Synthetic circular supercoiled DNA: an alternative to plasmids for the production of lentiviral vectors for cell and gene therapies

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. Minicircles plasmids 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. Nanoplasmid and MiniVecTM constructs lack antibiotic selection markers but still require E. coli fermentation for production. Doggybone DNAs (dbDNAs) comprised of linear double stranded DNA with circularized single-stranded ends are produced synthetically but are not supercoiled. Therefore, there is a pressing need to develop alternatives to plasmids to mitigate both production and safety concerns. We developed a bacteria-free technology that generates synthetic circular supercoiled DNA (SCSDNA). Cyclic heteroduplex thermostable ligation assembly (CHTLA) efficiently converts linear precursor DNAs <6 kb into circular ready-to-transfect molecules. We are nearing commercialization of this technology in the adeno-associated virus gene therapy industry pending success of a Phase 2 STTR project. However, CHTLA technology is not currently commercially viable for DNAs >10 kb due to the low linear DNA-to-circular DNA conversion rate. This limitation restricts our ability to enter the lucrative lentiviral gene therapy market wherein production requires 8-10 kb DNAs. The long-term goal of this project is to develop an efficient abacterial workflow to produce, at a commercially viable scale, and in a cost- competitive manner, DNAs >10 kb in length. The objectives of this proposal are 1) to increase the precursor conversion rate and final yield for SCSDNA production for larger (>10 kb) DNAs to a commercially viable level and 2) to demonstrate the utility of SCSDNA in lentivirus vector production. The rationale is that the optimization of CHTLA to produce large DNAs will allow us to serve the lucrative lentivirus gene therapy market segment and other clients requiring endotoxin and plasmid vector free DNAs of that size. 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, large DNAs with a supercoiled topology that are comprised exclusively of the sequence of interest. SCSDNA versions of three lentivirus helper plasmids and an 11 kb transfer plasmid will be generated using a highly diversified precursor DNA pool using conditions identified in Aim 1. SCSDNAs will be quantitatively compared to standard bacterially-sourced lentivirus helper and transfer plasmids by our commercial collaborator. Upon completion of these Aims we will have determined optimal conditions to generate functional SCSDNAs >10 kb at a scale that is commercially viable. In Phase 2, we will seek to further scale production toward the gram+ quantities that will be required to serve customers in the lentivirus gene therapy sector.