A vector, a method for producing linear covalent closed DNA using the same, a method for producing a parvovirus vector, and a method for producing parvovirus vector-producing cells.

Abstract

This vector has a nucleotide sequence encoding a protelomerase, a pair of nucleotide sequences that recognize the protelomerase, and a nucleotide sequence that is located between the pair of nucleotide sequences and that encodes a target protein. This method is for preparing a linear covalent bond closed DNA using the vector. This parvovirus vector preparing method comprises: a gene introduction step for introducing, into a first host, a vector that has a nucleotide sequence encoding a protelomerase, a pair of nucleotide sequences that recognize the protelomerase, and a nucleotide sequence that is located between the pair of nucleotide sequences and that encodes a protein; and a transfection step for transfecting a second host with a linear covalent bond closed DNA obtained in the gene introduction step. This cell is for producing a parvovirus vector that is obtained from linear covalent bond closed DNA and that causes expression of a nucleotide sequence encoding a protein. The proportion of the number of vector genomes having a sequence other than the nucleotide sequence encoding the protein with respect to the number of vector genomes having the nucleotide sequence encoding the protein as quantified using quantitative PCR, is not more than 10% in a culture supernatant of the parvovirus vector producing cells or is not more than 1% in a cell lysate of the parvovirus vector producing cells.

Claims

1. A pair of nucleic acid sequences recognized by protelomerase, A nucleic acid sequence located between the pair of nucleic acid sequences and encoding a protein, A vector characterized by having a nucleic acid sequence that encodes the protelomerase, located in a region other than between the pair of nucleic acid sequences having the nucleic acid sequence encoding the protein.

2. The vector according to claim 1, which is a vector for producing linear covalent closed DNA.

3. The vector according to claim 1 or 2, which is a double-stranded circular plasmid DNA vector.

4. The vector according to any one of claims 1 to 3, comprising a nucleic acid sequence for controlling the expression of the protelomerase.

5. A pair of nucleic acid sequences recognized by protelomerase, A nucleic acid sequence located between the pair of nucleic acid sequences and encoding a protein, A method for producing linear covalent closed DNA, characterized by a gene transfer step of introducing into a host a vector having a nucleic acid sequence that encodes the protelomerase and is located in a region other than between the pair of nucleic acid sequences having the nucleic acid sequence encoding the protein.

6. The method for producing linear covalent closed DNA according to claim 5, wherein the vector is a double-stranded circular plasmid DNA vector.

7. The method for producing linear covalent closed DNA according to claim 5 or 6, wherein the vector comprises a nucleic acid sequence for controlling the expression of the protelomerase.

8. A method for producing linear covalent closed DNA according to claim 7, further comprising an expression induction step of inducing the expression of the protelomerase after the gene introduction step.

9. A pair of nucleic acid sequences recognized by protelomerase, A nucleic acid sequence located between the pair of nucleic acid sequences and encoding a protein, A gene transfer step of introducing into a first host a vector having a nucleic acid sequence encoding the protelomerase, located in a region other than between the pair of nucleic acid sequences having the nucleic acid sequence encoding the protein, and A method for producing a parvovirus vector, characterized by including a transfection step of transfecting a second host with linear covalent closed DNA obtained in the gene transfer step.

10. The method for producing a parvovirus vector according to claim 9, wherein the nucleic acid sequence encoding the protein is one of the following: a nucleic acid sequence encoding a target protein to be introduced into the parvovirus vector produced by the method described above, a nucleic acid sequence encoding a packaging protein, and a nucleic acid sequence encoding a helper protein.

11. In the gene introduction step, The pair of nucleic acid sequences recognized by the protelomerase, A nucleic acid sequence located between the pair of nucleic acid sequences and encoding the target protein, A vector having a nucleic acid sequence encoding the protelomerase located in a region other than between the pair of nucleic acid sequences having the nucleic acid sequence encoding the target protein is introduced into a first host. The pair of nucleic acid sequences recognized by the protelomerase, A nucleic acid sequence located between the pair of nucleic acid sequences and encoding a packaging protein, A vector having a nucleic acid sequence encoding the protelomerase, located in a region other than between the pair of nucleic acid sequences having the nucleic acid sequence encoding the packaging protein, is introduced into the first host. The pair of nucleic acid sequences recognized by the protelomerase, A nucleic acid sequence located between the pair of nucleic acid sequences and encoding a helper protein, A vector having a nucleic acid sequence encoding the protelomerase, located in a region other than between the pair of nucleic acid sequences having the nucleic acid sequence encoding the helper protein, is introduced into the first host, and A method for producing a parvovirus vector according to claim 9 or 10, wherein in the transfection step, three linear covalent closed DNAs obtained in the gene transfer step, comprising a linear covalent closed DNA containing a nucleic acid sequence encoding the target protein, a linear covalent closed DNA containing a nucleic acid sequence encoding the packaging protein, and a linear covalent closed DNA containing a nucleic acid sequence encoding the helper protein, are transfected into a second host.

12. In the gene introduction step, The pair of nucleic acid sequences recognized by the protelomerase, A nucleic acid sequence located between the pair of nucleic acid sequences and encoding the target protein, A vector having a nucleic acid sequence encoding the protelomerase located in a region other than between the pair of nucleic acid sequences having the nucleic acid sequence encoding the target protein is introduced into a first host. The pair of nucleic acid sequences recognized by the protelomerase, A nucleic acid sequence located between the pair of nucleic acid sequences and encoding a packaging protein and a helper protein, A vector having a nucleic acid sequence encoding the protelomerase, located in a region other than between the pair of nucleic acid sequences encoding the packaging protein and the helper protein, is introduced into the first host, and A method for producing a parvovirus vector according to claim 9 or 10, wherein in the transfection step, two linear covalent closed DNAs, one containing a nucleic acid sequence encoding the target protein obtained in the gene transfer step, and the other containing a nucleic acid sequence encoding the packaging protein and a nucleic acid sequence encoding the helper protein, are transfected into a second host.

13. A method for producing a parvovirus vector according to any one of claims 9 to 12, wherein the vector is a double-stranded circular plasmid DNA vector.

14. A method for producing a parvovirus vector according to any one of claims 9 to 13, wherein the vector comprises a nucleic acid sequence for controlling the expression of the protelomerase.

15. A method for producing a parvovirus vector according to claim 14, further comprising an expression induction step of inducing the expression of the protelomerase after the gene introduction step.

16. A method for producing a parvovirus vector according to any one of claims 10 to 12, wherein the nucleic acid sequence encoding the packaging protein includes a Rep gene or a Cap gene.

17. A method for producing a parvovirus vector according to any one of claims 10 to 12, wherein the nucleic acid sequence encoding the helper protein includes an adenovirus helper gene.

18. A method for producing a parvovirus vector according to any one of claims 9 to 17, wherein, in the culture supernatant of the second host transfected with the linear covalent closed DNA obtained in the gene transfer step, the number of vector genomes of sequences other than the nucleic acid sequence encoding the protein, as quantified by quantitative PCR, is 10% or less of the number of vector genomes of the nucleic acid sequence encoding the protein, or, in the cell lysate of the second host transfected with the linear covalent closed DNA obtained in the gene transfer step, the number of vector genomes of sequences other than the nucleic acid sequence encoding the protein, as quantified by quantitative PCR, is 1% or less of the number of vector genomes of the nucleic acid sequence encoding the protein.

19. A method for producing a parvovirus vector according to any one of claims 9 to 18, wherein the parvovirus vector is an adeno-associated virus vector.

20. A method for producing parvovirus vector-producing cells, A pair of nucleic acid sequences recognized by protelomerase, A nucleic acid sequence located between the pair of nucleic acid sequences and encoding a protein, A gene transfer step of introducing into a first host a vector having a nucleic acid sequence encoding the protelomerase, located in a region other than between the pair of nucleic acid sequences having the nucleic acid sequence encoding the protein, and A method for producing parvovirus vector-producing cells, characterized by including a transfection step of transfecting a second host with linear covalent closed DNA obtained in the gene transfer step.

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