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Rational engineering of minimally immunogenic nucleases for gene therapy

Author

Listed:
  • Rumya Raghavan

    (Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research at MIT
    Massachusetts Institute of Technology
    Massachusetts Institute of Technology)

  • Mirco J. Friedrich

    (Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research at MIT
    Massachusetts Institute of Technology
    Massachusetts Institute of Technology)

  • Indigo King

    (Cyrus Biotechnology)

  • Samuel Chau-Duy-Tam Vo

    (Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research at MIT
    Massachusetts Institute of Technology
    Massachusetts Institute of Technology)

  • Daniel Strebinger

    (Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research at MIT
    Massachusetts Institute of Technology
    Massachusetts Institute of Technology)

  • Blake Lash

    (Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research at MIT
    Massachusetts Institute of Technology
    Massachusetts Institute of Technology)

  • Michael Kilian

    (DKFZ)

  • Michael Platten

    (DKFZ)

  • Rhiannon K. Macrae

    (Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research at MIT
    Massachusetts Institute of Technology
    Massachusetts Institute of Technology)

  • Yifan Song

    (Cyrus Biotechnology)

  • Lucas Nivon

    (Cyrus Biotechnology)

  • Feng Zhang

    (Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research at MIT
    Massachusetts Institute of Technology
    Massachusetts Institute of Technology)

Abstract

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases, SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis, we identify putative immunogenic epitopes on each nuclease. Using computational modeling, we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components, including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses, yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9, but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection.

Suggested Citation

  • Rumya Raghavan & Mirco J. Friedrich & Indigo King & Samuel Chau-Duy-Tam Vo & Daniel Strebinger & Blake Lash & Michael Kilian & Michael Platten & Rhiannon K. Macrae & Yifan Song & Lucas Nivon & Feng Zh, 2025. "Rational engineering of minimally immunogenic nucleases for gene therapy," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-024-55522-1
    DOI: 10.1038/s41467-024-55522-1
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    References listed on IDEAS

    as
    1. Julie M. Crudele & Jeffrey S. Chamberlain, 2018. "Cas9 immunity creates challenges for CRISPR gene editing therapies," Nature Communications, Nature, vol. 9(1), pages 1-3, December.
    2. Vijaya L. Simhadri & Louis Hopkins & Joseph R. McGill & Brian R. Duke & Swati Mukherjee & Kate Zhang & Zuben E. Sauna, 2021. "Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4+ T-cells," Nature Communications, Nature, vol. 12(1), pages 1-7, December.
    3. Keiichiro Suzuki & Yuji Tsunekawa & Reyna Hernandez-Benitez & Jun Wu & Jie Zhu & Euiseok J. Kim & Fumiyuki Hatanaka & Mako Yamamoto & Toshikazu Araoka & Zhe Li & Masakazu Kurita & Tomoaki Hishida & Mo, 2016. "In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration," Nature, Nature, vol. 540(7631), pages 144-149, December.
    4. F. Ann Ran & Le Cong & Winston X. Yan & David A. Scott & Jonathan S. Gootenberg & Andrea J. Kriz & Bernd Zetsche & Ophir Shalem & Xuebing Wu & Kira S. Makarova & Eugene V. Koonin & Phillip A. Sharp & , 2015. "In vivo genome editing using Staphylococcus aureus Cas9," Nature, Nature, vol. 520(7546), pages 186-191, April.
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