US 12,442,015 B2
Closed-ended DNA vectors obtainable from cell-free synthesis and process for obtaining ceDNA vectors
Ozan Alkan, Cambridge, MA (US); Robert Michael Kotin, Cambridge, MA (US); Matthew Stanton, Cambridge, MA (US); Douglas Anthony Kerr, Cambridge, MA (US); and Carolyn Pelletier, Cambridge, MA (US)
Assigned to Generation Bio Co., Cambridge, MA (US)
Appl. No. 16/962,005
Filed by Generation Bio Co., Cambridge, MA (US)
PCT Filed Jan. 18, 2019, PCT No. PCT/US2019/014122
§ 371(c)(1), (2) Date Jul. 14, 2020,
PCT Pub. No. WO2019/143885, PCT Pub. Date Jul. 25, 2019.
Claims priority of provisional application 62/619,392, filed on Jan. 19, 2018.
Prior Publication US 2021/0071197 A1, Mar. 11, 2021
Int. Cl. C12N 15/85 (2006.01)
CPC C12N 15/85 (2013.01) [C12N 2800/105 (2013.01)] 16 Claims
 
1. A cell-free method of preparing a non-viral, linear and closed-ended DNA (ceDNA) vector comprising:
providing a first single-stranded inverted terminal repeat (ITR) molecule comprising a first ITR;
providing a second single-stranded ITR molecule comprising a second ITR, wherein the first ITR and the second ITR are synthetic;
providing a double-stranded polynucleotide comprising a double-stranded expression cassette sequence, wherein the double-stranded polynucleotide comprising the double-stranded expression cassette is obtained by excision from a double-stranded DNA construct; and
ligating the 5′ and 3′ ends of the first ITR molecule to a first end of the double-stranded polynucleotide and ligating the 5′ and 3′ ends of the second ITR molecule to a second end of the double-stranded polynucleotide to form the ceDNA vector, wherein:
the first ITR molecule comprises a first overhang region that is complementary to an overhang region on the first end of the double-stranded polynucleotide, and the second ITR molecule comprises a second overhang region that is complementary to an overhang region on the second end of the double-stranded polynucleotide;
each of the first overhang region and the second overhang region is located in an A-stem of the respective ITR; and
ITR D regions are present in the double-stranded expression cassette sequence;
thereby preparing the ceDNA vector without using cells.