Synthetic production of single-stranded adeno associated viral DNA vectors
AssigneeGeneration Bio Co.
InventorsOzan Alkan, Robert Michael Kotin, Douglas Anthony Kerr, Russell Monds, Carolyn Pelletier, Matthew Stanton
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Abstract
The present application discloses methods for synthetic production and cell-free synthesis of single stranded adeno-associated virus (AAV) vectors, for delivery and expression of a transgene in host cells. The present invention also relates to an in vitro process for production of closed-ended DNA vectors and corresponding single stranded AAV DNA vector products synthesized from the closed-ended DNA vectors having nicks.
Claims23
A cell-free method of producing a single-stranded AAV vector using a double-stranded DNA construct, the method comprising:
- providing a double-stranded DNA construct comprising an expression cassette, wherein the expression cassette comprises a promoter linked to a transgene, and wherein said double-stranded DNA construct comprises overhang sequences on its 5′ and 3′ ends;
- providing a first inverted terminal repeat (ITR) with an overhang sequence that is a complement to the overhang sequence on the 5′ end of the double-stranded DNA construct;
- providing a second ITR with an overhang sequence that is a complement to the overhang sequence on the 3′ end of the double-stranded DNA construct;
- contacting said double-stranded DNA construct comprising the expression cassette with said first ITR, said second ITR, and a ligase, wherein ligation of the first ITR and the second ITR with the double-stranded DNA construct comprising the expression cassette produces a gapped closed-ended DNA vector having a first gap 5′ upstream of the expression cassette and a second gap 3′ downstream of the expression cassette, and
- removing one strand from the gapped closed-ended DNA vector by contacting the gapped closed-ended DNA vector with a strand-specific exonuclease, wherein the strand-specific exonuclease is T7 exonuclease or ExoV, thereby producing a single-stranded AAV vector.
A cell-free method of producing a single-stranded AAV vector, the method comprising:
- a) providing a double-stranded DNA construct comprising an expression cassette, wherein said double-stranded DNA construct comprises overhangs on its 5′ and 3 ends;
- b) providing a first inverted terminal repeat (ITR) with an overhang sequence that is a complement to the overhang sequence on the 5′ end of the said double-stranded DNA construct (5′ ITR);
- c) providing a second ITR with an overhang sequence that is a complement to the overhang sequence on the 3′ end of the said double-stranded DNA construct (3′ ITR);
- d) contacting said double-stranded DNA construct with said first ITR, said second ITR, and a ligase, wherein ligation produces a nicked closed-ended DNA (neDNA) having two gaps, a first gap present 5′ upstream of said expression cassette between a DD′ stem region of said 5′ ITR and said expression cassette, and a second gap present 3′ downstream of said expression cassette between said expression cassette and a DD′ stem region of said 3′ ITR;
- and
- e) contacting said neDNA with a strand-specific exonuclease capable of catalyzing removal of nucleotides from the nicked strand of the neDNA in the 5′ to 3′ direction and/or 3′ to 5′ direction, removing the entire sequence between the first gap and the second gap encompassing 5′ and 3′ ends of the expression cassette, wherein the strand-specific exonuclease is T7 exonuclease or ExoV, wherein said nicked strand is a sense strand of the expression cassette,
- thereby producing a single-stranded AAV vector.
Dependencies are inferred from the claim text.