Nucleic acid molecules containing asymmetrically modified ITRs for improving the expression rate of inserted genes and their applications

Abstract

We disclose a nucleic acid molecule containing an asymmetrically deformed inverse repeat (ITR). An AAV vector containing the nucleic acid molecule has advantages such as improved productivity and transgene expression efficiency, and reduced genotoxicity, because one of the two ITRs is a deformed asymmetric ITR. We also disclose a composition and a vector.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid molecule containing a modified ITR, an adeno-associated virus vector containing the same, and applications for gene therapy. [Background technology]

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

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

[0004] However, the aforementioned AAV has a problem in that, due to the capsidization of up to approximately 4.4 kb of protein-coding sequences by the reverse repeat sequence (ITR), it has the drawback of having low DNA packaging capacity. Furthermore, when genes inserted in the 3'→5' direction and genes inserted in the 5'→3' direction are expressed in the host cell genome, the expression rate of the delivered gene is reduced due to the inter-strand competition. In addition, there is a possibility that the delivered gene may induce cancer when it is inserted into the host cell genome.

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

[0006] One specific example relates to a nucleic acid molecule containing a gene expression cassette between a first inverted terminal repeat (ITR) and a second ITR, wherein the gene expression cassette contains a heterologous polynucleotide sequence, and at least one of the first and second ITRs contains a nucleotide sequence or a complementary sequence having approximately 75% or more sequence identity with one of the nucleotide sequences or a functional derivative of sequence numbers 1 to 9. In the nucleic acid molecule, one of the nucleotide sequences of the first and second ITRs has approximately 95% or more sequence identity with one of the nucleotide sequences or a functional derivative of sequence numbers 1 to 9. Alternatively, in the nucleic acid molecule, one of the nucleotide sequences of the first and second ITRs is selected from one of the nucleotide sequences or a functional derivative of sequence numbers 1 to 9. Alternatively, in the nucleic acid molecule, one of the first ITR and the second ITR is composed of a nucleotide sequence or a complementary sequence having approximately 75% or more sequence identity with one of the nucleotide sequences or functional derivatives of sequence numbers 1 to 9. The sequences of the first ITR and the second ITR can be based on ITR sequences of viruses belonging to the genus Dependovirus of the family Parvoviridae. The sequences of the first ITR and the second ITR can be based on ITR sequences of adeno-associated viruses (AAVs). The sequences of the first ITR and the second ITR can each be independently based on ITR sequences of AAV serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In the nucleic acid molecule, the first ITR is the wild-type ITR of AAV, and the second ITR includes at least one nucleotide sequence or a complementary sequence having approximately 75% or more sequence identity with any one of sequence numbers 1 to 9 or a functional derivative thereof.Alternatively, in the nucleic acid molecule, the first ITR is the wild-type ITR of AAV, and the second ITR is composed of a nucleotide sequence or a complementary sequence having approximately 75% or more sequence identity with any one of the nucleotide sequences of SEQ ID NOs. 1 to 9 or a functional derivative thereof. Alternatively, in the nucleic acid molecule, the first ITR is the wild-type ITR of AAV, and the second ITR is essentially composed of any one sequence selected from the nucleotide sequences of SEQ ID NOs. Alternatively, in the nucleic acid molecule, the first ITR is the wild-type ITR of AAV, and the second ITR is essentially composed of the nucleotide sequence of SEQ ID NO. 1. In the nucleic acid molecule, in either the first ITR or the second ITR, all or part of the stem-loop structure formed in the RBE (rep-binding element), RBE', A, A', B, B', C, C', and D regions is modified. Alternatively, in the nucleic acid molecule, one of the first ITR and the second ITR is modified so as not to form a stem-loop structure. Alternatively, in the nucleic acid molecule, all or part of the stem-loop structures formed in the RBE, RBE', A, A', B, B', C, C', and D regions may be deleted in one of the first ITR and the second ITR. In the nucleic acid molecule, the gene expression cassette may further include one or more of the promoter and polyadenylated sequences. In the nucleic acid molecule, the heterogeneous polynucleotide sequence may encode a therapeutic gene.

[0007] Other specific examples relate to vectors containing nucleic acid molecules as described above and throughout the invention. Such vectors may be AAV vectors.

[0008] Further specific examples relate to compositions comprising vectors and pharmaceutically acceptable carriers as described herein. Such compositions may be used for therapeutic gene delivery for gene therapy.

Means for Solving the Problem

[0009] Hereinafter, embodiments shown in the accompanying drawings will be referred to in detail, where the same reference numerals refer to the same elements. In this regard, this embodiment has various forms and should not be construed as being limited to the description presented herein. Therefore, the embodiments described with reference to the drawings are merely for explaining aspects of this specification. As used herein, the term "and / or" includes any at least one of the related listed items and any and all combinations thereof. When an expression such as "at least one of" comes before a list of elements, it applies to the entire list of elements, not to individual elements of the list.

[0010] One aspect relates to a nucleic acid molecule comprising a modified inverted repeat sequence.

[0011] Another aspect relates to a vector comprising the nucleic acid molecule.

[0012] Another aspect relates to a cell transformed with the vector.

[0013] Another aspect relates to a method of delivering a transgene, comprising the step of administering an effective amount of the vector.

[0014] Another aspect relates to a gene therapy method, comprising the step of administering an effective amount of the vector.

[0015] Another aspect relates to a method of treating a disease in an individual, comprising the step of administering an effective amount of the vector described herein to an individual who needs it.

[0016] Another aspect relates to a composition comprising the vector.

[0017] Another aspect relates to the use of the vector for gene therapy.

[0018] Additional specific examples are described in part in the following explanations, and in part will become clear from the explanations or may be learned by implementing the specific examples presented in the present invention.

[0019] One aspect relates to a nucleic acid molecule comprising a modified inverted terminal repeat (ITR).

[0020] As used herein, the term "nucleic acid molecule" is used interchangeably with "nucleic acid", "nucleotide", and "polynucleotide". The nucleic acid molecule refers to a phosphoester polymer form of deoxyribonucleoside or ribonucleoside or a phosphoester analog thereof.

[0021] In certain specific examples, the nucleic acid molecule comprises a first ITR, a gene expression cassette, and a second ITR.

[0022] In certain specific examples, the nucleic acid molecule comprises a gene expression cassette between the first ITR and the second ITR.

[0023] In certain specific examples, the gene expression cassette is operably arranged between the first ITR and the second ITR.

[0024] In certain specific examples, the gene expression cassette comprises a heterologous polynucleotide sequence.

[0025] In certain specific examples, the nucleic acid molecule may comprise an asymmetrically modified ITR.

[0026] In certain specific examples, the sequence of the ITR can be based on the ITR sequence of a virus belonging to the genus Dependovirus of the family Parvoviridae.

[0027] In certain specific examples, the sequence of the ITR can be based on the ITR sequence of AAV. The ITR sequence of AAV is known.

[0028] 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, and is a helper vector-dependent human parvovirus. The genome consists of ITRs at both ends and two ORFs (open reading frames), rep and cap. The N-terminus of the genome codes for the rep gene, which is involved in viral replication and viral gene expression, and the C-terminus codes for the cap gene, which encodes the viral capsid protein. The ITR is involved in the replication of the AAV genome and the packaging of AAV particles. The ITR includes the RBE (rep-binding element), RBE', A, A', B, B', C, C', and D regions, and forms a stem-loop structure (hairpin structure). The structure of AAV ITR is widely known in literature such as Goncalves, MA Virology Journal, 2(1):43(2005), which is incorporated herein by reference.

[0029] In specific examples, the AAV may include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and may also include other AAV serotypes that are currently known or may be discovered in the future. The AAV may include known AAV derivatives. And, or alternatively, the AAV may include modified or artificial AAVs.

[0030] Therefore, in a specific example, the sequence of the ITR may be based on an ITR sequence of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The sequence of the ITR may be based on an ITR sequence of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. The first ITR and the second ITR may be based on ITR sequences of the same or different AAV serotypes.

[0031] In a specific example, the nucleic acid molecule includes a gene expression cassette between a first ITR and a second ITR, the gene expression cassette includes heterogeneous polynucleotide sequences, and either the first ITR or the second ITR may include at least a nucleotide sequence or a complementary sequence having approximately 75% or more, approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, approximately 99% or more, or 100% sequence identity with any one of the nucleotide sequences from SEQ ID NOs: 1 to 9 or a functional derivative thereof.

[0032] In this specification, the term "functional derivative" means a derivative having substantially the same functional properties. Such derivative means a similar compound obtained by chemically altering part of the structure of a compound. Such derivative may also mean a compound in which a hydrogen atom or a specific group of atoms in the compound is substituted with another atom or group of atoms. Methods for producing derivatives of a compound while retaining substantially the same functional properties are known in the art.

[0033] In this specification, the term "sequence identity" refers to the degree of amino acid residue or base identity between sequences after alignment to make both sequences as similar as possible in a specific comparison region. The percentage of sequence identity is determined using a known sequence comparison program, such as BLASTN (NCBI), CLC Main Workbench (CLC bio), and MegAlign™ (DNASTAR Inc).

[0034] In this specification, the term "about" is used to mean a range of ±10% of the specified number.

[0035] In a specific example, a nucleic acid molecule containing a gene expression cassette between a first reverse repeat sequence (ITR) and a second ITR is disclosed, wherein the gene expression cassette contains heterogeneous polynucleotide sequences, and either the first ITR or the second ITR contains at least one nucleotide sequence or a complementary sequence having approximately 75% or more sequence identity with any one of sequence numbers 1 to 9 or a functional derivative thereof. Alternatively, either the first ITR or the second ITR has approximately 95% or more sequence identity with any one of sequence numbers 1 to 9 or a functional derivative thereof. Alternatively, either the first ITR or the second ITR is selected from any one of sequence numbers 1 to 9 or a functional derivative thereof. In a specific example, either the first ITR or the second ITR consists of a nucleotide sequence or a complementary sequence having approximately 75% or more sequence identity with any one of sequence numbers 1 to 9 or a functional derivative thereof. In the nucleic acid molecule, the sequences of the first ITR and the second ITR may be based on the ITR sequences of viruses belonging to the genus Dependovirus of the family Parvoviridae. In the nucleic acid molecule, the sequences of the first ITR and the second ITR may be based on the ITR sequences of adeno-associated viruses (AAVs). In the nucleic acid molecule, the sequences of the first ITR and the second ITR may each be independently based on the ITR sequences of AAV serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In this regard, the origins of the first ITR and the second ITR sequences may be the same or different from each other.

[0036] The sequence of sequence number 1 can be based on the AAV2 ITR sequence.

[0037] The sequence of sequence number 2 can be based on the AAV1 ITR sequence.

[0038] The sequence of sequence number 3 can be based on the AAV3 ITR sequence.

[0039] The sequence of sequence number 4 can be based on the AAV4 ITR sequence.

[0040] The sequence of sequence number 5 can be based on the AAV6 ITR sequence.

[0041] The sequence of sequence number 6 can be based on the AAV7 ITR sequence.

[0042] The sequence of sequence number 7 can be based on the AAV5 ITR sequence.

[0043] The sequence of sequence number 8 can be based on the AAV8 ITR sequence.

[0044] The sequence of sequence number 9 can be based on the AAV9 ITR sequence.

[0045] In certain specific examples, the sequences of sequence numbers 1 to 9 may be AAV ITR sequences with a portion deleted. The sequences of sequence numbers 1 to 9 may include the trs (terminal resolution site) sequence and the RBE sequence among the AAV ITR sequences. The sequences of sequence numbers 1 to 9 may have all of the C, C', B', B, RBE', A', and D sequences deleted from after the RBE.

[0046] In a specific example, either the first ITR or the second ITR may consist of a nucleotide sequence or a complementary sequence having approximately 75% or more, approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, approximately 99% or more, or 100% sequence identity with any one of the nucleotide sequences or functional derivatives of sequence numbers 1 to 9.

[0047] In certain specific examples, the nucleic acid molecule may contain asymmetrically deformed ITRs. In one specific example, the nucleic acid molecule has a deformed first ITR and / or second ITR. In another specific example, the nucleic acid molecule has a deformed first ITR and an undeformed second ITR. In yet another specific example, the nucleic acid molecule has an undeformed first ITR and a deformed second ITR. In yet another specific example, the nucleic acid molecule has an undeformed 5'-ITR on the (+) strand of the transgene and a deformed 3'-ITR on the (+) strand. In yet another specific example, the nucleic acid molecule has a deformed 5'-ITR on the (-) strand of the transgene and an undeformed 3'-ITR on the (-) strand. In other words, a deformed second ITR may mean that the 3'-ITR on the (+) strand and / or the 5'-ITR on the (-) strand of the transgene have been deformed.

[0048] In specific examples, AAV vectors containing nucleic acid molecules, by including asymmetrically modified ITRs, increase the productivity of the AAV vector and the expression rate of the transgene, while reducing genotoxicity. Therefore, AAV complexes can be used as AAV transmission platform for transmitting diverse genes into target cells. Furthermore, AAV vectors containing the aforementioned nucleic acid molecules can be used as target transmission devices for expressing transgenes efficiently for a short period while suppressing the long-term expression of the transgenes in host cells.

[0049] In a specific example, the non-modified ITR among the first and second ITRs may be a wild-type ITR. The non-modified ITR among the first and second ITRs may be an AAV wild-type ITR.

[0050] In certain specific examples, the non-deformed ITR among the first and second ITRs may be a functional derivative possessing substantially the same functional properties as the wild-type ITR (e.g., AAV wild-type ITR).

[0051] In one specific example, the first ITR is the wild-type ITR of AAV, and the second ITR may include at least one nucleotide sequence or a complementary sequence having approximately 75% or more, approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, approximately 99% or more, or 100% sequence identity with any one of the nucleotide sequences from sequence numbers 1 to 9 or a functional derivative thereof.

[0052] In other specific examples, the first ITR is the wild-type ITR of AAV, and the second ITR may consist of a nucleotide sequence or a complementary sequence having approximately 75% or more, approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, approximately 99% or more, or 100% sequence identity with any one of the nucleotide sequences from sequence numbers 1 to 9 or a functional derivative thereof.

[0053] In other specific examples, the first ITR is the wild-type ITR of AAV, and the second ITR may contain at least one nucleotide sequence that has approximately 75% or more, approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, approximately 99% or more, or 100% sequence identity with any one of the nucleotide sequences of sequence numbers 1 through 9.

[0054] In other specific examples, the first ITR may be the wild-type ITR of AAV, and the second ITR may consist of a nucleotide sequence having approximately 75% or more, approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, approximately 96% or more, approximately 97% or more, approximately 98% or more, approximately 99% or more, or 100% sequence identity with any one of the nucleotide sequences from sequence numbers 1 to 9.

[0055] In other specific examples, the first ITR is the AAV wild-type ITR, and the second ITR may contain at least one nucleotide sequence from sequence numbers 1 through 9.

[0056] In other specific examples, the first ITR is the AAV wild-type ITR, and the second ITR consists of, or may be composed of, any one nucleotide sequence from sequence numbers 1 through 9.

[0057] In other specific examples, the first ITR is the AAV wild-type ITR, and the second ITR consists of, or may be required to consist of, the nucleotide sequence of Sequence ID No. 1.

[0058] In a specific example, in either the first ITR or the second ITR, all or part of the stem-loop structure consisting of the RBE (rep-binding element), RBE', A, A', B, B', C, C', and D regions is deformed.

[0059] In one specific example, the deformation of the stem-loop structure (hairpin structure) may be selected from insertion, deletion, and substitution.

[0060] In one specific example, the deformation of the stem-loop structure (hairpin structure) includes deformation that includes a single stem and a single loop. For example, the deformed ITR may include the removal of the B-B' arm so that the C-C' arm remains, or the removal of the C-C' arm so that the B-B' arm remains.

[0061] In one specific example, the deformation of the stem-loop structure (hairpin structure) includes deformation to have a single stem instead of two loops. For example, the deformed ITR may include the absence of B-B' and C-C' arms.

[0062] In one specific example, the deformed ITR may include a deletion of the C' region such that the severed C loop and B-B' arm remain. Similarly, the deformed ITR may include a deletion of the B region such that the severed B loop and C-C' arm remain.

[0063] In one specific example, the modified ITR may include base pair deletions in at least one of the C, C', B, or B' portions such that complementary base pairing occurs between the C and B' portions and between the C' and B portions, generating a single arm.

[0064] In one specific example, the modified ITR may include one, two, three, four, five, or six nucleotide modifications (e.g., deletion, substitution, or addition) in at least one region selected between A' and C, C and C', C' and B, B and B', and B' and A.

[0065] In one specific example, the deformation of the stem-loop structure (hairpin structure) may include deformation of the structural elements. Specifically, deformation of the structural elements may include changes in the height of the stem and / or changes in the number of nucleotides in the loop. For example, the height of the stem may be approximately 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range of nucleotides within that number. In yet another example, the loop may have approximately 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range of nucleotides within that number.

[0066] In other specific examples, the interval between two elements (e.g., RBE and hairpin, not limited to) can be altered (e.g., increased or decreased) to change the functional interaction with the larger Rep protein. For example, the interval can be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides or more, or any range of nucleotides within that number.

[0067] In one specific example, either the first ITR or the second ITR is deformed so as not to form a stem-loop structure (hairpin structure). In another specific example, the first ITR is deformed so as not to form a stem-loop structure. In yet another specific example, the second ITR is deformed so as not to form a stem-loop structure. In yet another specific example, the first ITR may not be deformed, and the second ITR may be deformed so as not to form a stem-loop structure.

[0068] The expression "deformed so as not to form a stem-loop sequence (hairpin structure)" means that the ITR's structure is deformed by the alteration of its sequence so that it does not form a stem-loop sequence (hairpin structure) but exists as an open-end or free-end. The AAV vector containing the nucleic acid molecule described herein can suppress the formation of circular dimers and circular concatemers in infected cells and inhibit the integration into the host genome observed by AAV by deforming either the first ITR or the second ITR so as not to form a stem-loop sequence (hairpin structure). Furthermore, this deformation can increase the productivity of the AAV vector and the expression rate of the transgene.

[0069] In one specific example, either the first ITR or the second ITR is deformed into a blunt end. In another specific example, either the first ITR or the second ITR is deformed into a sticky end. In yet another specific example, the second ITR is deformed into a blunt end or a sticky end. In yet another specific example, the second ITR is deformed into a blunt end. The deformation to a blunt end or a sticky end can be performed by a person of ordinary skill using methods known to the art.

[0070] In one specific example, all or part of the stem-loop structure consisting of regions RBE, RBE', A, A', B, B', C, C', and D is deleted in either the first ITR or the second ITR. In another specific example, part of the stem-loop structure consisting of regions RBE, RBE', A, A', B, B', C, C', and D is deleted in either the first ITR or the second ITR. In yet another specific example, either the first ITR or the second ITR contains the trs sequence and the RBE sequence, with the sequences C, C', B', B, RBE', A', and D completely deleted from RBE onward. In yet another specific example, the first ITR may remain unchanged, while the second ITR contains the trs sequence and the RBE sequence, with the sequences C, C', B', B, RBE', A', and D completely deleted from RBE onward.

[0071] Generally, RNA polymerases produce mRNA with a sequence complementary to the target gene in the promoter region. This process is called "transcription," and it proceeds in the 5' to 3' direction. On the other hand, when inserting a target gene into an AAV vector, the gene is inserted into the double helix of the AAV vector DNA in the 5' to 3' direction and the 3' to 5' direction, respectively. Therefore, while the transcription process of the target gene is progressing, the AAV vector DNA double helix interferes with the transcription of the target gene, leading to a problem of reduced expression efficiency of the target gene. However, AAV vectors containing nucleic acid molecules as described herein can avoid interference from the double helix structure of the AAV vector DNA during the transcription of the target gene by modifying the stem-loop structure, specifically by deleting all or part of the stem-loop structure, thereby improving the expression efficiency of the target gene.

[0072] For example, in an AAV vector containing a nucleic acid molecule with asymmetrically deformed ITRs, the first ITR may remain undeformed, while the second ITR may be deformed in such a way that it does not form a stem-loop structure. As a result, the 5'-ITR on the (-) strand of the transgene does not form a hairpin structure, and transcription of the transgene proceeds complementary to that strand in the 5' to 3' direction. On the other hand, the 3'-ITR on the (+) strand of the transgene does not form a hairpin structure, and transcription of the transgene does not proceed on that strand. In other words, transcription of the transmission gene proceeds only in the 5' to 3' direction, and the competitive factor in the 3' to 5' direction is removed, thus increasing the efficiency of gene expression.

[0073] In one embodiment, an AAV complex (pAAV-GC ITR vector) was produced in which an asymmetrically deformed ITR was created by partially modifying the hairpin structure of the second ITR among the wild-type AAV ITRs contained in the vector, using an AAV vector containing diverse transgenes. The produced AAV complex was compared with an AAV complex in which the hairpin structure was not deformed (pAAV-WT ITR vector) and an AAV complex in which both ends of the hairpin structure were deformed to be symmetrical (pAAV-BC del.ITR vector), and the productivity, transgene expression rate, and genotoxicity of each were confirmed. As a result, it was confirmed that the AAV complex containing the asymmetrically deformed ITR (pAAV-GC ITR vector) showed a significant increase in viral productivity and transgene expression rate, and very low genotoxicity, compared with the AAV complex in which the ITR was not deformed (pAAV-WT ITR vector) and the AAV complex containing the symmetrically deformed ITR (pAAV-BC del.ITR vector).

[0074] In certain specific examples, the nucleic acid molecule described herein may include a first ITR, a gene expression cassette, and a second ITR in the 5' to 3' direction.

[0075] In certain specific examples, the gene expression cassette may further include one or more of a promoter and a polyadenylated sequence, in addition to a heterogeneous polynucleotide sequence. The gene expression cassette may include a promoter sequence, a heterogeneous nucleotide sequence, and a polyadenylated sequence in the 5' to 3' direction.

[0076] In certain specific examples, the gene expression cassette may further include post-transcriptional regulatory elements. The gene expression cassette may include a promoter sequence, a heterogeneous polynucleotide sequence, a post-transcriptional regulatory element, and a polyadenylated sequence in the 5' to 3' direction.

[0077] In this specification, the term "promoter" refers to a site that regulates the transcription of a gene. The promoter is operablely ligated to the coding sequence of a transgene. The promoter is either a tissue-specific promoter or an inducible promoter. The tissue-specific promoter is not limited to any type of promoter that induces the specific expression of a gene in a specific type of cell or tissue in vivo. The tissue-specific promoter can be appropriately selected to be specific to the target tissue so that the transgene is expressed according to the type of transgene. Non-restrictive examples of the aforementioned tissue-specific promoters include liver-specific TBG (thyroxin binding globulin) promoters, insulin promoters, glucagon promoters, somatostatin promoters, PPY (pancreatic polypeptide) promoters, Syn (synapsin-1) promoters, creatine kinase (MCK) promoters, mammalian desmin (DES) promoters, A-MHC (α-myosin heavy chain) promoters, cTnT (cardiac troponin T) promoters, and SPC (Surfactant Protein C) promoters.

[0078] In certain specific examples, the heterogeneous polynucleotide may encode a transgene.

[0079] In this specification, the term "transgene" refers to a gene that is transmitted from one organism to another, either naturally or through various genetic engineering techniques. The term "transgene" may be used interchangeably with "transfer gene" and "target gene."

[0080] The aforementioned transgene is not limited to a specific type, as long as it is the target gene to be introduced into the host cell. In specific examples, AAV vectors containing nucleic acid molecules can be used as AAV transmission platform for transmitting diverse transgenes, regardless of the type of transgene, because they offer the advantages of improved AAV productivity, improved transgene expression rates, and reduced genotoxicity.

[0081] In one specific example, the introduced gene is a therapeutic gene. When the introduced gene is a therapeutic gene, the AAV vector containing the nucleic acid molecule described herein can be used as a gene therapy agent. Therefore, the AAV vector containing the nucleic acid molecule is a gene therapy AAV vector.

[0082] In one specific example, the transgene may be GFP, luciferase, TP53, RPE65, TPP1, or FVIII, but is not limited to these. When the transgene is TP53, the AAV complex for transmitting the transgene can be used as a gene therapy agent for anti-cancer treatment. When the transgene is RPE65, the AAV complex for transmitting the transgene can be used as a gene therapy agent for inherited retinal disease (IRD). When the transgene is TPP1 (CKN2), the AAV complex for transmitting the transgene can be used as a gene therapy agent for Batten disease. When the transgene is FVIII, the AAV complex for transmitting the transgene can be used as a gene therapy agent for hemophilia.

[0083] In specific examples, the introduced gene is of human or animal origin.

[0084] In certain specific examples, the post-transcriptional regulatory element may include WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

[0085] In one specific example, the gene expression cassette may further include a gene junction between the promoter and the polynucleotide sequence that encodes the introduced gene.

[0086] In this specification, the term "gene junction" means an undefined portion of the sequence between the promoter end and the start of the target gene sequence. Specifically, a promoter is a site where transcription machinery complexes bind to regulate a gene, and the boundary between the known promoter sequence end boundary and the gene start site whose expression is to be regulated is generally ambiguous. Therefore, optimization of the junction may be necessary to establish a successful promoter-gene expression relationship. The sequence of the gene junction can be appropriately selected by a person skilled in the art using conventional methods.

[0087] Other specific examples relate to vectors containing nucleic acid molecules as described herein.

[0088] The specific contents of the nucleic acid molecule are as described above.

[0089] The term "vector" refers to any medium used for the transport and / or cloning of nucleic acid molecules into a host cell.

[0090] In a specific example, the vector may be an AAV vector.

[0091] In specific examples, as an alternative, a different type of virus belonging to the genus Dependovirus of the family Parvoviridae may be used instead of the aforementioned AAV.

[0092] In this specification, the term "AAV vector" may be used interchangeably with "AAV complex," "AAV carrier," "recombinant AAV," and "recombinant AAV vector."

[0093] The vector may be manipulated to encode a sorting marker or reporter that provides selection or confirmation of contaminated cells. The sorting marker or reporter is publicly known in the art. Non-limiting examples of sorting markers include genes that provide resistance to ampicillin, streptovidine, kanamycin, hygromycin, etc. Non-limiting examples of reporters include luciferase, green fluorescent protein (GFP), etc.

[0094] Other specific examples relate to cells transformed with the vectors described herein.

[0095] The specific contents of the aforementioned vector are as described above.

[0096] In a specific example, the cell is a host cell.

[0097] In this specification, the term "transformation" refers to the change in the genetic properties of an organism due to the introduction of DNA from an external source. Transformation is the phenomenon in which DNA, a type of nucleic acid extracted from cells of one strain of an organism, is introduced into living cells of another strain, causing the DNA to enter those cells and altering their genetic traits. In other words, "transformation" means making it possible to introduce genes into host cells and express them within those host cells.

[0098] In specific examples, methods for introducing the AAV complex into cell lines and transforming them include, but are not limited to, methods known to the art, such as transient transfection using lipofectamine, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, and electroporation. Preferably, transformation can be performed using the lipofectamine 2000 reagent.

[0099] In other specific examples, the present invention relates to a method for transmitting a transgene to an individual, comprising the step of administering an effective amount of the vector described herein to an individual in need.

[0100] Further specific examples include gene therapy methods comprising the step of administering an effective amount of the vector described herein to an individual in need.

[0101] Further specific examples include methods for treating a disease in an individual, comprising the step of administering an effective amount of the vector described herein to an individual in need.

[0102] In the method of the specific example described above, the specific content of the vector is as described above. The vector may be an AAV vector (AAV complex).

[0103] In certain specific examples, in the methods described above, the vector is administered to an organism either on its own or the vector is put into a dosage form that can be administered to an organism and then administered to the organism. In one specific example, the vector may be administered to an organism in the form of a composition containing the vector as described herein. For example, the vector may be put into a dosage form that contains the vector and a pharmaceutically acceptable carrier and then administered to an organism.

[0104] In specific examples, the individual is one that requires the expression of a transgene transmitted by the AAV complex. The individual is one that suffers from or is likely to suffer from a disease to which gene therapy is applicable. The individual is one that suffers from or is likely to suffer from a disease that is treatable by the expression of a transgene transmitted by the AAV complex. The individual may be a mammal, such as a human, cattle, horse, pig, dog, sheep, goat, or cat.

[0105] In this specification, the term "gene therapy" means a treatment that utilizes genes to treat or prevent a disease. AAV vectors for delivering therapeutic genes into cells can be used as gene therapy agents. Diseases to which gene therapy is readily applicable include, but are not limited to, diseases caused by defects in a single gene. Non-limiting examples of diseases to which gene therapy is applicable include cancer, cardiovascular diseases, genetic disorders such as inherited retinal dystrophy (IRD), Batten disease, haemophilia, cystic fibrosis, muscular dystophy, thalassemia, and sickle cell anemia, neurological disorders, infectious diseases (such as acquired immunodeficiency), and joint diseases.

[0106] In a specific example, the cancer is a cancer in which tumor suppressor genes are deactivated. The cancer is, for example, a KRAS-mutated cancer. In a specific example, the cancer is, for example, a KRAS-mutated solid tumor. In a specific example, the cancer is, for example, a KRAS-mutated lung cancer. In a specific example, if the activity of the tumor suppressor genes is restored, cancer cells are eliminated and normal cells are present, making it possible to treat KRAS-mutated lung cancer. The tumor suppressor genes may be, for example, sPD-1, VHL, MMAC1, DCC, p53, NF1, WT1, RB, BRAC1, BRAC2, and RUNX3 genes.

[0107] In one specific example, the lung cancer is either non-small cell lung cancer or small cell lung cancer. Non-small cell lung cancer includes, for example, squamous cell carcinoma, large cell carcinoma, and lung adenocarcinoma.

[0108] Therefore, in certain specific examples, the AAV complex described herein can be used in gene therapy because it can activate a specific gene in a disease caused by reduced activity of that gene, thereby preventing or treating the disease.

[0109] In this specification, the term "prevention" means all actions that suppress or delay the onset of a disease by administering the AAV complex. The term "treatment" means all actions that improve or beneficially modify the symptoms of a disease by administering the AAV complex.

[0110] In a particular example, the method may further include the step of administering a second active ingredient to the individual. The second active ingredient is an active ingredient for the prevention or treatment of a disease to be treated. The active ingredient may be administered simultaneously with, separately from, or sequentially with the AAV complex.

[0111] In certain specific examples, the AAV complex can be formulated as an injectable suitable for administration by any appropriate route, such as intravenously, intraarterially, subcutaneously, intradermally, intraperitoneally, intramuscularly, intraarticularly, or intrathecally, and can be administered to an individual. The AAV complex can be administered systemically or locally, alone or in combination with other pharmaceutically active compounds.

[0112] The desirable dosage of the AAV complex varies depending on the patient's condition and body weight, the degree of the disease, the drug form, the administration route, and the duration, but can be appropriately selected by those skilled in the art. In one specific example, the dosage of the AAV complex is about 1.0×10 6 vg / kg to about 1.0×10 16 vg / kg, about 1.0×10 8 vg / kg to about 1.0×10 16 vg / kg, about 1.0×10 10 vg / kg to about 1.0×10 16 vg / kg, about 1.0×10 10 vg / kg to about 1.0×10 14 vg / kg, about 1.0×10 12 vg / kg to about 1.0×10 14 vg / kg, for example, about 1.0×10 12 vg / kg, about 1.0×10 13 vg / kg, about 1.0×10 14 vg / kg. In certain specific examples, the dosage of the AAV complex is about 1.0×10 13 vg / kg. The administration can be once a day, multiple times a day, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

[0113] Other specific examples relate to compositions containing the vectors described herein.

[0114] Still other specific examples relate to the use of the vectors described herein for gene therapy.

[0115] The specific details of the vectors and gene therapy are as described above.

[0116] In certain specific examples, the composition may be a gene therapy composition. In certain specific examples, the composition may be a therapeutic gene delivery composition for gene therapy. In certain specific examples, the composition may be a pharmaceutical composition. The composition may further comprise a pharmaceutically acceptable carrier. The carrier may comprise excipients, diluents, or adjuvants. As the carrier, a carrier suitable for delivering the AAV complex into a living organism may be used. Specifically, the carrier may be selected to be suitable for formulation into a parenteral dosage form (e.g., an injectable preparation). For example, the carrier may be selected to be suitable for formulation into an intravenous injection preparation. The carrier may be an aqueous solution, such as water or a buffered saline solution.

[0117] In certain specific examples, the composition may be prepared in any dosage form by conventional methods. The composition may be formulated into a form suitable for delivering an AAV vector to an individual. In certain specific examples, the composition may be formulated in an aqueous solution, for example, in water or buffered saline solution. In certain specific examples, the composition may be formulated, for example, as a parenteral dosage form (e.g., an injection, e.g., bolus injection or continuous infusion). In one specific example, the pharmaceutical composition may be formulated into an injectable dosage form suitable for administration by any suitable route, such as intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, or intravertebral cavity. In certain specific examples, the composition may be formulated to be administered by intravenous or subcutaneous injection. The composition may also be manufactured in systemic or topical dosage forms. The composition may be provided as a unit dose form, such as an ampoule, pre-filled syringe, small injectable container, or multi-dose container with preservatives added.

[0118] In specific examples, when the composition is used for gene therapy for a specific disease, it may further contain a second therapeutic agent that has a preventive or therapeutic effect on that disease. The pharmaceutical composition may be a single composition or individual compositions.

[0119] In certain specific examples, the composition may contain the vector (e.g., the AAV complex) in an effective amount. In the context of the specific examples described, the term “effective amount” means an amount sufficient to produce the desired preventive or therapeutic effect when administered to an individual requiring prevention or treatment of a disease. The effective amount can be appropriately selected by those skilled in the art depending on the cell or individual. The effective amount may be determined depending on the severity of the disease, the patient’s age, weight, health, sex, the patient’s sensitivity to the drug, the time of administration, the route of administration, the elimination ratio, the duration of treatment, factors including drugs compounded or used in combination with the composition used, and other factors well known in the medical field. In one specific example, the composition contains about 1.0 × 10 6 vg / kg or approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 8 vg / kg or approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 10 vg / kg or approximately 1.0 × 10 16 vg / kg, approximately 1.0×10 10 vg / kg or approximately 1.0 × 10 14 vg / kg, approximately 1.0×10 12 vg / kg or approximately 1.0 × 10 14 vg / kg, for example, approximately 1.0 × 10⁻⁶ 12 vg / kg, approximately 1.0×10 13 vg / kg, approximately 1.0×10 14 The AAV complex may contain a dose of vg / kg. In a specific example, the pharmaceutical composition may contain approximately 1.0 × 10⁻⁶ 13 The administration may include an AAV complex in a dose of vg / kg. The administration may be once daily, multiple times daily, or once a week, once every two weeks, once every three weeks, or once every four weeks or once a year. [Effects of the Invention]

[0120] AAV vectors containing nucleic acid molecules as described herein can be used as AAV carrier platforms because they have an asymmetric ITR in which one of the two ITRs is deformed, offering advantages such as increased productivity and transgene expression efficiency, and reduced genotoxicity.

[0121] Specifically, AAV vectors containing nucleic acid molecules as described herein have the following advantages:

[0122] 1. Deletion of any one of the two ITRs partially omits the self-renewal step in the host cell (or packaging cell), increasing the efficiency of packaging to viral particles within the same production period, thus increasing self-renewal efficiency and improving the productivity of the AAV vector compared to existing AAV complexes.

[0123] 2. The expression rate (expression level) of the transgene is higher compared to typical wild-type (WT) AAV.

[0124] 3. The genome of WT AAV forms a concatemer structure essential for the recombination of the transgene within infected cells. Specifically, it has been reported that the transgene is inserted into the host cell's chromosome by forming a concatemer structure, which is a multimerized form in which multiple identical genomes are linked together. On the other hand, the AAV complex described herein has an asymmetrically deformed ITR, and therefore does not form a concatemer structure, thus suppressing the insertion of the transgene into the host chromosome (i.e., recombination). Therefore, it has the advantage of reducing genotoxicity.

[0125] 4. The AAV complex having an asymmetrically deformed ITR is an AAV transmission platform with improved productivity and transgene expression rates, and reduced genotoxicity, allowing for the transmission of diverse genes into target cells using this platform.

[0126] Specific examples, features, and advantages of the present invention will become even clearer from the following description, along with the accompanying drawings. [Brief explanation of the drawing]

[0127] [Figure 1] This shows a cleavage map of an adeno-associated virus (AAV) vector according to one embodiment.

[0128] [Figure 2] The structure of an AAV vector according to one embodiment is shown.

[0129] [Figure 3A] This is a schematic genome diagram of the AAV vector containing the asymmetrically deformed ITR from Example 1.

[0130] [Figure 3B] This is a schematic genome diagram of the AAV vector containing an ITR that is not symmetrically deformed, as in Comparative Example 1.

[0131] [Figure 3C] This is a schematic genome diagram of the AAV vector containing the symmetrically deformed ITR of Comparative Example 2.

[0132] [Figure 4] These are Western blot results confirming the AAV productivity of Example 1 (GC), Comparative Example 1 (WT), and Comparative Example 2 (BC-del) for GFP gene expression.

[0133] [Figure 5] These are the qPCR viral genome quantification results confirming AAV productivity for GFP gene expression in Example 1 (pAAV-GC), Comparative Example 1 (pAAV-WT), and Comparative Example 2 (pAAV-BC del).

[0134] [Figure 6]This is a qPCR viral genome quantification result confirming the AAV productivity of pAAV-GC vectors, pAAV-WT vectors, and pAAV-BC del vectors for the expression of various genes (GFP, Luciferase, TP53, RPE65, TPP1, or FVIII).

[0135] [Figure 7] This is a Western blot analysis of the expression of the transgene GFP in the AAV complex, based on the ITR type (ITR WT, ITR BC del, or ITR GC).

[0136] [Figure 8] This shows the relative expression rate (%) of transgenes in the AAV complex according to the ITR type (pAAV-GC, pAAV-BC del, or pAAV-WT).

[0137] [Figure 9A] The results of fluorescence microscopy imaging of GFP expression at passage numbers P3 and P10 are shown.

[0138] [Figure 9B] Table 4, which quantifies the percentage of GFP-expressing cells, is shown in a diagram.

[0139] [Figure 10] Table 5, which quantifies the relative gene expression levels in P7, is shown in a diagrammatic form.

[0140] [Figure 11] The PCR amplification results confirming the presence or absence of concatemer formation of the transgene in host cells are shown. [Modes for carrying out the invention]

[0141] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided only to further facilitate understanding of the present invention and do not limit the scope of the present invention. [Examples]

[0142] Example 1. Production of an adeno-associated virus complex for GFP gene expression containing an asymmetrically deformed ITR.

[0143] 1-1. Production of recombinant adeno-associated virus vectors into which the GFP gene has been introduced

[0144] A recombinant adeno-associated virus (AAV) vector into which the GFP gene was introduced was manufactured. Specifically, the CBA-GFP (Chicken Beta-Actin promoter-GFP) gene, cloned into the CS4-GFP vector (Chungbuk National University Institute of Oncology), was PCR amplified using the primers shown in Table 1 below to clone it into the AAV2 vector. These primers were prepared by synthesizing the restriction enzymes NdeI and HindIII. After treating the amplified CBA-GFP DNA with the restriction enzymes NdeI and HindIII, it was cloned into the NdeI-HindIII position of the MCS (Multi Cloning Site) of a wild-type adeno-associated virus (AAV) 2 empty vector using T4 DNA ligase to obtain the AAV2-CBA-GFP plasmid. The ampicillin resistance gene was removed from the AAV2-CBA-GFP plasmid using the BspHI restriction enzyme sites located at both ends of the ampicillin resistance gene. A kanamycin resistance gene was inserted into the site where the ampicillin resistance gene had been removed. The kanamycin resistance gene was recombined using the PCR primers shown in Table 1 below.

[0145] [Table 1]

[0146] 1-2. Deformation of ITR structure

[0147] Site-directed mutagenesis was induced to deform a portion of the hairpin structure of the second ITR, one of the AAV2 wild-type reverse repeat sequences (ITRs) contained in the vector prepared in Example 1-1. Specifically, using the 5'-phosphorylation primers shown in Table 2 below, all or one or more of the C, C', B', B, RBE', A', and D sequences were deleted from after the RBE of the 5'-direction ITR of the (-) strand of the GFP gene in the AAV2 wild-type ITR contained in the vector. For example, all of the C, C', B', B, RBE', A', and D sequences were deleted from after the RBE of the 5'-direction ITR of the (-) strand of the GFP gene. As a result, the second ITR was deformed so that it did not form a hairpin structure. Consequently, an adenovirus complex for GFP gene expression was obtained containing an asymmetrically deformed ITR, in which the first ITR was not deformed and the second ITR was deformed. The acquired AAV complex contains an asymmetrically deformed ITR, in which the first ITR remains undeformed and the second ITR is deformed to consist of the sequence of sequence number 1.

[0148] An AAV vector containing an asymmetrically deformed ITR, such as in Example 1, was named pAAV-GC ITR (abbreviated as pAAV-GC).

[0149] Figure 1 shows a crack map of an AAV vector according to one embodiment.

[0150] Figure 2 shows the structure of an AAV vector according to one embodiment.

[0151] Figure 3A is a schematic genome diagram of the AAV vector containing the asymmetrically deformed ITR from Example 1.

[0152] [Table 2]

[0153] Example 2. Preparation of an AAV vector for Luciferase gene expression containing an asymmetrically deformed ITR.

[0154] An AAV complex for Luciferase gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the Luciferase gene (2,387 bp, GenBank accession no. M15077.1) was introduced instead of the GFP gene.

[0155] Example 3. Preparation of an AAV vector for TP53 gene expression containing an asymmetrically deformed ITR.

[0156] An AAV complex for TP53 gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the TP53 gene (2,512 bp, GenBank accession no. NM_000546.6) was introduced instead of the GFP gene.

[0157] Example 4. Preparation of an AAV vector for RPE65 gene expression containing an asymmetrically deformed ITR.

[0158] An AAV complex for RPE65 gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the RPE65 gene (2,605 bp, GenBank accession no. NM_000329.3) was introduced instead of the GFP gene.

[0159] Example 5. Preparation of an AAV vector for TPP1 gene expression containing an asymmetrically deformed ITR.

[0160] An AAV complex for TPP1(CLN2) gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the TPP1(CLN2) gene (1,693 bp, GenBank accession no. NM_000391.4) was introduced instead of the GFP gene.

[0161] Example 6. Preparation of an AAV vector for FVIII gene expression containing an asymmetrically deformed ITR.

[0162] An AAV complex for FVIII gene expression containing an asymmetrically modified ITR was prepared using the same method as in Example 1, except that the FVIII gene (673 bp, GenBank accession no. NM_000132.4) was introduced instead of the GFP gene.

[0163] Comparative Example

[0164] Comparative Example 1. Production of an AAV complex for GFP gene expression containing an undeformed, symmetrical ITR.

[0165] An AAV composite containing an undeformed, symmetrical ITR was fabricated using the same method as in Example 1-1, except that the hairpin structure of the ITR was not deformed.

[0166] As in Comparative Example 1, an AAV vector containing an undeformed, symmetrical ITR was named pAAV-WT ITR (abbreviated as pAAV-WT).

[0167] Figure 3B is a schematic genome diagram of the unmodified, symmetrical ITR-containing AAV vector of Comparative Example 1.

[0168] Comparative Example 2. Production of an AAV complex for GFP gene expression containing a symmetrically deformed ITR.

[0169] An AAV complex for GFP gene expression was produced using the same method as in Example 1-2, except that the C-C'-B'-RBE sequence was deleted from both ends of the ITR of the AAV complex produced in Example 1-1.

[0170] As shown in Comparative Example 2, an AAV vector containing a symmetrically deformed ITR was named pAAV-BC del.ITR (abbreviated as pAAV-BC del).

[0171] Figure 3C is a schematic genome diagram of the AAV vector containing the symmetrically deformed ITR of Comparative Example 2.

[0172] Comparative Example 3. Production of an AAV complex for Luciferase gene expression containing an undeformed, symmetrical ITR.

[0173] A Luciferase gene expression AAV complex containing a symmetrical, unmodified ITR was prepared using the same method as in Comparative Example 1, except that the Luciferase gene was introduced instead of the GFP gene.

[0174] Comparative Example 4. Production of an AAV complex for Luciferase gene expression containing a symmetrically deformed ITR.

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

[0176] Comparative Example 5. Preparation of an AAV complex for TP53 gene expression containing an undeformed, symmetrical ITR.

[0177] An AAV complex for TP53 gene expression containing an unmodified, symmetrical ITR was prepared using the same method as in Comparative Example 1, except that the TP53 gene was introduced instead of the GFP gene.

[0178] Comparative Example 6. Preparation of an AAV complex for TP53 gene expression containing a symmetrically deformed ITR.

[0179] An AAV complex for TP53 gene expression containing a symmetrically modified ITR was prepared using the same method as in Comparative Example 2, except that the TP53 gene was introduced instead of the GFP gene.

[0180] Comparative Example 7. Preparation of an AAV complex for RPE65 gene expression containing an undeformed, symmetrical ITR.

[0181] An AAV complex for RPE65 gene expression containing an unmodified, symmetrical ITR was prepared using the same method as in Comparative Example 1, except that the RPE65 gene was introduced instead of the GFP gene.

[0182] Comparative Example 8. Preparation of an AAV complex for RPE65 gene expression containing a symmetrically deformed ITR.

[0183] An AAV complex for RPE65 gene expression containing a symmetrically modified ITR was prepared using the same method as in Comparative Example 2, except that the RPE65 gene was introduced instead of the GFP gene.

[0184] Comparative Example 9. Production of an AAV complex for TPP1 gene expression containing a non-deformed, symmetrical ITR.

[0185] An AAV complex for TPP1 gene expression containing a non-modified ITR was prepared using the same method as in Comparative Example 1, except that the TPP1 gene was introduced instead of the GFP gene.

[0186] Comparative Example 10. Preparation of an AAV complex for TPP1 gene expression containing a symmetrically deformed ITR.

[0187] An AAV complex for TPP1 gene expression containing a symmetrically modified ITR was prepared using the same method as in Comparative Example 2, except that the TPP1 gene was introduced instead of the GFP gene.

[0188] Comparative Example 11. Production of an AAV complex for FVIII gene expression containing a non-deformed, symmetrical ITR.

[0189] An AAV complex for FVIII gene expression containing a non-deformed, symmetrical ITR was prepared using the same method as in Comparative Example 1, except that the FVIII gene was introduced instead of the GFP gene.

[0190] Comparative Example 12. Preparation of an AAV complex for FVIII gene expression containing a symmetrically deformed ITR.

[0191] An AAV complex for FVIII gene expression containing a symmetrically modified ITR was prepared using the same method as in Comparative Example 2, except that the FVIII gene was introduced instead of the GFP gene.

[0192] Experimental example

[0193] Experimental Example 1. Confirmation of the productivity of the AAV complex.

[0194] The productivity of recombinant AAV complexes was confirmed using a specific example.

[0195] Specifically, the AAV complexes prepared in Examples 1 to 6 and Comparative Examples 1 to 12 were used to plasma-infect 293T cells, which are human embryonic kidney (HEK) cells. The molar ratio of the HEK293 cell line:REP / CAP plasmid (Agilent Corporation):AAV complex was 1:1:1, and the standard AAV production method was followed. The AAV produced from the HEK293 lysate was obtained by ultracentrifugation, and the amounts of viral protein (VP) and viral genome were quantified by Western blot (WB) and qPCR, respectively. qPCR results #1, 2, and 3 represent biological replicates.

[0196] Figure 4 shows the Western blot results confirming AAV productivity for GFP gene expression, Example 1 (GC), Comparative Example 1 (WT), and Comparative Example 2 (BC-del).

[0197] Figure 5 shows the qPCR viral genome quantification results confirming AAV productivity for GFP gene expression, Example 1 (pAAV-GC), Comparative Example 1 (pAAV-WT), and Comparative Example 2 (pAAV-BC del).

[0198] Figure 6 shows the qPCR viral genome quantification results confirming AAV productivity for various gene expression vectors (GFP, Luciferase, TP53, RPE65, TPP1, or FVIII) using pAAV-GC vectors, pAAV-WT vectors, and pAAV-BCdel vectors.

[0199] As a result, as shown in Figures 4 to 6, it was confirmed that the pAAV-GC vector exhibits significantly higher AAV productivity compared to the pAAV-WT vector and the pAAV-BCdel vector, even when various genes are introduced.

[0200] Therefore, we observed that pAAV-GC vectors containing asymmetrically modified ITRs partially omit the self-renewal step in host cells, increasing the packaging efficiency to viral particles within the same production period, thus increasing self-renewal efficiency and improving the productivity of pAAV-GC vectors compared to existing AAV transduction devices.

[0201] Experimental Example 2. Confirmation of gene expression rate of the AAV complex.

[0202] 2-1. Confirmation of GFP gene expression rate

[0203] The target gene expression rate of recombinant AAV complexes was confirmed using a specific example. Specifically, HEK293, a cell line derived from a normal fetal kidney, was infected with the same amount of AAV complex (Example 1, Comparative Example 1, or Comparative Example 2). After 72 hours, the GFP signal expressed by the cells was imaged using an inverted fluorescence microscope. Subsequently, each cell was disrupted, the protein was purified, loaded onto an SDS page gel, and the GFP gene expression rate was confirmed by Western blotting. Western blot band intensity was quantified using ImageJ software. The detection value of the GFP protein in the pAAV-GC vector-treated sample with the strongest band intensity was set to 100%, and the expression rates of each gene were quantified as relative values. Biological replication was performed twice for each group.

[0204] Figure 7 shows the results of Western blotting to confirm the expression of the transgene GFP in the AAV complex according to the ITR type (ITR WT, ITR BC del, or ITR GC).

[0205] Table 3 shows the results of quantifying the relative expression rate (%) of the transgene GFP according to ITR type in the AAV complex.

[0206] [Table 3]

[0207] As a result, as shown in Figure 7 and Table 3, the expression rate of the GFP gene was significantly higher when using the pAAV-GC vector (Example 1) compared to the pAAV-WT vector (Comparative Example 1) and the pAAV-BC del vector (Comparative Example 2).

[0208] 2-2. Confirmation of expression rates of diverse genes

[0209] Using the same method as in Experimental Example 2-1, experiments were conducted to confirm the expression rates of various transgenes, GFP, Luciferase, TP53, RPE65, TPP1, and FVIII, using Examples 1 to 6 and Comparative Examples 1 to 12.

[0210] For the GFP and Luciferase genes, the same cell line as in Experimental Example 2-1 was used. For the TP53, TPP1, RPE65, and FVIII genes, HEK293 cells were used in which each gene was knocked out using CRISPR / Cas9 (Synthego, USA) to eliminate the influence of endogenous genes unique to the target cells.

[0211] After confirming that no endogenous gene expression occurred in the cell line, the cells were cultured in a 6-well cell culture plate. 1.0 × 10 5 1.0 × 10 per cell 8The concentration of the virus solution was quantified and applied to cells so that individual virus particles were processed. Two days after AAV treatment, proteins expressed from each introduced gene were detected by Western blot (WB). The antibodies used were as follows: GFP (Invitrogen A-11122), Luciferase (Invitrogen PA1-179), TP53 (MA5-14067), TPP1 (PA5-102819), RPE65 (MA1-16578), and FVIII (PA5-104451). WB band intensity was quantified using ImageJ software. The result band of pAAV-GC was set to 100%, and pAAV-WT and pAAV-BCdel were quantified as relative values. Biological replication was performed three times for each group.

[0212] Table 4 shows the quantified relative expression rates (%) of transgenes in the AAV complex based on ITR type (pAAV-GC, pAAV-BC del, or pAAV-WT).

[0213] Figure 8 shows the relative expression rates (%) of transgenes in the AAV complex according to ITR type (pAAV-GC, pAAV-BC del, or pAAV-WT).

[0214] [Table 4]

[0215] As a result, as shown in Table 4 and Figure 8, the expression rates of all transgenes were approximately 2 to 10 times higher when using pAAV-GC vectors containing asymmetrically deformed ITRs compared to pAAV-WT vectors containing non-deformed symmetric ITRs and symmetrically deformed pAAV-BC del vectors.

[0216] Therefore, it was found that pAAV-GC vectors containing asymmetrically modified ITRs can be used to transmit diverse transgenes into target cells as a transduction platform with significantly increased transgene expression efficiency.

[0217] Experimental Example 3. Confirmation of the genotoxicity of the AAV complex.

[0218] 3-1. Confirmation of whether the transgene GFP is inserted into the host chromosome.

[0219] An experiment was conducted to confirm that, when using a recombinant AAV complex with a specific example, the transgene is not recombined into the chromosome of the target cell.

[0220] Specifically, H460 cells were infected with pAAV-GC (Example 1), pAAV-WT (Comparative Example 1), and pAAV-BC del (Comparative Example 2), each containing the GFP gene. Cells expressing GFP were isolated, and the same number of cells were seeded into culture plates. The cells were subcultured by diluting them by 1 / 3 every 3 days, and the expression rate of the GFP gene was tracked to confirm whether or not the GFP gene was genetically transmitted to the offspring.

[0221] Figure 9A shows the results of fluorescence microscopy imaging of GFP expression at passage numbers P3 and P10.

[0222] Table 5 shows the percentage of GFP-expressing cells at subculturing stages P1 to P10.

[0223] [Table 5]

[0224] Figure 9B is a diagrammatic representation of Table 4, which quantifies the percentage of GFP-expressing cells.

[0225] As a result, as shown in Figures 9A and 9B and Table 5, after P7, the expression of the transgene GFP was observed very rarely in cells infected with the pAAV-GC vector (Example 1). On the other hand, in cells infected with the pAAV-WT vector (Comparative Example 1) or the pAAV-BC del vector (Comparative Example 2), the transgene GFP was observed persistently.

[0226] Therefore, it was found that the AAV complex of Example 1 exhibited significantly reduced genotoxicity in gene transmission to offspring compared to the AAV complexes of Comparative Examples 1 and 2.

[0227] 3-2. Confirmation of insertion of diverse transgenes into host chromosomes

[0228] Using the same method as in Experimental Example 3-1, experiments were conducted using Examples 1 to 6 and Comparative Examples 1 to 12 to confirm the insertion of various transgenes, GFP, Luciferase, TP53, RPE65, TPP1, and FVIII, into host chromosomes.

[0229] For the GFP and Luciferase genes, the same cell line as in Experimental Example 3-1 was used. For the TP53, TPP1, RPE65, and FVIII genes, HEK293 cells were used in which each gene was knocked out using CRISPR / Cas9 (Synthego, USA) to eliminate the influence of endogenous genes unique to the target cells.

[0230] After confirming that endogenous gene expression did not occur in the cell line, proteins expressed from each gene introduced by AAV were detected by Western blotting. The antibodies used were as follows: GFP (Invitrogen A-11122), Luciferase (Invitrogen PA1-179), TP53 (MA5-14067), TPP1 (PA5-102819), RPE65 (MA1-16578), and FVIII (PA5-104451). WB band intensity was quantified using ImageJ software. The result band of pAAV-GC was set to 100%, and pAAV-WT and pAAV-BCdel were quantified as relative values.

[0231] Table 6 shows the results (%) of relative gene expression levels at passage number P7.

[0232] [Table 6]

[0233] Figure 10 shows a diagrammatic representation of Table 5, which quantifies the relative gene expression levels in P7.

[0234] As a result, as shown in Table 6 and Figure 10, transgene expression was observed very rarely in cells infected with the pAAV-GC vector in P7. On the other hand, it was confirmed that transgene expression was continuously observed in cells infected with the pAAV-WT vector or the pAAV-BC del vector.

[0235] Therefore, we found that pAAV-GC vectors containing asymmetrically deformed ITRs exhibited significantly reduced genotoxicity in the transmission of introduced genes to offspring compared to pAAV-WT vectors containing non-deformed symmetric ITRs and symmetrically deformed pAAV-BC del vectors.

[0236] 3-3. Confirmation of concatemer formation in target cells of the transgene.

[0237] When a transgene is transmitted to a host cell using an AAV vector, we conducted an experiment to confirm whether or not concatemers, which are found when a transgene is inserted into the host chromosome, are formed.

[0238] Specifically, in Experimental Example 3-1, DNA was extracted from each GFP-expressing cell in P7. The extracted DNA was amplified by PCR, and electrophoresis was performed to confirm the residual form of the DNA that was transmitted to the host cell.

[0239] Figure 11 shows the PCR amplification results confirming the presence or absence of concatemer formation of the transgene in host cells.

[0240] As a result, as shown in Figure 11, it was confirmed that in the case of the pAAV-GC vector (Example 1) lacking the hairpin structure of one ITR, concatemers, which are well known as a characteristic of AAV vectors, could not be formed. On the other hand, in the case of the pAAV-WT vector (Comparative Example 1) and the pAAV-BC del vector (Comparative Example 2), in which both ITRs had apin structures, the transgene was observed as polymers in various forms, such as monomers, dimers, and concatemers.

[0241] Therefore, we observed that when using the pAAV-GC vector, the transgene does not form a concatemer structure in the host cell, and insertion into the host cell's chromosome is suppressed.

[0242] In summary, it was found that the pAAV-GC vector containing an asymmetrically modified ITR is suitable as a target signaling medium for expressing a transgene efficiently for a short period while suppressing the long-term expression of the transgene in the host cell.

[0243] The foregoing description of the present invention is illustrative, and a person with ordinary skill in the art to which the invention pertains will understand that embodiments and specific examples can be easily modified without altering the technical idea or essential features of the invention. Therefore, the embodiments should be understood to be illustrative and not limiting in all respects.

[0244] The specific examples described herein should be considered descriptive and not restrictive. Descriptions of features or embodiments within each specific example should generally be considered applicable to other similar features or embodiments of other specific examples. While one or more specific examples have been described with reference to the drawings, those skilled in the art will understand that various modifications to form and detail are possible without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. In a nucleic acid molecule containing a gene expression cassette between a first inverted terminal repeat (ITR) and a second ITR, The gene expression cassette includes a heterogeneous polynucleotide sequence, A nucleic acid molecule wherein either the first ITR or the second ITR comprises at least one nucleotide sequence or a complementary sequence having approximately 75% or more sequence identity with any one of the nucleotide sequences or functional derivatives of sequence numbers 1 to 9.

2. The nucleic acid molecule according to claim 1, wherein one of the nucleotide sequences of the first ITR and the second ITR has about 95% or more sequence identity with one of the nucleotide sequences of sequence numbers 1 to 9 or a functional derivative thereof.

3. The nucleic acid molecule according to claim 1, wherein one of the nucleotide sequences of the first ITR and the second ITR is selected from one of the nucleotide sequences of sequence numbers 1 to 9 or a functional derivative thereof.

4. The nucleic acid molecule according to claim 1, wherein either the first ITR or the second ITR is composed of a nucleotide sequence or a complementary sequence having about 75% or more sequence identity with any one of the nucleotide sequences or functional derivatives of sequence numbers 1 to 9.

5. The nucleic acid molecule according to claim 1, wherein the sequence of the first ITR and the sequence of the second ITR are based on the ITR sequences of a virus belonging to the genus Dependovirus of the family Parvoviridae.

6. The nucleic acid molecule according to claim 1, wherein the sequence of the first ITR and the sequence of the second ITR are based on the ITR sequences of adeno-associated virus (AAV).

7. The nucleic acid molecule according to claim 1, wherein the sequence of the first ITR and the sequence of the second ITR are each independently based on the ITR sequence of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

8. The first ITR is a wild-type ITR of AAV, The nucleic acid molecule according to claim 1, wherein the second ITR comprises at least one nucleotide sequence or a complementary sequence having about 75% or more sequence identity with any one of sequence numbers 1 to 9 or a functional derivative thereof.

9. The first ITR is a wild-type ITR of AAV, The nucleic acid molecule according to claim 1, wherein the second ITR is composed of a nucleotide sequence or a complementary sequence having about 75% or more sequence identity with any one of the nucleotide sequences 1 to 9 or a functional derivative thereof.

10. The first ITR is an AAV wild-type ITR, The nucleic acid molecule according to claim 1, wherein the second ITR is essentially composed of any one sequence selected from the nucleotide sequences of sequence numbers 1 to 9.

11. The first ITR is an AAV wild-type ITR, The nucleic acid molecule according to claim 1, wherein the second ITR is essentially composed of the nucleotide sequence of SEQ ID NO:

1.

12. The nucleic acid molecule according to claim 1, wherein in either the first ITR or the second ITR, all or part of the stem-loop structure formed in the RBE (rep-binding element), RBE', A, A', B, B', C, C', and D regions is deformed.

13. The nucleic acid molecule according to claim 1, wherein either the first ITR or the second ITR is deformed so as not to form a stem-loop structure.

14. The nucleic acid molecule according to claim 1, wherein in either the first ITR or the second ITR, all or part of the stem-loop structure formed in the RBE, RBE', A, A', B, B', C, C', and D regions is deleted.

15. The nucleic acid molecule according to claim 1, wherein the gene expression cassette further comprises one or more of a promoter and a polyadenylated sequence.

16. The nucleic acid molecule according to claim 1, wherein the heterogeneous polynucleotide sequence encodes a therapeutic gene.

17. A vector comprising the nucleic acid molecule described in claim 1.

18. The vector according to claim 17, wherein the vector is an AAV vector.

19. A composition comprising the vector and a pharmaceutically acceptable carrier as described in claim 17.

20. The composition according to claim 19, for use in the delivery of therapeutic genes for gene therapy.

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