Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Biomedical Engineering volume 3, pages 806–816 (2019)Cite this article

Subjects

An Author Correction to this article was published on 27 August 2019

This article has been updated

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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 (https://github.com/natepalmer/immune-orthogonal). 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.

References

  1. Mingozzi, F. & High, K. A. Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122, 23–36 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Zaldumbide, A. & Hoeben, R. C. How not to be seen: immune-evasion strategies in gene therapy. Gene Ther. 15, 239–246 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Jawa, V. et al. T-cell dependent immunogenicity of protein therapeutics: preclinical assessment and mitigation. Clin. Immunol. 149, 534–555 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. 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).

    Article  CAS  PubMed  Google Scholar 

  6. 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).

    PubMed  PubMed Central  Google Scholar 

  7. 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).

    Article  CAS  PubMed  Google Scholar 

  8. 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).

    Article  CAS  PubMed  Google Scholar 

  9. 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).

    Article  CAS  PubMed  Google Scholar 

  10. 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).

    Article  CAS  PubMed  Google Scholar 

  11. Chew, W. L. Immunity to CRISPR Cas9 and Cas12a therapeutics. Wiley Interdiscip. Rev. Syst. Biol. Med. 10, e1408 (2018).

    Article  CAS  Google Scholar 

  12. Sathish, J. G. et al. Challenges and approaches for the development of safer immunomodulatory biologics. Nat. Rev. Drug Discov. 12, 306–324 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 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).

    Google Scholar 

  15. Tangri, S. et al. Rationally engineered therapeutic proteins with reduced immunogenicity. J. Immunol. 174, 3187–3196 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Ferdosi, S. R. et al. Multifunctional CRISPR/Cas9 with engineered immunosilenced human T cell epitopes. Nat. Comms 10, 1842 (2019).

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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).

    Article  PubMed  Google Scholar 

  19. 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).

    Article  CAS  PubMed  Google Scholar 

  20. Veronese, F. M. & Mero, A. The impact of PEGylation on biological therapies. BioDrugs 22, 315–329 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Jevševar, S., Kunstelj, M. & Porekar, V. G. PEGylation of therapeutic proteins. Biotechnol. J. 5, 113–128 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kok, C. Y. et al. Adeno-associated virus-mediated rescue of neonatal lethality in argininosuccinate synthetase-deficient mice. Mol. Ther. 21, 1823–1831 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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).

    CAS  PubMed  Google Scholar 

  25. Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptice bacterial immunity. Science 337, 816–822 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Moreno, A. M. & Mali, P. Therapeutic genome engineering via CRISPR-Cas systems. Wiley Interdiscip. Rev. Syst. Biol. Med. 9, e1380 (2017).

    Article  CAS  Google Scholar 

  28. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  PubMed  Google Scholar 

  36. Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Makarova, K. S. et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Shmakov, S. et al. Diversity and evolution of class 2 CRISPR–Cas systems. Nat. Rev. Microbiol. 15, 169–182 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Charlesworth, C. T. et al. Identification of pre-existing adaptive immunity to Cas9 proteins in humans. Nat Med. 25, 249–254 (2019).

  42. 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).

  43. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 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).

    Article  CAS  PubMed  Google Scholar 

  45. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 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).

    Article  CAS  PubMed  Google Scholar 

  48. 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).

    Article  CAS  PubMed  Google Scholar 

  49. 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).

    Article  CAS  PubMed  Google Scholar 

  50. Zhang, H. G. et al. Genetic analysis of the antibody response to AAV2 and factor IX. Mol. Ther. 11, 866–874 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Chew, W. L. et al. A multifunctional AAV–CRISPR–Cas9 and its host response. Nat. Methods 13, 868–874 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 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).

    Article  CAS  PubMed  Google Scholar 

  54. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 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).

    Article  CAS  PubMed  Google Scholar 

  57. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Gernoux, G., Wilson, J. M. & Mueller, C. Regulatory and exhausted T cell responses to AAV capsid. Hum. Gene Ther. 28, 338–349 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Kurosaki, T., Kometani, K. & Ise, W. Memory B cells. Nat. Rev. Immunol. 15, 149–159 (2015).

    Article  CAS  PubMed  Google Scholar 

  60. Zabel, F. et al. Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation. Immunology 150, 329–342 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Ding, Q. et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ. Res. 115, 488–492 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Zinn, E. et al. In silico reconstruction of the viral evolutionary lineage yields a potent gene therapy vector. Cell Rep. 12, 1056–1068 (2017).

    Article  CAS  Google Scholar 

  63. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 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).

    Article  CAS  PubMed  Google Scholar 

  67. 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).

    Article  CAS  PubMed  Google Scholar 

  68. 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).

    Article  CAS  Google Scholar 

  69. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Riechmann, L., Clark, M., Waldmann, H. & Winter, G. Reshaping human antibodies for therapy. Nature 332, 323–327 (1988).

    Article  CAS  PubMed  Google Scholar 

  71. Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  74. Adamopoulou, E. et al. Exploring the MHC-peptide matrix of central tolerance in the human thymus. Nat. Commun. 4, 2039 (2013).

    Article  CAS  PubMed  Google Scholar 

  75. Ruppert, J. et al. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 74, 929–937 (2017).

    Article  Google Scholar 

  76. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  78. EL-Manzalawy, Y., Dobbs, D. & Honavar, V. Predicting linear B-cell epitopes using string kernels. J. Mol. Recognit. 21, 243–255 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Larsen, J. E. P., Lund, O. & Nielsen, M. Improved method for predicting linear B-cell epitopes. Immunome Res. 2, 2 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Sollner, J. et al. Analysis and prediction of protective continuous B-cell epitopes on pathogen proteins. Immunome Res. 4, 1 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. 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).

    Article  CAS  Google Scholar 

  82. Sun, P. et al. Bioinformatics resources and tools for conformational B-cell epitope prediction. Comput. Math. Methods Med. 2013, 943636 (2013).

    PubMed  PubMed Central  Google Scholar 

  83. Liepe, J. et al. A large fraction of HLA class I ligands are proteasome-generated spliced peptides. Science 354, 354–358 (2016).

    Article  CAS  PubMed  Google Scholar 

  84. 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).

    Article  CAS  PubMed  Google Scholar 

  85. Burstein, D. et al. New CRISPR–Cas systems from uncultivated microbes. Nature 542, 237–241 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Shmakov, S. et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol. Cell 60, 385–397 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Andreatta, M. & Nielsen, M. Gapped sequence alignment using artificial neural networks: application to the MHC class I system. Bioinformatics 32, 511–517 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Vita, R. et al. The immune epitope database (IEDB) 3.0. Nucleic Acids Res. 43, D405–D412 (2015).

    Article  CAS  PubMed  Google Scholar 

  90. Truong, D.-J. J. et al. Development of an intein-mediated split–Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Moreno, A. M. et al. In situ gene therapy via AAV-CRISPR-Cas9-mediated targeted gene regulation. Mol. Ther. 26, 1818–1827 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Grieger, J. C., Choi, V. W. & Samulski, R. J. Production and characterization of adeno-associated viral vectors. Nat. Protoc. 1, 1412–1428 (2006).

    Article  CAS  PubMed  Google Scholar 

  93. Schubert, M., Lindgreen, S. & Orlando, L. AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Res. Notes 9, 88 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  94. Pinello, L. et al. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat. Biotechnol. 34, 695–697 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. 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).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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

Author notes

  1. These authors contributed equally: Ana M. Moreno, Nathan Palmer.

Authors and Affiliations

  1. Department of Bioengineering, University of California San Diego, San Diego, CA, USA

    Ana M. Moreno, Genghao Chen, Andrew Pla & Prashant Mali

  2. Division of Biological Sciences, University of California San Diego, San Diego, CA, USA

    Nathan Palmer

  3. Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA

    Fernando Alemán & Mansun Law

  4. Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA

    Ning Jiang

  5. Synthetic Biology, Genome Institute of Singapore, Singapore, Singapore

    Wei Leong Chew

Authors
  1. Ana M. Moreno
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nathan Palmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fernando Alemán
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Genghao Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrew Pla
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ning Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wei Leong Chew
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mansun Law
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Prashant Mali
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Prashant Mali.

Ethics declarations

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.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

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

Reporting Summary

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-019-0431-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research