DNA fragment production method

Abstract

The present invention addresses the problem of providing a method for producing, by single primer PCR, a DNA fragment having ITR sequences on both ends. In the present invention, a DNA fragment including a transgene is produced by single primer PCR with a DNA having ITR sequences serving as a template DNA, using: (A-i) a polynucleotide A comprising an artificially designed polynucleotide a1 comprising at least 5 bases which are any of the bases selected from adenine, guanine, cytosine, and thymine and a polynucleotide a2 linked to the 3' end of the polynucleotide a1, the polynucleotide a2 comprising the full length sequence from 1st to 54th of the base sequence represented by SEQ ID NO: 1 or comprising a consecutive partial sequence of at least 16 consecutive bases in the base sequence from the 12th to 39th of the base sequence represented by SEQ ID NO: 1; (A-ii) the polynucleotide A of (A-i) with one or more bases deleted, substituted, added or inserted; or the like.

Description

Method for preparing DNA fragments

[0001] This disclosure relates to a method for preparing DNA fragments and to a single primer for preparing DNA fragments.

[0002] In recent years, the development of nucleic acid drugs and viral vector therapies has progressed. Adeno-associated virus (AAV) vectors are among the most advanced viral vectors under development. Because AAV vectors can efficiently deliver genes into non-dividing cells, they are being applied to gene therapy. One method for producing AAV virus particles involves introducing a transfer vector and a helper vector into human cultured cells. Specifically, first, the target gene is cloned into a plasmid vector for E. coli, in the region between the inverted repeat sequence (ITR sequence) at the 5' end and the inverted repeat sequence at the 3' end. Next, this plasmid vector is used as a transfer vector and transfected together with a helper plasmid into virus-producing cells such as HEK293T cells. Here, plasmid vectors are produced in E. coli, but there has been a problem in that mass production of plasmid vectors is time-consuming and costly.

[0003] So far, several methods for detecting AAV vectors have been disclosed. For example, a method of amplifying an AAV vector region containing a foreign gene expression unit by PCR using primers set based on the ITR sequence important for virus particle production and detecting the AAV vector by agarose gel electrophoresis has been disclosed (see Non-Patent Document 1). Further, a primer set including a first primer having a sequence complementary to a part of a specific base sequence of an adeno-associated virus vector and a second primer having a sequence homologous to a part of the specific base sequence is used to amplify a nucleic acid containing the specific base sequence or a complementary sequence of the specific base sequence, and a method for detecting an adeno-associated virus vector has been disclosed (see Patent Document 1). According to these methods, although detection of DNA within the ITR region can be performed, the ITR region necessary as a virus is not included, and it was not possible to construct an AAV virus from the obtained amplification product.

[0004] In addition, a method of producing an AAV by preparing a DNA fragment not containing inverted repeat regions B and C using a primer set including a forward primer that anneals to a part of the ITR sequence and introducing it into HEK293 cells has been disclosed (see Non-Patent Document 2).

[0005] Japanese Unexamined Patent Application Publication No. 2023-7304

[0006] RIKEN homepage, Virus Bank SOP-AAV-001, "Standard Operating Procedure Manual for Highly Sensitive Detection Method of Adeno-Associated Virus (AAV) Vector by PCR", Kumi Adachi et al., August 2022, Gene Therapy 29(7-8):1-9 DOI:10.1038 / s41434-021-00299-x

[0007] An object of the present disclosure is to provide a method for producing a DNA fragment containing a transgene sandwiched between two ITR sequences by single-primer PCR.

[0008] Because ITR sequences contain nucleotide sequences that can form a hairpin structure, simply designing forward and reverse primers based on sequences that anneal to ITR sequences does not result in the intended amplification due to annealing between primers or between primers and PCR amplification products. For this reason, plasmid amplification using E. coli has been the mainstream method for amplifying DNA containing transgenes sandwiched between two ITR sequences. As a result of diligent research to solve the above problem, the inventors have found that DNA fragments having at least a portion of the ITR sequence at both ends of the transgene-containing DNA can be produced by single-primer PCR using a single primer consisting of a nucleotide sequence of a predetermined position and length in the ITR sequence.

[0009] In other words, this disclosure is as follows:[1] (A-i) A polynucleotide A formed by linking a polynucleotide a2, which consists of at least 16 consecutive bases in the nucleotide sequence from the 12th to the 39th bases in the nucleotide sequence described in Sequence ID No. 1, to the 3' end of an artificially designed polynucleotide a1 consisting of at least 5 bases selected from adenine, guanine, cytosine, and thymine; (A-ii) A polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide A of (A-i); or (A-iii) A polynucleotide having 90% or more identity with the polynucleotide A of (A-i); a single primer A containing any of the polynucleotides from (A-i) to (A-iii); (B-i) A polynucleotide B2 formed by linking a polynucleotide B1 consisting of at least 20 bases, comprising the full sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, including bases 89 to 108 in Sequence ID No. 1, or an artificially designed polynucleotide b1 consisting of at least 5 bases, comprising any base selected from adenine, guanine, cytosine, and thymine, to the 3' end of a polynucleotide B2 consisting of at least 16 consecutive bases, comprising the full sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, including bases 79 to 106 in Sequence ID No. 1; (B-ii) A polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide B1 or polynucleotide B2 of (B-i); or (B-iii) A polynucleotide having 90% or more identity with the polynucleotide B1 or polynucleotide B2 of (B-i); A single primer B containing any of the polynucleotides (B-i) to (B-iii); a method for producing a DNA fragment containing a transgene, characterized by performing single-primer PCR using single primer A or single primer B, with DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences as template DNA.[2] The method according to [1], characterized in that single primer A is used as a single primer and the length of polynucleotide A is 34 to 150 mer. [3] The method according to [1] or [2], characterized in that single primer A is used as a single primer and polynucleotide A is a full-length sequence or a continuous partial sequence of the nucleotide sequences 1 to 54 in the nucleotide sequence described in SEQ ID NO: 1, and is a polynucleotide containing the nucleotide sequences 3 to 35 in SEQ ID NO: 1. [4] The method according to [1], characterized in that single primer B is used as a single primer and the length of polynucleotide B1 or polynucleotide B2 is 34 to 130 mer. [5] The method according to [1] or [4], characterized in that single primer B is used as a single primer and polynucleotide B1 or polynucleotide B2 is a polynucleotide containing the nucleotide sequences 79 to 108 in SEQ ID NO: 1. [6] The method according to any one of [1] to [5], characterized in that the DNA containing the transgene contains DNA encoding a promoter sequence. [7] The method according to any one of [1] to [6] above, characterized in that PCR is performed in a two-step program consisting of a thermal denaturation step and an annealing and extension step. [8] A method for producing an adeno-associated virus (AAV) vector, characterized in that it includes the step of transfecting packaging cells with DNA containing a transgene obtained by the method according to any one of [1] to [7] above, DNA encoding E2A derived from adenovirus, DNA encoding E4 derived from adenovirus, DNA encoding VA derived from adenovirus, DNA encoding rep, and DNA encoding cap. [9] A pharmaceutical composition comprising an adeno-associated virus (AAV) vector obtained by the method according to [8] above and a pharmaceutically acceptable additive.

[10] A single primer A for use in a method of producing a DNA fragment containing a transgene by performing single-primer PCR using DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences as template DNA. The single primer A comprises any of the polynucleotides (A-i) to (A-iii). The single primer A comprises a polynucleotide A, which is formed by linking a full-length sequence or a continuous partial sequence of the 1st to 54th base sequence in the nucleotide sequence described in Sequence ID No. 1, and a polynucleotide A comprising a continuous sequence of at least 16 bases in the 12th to 39th base sequence in Sequence ID No. 1, to the 3' end of an artificially designed polynucleotide a1 consisting of at least five bases selected from adenine, guanine, cytosine, and thymine; a polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide A of (A-ii)(A-i); or a polynucleotide having 90% or more identity with the polynucleotide A of (A-iii)(A-i); and any of the polynucleotides (A-i) to (A-iii).

[11] A polynucleotide B2 for use in a method of producing a DNA fragment containing a transgene by performing single-primer PCR using DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences as template DNA, wherein (B-i) a polynucleotide B1 consisting of at least 20 bases, which is the full-length sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, including bases 89 to 108 in Sequence ID No. 1, or an artificially designed polynucleotide b1 consisting of at least 5 bases, which is any base selected from adenine, guanine, cytosine, and thymine, is linked to the 3' end of a polynucleotide B2 consisting of at least 16 continuous bases, which is the full-length sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, including bases 79 to 106 in Sequence ID No. 1; A polynucleotide in which one or more bases are deleted, substituted, added, or inserted in polynucleotide B1 or polynucleotide B2 of (B-ii)(B-i); or a polynucleotide having 90% or more identity with polynucleotide B1 or polynucleotide B2 of (B-iii)(B-i); a single primer B containing any polynucleotide from (B-i) to (B-iii). Another aspect of the present disclosure is as follows: <1> A method for producing an adeno-associated virus (AAV) vector, comprising the steps of producing a DNA fragment containing a transgene by any of the methods described in [1] to [7] above, and introducing the produced DNA fragment into a packaging cell.

[0010] This disclosure makes it possible to produce DNA fragments containing a transgene sandwiched between two ITR sequences by single-primer PCR.

[0011] Figure 1 shows the vector map of the pAAV-CMV vector used in Reference Example 1 and the annealing positions of the forward and reverse primers. Figure 2 shows the agarose electrophoresis results of the PCR amplification product in Reference Example 1. Figure 3 shows the agarose electrophoresis results of the PCR amplification product in Reference Example 2. Figure 4 shows the vector map of the pAAV-CMV vector used in Example 1 and the annealing positions of the primers. Figure 5 shows the agarose electrophoresis results of the single-primer PCR amplification product when the pAAV-CMV vector was used as the template DNA in Example 1. Figure 6 shows the sequence alignment of SEQ ID NO: 1 and the ITR sequence of AAV2 (SEQ ID NO: 2). Figure 7 shows the agarose electrophoresis results of the PCR amplification product when single-primer PCR annealing was performed at 75°C, 74°C, 73°C, 71°C, 69°C, 67°C, 66°C, or 65°C in Example 2. Figure 8 shows the results of agarose electrophoresis of the amplified PCR products when single-primer PCR annealing was performed at 73°C, 72.7°C, 72.1°C, 71.2°C, 70.0°C, 69.2°C, 68.5°C, and 68°C in Example 2. Figure 9 shows the results of agarose electrophoresis of the amplified PCR products when single-primer PCR was performed on a 10 mL scale using the VB010000-9394npt vector as the template DNA in Example 3. Figure 10 shows the results of agarose electrophoresis of the amplified PCR products when single-primer PCR was performed in Example 4 using pAAV-CMV-ITR+1(45), pAAV-CMV-ITR+1(50), pAAV-CMV-ITR+1(52), pAAV-CMV-ITR+1(54), and pAAV-CMV-ITR+1(55) as single primers. Figure 11 shows the results of agarose electrophoresis of the amplified PCR products when single-primer PCR was performed in Example 5 using pAAV-CMV-ITR+1(45), pAAV-CMV-ITR+1(45)Ca, pAAV-CMV-ITR+1(45)CaCa, and pAAV-CMV-ITR+1(45)CaCaGa as single primers.Figure 12 shows the results of agarose electrophoresis of the amplified PCR products when single-primer PCR was performed in Example 6 using pAAV-CMV-ITR+7(33), pAAV-CMV-ITR+7(38), pAAV-CMV-ITR+12(28), pAAV-CMV-ITR+12(30), pAAV-CMV-ITR+12(33), pAAV-CMV-ITR+12(35), pAAV-CMV-ITR+12(40), and pAAV-CMV-ITR+12(45) as single primers. Figure 13 shows the agarose electrophoresis results of the PCR amplification products when single-primer PCR was performed in Example 6 using pAAV-CMV-ITR+1 (45), pAAV-CMV-ITR+2 (44), pAAV-CMV-ITR+3 (43), pAAV-CMV-ITR+4 (42), pAAV-CMV-ITR+5 (41), pAAV-CMV-ITR+6 (40), and pAAV-CMV-ITR+7 (38) as single primers. Figure 14A shows the results of agarose electrophoresis of the amplified PCR products when single-primer PCR was performed in Example 7 using pAAV-CMV-ITR+79(30), pAAV-CMV-ITR+79(35), pAAV-CMV-ITR+79(40), pAAV-CMV-ITR+79(45), pAAV-CMV-ITR+79(50), pAAV-CMV-ITR+79(-7)(50), and pAAV-CMV-ITR+79(-17)(60) as single primers at an annealing temperature of 68°C. Figure 14B shows the results of agarose electrophoresis of the amplified PCR products when single-primer PCR was performed in Example 7 using pAAV-CMV-ITR+79(30), pAAV-CMV-ITR+79(35), pAAV-CMV-ITR+79(40), pAAV-CMV-ITR+79(45), pAAV-CMV-ITR+79(50), pAAV-CMV-ITR+79(-7)(50), and pAAV-CMV-ITR+79(-17)(60) as single primers at an annealing temperature of 71°C.

[0012] The method for preparing DNA fragments in this disclosure includes: a polynucleotide A formed by linking a polynucleotide a2, which consists of at least 16 consecutive bases from the 12th to 39th base sequence in SEQ ID NO: 1, to the 3' end of an artificially designed polynucleotide a1 consisting of at least five bases selected from adenine, guanine, cytosine, and thymine; a polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide A of (A-ii)(A-i); or a polynucleotide having 90% or more identity with the polynucleotide A of (A-iii)(A-i); and a single primer A containing any of the polynucleotides of (A-i) to (A-iii). (B-i) A polynucleotide B2 formed by linking a polynucleotide B1 consisting of at least 20 bases, comprising the full sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, including bases 89 to 108 in Sequence ID No. 1, or an artificially designed polynucleotide b1 consisting of at least 5 bases, comprising any base selected from adenine, guanine, cytosine, and thymine, to the 3' end of a polynucleotide B2 consisting of at least 16 consecutive bases, comprising the full sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, including bases 79 to 106 in Sequence ID No. 1; (B-ii) A polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide B1 or polynucleotide B2 of (B-i); or (B-iii) A polynucleotide having 90% or more identity with the polynucleotide B1 or polynucleotide B2 of (B-i); Single primer B containing any of the polynucleotides (B-i) to (B-iii);A method for producing a DNA fragment containing a transgene can be described, which involves performing single-primer PCR using either single primer A or single primer B, with DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences as the template DNA. This method will also be referred to hereinafter as "the method for producing the DNA fragment in question."

[0013] Furthermore, the method for producing an AAV vector in this disclosure is not particularly limited as long as it includes the step of transfecting packaging cells with DNA containing a transgene obtained by the method for producing the DNA fragments, DNA encoding E2A derived from adenovirus, DNA encoding E4 derived from adenovirus, DNA encoding VA derived from adenovirus, DNA encoding rep, and DNA encoding cap, and is hereinafter also referred to as "the method for producing the AAV vector."

[0014] Furthermore, the single primer A in this disclosure is a polynucleotide A formed by performing single-primer PCR using DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences as template DNA to produce a fragment of DNA containing the transgene, and includes: (A-i) a polynucleotide a2 formed by linking a full-length sequence or a continuous partial sequence of the 1st to 54th base sequences in the nucleotide sequence described in SEQ ID NO: 1, and a polynucleotide a2 formed by linking a full-length sequence or a continuous partial sequence of the 12th to 39th base sequences in SEQ ID NO: 1 to the 3' end of an artificially designed polynucleotide a1 consisting of at least five bases selected from adenine, guanine, cytosine, and thymine; (A-ii) a polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide A of (A-ii); or a polynucleotide having 90% or more identity with the polynucleotide A of (A-iii)(A-i); A single primer A containing any of the polynucleotides (A-i) to (A-iii) can be listed, and hereinafter referred to as "the single primer A in question."

[0015] Furthermore, single primer B in this disclosure is used in a method for producing a DNA fragment containing the transgene by performing single-primer PCR using DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences as template DNA, and comprises: (B-i) a polynucleotide B2 formed by linking a polynucleotide B1 consisting of at least 20 bases, which is the full-length sequence or a continuous partial sequence of bases 64 to 141 in the nucleotide sequence described in SEQ ID NO: 1, including bases 89 to 108 in SEQ ID NO: 1, or an artificially designed polynucleotide b1 consisting of at least 5 bases, which is any base selected from adenine, guanine, cytosine, and thymine, to the 3' end of a polynucleotide B2 consisting of at least 16 continuous bases, which is the full-length sequence or a continuous partial sequence of bases 64 to 141 in the nucleotide sequence described in SEQ ID NO: 1, including bases 89 to 108 in SEQ ID NO: 1; Examples of single primer B include polynucleotides in which one or more bases are deleted, substituted, added, or inserted in polynucleotide B1 or polynucleotide B2 of (B-ii)(B-i); or polynucleotides having 90% or more identity with polynucleotide B1 or polynucleotide B2 of (B-iii)(B-i); and single primer B containing any of the polynucleotides from (B-i) to (B-iii), hereinafter also referred to as "the single primer B in question".

[0016] [Single Primer] In this specification, a single primer means a primer that contains a nucleotide sequence that can anneal to at least two regions, preferably only two regions, of a given template DNA when performing a PCR reaction using that template DNA, and in which one primer can serve as either a forward primer or a reverse primer. Using this single primer, amplification by PCR is possible with substantially only one type of primer. Here, substantially one type of primer includes not only a single molecular species consisting of a specific nucleotide sequence, but also multiple molecular species that include mutant bases or mixed bases, or that include base deletions or insertions, as long as the primer set is designed to anneal to the same region.

[0017] In this specification, single-primer PCR refers to PCR using the single primer described above. Specifically, single-primer PCR is PCR performed by adding substantially only one type of primer to the reaction mixture.

[0018] Examples of single primers used herein include single primers A and B. Single primer A is one of the following polynucleotides: (A-i) a polynucleotide a2 consisting of at least 16 consecutive bases from the 12th to 39th bases in the sequence of sequence number 1, which is the full-length sequence or a continuous partial sequence of the 1st to 54th bases in the sequence of sequence number 1 described in Sequence ID No. 1, linked to the 3' end of an artificially designed polynucleotide a1 consisting of at least 5 bases selected from adenine, guanine, cytosine, and thymine; (A-ii) a polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide A of (A-i); or (A-iii) a polynucleotide having 90% or more identity with the polynucleotide A of (A-i). Furthermore, (A-ii) and (A-iii) above are polynucleotides consisting of sequences that differ from the natural ITR sequence or a subsequence thereof by one or more bases, preferably two or more bases, more preferably three or more bases, and more preferably four or more bases.

[0019] The polynucleotide A described in (A-i) above is a polynucleotide in which a polynucleotide a2, consisting of at least 16 consecutive bases from the 12th to 39th bases in the sequence number 1, is linked to the 3' end of an artificially designed polynucleotide a1 consisting of at least 5 bases selected from adenine (A), guanine (G), cytosine (C), and thymine (T). Here, the "full-length sequence or consecutive partial sequence of the 1st to 54th bases in the sequence number 1" is either the full-length sequence of the 1st to 54th bases in the sequence number 1, or a consecutive partial sequence of the 1st to 54th bases in the sequence number 1, in other words, a sequence in which the 5' end and / or 3' end of the 1st to 54th bases in the sequence number 1 is partially deleted.

[0020] The above-mentioned "polynucleotide a2" can be a polynucleotide that starts from any of the 1st to 5th bases in SEQ ID NO: 1, is a sequence that is continuous along the base sequence of SEQ ID NO: 1, and consists of at least 16 consecutive bases in the base sequence from the 12th to the 39th bases in SEQ ID NO: 1. The 5' end of polynucleotide a2 is preferably any of the 1st to 5th bases in SEQ ID NO: 1.

[0021] The artificially designed polynucleotide a1 in polynucleotide A described in (A-i) above, which consists of at least five bases selected from adenine, guanine, cytosine, and thymine, may be a polynucleotide consisting of at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen bases selected from adenine, guanine, cytosine, and thymine, or a polynucleotide consisting of six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen bases, or it may be 5 to 50 bases or 8 to 30 bases. In the case of artificially designed polynucleotide a1, "artificially designed polynucleotide" refers to a polynucleotide whose base sequence is designed such that the sequence attached to the 5' end of polynucleotide A differs by at least one base from the base sequence of the corresponding region in the ITR sequence of natural AAV.

[0022] It is preferable that the 3' end of the artificially designed polynucleotide a1 consisting of at least five bases, any base selected from adenine, guanine, cytosine, and thymine, in particular, has a sequence difference of a certain magnitude or more from the sequence commonly conserved at the 5' end in known natural AAV ITR sequences, in order to prevent nonspecific annealing to other regions in the ITR sequence, particularly regions corresponding to inverted repeat sequences. Here, as a sequence commonly conserved at the 5' end in known natural AAV ITR sequences, it is preferable, for example, the 5th to 16th polynucleotides in the base sequence of the AAV2 ITR sequence described in SEQ ID NO: 2 (SEQ ID NO: 3: 5'-CCACTCCCCTCTC-3'), or a polynucleotide in which one, two, three, four, five, or six bases are deleted at the 5' end of the polynucleotide described in SEQ ID NO: 3, in which two or more, three or more, four or more, or five or more bases are substituted with other bases. Alternatively, the 3' terminal sequence of an artificially designed polynucleotide a1 consisting of at least five bases, any of the above-mentioned bases, and the 3' terminal sequence of the polynucleotide described in SEQ ID NO: 3 or a polynucleotide in which one, two, three, four, five, or six bases are deleted from the 5' terminal of the polynucleotide described in SEQ ID NO: 3 can be 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 0%. The artificially designed polynucleotide a1 may include sequences recognized by restriction enzymes such as SbfI and sequences not included in the ITR sequences of natural AAVs. Figure 6 shows the sequence alignment of the nucleotide sequence of SEQ ID NO: 1 and the ITR sequence of AAV2 (Genbank accession number: NC_001401: SEQ ID NO: 2).

[0023] For example, in an artificially designed polynucleotide a1 consisting of at least five bases made up of the above arbitrary bases, if there are 12 bases, we can give an example of a polynucleotide in which the 12 bases from the 3' end are replaced with a base other than cytosine, a base other than thymine,cytosine, a base other than cytosine, a base other than thymine, a base other than cytosine, a base other than adenine, a base other than cytosine, and a base other than cytosine. Also, for example, we can give an example of a polynucleotide in which the bases of an artificially designed polynucleotide a1 consisting of at least five bases made up of the above arbitrary bases contain 20% or more, 30% or more, 40% or more, or 50% or more of adenine or guanine, or a polynucleotide in which the bases of an artificially designed polynucleotide a1 consisting of at least five bases made up of the above arbitrary bases contain 20-80% or 40-70% of adenine or guanine. Furthermore, as an artificially designed polynucleotide a1 consisting of at least five bases selected from adenine, guanine, cytosine, and thymine, examples include a polynucleotide having a cytosine or guanine content of 50-100%, 60-90%, or 70-80%, preferably a cytosine content, or a polynucleotide containing a sequence of bases consisting only of cytosine, guanine, adenine, or thymine, preferably a sequence of bases consisting only of cytosine or guanine, more preferably a sequence of bases consisting only of cytosine. Here, the length of the sequence of bases consisting only of cytosine, guanine, adenine, or thymine can be 5 bases or more, 8 bases or more, 10 bases or more, 12 bases or more, or 15 bases or more, and may be 5-50 bases, 8-30 bases, or 10-20 bases.

[0024] Specific examples of artificially designed polynucleotide a1 consisting of at least five bases from the above arbitrary bases include the artificially designed polynucleotides a1(12) to a1(5) listed in SEQ ID NOs. 4 to 11 in Table 1. By including these artificially designed polynucleotides a1 in polynucleotide A, it becomes possible to prevent the primer from annealing to a sequence different from the ITR sequence that it should anneal to during the PCR reaction, and to prevent the amplified DNA from annealing to itself.

[0025]

[0026] The 3' terminal base of polynucleotide a1 and the 5' terminal base of polynucleotide a2 may be directly linked, or they may be linked via a linker sequence consisting of any base sequence selected from adenine, guanine, cytosine, and thymine. The length of the linker sequence is not particularly limited, but examples include 1 to 20 bases, 1 to 10 bases, and 1 to 5 bases. The linker sequence may be an artificially designed sequence, a part of a known natural AAV ITR sequence, or a combination of these sequences. An example of a part of the natural ITR sequence in the linker sequence is the polynucleotide described as the 1st to 15th bases in the AAV2 ITR sequence described in SEQ ID NO: 2 (SEQ ID NO: 49: 5'-TTGGCCACTCCCCTCT-3') or a subsequence thereof. Examples of partial sequences of polynucleotides described as the 1st to 15th positions in the base sequence of the AAV2 ITR sequence described in Sequence ID No. 2 include sequences in which the 5' end and / or 3' end, preferably the 5' end, of the polynucleotide described as the 1st to 15th positions in the base sequence of the AAV2 ITR sequence described in Sequence ID No. 2 is partially deleted.

[0027] Examples of the above-mentioned "polynucleotide A" include polynucleotides containing the 5th to 30th base sequences in SEQ ID NO: 1, polynucleotides containing the 4th to 30th base sequences in SEQ ID NO: 1, polynucleotides containing the 3rd to 30th base sequences in SEQ ID NO: 1, polynucleotides containing the 2nd to 30th base sequences in SEQ ID NO: 1, and polynucleotides containing the 1st to 30th base sequences in SEQ ID NO: 1.

[0028] Furthermore, other embodiments of the above-mentioned "polynucleotide A" include a polynucleotide containing the 5th to 35th base sequence in SEQ ID NO: 1, a polynucleotide containing the 5th to 38th base sequence in SEQ ID NO: 1, a polynucleotide containing the 5th to 40th base sequence in SEQ ID NO: 1, a polynucleotide containing the 5th to 45th base sequence in SEQ ID NO: 1, a polynucleotide containing the 5th to 50th base sequence in SEQ ID NO: 1, a polynucleotide containing the 5th to 53rd base sequence in SEQ ID NO: 1, and a polynucleotide containing the 5th to 54th base sequence in SEQ ID NO: 1.

[0029] Furthermore, other embodiments of the "polynucleotide A" described above include a polynucleotide containing the 1st to 45th base sequences in SEQ ID NO: 1, a polynucleotide containing the 2nd to 45th base sequences in SEQ ID NO: 1, a polynucleotide containing the 3rd to 45th base sequences in SEQ ID NO: 1, and a polynucleotide containing the 4th to 45th base sequences in SEQ ID NO: 1.

[0030] One embodiment of the polynucleotide described in (A-ii) above is a polynucleotide in which one or more bases are deleted, substituted, added, or inserted in polynucleotide A of (A-i) above, as long as the entire sequence is annealable with the template DNA used in the PCR reaction. Preferably, one or more bases are deleted, substituted, added, or inserted in polynucleotide a2 contained in polynucleotide A of (A-i) above, but this is not particularly limited. Here, "one or more" can be 1 to 10, but 1 to 5 is preferred, 1 to 4 is more preferred, 1 to 3 is even more preferred, 1 to 2 is particularly preferred, and 1 is most preferred. Other embodiments of the polynucleotide described in (A-ii) above include polynucleotides in which one or more bases are deleted, substituted, added, or inserted in polynucleotide A of (A-i) above, and when PCR is performed using such polynucleotide as a single primer, the amount of DNA in the PCR amplification product is 1 / 2 or more, 1 / 3 or more, 1 / 5 or more, 1 / 10 or more, 1 / 20 or more, or 1 / 50 or more compared to when PCR is performed using the polynucleotide of (A-i) above as a single primer under the same conditions as above. More specifically, polynucleotides in which one or more bases are deleted, substituted, added, or inserted in polynucleotide a2 contained in polynucleotide A of (A-i) above, and when PCR is performed using such polynucleotide as a single primer, the amount of DNA in the PCR amplification product is 1 / 2 or more, 1 / 3 or more, 1 / 5 or more, 1 / 10 or more, 1 / 20 or more, or 1 / 50 or more compared to when PCR is performed using the polynucleotide of (A-i) above as a single primer under the same conditions as above, can be listed, but are not particularly limited. The above amount of DNA can be evaluated by values ​​based on bands obtained by electrophoresis or by the weight of DNA.

[0031] In one aspect of this disclosure, the polynucleotide A or polynucleotide a2 may be a polynucleotide consisting of a continuous base sequence from the Xth base to the Yth base in the base sequence described in Sequence ID No. 1. Here, X is an arbitrary integer selected from 1 to 12, indicating the base position at the 5' end of the polynucleotide A or polynucleotide a2. Specifically, X may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and more preferably 1, 2, 3, 4, or 5. Furthermore, Y is an arbitrary integer selected from 30 to 54, indicating the base position at the 3' end of the polynucleotide A or polynucleotide a2. Specifically, Y may be 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54. In this disclosure, all ranges consisting of the above combinations of X and Y, such as the 1st to 54th, 3rd to 45th, 5th to 40th sequences of Sequence ID No. 1, are disclosed individually and specifically.

[0032] One embodiment of the polynucleotide described in (A-iii) above is a polynucleotide having 90% or more identity with polynucleotide A in (A-i) above, insofar as the whole sequence can be annealed with the template DNA used in the PCR reaction. It may also be a polynucleotide having 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with polynucleotide A in (A-i) above. Other embodiments of the polynucleotide described in (A-iii) above include, but are not limited to, polynucleotides having 92% or more identity with polynucleotide A of (A-i) above, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and in which, when PCR is performed using such polynucleotide as a single primer, the amount of DNA in the PCR amplification product is 1 / 2 or more, 1 / 3 or more, 1 / 5 or more, 1 / 10 or more, 1 / 20 or more, or 1 / 50 or more compared to when PCR is performed using polynucleotide A of (A-i) above as a single primer under the same conditions as above. In this disclosure, "identity" of the base sequence means a value calculated using default parameters with sequence analysis software such as BLAST.

[0033] The single primer B in this case is: (B-i) a polynucleotide B1 consisting of at least 20 bases, which is the full sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in SEQ ID NO: 1, including bases 89 to 108 in SEQ ID NO: 1, or an artificially designed polynucleotide b1 consisting of at least 5 bases, which is any base selected from adenine, guanine, cytosine, and thymine, with a polynucleotide b2 consisting of at least 16 consecutive bases, which is the full sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in SEQ ID NO: 1, including bases 89 to 108 in SEQ ID NO: 1, linked to the 3' end of the polynucleotide b1; (B-ii) a polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide B1 or polynucleotide B2 of (B-i); (B-iii) a polynucleotide having 90% or more identity with the polynucleotide B1 or polynucleotide B2 of (B-i); This is a single primer containing any of the polynucleotides (B-i) to (B-iiii). Note that (B-ii) and (B-iiii) are sequences that differ from the natural ITR sequence or a subsequence thereof by one or more bases, preferably two or more bases, more preferably three or more bases, and more preferably four or more bases.

[0034] The first embodiment of the polynucleotide described in (B-i) above is a polynucleotide B1 consisting of at least 20 bases, which is the full-length sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, and includes bases 89 to 108 in Sequence ID No. 1. Here, the "full-length sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1" may be the full-length sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, in other words, a sequence in which the 5' end and / or 3' end of bases 64 to 141 in the base sequence described in Sequence ID No. 1 are partially deleted.

[0035] In the first embodiment of the polynucleotide described in (B-i) above, the "polynucleotide consisting of at least 20 bases including the base sequence of the 89th to 108th bases in SEQ ID NO: 1" can preferably be a polynucleotide including the base sequence of the 89th to 108th bases in SEQ ID NO: 1, a polynucleotide including the base sequence of the 86th to 108th bases in SEQ ID NO: 1, a polynucleotide including the base sequence of the 83rd to 108th bases in SEQ ID NO: 1, a polynucleotide including the base sequence of the 79th to 108th bases in SEQ ID NO: 1, or a polynucleotide including the base sequence of the 69th to 108th bases in SEQ ID NO: 1.

[0036] In the first embodiment of the polynucleotide described in (B-i) above, the "polynucleotide consisting of at least 20 bases including the base sequence of the 89th to 108th bases in SEQ ID NO: 1" can further include a polynucleotide including the base sequence of the 89th to 109th bases in SEQ ID NO: 1, a polynucleotide including the base sequence of the 86th to 111th bases in SEQ ID NO: 1, a polynucleotide including the base sequence of the 83rd to 118th bases in SEQ ID NO: 1, a polynucleotide including the base sequence of the 79th to 128th bases in SEQ ID NO: 1, and a polynucleotide including the base sequence of the 72nd to 135th bases in SEQ ID NO: 1.

[0037] A second embodiment of the polynucleotide described in (B-i) above is a polynucleotide B2 in which a polynucleotide b2 consisting of at least 16 consecutive bases from the 79th to 106th base sequence in SEQ ID NO: 1 is linked to the 3' end of an artificially designed polynucleotide b1 consisting of at least 5 bases selected from adenine, guanine, cytosine, and thymine, which is the full-length sequence or a continuous partial sequence of bases from the 64th to 141st base sequence in the SEQ ID NO: 1 sequence. Here, the "full-length sequence or a continuous partial sequence of bases from the 64th to 141st base sequence in the SEQ ID NO: 1 sequence" may be either the full-length sequence or a continuous partial sequence of bases from the 64th to 141st base sequence in the SEQ ID NO: 1 sequence.

[0038] In the second embodiment of the polynucleotide described in (B-i) above, the artificially designed polynucleotide b1 consisting of at least five bases made up of the arbitrary bases may be a polynucleotide consisting of at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen bases made up of any base selected from adenine, guanine, cytosine, and thymine; or it may be a polynucleotide consisting of six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen bases; or it may be 5 to 50 bases or 8 to 30 bases. In the artificially designed polynucleotide b1, "artificially designed polynucleotide" refers to a polynucleotide whose base sequence is designed to be different from the base sequence of the corresponding region in the ITR sequence of natural AAV as the sequence ligated to the 5' end of the polynucleotide B. Furthermore, the artificially designed polynucleotide b1 may include sequences recognized by restriction enzymes such as SbfI, but which are not included in the ITR sequences of natural AAV.

[0039] In the second embodiment of the polynucleotide described in (B-i) above, the artificially designed polynucleotide b1 consisting of at least five bases, any base selected from adenine, guanine, cytosine, and thymine, is preferable to have a certain degree of sequence difference from a sequence commonly conserved at the 5' end in the ITR sequences of several known natural AAVs, in order to prevent nonspecific annealing to other regions in the ITR sequence, particularly regions corresponding to inverted repeat sequences. Here, as a sequence commonly conserved at the 5' end in known natural AAV ITR sequences, it is preferable that, for example, the polynucleotide described at positions 54 to 44 in the base sequence of the AAV2 ITR sequence described in SEQ ID NO: 2 (SEQ ID NO: 48: 5'-GAAACCAGGG-3') or a polynucleotide in which one, two, three, four, five, or six bases are deleted from the 5' or 3' end of the polynucleotide described in SEQ ID NO: 48, in which two or more, three or more, four or more, or five or more bases are substituted with other bases. Alternatively, the identity between the 3' or 5' terminal sequence of an artificially designed polynucleotide b1 consisting of at least five bases, any of the above-mentioned bases, and the polynucleotide (5'-GAAACCAGGG-3') described in Sequence ID No. 48 or the nucleotide sequence of a polynucleotide in which one, two, three, four, five, or six bases are deleted from the 5' or 3' terminal of the polynucleotide described in Sequence ID No. 48 can be 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 0%.

[0040] For example, in an artificially designed polynucleotide b1 consisting of at least five bases selected from adenine, guanine, cytosine, and thymine, if there are 11 bases, the 11 bases from the 5' end can be, in order, a base other than guanine, a base other than adenine, a base other than adenine, a base other than adenine, a base other than cytosine, a base other than adenine, a base other than guanine, a base other than cytosine, a base other than guanine, and a base other than guanine. Also, for example, as an artificially designed polynucleotide b1 consisting of at least five bases selected from adenine, guanine, cytosine, and thymine, the adenine or thymine content can be 20% or more, 30% or more, 40% or more, and 50% or more, and as an artificially designed polynucleotide a1 consisting of at least five bases selected from adenine, guanine, cytosine, and thymine, the adenine or guanine content can be 20-80% and 40-70%. Furthermore, as an artificially designed polynucleotide b1 consisting of at least five bases of any of the above bases, examples include polynucleotides having a cytosine or guanine content of 50-100%, 60-90%, or 70-80%, preferably a cytosine content, or polynucleotides containing a sequence of bases consisting only of cytosine, guanine, adenine, or thymine, preferably a sequence of bases consisting only of cytosine or guanine, more preferably a sequence of bases consisting only of cytosine. Here, the length of the sequence of bases consisting only of cytosine, guanine, adenine, or thymine can be 5 bases or more, 8 bases or more, 10 bases or more, 12 bases or more, or 15 bases or more, and may be 5-50 bases, 8-30 bases, or 10-20 bases.

[0041] By including these artificially designed polynucleotide b1s in polynucleotide B, it becomes possible to prevent primers from annealing to sequences other than the ITR sequence they should be annealing to during the PCR reaction, and to prevent amplified DNA from annealing to each other.

[0042] The polynucleotide b1 and polynucleotide b2 may be directly linked, or they may be linked via a linker sequence consisting of any base sequence selected from adenine, guanine, cytosine, and thymine. The length of the linker sequence is not particularly limited, but examples include 1 to 20 bases, 1 to 10 bases, and 1 to 5 bases. The linker sequence may be an artificially designed sequence, a part of a known natural AAV ITR sequence, or a combination of these sequences. An example of a part of a natural ITR sequence is the polynucleotide described at positions 72 to 82 in the base sequence of the AAV2 ITR sequence described in SEQ ID NO: 2 (SEQ ID NO: 50: 5'-CTTTGCCCGGGG-3') or a subsequence thereof. A partial sequence of the polynucleotide (SEQ ID NO: 50) described at positions 72 to 82 in the base sequence of the AAV2 ITR sequence described in SEQ ID NO: 2 can be an example of a sequence in which the 5' end and / or 3' end, preferably the 5' end, of the polynucleotide described at positions 72 to 82 in the base sequence of the AAV2 ITR sequence described in SEQ ID NO: 2 is partially deleted.

[0043] In the second embodiment of the polynucleotide described in (B-i) above, the "polynucleotide B1 consisting of at least 20 bases including the base sequence of the 89th to 108th bases in SEQ ID NO: 1" can be a polynucleotide including the base sequence of the 79th to 113th bases in SEQ ID NO: 1, a polynucleotide including the base sequence of the 79th to 118th bases in SEQ ID NO: 1, a polynucleotide including the base sequence of the 79th to 123rd bases in SEQ ID NO: 1, or a polynucleotide including the base sequence of the 79th to 133rd bases in SEQ ID NO: 1.

[0044] Further, as the "polynucleotide consisting of at least 20 bases containing the base sequence of positions 89 to 108 in SEQ ID NO: 1" in the second aspect of the polynucleotide described in (B-i) above, preferably, polynucleotides containing the base sequence of positions 86 to 108 in SEQ ID NO: 1, polynucleotides containing the base sequence of positions 83 to 108 in SEQ ID NO: 1, polynucleotides containing the base sequence of positions 81 to 108 in SEQ ID NO: 1, and polynucleotides containing the base sequence of positions 79 to 108 in SEQ ID NO: 1 can be mentioned.

[0045] One aspect of the polynucleotide described in (B-ii) above includes polynucleotides in which one or several bases are deleted, substituted, added, or inserted in the polynucleotide B1 or polynucleotide B2 of (B-i) above, as long as they can anneal with the template DNA used in the PCR reaction as a whole sequence, and preferably includes polynucleotides in which one or several bases are deleted, substituted, added, or inserted in the polynucleotide b2 contained in the polynucleotide B2 of (B-i) above, but is not particularly limited. Here, the "one or several" can include 1 to 10, preferably 1 to 5, more preferably 1 to 4, still more preferably 1 to 3, particularly preferably 1 to 2, and most preferably 1. Another aspect of the polynucleotide described in (B-ii) above is a polynucleotide in which one or several bases are deleted, substituted, added, or inserted in the polynucleotide B1 or polynucleotide B2 of (B-i) above, and when PCR is performed using the polynucleotide as a single primer, compared with the case where PCR is performed under the same conditions using the polynucleotide B1 or B2 of (B-i) above as a single primer, polynucleotides in which the DNA amount of the PCR amplification product is 1 / 2 or more, 1 / 3 or more, 1 / 5 or more, 1 / 10 or more, 1 / 20 or more, 1 / 50 or more can be mentioned, but is not particularly limited. The DNA amount can be evaluated based on the value of the band by electrophoresis or the weight of the DNA.

[0046] One embodiment of the polynucleotide described in (B-iii) above is a polynucleotide having 90% or more identity with the polynucleotide in (B-i) above, insofar as the entire sequence can be annealed with the template DNA used in the PCR reaction. It may also be a polynucleotide having 92% or more identity with the polynucleotide in (A-i) above, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Other embodiments of the polynucleotide described in (B-iii) above include polynucleotides having 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the polynucleotide described in (B-i) above, wherein when PCR is performed using the polynucleotide as a single primer, the amount of DNA in the PCR amplification product is 1 / 2 or more, 1 / 3 or more, 1 / 5 or more, 1 / 10 or more, 1 / 20 or more, or 1 / 50 or more compared to when PCR is performed using the polynucleotide described in (B-i) above as a single primer under the same conditions as above, and preferably the above (B- i) Polynucleotides, preferably polynucleotide b2 contained in polynucleotide B2, that have identity of 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and when PCR is performed using such polynucleotide as a single primer, the amount of DNA in the PCR amplification product is 1 / 2 or more, 1 / 3 or more, 1 / 5 or more, 1 / 10 or more, 1 / 20 or more, or 1 / 50 or more compared to when PCR is performed using the polynucleotide of (B-i) above as a single primer under the same conditions as above. Examples of such polynucleotides include, but are not particularly limited.

[0047] In one aspect of the present disclosure, the polynucleotide B1 can be a polynucleotide consisting of a continuous nucleotide sequence from the S-th nucleotide to the T-th nucleotide in the nucleotide sequence set forth in SEQ ID NO: 1. Here, S indicates the nucleotide position at the 5'-end of the polynucleotide B1 and is any integer selected from 64 to 89. Specifically, examples of S include 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89, and preferably 79. Further, T indicates the nucleotide position at the 3'-end of the polynucleotide B1 and is any integer selected from 108 to 141. Specifically, examples of T include 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or 141. In the present disclosure, all ranges consisting of combinations of the above S and T are specifically disclosed individually.

[0048] Similarly, in one embodiment of the present disclosure, the polynucleotide b2 constituting the polynucleotide B2 may be a polynucleotide consisting of a continuous base sequence from the U-th base to the V-th base in the base sequence described in Sequence ID No. 1. Here, U indicates the base position at the 5' end of the polynucleotide b2 and is any integer selected from 64 to 89. Specifically, U can be 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or 89, and is preferably 79. V indicates the base position at the 3' end of the polynucleotide b2 and is any integer selected from 106 to 141. Examples of V include 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or 141. In this disclosure, all ranges consisting of the above combinations of U and V are disclosed individually and specifically.

[0049] Furthermore, the polynucleotides described in (A-i), (A-ii), or (A-iii) above, and (B-i), (B-ii), or (B-iii) above, are polynucleotides that can be annealed with template DNA used in PCR reactions.

[0050] Single primer A may have any base selected from adenine (A), guanine (G), cytosine (C), or thymine (T) attached to the 5' and / or 3' ends of any polynucleotide (A-i) to (A-iiii), as long as it is annealable to the template DNA used in the PCR reaction. Similarly, single primer B may have any base selected from adenine (A), guanine (G), cytosine (C), or thymine (T) attached to the 5' and / or 3' ends of any polynucleotide (B-i) to (B-iiii), as long as it is annealable to the template DNA used in the PCR reaction. Note that the base sequence with the above arbitrary bases may not be a sequence that anneals to the template DNA.

[0051] The polynucleotide lengths of single primer A and single primer B are not particularly limited as long as they are polynucleotides that can anneal to the template DNA used in the PCR reaction, but examples include 34-150 mer, 34-130 mer, 35-130 mer, 38-80 mer, 40-60 mer, 35-140 mer, 38-130 mer, 40-120 mer, 42-100 mer, 45-80 mer, and 45-60 mer, respectively.

[0052] The single primers A and B may have sequences complementary to the polynucleotides that constitute them, respectively.

[0053] [Template DNA] In this specification, template DNA can be defined as DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences, in other words, DNA having ITR sequences on both ends of the transgene, i.e., the 5' and 3' ends of the transgene. Using the single primer A or single primer B and the template DNA described above, DNA containing a transgene sandwiched between the full length of the two ITR sequences or a portion of the two ITR sequences can be used as the target for amplification.

[0054] Examples of ITR sequences include the ITR sequence described in Sequence ID No. 1, as well as ITR sequences derived from AAV. The serotype of AAV is not particularly limited and may be any of serotypes 1 to 12. Furthermore, the above ITR sequence may consist of a polynucleotide obtained by deleting, substituting, adding, or inserting one or more bases from the AAV-derived ITR sequence, depending on the desired purpose. Sequence information for the above ITR can be appropriately obtained by searching known literature or databases such as NCBI (www.ncbi.nlm.nih.gov / guide / ). The nucleotide sequences of the genomes of AAV serotypes 1-13 (AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13) are, respectively, represented by GenBank accession numbers: AAV1: NC_002077.1, AAV2: NC_001401.2, AAV3A: NC_001729.1, AAV3B: NC The sequence information for the above ITRs is as follows: _028705.1, AAV4:NC_001829.1, AAV5:NC_006152.1, AAV6:AF028704.1, AAV7:NC_006260.1, AAV8:NC_006261.1, AAV9:AX753250.1, AAV10:NC_AY631965.1, AAV11:AY631966.1, AAV12:DQ813647.1, AAV13:EU2775562.1. Furthermore, the sequence information for the above ITRs is incorporated herein by reference from the document disclosed in Japanese Patent Application Publication No. 2023-2483.

[0055] The single primer A or single primer B is not particularly limited as long as it is a polynucleotide containing a base sequence that can anneal to both ITR sequences in the template DNA. In other words, the base sequence of single primer A or B can be appropriately adjusted depending on the ITR sequences contained in the template DNA used.

[0056] The transgenes mentioned above are DNA or RNA that encode the target product. Examples of DNA or RNA that encode the target product include DNA that encodes a specific protein, and DNA that encodes RNA such as siRNA, shRNA, miRNA, antisense RNA, guide RNA, trans-splicing RNA, transfer RNA, and ribosomal RNA.

[0057] When an adeno-associated virus (AAV) vector is used to treat a specified disease, the target product may be a therapeutically effective protein or RNA. Examples of therapeutically effective proteins include those capable of treating the diseases targeted by the pharmaceutical composition described below, such as growth hormone, blood coagulation factors, cytokines (e.g., α-interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin-12), erythropoietin, albumin, insulin, mini-dystrophin, C1 esterase inhibitor, copper transporter type P ATPase (ATP7B), copper-zinc superoxide dismutase 1 (SOD1), myosin-binding protein C3, follicle-stimulating hormone, and LDL receptor. Furthermore, transgenes are not limited to those described above and include DNA encoding nucleases for genome editing (e.g., Cas9, Cpf1, etc.) or guide RNA, DNA encoding reporter proteins (e.g., GFP, luciferase, etc.), or DNA containing only expression regulatory sequences. In addition, the method described in Japanese Patent Application Publication No. 2023-2483 is incorporated into this disclosure by reference.

[0058] RNA effective for the above treatment can be cited as RNA that inhibits the expression of a specific gene related to the disease. The specific gene related to the above disease can be cited as the Huntington's (HTT) gene. Sequence information of proteins and RNAs effective for the above treatment is incorporated herein by reference from the contents disclosed in Japanese Patent Application Publication No. 2023-2483 and other literature.

[0059] Furthermore, the DNA containing the two ITR sequences and the transgene sandwiched between them may also contain, in addition to the transgene, DNA encoding a promoter sequence, DNA encoding an intron, DNA encoding a polyadenylated (polyA) sequence, a multicloning site, or a restriction enzyme site. Each of the above DNAs may be linked with any base selected from adenine, guanine, cytosine, and thymine, as long as the target product can be expressed in the cell into which it is introduced. Specifically, the DNA containing the two ITR sequences and the transgene sandwiched between them can be described as DNA in which the promoter sequence, the DNA encoding the target product, and the DNA encoding the polyadenylated sequence are sequentially linked from 5' to 3'. Furthermore, by performing PCR using single primer A or single primer B, a DNA fragment containing the two ITR sequences and a transgene sandwiched between them is produced. Depending on the single primer A or single primer B used, all or part of the ITR sequence is added to the 5' and 3' ends of the PCR product. Therefore, a DNA fragment encoding the target product containing all or part of the ITR sequence at both ends can be obtained for AAV vector production. The size of the DNA containing the transgene is not particularly limited, but it is preferably 4.7 kb or less, which is a size that can be packaged as an AAV vector. When producing a self-complementary AAV (scAAV) vector, it is preferably about 2.2 kb or less.

[0060] The length of the DNA to be amplified is not particularly limited as long as it can be accommodated in the capsid of the AAV virus vector, but examples include 30-4700 bp, 100-4000 bp, 200-3500 bp, and 500-3000 bp.

[0061] Examples of promoter sequences include the cytomegalovirus (CMV) promoter, the β-actin promoter, and the EF-1α promoter.

[0062] [Single-Primer PCR] Unlike conventional PCR which uses forward and reverse primers, single-primer PCR as described herein allows PCR to be performed using only one or substantially one type of primer. While the above single-primer PCR does not prevent the inclusion of multiple single primers A or B in the PCR reaction solution, it is preferable not to use single primer A and single primer B in combination. This is because single primer A and single primer B, or the amplification products of the PCR, may anneal, making it difficult to obtain the desired amplification product.

[0063] The PCR reaction solution can be any reaction solution used in known PCR methods. Possible volumes of the PCR reaction solution include 5 μL to 1 L, 10 μL to 1 L, 100 μL to 100 mL, 500 μL to 20 mL, 1 mL to 15 mL, 10 mL to 1 L, and 50 mL to 500 mL. Furthermore, as shown in the examples described later, even with large-volume PCR (thermal cycling with a slow temperature change rate in a 10 mL reaction solution), which is not typically used in genetic engineering techniques, using single primer A or single primer B makes it possible to produce fragments of the target DNA by single-primer PCR.

[0064] The PCR reaction may be a two-step program consisting of a thermal denaturation step and an annealing and extension step, or it may be a three-step program in which the annealing and extension steps are performed at different temperatures after the thermal denaturation step.

[0065] In this specification, the temperature for the thermal denaturation step in the PCR reaction can be 84 to 98°C, 86 to 97°C, 90 to 96°C, or 92 to 95°C. The thermal denaturation time can be 1 to 90 seconds, 5 to 60 seconds, or 7 to 30 seconds.

[0066] In this specification, possible annealing temperatures in the PCR reaction include 50-77°C, 55-76°C, 57-75°C, 60-73°C, and 65-72°C.

[0067] In this specification, possible temperatures for the extension reaction in the PCR reaction are 50-77°C, 55-76°C, 57-75°C, 60-73°C, and 65-72°C. Furthermore, possible extension reaction times are 10 seconds to 20 minutes, 30 seconds to 10 minutes, 1 to 5 minutes, and 1.5 to 3 minutes.

[0068] In this specification, when performing a PCR reaction using a two-step cycle, suitable temperatures for the annealing and extension reactions include 55-80°C, 60-75°C, and 65-72°C. Suitable durations for the annealing and extension reactions include 10 seconds to 20 minutes, 30 seconds to 10 minutes, 1 to 5 minutes, and 1.5 to 3 minutes.

[0069] Furthermore, the thermal cycles for the thermal denaturation, annealing, and extension reactions can range from 20 to 50 cycles, 15 to 40 cycles, and 24 to 36 cycles. The duration of one cycle, i.e., the time for each step in one cycle of the thermal denaturation, annealing, and extension reactions, and the temperature control time can range from 3 to 30 minutes, 5 to 25 minutes, 7 to 20 minutes, and 10 to 15 minutes, respectively.

[0070] In this specification, a typical two-step example of a PCR reaction is as follows: (1) Thermal denaturation at 94°C for 10 seconds (2) Annealing and extension reaction for 1.5 to 3 minutes (3) Repeat (1) and (2) for 30 cycles

[0071] [Method for producing AAV vectors] In the method for producing AAV vectors, DNA fragments obtained by the method for producing DNA fragments, along with DNA encoding E2A derived from adenovirus, DNA encoding E4 derived from adenovirus, DNA encoding VA derived from adenovirus, DNA encoding rep, and DNA encoding cap are introduced into packaging cells. Alternatively, DNA fragments obtained by the method for producing DNA fragments may be introduced into packaging cells that have already been introduced with DNA encoding E2A derived from adenovirus, DNA encoding E4 derived from adenovirus, DNA encoding VA derived from adenovirus, DNA encoding rep, and DNA encoding cap. Alternatively, DNA fragments obtained by the method for producing DNA fragments may be introduced into packaging cells first, and then DNA encoding E2A derived from adenovirus, DNA encoding E4 derived from adenovirus, DNA encoding VA derived from adenovirus, DNA encoding rep, and DNA encoding cap may be introduced into the packaging cells. The order in which the DNA is introduced is not particularly limited.

[0072] Furthermore, in the introduction of adenovirus-derived DNA encoding E2A, adenovirus-derived DNA encoding E4, and adenovirus-derived DNA encoding VA, DNA fragments or plasmids containing adenovirus-derived DNA encoding E2A, adenovirus-derived DNA encoding E4, and adenovirus-derived DNA encoding VA may be used. In the introduction of rep-coding DNA and cap-coding DNA, DNA fragments or plasmids containing rep-coding DNA and cap-coding DNA may be used.

[0073] The packaging cells can be any cells capable of producing recombinant AAV vectors, such as mammalian cells, insect cells, microorganisms, and yeast cells, with mammalian cells being preferred. Examples of mammalian cells include human fetal kidney-derived HEK293 cells, human cervical cancer-derived Hela cells, Chinese hamster ovary-derived CHO cells, hamster kidney cells BHK cells, mouse myeloma cells NS0 cells, mouse embryonic fibroblast cells NIH3T3 cells, human liver cancer cells Huh-7 cells, human hepatocellular carcinoma cells HepG2 cells, human liver cancer cells Hep1A cells, human fetal kidney cells 911 cells, African green monkey kidney cells COS cells, human melanoma cells MeWo cells, human fibrosarcoma cells HT1180 cells, monocytes, immature dendritic cells, mature dendritic cells, and derivatives of the above cells (for example, the human-derived HEK293 cell derivative HEK293T cells and the human-derived HEK293 cell derivative Pro10 cells). In addition, insect cells such as Sf9 (Spodoptera frugiperda) cells, Hi5 cells, and derivative strains of these cells can be mentioned.

[0074] As a method for introducing the DNA fragments obtained by the method for producing the DNA fragments in question, along with DNA encoding E2A from adenovirus, DNA encoding E4 from adenovirus, DNA encoding VA from adenovirus, DNA encoding rep, and DNA encoding cap, as well as the DNA fragments obtained by the method for producing the DNA fragments in question, along with a helper plasmid and the Rep-Cap plasmid, known nucleic acid delivery methods can be used, including methods using electroporation, calcium phosphate precipitation, microinjection, and liposome-mediated gene delivery.

[0075] AAV helper plasmids only need to contain genes that have helper functions necessary for viral replication, and examples include plasmids containing adenovirus E2A, E4, and VA RNA. Helper plasmids are known in the field of genetic engineering, as described in, for example, Japanese Patent Publication No. 2024-536223, and such literature is incorporated herein by reference.

[0076] Examples of Rep-Cap plasmids include plasmids containing Rep and Cap. The rep gene is a gene that codes for replicase proteins (Rep78, Rep68, Rep52, Rep40) necessary for the amplification and transcription of viral DNA, and the cap gene is a gene that codes for capsid proteins (VP1, VP2, VP3) that constitute the viral particle. The sequences of Rep and Cap are disclosed in Japanese Patent Publication No. 2024-536223 and are incorporated herein by reference. Sequence information for the above-mentioned Rep gene, cap gene, or replicase proteins Rep78, Rep68, Rep52, Rep40, and capsid proteins V1, V2, V3 can be appropriately obtained by searching known literature or databases such as NCBI (www.ncbi.nlm.nih.gov / guide / ).

[0077] The pharmaceutical composition in this disclosure is a pharmaceutical composition (hereinafter also referred to as "the pharmaceutical composition") comprising an adeno-associated virus (AAV) vector obtained by the method for producing the AAV vector and pharmaceutically acceptable additives. Examples of pharmaceutically acceptable additives in the pharmaceutical composition include water, physiological saline, dextrose, glycerol, and ethanol, and these may be used in combination. Furthermore, the pharmaceutical composition may also contain wetting agents, emulsifiers, pH buffers, stabilizers, etc.

[0078] This pharmaceutical composition can be used for gene therapy. Potential treatment targets include neurodegenerative diseases such as Alzheimer's disease, Canavan disease, and Parkinson's disease; genetic disorders such as spinal muscular atrophy (SMA), hemophilia, Fabry disease, Duchenne muscular dystrophy (DMD), Wilson's disease, amyotrophic lateral sclerosis (ALS), hereditary angioedema (HAE), Pompe disease, and hypertrophic cardiomyopathy; visual impairment disorders such as retinitis pigmentosa; cardiovascular diseases such as ischemic heart disease and heart failure; and cancer. Furthermore, this pharmaceutical composition can be used to treat those who require it. Methods of administration of this pharmaceutical composition include intravenous, intraperitoneal, subcutaneous, and intradermal administration.

[0079] All patent and non-patent documents cited herein are incorporated herein by reference as a whole.

[0080] The present disclosure will be described in more detail below with reference to examples, but the technical scope of the present disclosure is not limited to these examples.

[0081] [Reference Example 1] PCR amplification of the region containing the ITR First, the region containing the ITR at both ends was amplified by PCR using forward primers and reverse primers, which is a common method.

[0082] Using a pAAV-CMV vector containing two ITR regions (AAV Helper Free System: Takara Bio Inc.) as template DNA, PCR was performed using forward primer EcOri-1c(35) (SEQ ID NO: 12) and reverse primer AmpR+35c(35) (SEQ ID NO: 13) to amplify DNA regions containing ITR sequences at the 5' and 3' ends of the CMV promoter, β-globin introns, and human growth hormone (hGH) polyA.

[0083] The sequences of the primers used are shown in Table 2.

[0084]

[0085] PCR was performed using a two-step program: thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 68°C for 3 minutes. The program consisted of 30 cycles.

[0086] The PCR reaction mixture was prepared to the following composition: Water: 2.8 μL 2x HC buffer (Helixextension): 5.0 μL Forward primer 10 μM: 0.5 μL Reverse primer 10 μM: 0.5 μL Template DNA (1 μg / mL): 1.0 μL Helix DNA polymerase (Helixextension): 0.2 μL Total: 10.0 μL

[0087] Furthermore, Figure 1 shows the vector map of the pAAV-CMV vector, the annealing positions of the forward primer EcOri-1c(35) and the reverse primer AmpR+35c(35), and Figure 2 shows the agarose electrophoresis results of the PCR amplification product.

[0088] When PCR is performed using the pAAV-CMV vector as a template with the forward primer EcOri-1c(35) (SEQ ID NO: 12) and the reverse primer AmpR+35c(35) (SEQ ID NO: 13), a DNA fragment of 3414 bp should be obtained. However, as shown in Figure 2, a band of approximately 1.5 kb was detected. This was thought to be because, by using primers outside the ITR sequence, the primers annealed to a sequence different from the ITR sequence that should have been annealed during the PCR reaction, or the amplified DNA annealed, resulting in amplification of a DNA fragment from a region other than the one that should have been amplified.

[0089] [Reference Example 2] In Reference Example 1, the pAAV-CMV vector was used as the template DNA, but we attempted to amplify the ITR region using PCR with forward and reverse primers, which is a commonly performed method, using other vectors.

[0090] Using the VB010000-9394npt vector (VectorBuilder, containing two ITR regions, with ITR sequences positioned at both ends of the CMV promoter, EGFP, WPRE, and BGH polyA), PCR was performed using forward primer pVBss9394ITR+1(-35) (SEQ ID NO: 14) and reverse primer pVBss9394ITR+1(+35)c (SEQ ID NO: 15) as template DNA, the DNA regions with ITR sequences at the 5' and 3' ends of the CMV promoter, EGFP, WPRE, and BGH polyA were amplified. The forward primer pVBss9394ITR+1(-35) (SEQ ID NO: 14) and the reverse primer pVBss9394ITR+1(+35)c (SEQ ID NO: 15) are sequences that anneal to the region outside the respective ITR sequence.

[0091] The sequences of the primers used are shown in Table 3.

[0092]

[0093] PCR was performed using a two-step program: thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 68°C for 3 minutes. The program consisted of 30 cycles. 0.4 M Tris buffer was used for the PCR reaction mixture.

[0094] Figure 3 shows the results of agarose electrophoresis of the PCR amplification products.

[0095] If primer PCR is performed using the VB010000-9394npt vector as the template DNA and the forward primer pVBss9394ITR+1(-35) and reverse primer pVBss9394ITR+1(+35)c described above, a 2.5kb DNA fragment should be obtained. However, as shown in Figure 3, even when PCR was performed using the VB010000-9394npt vector as the template DNA and the forward primer pVBss9394ITR+1(-35) and reverse primer pVBss9394ITR+1(+35)c described above, the desired amplification product was not obtained.

[0096] [Example 1] From the above results, it was confirmed that it is difficult to obtain the desired amplification product even when attempting to amplify the region of the target gene sandwiched between ITR sequences from outside the ITR sequence using two common forward primers and reverse primers. Therefore, primers were designed based on the sequence within the ITR region, and single-primer PCR was attempted using a single primer.

[0097] As a single primer, we designed a 35-mer pAAV-CMV-ITR+1(35) (SEQ ID NO: 16) starting from the 5' end of the ITR sequence.

[0098] Using a pAAV-CMV vector (AAV Helper Free System: Takara Bio Inc.) containing two ITR regions as described in SEQ ID NO: 1 as the template DNA, single-primer PCR was performed using only primer pAAV-CMV-ITR+1(35) (SEQ ID NO: 16) as the primer. The region containing the ITR sequence, CMV promoter sequence, β-globin intron, and hGH poly(A) was amplified. Note that primer pAAV-CMV-ITR+1(35) is a sequence that includes a portion of the ITR region.

[0099] The sequences of the primers used are shown in Table 4.

[0100]

[0101] Single-primer PCR was performed using a two-step program: thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 68°C for 1.5 minutes. The program consisted of 30 cycles.

[0102] The PCR reaction mixture was prepared to the following composition: Water: 1.3 μL 2x HC buffer (Helixextension): 5.0 μL Primer 10 μM: 2.5 μL Template DNA (1 μg / mL): 1.0 μL Helix DNA polymerase (Helixextension): 0.2 μL Total: 10.0 μL

[0103] Furthermore, Figure 4 shows the vector map of the pAAV-CMV vector and the annealing positions of the primers, and Figure 5 shows the agarose electrophoresis results of the PCR amplification product.

[0104] Using the pAAV-CMV vector as template DNA, single-primer PCR with the primer pAAV-CMV-ITR+1(35) should yield a 1914 bp DNA fragment. As shown in Figure 5, a band of approximately 1.9 kb was detected. Therefore, it was confirmed that single-primer PCR using the primer pAAV-CMV-ITR+1(35) prevents abnormal annealing of the ITR sequence and allows for the acquisition of the desired PCR amplification product.

[0105] [Example 2] In Example 1, a 35-mer pAAV-CMV-ITR+1(35) primer was used, but PCR was attempted with primers of various lengths.

[0106] As single primers, we designed 25-mer pAAV-CMV-ITR+1(25) (SEQ ID NO: 17), 35-mer pAAV-CMV-ITR+1(35) (SEQ ID NO: 16), 40-mer pAAV-CMV-ITR+1(40) (SEQ ID NO: 18), 45-mer pAAV-CMV-ITR+1(45) (SEQ ID NO: 19), 50-mer pAAV-CMV-ITR+1(50) (SEQ ID NO: 20), and 55-mer pAAV-CMV-ITR+1(55) (SEQ ID NO: 21), starting from the 5' end of the ITR sequence.

[0107] Using a pAAV-CMV vector (AAV Helper Free System: Takara Bio Inc.) containing the above-mentioned ITR regions in two locations as template DNA, single-primer PCR was performed using the respective primers described above, and the region containing the ITR (1914 bp) was amplified.

[0108] Table 5 shows the sequences of the primers used. Note that the 1st to 12th polynucleotides in SEQ ID NOs. 16 to 21 (SEQ ID NO: 4: 5'-CCTGCAGGCAGC-3') are artificially designed polynucleotides, for example, the 7th to 16th bases in the 5th to 16th base sequence of the ITR sequence of AAV2 described in SEQ ID NO: 2 (SEQ ID NO: 3: 5'-CCACTCCCCTCTC-3') have been replaced with other bases. Note that the 1st to 8th polynucleotides in SEQ ID NOs. 16 to 21 are sequences recognized by the restriction enzyme SbfI.

[0109]

[0110] Single-primer PCR was performed using a gradient annealing and extension temperature from 75°C to 65°C. Specifically, a two-step program was used: denaturation at 94°C for 10 seconds, followed by annealing and extension at 75°C, 74°C, 73°C, 71°C, 69°C, 67.4°C, 66°C, or 65°C for 2 minutes. The process consisted of 30 cycles.

[0111] The PCR reaction mixture was prepared to the following composition: Water: 1.3 μL 2x HC buffer (Helixextension): 5.0 μL Primer 10 μM: 2.5 μL Template DNA (1 μg / mL): 1.0 μL Helix DNA polymerase (Helixextension): 0.2 μL Total: 10.0 μL

[0112] Figure 7 shows the results of agarose electrophoresis of the PCR amplification products.

[0113] Using the pAAV-CMV vector as a template, single-primer PCR with each primer should yield a 1914 bp DNA fragment. As shown in Figure 7, when pAAV-CMV-ITR+1 (35), pAAV-CMV-ITR+1 (40), pAAV-CMV-ITR+1 (45), and pAAV-CMV-ITR+1 (50) were used as single primers, a band of approximately 1.9 kb was detected. Furthermore, it was confirmed that the preferred annealing and extension temperatures were 71°C, 69°C, 67°C, and 66°C when using pAAV-CMV-ITR+1 (35) as a single primer; 71°C, 69°C, 67°C, and 66°C when using 40 mer pAAV-CMV-ITR+1 (40); 71°C, 69°C, 67°C, 66°C, and 65°C when using 45 mer pAAV-CMV-ITR+1 (45); and 71°C and 69°C when using 50 mer pAAV-CMV-ITR+1 (50). In addition, when using pAAV-CMV-ITR+1 (55), a band of approximately 1.9 kb was not detected, and bands of short DNA fragments were detected, with the bands becoming larger, especially at lower temperatures. This is thought to be due to the primer not annealing at the correct position. Therefore, it is preferable not to include the 55th base sequence and its 3' end in the ITR region of Sequence ID No. 1 in the primer.

[0114] Furthermore, by narrowing the temperature range, single-primer PCR was performed in the same manner as above using pAAV-CMV-ITR+1(35) (SEQ ID NO: 16), pAAV-CMV-ITR+1(40) (SEQ ID NO: 18), pAAV-CMV-ITR+1(45) (SEQ ID NO: 19), and pAAV-CMV-ITR+1(50) (SEQ ID NO: 20) listed in Table 5, and the results of agarose electrophoresis of the amplified PCR products are shown in Figure 8.

[0115] Bands were observed at annealing and extension temperatures of 72.1°C, 71.2°C, 70.0°C, 69.2°C, 68.5°C, and 68°C, with particularly thick bands observed between 69.2°C and 72.1°C. Therefore, it was confirmed that amplification is possible at annealing and extension temperatures of at least 68 to 72.1°C. [Example 3] In the production of nucleic acid drugs and viral vector therapeutics, it is necessary to amplify large amounts of DNA containing transgenes sandwiched between two ITR sequences. Therefore, we confirmed whether amplification by single-primer PCR can amplify transgenes even in large-volume PCR.

[0116] Using a pAAV-CMV vector containing two of the above-mentioned ITR regions (AAV Helper Free System: Takara Bio Inc.) as template DNA, single-primer PCR was performed using primer pAAV-CMV-ITR+1(45) (SEQ ID NO: 19) to amplify the region containing the ITR.

[0117] Single primers were tested on a 10 mL scale. The program consisted of two stages: thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 71°C for 2 minutes, for a total of 30 cycles.

[0118] The PCR reaction mixture was prepared to a total volume of 10 mL with the following composition: Water: 2.3 mL 2x HC buffer (Helixextension): 5.0 mL Single primer 10 μM: 1.5 mL Template DNA (1 μg / mL): 1.0 mL Helix DNA polymerase (Helixextension): 0.2 mL Total: 10.0 mL

[0119] Figure 9 shows the results of agarose electrophoresis of the PCR amplification products.

[0120] It was confirmed that even when performing single-primer PCR using the pAAV-CMV vector as template DNA and the primer pAAV-CMV-ITR+1(35), a large amount of DNA fragments sandwiched between two ITR sequences can be obtained, even when performing PCR in a large volume of 10 mL.

[0121] [Example 4] In Example 2 above, the primers listed in Table 5 were used as single primers. However, in order to confirm how many bases on the 3' side need to be included for amplification by PCR, we designed further primers and investigated whether it was possible to amplify DNA sandwiched between two ITR sequences by single-primer PCR.

[0122] First, in Table 5, primers with lengths of 25, 35, 40, 45, 50, and 55 mer were designed based on the base at position +1 of the ITR sequence. However, as shown in Table 6, primers with lengths between 50 and 55 mer, specifically 52 mer and 54 mer, were designed, and amplification by single-primer PCR was attempted.

[0123]

[0124] Single-primer PCR was performed using a two-step program: thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 71°C for 1.5 minutes. The program consisted of 30 cycles. The PCR reaction mixture was prepared to a total volume of 10 mL with the following composition: Water: 2.3 mL; 2x HC buffer (Helixextension): 5.0 mL; Single primer 10 μM: 1.5 mL; Template DNA (1 μg / mL): 1.0 mL; Helix DNA polymerase (Helixextension): 0.2 mL; Total: 10.0 mL

[0125] Figure 10 shows the results of agarose electrophoresis of the PCR amplification products. In Figure 10, +1 (45), +1 (50), +1 (52), +1 (54), and +1 (55) are lanes where amplification products from single-primer PCR using pAAV-CMV-ITR+1 (45), pAAV-CMV-ITR+1 (50), pAAV-CMV-ITR+1 (52), pAAV-CMV-ITR+1 (54), and pAAV-CMV-ITR+1 (55) were applied, respectively.

[0126] Figure 10 shows that the band gradually thinned from +1 (45) to +1 (54), and at +1 (55), the band was almost invisible. Therefore, it became clear that amplification by single-primer PCR decreased when the single primer included glycine (G), which is the 55th base in the nucleotide sequence described in Sequence ID No. 1, or the base at the 3' end of that 55th base. This is thought to be due to the interaction between the 55th base and its 3' end in Sequence ID No. 1 and the 41st base and its 5' end in Sequence ID No. 1.

[0127] [Example 5] The effect of single-primer PCR amplification when there was a 1-3 base substitution as the single primer was investigated.

[0128] As shown in Table 7, along with pAAV-CMV-ITR+1(45) (SEQ ID NO: 19), we designed pAAV-CMV-ITR+1(45)Ca (SEQ ID NO: 24) in which the 17th cytosine (C) of SEQ ID NO: 19 was replaced with alanine (a), pAAV-CMV-ITR+1(45)CaCa (SEQ ID NO: 25) in which the 17th cytosine (C) of SEQ ID NO: 19 was replaced with alanine (a) and the 21st cytosine (C) was replaced with alanine (a), and pAAV-CMV-ITR+1(45)CaCaGa (SEQ ID NO: 26) in which the 17th cytosine (C) of SEQ ID NO: 19 was replaced with alanine (a), the 21st cytosine (C) was replaced with alanine (a), and the 26th glycine (G) was replaced with alanine (a), and attempted amplification by single-primer PCR.

[0129]

[0130] Single-primer PCR was performed using a two-step program: thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 71°C for 1.5 minutes. The program consisted of 30 cycles. The composition of the PCR reaction solution was the same as in Example 4 above.

[0131] Figure 11 shows the results of agarose electrophoresis of the PCR amplification products. In Figure 11, +1(45), +1(45)Ca, +1(45)CaCa, and +1(45)CaCaGa are the lanes to which the amplification products of single-primer PCR using pAAV-CMV-ITR+1(45), pAAV-CMV-ITR+1(45)Ca, pAAV-CMV-ITR+1(45)CaCa, and pAAV-CMV-ITR+1(45)CaCaGa were applied, respectively.

[0132] Figure 11 shows that with a single primer, a single base substitution resulted in almost the same amplification as no substitution, and with two substitutions, amplification was still possible, although slightly reduced. On the other hand, no band was detected with a three-base substitution.

[0133] [Example 6] Up to Example 5, the sequence included the +1 position of the ITR sequence described in Sequence ID No. 1. Here, in order to confirm which base of the 5' end should be included as a single primer for amplification, we designed further primers and investigated whether amplification of DNA sandwiched between two ITR sequences was possible by single-primer PCR.

[0134] As shown in Table 8, single primers of a predetermined length were designed from the +7 or +12 position of the ITR sequence, and amplification by single-primer PCR was attempted.

[0135]

[0136] Single-primer PCR was performed using a two-step program: thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 71°C for 1.5 minutes. The program consisted of 30 cycles. The composition of the PCR reaction solution was the same as in Example 4 above.

[0137] Figure 12 shows the results of agarose electrophoresis of the PCR amplification products.

[0138] As shown in Figure 12, no band was detected in single primers consisting of nucleotide sequences of 28, 30, 33, 35, 40, and 45 bases consecutively from position +7 of the ITR sequence described in SEQ ID NO: 1, or nucleotide sequences of 33 and 38 bases consecutively from position +12. Therefore, it was confirmed that for amplification by single-primer PCR, the sequence 5' end of position +7 of the ITR sequence described in SEQ ID NO: 1 is necessary.

[0139] Based on the above results, we further designed primers starting from positions +1, +2, +3, +4, +5, +6, and +7 of the ITR sequence described in Sequence ID No. 1 as single primers, and investigated whether amplification of DNA sandwiched between two ITR sequences was possible by single-primer PCR.

[0140] As shown in Table 9, single primers of a predetermined length were designed from the +1 or +7 position of the ITR sequence, and amplification by single-primer PCR was attempted.

[0141]

[0142] Single-primer PCR was performed using a two-step program: thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 71°C for 1.5 minutes. The program consisted of 30 cycles. The composition of the PCR reaction solution was the same as in Example 4 above.

[0143] Figure 13 shows the results of agarose electrophoresis of the PCR amplification products. In Figure 13, +1 (45), +2 (44), +3 (43), +4 (42), +5 (41), +6 (40), and +7 (38) are the lanes to which amplification products from single-primer PCR using pAAV-CMV-ITR+1 (45), pAAV-CMV-ITR+2 (44), pAAV-CMV-ITR+3 (43), pAAV-CMV-ITR+4 (42), pAAV-CMV-ITR+5 (41), pAAV-CMV-ITR+6 (40), and pAAV-CMV-ITR+7 (38) were applied, respectively.

[0144] Figure 13 shows that in the single primer, a faint band was visible in the +5 (41) lane, and the band became darker as the primers progressed to +4 (42), +3 (43), +2 (44), and +1 (45). Therefore, it was confirmed that including the +5 position or the 5' end of the ITR sequence described in Sequence ID No. 1 is effective in increasing amplification efficiency when performing amplification by single-primer PCR.

[0145] [Example 7] Up to Example 6, single primers were designed by focusing on the 5' terminal sequence of the ITR sequence described in SEQ ID NO: 1. Next, as the 3' terminal sequence of the ITR sequence, single primers were designed by focusing on the 79th base or the base 5' to it in the ITR sequence described in SEQ ID NO: 1.

[0146] As shown in Table 10, single primers of predetermined lengths were designed from positions +79, +79(-7), and +79(-17) of the ITR sequence described in Sequence ID No. 1, and amplification by single-primer PCR was attempted. Note that +79(-7) refers to position +72 of the ITR sequence described in Sequence ID No. 1, and +79(-17) refers to position +62 of the ITR sequence described in Sequence ID No. 1.

[0147]

[0148] Single-primer PCR was performed using a two-step program consisting of thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 68°C for 1.5 minutes. Alternatively, PCR was performed using two different programs: thermal denaturation at 94°C for 10 seconds, followed by annealing and extension at 71°C for 1.5 minutes. The number of cycles was 30. The composition of the PCR reaction solution was the same as in Example 2 above.

[0149] Figures 14A and 14B show the results of agarose electrophoresis of the PCR amplification products. In the figures, Figure 14A shows the lane where the amplification product was applied when amplification was performed using a two-step program of heat denaturation at 94°C for 10 seconds, followed by annealing and extension at 68°C for 1.5 minutes as a single-primer PCR. Figure 14B shows the lane where the amplification product was applied when amplification was performed using a two-step program of heat denaturation at 94°C for 10 seconds, followed by annealing and extension at 71°C for 1.5 minutes as a single-primer PCR.

[0150] Figures 14A and 14B show that bands were detected in single primers consisting of the 30, 35, 40, 45, and 50 base sequences consecutively from position +79 of the ITR sequence described in SEQ ID NO: 1, and the 50 base sequence consecutively from position +72 of the ITR sequence described in SEQ ID NO: 1. On the other hand, no bands were detected in the 60 base sequence consecutively from position +62 of the ITR sequence described in SEQ ID NO: 1. Therefore, it was confirmed that amplification by single-primer PCR is possible using the nucleotide sequence at position +79 and its 3' end of the ITR sequence described in SEQ ID NO: 1, as well as the nucleotide sequence at position +72 and its 3' end of the ITR sequence described in SEQ ID NO: 1. On the other hand, no bands were detected in single-primer PCR using the nucleotide sequence at position +62 and its 3' end of the ITR sequence described in SEQ ID NO: 1. This is thought to be due to the interaction between the 62nd base and its 5' end in SEQ ID NO: 1 and the 78th base and its 3' end in SEQ ID NO: 1.

Claims

1. Polynucleotide A, in which polynucleotide a2, consisting of at least 16 consecutive bases from the 12th to 39th base sequence in SEQ ID NO: 1, is linked to the 3' end of an artificially designed polynucleotide a1 consisting of at least 5 bases selected from adenine, guanine, cytosine, and thymine; (A-ii) a polynucleotide in which one or more bases are deleted, substituted, added, or inserted in polynucleotide A of (A-i); or a polynucleotide having 90% or more identity with polynucleotide A of (A-iii); single primer A containing any of the polynucleotides (A-i) to (A-iii); (B-i) A polynucleotide B2 formed by linking a polynucleotide B1 consisting of at least 20 bases, comprising the full sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, including bases 89 to 108 in Sequence ID No. 1, or an artificially designed polynucleotide b1 consisting of at least 5 bases, comprising any base selected from adenine, guanine, cytosine, and thymine, to the 3' end of a polynucleotide B2 consisting of at least 16 consecutive bases, comprising the full sequence or a continuous partial sequence of bases 64 to 141 in the base sequence described in Sequence ID No. 1, including bases 79 to 106 in Sequence ID No. 1; (B-ii) A polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide B1 or polynucleotide B2 of (B-i); or (B-iii) A polynucleotide having 90% or more identity with the polynucleotide B1 or polynucleotide B2 of (B-i); A single primer B containing any of the polynucleotides (B-i) to (B-iii); a method for producing a DNA fragment containing a transgene, characterized by performing single-primer PCR using single primer A or single primer B, with DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences as template DNA.

2. The method according to claim 1, characterized in that the single primer A is used as the single primer, and the length of the polynucleotide A is 34 to 150 mer.

3. The method according to claim 1 or 2, characterized in that single primer A is used as a single primer, and polynucleotide A is a polynucleotide that is a full-length sequence or a continuous partial sequence of the nucleotide sequences 1 to 54 in the nucleotide sequence described in SEQ ID NO: 1, and includes the nucleotide sequences 3 to 35 in SEQ ID NO:

1.

4. The method according to claim 1, characterized in that the single primer B is used as the single primer, and the length of the polynucleotide B1 or the polynucleotide B2 is 34 to 130 mer.

5. The method according to claim 1 or 4, characterized in that single primer B is used as a single primer, and polynucleotide B1 or polynucleotide B2 is a polynucleotide containing the base sequence from the 79th to the 108th base in SEQ ID NO:

1.

6. The method according to any one of claims 1 to 5, wherein the DNA containing the transgene includes DNA encoding a promoter sequence.

7. The method according to any one of claims 1 to 6, characterized in that PCR is performed in a two-step program consisting of a thermal denaturation step and an annealing and extension step.

8. A method for producing an adeno-associated virus (AAV) vector, comprising the step of transfecting packaging cells with DNA containing a transgene obtained by the method of any one of claims 1 to 7, DNA encoding E2A derived from adenovirus, DNA encoding E4 derived from adenovirus, DNA encoding VA derived from adenovirus, DNA encoding rep, and DNA encoding cap.

9. A pharmaceutical composition comprising an adeno-associated virus (AAV) vector obtained by the method of claim 8 and a pharmaceutically acceptable additive.

10. A single primer A for use in a method of producing a DNA fragment containing a transgene by performing single-primer PCR using DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences as template DNA. The single primer A comprises (A-i) a polynucleotide a2 consisting of at least 16 consecutive bases from the 12th to 39th base sequences in the sequence number 1, which is a full-length sequence or a continuous partial sequence of the 1st to 54th base sequences in the sequence number 1 described in Sequence ID No. 1, linked to the 3' end of an artificially designed polynucleotide a1 consisting of at least 5 bases selected from adenine, guanine, cytosine, and thymine; (A-i) a polynucleotide in which one or more bases are deleted, substituted, added, or inserted in the polynucleotide A of (A-i); or a polynucleotide having 90% or more identity with the polynucleotide A of (A-iii); and any of the polynucleotides from (A-i) to (A-iii).

11. A polynucleotide B2 for use in a method of producing a DNA fragment containing a transgene by performing single-primer PCR using DNA containing two terminal inverted repeat (ITR) sequences and a transgene sandwiched between the two ITR sequences as template DNA; (B-i) a polynucleotide B1 consisting of at least 20 bases, which is the full-length sequence or a continuous partial sequence of bases 64 to 141 in the nucleotide sequence described in Sequence ID No. 1, including bases 89 to 108 in Sequence ID No. 1, or an artificially designed polynucleotide b1 consisting of at least 5 bases, which is any base selected from adenine, guanine, cytosine, and thymine, with a polynucleotide b2 consisting of at least 16 continuous bases, which is the full-length sequence or a continuous partial sequence of bases 64 to 141 in the nucleotide sequence described in Sequence ID No. 1, including bases 79 to 106 in Sequence ID No. 1, ligated to the 3' end of the polynucleotide B1; A polynucleotide in which one or more bases are deleted, substituted, added, or inserted in polynucleotide B1 or polynucleotide B2 of (B-ii)(B-i); or a polynucleotide having 90% or more identity with polynucleotide B1 or polynucleotide B2 of (B-iii)(B-i); a single primer B containing any polynucleotide from (B-i) to (B-iii).

Images