TAAV-manufactured enzymatic DNA¹ can be used as starting material to produce rAAV, providing comparable productivity and potency to plasmid-derived vectors, and importantly, increased effectiveness as less input DNA is required in transfections.

The Enzymatic DNA manufacturing process essentially combines the use of two enzymes, Phi29 polymerase and TelN protelomerase. High throughput sequencing (HTP) analyses have demonstrated that an intended sequence is amplified with high fidelity, including complex molecular structures such as AAV-ITRs. The results obtained showed no mutations in enzymatic DNA final product compared to the precursor template used in the process.

The removal of bacterial sequences is crucial from a regulatory perspective for improving the safety profile of rAAV products. Moving from plasmid towards enzymatic DNA reduces residual host cell DNA and eliminates antibiotic resistance genes. HTP and quantitative PCR  analyses of TAAV-enzymatic DNA product have consistently shown <1% Kanᴿ residual sequences. Enzymatic DNA is comprised of the DNA sequence of interest, except for a small flanking site with the TelN recognition site, and a stuffer sequence, that allows the measure of any residual DNA in final rAAV products. Asklepios BioPharmaceutical has proven in vivo that the stuffer sequence does not have any transcriptional activity. 

The current study focuses on highlighting the advantages of using TAAV-manufactured enzymatic DNA as critical starting material to resolve the challenges in rAAV production by providing rapid production, scalability, robustness, effectiveness, and a likely safer profile of a high purity product.

¹ Technology for making TAAV-manufactured enzymatic DNA is licensed from Touchlight IP Ltd.