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Rational engineering of a functional CpG-free ITR for AAV gene therapy

Gene Therapy volume 29, pages 333–345 (2022)Cite this article

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Abstract

Inverted terminal repeats (ITRs) are the only wild-type components retained in the genome of adeno-associated virus (AAV) vectors. To determine whether ITR modification is a viable approach for AAV vector engineering, we rationally deleted all CpG motifs in the ITR and examined whether CpG elimination compromises AAV-vector production and transduction. Modified ITRs were stable in the plasmid and maintained the CpG-free nature in purified vectors. Replacing the wild-type ITR with the CpG-free ITR did not affect vector genome encapsidation. However, the vector yield was decreased by approximately 3-fold due to reduced vector genome replication. To study the biological potency, we made micro-dystrophin (μDys) AAV vectors carrying either the wild-type ITR or the CpG-free ITR. We delivered the CpG-free μDys vector to one side of the tibialis anterior muscle of dystrophin-null mdx mice and the wild-type μDys vector to the contralateral side. Evaluation at four months after injection showed no difference in the vector genome copy number, microdystrophin expression, and muscle histology and force. Our results suggest that the complete elimination of the CpG motif in the ITR does not affect the biological activity of the AAV vector. CpG-free ITRs could be useful in engineering therapeutic AAV vectors.

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Fig. 1: Design of the version-1 CpG-free ITR.
Fig. 2: Confirmation of the CpG-free ITR in the cis-plasmid.
Fig. 3: SMRT sequencing evaluation of the ITR sequence in the purified AAV vector.
Fig. 4: Quantitative evaluation of AAV production.
Fig. 5: Evaluation of vector-genome replication during AAV production.
Fig. 6: The CpG-free ITR optimization and the yield from the vector carrying the modified CpG-free ITR.
Fig. 7: Evaluation of microdystrophin expression.
Fig. 8: Evaluation of centronucleation and myofiber size distribution.
Fig. 9: Quantitative evaluation of the tibialis anterior muscle contractility from mdx mice that were treated with CpG-free and the wild-type μDys vectors.

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Acknowledgements

This work was supported in part by grants from National Institutes of Health (NS-90634 and AR-70517 to DD, AI-116595 to DJP), Department of Defense (MD130014 to DD), Jesse’s Journey—The Foundation for Gene and Cell Therapy (to DD), and Jackson Freel DMD Research Fund (to DD). The authors thank Dr. Arun Srivastava (University of Florida) for providing the AAV9 tyrosine mutant Rep–Cap packaging plasmid and Dr. Glenn Morris (The Rober Jones and Agnes Hunt Orthopedic Hospital, UK) for providing the MANNEX 44 A antidystrophin antibody. The authors thank Duan lab members for helpful discussion. The authors thank Lisa Burger for expert technical assistance.

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Authors and Affiliations

  1. Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, 65212, USA

    Xiufang Pan, Yongping Yue, Lakmini P. Wasala, Keqing Zhang, David J. Pintel & Dongsheng Duan

  2. Pathobiology Area Graduate Program, University of Missouri, Columbia, MO, 65212, USA

    Maria Boftsi & Lakmini P. Wasala

  3. Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, 65212, USA

    Maria Boftsi & David J. Pintel

  4. Horae Gene Therapy Center, UMass Chan Medical School, Worcester, MA, 01605, USA

    Ngoc Tam Tran & Phillip W. L. Tai

  5. Department of Microbiology and Physiological Systems, UMass Chan Medical School, Worcester, MA, 01605, USA

    Ngoc Tam Tran & Phillip W. L. Tai

  6. Department of Biomedical Sciences, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65212, USA

    Dongsheng Duan

  7. Department of Neurology, School of Medicine, University of Missouri, Columbia, MO, 65212, USA

    Dongsheng Duan

  8. Department of Biomedical, Biological & Chemical Engineering, College of Engineering, University of Missouri, Columbia, MO, 65212, USA

    Dongsheng Duan

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Contributions

Conceived and designed experiments: XP and DD. Performed the experiments: XP, YY, MB, LPW, NTT, KZ, and PWLT. Analyzed the data: XW, YY, MB, LPW, DJP, PWLT, and DD. Wrote the paper: XP and DD. All authors edited the paper and approved submission.

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Correspondence to Dongsheng Duan.

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DD is a member of the scientific advisory board for Solid Biosciences and equity holders of Solid Biosciences. DD and YY are inventors on patents that were licensed to Solid Biosciences. The Duan lab has received research support (unrelated to this project) from Solid Biosciences and Edgewise Therapeutics in the last three years.

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Pan, X., Yue, Y., Boftsi, M. et al. Rational engineering of a functional CpG-free ITR for AAV gene therapy. Gene Ther 29, 333–345 (2022). https://doi.org/10.1038/s41434-021-00296-0

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