Modulation of rep protein activity in closed-ended dna (cedna) production
AssigneeGeneration Bio Co.
InventorsRobert Michael Kotin, Anna Ucher, Ara Karl Malakian
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Abstract
Provided herein are methods for producing DNA vectors comprising incubating a population of cells harboring the vector polynucleotide encoding a heterologous nucleic acid operatively positioned between a first and a second AAV inverted terminal repeat DNA polynucleotide sequence (ITRs), with at least one of the ITRs having nucleotide sequences corresponding to AAV wild type ITR in the presence of only a single species of Rep protein having at least DNA binding and DNA nicking functionality, under conditions effective and for a time sufficient to induce production of the DNA within the cells and harvesting and isolating the resultant DNA with the ITRs from the cells.
Claims85
A DNA vector obtained from a vector polynucleotide, wherein the vector polynucleotide encodes a heterologous nucleic acid operatively positioned between a first inverted terminal repeat DNA polynucleotide sequence (ITR) and a second ITR, wherein at least one of the first ITR and the second ITR comprises a nucleotide sequence corresponding to an AAV Rep binding sequence to induce replication of the DNA vector in a cell in the presence of a single species of Rep protein, the DNA vector being obtainable from a method comprising the steps of:
- a. incubating a population of cells harboring the vector polynucleotide, which is devoid of viral capsid coding sequences, in the presence of a single species of Rep protein having at least DNA binding and DNA nicking functionality, under conditions effective and for a time sufficient to induce production of the DNA vector within the cells, wherein the cells do not comprise viral capsid coding sequences, and wherein no other species of Rep proteins are present;
- and
- b. harvesting and isolating the resultant DNA vector from the cells.
A DNA vector obtained from a vector polynucleotide, wherein the vector polynucleotide encodes a heterologous nucleic acid operatively positioned between two different inverted terminal repeat sequences (ITRs), wherein at least one of the ITRs is a functional ITR comprising a functional terminal resolution site and a Rep binding site;
- the presence of a single species of Rep protein inducing replication of the vector polynucleotide and production of the DNA vector in a cell, the DNA vector being obtainable from a method comprising the steps of:
- a. incubating a population of cells harboring the vector polynucleotide, which is devoid of viral capsid coding sequences, in the presence of a single species of Rep protein that has at least DNA binding and DNA nicking functionality under conditions effective and for time sufficient to induce production of the DNA vector within the cells, wherein the cells do not comprise any nucleic acid encoding Rep52 or Rep40 within the cells, wherein no other species of Rep are present in the cell;
- and
- b. harvesting and isolating the DNA vector from the cells.
A polynucleotide for generating a DNA vector comprising a nucleotide sequence encoding a single species of Rep protein amino acid sequence that has at least DNA binding and DNA nicking functionality operatively linked to at least one expression control sequence.
A method of producing a DNA vector, the method comprising
- contacting a cell with:
- (1) a nucleotide sequence encoding a single species of AAV Rep protein (Rep78 and/or Rep68) that has at least DNA binding and DNA nicking functionality, linked to at least one expression control sequence, wherein the cell does not express any other species of Rep protein and is not contacted with any other species of Rep protein;
- (2) a double-stranded DNA construct comprising:
- an expression cassette;
- a first ITR on the upstream (5′-end) of the expression cassette;
- and
- a second ITR on the downstream (3′-end) of the expression cassette, and
- (3) harvesting the DNA vector.
A kit for producing a DNA vector, the kit comprising:
- an expression construct comprising at least one restriction site for insertion of at least one heterologous nucleotide sequence, or regulatory switch, or both, the at least one restriction site operatively positioned between asymmetric inverted terminal repeat sequences (asymmetric ITRs), wherein at least one of the asymmetric ITRs comprises a functional terminal resolution site and a Rep binding site;
- and
- a vector comprising a polynucleotide sequence that encodes a single species of Rep protein, wherein the vector is suitable for expressing the single species of Rep protein in an insect cell.
A cell comprising:
- a nucleotide sequence encoding a single species of AAV Rep protein (Rep78 and/or Rep68) that has at least DNA binding and DNA nicking functionality, operably linked to at least one expression control sequence, wherein the cell does not express any other parvovirus Rep protein (Rep52 or Rep40) and is not contacted with any other species of Rep protein;
- and optionally a double-stranded DNA construct comprising an expression cassette;
- a first ITR on the upstream (5′-end) of the expression cassette;
- and a second ITR on the downstream (3′-end) of the expression cassette.
Dependencies are inferred from the claim text.