Adeno-associated virus complex with enhanced expression of the RUNX3 gene, use thereof for the treatment of KRAS mutation lung cancer and pharmaceutical composition.

Description

1 / 37 Adeno-associated virus complex with enhanced expression of the RUNX3 gene, use thereof for the treatment of cancer of LUNG WITH KRAS MUTATION AND PHARMACEUTICAL COMPOSITION Technical Field

[001] The present disclosure relates to an adeno-associated virus complex with enhanced expression of RUNX3, and uses for the prevention or treatment of KRAS-mutated lung cancer. Technical Background

[002] A KRAS gene is a substance involved in the function of a GTP enzymatic protein, which plays an important role in a signal transduction system related to cell differentiation, proliferation, and survival. When a signal is transmitted in a cellular unit, a KRAS protein binds to GTP and helps cancer cells grow.

[003] KRAS mutations are relatively common oncogenic mutations found in about 20% of solid cancers. In particular, this mutation is most commonly found in adenocarcinomas of the pancreas and colon, lung cancer, and similar cancers. Specifically, when observing the prognosis after treatment of patients with lung cancer, pancreatic cancer, colon cancer, etc., caused by a KRAS mutation, most patients have a worse prognosis than patients without a KRAS mutation. Furthermore, unlike other non-small cell lung cancers (NSCLCs), all therapies targeting cancer with the KRAS mutation have been discontinued in clinical trials due to side effects.

[004] On the other hand, an AAV gene delivery vehicle is safe as a delivery vehicle derived from a non-pathogenic human virus and has a wide host range without inducing a cellular immune response. Furthermore, an AAV gene delivery vehicle is capable of delivering genes to Petition 870250120661, dated 12 / 29 / 2025, page 13 / 94 2 / 37 undivided cells and dividing cells, and in particular, the expression of a gene released by an AAV gene delivery vehicle is characterized by long-term persistence in vivo.

[005] However, due to its inverted terminal repeat (ITR), AAV has a problem with low DNA packaging capacity, because a protein-coding sequence of up to about 4.4 kb can be encapsulated. Furthermore, it is preferable that a gene therapy for anticancer treatment not be expressed for a long period of time, but that the introduced gene disappear simultaneously with the death of cancer cells.

[006] Therefore, there is a need to develop, as a gene delivery system for anticancer therapy, an AAV complex useful for the prevention or treatment of KRAS-mutated solid cancers, in which the AAV complex, due to the ITR modification, which is a characteristic of AAVs, has: improved DNA packaging capacity; lower probability of being inserted into the chromosome of infected cells; improved productivity and expression efficiency; and selectivity in eliminating KRAS-mutated solid cancers. Revealing the Invention Technical Problem

[007] In one embodiment, the present invention describes an adeno-associated virus (AAV) complex comprising a polynucleotide sequence encoding a runt-related transcription factor 3 (RUNX3) protein between a first inverted terminal repeat (ITR) and a second ITR, wherein in either the first ITR or the second ITR, all or part of a stem-loop structure, which is formed by a repeat-binding element (RBE), RBE', A, A', B, B', C, C' and D regions, is modified. The adeno-associated virus complex may comprise an SPC promoter. Petition 870250120661, dated 12 / 29 / 2025, page 14 / 94 Operationally linked, 3 / 37 consists of a polynucleotide sequence encoding a RUNX3 protein and a polyadenylation sequence between the first and second ITRs. The AAV can be of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In certain embodiments, the first ITR is not modified and the second ITR is modified. The stem-loop structure modification can be insertion, deletion, or substitution. In the complex, either the first or second ITR can be modified to not form a stem-loop structure. In the complex, in either the first ITR or the second ITR, all or part of a stem-loop structure, which is formed by rep-linked elements (RBE), regions RBE', A, A', B, B', C, C' and D, can be deleted.Where either the first ITR or the second ITR comprises a terminal resolution site (trs) sequence and an RBE sequence, and exhibits deletions of all C, C', B', B, and RBE' sequences, as well as the A or A' region, whichever is closest to the 3' end of the (+) strand of the transgene or the 5' end of the (-) strand of the transgene after RBE. In the complex, the first ITR may be a wild-type AAV ITR, and the second ITR may consist of, or consist essentially of, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 9. In the complex, the first ITR may be a wild-type AAV ITR, and the second ITR may consist of, or consist essentially of, a nucleotide sequence of SEQ ID NO: 1. The adeno-associated virus complex may additionally comprise a gene junction comprising SEQ ID NO: 10 between the SPC promoter and the nucleotide sequence encoding RUNX3.

[008] In another embodiment, the present invention describes a method for treating KRAS-mutant lung cancer, comprising administering an effective amount of the adeno-associated virus complex. Petition 870250120661, dated 12 / 29 / 2025, page 15 / 94 4 / 37 described in the present invention to an individual in need thereof. Lung cancer can be non-small cell lung cancer or small cell lung cancer, where non-small cell lung cancer can be selected from the group consisting of squamous cell carcinoma, large cell carcinoma and lung adenocarcinoma.

[009] In yet another embodiment, a pharmaceutical composition for the prevention or treatment of KRAS-mutant lung cancer is described in the present invention, comprising the adeno-associated virus complex described in the present invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Solution to the Problem

[010] Detailed reference will now be made to the embodiments, which are illustrated in the accompanying drawings, where similar reference numbers refer to similar elements throughout the document. In this sense, the present embodiments may have different forms and should not be interpreted as limited to the descriptions set forth in the present invention. Thus, the embodiments are merely described below, with reference to the figures, to explain aspects of the present description. As used in the present invention, the term and / or includes any and all combinations of at least one of the associated listed items. Expressions such as at least one of, when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[011] Several modalities will be presented in part in the following description and, in part, will be apparent from the description or can be learned by practicing the presented modalities of revelation.

[012] In one embodiment, an adeno-associated virus complex including a modified inverted terminal repeat is described in the present invention. Petition 870250120661, dated 12 / 29 / 2025, page 16 / 94 5 / 37

[013] In another embodiment, a cell transformed by the adeno-associated virus complex is described in the present invention.

[014] In yet another embodiment, the present invention describes a method for treating cancer with KRAS mutation, including administering an effective amount of the described adeno-associated virus complex.

[015] In yet another embodiment, the present invention describes a pharmaceutical composition for the prevention or treatment of KRAS-mutant lung cancer, including the adeno-associated virus complex.

[016] In yet another embodiment described in the present invention is a use of the adeno-associated virus complex for the preparation of a therapeutic agent for the treatment of KRAS-mutant lung cancer.

[017] In another embodiment, an adeno-associated virus (AAV) complex including a modified inverted terminal repeat (ITR) is described in the present invention.

[018] The term adeno-associated virus (AAV), as used in the present invention, refers to a single-stranded DNA virus with a genome size of about 4.6 kbp, which is a human parvovirus dependent on a helper vector. The genome consists of ITRs at both ends and two open reading frames (ORFs), rep and cap. The N-terminal region of the genome encodes a rep gene involved in viral replication and viral gene expression, and the C-terminal region encodes a cap gene that encodes a viral capsid protein. The ITRs are involved in the replication of an AAV genome and the packaging of AAV particles. The ITR includes a rep-binding element (RBE), RBE', A, A', B, B', C, C', and D regions to form a stem-loop structure. The structure of AAV ITRs is well described, for example, in Goncalves, MA Virology Journal, 2(1):43 (2005), which is incorporated into the present invention by reference. Petition 870250120661, dated 12 / 29 / 2025, page 17 / 94 6 / 37

[019] In certain embodiments, the ITR sequence may be based on an ITR sequence of a virus belonging to the genus Dependovirus of the family Parvoviridae.

[020] In certain alternative embodiments, the ITR sequence may be based on an AAV ITR sequence. The AAV ITR sequence is publicly known.

[021] AAV includes, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, etc., and may also include other AAV serotypes currently known or to be discovered later. AAV may include derivatives of known AAV. AAV may include modified or artificial AAV.

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

[023] Alternatively, other types of viruses belonging to the genus Dependovirus of the family Parvoviridae may be used in place of AAV.

[024] The term AAV complex, used in the present invention, can be used interchangeably with AAV vector, AAV delivery vehicle, recombinant AAV and recombinant AAV vector.

[025] In certain embodiments, the AAV complex includes a polynucleotide sequence that encodes a runt-related transcription factor 3 (RUNX3) protein between the first ITR and the second ITR.

[026] The polynucleotide sequence that codes for a RUNX3 protein Petition 870250120661, dated 12 / 29 / 2025, page 18 / 94 7 / 37 can be operationally organized between the first ITR and the second ITR.

[027] In certain embodiments, the AAV complex genome includes a first ITR (5'-ITR), an SPC promoter, a polynucleotide sequence encoding the RUNX3 protein, a polyadenylation sequence, and a second ITR (3'-ITR) in a 5' to 3' direction.

[028] In certain embodiments, the AAV complex includes a polynucleotide sequence that encodes a 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, which is formed by the RBE, RBE', A, A', B, B', C, C' and D regions, may be modified.

[029] In an embodiment described in the present invention, the AAV complex includes an operationally linked surfactant protein C (SPC) promoter, a polynucleotide sequence encoding a RUNX3 protein, and a polyadenylation sequence between a first ITR and a second ITR.

[030] In certain embodiments, an AAV complex genome includes a first ITR (5'-ITR), an SPC promoter, a polynucleotide sequence encoding a RUNX3 protein, a polyadenylation sequence, and a second ITR (3'-ITR) in a 5' to 3' direction.

[031] In certain other embodiments, the AAV complex includes an operationally linked SPC promoter, a polynucleotide sequence encoding a RUNX3 protein and a polyadenylation sequence, between the first ITR and the second ITR, and in either of the first ITR and the second ITR, all or part of a stem-loop structure, which is formed by the RBE, RBE', A, A', B, B', C, C' and D regions, may be modified.

[032] In certain other embodiments, the AAV complex may include an asymmetrically modified ITR. In one embodiment, the AAV complex may have either a first ITR or a second modified ITR. Petition 870250120661, dated 12 / 29 / 2025, p. 19 / 94 8 / 37 In another embodiment, the AAV complex may have a first modified ITR and a second unmodified ITR. In another embodiment, the AAV complex may have a first unmodified ITR and a second modified ITR. In another embodiment, the AAV complex may have an unmodified 5'-ITR of the (+) strand of a target gene and a modified 3'-ITR of the (+) strand. In another embodiment, the AAV complex may have a modified 5'-ITR of the (-) strand of the target gene and an unmodified 3'-ITR of the (-) strand. In other words, the fact that the second ITR is modified may mean that the 3'-ITR of the (+) strand and / or the 5'-ITR of the (-) strand of the target gene is modified. An AAV complex, according to one aspect, may have higher AAV complex productivity and a higher RUNX3 gene expression rate, including the asymmetrically modified ITR.

[033] Between the first ITR and the second ITR, the unmodified ITR may be a wild-type ITR. Between the first ITR and the second ITR, the unmodified ITR may be a wild-type ITR AAV.

[034] Between the first ITR and the second ITR, the unmodified ITR may be a functional derivative with substantially the same functional properties as a wild-type ITR (e.g., wild-type ITR AAV).

[035] The term functional derivative, as used in the present invention, may mean a derivative with substantially the same functional properties. The derivative refers to a similar compound obtained by chemically altering a part of the structure of a compound. The derivative may refer to a compound in which a hydrogen atom or a specific atomic group in a compound is replaced by another atom or atomic group. A method for preparing derivatives of a compound while maintaining substantially the same functional properties is known in the art.

[036] In either the first ITR or the second ITR, all or part of a stem-loop structure, which is formed by linking elements to rep Petition 870250120661, dated 12 / 29 / 2025, page 20 / 94 9 / 37 (RBE), regions RBE', A, A', B, B', C, C' and D, is modified.

[037] In one embodiment, the modification of the stem-loop structure can be selected from insertion, deletion, and replacement.

[038] In one embodiment, the modification of the stem-loop structure includes modification to include a single stem and a single loop. For example, the modified ITR may include the deletion of a BB' arm so that a CC' arm remains, or the deletion of the CC' arm so that the BB' arm remains.

[039] In one embodiment, the modification of the stem-loop structure includes modification to include a single stem instead of two loops. For example, a modified ITR might include the deletion of a BB' arm and a C-C' arm.

[040] In one embodiment, the modified ITR may include a deletion of a C' region so that a truncated C-loop and a BB' arm remain. Similarly, the modified ITR may include a deletion of a B region so that a truncated B-loop and a CC' arm remain.

[041] In one embodiment, the modified ITR may include a deletion of a base pair in at least one selected portion from a C portion, a C' portion, a B portion, or a B' portion, so that a single arm can be formed, since complementary base pairs occur between a C portion and a B' portion and between a C' portion and a B portion.

[042] In one embodiment, the modified ITR may include a modification (e.g., deletion, substitution, or addition) of 1, 2, 3, 4, 5, or 6 nucleotides in at least one region selected from between A' and C, between C and C', between C' and B, between B and B', and between B' and A.

[043] In one embodiment, the modification of the stem-loop structure Petition 870250120661, dated 12 / 29 / 2025, page 21 / 94 10 / 37 (hairpin structure) may involve a modification of the structure of a structural element. Specifically, the modification of the structure of a structural element may include a change in the height of a stem and / or a change in the number of nucleotides in a loop. For example, the stem height may be approximately 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range of nucleotides contained therein. In another example, the loop may have approximately 3, 4, 5, 6, 7, 8, 9, 10 nucleotides or more, or any range of nucleotides contained therein.

[044] In another embodiment, by altering (for example, increasing or decreasing) a distance between two elements (as a non-limiting example, RBE and a staple), the functional interaction with a large Rep protein can be altered. For example, the distance can be about 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 contained therein.

[045] In one embodiment, either the first ITR or the second ITR can be modified so as not to form a stem-loop structure. In another embodiment, the first ITR can be modified so as not to form a stem-loop structure. In another embodiment, the second ITR can be modified so as not to form a stem-loop structure. In another embodiment, the first ITR cannot be modified, and the second ITR can be modified so as not to form a stem-loop structure.

[046] The expression modified to not form a stem-loop sequence (hairpin structure) may mean that an ITR structure is modified to exist as an open or free end without forming a stem-loop structure (hairpin structure), due to a modification of an ITR sequence. An aspect-according AAV complex is modified so that neither the first ITR nor the second ITR forms a Petition 870250120661, dated 12 / 29 / 2025, page 22 / 94 11 / 37 stem-loop structure, to suppress the formation of a circular dimer and a circular concatemer in infected cells and to suppress integration into the host genome, which is observed in AAV. Furthermore, the productivity of the AAV complex and the expression rate of the RUNX3 gene can be increased by the modification.

[047] In one embodiment, either the first ITR or the second ITR can be modified to a blunt end. In another embodiment, either the first ITR or the second ITR can be modified to an adhesive end. In another embodiment, the second ITR can be modified to a blunt end or a sticky end. In another embodiment, the second ITR can be modified to a blunt end. The modification to a blunt end or a sticky end can be performed using a method known to a person skilled in the art.

[048] In one embodiment, in either the first ITR or the second ITR, all or part of a stem-loop structure, which is formed by the regions RBE, RBE', A, A', B, B', C, C', and D, may be deleted. In another embodiment, in either the first ITR or the second ITR, part of a stem-loop structure, which is formed by the regions RBE, RBE', A, A', B, B', C, C', and D, may be deleted. In yet another embodiment, either the first ITR or the second ITR may include a terminal resolution site (trs) sequence and an RBE sequence, and may exhibit deletions of all sequences C, C', B', B, and RBE', as well as the A or A' region, whichever is closest to the 3' end of the (+) strand of the transgene or the 5' end of the (-) strand of the transgene after RBE.In another embodiment, the first ITR may not be modified, and the second ITR may include a sequence of trs and a sequence of RBE, and may exhibit deletions of all sequences C, C', B', B and RBE', as well as the A or A' region, whichever is closest to the 3' end of the tape. Petition 870250120661, dated 12 / 29 / 2025, p. 23 / 94 12 / 37 (+) of the transgene or the 5' end of the (-) strand of the transgene after RBE.

[049] In another embodiment, either the first ITR or the second ITR may include, consist essentially of, or consist of any nucleotide sequence selected from the sequences with SEQ ID NOs: 1 to 9, or a sequence complementary thereto. In another embodiment, the first ITR may not be modified, and the second ITR may include, consist essentially of, or consist of any nucleotide sequence selected from the sequences with SEQ ID NOs: 1 to 9, or a sequence complementary thereto.

[050] The sequence with SEQ ID NO: 1 may be based on an ITR sequence from AAV2.

[051] The sequence with SEQ ID NO: 2 may be based on an ITR sequence from AAV1.

[052] The SEQ ID NO: 3 sequence may be based on an ITR sequence of AAV3.

[053] The sequence with SEQ ID NO: 4 may be based on an ITR sequence from AAV4.

[054] The sequence with SEQ ID NO: 5 may be based on an ITR sequence from AAV6.

[055] The sequence with SEQ ID NO: 6 may be based on an ITR sequence from AAV7.

[056] The sequence with SEQ ID NO: 7 may be based on an ITR sequence from AAV5.

[057] The SEQ ID NO: 8 sequence may be based on an ITR sequence of AAV8.

[058] The SEQ ID NO: 9 sequence may be based on an ITR sequence of AAV9. Petition 870250120661, dated 12 / 29 / 2025, p. 24 / 94 13 / 37

[059] SEQ ID sequences NOS: 1 to 9 may have a portion of an AAV ITR sequence deleted. SEQ ID sequences NOS: 1 to 9 may include a trs sequence and an RBE sequence between the AAV ITR sequences. SEQ ID sequences NOS: 1 to 9 may exhibit deletions of all C, C', B', B and RBE' sequences, as well as the A or A' region, whichever is closest to the 3' end of the (+) strand of the transgene or the 5' end of the (-) strand of the transgene after RBE.

[060] In general, an RNA polymerase generates mRNA with a sequence complementary to a target gene in a promoter region. The process is called transcription, and transcription occurs in the 5' to 3' direction. Meanwhile, when a target gene is inserted into the AAV complex, the gene is inserted in the 5' to 3' and 3' to 5' directions, respectively, relative to the DNA double helix of the AAV complex. Therefore, while transcription of the target gene is underway, transcription of the target gene occurs in both directions due to the DNA double helix of the AAV complex, and thus there is a problem that the efficiency of target gene expression is reduced due to interference.However, an aspect-specific AAV complex can increase the expression efficiency of a target gene by preventing interference from the double helix structure of the AAV complex's DNA during target gene transcription, through a modification of the stem-loop structure, specifically, by deleting all or part of the stem-loop structure.

[061] For example, in an AAV complex including an asymmetrically modified ITR, a first ITR may not be modified, and a second ITR may be modified not to form a stem-loop structure. Consequently, the 5'-ITR of the (-) strand of the RUNX3 gene does not form a hairpin structure, and transcription of the RUNX3 gene proceeds complementarily to the corresponding strand in the 5' to 3' direction. Meanwhile, on the (+) strand of the gene Petition 870250120661, dated 12 / 29 / 2025, page 25 / 94 14 / 37 In RUNX3, the 3'-ITR does not form a hairpin structure, and transcription of the RUNX3 gene does not occur on the corresponding strand. That is, since only transcription in the 5' to 3' direction of the released gene proceeds, and a competitor in the 3' to 5' direction is removed, the efficiency of gene expression can be increased.

[062] In one example, in an AAV vector including a RUNX3 gene, an AAV complex (Example 1) including an asymmetrically modified ITR was prepared by partially modifying a hairpin structure of a second ITR between wild-type AAV ITRs included in the vector. The prepared AAV complex was compared with an AAV complex (Comparative Example 1), in which the hairpin structure is not modified, and with an AAV complex (Comparative Example 2) modified so that both ends of the hairpin structure are symmetrical, and the productivity and expression rate of the RUNX3 gene of each AAV complex were confirmed.As a result, it was confirmed that the AAV complex (Example 1), including the asymmetrically modified ITR, had viral productivity increased by 3 times or more, and a RUNX3 gene expression rate increased by 6 times or more, compared to the AAV complex without ITR modification (Comparative Example 1) and the AAV complex containing the symmetrically modified ITR (Comparative Example 2).

[063] The term runt-related transcription factor protein 3 (RUNX3), used in the present invention, refers to a protein expressed by a RUNX3 gene and is a member of the RUNX family, composed of RUNX1, RUNX2, and RUNX3. Genes belonging to the RUNX family play an important role in normal development and tumorigenesis, and function as regulatory factors for the transcription of TGF-β and the Smad family, a subfactor that mediates TGF-β signal transduction.

[064] The RUNX3 protein may include at least one sequence of Petition 870250120661, dated 12 / 29 / 2025, page 26 / 94 15 / 37 amino acids selected from SEQ ID NO: 19 and SEQ ID NO: 20. Additionally, a polynucleotide sequence encoding the RUNX3 protein can be selected from SEQ ID NOS: 21 and 22.

[065] The RUNX3 protein can be derived from a human or an animal.

[066] The RUNX3 protein can be synthesized by a chemical synthesis method (WH Freeman and Co., Proteins; structures and molecular principles, 1983) in the art and can be prepared by a genetic engineering method (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual; etc.) in the art.

[067] The RUNX3 protein may be an amino acid variant with a different sequence due to deletion, insertion, substitution, or a combination of the same amino acid residues, within a range that does not affect the protein's function. Amino acid exchanges in proteins that do not completely alter the activity of the molecules are known in the art. In some cases, amino acids may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or similar processes.

[068] Therefore, in one embodiment, the RUNX protein may include a peptide and a variant or fragment thereof, the peptide having an amino acid sequence substantially identical to a protein including any one or more amino acid sequences selected from SEQ ID NOS: 19 and 20. The substantially identical protein may have homology of 80% or more, specifically 90% or more, or more specifically 95% or more, with the RUNX3 protein.

[069] In one embodiment, the AAV complex may additionally include post-transcriptional regulatory elements. The AAV complex Petition 870250120661, dated 12 / 29 / 2025, page 27 / 94 16 / 37 may include a first ITR, an SPC promoter sequence, a polynucleotide sequence encoding a RUNX3 protein, post-transcriptional regulatory elements, a polyadenylation sequence, and a second ITR in the 5' to 3' direction.

[070] The post-transcriptional regulatory element may include a post-transcriptional regulatory element of the marmot hepatitis virus (WPRE).

[071] In one embodiment, the AAV complex may additionally include a gene junction between the SPC promoter and the polynucleotide sequence encoding the RUNX3 protein.

[072] The term gene junction used in the present invention refers to a sequence that is not defined and is located between the end of a promoter and the beginning of a target gene sequence. Specifically, a promoter is a site where the transcription machinery is intricately linked to regulate genes, and a boundary, between the end of a known promoter sequence and the initial region of a gene whose expression is to be controlled, is generally ambiguous. Thus, optimization of the junction may be necessary to build a successful relationship between promoter and gene expression.

[073] Therefore, to regulate the expression of the RUNX3 gene by an SPC promoter, the present inventors found optimized sequences by replacing them with various polynucleotide combinations and confirmed that a polynucleotide in a range of 22 bp to 28 bp was suitable. Among the optimized sequences, as the promoter DNA and RUNX3 DNA sequences can be prepared by PCR using primers designed to have restriction enzymes that are not included in the vector body or the DNA sequence to be amplified, the junctions, which have an additional site for a restriction enzyme that does not cleave the vector, the promoter or the gene sequences, were finally selected. Petition 870250120661, dated 12 / 29 / 2025, p. 28 / 94 17 / 37

[074] Thus, in one embodiment, gene junction may include SEQ ID NO: 11. Furthermore, gene junction may determine whether or not the RUNX3 gene should be expressed or affect the expression efficiency of the RUNX3 gene, depending on its length or structure.

[075] The AAV complex can be designed to encode selectable markers or reporters that provide means for selection or identification of cells that have incorporated them. Selectable markers or reporters are known in the art. Non-limiting examples of selectable markers include genes that confer resistance to ampicillin, streptavidin, kanamycin, hygromycin, and the like. Non-limiting examples of reporters include luciferase, green fluorescent protein (GFP), and the like.

[076] Another aspect provides a cell transformed by an adeno-associated virus complex according to one aspect.

[077] Details of the adeno-associated virus complex are described above.

[078] The term transformation, as used in the present invention, means that the genetic properties of an organism are altered by DNA introduced from outside. Transformation is a phenomenon in which DNA enters a cell and alters hereditary characteristics when DNA, a type of nucleic acid extracted from a cell line of one organism, is injected into a living cell of another cell line. That is, transformation means introducing a gene into a host cell so that it can be expressed in the host cell.

[079] In certain embodiments, a method of transforming a cell line by introducing an AAV complex may be a method known in the art, for example, transient transfection using lipofectamine, etc. microinjection, transduction, cell fusion, precipitation of Petition 870250120661, dated 12 / 29 / 2025, page 29 / 94 18 / 37 calcium phosphate, liposome-mediated transfection, DEAE-mediated dextran transfection, polybrene-mediated transfection, electroporation, etc., but it is not limited to these, and preferably, the lipofectamine 2000 reagent can be used for transformation.

[080] Another aspect provides a method of preventing or treating lung cancer, including administering an effective amount of the adeno-associated virus complex according to an aspect to a cell or individual.

[081] Details of the adeno-associated virus complex are described above.

[082] In the method described in the present invention, the AAV complex can be administered to an individual by itself, or the AAV complex can be formulated in a form administrable to an individual and administered to the individual. In one embodiment, the AAV complex can be administered to an individual in the form of a composition including an AAV complex according to the following aspect. For example, the AAV complex can be formulated in a composition including the AAV complex and a pharmaceutically acceptable carrier and administered to an individual.

[083] The individual may need the expression of a RUNX3 gene provided by the AAV complex. The individual may suffer from, or be highly likely to suffer from, a disease to which gene therapy is applicable. The individual may suffer from, or be highly likely to suffer from, a disease that can be treated by the expression of a RUNX3 gene administered by an AAV complex. The individual may be a mammal, such as a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The individual may be someone who suffers from, or is highly likely to suffer from, cancer. Petition 870250120661, dated 12 / 29 / 2025, page 30 / 94 19 / 37

[084] Lung cancer can be a KRAS-mutant lung cancer.

[085] The term 'KRAS-mutant lung cancer', as used in the present invention, refers to lung cancer in which a mutated KRAS gene is activated and cancer suppressor genes are inactivated. When cancer suppressor gene activity is restored, as lung cancer cells are removed and normal cells remain, KRAS-mutant lung cancer can be treated. The cancer suppressor gene may be, for example, the sPD-1, VHL, MMAC1, DCC, p53, NF1, WT1, Rb, BRAC1, BRAC2 or RUNX3 genes.

[086] In one modality, lung cancer can be non-small cell lung cancer or small cell lung cancer. Non-small cell lung cancer includes, for example, squamous cell carcinoma, large cell carcinoma, lung adenocarcinoma, and the like.

[087] In one example, it was confirmed that when a recombinant AAV including a RUNX3 gene was injected into mouse models of non-small cell lung cancer in which the KRAS mutation was activated, lung cancer cell death was promoted and cancer development was inhibited. Furthermore, as a result of infecting a normal lung epithelial cell line and a non-small cell lung cancer cell line, it was confirmed that RUNX3 and cell death markers increased significantly in the lung cancer cell line, while there was no change in the cell death rate in normal lung epithelial cells.

[088] Therefore, an AAV complex according to one aspect can specifically remove lung cancer cells by activating the RUNX3 gene. Petition 870250120661, dated 12 / 29 / 2025, page 31 / 94 20 / 37 in lung cancer caused by decreased activity of RUNX3 proteins and, therefore, can be used for prevention or treatment of KRAS-mutant lung cancer. Furthermore, recurrence of KRAS-mutant lung cancer can be prevented.

[089] The term 'prevention', as used in the present invention, refers to all actions that suppress or delay the onset of a disease by administering the AAV complex. The term 'treatment' refers to all actions that improve or beneficially alter the symptoms of a disease by administering the AAV complex.

[090] In certain embodiments, the method may additionally include the administration of a second active ingredient to the individual. The second active ingredient may be an active ingredient for the prevention or treatment of lung cancer. The active ingredient may be administered concomitantly, separately, or sequentially with the AAV complex.

[091] The AAV complex can be formulated into an injectable formulation suitable for administration by any suitable route, such as intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular or intrathecal, and can be administered to an individual. The AAV complex can be administered systemically or locally and can be administered alone or in combination with other pharmaceutically active compounds.

[092] A preferred dosage of the AAV complex may vary depending on the patient's condition and body weight, the severity of the disease, the formulation of the therapeutic agent, the route and duration of administration, etc., and may be appropriately selected by those skilled in the art. In one embodiment, a dosage of the AAV complex may be Petition 870250120661, dated 12 / 29 / 2025, page 32 / 94 21 / 37 approximately 1.0x10⁶vg / kg to approximately 1.0x10¹⁶vg / kg, approximately 1.0x10⁸vg / kg to approximately 1.0x10¹⁶vg / kg, approximately 1.0x10¹⁰vg / kg to approximately 1.0x10¹⁶vg / kg, approximately 1.0x10¹⁰vg / kg to approximately 1.0x10¹⁴vg / kg, approximately 1.0x10¹²vg / kg to approximately 1.0x10¹⁴vg / kg, for example, approximately 1.0x10¹²vg / kg, approximately 1.0x10¹³vg / kg or approximately 1.0x10¹⁴vg / kg. In one particular dosage, the AAV complex may be approximately 1.0 x 10¹³ g / kg. Administration may be once daily, several times daily, once weekly, once every 2 weeks, once every 3 weeks, or once every 4 weeks up to once a year.

[093] The term about, as used in the present invention, is used to include a range of ±10% of a designated numerical value.

[094] Another aspect provides a pharmaceutical composition for the prevention or treatment of lung cancer, including an adeno-associated virus complex, according to one aspect. Yet another aspect provides a use of an adeno-associated virus complex according to one aspect, for the preparation of a therapeutic agent for lung cancer.

[095] Details of the adeno-associated virus complex and lung cancer are described above.

[096] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier includes an excipient, diluent, or auxiliary agent. As the carrier, a suitable carrier may be used to deliver the AAV complex into a living body. Specifically, for the carrier, a suitable carrier for formulation in a parenteral formulation (e.g., an injection formulation) may be selected. For example, for the carrier, a suitable carrier for formulation in an intravenous formulation may be selected. The carrier may be an aqueous solution, such as water or buffered saline solution and the like. Petition 870250120661, dated 12 / 29 / 2025, page 33 / 94 22 / 37

[097] The pharmaceutical composition may be prepared in any formulation according to a method in the art. The composition may be formulated in 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 in a buffered saline solution. The composition may be formulated, for example, as a parenteral formulation (e.g., as an injection, for example, for bolus injection or continuous infusion). In one embodiment, the pharmaceutical composition may be formulated as an injectable formulation suitable for administration by any suitable route, such as intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, or intrathecal. In a particular embodiment, the composition may be formulated for administration by intravenous injection or subcutaneous injection. Furthermore, the composition may be prepared as a systemic formulation or a topical formulation.The composition may be supplied as ampoules, pre-filled syringes, small injection containers, or as a unit-dose form in multidose containers with added preservatives.

[098] In certain embodiments, the pharmaceutical composition may additionally include one or more anticancer agents. Exemplary anticancer agents 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 separate compositions. For example, the antibody composition or antigen-binding fragments thereof may be a composition of a parenteral dosage form, and the anticancer agent may be a composition of an oral dosage form.

[099] The pharmaceutical composition may contain an effective amount of Petition 870250120661, dated 12 / 29 / 2025, page 34 / 94 23 / 37 AAV complex. The term effective amount refers to an amount that is sufficient to bring about a desired preventive or therapeutic effect when administered to an individual who needs it. The effective amount may be selected by the person skilled in the art depending on the cell or individual. The effective amount may be determined according to the severity of the disease, age, weight, health, sex and sensitivity to a therapeutic agent of a patient, time of administration, route of administration, excretion rate, duration of treatment, factors including therapeutic agents used in combination with or concomitantly with the composition used and other factors well known in the medical field.

[100] In one embodiment, the pharmaceutical composition may include the AAV complex at a dosage of about 1.0x106 vg / kg to about 1.0x1016 vg / kg, about 1.0x108 vg / kg to about 1.0x1016 vg / kg, about 1.0x1010 vg / kg to about 1.0x1016 vg / kg, about 1.0x1010 vg / kg to about 1.0x1014 vg / kg, about 1.0x1012 vg / kg to about 1.0x1014 vg / kg, for example, about 1.0x1012 vg / kg, about 1.0x1013 vg / kg or about 1.0x1014 vg / kg. In one particular embodiment, the pharmaceutical composition may include an AAV complex at a dosage of approximately 1.0 x 10¹³ g / kg. Administration may be once daily, several times daily, once weekly, once every 2 weeks, once every 3 weeks, or once every 4 weeks up to once a year. Advantageous Effects of the Invention

[101] The adeno-associated virus complex according to one aspect has asymmetric ITRs in which one of the two ITRs is modified, thus increasing the efficiency of self-replication in host cells and increasing the efficiency of expression of a released gene and, therefore, compared to existing AAV complexes, the adeno-associated virus complex has a Petition 870250120661, dated 12 / 29 / 2025, page 35 / 94 24 / 37 advantage in productivity and improved gene expression efficiency. Brief Description of the Drawings

[102] The described modalities, characteristics and advantages of certain modalities of revelation will be more evident from the following description, taken together with the attached drawings.

[103] FIG. 1 shows a cleavage map of an adeno-associated virus (AAV) vector according to an example.

[104] FIG. 2 shows a structure of an AAV vector according to an example.

[105] FIG. 3A is a schematic diagram of an AAV vector genome including an asymmetrically modified ITR from Example 1.

[106] FIG. 3B is a schematic diagram of an AAV vector genome including an unmodified symmetric ITR from Comparative Example 1.

[107] FIG. 3C is a schematic diagram of an AAV vector genome including a symmetrically modified ITR from Comparative Example 2.

[108] FIG. 4A shows the results of the Western blot confirmation of the expression of a target gene in an AAV complex according to one aspect, 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 cell 293: HEK293 cell line not infected with AAV complex, empty: culture medium without any addition (double negative control), positive control: purified RUNX3 protein.

[109] FIG. 4B shows the results of quantifying the expression rate of a target gene in an AAV complex according to an aspect, C1: AAV complex from Comparative Example 1, C2: AAV complex from Comparative Example 2, WT: wild-type AAV complex, RX001: Petition 870250120661, dated 12 / 29 / 2025, page 36 / 94 25 / 37 AAV complex from Example 1, control cell 293: HEK293 cell line not infected with AAV complex, empty: culture medium with no addition of anything (double negative control), positive control: purified RUNX3 protein.

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

[111] FIG. 6A shows photos taken with a microscope 1 day, 3 days and 5 days after infection of KRAS-mutant lung cancer cell lines (H460, Calu6) and normal lung epithelial cells (WI38) with Example 1.

[112] FIG. 6B shows the results of a fluorescence-activated cell classification analysis (FACS), 0 hours (top), 16 hours (middle) and 32 hours (bottom) after infection of KRAS-mutant lung cancer cell lines (H460, Calu6) and normal lung epithelial cells (WI38) with Example 1.

[113] FIG. 6C shows the results of a FACS analysis 0 hours (top), 16 hours (middle) and 32 hours (bottom) after infection of a KRAS-mutant lung cancer cell line (Calu6) with Example 1.

[114] FIG. 6D shows a graph quantifying the results of FACS analyses, 1 day and 3 days after infection of a KRAS-mutant lung cancer cell line (H460) and normal lung epithelial cells (WI38) with Example 1.

[115] FIG. 6E shows the results of the detection of RUNX3 and cleaved caspase 3 expressed in KRAS-mutant lung cancer cell lines infected with Example 1.

[116] FIG. 7A shows the results of confirming the interior of cancerous tissues cut to show cross-sections, after injection of Petition 870250120661, dated 12 / 29 / 2025, page 37 / 94 26 / 37 Example 1 in non-small cell lung cancer xenograft models and reproduction for 12 days, AAV2-Control: injected with AAV having a genome that does not contain RUNX3 (AAV-empty), AAV2-SPC-RUNX3: injected with the AAV complex from Example 1.

[117] FIG. 7B shows the results of measuring cancerous tissue volumes after injection of Example 1 into non-small cell lung cancer xenograft models and reproduction for 12 days; Con1~4: mice treated with AAV-control (Empty), DI1~4: mice treated with AAV2-SPC-RUNX3 (RX001). Best Way to Carry Out the Invention

[118] The following are preferred examples to aid understanding of the present revelation. However, the following examples are provided only to facilitate understanding of the present revelation, and the content of the present revelation is not limited by the following examples. EXAMPLES Example 1. Preparation of an adeno-associated virus complex including asymmetrically modified ITR, for RUNX3 gene expression. 1-1. Preparation of an adeno-associated virus vector introduced with the RUNX3 gene and SPC promoter.

[119] Adeno-associated virus (AAV) vectors introduced with a RUNX3 gene were prepared. Specifically, after synthesizing the human RUNX3 gene (NCBI reference: NM_004350.2, 412 nt to 1659 nt), PCR amplification was performed using primers shown in Table 1 below. In this regard, the primers were prepared by synthesizing the restriction enzymes KpnI and HindlII. In addition, a surfactant protein C (SPC) promoter (GenBank accession number AC122268, 148366 nt to 149406 nt), specifically expressed in lung epithelial cells, was amplified by Petition 870250120661, dated 12 / 29 / 2025, page 38 / 94 27 / 37 PCR was performed using the primers shown in Table 1 below. The primers were prepared by synthesizing the restriction enzymes NheI and KpnI. Subsequently, each of the amplified RUNX3 and SPC promoter DNAs was treated with the restriction enzyme KpnI, and the KpnI sites were ligated using T4 DNA ligases to prepare NheI-SPC-KpnI-RUNX3HindIII DNA strands. Then, an NheI-SPC-KpnI-RUNX3-HindIII DNA strand was cloned by removing a GFP gene present at an NheI-HindIII site from a multiple cloning site (MCS) of adeno-associated virus (AAV) 2 GFP vectors (National Chungbuk University Tumor Research Institute). To induce SPC promoter expression in AAV2-GFP vectors, chicken beta-actin promoters were removed using the restriction enzymes NdeI and Bglll.Next, in the AAV2-SPC-RUNX3 plasmids from which the chicken beta-actin promoters were removed, the ampicillin resistance genes were removed using BspHI restriction enzyme sites present at both ends of the ampicillin resistance genes. A kanamycin resistance gene was inserted into a site from which the ampicillin resistance gene had been removed. The kanamycin resistance gene was then recombined using PCR primers as shown in Table 1 below. [Table 1] Gene SEQ ID NO: Direction Sequence RUNX3 11 Forward 5'- tggtaccgcggccaccatgcgtattcccgtaga-3' 12 Reverse 5'- aagctttactcgagtcagtagggccgccaca-3' SPC 13 Forward 5'-ctgctagcagaaggcagc-3' 14 Reverse 5'- ggtaccactagtgatatcttttgtaaggtttc-3' Kanamycin 15 Forward 5'-TG TATCCG CTCATGAG AG CTCG GTCATAG CTGTTTCCTG 3' 16 Reverse 5'- GGAII HGGTCATGAGCATGCTTAGAAAAACTCATCGAGC3' 1-2. Modification of the ITR structure

[120] Site-directed mutagenesis was induced to modify a Petition 870250120661, dated 12 / 29 / 2025, p. 39 / 94 28 / 37 part of a hairpin structure of a second ITR between wild-type AAV2 inverted terminal repeats (ITRs) included in the vectors prepared in Example 1-1. Specifically, between the wild-type AAV2 ITRs included in the vector, from a rep-linking element (RBE) of the 5'-directing ITR of the (-) strand of the RUNX3 gene, all or at least one of the C, C', B', B, RBE', and A sequences was deleted using 5'-phosphorylated primers from Table 2 below. For example, all B', B, C', C, RBE', and A sequences were deleted from the RBE of the 5'-directing ITR of the (-) strand of the RUNX3 gene. In this way, the second ITR was modified so as not to form a hairpin structure. As a result, an adeno-associated virus complex for RUNX3 gene expression was obtained, including an asymmetrically modified ITR, in which the first ITR was not modified and the second ITR was modified.

[121] FIG. 1 shows a cleavage map of an adeno-associated virus vector according to an example.

[122] FIG. 2 shows a structure of an adeno-associated virus vector according to an example.

[123] FIG. 3A is a schematic diagram of an adeno-associated virus vector genome including an asymmetrically modified ITR from Example 1. [Table 2] Name SEQ ID NO: Direction Sequence 5'-Phosphorylation 17 Forward 5'-P- cactgactcgctgcgctcggtcgtt-3' 18 Reverse 5'-P- agcgagtcagtgagcgagcgagcgc-3' Comparative Examples Comparative Example 1. Preparation of the AAV complex including symmetrically unmodified ITR, for RUNX3 gene expression.

[124] An AAV complex including an unmodified symmetric ITR was prepared in the same manner as in Example 1-1, except that the structure Petition 870250120661, dated 12 / 29 / 2025, page 40 / 94 29 / 37 in the ITR clamp was not modified.

[125] FIG. 3B is a schematic diagram of an AAV vector genome including the unmodified symmetric ITR from Comparative Example 1. Comparative Example 2. Preparation of the AAV complex including symmetrically modified ITR, for RUNX3 gene expression.

[126] An AAV complex including an ITR modified so that both ends are symmetric to each other was prepared in the same way as in Example 1-2, except that a C-C'-B'-RBE sequence was deleted from both ends of the ITR of the AAV complex prepared in Example 1-1.

[127] FIG. 3C is a schematic diagram of an AAV vector genome including a symmetrically modified ITR from Comparative Example 2. Experimental Examples Experimental Example 1. Confirmation of the productivity of the AAV complex.

[128] The productivity of recombinant AAV complexes was confirmed in one respect. Specifically, the AAV complexes prepared in Example 1 and Comparative Examples 1 and 2 were transformed into 293T cells, which are human embryonic kidney cells (HEKs), and the number of viral particles expressed in the cells was measured. A wild-type AAV complex was used as a negative control group.

[129] First, 293T cells (Chungbuk National University Tumor Research Center) cultured in Dulbecco's modified Eagle medium (DMEM, Welgene, LM001-05) supplemented with 10% fetal bovine serum (Welgene, S001-01) and 1X antibiotic (Welgene, LS203-01) were split into 1 x 106 in 75 T flasks (SPL, 70075) and cultured for 24 hours. Then, for transformation, the viral complexes prepared in Example 1 Petition 870250120661, dated 12 / 29 / 2025, page 41 / 94 In Examples 30 / 37 and Comparative Examples 1 and 2, auxiliary plasmids (aldevron) and AAV2 rep / cap plasmids (aldevron) were mixed at a 1:3:1 ratio (9 μg: 27 μg: 9 μg) to prepare a mixture. Next, 100 μl of TOMTMT (Welgene, TR 004-01) was added, and after the addition of Transfection Grade Linear Polyethyleneimine Hydrochloride (PEI, MW 40,000) (Polysciences Inc, 24765-1) at a ratio of 1:2 (45:90 μl relative to the total amount of plasmids), the mixture was left at room temperature for 15 minutes. After renewing the cell culture medium, the above mixture was added, and after 48 or 72 hours, 0.5 M EDTA, pH 8.0 (TransLab, 15-10ED18), corresponding to 1 / 80 of the total volume, was added, and left to stand for 10 minutes at room temperature for cell flotation. The suspended cells were collected in a 50 ml centrifuge tube (SPL, 50050), a first centrifugation was performed for 10 minutes at 2.000 g e at 4 °C, and then a second centrifugation was performed for 1 minute under the same conditions for complete removal of the supernatant. Viral DNA was then extracted using RT-PCR, and the extracted DNA was quantified using qPCR. Table 3 shows the number of viral particles expressed in 293T cells. [Table 3] Virus Productivity (number of particles / mL) Negative control group 2.2X109 Example 1 9.6X109 Example Comparative 1 3.3X109 Example Comparative 2 2.3X109

[130] As a result, as shown in Table 3, it was confirmed that Example 1 had a significantly higher number of AAV complex particles than the negative control group and Comparative Examples 1 and 2. Specifically, the number of particles in Example 1 was about 4 times greater than that of the negative control group and Comparative Example 2, and about 3 times greater than that of Comparative Example 1. Petition 870250120661, dated 12 / 29 / 2025, page 42 / 94 31 / 37

[131] That is, the productivity of the AAV complex according to one aspect can be improved by asymmetric modification of the ITR. Experimental Example 2. Confirmation of the gene expression rate of the AAV complex.

[132] A target gene expression rate of a recombinant AAV complex according to one aspect was confirmed. Specifically, H460, a non-small cell lung cancer cell line, was infected with the same amount of the complexes prepared in Example 1 and Comparative Examples 1 and 2. After 48 hours, the cells were ruptured to purify the protein, loaded onto an SDS page gel, and the RUNX3 gene expression rates were confirmed by Western blotting. Then, the gene expression rates of Example 1 and Comparative Examples 1 and 2 were calculated, defining a detection rate of the purified RUNX3 proteins as 100%. As a negative control group, a wild-type AAV complex was used. In addition, as an additional negative control, an HEK293 cell line not infected with an AAV complex was used.Unlike cancer cells, which do not express RUNX3 well, since HEK293 cells are normal, non-cancerous cells, a large amount of endogenous RUNX3 gene expression was detected.

[133] FIG. 4A shows the results of confirming the expression of a target gene in an AAV complex according to an aspect with Western blot.

[134] FIG. 4B shows the results of quantifying the expression rate of a target gene in an AAV complex according to one aspect.

[135] As a result, as shown in FIGS. 4A and 4B, it was confirmed that the expression rate of the RUNX3 gene in the virus produced in Example 1 was significantly increased by more than two times in Petition 870250120661, dated 12 / 29 / 2025, page 43 / 94 32 / 37 comparison to the negative control group and to Comparative Examples 1 and 2.

[136] In conjunction with the results of Experimental Example 1, the AAV complex according to one aspect had productivity increased by 3 times or more, and the expression rate of the RUNX3 gene increased by more than 2 times and, as a result, it can be observed that efficiency increased by about 6 times or more. Experimental Example 3. Confirmation of the effect of the AAV complex in the treatment of lung cancer. 3-1. Confirmation of cell death in non-small cell lung cancer with KRAS mutation.

[137] A cell death induction effect of the recombinant AAV complex in accordance with one aspect in KRAS-mutant non-small cell lung cancer was confirmed. Specifically, C57B6 mice (6 to 8 weeks old) with Runx3lox / lox;KRASwt / LSL genotype were infected with adeno5-CRE 2.5X10e7 particles, an inducer capable of depleting RUNX3 while activating KRAS mutations, via the respiratory tract. Subsequently, after breeding the mice for 6 weeks to determine whether or not they developed non-small cell lung cancer, the AAV complexes from Example 1 or Comparative Example 1 were infected via the respiratory tract of mice with confirmed lung cancer development. After 2 weeks, the mice were sacrificed and lung tissues were extracted to prepare specimens for histopathological examination.Next, the samples were stained with hematoxylin and eosin (H&E) and labeled with deoxynucleotidyl transferase (TUNEL) DNA nick ends, and the stained tissues were observed under a microscope.

[138] FIGS. 5A and 5B show the results of staining with H&E and Petition 870250120661, dated 12 / 29 / 2025, page 44 / 94 33 / 37 TUNEL of lung cancer tissues infected with Example 1 and Comparative Example 1, respectively.

[139] As a result, as shown in FIG. 5A, for lung cancer tissue infected with the AAV complex of Example 1, a high level of TUNEL staining was observed in cancerous regions, and it was confirmed that the bands between the H&E-stained regions were relatively wide. On the other hand, as shown in FIG. 5B, for lung cancer tissue infected with the AAV complex of Comparative Example 1, TUNEL staining was rarely observed, and it was confirmed that the bands between the H&E-stained regions were relatively narrow.

[140] That is, it can be observed that in lung cancer tissue infected by Example 1, cell death occurred at the cancerous site, and no cancer mass was observed, and an original lung structure was maintained. Therefore, the AAV complex, according to one aspect, promotes the death of lung cancer cells and suppresses the occurrence of cancer and, therefore, may be useful for the prevention or treatment of lung cancer. 3-2. Confirmation of cell death in human non-small cell lung cancer

[141] A cell death induction effect of the recombinant AAV complex on non-small cell lung cancer was confirmed. Specifically, the non-small cell lung cancer cell lines H460 and Calu6 and a normal lung cell line WI38 were infected with the AAV complex from Example 1. After that, each of the cell lines was cultured for 1 to 7 days, and the occurrence of cell death was confirmed by flow cytometry (fluorescence-activated cell sorting; FACS) every 1 to 3 days from day 0 of culture for 3 to 7 days. Furthermore, after protein extraction from the cell line Petition 870250120661, dated 12 / 29 / 2025, page 45 / 94 In 34 / 37 H460 cells, the expression of RUNX3 and cleaved caspase 3 was confirmed by Western blotting.

[142] FIG. 6A shows micrographs taken 1 day, 3 days and 5 days after infection of KRAS-mutant lung cancer cell lines (H460, Calu6) and normal lung epithelial cells (WI38) with Example 1.

[143] FIG. 6B shows the results of a FACS analysis, 0 hours (top), 16 hours (middle) and 32 hours (bottom) after infection of normal lung epithelial cells (WI38) with Example 1.

[144] FIG. 6C shows the results of a FACS analysis 0 hours (top), 16 hours (middle) and 32 hours (bottom) after infection of a KRAS-mutant lung cancer cell line (Calu6) with Example 1.

[145] FIG. 6D shows a graph quantifying the results of FACS analyses, 1 day and 3 days after infection of a KRAS-mutant lung cancer cell line (H460) and normal lung epithelial cells (WI38) with Example 1.

[146] FIG. 6E shows the results of the detection of RUNX3 and cleaved caspase 3 expressed in KRAS-mutant lung cancer cell lines infected with Example 1.

[147] As a result, as shown in FIG. 6A, for the lung cancer cell line infected with the Example 1 complex, it was confirmed that most cells died on the 5th day of infection. On the other hand, it was confirmed that no cell death was observed in normal lung epithelial cells.

[148] Furthermore, as shown in FIGS. 6B to 6D, for the lung cancer cell line (H460) infected with the Example 1 complex, it was confirmed that the cell death rate increased approximately 4 times or more on day 3 of infection compared to day 1 of infection. On the other hand, it was Petition 870250120661, dated 12 / 29 / 2025, page 46 / 94 35 / 37 confirmed that there was no change in the rate of cell death over time in normal lung epithelial cells (WI38).

[149] Furthermore, as shown in FIG. 6E, for the lung cancer cell line infected with the Example 1 complex, it was confirmed that the expression of RUNX3 and cleaved caspase 3 increased continuously from day 2 after infection.

[150] That is, since the AAV complex, according to one aspect, is not toxic to normal cells and selectively kills only KRAS-mutant lung cancer cell lines, it may be useful for prevention or treatment of KRAS-mutant lung cancer. 3-3. Confirmation of cancerous tissue necrosis in xenograft models of non-small cell lung cancer

[151] A necrotic effect of the recombinant AAV complex on cancerous tissues in non-small cell lung cancer xenograft models was confirmed. Specifically, the H460 non-small cell lung cancer cell line was injected subcutaneously into the dorsal flank of 6-week-old nude mice at 2x10⁵ cells / mice to prepare non-small cell lung cancer xenograft models. Example 1 was mixed and diluted with the same volume of PBS to 2x10⁷ viral genome / mm³. After that, the xenografted model mice were reared for 2 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. A control group was injected with a dilution of an AAV complex (empty AAV) with a genome that did not include RUNX3.After the injection, the mice were raised for another 12 days, and the cancer volume increased by an average of 100% or more in comparison. Petition 870250120661, dated 12 / 29 / 2025, page 47 / 94 On day 0, mice 36 / 37 were sacrificed. After that, the cancerous tissue was removed from the mouse and cut to show a cross-section. The experiment was performed using a total of eight mice, among which four mice (DI1, DI2, DI3, DI4) were injected with Example 1, and four mice (Con1, Con2, Con3, Con4) were injected with empty AAV, under the same conditions.

[152] FIG. 7A shows the results of confirmation of the interior of cancerous tissues cut to show cross-sections, after injection of Example 1 into non-small cell lung cancer xenograft models and reproduction for 12 days.

[153] FIG. 7B shows the results of measuring cancerous tissue volumes after injecting Example 1 into non-small cell lung cancer xenograft models and breeding for 12 days.

[154] As a result, as shown in FIG. 7A, for the xenograft animal models injected with Example 1, it was confirmed that the cancer was necrotic or that the binding strength of the cancer mass was loosely maintained, due to RUNX3.

[155] Furthermore, as shown in FIG. 7B, for the xenograft animal models injected with Example 1, there was little change in the volume of cancerous tissue up to 12 days after injection. On the other hand, for the control group, it was confirmed that the volume of cancerous tissue increased by up to 150% on day 12 after injection, compared to day 1.

[156] That is, the AAV complex according to one aspect expresses the RUNX3 gene specifically for lung cancer, thus inhibiting tumor growth and inducing cell death by the RUNX3 gene and therefore the AAV complex may be useful for prevention or treatment of lung cancer with KRAS mutation. Petition 870250120661, dated 12 / 29 / 2025, page 48 / 94 37 / 37

[157] The above description of the present disclosure is for illustrative purposes, and those skilled in the art to which the present disclosure pertains will be able to understand that the examples and embodiments can be easily modified without altering the technical idea or the essential features of the disclosure. Therefore, it should be understood that the above examples are not limiting, but illustrative in all respects.

[158] It should be understood that the modalities described herein are to be regarded only in a descriptive sense and not for purposes of limitation. Descriptions of features or aspects within each modality are normally to be regarded as available to other similar features or aspects in other modalities. Although one or more modalities have been described with reference to the figures, it will be understood by those skilled in the art that various alterations in form and detail may be made to the same without departing from the spirit and scope of the revelation as defined by the following claims. Petition 870250120661, dated 12 / 29 / 2025, p. 49 / 94

Claims

1 / 3 CLAIMS 1. Adeno-associated virus (AAV) complex, characterized in that it comprises a polynucleotide sequence encoding a runt-related transcription factor 3 (RUNX3) protein between a first inverted terminal repeat (ITR) and a second ITR, wherein in either the first ITR or the second ITR, all or part of a stem-loop structure, which is formed of rep-binding element (RBE) regions, RBE', A, A', B, B', C, C' and D, is modified.

2. Adeno-associated virus complex according to claim 1, characterized in that it comprises: an operationally linked SPC promoter, a polynucleotide sequence encoding a RUNX3 protein, and a polyadenylation sequence between the first ITR and the second ITR.

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

4. Adeno-associated virus complex according to claim 1, characterized in that the first ITR is not modified and the second ITR is modified.

5. Adeno-associated virus complex according to claim 1, characterized in that the stem-loop structure modification is insertion, deletion, or substitution.

6. Adeno-associated virus complex according to claim 1, characterized in that either the first ITR or the second ITR is modified so as not to form a stem-loop structure.

7. Adeno-associated virus complex according to claim 1, Petition 870250120661, dated 12 / 29 / 2025, p. 87 / 94 2 / 3 characterized in that, in either the first ITR or the second ITR, all regions B, B', C, C and RBE' are deleted, as well as region A or A', whichever is closest to the 3' end of the (+) strand of the transgene or the 5' end of the (-) strand of the transgene, is deleted.

8. Adeno-associated virus complex according to claim 1, characterized in that either the first ITR or the second ITR comprises a terminal resolution site (trs) sequence and an RBE sequence.

9. Adeno-associated virus complex according to claim 1, characterized in that the first ITR is a wild-type AAV ITR, and the second ITR consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 9.

10. Adeno-associated virus complex according to claim 1, characterized in that the first ITR is a wild-type AAV ITR, and the second ITR consists essentially of a nucleotide sequence with SEQ ID NO:

1.

11. Adeno-associated virus complex according to claim 1, characterized in that it further comprises a gene junction comprising SEQ ID NO: 10 between the SPC promoter and the nucleotide sequence encoding RUNX3.

12. Use of the adeno-associated virus complex defined in claim 1, characterized by the fact that it is for the manufacture of a medicament for the treatment of KRAS-mutant lung cancer.

13. Use according to claim 12, characterized in that the lung cancer is non-small cell lung cancer or small cell lung cancer.

14. Use according to claim 13, characterized in that Petition 870250120661, dated 12 / 29 / 2025, page 88 / 94 3 / 3 non-small cell lung cancer is selected from the group consisting of squamous cell carcinoma, large cell carcinoma and lung adenocarcinoma.

15. Pharmaceutical composition, characterized by being for the prevention or treatment of lung cancer with KRAS mutation, comprising the adeno-associated virus complex defined in claim 1.

16. Pharmaceutical composition according to claim 15, characterized in that it further comprises a pharmaceutically acceptable carrier. Petition 870250120661, dated 12 / 29 / 2025, pp. 89 / 94

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