Abstract: Described herein are ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette comprises a codon optimized nucleic acid sequence encoding a PAH protein, in combination with particular promoter sequences and cis-regulatory elements. Further provided herein are methods and cell lines for reliable gene expression of PAH protein in vitro, ex vivo and in vivo using the ceDNA vectors. Also provided herein are methods and compositions comprising ceDNA vectors useful for the expression of PAH protein in a cell, tissue or subject, and methods of treatment of diseases with said ceDNA vectors expressing PAH protein. Such PAH protein can be expressed for treating disease, e.g., Phenylketonuria (PKU).

Abstract: The application describes ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette encodes a transgene encoding FVIII protein. Some ceDNA vectors further comprise cis-regulatory elements, including regulatory switches. Further provided herein are methods and cell lines for reliable gene expression of FVIII protein in vitro, ex vivo and in vivo using the ceDNA vectors. Provided herein are methods and compositions comprising ceDNA vectors useful for the expression of FVIII protein in a cell, tissue or subject, and methods of treatment of diseases with said ceDNA vectors expressing FVIII protein. Such FVIII protein can be expressed for treating disease, e.g., hemophilia A.

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.