Abstract:

Developing novel editing systems that circumvent double-strand breaks (DSBs) represents a rapidly growing area of genetic research. Prime editing was developed as a “search-and-replace” genome editing technology and comprises three key components: (1) Prime Editing Guide RNA (pegRNA); (2) Cas9 H840A Nickase; and (3) Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT). Despite its potential, the widespread application of prime editing has been limited by suboptimal editing efficiency. While studies have demonstrated that stabilizing the 3’ moiety through structural modifications (e.g., TeveproQ, G-quadruplex, or hairpin motifs) can enhance efficiency, sequence length extension may become problematic during the CMC stage. PegRNAs typically range from approximately 130-150nt, making it challenging to develop corresponding production processes and analytical methods. To address this issue, we screened novel modification combinations that do not elongate sequence length in order to improve editing efficiency.

Abstract:

Non-viral DNA delivery systems offer key advantages over viral vectors, including lower immunogenicity, no cargo size limits, and simpler manufacturing, yet conventional plasmids remain hampered by immune activation and transcriptional silencing. Here we present GenCircle™ DNA, a minimized circular double-stranded DNA vector, and a companion circular single-stranded DNA (cssDNA) platform, evaluated for in vivo gene delivery and ex vivo gene editing.

Delivered via lipid nanoparticles (LNPs) in mice, GenCircle™ DNA increased luciferase expression over 100-fold versus conventional plasmid, with sustained expression beyond 200 days post-injection. Optimized transcriptional elements and LNP formulations further elevated expression by approximately two orders of magnitude. cssDNA achieved comparable transgene expression while eliciting significantly reduced IL-6 levels and minimal body weight loss, confirming a superior safety profile. Both platforms also demonstrated markedly improved large-fragment insertion efficiency when used as HDR and transposon donor templates in vitro. These results establish GenCircle™ DNA and cssDNA as versatile, high-performance platforms for durable, safe gene delivery and gene writing applications, including CAR-T cell generation.