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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