Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy

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Protein-based therapeutics can activate the adaptive immune system, leading to the production of neutralizing antibodies and the clearance of the treated cells mediated by cytotoxic T cells. Here, we show that the sequential use of immune-orthogonal orthologues of CRISPR-associated protein 9 (Cas9) and adeno-associated viruses (AAVs) evades adaptive immune responses and enables effective gene editing using repeated dosing. We compared total sequence similarities and predicted binding strengths to class-I and class-II major histocompatibility complex (MHC) proteins for 284 DNA-targeting and 84 RNA-targeting CRISPR effectors and 167 AAV VP1-capsid-protein orthologues. We predict the absence of cross-reactive immune responses for 79% of the DNA-targeting Cas orthologues—which we validated for three Cas9 orthologues in mice—yet we anticipate broad immune cross-reactivity among the AAV serotypes. We also show that efficacious in vivo gene editing is uncompromised when using multiple dosing with orthologues of AAVs and Cas9 in mice that were previously immunized against the AAV vector and the Cas9 cargo. Multiple dosing with protein orthologues may allow for sequential regimens of protein therapeutics that circumvent pre-existing immunity or induced immunity.

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Fig. 1: Protein-based therapeutics elicit an adaptive immune response: experimental and in silico analyses.
Fig. 2: Experimental validation of Cas9 and AAV immunogenicity predictions.
Fig. 3: Engineering re-dosing with immune orthogonal orthologues.

Data availability

The authors declare that the main data supporting the results of this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request.

Code availability

All code, input and output files used in this study are publicly available on GitHub ( Additional modified scripts can be accessed on request.

Change history

  • 27 August 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.


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We thank members of the Mali laboratory for advice and help with experiments and the Salk GT3 viral core for help with the production of AAVs. This research was supported by UCSD Institutional Funds, the Burroughs Wellcome Fund (1013926), the March of Dimes Foundation (5-FY15-450), the Kimmel Foundation (SKF-16-150), and NIH grants (R01HG009285, RO1CA222826, RO1GM123313, R01AI079031 and R01AI106005). A.M.M. acknowledges a graduate fellowship from CONACYT and UCMEXUS. W.L.C. acknowledges the IAF-PP grant (H17/01/a0/012).

Author information

A.M.M. designed and performed experiments, analysed the data and wrote the manuscript. N.P. performed the in silico analysis, designed and performed experiments, analysed the data and wrote the manuscript. F.A. performed the ELISPOT experiments and analysed the data. G.C. and A.P. helped to perform experiments. N.J., W.L.C. and M.L. helped to design experiments. P.M. supervised the project, designed and helped to perform experiments and wrote the manuscript.

Correspondence to Prashant Mali.

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Competing interests

A.M.M., N.P. and P.M. have filed patents on the basis of this research. P.M. is a scientific co-founder of Navega Therapeutics, Pretzel Therapeutics, Seven Therapeutics, Engine Biosciences and Shape Therapeutics. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its policies regarding conflicts of interest. W.L.C. is a scientific co-founder of Seven Therapeutics. N.J. is a scientific advisor of ImmuDX, LLC and Immune Arch, Inc.

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Supplementary Information

Supplementary Figs. 1–9, Supplementary Tables 1–4.

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Supplementary Tables

Lists of all DNA and RNA targeting CRISPR orthologues, of all AAV VP1 orthologues, and of SpCas9 and SaCas9 peptides and AAV2, AAV5, AAV8 and AAVDJ peptides predicted to bind to human MHC proteins.

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Moreno, A.M., Palmer, N., Alemán, F. et al. Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy. Nat Biomed Eng 3, 806–816 (2019) doi:10.1038/s41551-019-0431-2

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