Manufacturing DNA in E. coli yields higher-fidelity DNA than in vitro enzymatic synthesis

Steven J Hersch; Siddarth Chandrasekaran; Jamie Lam; Nafiseh Nafissi; Roderick A Slavcev

doi:10.1016/j.omtm.2024.101227

Manufacturing DNA in E. coli yields higher-fidelity DNA than in vitro enzymatic synthesis

Mol Ther Methods Clin Dev. 2024 Feb 28;32(2):101227. doi: 10.1016/j.omtm.2024.101227. eCollection 2024 Jun 13.

Authors

Steven J Hersch¹, Siddarth Chandrasekaran¹, Jamie Lam¹, Nafiseh Nafissi^{1

2}, Roderick A Slavcev^{1

2

3}

Affiliations

¹ Mediphage Bioceuticals, Inc, Toronto, ON, Canada.
² School of Pharmacy, University of Waterloo, Waterloo, ON, Canada.
³ Centre for Eye and Vision Research, HKSTP, Ma Liu Shui, Hong Kong.

Abstract

Biotechnologies such as gene therapy have brought DNA vectors to the forefront of pharmaceuticals. The quality of starting material plays a pivotal role in determining final product quality. Here, we examined the fidelity of DNA replication using enzymatic methods (in vitro) compared to plasmid DNA produced in vivo in E. coli. Next-generation sequencing approaches rely on in vitro polymerases, which have inherent limitations in sensitivity. To address this challenge, we introduce a novel assay based on loss-of-function (LOF) mutations in the conditionally toxic sacB gene. Our findings show that DNA production in E. coli results in significantly fewer LOF mutations (80- to 3,000-fold less) compared to enzymatic DNA replication methods such as polymerase chain reaction (PCR) and rolling circle amplification (RCA). These results suggest that using DNA produced by PCR or RCA may introduce a substantial number of mutation impurities, potentially affecting the quality and yield of final pharmaceutical products. Our study underscores that DNA synthesized in vitro has a significantly higher mutation rate than DNA produced traditionally in E. coli. Therefore, utilizing in vitro enzymatically produced DNA in biotechnology and biomanufacturing may entail considerable fidelity-related risks, while using DNA starting material derived from E. coli substantially mitigates this risk.

Keywords: AAV manufacturing; DNA manufacturing; DNA replication fidelity; DNA synthesis; E. coli; SacB; loss of function; mutation rate; polymerase chain reaction; rolling circle amplification.