Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing

Abstract

The expanding applications of nonviral genomic medicines in the lung remain restricted by delivery challenges. Here, leveraging a high-throughput platform, we synthesize and screen a combinatorial library of biodegradable ionizable lipids to build inhalable delivery vehicles for messenger RNA and CRISPR-Cas9 gene editors. Lead lipid nanoparticles are amenable for repeated intratracheal dosing and could achieve efficient gene editing in lung epithelium, providing avenues for gene therapy of congenital lung diseases.

Fig. 1 |. Overview of the lipid nanoparticle synthesis and screening.

a, A illustration shows the three-component reaction (3-CR) of amine (headgroup), ricinoleic acrylate (linker), and alcohols (tail) for the high-throughput synthesis of lipids. b, The synthesis route of carbonate-based ionizable biodegradable lipids from Nitro Ricinoleic Acrylate (NRA). c, Structures of amine headgroups (72) and alcohol tails (10) in a combinatorial library of 720 biodegradable ionizable lipids. d, A schematic to illustrate the formulation of a newly designed RCB-4-8 LNP for pulmonary delivery of Cre-mRNA and CRISPR-Cas9 gene-editing tools. e, A549 cells were treated with mLuc-loaded LNPs. The relative luciferase expression after incubating with mLuc LNPs overnight is shown in a heat map. f, The hit rate of each amine headgroup was calculated by the percentage of LNPs with luciferase expression > 50,000. g-j, Results of the in vivo batch-based testing, where batch 1 analysis (g) determined the top-performing amine headgroup structures (intramuscular injection, 0.5 mg/kg mLuc, 10 LNP mixtures per mouse); batch 2 analysis (h) identified the top-performing lipid tail structures (intramuscular injection, 0.4 mg/kg, 8 LNP mixtures per mouse); batch 3 analysis (i) identified the individual lipids with top-performing mRNA delivery efficiency in the muscle (intramuscular injection, 0.25 mg/kg mLuc per mouse). Finally, batch 4 analysis (j) identified the individual lipids with top-performing mRNA delivery efficiency in the lung (intratracheal administration, 0.25 mg/kg mLuc per mouse). For all in vivo screening studies in c–f, results were obtained from two mice per group and presented as mean ± SD. k, Representative IVIS images of mouse lungs at 6 hr following intratracheal administration of MC3 or RCB-4-8 mLuc LNPs (0.125, 0.25, 0.5 mg/kg mLuc per mouse).

Fig. 2 |. Gene editing in mouse lung with RCB-4-8 LNP-mRNA.

a, Measuring RCB-4-8 LNP-Cre-mRNA mediated editing using Lox-STOP-Lox tdTomato (Ai9) reporter mice. The Cre recombinase will delete the STOP cassettes and activate the tdTomato reporter. b, Ai9 mice were intratracheally administered with either one or three doses (over four days) of Cre-mRNA. To study the impact of AAV5 immunogenicity on the LNP, we delivered 6×10¹⁰ AAV5 with a dummy sequence in the mouse lung one week before dosing LNP-mRNA. Three days after the last dose, the lungs were collected and analyzed by flow cytometry (n=3 mice for each group). Results were obtained from three mice and presented as mean ± SD. c, Representative native fluorescence images of lung sections are shown. Results were obtained from 3 independent experiments. Saline serves as a negative control. Scale bar: 100μm. d, Paraffin-embedded lung sections were stained with antibodies for tdTomato. e, Quantification of IHC in d. Results were obtained from three mice (n=5 sections per mouse) and presented as mean ± SD. ***P<0.001 by one-way ANOVA with Tukey’s multiple comparisons test. f. Genomic DNA was collected from mice (n=3/group) treated with LNP-Cre mRNA or controls. PCR was performed with primers flanking the Ai9 locus. WT: wide type. M, marker. g, Quantification of tdTomato⁺ club cells. Results were obtained from three mice (3 airways per mouse) and presented as mean ± SD. h, Quantification of tdTomato⁺ ciliated cells. Results were obtained from three mice (3 airways per mouse) and presented as mean ± SD. i, Measuring RCB-4-8 LNP-SpCas9-mRNA/sgRNA mediated NHEJ using Ai9 reporter mice. The sgAi9 or sgA and sgB will delete the STOP cassettes and activate the tdTomato reporter. Ai9 mice were intratracheally administered with low or high doses of SpCas9-mRNA/sgAi9. To develop a combined viral and non-viral delivery of CRISPR system components in vivo, we intratracheally delivered 6×10¹⁰ scAAV5-sgA-sgB-GFP one week before dosing LNP-SpCas9-mRNA. Three days after the last dose, the lungs were collected. j-k, Quantification of tdTomato positive cells (k) and representative native fluorescence images of lung sections (j) are shown. LNP_high-SpCas9mRNA serves as a negative control. Scale bar: 100μm. n=9 sections from 3 mice. Error bars are S.D. Results were obtained from three mice (3 airways per mouse) and presented as mean ± SD.