Process for the production of closed linear DNA
The present invention provides a process for the production of a closed linear DNA comprising the steps of (a) providing a DNA template comprising a DNA sequence of interest; (b) amplifying DNA from the DNA template of step (a) wherein the amplification is primed with a primase/polymerase enzyme; (c) generating a closed linear DNA with the amplified DNA produced in step (b); and (d) purifying the closed linear DNA produced in step (c). The invention also provides a closed linear DNA obtainable according to the process of the invention, a pharmaceutical composition comprising a therapeutically effective amount of the closed linear DNA of the invention, and a concatameric DNA comprising repeats of a DNA sequence of interest.
1 . A process for the production of a closed linear DNA comprising the steps of:
a) providing a closed linear DNA template comprising a double-stranded middle segment comprising a DNA sequence of interest flanked by two single-stranded loops, wherein the single-stranded loops do not comprise a primase/polymerase priming site;
b) amplifying DNA from the closed linear DNA template of step (a) wherein the amplification is primed with a primase/polymerase enzyme of SEQ ID NO:1;
c) generating a closed linear DNA with the amplified DNA produced in step (b); and
d) purifying the closed linear DNA produced in step (c).
2 . The process according to claim 1 , wherein the amplification performed in step (b) is a rolling-circle amplification.
3 . The process according to claim 1 , wherein the amplification of step (b) is carried out with a strand displacement DNA polymerase.
4 . The process according to claim 1 , wherein step (a) is performed by contacting a plasmid vector comprising at least two restriction sites flanking the DNA sequence of interest with at least one restriction enzyme thereby producing open double stranded DNA containing the DNA sequence of interest, and attaching single stranded DNA adaptors to both ends of the open double stranded DNA containing the DNA sequence of interest.
5 . The process according to claim 1 , wherein step (a) is performed by contacting a plasmid vector comprising at least two protelomerase target sequences flanking the DNA sequence of interest with a protelomerase.
6 . The process according to claim 1 , wherein the amplified DNA resulting from step (b) is a concatameric DNA comprising repeats of the DNA sequence of interest, wherein each one of the repeated DNA sequences of interest is flanked by restriction sites and/or protelomerase target sequences.
7 . The process according to claim 6 ,
wherein when the concatameric DNA comprises repeats of the DNA sequence of interest flanked by at least restriction sites, then step (c) is performed by:
(c.1) contacting the concatameric DNA with at least one restriction enzyme thereby producing a plurality of open double stranded DNA fragments each containing the DNA sequence of interest, and
(c.2) attaching a single stranded DNA adaptor to each of both ends of the open double stranded DNA fragments.
8 . The process according to claim 6 , wherein when the concatameric DNA comprises repeats of the DNA sequence of interest flanked by at least a protelomerase target sequence, then step (c) is performed by contacting the concatameric DNA with a protelomerase.
9 . The process according to claim 1 ,
wherein step (a) is performed by contacting a plasmid vector comprising two protelomerase target sequences flanking at least two restriction sites flanking the DNA sequence of interest with a protelomerase; and
wherein step (c) is performed by:
(c.1) contacting the concatameric DNA with at least one restriction enzyme thereby producing a plurality of open double stranded DNA fragments each containing the DNA sequence of interest, and
(c.2) attaching single stranded DNA adaptors to both ends of the open double stranded DNA fragments.
10 . The process according to claim 1 ,
wherein step (a) is performed by contacting a plasmid vector comprising at least two restriction sites flanking the DNA sequence of interest and no protelomerase target sites with at least one restriction enzyme thereby producing open double stranded DNA containing the DNA sequence of interest, and attaching single stranded DNA adaptors to both ends of the open double stranded DNA containing the DNA sequence of interest, with the proviso that the single stranded DNA adaptors do not contain protelomerase target sites; and
wherein step (c) is performed by:
(c.1) contacting the concatameric DNA with at least one restriction enzyme thereby producing a plurality of open double stranded DNA fragments each containing the DNA sequence of interest, and
(c.2) attaching a single stranded DNA adaptor to each of both ends of the open double stranded DNA fragments.
11 . The process according to claim 1 , wherein the sequence of interest comprises inverted terminal repeats (ITRs) and an expression cassette, wherein the expression cassette is flanked by the inverted terminal repeats (ITRs).
12 . The process according to claim 7 , wherein the single stranded DNA adaptor comprises modified oligonucleotides.
13 . The process according to claim 1 , which is a cell-free in vitro process.
14 . A closed linear DNA obtainable according to the process as defined in claim 1 .
15 . A pharmaceutical composition comprising a therapeutically effective amount of the closed linear DNA according to claim 14 and a pharmaceutically acceptable carrier or an excipient.