Modified complex platform loaded with adeno-associated viruses with increased gene expression rate and reduced genotoxicity

Abstract

An adeno-associated virus (AAV) complex platform is disclosed that includes an asymmetrically modified terminal inverted repeat (ITR). The AAV complex has an asymmetric ITR in which any one of two ITRs is modified, and thus has the advantages of increased productivity and transgene expression efficiency, and reduced genetic toxicity. Also disclosed are compositions comprising the adeno-associated virus complexes and gene therapy methods.

Description

Technical Field

[0001] This disclosure relates to a modified complex platform for adeno-associated virus and its use. Background Technology

[0002] Adeno-associated virus (AAV) is a single-stranded deoxyribonucleic acid (DNA) virus with a genome size of approximately 4.6 kbp. It is a helper-dependent human parvovirus. The genome consists of two terminal ITRs and two open reading frames (ORFs): rep and cap. The N-terminal region of the genome encodes the rep gene, which is involved in viral replication and viral gene expression, while the C-terminal region encodes the capsid protein, the cap gene. The ITRs are involved in AAV genome replication and AAV particle packaging.

[0003] In contrast, AAV gene delivery vectors are delivery vectors derived from non-pathogenic human viruses. They are not only safe and do not induce cellular immune responses, but also have a broad host range. Furthermore, AAV gene delivery vectors are characterized by their ability to deliver genes to both non-dividing and dividing cells, and, in particular, the expression of genes delivered by AAV gene delivery vectors persists in vivo for a long period of time.

[0004] However, a drawback of the aforementioned AAV is that its terminal inverted repeat (ITR) sequence can lead to capsidation of up to approximately 4.4 kb of protein-coding sequences, resulting in poor DNA packaging ability. Furthermore, a further drawback is that along the... Directional insertion of genes and along When a gene inserted in a specific direction is expressed on the host cell's genome, competition occurs between the two strands, resulting in reduced expression of the delivered gene. Furthermore, the delivered gene has the potential to cause cancer during its insertion into the host cell's genome.

[0005] Therefore, there is a need to develop an AAV complex as a gene delivery vector, which, due to the ITR modification in AAV properties, has improved DNA packaging capabilities, reduced insertion into infected cell chromosomes, and improved productivity and expression efficiency. Summary of the Invention

[0006] Technical issues

[0007] One embodiment described in this specification relates to an adeno-associated virus (AAV) complex comprising a polynucleotide sequence encoding a transgene between a first inverted terminal repeat (ITR) and a second ITR, wherein, in either the first or second ITR, all or part of a stem-loop structure formed by a rep-binding element (RBE), RBE', A, A', B, B', C, C', and D regions is modified. The AAV complex may further comprise an operatively linked promoter, a polynucleotide sequence encoding the transgene, and a polyadenylated sequence between the first and second ITRs. The AAV may be an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In the adeno-associated virus complex, the first ITR may be unmodified, while the second ITR may be modified. The modification of the stem-loop structure may be at least one of insertion, deletion, or substitution. In the adeno-associated virus complex, either the first ITR or the second ITR may be modified to not form a stem-loop structure. In the adeno-associated virus complex, in either the first ITR or the second ITR, all or part of the stem-loop structure formed by the RBE, RBE', A, A', B, B', C, C', and D regions may be deleted. In the adeno-associated virus complex, either the first ITR or the second ITR may include a terminal resolution site (TRS) sequence and an RBE sequence; the C, C', B', B, RBE', A', and D sequences following the RBE may all be deleted. The promoter may be a tissue-specific promoter. In the adeno-associated virus complex, the transgene may be a therapeutic gene. In the adeno-associated virus complex, the transgene may be green fluorescent protein (GFP), luciferase, TP53, RPE65, TPP1, or FVIII.

[0008] Another embodiment relates to a gene therapy method comprising administering an effective amount of the adeno-associated virus complex described herein to an individual in need of it.

[0009] Another embodiment relates to a composition comprising the adeno-associated virus complex described herein. The composition may further comprise a pharmaceutically acceptable vector. The composition can be used to deliver therapeutic genes for gene therapy.

[0010] Technical solution

[0011] Hereinafter, embodiments will be described with reference to the accompanying drawings, in which the same reference numerals refer to the same elements. In this respect, the embodiments may take various forms and should not be construed as limited to the description presented herein. Therefore, the embodiments are described only with reference to the accompanying drawings, and their purpose is to describe the described embodiments. The term "and / or" as used in this specification includes any and all combinations of at least one of the listed related items. When expressions such as "at least one of" are used before an element list, such expressions are used to modify the entire element list, rather than individual elements of the list.

[0012] One implementation relates to an adeno-associated virus complex comprising a terminal inverted repeat sequence.

[0013] Another embodiment involves cells transformed by the adeno-associated virus complex.

[0014] Another embodiment relates to a method for delivering a genetically modified organism (GMO), which includes the step of administering an effective amount of the adeno-associated virus complex.

[0015] Another embodiment relates to a gene therapy method comprising the step of administering an effective amount of the adeno-associated virus complex.

[0016] Another embodiment relates to a method of treating an individual disease, which includes administering an effective amount of an adeno-associated virus complex according to one aspect to the individual in need.

[0017] Another embodiment relates to a composition comprising the adeno-associated virus complex.

[0018] Another embodiment relates to the use of the adeno-associated virus complex in gene therapy.

[0019] Additional embodiments will be described below, some of which will become apparent from the description or may be learned by practicing the embodiments shown in this disclosure.

[0020] According to one embodiment, an adeno-associated virus (AAV) complex is disclosed, which includes a modified inverted terminal repeat (ITR) sequence.

[0021] In this specification, 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-dependent human parvovirus. The genome consists of two terminal ITRs and two open reading frames (ORFs): rep and cap. The N-terminal region of the genome encodes the rep gene, which is involved in viral replication and viral gene expression, while the C-terminal region encodes the cap gene, which codifies the viral capsid proteins. The ITRs are involved in AAV genome replication and AAV particle packaging. The ITRs include rep-binding elements (RBEs), RBE', A, A', B, B', C, C', and the D region, forming a stem-loop structure (hairpin structure). The AAVITR structure is widely known, for example, in literature such as Goncalves, MAVIrology Journal, 2(1):43 (2005), which are incorporated herein by reference.

[0022] The ITR sequence may be based on the ITR sequence of a virus belonging to the genus Dependovirus of the family Parvoviridae.

[0023] The ITR sequence may be based on the ITR sequence of AAV. The ITR sequence of AAV is well known.

[0024] Examples of AAVs include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12, and may also include other known or to be discovered AAV serotypes. AAVs may include known AAV derivatives. AAVs may include modified or artificial AAVs.

[0025] Therefore, the ITR sequence can be based on the ITR sequence of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The ITR sequence can also be based on the 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.

[0026] Alternatively, another virus belonging to the genus Dependovirus of the family Parvoviridae can be used instead of the AAV.

[0027] In this specification, the term "AAV complex" may be used interchangeably with "AAV vector", "AAV delivery vector", "recombinant AAV", and "recombinant AAV vector".

[0028] In one embodiment, the AAV complex includes a heteropolynucleotide sequence between the first ITR and the second ITR.

[0029] The heteropolynucleotide sequence can be operatively arranged between the first ITR and the second ITR.

[0030] In one embodiment, the genome of the AAV complex includes a first ITR (5'-ITR), a polynucleotide sequence encoding a transgene, and a second ITR (3'-ITR) along the 5'→3' direction.

[0031] In some specific embodiments, the AAV complex may include a polynucleotide sequence encoding a transgene between the first ITR and the second ITR, and in either the first ITR or the second ITR, all or part of the stem-loop structure formed by the RBE, RBE', A, A', B, B', C, C', D regions may be modified.

[0032] In some specific embodiments, the AAV complex may include an operatively linked promoter, a polynucleotide sequence encoding a transgene, and a polyadenylated sequence between the first ITR and the second ITR.

[0033] In some specific embodiments, the genome of the AAV complex includes a first ITR (5'-ITR), a promoter, a polynucleotide sequence encoding the transgene, a polyadenylated sequence, and a second ITR (3'-ITR) along the 5'→3' direction.

[0034] In some additional specific embodiments, the AAV complex includes an operatively linked promoter, a transgene-encoding polynucleotide sequence, and a polyadenylated sequence between the first ITR and the second ITR, and all or part of the stem-loop structure formed by the RBE, RBE', A, A', B, B', C, C', and D regions in either the first ITR or the second ITR may be modified.

[0035] In some specific embodiments, the AAV complex may include an asymmetrically modified ITR. In one embodiment, either the first ITR or the second ITR in the AAV complex may be modified. In another embodiment, the AAV complex may have a modified first ITR and an unmodified second ITR. In yet another embodiment, the AAV complex may have an unmodified first ITR and a modified second ITR. In yet another embodiment, the 5'-ITR of the (+) strand of the transgene in the AAV complex may be unmodified, while the 3'-ITR of the (+) strand may be modified. In yet another embodiment, the 5'-ITR of the (-) strand of the transgene in the AAV complex may be modified, while the 3'-ITR of the (-) strand may be unmodified. In other words, modification of the second ITR may mean that the 3'-ITR of the (+) strand and / or the 5'-ITR of the (-) strand in the transgene are modified.

[0036] Because the AAV complex described in this specification includes an asymmetrically modified ITR, its productivity can be increased, transgene expression rate can be increased, and genotoxicity can be reduced. Therefore, it can be used as an AAV delivery vector platform for delivering various genes into target cells. Furthermore, the AAV complex described in this specification can be used as a delivery vector to inhibit long-term transgene expression in host cells while simultaneously enabling efficient transgene expression in a short time.

[0037] In both the first and second ITRs, the unmodified ITR can be a wild-type ITR. Alternatively, the unmodified ITR in both the first and second ITRs can be an AAV wild-type ITR.

[0038] In some specific implementations, the unmodified ITR in the first ITR and the second ITR may be a functional derivative having substantially the same functional properties as the wild-type ITR (e.g., AAV wild-type ITR).

[0039] In this specification, the term "functional derivative" can refer to a derivative having substantially the same functional properties. The derivative refers to a similar compound obtained by chemically altering a portion of the structure of a compound. The derivative can refer to a compound in which hydrogen atoms or specific atomic groups are replaced by other atoms or atomic groups. Methods for preparing compound derivatives while maintaining substantially the same functional properties are well known in the art.

[0040] In some specific embodiments, in either the first ITR or the second ITR, all or part of the stem-loop structure formed by the regions RBE, RBE', A, A', B, B', C, C', and D can be modified.

[0041] In one embodiment, the modification of the stem-ring structure (hairpin structure) can be selected from insertion, deletion, and replacement.

[0042] In one embodiment, the modification of the stem-ring structure (hairpin structure) includes being modified to include a single stem and a single ring. For example, the modified ITR may include the removal of the B-B' arm to retain the C-C' arm, or the removal of the C-C' arm to retain the B-B' arm.

[0043] In one embodiment, modification of the stem-ring structure (hairpin structure) includes modifying it to have a single stem instead of a double ring. For example, a modified ITR may include the removal of the B-B' arm and the C-C' arm.

[0044] In one implementation, the modified ITR may include the absence of the C' region to preserve the cut C-loop and B-B' arm. Similarly, the modified ITR may include the absence of the B region to preserve the cut B-loop and C-C' arm.

[0045] In one embodiment, the modified ITR may include base pairings in at least one of the C portion, C' portion, B portion, or B' portion, such that complementary base pairings occur between the C portion and the B' portion and between the C' portion and the B portion, forming a single arm.

[0046] In one embodiment, the modified ITR may include modifications (e.g., deletion, substitution, or addition) of one, two, three, four, five, or six nucleotides in at least one region selected between A' and C, C and C', C' and B, B and B', and B' and A.

[0047] In one embodiment, modification of the stem-loop structure (hairpin structure) may include modification of the structure of the structural elements. Specifically, modification of the structure of the structural elements may include changes in the stem height and / or changes in the number of nucleotides within the loop. For example, the stem height may be equal to about 2, 3, 4, 5, 6, 7, 8, or 9 or more nucleotides, or any range of said numbers. In another embodiment, the loop may have about 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides, or any range of said numbers.

[0048] In another embodiment, the functional interaction of large Rep proteins can be altered by changing (e.g., increasing or decreasing) the interval between two elements (e.g., RBE and hairpin, as a non-limiting example). For example, the interval 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 or more nucleotides, or any range of said numbers.

[0049] In one embodiment, either the first ITR or the second ITR may be modified to not form a stem-loop structure (hairpin structure). In another embodiment, the first ITR may be modified to not form a stem-loop structure. In another embodiment, the second ITR may be modified to not form a stem-loop structure. In yet another embodiment, the first ITR may be left unmodified, and the second ITR may be modified to not form a stem-loop structure.

[0050] The expression "modified to not form a stem-loop sequence (hairpin structure)" can refer to ITR sequence modification that causes the ITR structure to exist with open-ends or free-ends, without forming a stem-loop structure (hairpin structure). In some specific embodiments, the AAV complex is modified so that either the first ITR or the second ITR does not form a stem-loop structure (hairpin structure), thereby inhibiting the formation of circular dimers and circular concatenators within infected cells to integrate with the host genome observed in AAV. Furthermore, through this modification, the productivity of the AAV complex and the expression rate of transgenes can be increased.

[0051] In one embodiment, either the first ITR or the second ITR may be modified to have a blunt end. In another embodiment, either the first ITR or the second ITR may be modified to have a sticky end. In yet another embodiment, the second ITR may be modified to have either a blunt end or a sticky end. In yet another embodiment, the second ITR may be modified to have a blunt end. Modifications to the blunt or sticky ends can be performed by those skilled in the art using known methods.

[0052] In one embodiment, in either the first ITR or the second ITR, all or part of the stem-loop structure formed by the RBE, RBE', A, A', B, B', C, C', and D regions may be deleted. In another embodiment, in either the first ITR or the second ITR, a portion of the stem-loop structure formed by the RBE, RBE', A, A', B, B', C, C', and D regions 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 the C, C', B', B, RBE', A', and D sequences following the RBE may be completely deleted. In yet another embodiment, the first ITR may be unmodified, and the second ITR may include a trs sequence and an RBE sequence, and the C, C', B', B, RBE', A', and D sequences following the RBE may be completely deleted.

[0053] In another embodiment, either the first ITR or the second ITR may comprise, substantially constitute, or may comprise, a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 9 or its complementary sequence. In another embodiment, the first ITR may be unmodified, and the second ITR may comprise, substantially constitute, or may comprise, a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 9 or its complementary sequence.

[0054] The sequence of SEQ ID NO: 1 may be based on an AAV2 ITR sequence.

[0055] The sequence of SEQ ID NO: 2 may be based on an AAV1 ITR sequence.

[0056] The sequence of SEQ ID NO: 3 may be based on an AAV3 ITR sequence.

[0057] The sequence of SEQ ID NO: 4 may be based on an AAV4 ITR sequence.

[0058] The sequence of SEQ ID NO: 5 may be based on an AAV6 ITR sequence.

[0059] The sequence of SEQ ID NO: 6 may be based on the AAV7 ITR sequence.

[0060] The sequence of SEQ ID NO: 7 may be based on an AAV5 ITR sequence.

[0061] The sequence of SEQ ID NO: 8 may be based on an AAV8 ITR sequence.

[0062] The sequence of SEQ ID NO: 9 may be based on the AAV9 ITR sequence.

[0063] In some specific embodiments, a portion of the AAV ITR sequence may be deleted from the sequences represented by SEQ ID NOs: 1 to 9. The sequences represented by SEQ ID NOs: 1 to 9 may include the trs sequence and the RBE sequence in the AAV ITR sequence. In the sequences represented by SEQ ID NOs: 1 to 9, the C, C', B', B, RBE', A', and D sequences following the RBE sequence may be completely deleted.

[0064] Typically, RNA polymerase generates mRNA with a sequence complementary to the target gene within the promoter region. This process is called transcription, which occurs along the 5'→3' direction. When the target gene is inserted into the AAV complex, the gene is inserted along both the 5'→3' and 3'→5' directions relative to the double helix of the AAV complex DNA. Therefore, during transcription of the target gene, the double helix of the AAV complex DNA causes bidirectional transcription, resulting in interference and reduced target gene expression efficiency. However, the AAV complex described in this specification can be modified by altering the stem-loop structure, specifically by deleting all or part of the stem-loop structure, to avoid interference during target gene transcription caused by the double helix structure of the AAV complex DNA, thereby improving the target gene expression efficiency.

[0065] For example, in AAV complexes including those with asymmetric modifications, the first ITR may be unmodified, while the second ITR may be modified to not form a stem-loop structure. Therefore, the 5'-ITR of the transgene's (-) strand does not form a hairpin structure, and transcription of the transgene along the 5'→3' direction occurs complementaryly with this strand. Conversely, in the transgene's (+) strand, the 3'-ITR does not form a hairpin structure, and transcription of the transgene does not occur in this strand. Furthermore, since transcription of the target gene occurs only along the 5'→3' direction and competitors along the 3'→5' direction are removed, gene expression efficiency can be increased.

[0066] In one embodiment, in an AAV vector comprising various transgenes, a portion of the hairpin structure of the second ITR in the wild-type AAV ITR included in said vector is modified to prepare an AAV complex comprising an asymmetrically modified ITR (pAAV-GC ITR vector). The prepared AAV complex is compared with an AAV complex without modified hairpin structure (pAAV-WT ITR vector) and an AAV complex with two symmetrically symmetrical ends modified to hairpin structure (pAAV-BC del.ITR vector) to confirm their respective productivity, transgene expression rate, and genotoxicity. The results show that the AAV complex comprising an asymmetrically modified ITR (pAAV-GC ITR vector) exhibits significantly increased viral productivity and transgene expression rate, and extremely low genotoxicity, compared to the unmodified AAV complex (PAAV WT ITR vector) and the symmetrically modified AAV complex (pAAV-BC del.ITR vector).

[0067] In this specification, the term "promoter" refers to the region that regulates gene transcription. The promoter is operatively linked to the coding sequence of a transgene. The promoter can be a tissue-specific promoter or an inducible promoter. The type of tissue-specific promoter is not limited, as long as it induces specific expression of the gene in a specific type of cell or tissue in vivo. As a tissue-specific promoter, a promoter that is specific to the desired tissue can be appropriately selected so that the transgene is expressed according to that type of transgene. Non-limiting examples of tissue-specific promoters include the liver-specific thyroxin-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian endothelin (DES) promoter, α-myosin heavy chain (α-MHC) promoter, cardiac troponin T (cTnT) promoter, and surfactant protein C (SPC) promoter.

[0068] The heteropolynucleotide can encode a transgene.

[0069] In this specification, the term "transgene" is a gene that is delivered from one organism to another naturally or through various genetic engineering techniques. The term "transgene" may be used interchangeably with "delivering gene" and "target gene".

[0070] The transgene is not limited to a specific type; it can be any gene that will be introduced into the host cell. In some specific embodiments, the AAV complex has advantages such as increased AAV productivity, increased transgene expression rate, and reduced genotoxicity, regardless of the type of transgene, and therefore can be used as an AAV delivery vector platform for delivering various transgenes.

[0071] In one embodiment, the transgene can be a therapeutic gene. When the transgene is a therapeutic gene, an AAV complex according to one aspect can be used as a gene therapy agent. Therefore, the AAV complex can be an AAV vector for gene therapy.

[0072] In one embodiment, the transgene can be green fluorescent protein (GFP), luciferase, TP53, RPE65, TPP1, or FVIII, but is not limited thereto. When the transgene is TP53, the AAV complex used to deliver the transgene can be used as a gene therapy agent for cancer treatment. When the transgene is RPE65, the AAV complex used to deliver the transgene can be used as a gene therapy agent for treating inherited retinal disease (IRD). When the transgene is TPP1 (CLN2), the AAV complex used to deliver the transgene can be used as a gene therapy agent for treating Batten disease. When the transgene is FVIII, the AAV complex used to deliver the transgene can be used as a gene therapy agent for hemophilia.

[0073] The genetically modified organisms can be derived from humans or animals.

[0074] In one embodiment, the AAV complex may further include post-transcriptional regulatory elements. The AAV complex may include, along the 5'-3' direction, a first ITR, a promoter sequence, a polynucleotide sequence encoding a transgene, post-transcriptional regulatory elements, a polyadenylated sequence, and a second ITR.

[0075] The post-transcriptional regulatory elements may include the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0076] In one embodiment, the AAV complex may further include a gene junction between the promoter and the polynucleotide sequence encoding the transgene.

[0077] In this specification, the term "gene junction" refers to a portion of the sequence that is not defined and located between the end of a promoter and the start region of a target gene sequence. Specifically, a promoter is the site where transcription machinery complexes bind to regulate gene expression, and the boundary between the known end of a promoter sequence and the start region of the gene whose expression will be regulated is often ambiguous. Therefore, optimization of the junction may be necessary to establish a successful promoter-gene expression relationship. The sequence of the gene junction is appropriately selected by those skilled in the art using conventional methods.

[0078] The AAV complex can be engineered to encode a selection marker or reporter that provides selection or confirmation for inclusion in cells. Selection markers or reporters are well known in the art. Non-limiting examples of selection markers include genes that provide resistance to ampicillin, streptavidin, kanamycin, hygromycin, etc. Non-limiting examples of reporters include luciferase and green fluorescent protein (GFP).

[0079] Furthermore, cells transformed by adeno-associated virus complex were disclosed.

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

[0081] In this specification, the term "transformation" refers to an alteration of an organism's genetic characteristics due to the introduction of DNA from an external source. Transformation is the phenomenon where DNA, i.e., a nucleic acid extracted from the cells of one system of an organism, is injected into a living cell of another system, causing the DNA to enter that cell and alter its genetic characteristics. In other words, "transformation" refers to the introduction of a gene into a host cell so that the gene can be expressed within the host cell.

[0082] The method of transforming a cell line by introducing the AAV complex described in this specification can be any method known in the art, such as, but not limited to, transient transfection using cationic liposomes (Lipofectamine), microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, etc. Preferably, cationic liposome 2000 reagent can be used for transformation.

[0083] In another embodiment, a method for delivering a transgene to an individual is disclosed, comprising the step of administering an effective amount of an adeno-associated virus complex according to one aspect to the individual in need of it.

[0084] Another implementation relates to a gene therapy method that includes the step of administering an effective amount of an adeno-associated virus complex to an individual in need of it.

[0085] Another implementation relates to a method of treating an individual disease, which includes administering an effective amount of an adeno-associated virus complex to the individual in need of it.

[0086] In the methods described above, the specific details regarding the adeno-associated virus complex are as described above.

[0087] In the methods described above, the AAV complex can be applied directly to an individual, or it can be prepared into an applicable form and applied to an individual. In one embodiment, the AAV complex can be applied to an individual as a composition comprising the AAV complex. For example, the AAV complex can be prepared into a composition comprising the AAV complex and a pharmaceutically acceptable carrier and applied to an individual.

[0088] The individual may be an individual that requires expression of a transgene delivered by the AAV complex. The individual may be an individual who already has or is likely to have a disease that can be treated with gene therapy. The individual may be an individual who already has or is likely to have a disease that can be treated with expression of a transgene delivered by the AAV complex. The individual may be a mammal, such as a human, cattle, horse, pig, dog, sheep, goat, or cat.

[0089] In this specification, the term "gene therapy" refers to treatment using genes for the purpose of treating or preventing disease. AAV vectors used to deliver therapeutic genes into cells can be used as gene therapy agents. Diseases that can be readily treated with gene therapy include, but are not limited to, diseases caused by defects in a single gene. Non-limiting examples of diseases to which gene therapy can be applied include cancer; cardiovascular diseases; inherited retinal dystrophy (IRD), Batten disease, hemophilia, cystic fibrosis, muscular dystrophy, thalassemia, and sickle cell anemia; cranial nervous system diseases; infectious diseases (such as acquired immunodeficiency); and joint diseases.

[0090] The cancer may be a cancer in which tumor suppressor genes are inactivated. The cancer may be, for example, a KRAS-mutated cancer. The cancer may be, for example, a KRAS-mutated solid tumor. The cancer may be, for example, a KRAS-mutated lung cancer. In some embodiments, when the activity of the tumor suppressor gene is restored, cancer cells are eliminated and normal cells survive; therefore, KRAS-mutated lung cancer can be treated. The tumor suppressor gene may be, for example, sPD-1, VHL, MMAC1, DCC, p53, NF1, WT1, Rb, BRAC1, BRAC2, or RUNX3 genes.

[0091] In one embodiment, the lung cancer may 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, and lung adenocarcinoma.

[0092] Therefore, in some specific embodiments, the AAV complex can activate the gene in diseases caused by reduced activity of a specific gene to prevent or treat the disease, and thus can be used for gene therapy.

[0093] In this specification, the term "prevention" refers to all actions that suppress or delay the onset of a disease by using the AAV complex. The term "treatment" refers to all actions that improve or make beneficial to the symptoms of a disease by administering the AAV complex.

[0094] In some specific embodiments, the method may further include the step of administering a second active ingredient to the individual. The second active ingredient may be an active ingredient used for the prevention or treatment of a disease to be treated. The active ingredient may be administered simultaneously, separately, or sequentially with the AAV complex.

[0095] In some specific embodiments, the AAV complex is prepared, for example, into an injectable formulation suitable for administration to an individual via any suitable route such as intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, or intrathecal administration. The AAV complex can be administered systemically or locally, and can be administered alone or in combination with other pharmaceutically active compounds.

[0096] The preferred dosage of the AAV complex varies depending on the patient's condition, weight, disease severity, drug form, route of administration, and duration; however, it can be appropriately selected by those skilled in the art. In one embodiment, the dosage of the AAV complex may be approximately 1.0 × 10⁻⁶. 6 vg / kg to approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 8 vg / kg to approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 10 vg / kg to approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 10 vg / kg to approximately 1.0 × 10 14 vg / kg, approximately 1.0×10 12 vg / kg to approximately 1.0 × 10 14 vg / kg, for example, approximately 1.0 × 10 12 vg / kg, approximately 1.0×10 13 vg / kg, approximately 1.0×10 14 vg / kg. In a particular embodiment, the dosage of the AAV complex may be approximately 1.0 × 10⁻⁶. 13 vg / kg. The application can be performed once a day, multiple times a day, once a week, once every two weeks, once every three weeks, once every four weeks, or once a year.

[0097] In this specification, the term "about" is used to include a range of ±10% of the specified value.

[0098] On the other hand, it relates to a composition comprising an adeno-associated virus complex.

[0099] On the other hand, it relates to the use of the adeno-associated virus complex in gene therapy.

[0100] The specific details regarding the adeno-associated virus complex and gene therapy are as described above.

[0101] In certain embodiments, the composition may be a composition for gene therapy. The composition may be a composition for delivering a therapeutic gene for gene therapy. The composition may be a pharmaceutical composition. The composition may further include a pharmaceutically acceptable carrier. The carrier includes excipients, diluents, or adjuvants. A carrier suitable for delivering the AAV complex into a living organism may be used as the carrier. Specifically, a carrier suitable for preparation into a non-parenteral dosage form (e.g., an injectable dosage form) may be selected as the carrier. For example, a carrier suitable for preparation into an intravenous dosage form may be selected as the carrier. The carrier may be an aqueous solution, such as water or a buffered saline solution.

[0102] In some specific embodiments, the composition can be prepared into any dosage form using conventional methods. The composition can be formulated to suit the delivery of the AAV carrier to an individual. The composition can be prepared in an aqueous solution, for example, in water or a buffered saline solution. Alternatively, the composition can be formulated, for example, into a parenteral dosage form (e.g., an injection, such as a dose-dependent injection or continuous infusion). In one embodiment, the pharmaceutical composition can be formulated into an injectable dosage form suitable for administration via any suitable route, such as intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, or intrathecal. In one specific embodiment, the composition is formulated for administration via intravenous or subcutaneous injection. Furthermore, the composition can be formulated into a systemic or local dosage form. The composition can be provided in unit administration form in ampoules, pre-filled syringes, small-volume injection containers, or multiple-use containers with added preservatives.

[0103] In some specific implementations, when used for gene therapy of a specific disease, the composition may further include a second therapeutic agent having a preventive or therapeutic effect on the disease. The pharmaceutical composition may be a single composition or a combination of individual compositions.

[0104] In some specific embodiments, the composition may include an effective amount of the AAV complex. The term "effective amount" refers to an amount sufficient to produce the desired preventive or therapeutic effect when administered to an individual requiring prevention or treatment of a disease. The effective amount may be appropriately selected based on the chosen cells or individual. The effective amount may be determined based on disease severity, patient age, weight, health status, sex, patient sensitivity to the drug, timing of administration, route of administration, elimination rate, duration of treatment, factors including drugs administered in combination with or concurrently with the applied composition, and other pharmaceutically known factors. In one embodiment, the composition may include about 1.0 × 10⁻⁶. 6vg / kg to approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 8 vg / kg to approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 10 vg / kg to approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 10 vg / kg to approximately 1.0 × 10 14 vg / kg, approximately 1.0×10 12 vg / kg to approximately 1.0 × 10 14 vg / kg, for example, approximately 1.0 × 10 12 vg / kg, approximately 1.0×10 13 vg / kg, approximately 1.0×10 14 The AAV complex is administered at an dosage of vg / kg. In a particular embodiment, the composition may include about 1.0 × 10⁻⁶. 13 The AAV complex is administered at a dosage of vg / kg. The administration can be once a day, multiple times a day, once a week, once every two weeks, once every three weeks, once every four weeks, or once a year.

[0105] Beneficial effects

[0106] One type of AAV complex has an asymmetric ITR in which either of the two ITRs is modified, thus having the advantages of increased productivity and transgene expression efficiency and reduced genotoxicity, and therefore can be used as an AAV delivery vector platform.

[0107] Specifically, the AAV complex according to one aspect has the following advantages:

[0108] 1. Because either of the two ITRs is deleted, the AAV vector partially omits the self-replication step in the host cell (or production cell; packaging cell) and thus increases the packaging efficiency to viral particles within the same production period, resulting in increased self-replication efficiency, and the productivity of the AAV complex is increased compared to existing AAV complexes.

[0109] 2. The expression rate (expression level) of the transgene is higher than that of the common wild-type (WT) AAV.

[0110] 3. The WT AAV genome generates a concatenator structure within infected cells, essential for transgene recombination. That is, there are reports of transgenes inserting into host chromosomes by forming a concatenator structure—a polymorphic form composed of multiple identical genomes linked together. Conversely, due to the asymmetric modification of ITRs in some AAV complexes, a concatenator structure is not formed, thus inhibiting transgene insertion into the host chromosome (i.e., recombination). This offers the advantage of reduced genotoxicity.

[0111] 4. According to one aspect, the AAV complex with asymmetrically modified ITR is an AAV delivery vector platform that increases productivity and transgene expression rate while reducing genotoxicity. Therefore, the platform can be used to deliver various genes into target cells. Attached Figure Description

[0112] The described embodiments, features, and advantages of particular embodiments of this disclosure will become more apparent from the following description in conjunction with the accompanying drawings.

[0113] Figure 1 A septal diagram of an adeno-associated virus (AAV) vector according to one embodiment is shown.

[0114] Figure 2 The structure of an AAV carrier according to one embodiment is shown.

[0115] Figure 3A This is a schematic diagram of the genome of the AAV vector including the asymmetrically modified ITR in Example 1.

[0116] Figure 3B This is a schematic diagram of the genome of the AAV vector including the symmetrical unmodified ITR in Comparative Example 1.

[0117] Figure 3C This is a schematic diagram of the genome of the AAV vector including the symmetrically modified ITR in Comparative Example 2.

[0118] Figure 4 Western blot results confirming AAV productivity in Example 1 (GC), Comparative Example 1 (WT), and Comparative Example 2 (BC-del) for green fluorescent protein (GFP) gene expression are shown.

[0119] Figure 5 The results of qPCR viral genome quantification for AAV productivity in Example 1 (pAAV-GC), Comparative Example 1 (pAAV-WT), and Comparative Example 2 (pAAV-BC del) used for GFP gene expression are shown.

[0120] Figure 6 The results show qPCR viral genome quantification of AAV productivity using pAAV-GC, pAAV-WT, and pAAV-BC del vectors to confirm the expression of various genes (GFP, Luciferase, TP53, RPE65, TPP1, FVIII).

[0121] Figure 7 The results show the results of Western blotting confirmation of transgenic GFP expression in the AAV complex according to ITR type (ITR WT, ITR BC del, or ITR GC).

[0122] Figure 8 The relative expression rate (%) of transgenes in the AAV complex according to ITR type (pAAV-GC, pAAV-BC del, or pAAV-WT) is shown.

[0123] Figure 9A The results of GFP expression were shown using fluorescence microscopy at passage numbers P3 and P10.

[0124] Figure 9B Table 4 shows the results of quantifying the ratio of GFP-expressing cells.

[0125] Figure 10 Table 5 shows the results of quantifying the relative gene expression levels in P7.

[0126] Figure 11 The results show PCR amplification to confirm whether the transgene forms a concatenator in the host cell. Detailed Implementation

[0127] The following preferred embodiments are presented to aid in understanding the implementation methods described herein. However, these embodiments are provided merely for the purpose of facilitating a better understanding of this disclosure, and the disclosure is not limited to these embodiments.

[0128] Example

[0129] Example 1: Preparation of an adeno-associated virus complex containing asymmetrically modified ITR for expressing the GFP gene.

[0130] 1-1. Preparation of recombinant adeno-associated virus vector with GFP gene introduced

[0131] A recombinant adeno-associated virus (AAV) vector carrying the GFP gene was prepared. Specifically, to copy the chicken β-actin promoter-GFP (CBA-GFP) gene, which was replicated in the CS4-GPF vector (Chungbuk National University Cancer Institute), to the AAV2 vector, PCR amplification was performed using primers listed in Table 1 below. These primers were prepared using restriction enzymes NdeI and HindIII. After treating the amplified CBA-GFP DNA with restriction enzymes NdeI and HindIII, the amplified GBA-GFP DNA was copied to the NdeI-HindIII position at the multicloning site (MCS) of the wild-type adeno-associated virus (AAV) 2 co-vector using T4 DNA ligase to obtain the AAV2-CBA-GFP plasmid. The ampicillin resistance gene was removed using the BspHI restriction enzyme sites at both ends of the ampicillin resistance gene present in the AAV2-CBA-GFP plasmid. The kanamycin resistance gene was inserted into the site where the ampicillin resistance gene was removed. Then, the kanamycin resistance gene was recombined using the PCR primers listed in Table 1 below.

[0132] [Table 1]

[0133]

[0134] 1-2. Modification of the ITR structure

[0135] Site-directed mutagenesis was induced to alter a portion of the hairpin structure of the second ITR in the AAV2 wild-type inverted terminal repeat (ITR) included in the vector prepared in Examples 1-1. Specifically, 5'-phosphorylated primers from Table 2 below were used to include deletions of all or at least one of the C, C', B', B, RBE', A', and D sequences following the RBE of the 5'-direction ITR of the GFP gene (-) chain in the AAV2 wild-type ITR of the vector. For example, all of the C, C', B'', B, RBE', A', and D sequences following the RBE of the 5'-direction ITR of the GFP gene (-) chain were deleted. Thus, the second ITR was modified to not form a hairpin structure. As a result, an AAV complex for expressing the GFP gene was obtained, comprising an asymmetrically modified ITR, wherein the first ITR was unmodified and the second ITR was modified.

[0136] The AAV vector that includes the asymmetric modified ITR as in Example 1 is named pAAV-GC ITR (abbreviated as pAAV-GC).

[0137] Figure 1 A slit diagram of an AAV carrier according to one embodiment is shown.

[0138] Figure 2 The structure of an AAV carrier according to one embodiment is shown.

[0139] Figure 3A This is a schematic diagram of the genome of the AAV vector including the asymmetrically modified ITR in Example 1.

[0140] [Table 2]

[0141]

[0142] Example 2: Preparation of an AAV vector containing asymmetrically modified ITR for expressing the Luciferase gene.

[0143] Except for replacing the GFP gene with the luciferase gene (2,387 bp; Genebank accession number M15077.1), an AAV complex for expressing the luciferase gene was prepared according to the same method as in Example 1, which included an asymmetrically modified ITR.

[0144] Example 3: Preparation of an AAV vector for expressing the TP53 gene, comprising asymmetrically modified ITR.

[0145] Except for the substitution of GFP with the TP53 gene (2,512 bp; GenBank accession number NM_000546.6), an AAV complex for expressing the TP53 gene, comprising an asymmetrically modified ITR, was prepared according to the same method as in Example 1.

[0146] Example 4: Preparation of an AAV vector containing asymmetrically modified ITR for expressing the RPE65 gene.

[0147] Except for replacing the GFP gene with the RPE65 gene (2,605 bp; GenBank accession number NM_000329.3), an AAV complex for expressing the RPE65 gene was prepared according to the same method as in Example 1, which included an asymmetrically modified ITR.

[0148] Example 5: Preparation of an AAV vector containing asymmetrically modified ITR for expressing the TPP1 gene.

[0149] Except for the introduction of the TPP1 (CLN2) gene (1,693 bp; GenBank accession number NM_000391.4) in place of the GFP gene, an AAV complex for expressing the TPP1 (CLN2) gene, comprising an asymmetrically modified ITR, was prepared according to the same method as in Example 1.

[0150] Example 6: Preparation of an AAV vector containing asymmetrically modified ITR for expressing the FVIII gene.

[0151] Except for replacing the GFP gene with the FVIII gene (673 bp; GenBank accession number NM_000132.4), a complex for expressing the FVIII gene was prepared according to the same method as in Example 1, which included an asymmetrically modified ITR.

[0152] Comparative example

[0153] Comparative Example 1: Preparation of an AAV complex containing unmodified symmetric ITR for expressing the GFP gene.

[0154] Except for the hairpin structure without modified ITR, an AAV carrier including unmodified symmetrical ITR was prepared according to the same method as in Example 1.

[0155] The AAV vector that includes an unmodified symmetric ITR, as in Comparative Example 1, is named pAAV-WT (abbreviated as pAAV-WT).

[0156] Figure 3B This is a schematic diagram of the genome of the AAV vector including the asymmetrically modified ITR in Comparative Example 1.

[0157] Comparative Example 2: Preparation of an AAV complex containing a symmetrically modified ITR for expressing the GFP gene.

[0158] Except for the deletion of the C-C'-B'-RBE sequence from both ends of the ITR of the AAV complex prepared in Examples 1-1, an AAV complex for expressing the GFP gene was prepared according to the same method as in Examples 1-2, which included an ITR modified to be symmetrical at both ends.

[0159] The AAV vector including the symmetrically modified ITR as in Comparative Example 2 is named pAAV-BC del.ITR (pAAV-BC del).

[0160] Figure 3C This is a schematic diagram of the genome of the AAV vector including the symmetrically modified ITR in Comparative Example 2.

[0161] Comparative Example 3: Preparation of an AAV complex containing unmodified symmetric ITR for expressing the Luciferase gene.

[0162] In addition to replacing the GFP gene with the Luciferase gene, an AAV complex for expressing the Luciferase gene was prepared using the same method as in Comparative Example 1, which included an unmodified symmetric ITR.

[0163] Comparative Example 4: Preparation of an AAV complex containing symmetrically modified ITR for expressing the Luciferase gene.

[0164] In addition to replacing the CFP gene with the Luciferase gene, an AAV complex for expressing the Luciferase gene was prepared using the same method as in Comparative Example 2, which included a symmetrically modified ITR.

[0165] Comparative Example 5: Preparation of an AAV complex containing unmodified symmetric ITR for expressing the TP53 gene.

[0166] In addition to replacing the GFP gene with the TP53 gene, an AAV complex for expressing the TP53 gene was prepared using the same method as in Comparative Example 1, which included an unmodified symmetric ITR.

[0167] Comparative Example 6: Preparation of an AAV complex containing a symmetrically modified ITR for expressing the TP53 gene.

[0168] In addition to replacing the GFP gene with the TP53 gene, an AAV complex for expressing the TP53 gene was prepared using the same method as in Comparative Example 2, which included a symmetrically modified ITR.

[0169] Comparative Example 7: Preparation of an AAV complex containing unmodified symmetric ITR for expressing the RPE65 gene.

[0170] In addition to replacing the GFP gene with the RPE65 gene, an AAV complex for expressing the RPE65 gene was prepared using the same method as in Comparative Example 1, which included an unmodified symmetric ITR.

[0171] Comparative Example 8: Preparation of an AAV complex containing symmetrically modified ITR for expressing the RPE65 gene.

[0172] In addition to replacing the GFP gene with the RPE65 gene, an AAV complex for expressing the RPE65 gene was prepared using the same method as in Comparative Example 2, which included a symmetrically modified ITR.

[0173] Comparative Example 9: Preparation of an AAV complex containing unmodified symmetric ITR for expressing the TPP1 gene.

[0174] In addition to replacing the GFP gene with the TPP1 gene, an AAV complex for expressing the TPP1 gene was prepared using the same method as in Comparative Example 1, which included an unmodified symmetric ITR.

[0175] Comparative Example 10: Preparation of an AAV complex containing a symmetrically modified ITR for expressing the TPP1 gene.

[0176] In addition to replacing the GFP gene with the TPP1 gene, an AAV complex for expressing the TPP1 gene was prepared according to the same method as in Example 2, which included a symmetrically modified ITR.

[0177] Comparative Example 11: Preparation of an AAV complex containing unmodified symmetric ITR for expressing the FVIII gene.

[0178] In addition to replacing the GFP gene with the FVIII gene, an AAV complex for expressing the FVIII gene was prepared using the same method as in Comparative Example 1, which included an unmodified symmetric ITR.

[0179] Comparative Example 12: Preparation of an AAV complex containing a symmetrically modified ITR for expressing the FVIII gene.

[0180] In addition to replacing the GFP gene with the FVIII gene, an AAV complex for expressing the FVIII gene was prepared using the same method as in Comparative Example 2, which included a symmetrically modified ITR.

[0181] Test case

[0182] Experimental Example 1: Confirmation of the productivity of AAV complex

[0183] The productivity of the recombinant AAV complex according to one embodiment was confirmed.

[0184] Specifically, the AAV complexes obtained in Examples 1 to 6 and Comparative Examples 1 to 12 were transfected into 293T cells, i.e., human embryonic kidney (HEK) cells. The molar ratio of HEK293 cell line: REP / CAP plasmid (Agilent): AAV complex was set at 1:1:1, following conventional AAV production methods in the art. AAV from HEK293 lysates was obtained by ultracentrifugation, and the amounts of viral protein (VP) and viral genome were quantified by Western blot (WB) and qPCR. qPCR results #1, #2, and #3 were biological replicates.

[0185] Figure 4 Western blot results confirming AAV productivity in Example 1 (GC), Comparative Example 1 (WT), and Comparative Example 2 (BC-del) for green fluorescent protein (GFP) gene expression are shown.

[0186] Figure 5 The results of qPCR viral genome quantification for AAV productivity in Example 1 (pAAV-GC), Comparative Example 1 (pAAV-WT), and Comparative Example 2 (pAAV-BC del) used for GFP gene expression are shown.

[0187] Figure 6 The results show qPCR viral genome quantification of AAV productivity using pAAV-GC, pAAV-WT, and pAAV-BC del vectors to confirm the expression of various genes (GFP, Luciferase, TP53, RPE65, TPP1, FVIII).

[0188] As a result, Figures 4 to 6As shown, even with the introduction of various genes, the AAV productivity of the pAAV-GC vector is significantly higher than that of the pAAV-WT and pAAV-BC del vectors.

[0189] It is evident that the pAAV-GC vector, including the asymmetrically modified ITR, partially omits self-replication in the host cell, increasing the encapsulation efficiency of viral particles during the same production period. Therefore, due to the increased self-replication efficiency, the productivity of the pAAV-GT vector is increased compared to existing AAV delivery vectors.

[0190] Experiment 2: Confirming the gene expression rate of the AAV complex

[0191] 2-1. Confirm the expression rate of the GFP gene.

[0192] The expression rates of the recombinant AAV complex according to one embodiment were confirmed. Specifically, HEK293, a normal fetal kidney-derived cell line, was infected with the same amount of AAV complex (Example 1, Comparative Example 1, or Comparative Example 2). After 72 hours, the GFP signal expressed by the cells was photographed using an inverted fluorescence microscope. The cells were then lysed to purify the protein, and the expression rate of the GFP gene was confirmed by Western blotting after loading the sample onto an SDS-PAGE gel. The intensity of the immunoblotting protein bands was measured using ImageJ software. The detection level of GFP protein in the pAAV-GC vector-treated sample with the strongest band intensity was set to 100%, and the expression rate of each gene was quantified as a relative value. Two biological replicates were used for each group.

[0193] Figure 7 The results show the results of Western blotting confirmation of transgenic GFP expression in the AAV complex according to ITR type (ITR WT, ITR BC del, or ITR GC).

[0194] Table 3 shows the results of quantifying the relative expression rate (%) of transgenic GFP in the AAV complex according to ITR type.

[0195] [Table 3]

[0196]

[0197] As a result, Figure 7 As shown in Table 3, the expression rate of the GFP gene was significantly higher when using the pAAV-GC vector (Example 1) compared with the pAAV-WT vector (Comparative Example 1) and the pAAV-BC del vector (Comparative Example 2).

[0198] 2-2. Confirm the expression rates of various genes.

[0199] Experiments were conducted using Examples 1 to 6 and Comparative Examples 1 to 12, following the same method as in Experimental Example 2-1, to confirm the expression rates of various transgenic GFP, Luciferase, TP53, RPE65, TPP1, and PVⅢ.

[0200] For the GFP and Luciferase genes, the same cell lines as those used in Experiment 2-1 were used. For the TP53, TPP1, RPE65, and FVIII genes, HEK293 cells with each gene knocked out using CRISPR / Cas9 (Synthego, USA) were used to remove the effects caused by endogenous genes inherent in the target cells.

[0201] After confirming that no endogenous gene expression occurred in the cells, the cells were cultured in 6-well cell culture plates. The concentration of the virus solution was adjusted to 1.0 × 10⁻⁶ cells per well. 5 Cell treatment 1.0 × 10 8 Viral particles were collected, and cells were treated with a viral solution. Two days after AAV treatment, the proteins expressed in the introduced genes were detected by Western blot (WB). The antibodies used at this time were: GFP (Invitrogen A-11122), Luciferase (Invitrogen PA1-179), TP53 (MA5-14067), TPP1 (PA5-102819), RPE65 (MA1-16578), and FVIII (PA5-104451). The intensity of the WB bands was numerically calculated using ImageJ software. The band of pAAV-GC was considered as 100%, and the values ​​of pAAV-WT and pAAV-BC del were converted to relative values. Three biological replicates were performed for each group.

[0202] Table 4 shows the results of quantifying the relative expression rate (%) of transgenes in AAV according to ITR type (pAAV-GC, pAAV-BC del, or pAAV-WT).

[0203] Figure 8 The relative expression rate (%) of transgenes in the AAV complex according to ITR type (pAAV-GC, pAAV-BC del, or pAAV-WT) is shown.

[0204] [Table 4]

[0205]

[0206] The results are shown in Table 4 and Figure 8 As shown, compared with the pAAV-WT vector containing symmetrically unmodified ITR and the symmetrically modified pAAV-BC del vector, the expression rate of all transgenes was about 2-fold to about 10-fold higher when using the pAAV-GC vector containing asymmetrically modified ITR.

[0207] Therefore, it is evident that pAAV-GC, including asymmetrically modified ITRs, can be used as a delivery vector platform in which transgene expression efficiency is significantly increased and for delivering various transgenes into target cells.

[0208] Experimental Example 3: Confirmation of the genotoxicity of the AAV complex

[0209] 3-1. Confirm whether the transgenic GFP has been inserted into the host chromosome.

[0210] Experiments were conducted to confirm that the transgene did not recombine with the target cell chromosome when the recombinant AAV complex according to one embodiment was used.

[0211] Specifically, H460 cells were infected with pAAV-GC (Example 1), pAAV-WT (Comparative Example 1), and pAAV-BC del (Comparative Example 2), each loaded with the GFP gene. GFP-expressing cells were isolated and seeded in equal numbers into culture dishes. Cells were subcultured every 3 days at a 1 / 3 ratio, and the GFP gene expression rate was monitored to confirm whether the GFP gene was heritably passed on to offspring.

[0212] Figure 9A The results of GFP expression were shown using fluorescence microscopy at passage numbers P3 and P10.

[0213] Table 5 shows the results of the percentage of GFP-expressing cells at subculture numbers P1 to P10.

[0214] [Table 5]

[0215]

[0216] Figure 9B Table 4 shows the results of the quantitative GFP-expressing cell ratio.

[0217] As a result, Figure 9A and 9BAs shown in Table 5, after P7, expression of GFP as a transgene was rarely observed in cells infected with the pAAV-GC vector (Example 1). Conversely, GFP as a transgene was continued to be observed in cells infected with the pAAV-WT vector (Comparative Example 1) or the pAAV-BCdel vector (Comparative Example 2).

[0218] Therefore, it was confirmed that, compared with the AAV complexes in Comparative Examples 1 and 2, the genotoxicity of the genes transmitted to offspring was significantly reduced in the AAV complex of Example 1.

[0219] 3-2. Confirm whether various transgenes have been inserted into the host chromosome.

[0220] Experiments were conducted using Examples 1 to 6 and Comparative Examples 1 to 12, following the same method as in Experimental Example 3-1, to confirm the insertion of various transgenic GFP, Luciferase, TP53, RPE65, TPP1, and FVIII into the host chromosome.

[0221] For the GFP and Luciferase genes, the same cell lines as those used in Experiment 3-1 were employed. For the TP53, TPP1, RPE65, and FVIII genes, HEK293 cells with each gene knocked out using CRISPR / Cas9 (Synthego, USA) were used to remove the effects caused by endogenous genes inherent in the target cells.

[0222] After confirming that no endogenous gene expression occurred in the cell lines, the proteins expressed in each gene introduced by AAV were detected by Western blotting. The antibodies used were: GFP (Invitrogen A-11122), Luciferase (Invitrogen PA1-179), TP53 (MA5-14067), TPP1 (PA5-102819), RPE65 (MA1-16578), and FVIII (PA5-104451). ImageJ software was used to numerically represent the intensity of the Western blotting bands. The band values ​​of pAAV-WT were considered 100%, and the band values ​​of pAAV-GC and pAAV-BC del were converted to relative values.

[0223] Table 6 shows the results of relative gene expression levels in subculture number P7.

[0224] [Table 6]

[0225]

[0226] Figure 10 Table 5 shows the results of quantifying the relative gene expression levels in P7.

[0227] The results are shown in Table 6 and Figure 10 As shown, transgene expression was rarely observed in cells infected with the pAAV-GC vector in P7. Conversely, transgene expression was confirmed to continue to be observed in cells infected with the pAAV-WT or pAAV-BC del vectors.

[0228] Therefore, it was confirmed that the genotoxicity of transgenes transmitted to offspring was significantly reduced in the pAAV-GC vector, which includes an asymmetrically modified ITR, compared with the pAAV-WT vector, which includes a symmetrically unmodified ITR, and the pAAV-BCdel vector, which includes a symmetrically modified ITR.

[0229] 3-3. Confirm whether the transgene forms a tandem structure in the target cells.

[0230] Experiments were conducted to confirm whether concatenators, which are found when transgenes are inserted into host chromosomes, are formed when transgenes are delivered to host cells using AAV vectors.

[0231] Specifically, in P7 of Experiment 3-1, deoxyribonucleic acid (DNA) was extracted from each GFP-expressing cell. The extracted DNA was amplified by polymerase chain reaction (PCR) and electrophoresis was performed to confirm the residual form of the DNA delivered to the host cell.

[0232] Figure 11 The results show PCR amplification to confirm whether the transgene forms a concatenator in the host cell.

[0233] As a result, it was confirmed that... Figure 11 As shown, for the pAAV-GC vector (Example 1) which lacks a hairpin structure on one side of its ITR, it is impossible to form a tandem structure, which is known to be characteristic of AAV vectors. Conversely, for the pAAV-WT vector (Comparative Example 1) and the pAAV-BC del vector (Comparative Example 2) which have hairpin structures on both sides of their ITRs, transgenes in various polymeric forms, such as monomers, dimers, and tandem structures, were observed.

[0234] Therefore, it was confirmed that if the pAAV-GC vector is used, the transgene does not form a tandem structure in the host cell, thus inhibiting insertion into the host cell chromosome.

[0235] In summary, pAAV-GC vectors with asymmetrically modified ITRs are suitable as delivery vectors that can suppress transgene expression in host cells for a long period and express transgenes efficiently in a short period.

[0236] The foregoing description of this disclosure is for illustrative purposes only. Those skilled in the art to which this disclosure pertains will understand that embodiments and implementations can be readily modified without altering the technical concept or essential features of this disclosure. Therefore, the embodiments described should be understood in all respects as exemplary, not restrictive.

[0237] The embodiments described in this specification should be considered illustrative only and not for limiting purposes. Features or aspects of each embodiment are generally considered to be applicable to other similar features or aspects of other embodiments. Although one or more embodiments have been described with reference to the accompanying drawings, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of this disclosure as defined by the following claims.

Claims

1. An adeno-associated virus (AAV) complex comprising a polynucleotide sequence encoding a transgene between a first inverted terminal repeat (ITR) and a second ITR, wherein, all or a portion of a stem-loop structure formed by RBE (rep-binding element), RBE', A, A', B, B', C, C', and D regions in either of the first ITR and the second ITR is modified.

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

3. The adeno-associated viral complex of 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 viral complex of claim 1, wherein, the first ITR is not modified and the second ITR is modified.

5. The adeno-associated viral complex of claim 1, wherein, the modification of the stem-loop structure is at least one of an insertion, a deletion, or a substitution.

6. The adeno-associated virus complex of claim 1, wherein either of the first ITR and the second ITR is modified to not form a stem-loop structure.

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

8. The adeno-associated viral complex of claim 1, wherein, either of the first ITR and the second ITR comprises a trs (terminal resolution site) sequence and a RBE sequence, and all of the C, C', B', B, RBE', A', and D sequences after the RBE are deleted.

9. The adeno-associated viral complex of claim 2, wherein, the promoter is a tissue-specific promoter.

10. The adeno-associated viral complex of claim 1, wherein, the transgene is a therapeutic gene.

11. The adeno-associated viral complex of claim 1, wherein, the transgene is green fluorescent protein (GFP), Luciferase, TP53, RPE65, TPPl, or FVIII.

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

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

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

15. The composition of claim 13 for use in delivering a therapeutic gene for gene therapy.

Images