Closed-ended DNA vectors obtainable from cell-free synthesis and process for obtaining ceDNA vectors

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.