Oligonucleotide primers for detecting adeno-associated virus vectors and method for detecting the vectors using the primers.

Abstract

To provide oligonucleotide primers capable of amplifying adeno associated virus (AAV) vectors with speed and high sensitivity and methods for specifically detecting the vectors using the primers.SOLUTION: Disclosed is a primer set comprising a first primer having a sequence complementary to a part of a specific base sequence of an AAV vector and a second primer having a sequence homologous to a part of the specific base sequence. The primer set further comprises a third primer having a sequence complementary or homologous to a part of the specific base sequence positioned at 5' terminal side of the first or second primer, where a promoter of an enzyme having RNA polymerase activity is further added at the 5' terminal side of anyone of the first and second primers, and the base sequences of the first and second primers are optimized.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an oligonucleotide primer for detecting an adeno-associated virus vector and a method for specifically detecting the vector using the primer.

Background Art

[0002] In the treatment of genetic diseases, a method of introducing into cells a viral vector containing a gene for the treatment is widely used in terms of good introduction efficiency into the cells. Among viral vectors, adeno-associated virus (AAV) vectors have attracted particular attention in recent years (Non-Patent Document 1) because AAV itself is not pathogenic, can efficiently introduce a gene inserted into the vector into non-dividing cells, and the expression of the introduced gene persists for a long time.

[0003] AAV is a particle with a diameter of 20 nm to 26 nm whose genome is encapsulated in a capsid protein. The genome of AAV is a linear single-stranded DNA of 4.7 kb, and there are T-shaped loop structures called inverted terminal repeat (ITR) sequences at the 3'-terminal side and 5'-terminal side thereof. When using the genome as an AAV vector for gene therapy, the Rep gene and Cap gene sandwiched between the ITR sequences at both ends of the genome are replaced with a promoter sequence specific to the tissue into which the gene is to be introduced, a gene used for gene therapy, a polyA signal sequence, and the like.

[0004] The production of AAV vectors is carried out by simultaneously transfecting a host cell (such as HEK293 cells or HeLa cells) with an AAV genome substituted with a gene used for gene therapy by the method described above, a vector containing the Rep gene and the Cap gene, and a helper virus such as an adenovirus or herpes simplex virus. In recent years, a method using an Adv helper plasmid in which the replication gene of an adenovirus is cloned, which is safer, instead of the helper virus, has also been carried out.

[0005] Among the AAV vectors produced using the method described above, some vectors lack the AAV genome necessary for gene therapy (so-called empty vectors). Therefore, in order to use the produced AAV vectors for gene therapy, it is necessary to confirm whether the AAV genome is present in the vector, or to quantify the amount of AAV genome contained in the sample (such as cell culture medium) containing the AAV vector.

[0006] Qualitative and / or quantitative measurement methods for AAV genomes contained in AAV vectors include image analysis of AAV vector particles using transmission electron microscopy, analytical ultracentrifugation, quantitative PCR (qPCR), digital droplet PCR (ddPCR), dot blotting, and electrophoresis. Of these, qPCR is widely used for the quantification of AAV vectors due to its ease of operation and speed. However, there are problems such as the lack of correlation between quantitative results from qPCR and other methods such as electrophoresis, and the fact that quantitative results differ depending on the gene region being measured (Non-patent documents 2 to 4, and patent documents 1 and 2). Furthermore, PCR-based methods such as qPCR and ddPCR require rapid increases and decreases in reaction temperature, which poses problems in terms of reducing the labor involved in the reaction process and the cost of reaction equipment when automating the process.

[0007] As nucleic acid amplification methods that allow for relatively easy reduction of reaction process labor and cost reduction of reaction equipment, the NASBA (Nucleic Acid Sequence Based Amplification) method, the TMA (Transcription Mediated Amplification) method, and the TRC (Transcription Reverse Transcription Concerted) method are known, which enable nucleic acid amplification at a constant temperature of relatively low temperatures (e.g., in the range of 40°C to 50°C). These amplification methods usually target single-stranded RNA, but DNA amplification is also possible by modifying the reaction system.

[0008] For example, Patent Document 3 discloses the amplification of hepatitis B virus nucleic acid using a reagent that includes an enzyme group and primer set used in RNA amplification by the TRC method, as well as a strand substitution enzyme and / or a primer for unifying the amplified nucleic acid, while Patent Document 4 discloses the amplification of single-stranded nucleic acid having a complementary region at the end using the aforementioned reagent. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2003-235562 [Patent Document 2] WO2015 / 080223 issue [Patent Document 3] Japanese Patent Publication No. 2015-116136 [Patent Document 4] Japanese Patent Publication No. 2020-162550 [Non-patent literature]

[0010] [Non-Patent Document 1] Keiya Ozawa, Virus, 57(1), 47-56 (2007) [Non-Patent Document 2] P.Fagone et al.,Human Gene Therapy Methods:B,23,1-7(2012) [Non-Patent Document 3] M.Lock et al.,Human Gene Therapy Methods,25,115-125(2014) [Non-Patent Document 4] S.D'Costa et al.,Methods And Clinical Development 5,16019(2016) [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The object of the present invention is to provide oligonucleotide primers capable of rapidly and sensitively amplifying adeno-associated virus vectors, and a method for the specific detection of said vectors using said primers. [Means for solving the problem]

[0012] As a result of diligent research to solve the above problems, the inventors have discovered a combination of oligonucleotide primers (primer set) that can rapidly and sensitively amplify adeno-associated virus (AAV) vectors, and have completed the present invention.

[0013] In other words, the first aspect of the present invention is A primer set for amplifying nucleic acids containing the specific nucleotide sequence or a complementary sequence of the specific nucleotide sequence, comprising a first primer having a sequence complementary to a portion of the specific nucleotide sequence of an AAV vector, and a second primer having a sequence homologous to a portion of the specific nucleotide sequence, The present invention further includes a third primer having a sequence complementary or homologous to a portion of the vector, located at the 5' end of the first or second primer, A promoter of an enzyme having RNA polymerase activity is further added to the 5' end of either the first or the second primer, and The primer set wherein the combination of the first and second primers is one of the combinations shown in (1) to (7) below; (1) First primer: Oligonucleotide that can specifically hybridize with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 17 under stringent conditions, Second primer: Oligonucleotide that can specifically hybridize with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 48 under stringent conditions, (2) First primer: an oligonucleotide that can specifically hybridize with the complementary sequence of the base sequence described in SEQ ID NO: 8 under stringent conditions; Second primer: an oligonucleotide consisting of the base sequence described in SEQ ID NO: 23 or 25. (3) First primer: an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 10; second primer: an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 21 or 24 (4) First primer: an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 12; second primer: an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 47 (5) First primer: an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 14; second primer: an oligonucleotide consisting of the nucleotide sequence set forth in any of SEQ ID NOs: 26, 27, and 30 (6) First primer: an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 16; second primer: an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 29 (7) First primer: an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 18; second primer: an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 48

[0014] Further, a second aspect of the present invention is the primer set according to the first aspect, wherein the third primer is an oligonucleotide further having a promoter of an enzyme having RNA polymerase activity added to the 5'-end side thereof.

[0015] Furthermore, a third aspect of the present invention is a method for detecting an AAV vector using a probe capable of specifically hybridizing under stringent conditions with a nucleic acid containing a specific nucleotide sequence or a complementary sequence of the specific nucleotide sequence, which is amplified by the primer set according to the first or second aspect, with a part of the nucleic acid.

[0016] The present invention will be described in detail below.

[0017] In this invention, the specific nucleotide sequence refers to the nucleotide sequence of the AAV vector from the 3' end of the complementary region with the first primer to the 5' end of the homologous region with the second primer. In other words, in this invention, nucleic acids containing the specific nucleotide sequence or the complementary sequence of the specific nucleotide sequence are amplified.

[0018] In the present invention, a complementary sequence refers to a sequence that can specifically hybridize under stringent conditions, and a homologous sequence refers to a sequence that can specifically hybridize to the complementary sequence of a specific base sequence under stringent conditions. An example of "stringent conditions" here is a condition in which so-called specific hybrids are formed and nonspecific hybrids are not formed. To give one example, this is a condition in which polynucleotides with high homology (e.g., identity or similarity), for example, polynucleotides with 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more homology, hybridize, and polynucleotides with lower homology do not hybridize. While not limited to these, specific hybridization conditions include those at 42°C in the presence of 50% (v / v) formamide, 0.1% (w / v) bovine serum albumin, 0.1% (w / v) Ficol (trade name), 0.1% (w / v) polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate, as well as the nucleic acid amplification conditions described in the examples of this specification. Furthermore, examples of washing conditions include washing once, preferably two to three times, at a salt concentration and temperature equivalent to stringent conditions such as 60°C, 1×SSC (Saline Sodium Citrate Buffer), 0.1% (w / v)SDS (Sodium Dodecyl Sulfate), preferably 0.1×SSC, 0.1% (w / v)SDS, more preferably 65°C, 0.1×SSC, 0.1% (w / v)SDS, and more preferably 68°C, 0.1×SSC, 0.1% (w / v)SDS.Furthermore, those skilled in the art can easily obtain the first primer of the present invention (an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of any of the nucleotide sequences described in SEQ ID NOs: 8, 10, 12, 14, 16, 17, and 18) by referring to the description herein and Molecular Cloning (Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press, Woodbury, NY 2001), etc.

[0019] The primer set of the present invention is characterized in that the combination of a first primer having a sequence complementary to a portion of a specific nucleotide sequence of the AAV vector and a second primer having a sequence homologous to the portion of the specific nucleotide sequence is one of the following combinations (1) to (7). The first primer may have substitutions, deletions, additions, or modifications to the nucleotide sequence, as long as it can hybridize with the target nucleotide sequence sufficiently specifically and efficiently under the stringent conditions described above. The length of the first primer can be set arbitrarily, but is preferably 10 nucleotides or more. (1) First primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 17 (the nucleotide sequence from position 133 to 150 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). Second primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 48 (the nucleotide sequence from the 11th to the 38th nucleotide of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). (2) First primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 8 (the nucleotide sequence from position 525 to 542 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). Second primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 23 (from the nucleotide sequence described in SEQ ID NO: 37, from nucleotides 291 to 308) or SEQ ID NO: 25 (from the nucleotide sequence described in SEQ ID NO: 37, from nucleotides 201 to 218). (3) First primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 10 (nucleotide sequences from position 441 to 458 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). Second primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 21 (from nucleotides 381 to 398 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37) or SEQ ID NO: 24 (from nucleotides 275 to 292 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). (4) First primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 12 (nucleotide sequences from position 385 to 402 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). Second primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 47 (the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 11 to the 308th nucleotide). (5) First primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 14 (nucleotide sequences from position 301 to 318 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). Second primer: Oligonucleotides consisting of any of the nucleotide sequences described in SEQ ID NO: 26 (from the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 196 to 213), 27 (from the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 111 to 128), and 30 (from the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 11 to 28). (6) First primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 16 (the nucleotide sequence from position 189 to 206 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). Second primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 29 (the nucleotide sequence from the 21st to the 38th nucleotide of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). (7) First primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 18 (nucleotide sequences from position 105 to 122 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37). Second primer: Oligonucleotides that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 48. A preferred embodiment of the second primer in (1) and (7) above is an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 29 or 30. A preferred embodiment of the second primer in (4) above is an oligonucleotide consisting of the nucleotide sequence described in any of SEQ ID NOs: 23, 24, 25, 26, 27, 28 (from the 105th to the 122nd nucleotide of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 37), 29, and 30.

[0020] The primer set of the present invention is characterized by further adding a promoter of an enzyme having RNA polymerase activity to the 5' end of either the first or second primer described above. The promoter to be added may be one that corresponds to an enzyme having RNA polymerase activity used for RNA amplification (for example, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, which are commonly used in the field of molecular biology). Furthermore, a transcription start region known to affect transcription efficiency may be added to the promoter. As a specific example of a promoter (T7 promoter) to be added to the 5' end of the primer when T7 RNA polymerase is used as the RNA polymerase for RNA amplification, an oligonucleotide consisting of the nucleotide sequence described in Sequence ID No. 38 can be mentioned.

[0021] Furthermore, the primer set of the present invention is characterized by further comprising a third primer located at the 5' end of the first or second primer described above, having a sequence complementary or homologous to a portion of the AAV vector. When the third primer is located at the 5' end (3' end in the AAV vector) of the first primer, it becomes an oligonucleotide having a base sequence complementary to a portion of the vector. When the third primer is located at the 5' end (5' end in the AAV vector) of the second primer, it becomes an oligonucleotide having a base sequence homologous to a portion of the vector. The base length from the third primer to the first or second primer can be determined as appropriate, for example, from the 5' end of the third primer to the 5' end of the first or second primer. The length up to this point should be 450 bases or less. The third primer is the nucleic acid amplification primer of the present invention. Multiple types may be added to the system, and the 5' end of each may contain an enzyme with RNA polymerase activity. A promoter sequence may be added. Adding the promoter sequence to the third primer is preferable because the third primer also contributes to the nucleic acid amplification reaction, allowing for more rapid detection of the AAV vector. As an example of a third primer, If an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 17 is used as the first primer, then an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 46 (the nucleotide sequence from position 161 to 178 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37) is used. When using an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 8 as the first primer, the oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 7 (the nucleotide sequence from position 553 to 570 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37) is used. When using an oligonucleotide that can specifically hybridize with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 10 under stringent conditions as the first primer, the oligonucleotide that can specifically hybridize with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 9 (the nucleotide sequence from position 469 to 486 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37) under stringent conditions is used. When using an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 12 as the first primer, the oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 11 (the nucleotide sequence from position 413 to 430 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37) is used. When using an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 14 as the first primer, the oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 13 (the nucleotide sequence from position 329 to 346 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37) is used. When using an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 16 as the first primer, the oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 15 (the nucleotide sequence from position 217 to 234 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37) is used. When using an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 18 as the first primer, the oligonucleotide that can specifically hybridize under stringent conditions with any of the complementary sequences of the nucleotide sequences described in SEQ ID NOs: 7 to 17, the complementary sequence of the nucleotide sequence described in SEQ ID NO: 42 (nucleotide sequences from position 497 to 514 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37), the complementary sequence of the nucleotide sequence described in SEQ ID NO: 43 (nucleotide sequences from position 357 to 374 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37), the complementary sequence of the nucleotide sequence described in SEQ ID NO: 44 (nucleotide sequences from position 273 to 290 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37), the complementary sequence of the nucleotide sequence described in SEQ ID NO: 45 (nucleotide sequences from position 245 to 262 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 37), and the complementary sequence of the nucleotide sequence described in SEQ ID NO: 46 is used. They can be listed individually.

[0022] The detection of nucleic acids containing a specific nucleotide sequence or its complementary sequence in an AAV vector amplified using the primer set of the present invention can be performed using conventionally known nucleic acid detection methods. Specifically, (A) Methods using electrophoresis or liquid chromatography, (B) A hybridization method using an oligonucleotide probe that is labeled with a detectable label and is specifically hybridizable with a portion of the nucleic acid under stringent conditions. (C) A method using a fluorescent dye-labeled oligonucleotide probe designed to change its fluorescence properties by hybridizing with a portion of the nucleic acid under stringent conditions. These are some examples. Among them, method (C) is preferred because it allows the amplification and detection of the nucleic acid to be performed in a single step and in a sealed container.

[0023] Examples of fluorescent dye-labeled probes in (C) above include fluorescently labeled probes utilizing FRET (fluorescence resonance energy transfer), oligonucleotide probes labeled with intercalator fluorescent dyes, TaqMan (trade name) probes, and molecular beacon probes.

[0024] Examples of oligonucleotides constituting the probe include oligonucleotides that can specifically hybridize under stringent conditions with the nucleotide sequences described in any of the following sequences or their complementary sequences: SEQ ID NO: 31 (from the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 508 to 523), SEQ ID NO: 32 (from the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 409 to 424), SEQ ID NO: 33 (from the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 351 to 366), SEQ ID NO: 34 (from the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 258 to 273), SEQ ID NO: 35 (from the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 169 to 184), and SEQ ID NO: 36 (from the nucleotide sequence described in SEQ ID NO: 37, from the nucleotide sequence 66 to 81). The probe should be appropriately selected to correspond to a specific nucleotide sequence or its complementary sequence region of the amplified AAV vector.

[0025] When an oligonucleotide having a nucleotide sequence complementary to a portion of the AAV vector located at the 5' end relative to the first primer is used as the third primer constituting the primer set of the present invention, the amplification of a specific nucleotide sequence or its complementary sequence of the AAV vector using the primer set of the present invention can be carried out, for example, by the steps shown in (I) to (VIII) below. (I) A step of synthesizing DNA complementary to the nucleic acid using a first primer and an enzyme having DNA-dependent DNA polymerase activity from among the AAV vector nucleic acids. (II) Using an enzyme having strand displacement activity and a third primer, the DNA synthesized in (I) is converted into single-stranded DNA having a complementary region at the 3' end. (III) A step of synthesizing DNA homologous to the nucleic acid using a second primer, an enzyme having DNA-dependent DNA polymerase activity, and an enzyme having strand displacement activity. (IV) A step of synthesizing double-stranded DNA from the DNA synthesized in (III) to which the promoter sequence of the RNA polymerase present in either the first or second primer has been added, using the first primer and an enzyme having DNA-dependent DNA polymerase activity. (V) A step of synthesizing an RNA transcript from the double-stranded DNA synthesized in (4) using an enzyme having RNA polymerase activity corresponding to the promoter sequence. (VI) A step of synthesizing a cDNA complementary to the RNA transcript using a primer having a sequence complementary to the RNA transcript and an enzyme having RNA-dependent DNA polymerase activity. (VII) A step of converting the cDNA into single-stranded DNA using an enzyme having ribonuclease H activity. (VIII) A step of continuously synthesizing RNA transcripts using the single-stranded DNA obtained in (VII) above as a template.

[0026] The enzymes having DNA-dependent DNA polymerase activity used in steps (I), (III), and (IV), the enzyme having RNA-dependent DNA polymerase activity used in step (VI), and the enzyme having ribonuclease H activity used in step (VII) may be added individually or in various combinations, but it is preferable to use a retrovirus-derived reverse transcriptase that possesses the aforementioned activities. Examples of such reverse transcriptases include AMV (Avian Myeloblastosis Virus) reverse transcriptase, MMLV (Molony Murine Leukemia Virus) reverse transcriptase, RAV (Rous Associated Virus) reverse transcriptase, and HIV (Human Immunodeficiency Virus) reverse transcriptase, which are commonly used in the field of molecular biology. Among these, AMV reverse transcriptase and its derivatives are particularly preferred. These reverse transcriptases may be used individually or in combination of two or more. The amount of each enzyme added to the reaction system should be determined appropriately according to the reaction system, referring to the description herein and known nucleic acid amplification techniques.

[0027] The enzymes having strand displacement activity used in steps (II) and (III) above refer to enzymes that synthesize a new DNA strand while simultaneously dissociating hydrogen bonds in the template double-stranded DNA. If the enzyme having DNA-dependent DNA polymerase activity also functions as an enzyme having strand displacement activity, then such enzymes may be used as both DNA-dependent DNA polymerase and an enzyme having strand displacement activity. Examples of enzymes having strand displacement activity include E. coli DNA polymerase I, Klenow fragments of DNA polymerase I, T7 or T5 bacteriophage DNA polymerase, AMV reverse transcriptase, MMLV reverse transcriptase, HIV reverse transcriptase, Bsu DNA polymerase, Bst DNA polymerase, Aac DNA polymerase, phi29 DNA polymerase, 96-7 DNA polymerase, and Bca DNA polymerase. Among these, 96-7 DNA polymerase is preferred. Helicases can also be used as enzymes with chain displacement activity in this invention, as they produce a chain displacement effect, that is, the substitution of nucleic acids linked to the synthesis of nucleic acids with the same sequence. Furthermore, RecA and single-strand binding proteins can also be used as enzymes with chain displacement activity in this invention. These reverse transcriptases may be used individually or in combination of two or more. The amount of enzyme with chain displacement activity to be added should be appropriately determined according to the reaction system, referring to the description herein and known nucleic acid amplification techniques.

[0028] The reaction temperature in the method for amplifying a specific base sequence of an AAV vector according to the above-described embodiment depends on the heat resistance and activity of each enzyme used, as well as the Tm of the primer / probe, etc. However, if the enzymes used are AMV reverse transcriptase, T7 RNA polymerase, and 96-7 DNA polymerase, and the length of the primer / probe is between 10 and 30 bases, the reaction temperature can be set between 35°C and 65°C, and more preferably between 40°C and 50°C. [Effects of the Invention]

[0029] The primer set of the present invention can rapidly and sensitively amplify a specific nucleotide sequence or its complementary sequence of an adeno-associated virus (AAV) vector by hybridizing it to a specific nucleotide sequence (specific nucleotide sequence) and a portion of its complementary sequence, respectively.

[0030] The AAV vector detection method using the primer set of the present invention enables rapid, highly sensitive, and highly specific detection of the vector. [Examples]

[0031] The embodiments of the present invention will be described in detail below using examples, but these examples are for illustrating one form of implementation of the present invention and do not limit the present invention.

[0032] Example 1: Preparation of single-stranded DNA from an adeno-associated virus (AAV) vector model AAV vector model single-stranded DNA (hereinafter also simply referred to as "AAV single-stranded DNA") was synthesized using the Long ssDNA Preparation Kit for 3.0 kb (BioDynamics Laboratory) by the following method.

[0033] (1) Nucleic acid amplification was performed by PCR using pLSODN-3 (SEQ ID NO: 3) included in the kit as a template and oligonucleotides consisting of the nucleotide sequences described in SEQ ID NOs: 1 and 2 as primers. Specifically, a reaction solution with the following composition was prepared in 16 PCR tubes to a volume of 20 μL each with sterile water, heated at 98°C for 2 minutes, then subjected to PCR by repeating a cycle of 98°C for 10 seconds - 65°C for 5 seconds - 72°C for 35 seconds 40 times, and then heated at 72°C for 1 minute.

[0034] Composition of the reaction solution: 1 x PrimeSTAR MAX Premix (manufactured by Takara Bio) 0.2 μM forward primer (SEQ ID NO: 1) 0.2 μM Reverse Primer (SEQ ID NO: 2) 1ng template (SEQ ID NO: 3) (2) The obtained PCR products were subjected to electrophoresis using a 0.8% (w / v) agarose gel, and the band corresponding to the PCR product was excised and then purified using the QIAquick Gel Extraction Kit (QIAGEN).

[0035] (3) Nucleic acid amplification was performed by PCR using pAAV-CMV (SEQ ID NO: 4) as a template and oligonucleotides consisting of the nucleotide sequences described in SEQ ID NO: 5 and SEQ ID NO: 6 as primers. Specifically, a reaction solution with the following composition was prepared in 20 μL each of eight PCR tubes with sterile water, heated at 98°C for 2 minutes, then subjected to PCR by repeating the cycle of 98°C for 10 seconds - 60.4°C for 5 seconds - 72°C for 12 seconds 40 times, and then heated at 72°C for 1 minute. The amplified PCR product was purified in the same manner as in (2) above.

[0036] Composition of the reaction solution: 1 x PrimeSTAR MAX Premix (manufactured by Takara Bio) 0.2 μM forward primer (SEQ ID NO: 5) 0.2 μM Reverse Primer (SEQ ID NO: 6) 1ng template (SEQ ID NO: 4) (4) After mixing the purified PCR products obtained in (1) and (3) above, plasmids were synthesized using In Fusion reagent (manufactured by Takara Bio) in the method shown below. This method yielded plasmids containing the target insert (AAV single-stranded DNA). (4-1) In-Fusion HD Enzyme (manufactured by Takara Bio) was added to the purified PCR mixture solution and reacted at 50°C for 15 minutes, after which a portion of the solution was used to transform E. coli strain JM109. (4-2) The samples were spread on agar plates containing carbenicillin sodium and cultured at 37°C overnight. (4-3) Colonies containing the target insert were cultured again by colony direct PCR, and the plasmid was extracted. The base sequence was then analyzed to confirm the presence of the target insert. The base sequence of the insert (AAV single-stranded DNA) is shown in Sequence ID No. 37.

[0037] (5) Nicks were inserted into the insert sites of each plasmid obtained in (4) using the nicking enzymes Nt BspQI and Nb BsrDI (both from New England Biolabs).

[0038] (6) After ethanol precipitation, Denaturing Gel-Loading buffer (BioDynamics Laboratory) was added, and the single-stranded DNA was released and electrophoresed by agarose gel. The band thought to be single-stranded DNA was excised to obtain AAV single-stranded DNA.

[0039] (7) The concentration of the single-stranded DNA obtained in (6) above was determined by measuring the absorbance using NanoDrop (manufactured by Thermo Fisher Scientific), and the pseudocopy number was calculated from the base length.

[0040] Example 2: Copy number calculation of AAV single-stranded DNA by qPCR (1) The concentration of the AAV single-stranded DNA solution prepared in Example 1 was calculated by qPCR using the Positive Control contained in the AAVpro Titration Kit for Real Time PCR (manufactured by Takara Bio) as the standard concentration. Specifically, 2 μL of the Positive Control diluted to various concentrations and the single-stranded DNA or plasmid were added to 18 μL of a reaction mixture having the following composition, and qPCR was performed under the following conditions: heating at 25°C for 2 minutes, 53°C for 10 minutes, 95°C for 2 minutes, followed by 40 cycles of heating at 95°C for 3 seconds and heating at 60°C for 30 seconds.

[0041] Composition of reaction solution: Final concentration in 20 μL TaqPath qPCR Master Mix 900nM forward primer (SEQ ID NO: 40) 900nM Reverse Primer (SEQ ID NO: 41) 250nM TaqMan probe (SEQ ID NO: 39) (2) The concentrations of the single-stranded DNA and plasmid were calculated from the calibration curve of the Positive Control. In the following examples, the copy numbers used are those calculated in this example.

[0042] Example 3: Detection of AAV single-stranded DNA (Part 1) The AAV single-stranded DNA solution prepared in Example 1 and whose concentration was determined in Example 2 was used as a sample, and the single-stranded DNA contained in the sample was attempted to be amplified by the TRC method and detected by a molecular beacon probe.

[0043] (1) The AAV single-stranded DNA solution (hereinafter also referred to as "CC4 solution") is diluted 1.1 × 10⁻¹ using TE (Tris-EDTA) buffer containing 0.01% (w / v) sodium cholate and 0.01% (w / v) sodium azide. 5 To make it copy / 2μL The sample was diluted and used as the DNA sample.

[0044] (2) 10 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and then 2 μL of the DNA sample prepared in (1) above was added. Note that the first primer and the second primer The imer has a T7 promoter at its 5' end, which is the nucleotide sequence described in SEQ ID NO: 38. It has several oligonucleotides attached.

[0045] Composition of the reaction solution: The concentration is the final concentration after the addition of the initiator (in 20 μL), as described below. 60mM Tris-HCl buffer (pH8.65) 0.3mM each dATP, dCTP, dGTP, dTTP 3.0mM each ATP, CTP, GTP, UTP 3.4mM ITP 67 mM Trehalose 75nM Molecular Beacon probe (one of SEQ ID NOs. 31 to 36, synthesized by Integrated DNA Technologies) 1.0 μM First primer (one of SEQ ID NOs: 8, 10, 12, 14, 16, 17, and 18) 0.1 μM third primer (SEQ ID NOs: 7, 9, 11, 13, 15, 16 and 1) (One of the 7) 1.0 μM Second primer (any of SEQ ID NOs. 19 to 30) 1.33U 96-7 DNA polymerase 4.3U AMV reverse transcriptase 95U T7 RNA polymerase (3) After the above reaction solution was kept warm at 46°C for 3 minutes, 8 μL of an initiator having the following composition was added.

[0046] Initiator composition: Concentration is the final concentration after adding the initiator (in 20 μL). 19.0 mM magnesium chloride 95.0 mM potassium chloride 3.8% (w / v) Glycerol 10.5% (v / v) DMSO (4) Subsequently, using a temperature-controlled fluorescence spectrophotometer capable of directly measuring the PCR tubes, the reaction was carried out at 46°C, and the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm) was measured over time for 30 minutes.

[0047] (5) With the addition of the initiator set as 0 minutes, primer combinations (primer sets) in which the difference between the fluorescence intensity ratio of the reaction solution after 30 minutes (the value obtained by dividing the fluorescence intensity value at a predetermined time by the background fluorescence intensity value) and the fluorescence intensity ratio when a negative control (0 copy) was measured under similar conditions exceeded 0.5 were judged to be "detectable".

[0048] The results are shown in Table 1. When an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 8 is used as the first primer, and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 23 or 25 is used as the second primer, When an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 10 is used as the first primer, and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 21 or 24 is used as the second primer, When an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 12 is used as the first primer, and an oligonucleotide consisting of the nucleotide sequence described in any of SEQ ID NOs: 23 to 30 is used as the second primer, When an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 14 is used as the first primer, and an oligonucleotide consisting of the nucleotide sequence described in any of SEQ ID NOs: 26, 27, and 30 is used as the second primer, When an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 16 is used as the first primer, and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 29 is used as the second primer, When an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 17 is used as the first primer, and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 29 or 30 is used as the second primer, and It can be seen that AAV single-stranded DNA can be detected when an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 18 is used as the first primer, and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 29 or 30 is used as the second primer.

[0049] [Table 1]

[0050] Example 4: Detection of AAV single-stranded DNA (Part 2) (1) Dissolve the CC4 solution in 1.1 × 10⁻¹⁶ solution using TE buffer containing 0.01% (w / v) sodium cholate and 0.01% (w / v) sodium azide. 5 Copy / 2μL or 1.1×10 4 The samples were diluted to a copy / 2μL ratio and used as DNA samples.

[0051] (2) 10 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and then 2 μL of the DNA sample prepared in (1) above was added. The first primer had an oligonucleotide consisting of the base sequence described in Sequence ID No. 38, which is the T7 promoter, attached to its 5' end.

[0052] Composition of the reaction solution: The concentration is the final concentration after the addition of the initiator (in 20 μL), as described below. 60mM Tris-HCl buffer (pH8.65) 0.3mM each dATP, dCTP, dGTP, dTTP 2.75mM each ATP, CTP, GTP, UTP 3.4mM ITP 67 mM Trehalose 100nM Molecular Beacon probe (SEQ ID NO: 36) 1.0 μM First primer (SEQ ID NO: 18) 0.1 μM third primer (one of SEQ ID NOs. 7-17 and 42-46) 1.0 μM Second primer (SEQ ID NO: 30) 1.33U 96-7 DNA polymerase 1.42U AMV reverse transcriptase 95U T7 RNA polymerase (3) After the above reaction solution was kept warm at 46°C for 3 minutes, 8 μL of an initiator having the composition described in Example 3(3) was added.

[0053] (4) Measurements were taken for 30 minutes using the same method as in Example 3(4). With the addition of the initiator set as 0 minutes, the detection time was defined as the time when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the background fluorescence intensity ratio) exceeded 2.8.

[0054] (5) A combination of primers (primer sets) that could detect DNA samples at each concentration and where the negative control (0 copies) was not detected (fluorescence intensity ratio less than 2.8 after 30 minutes of measurement) or where the value obtained by dividing the fluorescence intensity value of the DNA sample after 30 minutes of measurement by the fluorescence intensity value of the negative control after 30 minutes of measurement (S / N ratio) was 7 or higher was determined to be "detectable", and all other combinations were determined to be "undetectable".

[0055] 1.1 × 10⁶ DNA sample 5 Table 2 shows the results when using CC4 solution at a rate of 1.1 × 10⁻¹⁰ / 2 μL. 4 The results when using CC4 solution at a copy / 2 μL are shown in Table 3. Regardless of which of the oligonucleotide sequences examined was used as the third primer, the result was 1.1 × 10⁻⁶. 5 It can be seen that the copy of AAV single-stranded DNA can be detected within 6 minutes (Table 2). On the other hand, 1.1 × 10 4 Copies of AAV single-stranded DNA could be detected when an oligonucleotide consisting of any of the nucleotide sequences described in SEQ ID NOs. 7 to 17 and 42 to 45 was used as a third primer (Table 3).

[0056] [Table 2]

[0057] [Table 3]

[0058] Example 5: Detection of AAV single-stranded DNA (Part 3) (1) Dissolve the CC4 solution in 1.1 × 10⁻¹⁶ solution using TE buffer containing 0.01% (w / v) sodium cholate and 0.01% (w / v) sodium azide.5 The sample was diluted to a copy / 2μL ratio and used as the DNA sample.

[0059] (2) 10 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and 2 μL of the DNA sample prepared in (1) above was added. The first primer had an oligonucleotide consisting of the base sequence described in Sequence ID No. 38, which is the T7 promoter, attached to its 5' end. The third primer used either an oligonucleotide with the T7 promoter attached to its 5' end or an oligonucleotide without the T7 promoter attached.

[0060] Composition of the reaction solution: The concentration is the final concentration after the addition of the initiator (in 20 μL), as described below. 60mM Tris-HCl buffer (pH8.65) 0.3mM each dATP, dCTP, dGTP, dTTP 2.75mM each ATP, CTP, GTP, UTP 3.4mM ITP 67 mM Trehalose 100nM Molecular Beacon probe (SEQ ID NO: 36) 1.0 μM First primer (SEQ ID NO: 17) 0.1 μM Third Primer (SEQ ID NO: 46) 1.0 μM Second primer (SEQ ID NO: 29 or 30) 1.33U 96-7 DNA polymerase 1.42U AMV reverse transcriptase 95U T7 RNA polymerase (3) After the above reaction solution was kept warm at 46°C for 3 minutes, 8 μL of an initiator having the composition described in Example 3(3) was added.

[0061] (4) Measurements were taken for 30 minutes using the same method as in Example 3(4). With the addition of the initiator set as 0 minutes, the detection time was defined as the time when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the background fluorescence intensity ratio) exceeded 2.8.

[0062] The results are shown in Table 4. Adding the T7 promoter sequence to the 5' end of the third primer shortened the detection time. This indicates that the third primer can also contribute to the nucleic acid amplification reaction.

[0063] [Table 4]

Claims

1. A primer set for amplifying nucleic acids containing the specific nucleotide sequence or a complementary sequence of the specific nucleotide sequence, comprising: a first primer having a sequence complementary to a portion of the specific nucleotide sequence of an adeno-associated virus vector; and a second primer having a sequence homologous to a portion of the specific nucleotide sequence. The method for amplifying the nucleic acid is the TRC method using Avian Myeloblastosis Virus reverse transcriptase. The present invention further includes a third primer located at the 3' end of the vector relative to the first primer and having a complementary arrangement to a portion of the vector, or located at the 5' end of the vector relative to the second primer and having a homologous arrangement to a portion of the vector. A promoter of an enzyme having RNA polymerase activity is further added to the 5' end of either the first or the second primer, and The primer set wherein the combination of the first, second, and third primers is one of the combinations shown in (1) to (7) below; (1) First primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 17, Second primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 29 or 30, Third primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 46, (2) First primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 8, Second primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 23 or 25, Third primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 7, (3) First primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 10, Second primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 21 or 24, Third primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 9 (4) First primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 12, Second primer: Oligonucleotide consisting of the nucleotide sequence described in any of SEQ ID NOs: 23 to 30, Third primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 11, (5) First primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 14, Second primer: Oligonucleotide consisting of the nucleotide sequence described in any of SEQ ID NOs: 26, 27, and 30, Third primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 13, (6) First primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 16, Second primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 29, Third primer: Oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 15, (7) First primer: oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 18, second primer: oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 29 or 30, third primer: oligonucleotide consisting of the nucleotide sequence described in any of SEQ ID NOs: 7 to 17 and SEQ ID NOs: 42 to 46.

2. A method for detecting an adeno-associated virus vector using a nucleic acid amplified with the primer set described in claim 1, which includes a specific base sequence or a complementary sequence of the specific base sequence, and a probe that can specifically hybridize a portion of the nucleic acid under stringent conditions.

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