High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing

Abstract

Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein. Using this system (named FIND), we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells. The 7C3 delivered Cas9 mRNA and sgRNA to splenic endothelial cells as efficiently as hepatocytes, distinguishing it from LNPs that deliver Cas9 mRNA and sgRNA to hepatocytes more than other cell types. These data demonstrate that FIND can identify nanoparticles with novel tropisms in vivo.

Keywords: CRISPR; RNAi; barcoded nanoparticle; mRNA; nanoparticle.

Fig. 1.

FIND is a high-throughput screen for functional mRNA delivery. (A) Unlike previous biodistribution screens, which cannot distinguish between bound particles, particles stuck in endosomes, and particles that delivered RNA into the cytoplasm, FIND identifies LNPs that functionally deliver RNA. (B and C) Nanoparticles were formulated to carry Cre mRNA and a DNA barcode, before they were administered to Cre reporter cell lines or mice. Cells that underwent Cre-mediated genetic changes were isolated using FACS, and the DNA barcode was sequenced to identify the LNP that delivered the mRNA. (D) LNP barcodes were ranked by “normalized delivery”; each sample (e.g., lung 1 vs. heart 1) was analyzed individually on a single sequencing run. Using these data, we would hypothesize that LNP-1 delivered nucleic acids more efficiently to the lungs than LNP-2 and LNP-N. (E) LGSL-R cells were treated with naked Cre mRNA or Cre mRNA carrried by Lipofectamine 2000 (L2K). RFP expression indicates cytoplasmic Cre mRNA delivery. (F) Alexa 647 and RFP intensities after treatment with L2K carrying Cre mRNA and Alexa 647-labeled DNA barcode. Compared with untreated cells, there are no Alexa 647-RFP+ cells, demonstrating that biodistribution is necessary, but not sufficient, for cytoplasmic delivery. (G) RFP+ HEK cells as a function of the administered Cre mRNA, which was delivered with L2K. (H) Normalized DNA barcode delivery for 54 LNPs sequenced from RFP+ HEK cells after the administration of 10 ng or 100 ng total mRNA. A high degree of correlation between samples suggests that LNPs which deliver mRNA at the first dose deliver mRNA at the second dose. (I) RFP+ HEK cells following the administration of 54 LNPs (100 ng total mRNA), after cells were treated with endocytosis inhibitors. n = 3–4 wells per group. **P < 0.01, ****P < 0.0001, two-tailed t test.

Fig. 2.

FIND can quantify LNP delivery in vivo. (A) Cre recombinase leads to the deletion of a stop codon allowing for the expression of tdTomato driven by a CAG promoter. (B) Libraries of LNPs containing Cre mRNA and DNA barcodes are administered to LSL-tdTom (Ai14) mice. tdTomato+ cells are isolated by FACS and DNA barcodes are sequenced. (C) Screen 1 consisted of a library of 112 distinct LNPs created by formulating seven compounds with cholesterol, DOPE, and C14-PEG2000 at 16 different mole ratios. (D) DLS analysis of all 112 LNPs from this library; 71 formed stable LNPs and were included. (E) Normalized DNA delivery in kidney and lung endothelial cells (CD31+CD45−) after LNPs were i.v. injected, as well as CD45+ and CD45− cells isolated following intramuscular injection. (F) In vivo LNP targeting heat map generated by unbiased, Euclidean clustering. In vitro LNP delivery, in vivo intramuscular delivery, and in vivo i.v. delivery cluster separately, as expected.

Fig. 3.

Characterization of lead nanoparticles discovered by FIND. (A) The top two particles, 7C2 and 7C3, discovered following three rounds of FIND are characterized by measuring biodistribution, delivery of siRNA, sgRNA. (B) The 7C2 and 7C3 were composed of 7C1 compound, cholesterol, C14-PEG2000, and helper lipids, 18:1 Lyso PC and DOPE, respectively. (C) Following a 0.75-mg/kg Barcode-Cy5.5 administration, biodistribution of 7C2 and 7C3 was quantified and normalized to tissue weight. n = 4 miceper group. *P < 0.05, **P < 0.01, two-way ANOVA. (D) Following a 1-mg/kg siICAM2 administration, both 7C2 and 7C3 induced ICAM2 protein silencing in endothelial cells by flow cytometry. The 7C2 demonstrates more robust protein silencing in lung endothelial cells than 7C3. n = 4 mice per group. **P < 0.01, two tailed t test. (E–G) Following repeat administration of 7C2 and 7C3 at 1.5 mg/kg sgICAM2a and sgICAM2b, ICAM2 (E) protein and (F and G) indels were measured in endothelial cells isolated from multiple organs using FACS. n = 3–4 mice per group. *P < 0.05, **P < 0.01, two-tailed t test.

Fig. 4.

(A) The top two particles, 7C2 and 7C3, are characterized by delivery of Cre mRNA and codelivery of Cas9 mRNA and sgRNA. (B) Percentage of tdTomato+ endothelial cells in LSL-Tom mice 72 h after a single 1.5-mg/kg injection of Cre mRNA delivered by 7C2 or 7C3. n = 4 mice per group. **P < 0.01, two-tailed t test. (C) Percentage of tdTomato+ splenic endothelial cells and hepatocytes in LSL-Tom mice following a single 1.5-mg/kg injection of Cre mRNA delivered by 7C3 from eight independent experiments representing 34 mice. (D) Percentages from all eight experiments of tdTomato+ splenic endothelial cells and hepatocytes in LSL-Tom mice following a single 1.5-mg/kg injection of Cre mRNA delivered by 7C3. n = 34. ****P < 0.0001, two-tailed t test. (E) Indel percentage in splenic ECs following two injections of 7C3 carrying Cas9 mRNA and e-sgICAM2 at a mass ratio of 1:1, 3:1, and 5:1. (F) Indel percentage in hepatocytes following two injections of 7C3 carrying Cas9 mRNA and e-sgICAM2 at a mass ratio of 1:1, 3:1, and 5:1.