Abstract: The present application discloses methods for synthetic production and cell-free synthesis of DNA vectors, particularly closed-ended linear DNA vectors having one or more gaps (e.g., nicked ceDNA vectors, “neDNA”) and adenoassociated-virus (AAV) vector which is single strand DNA having linear and continuous structure, for delivery and expression of a transgene in the host cell. The present invention also relates to an in vitro process for production of closed-ended DNA vectors, corresponding DNA vector products produced by the methods and uses thereof, and oligonucleotides and kits useful in the process of the present invention.

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.

Abstract: The application describes methods for synthetic synthesis and cell-free synthesis of DNA vectors, particularly closed-ended DNA vectors (e.g., ceDNA vectors) having linear and continuous structure for delivery and expression of a transgene. The present invention relates to an in vitro process for production of closed-ended DNA vectors, corresponding DNA vector products produced by the methods and uses thereof, and oligonucleotides and kits useful in the process of the invention. DNA vectors produced using the methods described herein are free from unwanted side effects due to contaminants introduced during production in cell lines, for example, bacterial or insect cell lines. Further provided herein are methods and cell lines for reliable gene expression in vitro, ex vivo and in vivo using the ceDNA vectors synthesized using the methods herein.