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.

Grant Number: 1R41GM161305-01
NIH Institute/Center: NIH
Principal Investigator: Haibo Bai