Megabulb DNA (mbDNA) is a cell-free circular single-stranded DNA (ssDNA) template developed by Touchlight for non-viral gene editing and transient expression. Compared to linear ssDNA and double-stranded DNA (dsDNA), mbDNA offers higher homology-directed repair (HDR) efficiency, lower toxicity, and reduced immunogenicity. Its covalently closed design increases stability, enables nuclease complex formation, and enhances nuclear transport. In primary T cells, mbDNA achieves up to 70% CRISPR knock-in efficiency—matching viral vector performance—while supporting better cell recovery and expansion. Lipid nanoparticle delivery further mitigates electroporation toxicity. Capable of carrying multi-kilobase sequences without viral packaging limits, mbDNA is scalable to GMP-grade multi-milligram quantities. Leading industry evaluations in T cells and stem cells indicate broad potential for safer, efficient, and adaptable gene and cell therapy applications. More recently, mbDNA has shown to be a potent episomal expression vector, which promises reduced immunogenicity for in vivo non-viral gene therapy approaches. 

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Recombinant adeno-associated virus (rAAV) clinical translation remains constrained by significant manufacturing and safety challenges. One approach to address these limitations is to substitute the E. coli derived plasmid DNA with cell-free DNA. Touchlight’s linear dbDNA, an enzymatically amplified DNA construct, offers rapid, scalable generation of GMP-grade material in a fraction of the time of conventional plasmid DNA. dbDNA lacks bacterial sequences, making it an ideal DNA template for AAV production. To characterise the purity and quality of dbDNA-derived AAV vectors and plasmid-derived AAV vectors, nanopore sequencing was employed to profile the encapsidated DNA. Successful packaging of the ITR-to-ITR AAV genome using dbDNA was confirmed. Furthermore, the dbDNA-derived AAV population contained significantly fewer process- and product-related DNA impurities. While the reverse packaged plasmid sequences contained the antibiotic resistance and bacterial replication cassette, reverse packaged dbDNA comprised short inert stuffer sequences. Thus, dbDNA offers safety advantages over plasmid as an AAV template. Illumina sequencing confirmed full length ITRs are maintained during the production of dbDNA. Nanopore ITR characterisation of AAV genomes revealed high levels of intact ITRs in both dbDNA- and plasmid-derived AAV preparations. The dbDNA-derived AAV vectors contained lower levels of partial genomes compared to plasmid-derived AAV vectors. Thus, the high vector batch quality and low DNA impurity levels observed in dbDNA-produced AAVs further support their enhanced safety profile.

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mbDNA (megabulb DNA) represents a breakthrough in non-viral gene-editing technology. Its unique circular single-stranded DNA architecture, with a modifiable double stranded stem region and a fully customisable sequence free of phage or bacterial-derived elements, is specifically engineered to serve as an exceptionally efficient template for HDR. Following nuclear delivery, mbDNA consistently enables enhanced knock-in (KI) efficiency in both immune and stem cells whilst maintaining high viability. Compatible with electroporation and lipid nanoparticle (LNP) formulations, this versatile vector exemplifies the promise of next-generation non-viral systems optimised for safe, efficient, and flexible gene-editing applications.

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