PROJECT SUMMARY/ ABSTRACT Bacterial plasmid manufacture is now a major bottleneck in viral gene therapy production workflows. Although supercoiled plasmid-based vectors are the current industry standard for transient transfection of packaging cells, minimized DNAs offer substantial safety and efficiency advantages. Currently, there is a dearth of technologies to produce packaging and payload DNAs in a completely synthetic, abacterial manner. In fact, no minimized DNA technology capable of scalable synthetic production of supercoiled and completely scarless DNAs exists. Supercoiling is the preferred topology for circular DNAs given that their performance in transient transfections is superior to other DNA forms. Minicircles or minivectors generated by recombination in bacteria are supercoiled but require extensive and expensive purification and yield a final product ‘scarred’ by a variable length prokaryotic sequence. Doggybone DNAs (dbDNAs) comprised of linear double stranded DNA with circularized single-stranded ends are produced synthetically but are not supercoiled. The long-term goal of this project is to develop an efficient bacteria- independent workflow to produce, at a commercially viable scale, completely vectorless supercoiled DNAs (VLSDNAs). This will be achieved using a new DNA assembly technology we have developed termed cyclic heteroduplex thermostable ligation assembly (CHTLA). The objectives of the work proposed here are to 1) optimize CHTLA reaction conditions to maximize supercoil production and eliminate byproducts; and 2) to produce VLSDNAs encoding all packaging, replication, and payload components to generate adeno-associated virus (AAV) particles loaded with a green fluorescent protein (GFP) reporter gene. Rolling circle amplification (RCA) of a standardized green fluorescent protein (GFP) gene expression cassette will be used to generate milligram quantities of overlapping and offset DNA precursors for CHTLA reactions. To maximize supercoiling of CHTLA products, a two-enzyme, one-pot system will be developed containing both thermostable DNA ligase and thermostable DNA gyrase activity. The work proposed here is highly innovative because it represents a substantial departure from the status quo by developing a robust new technology to produce, entirely in vitro, DNAs with a supercoiled topology that are comprised exclusively of the sequence of interest. VLSDNA versions of pHelper-Kan, pAAV-Rep- Cap, and pAAV ITR-GFP encoding adenovirus E1A, E1B, E2A, E4 and VA RNA open reading frames will be generated using standard as well as optimized conditions identified in Aim 1. VLSDNAs will be quantitatively compared to standard bacterially-sourced pHelper-Kan, pAAV-Rep-Cap, and pAAV ITR- GFP triple plasmid transfection by commercial collaborator. Upon completion of these Aims we will have determined optimal conditions to generate functional VLSDNAs at a scale that is commercially viable. In Phase 2, we will seek to further scale prod...