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.
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
All code, input and output files used in this study are publicly available on GitHub (https://github.com/natepalmer/immune-orthogonal). Additional modified scripts can be accessed on request.
Mingozzi, F. & High, K. A. Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122, 23–36 (2013).
Zaldumbide, A. & Hoeben, R. C. How not to be seen: immune-evasion strategies in gene therapy. Gene Ther. 15, 239–246 (2008).
Yang, Y., Li, Q., Ertl, H. C. & Wilson, J. M. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69, 2004–2015 (1995).
Jawa, V. et al. T-cell dependent immunogenicity of protein therapeutics: preclinical assessment and mitigation. Clin. Immunol. 149, 534–555 (2013).
Mays, L. E. & Wilson, J. M. The complex and evolving story of T cell activation to AAV vector-encoded transgene products. Mol. Ther. 19, 16–27 (2011).
Basner-Tschakarjan, E., Bijjiga, E. & Martino, A. T. Pre-clinical assessment of immune responses to adeno-associated virus (AAV) vectors. Front. Immunol. 5, 28 (2014).
Ertl, H. C. J. & High, K. A. Impact of AAV capsid-specific T-cell responses on design and outcome of clinical gene transfer trials with recombinant adeno-associated viral vectors: an evolving controversy. Hum. Gene Ther. 28, 328–337 (2017).
Kotterman, M. A., Chalberg, T. W. & Schaffer, D. V. Viral vectors for gene therapy: translational and clinical outlook. Annu. Rev. Biomed. Eng. 17, 63–89 (2015).
Mingozzi, F. & High, K. A. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat. Rev. Genet. 12, 341–355 (2011).
Manno, C. S. et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat. Med. 12, 342–347 (2006).
Chew, W. L. Immunity to CRISPR Cas9 and Cas12a therapeutics. Wiley Interdiscip. Rev. Syst. Biol. Med. 10, e1408 (2018).
Sathish, J. G. et al. Challenges and approaches for the development of safer immunomodulatory biologics. Nat. Rev. Drug Discov. 12, 306–324 (2013).
Harding, F. A., Stickler, M. M., Razo, J. & DuBridge, R. B. The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions. MAbs 2, 256–265 (2010).
De Groot, A. S., Knopp, P. M. & Martin, W. De-immunization of therapeutic proteins by T-cell epitope modification. Dev. Biol. 122, 171–194 (2005).
Tangri, S. et al. Rationally engineered therapeutic proteins with reduced immunogenicity. J. Immunol. 174, 3187–3196 (2005).
Ferdosi, S. R. et al. Multifunctional CRISPR/Cas9 with engineered immunosilenced human T cell epitopes. Nat. Comms 10, 1842 (2019).
Salvat, R. S., Choi, Y., Bishop, A., Bailey-Kellogg, C. & Griswold, K. E. Protein deimmunization via structure-based design enables efficient epitope deletion at high mutational loads. Biotechnol. Bioeng. 112, 1306–1318 (2015).
Armstrong, J. K. et al. Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients. Cancer 110, 103–111 (2007).
Ganson, N. J., Kelly, S. J., Scarlett, E., Sundy, J. S. & Hershfield, M. S. Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase. Arthritis Res. Ther. 8, R12 (2006).
Veronese, F. M. & Mero, A. The impact of PEGylation on biological therapies. BioDrugs 22, 315–329 (2008).
Jevševar, S., Kunstelj, M. & Porekar, V. G. PEGylation of therapeutic proteins. Biotechnol. J. 5, 113–128 (2010).
Jacobs, F., Gordts, S. C., Muthuramu, I. & De Geest, B. The liver as a target organ for gene therapy: state of the art, challenges, and future perspectives. Pharmaceuticals 5, 1372–1392 (2012).
Kok, C. Y. et al. Adeno-associated virus-mediated rescue of neonatal lethality in argininosuccinate synthetase-deficient mice. Mol. Ther. 21, 1823–1831 (2013).
Courtenay-Luck, N. S., Epenetos, A. A. & Moore, R. Development of primary and secondary immune responses to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasms. Cancer Res. 46, 6489–6493 (1986).
Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptice bacterial immunity. Science 337, 816–822 (2012).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Moreno, A. M. & Mali, P. Therapeutic genome engineering via CRISPR-Cas systems. Wiley Interdiscip. Rev. Syst. Biol. Med. 9, e1380 (2017).
Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).
Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).
Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
Kelton, W. J., Pesch, T., Matile, S. & Reddy, S. T. Surveying the delivery methods of CRISPR/Cas9 for ex vivo mammalian cell engineering. Chim. Int. J. Chem. 70, 439–442 (2016).
Cho, S. W., Kim, S., Kim, J. M. & KimJ.-S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).
Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).
Makarova, K. S. et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).
Chylinski, K., Makarova, K. S., Charpentier, E. & Koonin, E. V. Classification and evolution of type II CRISPR-Cas systems. Nucleic Acids Res. 42, 6091–6105 (2014).
Shmakov, S. et al. Diversity and evolution of class 2 CRISPR–Cas systems. Nat. Rev. Microbiol. 15, 169–182 (2017).
Crawley, A. B., Henriksen, J. R. & Barrangou, R. CRISPRdisco: an automated pipeline for the discovery and analysis of CRISPR-Cas systems. CRISPR J. 1, 171–181 (2018).
Charlesworth, C. T. et al. Identification of pre-existing adaptive immunity to Cas9 proteins in humans. Nat Med. 25, 249–254 (2019).
Wagner, D. L. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat. Med. 25, 242–248 (2019).
Simhadri, V. L. et al. Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the US population. Mol. Ther. Methods Clin. Dev. 10, 105–112 (2018).
Wagner, J. A. et al. Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus. Laryngoscope 109, 266–274 (1999).
Song, S. et al. Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proc. Natl Acad. Sci. USA 95, 14384–14388 (1998).
Chirmule, N. et al. Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle. J. Virol. 74, 2420–2425 (2000).
Fields, P. A. et al. Risk and prevention of anti-factor IX formation in AAV-mediated gene transfer in the context of a large deletion of F9. Mol. Ther. 4, 201–210 (2001).
Herzog, R. W. et al. Influence of vector dose on factor IX-specific T and B cell responses in muscle-directed gene therapy. Hum. Gene Ther. 13, 1281–1291 (2002).
Lozier, J. N., Tayebi, N. & Zhang, P. Mapping of genes that control the antibody response to human factor IX in mice. Blood 105, 1029–1035 (2005).
Zhang, H. G. et al. Genetic analysis of the antibody response to AAV2 and factor IX. Mol. Ther. 11, 866–874 (2005).
Tam, H. H. et al. Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination. Proc. Natl Acad. Sci. USA 113, E6639–E6648 (2016).
Chew, W. L. et al. A multifunctional AAV–CRISPR–Cas9 and its host response. Nat. Methods 13, 868–874 (2016).
Benveniste, O. et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum. Gene Ther. 21, 704–712 (2010).
Gao, G.-P. et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl Acad. Sci. USA 99, 11854–11859 (2002).
Jooss, K., Yang, Y., Fisher, K. J. & Wilson, J. M. Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J. Virol. 72, 4212–4223 (1998).
Gernoux, G. et al. Early interaction of adeno-associated virus serotype 8 vector with the host immune system following intramuscular delivery results in weak but detectable lymphocyte and dendritic cell transduction. Hum. Gene Ther. 26, 1–13 (2015).
Zhu, J., Huang, X. & Yang, Y. The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice. J. Clin. Invest. 119, 2388–2398 (2009).
Gernoux, G., Wilson, J. M. & Mueller, C. Regulatory and exhausted T cell responses to AAV capsid. Hum. Gene Ther. 28, 338–349 (2017).
Kurosaki, T., Kometani, K. & Ise, W. Memory B cells. Nat. Rev. Immunol. 15, 149–159 (2015).
Zabel, F. et al. Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation. Immunology 150, 329–342 (2017).
Ding, Q. et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ. Res. 115, 488–492 (2014).
Zinn, E. et al. In silico reconstruction of the viral evolutionary lineage yields a potent gene therapy vector. Cell Rep. 12, 1056–1068 (2017).
Calcedo, R. & Wilson, J. M. AAV natural infection induces broad cross-neutralizing antibody responses to multiple AAV serotypes in chimpanzees. Hum. Gene Ther. Clin. Dev. 27, 79–82 (2016).
Harbison, C. E. et al. Examining the cross-reactivity and neutralization mechanisms of a panel of mAbs against adeno-associated virus serotypes 1 and 5. J. Gen. Virol. 93, 347–355 (2012).
Majowicz, A. et al. Successful repeated hepatic gene delivery in mice and non-human primates achieved by sequential administration of AAV5ch and AAV1. Mol. Ther. 25, 1831–1842 (2017).
McIntosh, J. H. et al. Successful attenuation of humoral immunity to viral capsid and transgenic protein following AAV-mediated gene transfer with a non-depleting CD4 antibody and cyclosporine. Gene Ther. 19, 78–85 (2012).
Mingozzi, F. et al. Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue. Gene Ther. 20, 417–424 (2013).
Mingozzi, F. et al. Pharmacological modulation of humoral immunity in a nonhuman primate model of AAV gene transfer for hemophilia B. Mol. Ther. 20, 1410–1416 (2017).
Unzu, C. et al. Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates. J. Transl. Med. 10, 122 (2012).
Riechmann, L., Clark, M., Waldmann, H. & Winter, G. Reshaping human antibodies for therapy. Nature 332, 323–327 (1988).
Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232 (2016).
Hinderer, C. et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an AAV vector expressing human SMN. Hum. Gene Ther. 29, 285–298 (2018).
Vollmers, C., Sit, R. V., Weinstein, J. A., Dekker, C. L. & Quake, S. R. Genetic measurement of memory B-cell recall using antibody repertoire sequencing. Proc. Natl Acad. Sci. USA 110, 13463–13468 (2013).
Adamopoulou, E. et al. Exploring the MHC-peptide matrix of central tolerance in the human thymus. Nat. Commun. 4, 2039 (2013).
Ruppert, J. et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 74, 929–937 (2017).
Zhang, S.-Q. et al. Direct measurement of T cell receptor affinity and sequence from naïve antiviral T cells. Sci. Transl. Med. 8, 341ra77 (2016).
Baker, M. P., Reynolds, H. M., Lumicisi, B. & Bryson, C. J. Immunogenicity of protein therapeutics: the key causes, consequences and challenges. Self Nonself 1, 314–322 (2010).
EL-Manzalawy, Y., Dobbs, D. & Honavar, V. Predicting linear B-cell epitopes using string kernels. J. Mol. Recognit. 21, 243–255 (2008).
Larsen, J. E. P., Lund, O. & Nielsen, M. Improved method for predicting linear B-cell epitopes. Immunome Res. 2, 2 (2006).
Sollner, J. et al. Analysis and prediction of protective continuous B-cell epitopes on pathogen proteins. Immunome Res. 4, 1 (2008).
Dalkas, G. A. & Rooman, M. SEPIa, a knowledge-driven algorithm for predicting conformational B-cell epitopes from the amino acid sequence. BMC Bioinform. 18, 95 (2017).
Sun, P. et al. Bioinformatics resources and tools for conformational B-cell epitope prediction. Comput. Math. Methods Med. 2013, 943636 (2013).
Liepe, J. et al. A large fraction of HLA class I ligands are proteasome-generated spliced peptides. Science 354, 354–358 (2016).
Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 42, 2577–2590 (2014).
Burstein, D. et al. New CRISPR–Cas systems from uncultivated microbes. Nature 542, 237–241 (2016).
Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol. Cell 60, 385–397 (2015).
Andreatta, M. & Nielsen, M. Gapped sequence alignment using artificial neural networks: application to the MHC class I system. Bioinformatics 32, 511–517 (2015).
Andreatta, M. et al. Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification. Immunogenetics 67, 641–650 (2015).
Vita, R. et al. The immune epitope database (IEDB) 3.0. Nucleic Acids Res. 43, D405–D412 (2015).
Truong, D.-J. J. et al. Development of an intein-mediated split–Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015).
Moreno, A. M. et al. In situ gene therapy via AAV-CRISPR-Cas9-mediated targeted gene regulation. Mol. Ther. 26, 1818–1827 (2018).
Grieger, J. C., Choi, V. W. & Samulski, R. J. Production and characterization of adeno-associated viral vectors. Nat. Protoc. 1, 1412–1428 (2006).
Schubert, M., Lindgreen, S. & Orlando, L. AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Res. Notes 9, 88 (2016).
Pinello, L. et al. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat. Biotechnol. 34, 695–697 (2016).
Clemente, T., Dominguez, M. R., Vieira, N. J., Rodrigues, M. M. & Amarante-Mendes, G. P. In vivo assessment of specific cytotoxic T lymphocyte killing. Methods 61, 105–109 (2013).
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).
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.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–9, Supplementary Tables 1–4.
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.
About this article
Cite this article
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). https://doi.org/10.1038/s41551-019-0431-2
Photoacoustic molecular imaging-escorted adipose photodynamic–browning synergy for fighting obesity with virus-like complexes
Nature Nanotechnology (2021)
Molecular Therapy - Methods & Clinical Development (2021)
Safe and Effective In Vivo Targeting and Gene Editing in Hematopoietic Stem Cells: Strategies for Accelerating Development
Human Gene Therapy (2021)
Gene Therapy (2021)
Molecular Therapy - Methods & Clinical Development (2021)