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 modified single-stranded DNA molecules, as well as their cell-free methods of synthesis and their use as therapeutic agents.

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 disclosure provides cleavable closed-ended DNA (ceDNA). In some embodiments, the cleavable ceDNA may be used as donor or repair template for editing of a target sequence in the genome.

Abstract: This disclosure provides methods for scalable and high-purity 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 cell-free synthesis includes digesting a double-stranded DNA construct with at least one restriction endonuclease that is capable of cleaving the construct at cleavage sites, which are distinct from the recognition sites, to release an insert having unique overhangs that regulate the high specificity of the subsequent ligation reaction. The insert is then ligated with inverted terminal repeat (ITR) oligonucleotides to form the closed-ended DNA vector. Corresponding DNA vectors prepared by these methods and related products as well other base and intermediate vectors and constructs associated with the methods are also provided in this disclosure.

Abstract: The present disclosure provides liver-specific expression cassettes, vectors comprising the expression cassettes, and uses in gene therapy, particularly liver-directed gene therapy.

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.