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Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver

Nature Biomedical Engineering volume 6, pages 157–167 (2022)Cite this article

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Abstract

Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.

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Fig. 1: Synthetic antisense oligonucleotides termed iOligos reduce Cas9 activity in cell culture.
Fig. 2: iOligo reduces gene editing by interacting with sgRNA.
Fig. 3: iOligo controls systemic gene editing in vivo.
Fig. 4: siRNA-mediated reduction of Cas9 expression controls gene editing in vitro and in vivo.
Fig. 5: Combining iOligo- and siGFP-based approaches markedly reduces cell-type-specific Cas9 gene editing.
Fig. 6: iOligo-based approaches reduced cell-type-specific Cas9 gene editing in models of lung inflammation.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, but they are available for research purposes from the corresponding author on reasonable request.

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Acknowledgements

We thank J. E. Cattie at Emory University and T. E. Shaw.

Author information

Author notes

  1. Cory D. Sago

    Present address: Beam Therapeutics, Atlanta, GA, USA

  2. Melissa P. Lokugamage

    Present address: Alloy Therapeutics, Lexington, MA, USA

  3. These authors contributed equally: Cory D. Sago, Melissa P. Lokugamage.

Authors and Affiliations

  1. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA

    Cory D. Sago, Melissa P. Lokugamage, David Loughrey, Kevin E. Lindsay, Brandon R. Krupczak, Sujay Kalathoor, Manaka Sato, Elisa Schrader Echeverri, Jordan P. Fitzgerald, Zubao Gan, Lena Gamboa, Kalina Paunovska, Marine Z. C. Hatit, Philip J. Santangelo & James E. Dahlman

  2. School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA

    Robert Hincapie, Carlos A. Sanhueza & M. G. Finn

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Contributions

C.D.S. and J.E.D. conceived the experiments. C.D.S., P.J.S., M.G.F. and J.E.D. designed the experiments. C.D.S., M.P.L., D.L., K.E.L., R.H., B.R.K., S.K., M.S., E.S.E., J.P.F., Z.G., L.G., K.P., C.A.S. and J.E.D. performed the experiments. C.D.S. and J.E.D. wrote the initial draft of the paper, which was edited by all authors.

Corresponding author

Correspondence to James E. Dahlman.

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Competing interests

Patents describing the system documented in this Article have been filed with the US Patent Office. C.D.S. and J.E.D. are listed as inventors on patent (International publication no. WO2021021636A1). C.D.S. works at Beam Therapeutics. J.E.D. consults for GV. All other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 iOligo variants also inhibit Cas12a-mediated gene editing.

(a,b) Sequence and chemical modifications for crGFP and (b) previously reported full-length 41 nucleotide antisense sequence targeting Cas12a. (c) The 41 nucleotide sequence Cas12a reduced indel formation at doses of 150 nM and 50 nM when delivered to cells before crGFP and Cas12a mRNA transfection. (d) Schematic of the proposed toehold mechanism for iOligo targeting Cas12a. The iOligo Cas12a is designed to complement to full- length crGFP RNA, providing the 5’ toe-hold and disrupting RNA secondary structure. (e) The sequences of the truncated iOligo Cas12a, and proposed binding to complementary regions on crGFP. The linear region of the crRNA is required to mediate a strand displacement reaction and facilitate iOligo binding. (F,G) Normalized indels in HEK293T cells after treatment with varying concentrations of full-length and truncated versions of iOligo Cas12a, before crGFP and Cas12a mRNA transfection at (f) 80 nM iOligo, *p = 0.015, **p = 0.008, and (g) 40 nM iOligo, *p = 0.022, **p = 0.0026, one-way ANOVA. All error bars show the average + /- SEM.

Extended Data Fig. 2 iOligo chemical modifications affect gene editing.

(a) Sequence and chemical modifications patterns for iOligo D with various modification patterns. (b) Normalized indel inhibition of various chemical modification patterns as compared to fully 2’ O-methyl, fully phosphorothioated iOligo-D. (c) Sequence and chemical modifications patterns for iOligo D with 2’ O-methyl and 2’ Methoxyethyl (MOE). (d) Normalized indels of 2’ O-methyl and 2’ Methoxyethyl modified iOligo. All error bars show the average + /- SEM.

Extended Data Fig. 3 siRNA-mediated reduction of Cas9 expression controls gene editing in vitro and in vivo.

(a) Engineered 3’ UTR with 5 siGFP-binding sites. An engineered luciferase-encoding mRNA with the custom 3’ UTR will be degraded in the presence of siGFP, leading to decreased luciferase protein production as measured by luminescence. (b) The engineered luciferase-encoding mRNA with the custom 3’ UTR led to dose-dependent normalized expression in the presence of siGFP, compared to cells treated with siICAM-2. (c) Mice were pre- treated with either siGFP or siICAM-2 delivered by a hepatocyte-trophic LNP. 14 hours later, the engineered luciferase mRNA was delivered by a hepatocyte LNP. Liver luminescence is measured ex vivo. (d) Normalized ex vivo luminescence the liver from mice pretreated with either siICAM- 2 or siGFP. All error bars show the average + /- SEM.

Extended Data Fig. 4 iOligo-based approaches can be used to reduce cell type-specific Cas9 gene editing in models of lung inflammation.

(a) The percentage of ICAM-2 indels in hepatocytes and (b) lung endothelial cells following treatment of Ova, iOligo / Ctrl, and sgICAM-2 / sgCtrl, *p = 0.04, **p = 0.009 one-way ANOVA, average + /- SEM.

Supplementary information

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Supplementary Table 1

In vivo editing in specific cell types.

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Sago, C.D., Lokugamage, M.P., Loughrey, D. et al. Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver. Nat Biomed Eng 6, 157–167 (2022). https://doi.org/10.1038/s41551-022-00847-9

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