of PBS. The xenograft model mice were then raised for two weeks, and when the diameter of the subcutaneously transplanted cell mass reached approximately 5 mm to 10 mm, the diluted solution was injected directly into the cancerous tissue. The control group was injected with a diluted solution of the AAV complex (AAV-empty) which had a genome that did not contain RUNX3. After injection, the mice were raised for a further 12 days, and when the volume of the tumor increased by an average of more than 100% compared to day 0, the mice were euthanized. Subsequently, the cancerous tissue was excised from the mice and the cross-section was cut. The experiment was carried out under the same conditions for a total of eight mice: four mice (DI1, DI2, DI3, DI4) injected with Example 1 and four mice (Con1, Con2, Con3, Con4) injected with AAV-empty.

[0169] Figure 7A shows the results of examining the internal structure of a cross-section of cancerous tissue after injecting Example 1 into a non-small cell lung cancer xenograft model and raising it for 12 days.

[0170] Figure 7B shows the results of measuring the volume of cancerous tissue after injecting a non-small cell lung cancer xenograft model with Example 1 and raising it for 12 days.

[0171] As a result, as shown in Figure 7A, in the case of the xenograft animal model injected with Example 1, it was confirmed that RUNX3 either caused necrosis of the cancer or loosely maintained the binding force of the tumor mass.

[0172] Furthermore, as shown in Figure 7B, in the xenograft animal model injected with Example 1, there was almost no change in the volume of cancerous tissue until 12 days after injection. On the other hand, in the control group, the volume gradually increased after injection, and by day 12, it was confirmed that the volume of cancerous tissue had increased by up to 150% compared to day 1.

[0173] In other words, an AAV complex according to one embodiment can be usefully used for the prevention or treatment of KRAS-mutated lung cancer because it specifically expresses the RUNX3 gene, thereby suppressing tumor growth and inducing cell death through the RUNX3 gene.

[0174] The above 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 described above should be understood to be illustrative and not limiting in all respects.

[0175] 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 the RUNX3 (RUNt-related transcription factor 3) protein between a first inverted terminal repeat (ITR) and a second inverted terminal repeat, 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 regions is deformed in either the first ITR or the second ITR.

2. The adeno-associated virus complex according to claim 1, comprising an SPC (Surfactant Protein C) promoter operably linked between a first ITR and a second ITR, a polynucleotide sequence encoding the RUNX3 protein, 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 an 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 the sequence of a stem-loop structure consisting of the RBE (rep-binding element), 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 1, wherein the first ITR is an AAV wild-type ITR, and the second ITR is essentially composed of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 9.

10. The adeno-associated virus complex according to claim 1, wherein the first ITR is an AAV wild-type ITR, and the second ITR is essentially composed of the nucleotide sequence of SEQ ID NO:

1.

11. The adeno-associated virus complex according to claim 1, further comprising a gene junction containing Sequence ID No. 10 between the nucleotide sequences that encrypt the SPC promoter and RUNX3.

12. A method for treating KRAS-mutated lung cancer, comprising the step of administering an effective amount of the adeno-associated virus complex described in claim 1.

13. The treatment method according to claim 12, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

14. The treatment method according to claim 13, wherein the non-small cell lung cancer is selected from the group consisting of squamous cell carcinoma, large cell carcinoma, and lung adenocarcinoma.

15. A pharmaceutical composition for the prevention or treatment of KRAS-mutated lung cancer, comprising the adeno-associated virus complex described in claim 1.

16. The pharmaceutical composition according to claim 15, further comprising a pharmaceutically acceptable carrier.

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