Abstract: Provided herein are methods for producing DNA vectors comprising incubating a population of cells harboring the vector polynucleotide encoding a heterologous nucleic acid operatively positioned between a first and a second AAV inverted terminal repeat DNA polynucleotide sequence (ITRs), with at least one of the ITRs having nucleotide sequences corresponding to AAV wild type ITR in the presence of only a single species of Rep protein having at least DNA binding and DNA nicking functionality, under conditions effective and for a time sufficient to induce production of the DNA within the cells and harvesting and isolating the resultant DNA with the ITRs from the cells.

Abstract: Provided herein are lipid nanoparticle formulations that comprise an ionizable lipid and non-viral, capsid-free DNA vectors with covalently-closed ends.

Abstract: CeDNA vectors having linear and continuous structure can be produced in high yields and used for effective transfer and expression of a transgene. ceDNA vectors comprise an expression cassette and two different ITR sequences derived from AAV genomes in a specified order. Some ceDNA vectors provided herein further comprise cis-regulatory elements and provide high gene expression efficiencies. Further provided herein are methods and cell lines for reliable and efficient production of the linear, continuous and capsid-free DNA vectors.