In Vivo RNA Delivery to Hematopoietic Stem and Progenitor Cells via Targeted Lipid Nanoparticles

Abstract

Ex vivo autologous hematopoietic stem cell (HSC) gene therapy has provided new therapies for the treatment of hematological disorders. However, these therapies have several limitations owing to the manufacturing complexities and toxicity resulting from required conditioning regimens. Here, we developed a c-kit (CD117) antibody-targeted lipid nanoparticle (LNP) that, following a single intravenous injection, can deliver RNA (both siRNA and mRNA) to HSCs in vivo in rodents. This targeted delivery system does not require stem cell harvest, culture, or mobilization of HSCs to facilitate delivery. We also show that delivery of Cre recombinase mRNA at a dose of 1 mg kg^-1 can facilitate gene editing to almost all (∼90%) hematopoietic stem and progenitor cells (HSPCs) in vivo, and edited cells retain their stemness and functionality to generate high levels of edited mature immune cells.

Keywords: RNA; antibody targeting; blood disorders; hematopoietic stem cells; lipid nanoparticle.

Figure 1

Anti-CD117 LNPs can deliver siCD45 both in vitro and in vivo. (a) DLS of unconjugated LNP vs conjugated LNP. (b) Anti-CD117 LNPs (dosed at 50 ng siCD45 per well) showed potent knockdown in vitro in EML cell line (n = 3 wells). Statistics were performed with multiple unpaired two-tailed t tests comparing the isotype control of each group. (c) Representative FACS scatter plots for the DiR signal in bone marrow HSPCs for different PEG-lipids at 1 mg kg^–1 siCD45. (d) Quantification of the level of LNP uptake in HSPCs and LT-HSCs. LNPs were administered at a dose of 1 mg kg^–1 siCD45 (n = 2 mice, initial screening, two-way ANOVA with Tukey’s multiple comparison test). (e) Dose–response of anti-CD117 LNP using the C18-PEG lipid. Bone marrow LSK cells were analyzed 72 h after LNP administration for uptake (left) and functional CD45 knockdown (right) (n = 2 mice, screening, one-way ANOVA with Tukey’s multiple comparison test). (f) Screening of alternate markers for in vivo HSPC delivery at a dose of 1 mg kg^–1 siCD45 (n = 3 mice for anti-CD49d condition, n = 2 mice for all other conditions, one-way ANOVA with Dunnett’s multiple comparison test). Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2

Anti-CD117 LNPs encapsulating Cre mRNA shows high levels of editing in vivo. (a) Schematic of the Ai14 transgenic mouse LoxP-flanked stop cassette preventing the transcription of TdTomato. Upon delivery of Cre recombinase via Cre mRNA, the stop cassette is excised, and the cell expresses TdTomato. (b) Timeline of experimental workflow for bone marrow and blood analysis. Panels a and b were created with BioRender.com. (c) Representative flow cytometry scatter plots of TdTomato expression in LT-HSCs with varying doses of Cre mRNA. (d) Quantification of the dose–response in both HSPCs and LT-HSCs (n = 4 mice for 1 mg kg^–1Ab-LNP group, n = 3 mice for others). Statistics were performed with two-way ANOVA with Tukey’s multiple comparison test. (e) Time course monitoring of TdTomato expression in mature immune cells (n = 3 mice). (f) TdTomato expression in T cell subsets (n = 3 mice). (g) TdTomato expression in erythrocytes (TER-119⁺) (n = 3 mice). Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

Tissue level biodistribution of LNPs conjugated to anti-CD117 (clone 2B8), anti-CD117 (clone ACK2), or rat IgG2b isotype control. (a) Representative IVIS bioluminescence images of all organs. LNPs containing firefly luciferase mRNA were injected at a dose of 6 μg of mRNA (0.3 mg kg ^–1). Tissues were collected and imaged 6 h after LNP administration. (b) Quantification of the average radiance. Data represents mean ± SD (n = 4 mice). Statistics performed using two-way ANOVA with Dunnett’s multiple comparison test comparing to the isotype control. (*P < 0.05, **P < 0.01, ***P < 0.001).