Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing

Abstract

CRISPR–Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and gene correction therapeutics in targeted tissues.

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Fig. 1: SORT allows LNPs to be systematically and predictably engineered to accurately deliver mRNA into specific organs.
Fig. 2: SORT relies on general biophysical properties and not exact chemical structures to deliver mRNAs encoding for therapeutically relevant proteins.
Fig. 3: SORT LNPs enabled tissue-specific tdTom activation by Cre mRNA delivery.
Fig. 4: SORT LNPs mediated tissue-specific CRISPR–Cas gene editing of tdTom transgenic mice and C57/BL6 wild-type mice by delivering Cas9 RNPs and co-delivering Cas9 mRNA and sgRNA.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

References

  1. 1.

    Doudna, J. A. & Charpentier, E. Genome editing: the new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

    Google Scholar 

  2. 2.

    Wang, H. X. et al. CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery. Chem. Rev. 117, 9874–9906 (2017).

    CAS  Google Scholar 

  3. 3.

    Sander, J. D. & Joung, J. K. CRISPR–Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    CAS  Google Scholar 

  4. 4.

    Hajj, K. A. & Whitehead, K. A. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2, 17056 (2017).

    CAS  Google Scholar 

  5. 5.

    Sahin, U., Kariko, K. & Tureci, O. mRNA-based therapeutics: developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014).

    CAS  Google Scholar 

  6. 6.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Google Scholar 

  7. 7.

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

    CAS  Google Scholar 

  8. 8.

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

    CAS  Google Scholar 

  9. 9.

    Kanasty, R., Dorkin, J. R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967–977 (2013).

    CAS  Google Scholar 

  10. 10.

    Wood, H. FDA approves patisiran to treat hereditary transthyretin amyloidosis. Nat. Rev. Neurol. 14, 570 (2018).

    Google Scholar 

  11. 11.

    Jayaraman, M. et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem. Int. Ed. 51, 8529–8533 (2012).

    CAS  Google Scholar 

  12. 12.

    Nelson, C. E. et al. Balancing cationic and hydrophobic content of PEGylated siRNA polyplexes enhances endosome escape, stability, blood circulation time, and bioactivity in vivo. ACS Nano 7, 8870–8880 (2013).

    CAS  Google Scholar 

  13. 13.

    Hao, J. et al. Rapid synthesis of a lipocationic polyester library via ring-opening polymerization of functional valerolactones for efficacious siRNA delivery. J. Am. Chem. Soc. 137, 9206–9209 (2015).

    CAS  Google Scholar 

  14. 14.

    Miller, J. B. et al. Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA. Angew. Chem. Int. Ed. 56, 1059–1063 (2017).

    CAS  Google Scholar 

  15. 15.

    Kranz, L. M. et al. Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 534, 396–401 (2016).

    Google Scholar 

  16. 16.

    Wilhelm, S. et al. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 1, 16014 (2016).

    CAS  Google Scholar 

  17. 17.

    Gustafson, H. H., Holt-Casper, D., Grainger, D. W. & Ghandehari, H. Nanoparticle uptake: the phagocyte problem. Nano Today 10, 487–510 (2015).

    CAS  Google Scholar 

  18. 18.

    Fehring, V. et al. Delivery of therapeutic siRNA to the lung endothelium via novel lipoplex formulation DACC. Mol. Ther. 22, 811–820 (2014).

    CAS  Google Scholar 

  19. 19.

    Dahlman, J. E. et al. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight. Nat. Nanotechnol. 9, 648–655 (2014).

    CAS  Google Scholar 

  20. 20.

    Fenton, O. S. et al. Synthesis and biological evaluation of ionizable lipid materials for the in vivo delivery of messenger RNA to B lymphocytes. Adv. Mater. 29, 1606944 (2017).

    Google Scholar 

  21. 21.

    Kowalski, P. S. et al. Ionizable amino-polyesters synthesized via ring opening polymerization of tertiary amino-alcohols for tissue selective mRNA delivery. Adv. Mater. 30, e1801151 (2018).

    Google Scholar 

  22. 22.

    Sago, C. D. et al. High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing. Proc. Natl Acad. Sci. USA 115, E9944–E9952 (2018).

    CAS  Google Scholar 

  23. 23.

    Kaczmarek, J. C. et al. Polymer-lipid nanoparticles for systemic delivery of mRNA to the lungs. Angew. Chem. Int. Ed. 55, 13808–13812 (2016).

    CAS  Google Scholar 

  24. 24.

    Yan, Y., Xiong, H., Zhang, X., Cheng, Q. & Siegwart, D. J. Systemic mRNA delivery to the lungs by functional polyester-based carriers. Biomacromolecules 18, 4307–4315 (2017).

    CAS  Google Scholar 

  25. 25.

    Kazi, D. S. et al. Cost-effectiveness of PCSK9 inhibitor therapy in patients with heterozygous familial hypercholesterolemia or atherosclerotic cardiovascular disease. JAMA 316, 743–753 (2016).

    CAS  Google Scholar 

  26. 26.

    Wittrup, A. et al. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat. Biotechnol. 33, 870–976 (2015).

    CAS  Google Scholar 

  27. 27.

    Cheng, Q. et al. Dendrimer-based lipid nanoparticles deliver therapeutic FAH mRNA to normalize liver function and extend survival in a mouse model of hepatorenal tyrosinemia type I. Adv. Mater. 30, e1805308 (2018).

    Google Scholar 

  28. 28.

    Zhou, K. et al. Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc. Natl Acad. Sci. USA 113, 520–525 (2016).

    CAS  Google Scholar 

  29. 29.

    Zhang, S. et al. Knockdown of anillin actin binding protein blocks cytokinesis in hepatocytes and reduces liver tumor development in mice without affecting regeneration. Gastroenterology 154, 1421–1434 (2018).

    CAS  Google Scholar 

  30. 30.

    Zhang, S. et al. The polyploid state plays a tumor suppressive role in the liver. Dev. Cell 44, 447–459 (2018).

    CAS  Google Scholar 

  31. 31.

    Akinc, A. et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat. Nanotechnol. 14, 1084–1087 (2019).

    CAS  Google Scholar 

  32. 32.

    Sabnis, S. et al. A novel amino lipid series for mRNA delivery: improved endosomal escape and sustained pharmacology and safety in non-human primates. Mol. Ther. 26, 1509–1519 (2018).

    CAS  Google Scholar 

  33. 33.

    Hassett, K. J. et al. Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines. Mol. Ther. - Nucl. Acids 15, 1–11 (2019).

    CAS  Google Scholar 

  34. 34.

    Ramaswamy, S. et al. Systemic delivery of factor IX messenger RNA for protein replacement therapy. Proc. Natl Acad. Sci. USA 114, E1941–E1950 (2017).

    CAS  Google Scholar 

  35. 35.

    Love, K. et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl Acad. Sci. USA 107, 1864–1869 (2010).

    CAS  Google Scholar 

  36. 36.

    Kauffman, K. J. et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett. 15, 7300–7306 (2015).

    CAS  Google Scholar 

  37. 37.

    Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR–Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).

    CAS  Google Scholar 

  38. 38.

    Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328–333 (2016).

    CAS  Google Scholar 

  39. 39.

    Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179 (2017).

    CAS  Google Scholar 

  40. 40.

    Wang, L. et al. Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol. Nat. Biotechnol. 36, 717–725 (2018).

    CAS  Google Scholar 

  41. 41.

    Amoasii, L. et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science 362, 86–91 (2018).

    CAS  Google Scholar 

  42. 42.

    Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

    CAS  Google Scholar 

  43. 43.

    Sun, W. J. et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew. Chem. Int. Ed. 54, 12029–12033 (2015).

    CAS  Google Scholar 

  44. 44.

    Staahl, B. T. et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat. Biotechnol. 35, 431–434 (2017).

    CAS  Google Scholar 

  45. 45.

    Finn, J. D. et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22, 2227–2235 (2018).

    CAS  Google Scholar 

  46. 46.

    Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016).

    CAS  Google Scholar 

  47. 47.

    Kamath, A. T. et al. The development, maturation, and turnover rate of mouse spleen dendritic cell populations. J. Immunol. 165, 6762–6770 (2000).

    CAS  Google Scholar 

  48. 48.

    Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).

    CAS  Google Scholar 

  49. 49.

    Monopoli, M. P., Aberg, C., Salvati, A. & Dawson, K. A. Biomolecular coronas provide the biological identity of nanosized materials. Nat. Nanotechnol. 7, 779–786 (2012).

    CAS  Google Scholar 

  50. 50.

    Akinc, A. et al. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther. 18, 1357–1364 (2010).

    CAS  Google Scholar 

  51. 51.

    Liu, J. J. et al. CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566, 218–223 (2019).

    CAS  Google Scholar 

  52. 52.

    Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR–Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).

    CAS  Google Scholar 

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Acknowledgements

D.J.S. acknowledges financial support from the National Institutes of Health (NIH) National Institute of Biomedical Imaging and Bioengineering (NIBIB) (grant no. R01 EB025192-01A1), the Cystic Fibrosis Foundation (CFF) (grant no. SIEGWA18XX0), the American Cancer Society (ACS) (grant no. RSG-17-012-01) and the Welch Foundation (grant no. I-1855). We acknowledge the UTSW Tissue Resource, supported in part by the National Cancer Institute (grant no. 5P30CA142543), the Moody Foundation Flow Cytometry Facility and the UTSW Proteomics Core. We thank Y. Jia, Y.-H. Lin, Y. Wei and H. Zhu for assistance with tissue processing and analyses.

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Q.C., T.W. and D.J.S. conceived and designed the experiments and wrote the manuscript. Q.C., T.W., L.F., L.T.J. and S.A.D. performed experiments. All authors discussed the results and commented on the manuscript. D.J.S. directed the research.

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Correspondence to Daniel J. Siegwart.

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Competing interests

D.J.S., Q.C., T.W. and the Reagents of the University of Texas System have filed patent applications on SORT and related technologies. D.J.S. is a co-founder of ReCode Therapeutics, which has licensed intellectual property from UT Southwestern.

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Peer review information Nature Nanotechnology thanks Roy van der Meel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Cheng, Q., Wei, T., Farbiak, L. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol. 15, 313–320 (2020). https://doi.org/10.1038/s41565-020-0669-6

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