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

Results

FIND combined rationally designed DNA barcodes and the Cre-Lox system to generate a multiplexed readout of functional mRNA delivery. FIND is distinct from previous barcoding systems we developed, which measure biodistribution (Fig. 1A). Using high-throughput microfluidics (26), we coformulated LNPs with Cre mRNA and a unique DNA barcode. LNP-1, with chemical structure 1, carried DNA barcode 1 and Cre mRNA; LNP-N, with chemical structure N, carried DNA barcode N and Cre mRNA (Fig. 1 A–C). We characterized the size and stability of each LNP using dynamic light scattering (DLS) and discarded LNPs that did not meet two criteria: (i) diameters between 20 and 200 nm and (ii) an autocorrelation curve with one inflection point (SI Appendix, Fig. S1A). LNPs that met these criteria were pooled together and administered to Lox-Stop-Lox-tdTomato (Ai14) mice (27). Ai14 cells fluoresce if Cre mRNA is translated into Cre protein, which then translocates to the nucleus and edits target DNA. We isolated tdTomato⁺ cells with FACS and deep-sequenced the cells to identify LNPs that delivered mRNA. We reasoned the codelivery of mRNA and a 56-nt ssDNA could approximate the codelivery of an mRNA encoding a nuclease and guide RNA (28⇓–30). After sequencing tdTomato⁺ cells (Fig. 1C), we ranked LNPs by calculating the “normalized delivery” of each barcode. Normalized delivery is analogous to counts per million in RNA-sequencing data (Fig. 1D and SI Appendix, Fig. S1B). We used barcodes we previously described and validated with a number of control experiments (SI Appendix, Fig. S1 C–E) (17, 20, 21).

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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.

We characterized FIND using in vitro and in vivo experiments. First, we cultured HEK.293 cells that expressed LoxP-GFP-Stop-LoxP-RFP (LGSL-RFP) under a CMV promoter (Fig. 1B). These cells became RFP⁺ 72 h after treatment with Cre mRNA carried by Lipofectamine 2000 (L2K), but not with naked Cre mRNA, which served as the negative control (Fig. 1E and SI Appendix, Fig. S1F). The number of RFP⁺ cells after L2K treatment increased with dose and time, up to 3 d (SI Appendix, Fig. S1G). We then coformulated L2K with Cre mRNA and an Alexa 647-labeled DNA barcode. After 24 h, 62, 36, 2, and 0% of the cells were 647⁺RFP⁻, 647⁺RFP⁺, 647⁻RFP⁻, and 647⁻RFP⁺, respectively. This indicates that biodistribution is required, but not sufficient for functional cytosolic delivery, as we expected (Fig. 1F). Untreated cells—used to control for autofluorescence—were 647⁻RFP⁻ (Fig. 1F and SI Appendix, Fig. S1H).

We then formulated 54 chemically distinct LNPs (SI Appendix, Fig. S1 I and J) carrying Cre mRNA and a unique DNA barcode. We administered the LNPs to LGSL-RFP cells with mRNA doses of 10, 100, or 1,000 ng and an mRNA:DNA barcode mass ratio of 10:1. We observed a dose-dependent increase in RFP⁺ cells, with over 80% of the cells RFP⁺ 3 d after the 1,000-ng transfection (Fig. 1G). We sequenced barcodes at all three doses, reasoning that barcode delivery at the lowest dose (4% RFP⁺ cells) would predict delivery at the middle dose (20% RFP⁺ cells); we observed a strong correlation (R² > 0.9) between normalized delivery at 10-ng and 100-ng doses, suggesting this was the case (Fig. 1H). As a control, we sequenced cells treated with 1,000 ng mRNA; in this case, the system was saturated (>80% of the cells were RFP⁺). As expected, the correlation between 1,000 ng and either 10 or 100 ng was weaker (SI Appendix, Fig. S1K) than the correlation between the two lower doses; importantly, these data indicate that FIND can become saturated as the percent of RFP⁺ increases. Therefore, varying the dose by the potency of the nanoparticles pooled may be critical. We then evaluated whether the number of RFP⁺ cells decreased after pretreatment with chlorpromazine, genistein, or ethylisopropyl amiloride (EIPA), which inhibit clathrin-, caveolin-, and macropinocytosis-mediated endocytosis, respectively. Compared with control cells that were not treated with inhibitors, RFP⁺ cells decreased by 40–60%, recapitulating results previously obtained with individual LNPs (24, 31) (Fig. 1I). Taken together, these results led us to conclude that FIND could analyze mRNA delivery in vitro.

We investigated whether FIND could identify nanoparticles that deliver mRNA in vivo (Fig. 2 A and B). We formulated 112 LNPs, varying four chemical traits that influence LNP delivery in vitro: the lipid-amine compound, the molar amount of PEG, the structure of PEG, and the molar amount of cholesterol (Fig. 2C and SI Appendix, Fig. S2 A and B). Seventy-one LNPs met our two criteria for inclusion and were pooled (Fig. 2D). Naked DNA barcode, which should not be delivered as efficiently as DNA barcodes in LNPs, was included as a negative control. We i.v. injected the pooled LNPs and naked barcode into Ai14 mice at a 1.5 mg/kg total mRNA dose (average dose 0.021 mg/kg per particle). Three days later, we isolated tdTomato⁺ lung and kidney endothelial cells (tdTomato⁺CD31⁺CD45⁻). Separately, we injected 1.0 mg/kg total mRNA into mice intramuscularly and isolated immune (tdTomato⁺CD45⁺) and nonimmune (tdTomato⁺CD45⁻) cells from muscle. We gated on PBS-injected Ai14 mice for i.v. injected mice and the contralateral limb for intramuscular administration. It was important to gate on untreated Ai14 mice instead of C57BL/6J mice; gating on C57BL/6J mice could lead to inflated values of tdTomato⁺ cells (SI Appendix, Fig. S2C). Separately, we administered the same LNPs to LGSL-RFP cells in vitro. Several lines of evidence suggested the data were robust. In all routes of administration, the naked barcode was delivered less efficiently than every LNP (Fig. 2E). Second, 7C1-based LNPs, which we previously optimized for in vivo RNA delivery (32), were enriched in the top 20% LNPs, compared with the other six lipid-amine compounds. (SI Appendix, Fig. S2 D and E). Finally, we reasoned that the route of administration would affect delivery; Euclidean clustering, a common bioinformatics technique (33) that compares how similar/dissimilar groups are to one another, and can be used to study nanoparticles in vivo (17, 20), separated intravenous, intramuscular, and in vitro delivery into three distinct clusters, as expected (Fig. 2F).

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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.

We formulated a second LNP library to study how PEG, cholesterol, and helper lipids [e.g., dioleoylphosphatidylethanolamine (DOPE)] influence delivery to cells in vivo. We focused on 7C1-based LNPs, since 7C1 was enriched in the first screen. We varied the PEG molar amount, as well as the alkyl length on the PEG-lipid (SI Appendix, Fig. S2F). Seventy-eight out of 108 LNPs met our two criteria for inclusion; we i.v. administered the pool to the mice at a total mRNA dose of 1.5 mg/kg. Three days later, we sequenced barcodes from tdTomato⁺ lung and kidney endothelial cells. LNPs containing C₁₄ alkyl PEG were enriched in lung endothelial cells, whereas LNPs containing C₁₈ alkyl PEG were enriched in kidney endothelial cells (SI Appendix, Fig. S2 G and H). To validate the relationship between lung delivery, kidney delivery, and PEG structure, we formulated a third LNP library. We formulated 158 LNPs designed to improve lung delivery relative to kidney delivery by only using C₁₄ alkyl-tail PEG varying helper lipid composition (SI Appendix, Fig. S2I). We found 3.75 times more lung endothelial cells were tdTomato⁺ than kidney endothelial cells, a ratio that was significantly higher than the second screen (SI Appendix, Fig. S2J), as expected. Consistent with previous results (17, 20), we did not observe a relationship between LNP size and delivery to endothelial cells between the range of 20–200 nm (SI Appendix, Fig. S2K). The poor performance of the naked barcode, enrichment of 7C1, the fact that unbiased clustering separated the three routes of administration, and the PEG data convinced us FIND was capable of quantifying delivery in vivo.

After completing these in vitro and in vivo validation experiments, we selected two LNPs (named 7C2 and 7C3) for more thorough characterization (Fig. 3 A and B and SI Appendix, Fig. S3 A and B). The 7C2 was formulated with 7C1:cholesterol:C14-PEG2000:18:1 Lyso PC at a molar ratio of 50:23.5:6.5:20. The 7C3 was formulated with 7C1:cholesterol:C14-PEG2000:DOPE at a molar ratio of 60:10:25:5. The 7C2 and 7C3 formed stable LNPs with average diameters between 50 and 80 nm when formulated with siRNA, single-guide RNA (sgRNA), or mRNA (SI Appendix, Fig. S3 C and D). The in vitro endocytosis and functional Cre mRNA delivery of both LNPs in LGSL-RFP cells decreased by at least 40% when cells were pretreated with chlorpromazine and genistein, relative to cells that were not treated with inhibitors. EIPA impacted LNP uptake but did not significantly decrease functional delivery in vitro (SI Appendix, Fig. S3 E–H). These results suggest 7C2 and 7C3 are endocytosed in vitro via several pathways. To study biodistribution, 7C2 and 7C3 were formulated with a Cy5.5-conjugated DNA barcode and i.v. injected into separate mice at a dose of 0.75 mg/kg DNA. Three hours later—a time point that is sufficiently long for LNPs to be cleared from the blood (34)—Cy5.5 ex vivo fluorescence, normalized by tissue weight, was highest in the spleen, kidney, and liver, suggesting 7C2 and 7C3 distributed to these tissues (Fig. 3C and SI Appendix, Fig. S3I).

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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.

We tested whether 7C2 and 7C3 functionally delivered siRNA, sgRNA, and mRNA, which are all clinically relevant. We performed our individual LNP proof-of-concept experiments with nucleic acids targeting ICAM-2. We chose this target for three reasons. First, ICAM-2 is related to a number of inflammatory diseases (35, 36). Second, ICAM-2 expression is robust on endothelial cells. Third, there is a well-validated monoclonal antibody to ICAM-2, which allows us to quantify protein expression robustly using FACS.

First, we formulated the LNPs to carry siRNA targeting ICAM-2. Three days after i.v. injecting mice with PBS, 2.0 mg/kg siGFP (a higher dose was used to test tolerability), or 1.0 mg/kg siICAM-2, we quantified ICAM-2 protein mean fluorescent intensity (MFI) in lung, kidney, and splenic endothelial cells using flow cytometry (32, 37). Both siRNAs were previously validated and chemically modified to reduce immunostimulation and promote on-target mRNA degradation (32, 37) (SI Appendix, Fig. S3J). ICAM-2 MFI was constant in PBS- and siGFP-treated mice but decreased by 60% in lung endothelial cells isolated from mice injected with siICAM-2. The 7C2-mediated delivery of siRNA reduced ICAM-2 expression in lung endothelial cells more than in splenic endothelial cells, whereas 7C3 silencing was more robust in splenic endothelial cells (Fig. 3D and SI Appendix, Fig. S3K). Compared with PBS-treated mice, mice injected with 2.0 mg/kg siGFP did not show any weight loss or changes in organ weight (SI Appendix, Fig. S3 L–N).

We then quantified 7C2- and 7C3-mediated sgRNA delivery. We coformulated two sgRNAs that were chemically modified at the 5′ and 3′ termini (38) and injected each at a dose of 0.75 mg/kg into mice that constitutively express SpCas9 (39) (SI Appendix, Fig. S3O). After three injections, ICAM-2 MFI decreased by up to 90, 75, and 59% in lung, spleen, and kidney endothelial cells, respectively for 7C2 (Fig. 3E). To confirm protein silencing was mediated by gene editing, we measured (40) ICAM-2 insertions and deletions (indels) in lung, spleen, and kidney endothelial cells and found between 30 and 70% editing per locus, leading to overall ICAM-2 indel percentages of 135, 95, and 123% indel, respectively for 7C2 (Fig. 3 F and G and SI Appendix, Fig. S3 P and Q). In these experiments, we utilized two sgRNAs per gene; thus, the total indel percentage was 200% (100% per site). Compared with PBS mice, mice treated with LNPs did not lose weight (SI Appendix, Fig. S3 R and S). Based on these experiments, we concluded that 7C2 potently delivered small RNAs to pulmonary endothelial cells in vivo.

In our siRNA and sgRNA experiments, 7C2 outperformed 7C3 and potently delivered RNAs to pulmonary endothelial cells. We then investigated whether the same observations were true for the delivery of large RNAs (mRNA). We formulated 7C2 and 7C3 to carry Cre mRNA and injected the LNPs into separate mice at a dose of 1.5 mg/kg mRNA (Fig. 4A). Three days later, we isolated tdTomato⁺ immune cells and endothelial cells from the spleen, kidney, heart, lung, and liver, as well as hepatocytes. We made two interesting observations. First, contrasting the small RNA delivery experiments, 7C3 outperformed 7C2. Second, splenic endothelial cells were targeted efficiently (Fig. 4B). Notably, relative to splenic endothelial cells, very few immune cells and hepatocytes were tdTomato⁺ (SI Appendix, Fig. S3 T and U). Given this degree of specificity has not been reported to date, we repeated the experiment using 34 Ai14 mice over the course of several months. The 7C3 delivered Cre mRNA to splenic endothelial cells in every experiment (Fig. 4C). However, we did observe batch-to-batch variability with respect to hepatocyte targeting (Fig. 4 C and D). In 22 of the 34 mice, a low percentage of hepatocytes were tdTomato⁺ (<13%); in 12 of the 34 mice, we observed high percentages of tdTomato⁺ cells. Averaging data from all 34 mice, the number of tdTomato⁺ splenic endothelial cells (43%) was 2.2 times higher than the number of tdTomato⁺ hepatocytes (20%) (Fig. 4D). Splenic endothelial cell delivery was also dose-dependent (SI Appendix, Fig. S3V). The variable delivery to hepatocytes was unlikely to be driven by toxicity; throughout our studies, we injected 34 mice with 7C3 and did not observe changes in mouse weight or obvious signs of toxicity (SI Appendix, Fig. S3W). We were unable to identify the source of batch variability; however, we hypothesize it is driven by the polymeric backbone of 7C3 and the resulting heterogenous chemical structure. Our data strongly suggest that future FIND screens should focus on compounds with defined chemical structures, which may be less likely to exhibit biological heterogeneity. The data also demonstrate that 7C3 can efficiently deliver mRNA to splenic endothelial cells in vivo. Importantly, the 7C2 and 7C3 formulations both included the lipomer 7C1 but had variable molar compositions of helper lipids (18:1 Lyso PC and DOPE), which distinguished them chemically from previously described endothelial-targeting nanoparticles. The differences in delivery between 7C2 and 7C3 provide interesting preliminary evidence that suggests changing the composition of helper lipids added to a given ionizable amine can alter tropism. The compositions and efficacy of 7C2 and 7C3 are included in SI Appendix, Fig. S4.

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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.

Given the difference between small and large RNA delivery, we quantified how LNPs codelivered CRISPR-Cas9 mRNA and sgRNA. We formulated Cas9 mRNA and two guides targeting ICAM-2 into LNPs at a 3:1 mass ratio and injected a 2.0 mg/kg total i.v. on day 0 and 2. On day 7, we isolated endothelial cells from the spleen, lung, kidney, and liver, as well as hepatocytes (SI Appendix, Fig. S5A). Splenic endothelial cell editing mediated by 7C3 was more robust than editing in hepatocytes (SI Appendix, Fig. S5B). Mice injected with 7C2 and 7C3 did not lose weight, compared with PBS-treated mice (SI Appendix, Fig. S5C). These studies were also conducted with sgRNAs that were chemically modified at the 5′ and 3′ termini (38); during the course of the experiments, two groups demonstrated that heavily modified sgRNAs improved in vivo editing (7, 8). We redesigned sgICAM2b with “enhanced” modifications (8) (referred to as e-sgICAM2) (SI Appendix, Fig. S5D) and formulated 7C3 to carry Cas9 mRNA and e-sgICAM2. Given that e-sgICAM2 was more likely to resist degradation in vivo (8), we hypothesized that the optimal mass ratio of Cas9 mRNA:sgRNA would decrease from 3:1, which is the optimized ratio with minimally stablized sgRNAs. To test this, we formulated 7C3 with Cas9 mRNA:e-sgRNA ratios of 5:1, 3:1, and 1:1, keeping the total injected dose constant at 2.0 mg/kg. After injecting mice i.v. with 2.0 mg/kg total RNA (Cas9 mRNA + sgRNA) on days 0 and 2, we isolated splenic endothelial cells and hepatocytes using FACS on day 7. We observed gene editing in splenic endothelial cells and hepatocytes at all mRNA-to-sgRNA ratios, with an average of 20% editing in the 1:1 mass ratio group (Fig. 4 E and F). As expected, the lower Cas9 mRNA:e-sgRNA mass ratio led to increased editing. Once again, mice injected with 7C3 did not lose weight compared with PBS-treated controls (SI Appendix, Fig. S5E). This is a demonstration of systemic Cas9-mediated editing of endothelial cells in wild-type mice. These data demonstrate that systemically administered nanoparticles can edit endothelial cells, and that the ideal Cas9:sgRNA ratio will depend on the relative stability of the two molecules.