Abstract
The application of recombinant adeno-associated viruses (rAAVs) for gene therapy faces certain challenges, including genome packaging of non-vector sequences. Inverted terminal repeats (ITRs) flanking the rAAV genome, comprising three inverted repeat regions (A, B, and C) and a non-inverted repeat region (D), contribute to non-vector genome packaging. We aimed to circumvent this issue by comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, including a single copy of the AD sequence (rAAV-pAD/L-AD, respectively), which is a truncated form of ITR, with those of wild-type ITR genome (single-stranded and self-complementary AAV; ssAAV and scAAV). The packaging efficiency of rAAV-pAD/L-AD was found to be comparable to that of scAAV, whereas the transduction efficiency of rAAV-pAD/L-AD was lower than that of ss/scAAV. Remarkably, rAAV-L-AD reduced the plasmid backbone packaging contamination compared to ss/scAAV. Furthermore, to confirm the functionality of this system, we generated a rAAV-L-AD harboring a short hairpin RNA targeting ATP5B (rAAV-L-AD-shATP5B) and found that it caused a significant decrease in ATP5B mRNA levels when transduced into HEK293EB cells, suggesting that it was functional. Thus, our system successfully packaged L-AD into capsids with minimal contamination of plasmid DNA, offering a novel functional packaging platform without causing plasmid backbone encapsidation.
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Acknowledgements
We thank Dr. James Wilson from the University of Pennsylvania for providing the AAV packaging plasmid (pAAV2/1) and Dr. Arun Srivastava from the University of Florida for providing the cis AAV vector plasmid (pdsAAV-CB-EGFP).
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KA designed the experiments, performed most of the experiments, and analyzed the data. TT designed the experiments, analyzed the data, and wrote the paper. HO and YS conceived the study. YM and MY designed and conducted the experiments, constructed the plasmids, analyzed the data, and wrote the paper. TO designed the experiments. All authors have read and approved the submitted paper.
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Adachi, K., Tomono, T., Okada, H. et al. A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging. Gene Ther 29, 449–457 (2022). https://doi.org/10.1038/s41434-021-00299-x
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DOI: https://doi.org/10.1038/s41434-021-00299-x