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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 Therapy volume 29, pages 449–457 (2022)Cite this article

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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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Fig. 1: Structure of the rAAV vector plasmids and characterization of rAAV carrying a single copy of the AD sequence.
Fig. 2: Structure of the PCR-amplified linear rAAV genomes with a single copy of the AD sequence and properties of rAAVs.
Fig. 3: Transduction efficiency of rAAV1-L-AD.
Fig. 4: Knockdown efficiency of endogenous ATP5B by rAAV carrying pAD-shRNA/L-AD-shRNA transduction.

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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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Author notes

  1. These authors contributed equally: Kumi Adachi, Taro Tomono.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan

    Kumi Adachi, Taro Tomono, Hironori Okada, Motoko Yamamoto & Yoshitaka Miyagawa

  2. Department of Neurology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan

    Taro Tomono

  3. Laboratory of Molecular Analysis, Nippon Medical School, Tokyo, Japan

    Yusuke Shiozawa

  4. Division of Molecular and Medical Genetics, Center for Gene and Cell Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Takashi Okada

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  1. Kumi Adachi
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Contributions

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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Correspondence to Yoshitaka Miyagawa or Takashi Okada.

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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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