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mRNA-encoded Cas13 can be used to treat dengue infections in mice

Abstract

Dengue is a major global health threat, and there are no approved antiviral agents. Prior research using Cas13 only demonstrated dengue mitigation in vitro. Here we demonstrate that systemic delivery of mRNA-encoded Cas13a and guide RNAs formulated in lipid nanoparticles can be used to treat dengue virus (DENV) 2 and 3 in mice. First, we identified guides against DENV 2 and 3 that demonstrated in vitro efficacy. Next, we confirmed that Cas13 enzymatic activity is necessary for DENV 2 or DENV 3 mitigation in vitro. Last, we show that a single dose of lipid-nanoparticle-formulated mRNA-encoded Cas13a and guide RNA, administered 1 day post-infection, promotes survival of all infected animals and serum viral titre decreases on days 2 and 3 post-infection after lethal challenge in mice. Off-target analysis in mice using RNA sequencing showed no collateral cleavage. Overall, these data demonstrate the potential of mRNA-encoded Cas13 as a pan-DENV drug.

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Fig. 1: Schematic of the composition and mechanism of action of a mRNA-expressed anti-DENV LbuCas13a drug.
Fig. 2: Screening Lbu Cas13a crRNAs against DENV 2.
Fig. 3: RNA-seq reveals a reduced cell antiviral response with minimal off-target effects in vitro.
Fig. 4: Screening and assessment of guides against DENV 3.
Fig. 5: mRNA-expressed LbuCas13a treats DENV 2 infection in vivo.
Fig. 6: mRNA-expressed LbuCas13a treats DENV 3 infection in vivo.

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Data availability

All data supporting the findings of this study are available within the article, extended data, source data and supplementary information. RNA-seq data are available through BioProject PRJNA1101024. Source data are provided with this paper.

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Acknowledgements

We acknowledge the animal support staff at the Georgia Institute of Technology Physiological Research Laboratory (PRL) and at the GSU Department for Animal Research for their assistance with the animal experiments. We thank S. Wold for technical assistance. Figures 1a,b, 2b,e, 3a, 4b, 5a and 6a, Extended Data Fig. 1b and Supplementary Figs. 3a and 4a were made, in part, using BioRender.com. Funding for this work was provided by Defense Advanced Research Projects Agency grant HR0011-19-2-0008 (P.J.S. and M.A.B.). This study was supported in part by the Emory Integrated Computational Core (EICC), which is subsidized by the Emory University School of Medicine (RRID:SCR_023525).

Author information

Authors and Affiliations

Authors

Contributions

J.P.B., L.E.S. and J.A.W. formulated LNPs for the in vivo experiments. J.P.B. performed the LNP targeting screen. H.P. synthesized all mRNA used in the study. D.V. and L.C.S.C. designed the crRNAs. L.C.S.C. and V.M. performed the cell-free detection assay. M.B. performed cell culture infection/transfection experiments and did the IFAs. H.S. extracted RNAs and performed the qPCR assays. R.B. grew virus pools and titred virus infectivity in samples from in vitro and in vivo experiments. O.O.A. titred virus infectivity for in vitro experiments. D.V. and L.C.S.C. analysed the RNA-seq data. C.Z. acquired and analysed the dsRNA imaging data. T.T.A., H.P. and A.E. conducted mouse experiments, including virus inoculation, LNP injections and tail bleeding and performed the cytokine panels experiment. T.T.A. and A.E. bred and genotyped the A129 and AG129 mice. R.A.A. and H.C. performed analysis of RNA-seq data. D.V., L.C.S.C. and C.Z. made and edited the figures. M.K. designed the in vivo experiments at GSU and analysed the data. M.A.B. proposed targeting DENV with Cas13a, oversaw the research activities at Georgia State University and contributed to the experimental design and the writing of the manuscript. P.J.S. wrote the manuscript. C.Z. and P.J.S. oversaw the research activities at GT/Emory. All authors edited the manuscript.

Corresponding authors

Correspondence to Mukesh Kumar, Margo A. Brinton or Philip J. Santangelo.

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Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Five DENV 2 PCR primer sets were used for screening.

a) Schematic of the DENV 2 NS5 gene with the positions of the PCR primer sets (red) and the crRNA target region (grey) indicated. b) Schematic of in vitro experimental design for prophylaxis guide screening using mRNA-expressed Cas13a and crRNA against DENV 2 NGC infection (MOI = 1) in Vero cells. Cells were processed for qPCR 48 h post-infection. c) Fold change of DENV 2 genomic RNA levels in Vero cells prophylactically treated with Cas13a and the indicated guide from the virus only condition using each primer set. The bars represent the geometric mean of n = 2 independent wells per treatment. ****p < 0.0001 by two-way ANOVA with Tuckey’s multiple comparisons on log-transformed data.

Source data

Extended Data Fig. 2 Delivery of mRNA-encoded Cas13 alone or with guide 8645 G does not alter cell viability or induce cell toxicity.

Vero cells were plated at the indicated levels of confluency, transfected with appropriate amounts of Cas13 mRNA alone or Cas13 mRNA and guide 8645 G and assayed at 48 h post-transfection. Untreated cells and cells treated with staurosporine were used as control. a) CellTiter-blue reagent (Promega, #G8080) was added to the wells and after a 1 h incubation at 37 °C, fluorescence was recorded at 555/595 nm. b) CytoTox-Glo reagent (Promega, # G9291) was added to replicate wells and after a 15 min incubation at RT, lysis buffer was added, incubated for 15 min and luminescence measured. Bars represent geometric mean. n = 2 independent wells per treatment. ****p < 0.0001 by two-way ANOVA with Tuckey’s multiple comparisons.

Source data

Extended Data Fig. 3 Representative cell free assay results and dsRNA detection by IFA in infected cells for the control treatment groups.

a) Raw fluorescence reads over time from the cell-free detection assay using Cas13a, dCas13a, or GFP protein and crRNA. Data represented as mean of n = 3 wells per condition. Bars indicate mean ± SD. Vero cells were infected with DENV2 (MOI = 0.1) for 24 h, transfected with the indicated mRNA and crRNA and processed for dsRNA staining at 72 h post-infection b) Representative dsRNA staining (white) of DENV 2 infected Vero cells treated with Cas13a, dCas13a, or GFP mRNA (green) and the indicated crRNA. Scale bar represents 150 µm.

Source data

Extended Data Fig. 4 Absolute RNAseq read coverage of DENV 2 genome RNA.

Vero cells were infected with DENV2 (MOI = 1) for 24 h, transfected with the indicated mRNA and crRNA and processed for total RNA extraction at 72 h post-infection. Coverage of RNAseq reads for DENV 2 NGC (a) polyprotein ORF and the (b) NS5 gene region.

Source data

Extended Data Fig. 5 Comparison of differential gene expression analyses for the additional control groups.

Vero cells were infected with DENV2 (MOI = 1) for 24 h, transfected with the indicated mRNA and crRNA and processed for total RNA extraction at 72 h post-infection. Differential gene expression analysis of Cas13a + 8645 G treatment compared to the dCas13a + 8645 G treatment group (top), of Cas13a + 8645 G treatment compared to the GFP + 8645 G treatment group (middle), and of the virus only control compared to the mock-infected group (bottom). Genes are indicated as significantly up (red) or down (blue) regulated or with no significant difference (gray). Vertical lines indicate fold change of ±2. Horizontal line indicates p-value of 0.05. Fold-changes and p- values by two-sided Wald Test with multiple comparison adjustment are listed in Supplemental Tables 57, respectively.

Source data

Extended Data Fig. 6 Viral RNA, cytokine expression and viral titers are undetectable upon treatment with mRNA-expressed Cas13a in mice surviving DENV lethal challenges.

a) Viral RNA in the indicated organs and b) cytokines levels in the brain from the DENV 2 mouse experiment in Fig. 5 were measured by qPCR and at the indicated timepoints post-infection. Treated A129 mice (n = 6) received 2 mg/kg LNP-formulated Cas13a and guide 8645 G and were euthanized 14 days after infection. Untreated, infected mice that received PBS (n = 5) were euthanized at day 6 post infection (Supp. Table 11). The control group (n = 5) refers to naïve mice. Bars indicate mean ± SD. c) Viral RNA in the indicated organs from the DENV 2 mouse experiment in Fig. 6 were measured by qPCR and at the indicated timepoints post-infection. Treated AG129 mice (n = 8) received 2 mg/kg LNP-formulated Cas13a and guide 8645 G and were euthanized 7 days after infection. Untreated, infected mice that received PBS (n = 12) were euthanized at day 4 post infection (Supp. Table 13). The control group (n = 6) refers to naïve mice. Bars indicate mean ± SD. p-values for each comparison are listed in Supplemental Table 2 as determined by one-way ANOVA with Tuckey’s multiple comparisons.

Source data

Extended Data Fig. 7 Changes in cytokine expression in serum during DENV 3 infection and treatment with mRNA-expressed Cas13a in AG129 mice.

Cytokine and chemokine levels in serum samples from the DENV 3 mouse experiment in Fig. 6 were measured using a MILLIPLEX Mouse Cytokine/Chemokine Magnetic Bead Panel. Expression levels of the indicated cytokines were assayed in sera collected at day 3 and day 7 after delivery of mRNA-encoded Cas13 and guide 8645 G. Treated mice (n = 9) received 2 mg/kg LNP-formulated Cas13a and guide 8645 G. Control mice were infected and received PBS or were untreated. Bars indicate mean ± SD. p-values for each comparison are listed in Supplemental Table 2 as determined by one-way ANOVA with Tuckey’s multiple comparisons.

Source data

Supplementary information

Supplementary Information

Supplementary results, discussion and Figs. 1–4.

Reporting Summary

Supplementary Tables 1–18

Supplementary Tables 1–18.

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Basu, M., Zurla, C., Auroni, T.T. et al. mRNA-encoded Cas13 can be used to treat dengue infections in mice. Nat Microbiol (2024). https://doi.org/10.1038/s41564-024-01726-6

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