CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy

Abstract

Harnessing CRISPR-Cas9 technology for cancer therapeutics has been hampered by low editing efficiency in tumors and potential toxicity of existing delivery systems. Here, we describe a safe and efficient lipid nanoparticle (LNP) for the delivery of Cas9 mRNA and sgRNAs that use a novel amino-ionizable lipid. A single intracerebral injection of CRISPR-LNPs against PLK1 (sgPLK1-cLNPs) into aggressive orthotopic glioblastoma enabled up to ~70% gene editing in vivo, which caused tumor cell apoptosis, inhibited tumor growth by 50%, and improved survival by 30%. To reach disseminated tumors, cLNPs were also engineered for antibody-targeted delivery. Intraperitoneal injections of EGFR-targeted sgPLK1-cLNPs caused their selective uptake into disseminated ovarian tumors, enabled up to ~80% gene editing in vivo, inhibited tumor growth, and increased survival by 80%. The ability to disrupt gene expression in vivo in tumors opens new avenues for cancer treatment and research and potential applications for targeted gene editing of noncancerous tissues.

Fig. 1. Design and construction of CRISPR LNPs (cLNPs).

(A) Schematic illustration of cLNP preparation. A microfluidic-based mixing of lipids to construct cLNPs encapsulating Cas9 mRNA and sgRNA. (B) Chemical structures of the selected ionizable amino lipids from the library screen. (C) Physicochemical characterization of cLNP by dynamic light scattering and ζ sizer. Data are means ± SD of five independent preparations. (D) Encapsulation efficiency as measured using a RiboGreen assay. (E) GFP disruption assay: HEK293 cells were transfected with cLNPs at different concentrations (0.1 to 1 μg/ml, 0.7 to 7 nM total RNA), and 72 hours after transfection, the percentage of GFP⁺ cells were analyzed by flow cytometry. Data are means ± SD of three independent experiments. (F and G) Percentage of gene editing events upon either GFP or PLK1-cLNP transfection (F) and allelic frequencies (G) in the GFP loci as determined by NGS analysis (allelic frequencies of >2% are presented). (H) GFP disruption assay in multiple cancer cell lines compared to mock-treated cells. Cells were transfected with L8-cLNPs, and 72 hours after transfection, the percentage of GFP⁺ cells were analyzed by flow cytometry. Data are means ± SD of three independent experiments.

Fig. 2. Therapeutic genome editing in HEK293 cells in vitro.

(A and B) Percentage of gene editing events (A) and allelic frequencies (B) in the PLK1 loci as determined by NGS analysis (allelic frequencies of >2% are presented). (C and D) Cell cycle analysis of HEK293 cells treated with mock, sgGFP, or sgPLK1-cLNPs (0.5 μg/ml, 3.5 nM of total RNA) for 48 hours and analyzed by flow cytometry. (C) Bar charts representing the percentage of G₁-S and G₂-M cell cycle phases. Data are means ± SD of three independent experiments. (D) Representative cell cycle analysis diagram. (E and F) DAPI/annexin V assay of HEK293 cells treated with mock, sgGFP, or sgPLK1-cLNPs (0.5 μg/ml, 3.5 nM total RNA) for 96 hours and analyzed by flow cytometry. (E) Bar charts representing the percentage of live cells normalized to mock-treated cells. Data are means ± SD of three independent experiments. (F) Representative DAPI/annexin assay diagram. (G) XTT cell viability assay of HEK293 cells treated with mock, sgGFP, or sgPLK1-cLNPs (0.5 μg/ml, 3.5 nM total RNA) 96 hours after treatment. Bar charts representing the % of cell viability normalized to mock-treated cells. Data are means ± SD of three independent experiments. (E and G) One-way analysis of variance (ANOVA) with Tukey multiple comparison test was used to assess the significance. ****P < 0.0001.

Fig. 3. Therapeutic genome editing in 005 (murine GBM) and OV8 (human ovarian carcinoma) cells in vitro.

(A and F) Percentage of gene editing events in the PLK1 loci in 005 and OV8 as determined by NGS analysis. (B and G) Bright-field microscopy representative images of 005 and OV8 cells treated with mock, sgGFP, or sgPLK1-cLNPs [005: 0.5 μg/ml (3.5 nM total RNA); OV8: 1 μg/ml (7 nM total RNA)], 72 hours after treatment. (C and H) Cell cycle analysis of 005 and OV8 cells, treated with mock, sgGFP, or sgPLK1-cLNPs [005: 0.5 μg/ml (3.5 nM total RNA); OV8: 1 μg/ml (7 nM total RNA)] for 48 hours and analyzed by flow cytometry. Bar charts representing the % of G₁, S, and G₂-M cell cycle phases. Data are means ± SD of three independent experiments. (D and I) XTT cell viability assay of 005 and OV8 cells treated with mock, sgGFP, or sgPLK1-cLNPs [005: 0.5 μg/ml (3.5 nM total RNA), OV8: 1 μg/ml (7 nM total RNA)] for 96 hours. Bar charts representing the % of cell viability normalized to mock-treated cells. Data are means ± SD of three independent experiments. (E and J) DAPI/annexin V apoptosis analysis of 005 or OV8 cells treated with mock, sgGFP, or sgPLK1-cLNPs [005: 0.5 μg/ml (3.5 nM total RNA); OV8: 1 μg/ml (7 nM total RNA)] for 96 hours and analyzed by flow cytometry. Bar charts representing the percentage of live cells normalized to mock-treated cells. Data are means ± SD of three independent experiments. (C, E, and H to J) One-way ANOVA with Tukey multiple comparison test was used to assess the significance. ***P < 0.001 and ****P < 0.0001.

Fig. 4. Therapeutic genome editing in 005 GBM bearing mice.

(A) Schematic illustration of intracerebral injection to mouse brain. (B) cLNP dispersion through the tumor lesion upon intracerebral injection of Cy5.5-cLNPs to the tumor bed of 005 GBM–bearing mice. Brain sections were analyzed by confocal microscopy, 6 hours after injection. Blue, DAPI; green, 005 GFP cells; yellow, Cy5.5 cLNPs. Scale bars, 50 μm. (C) Percentage of gene editing events in the PLK1 locus as determined by NGS analysis, 48 hours after injection of PBS or 0.05 mg/kg of sgGFP-cLNPs or sgPLK1-cLNPs. (D) In vivo apoptosis induction using activated caspase 3 staining upon injection of either PBS or 0.05 mg/kg of sgGFP-cLNPs or sgPLK1-cLNPs. Brain sections were analyzed by confocal microscopy 3 days after injection. Blue, DAPI; green, 005 GFP cells; red, cleaved caspase 3. Scale bars, 50 μm. (E) Experimental design. Ten days after tumor inoculation, sgGFP-cLNPs, sgPLK1-cLNPs, or PBS (0.05 mg/kg) was injected into the tumor bed. Tumor growth was monitored using bioluminescence of 005-GFP-Luc cells by the IVIS in vivo imaging system. (F and G) Tumor growth inhibition by single-dose treatment with cLNPs. (F) Representative bioluminescence imaging of 005 GBM–bearing mice. (G) 005 tumor growth curve quantification. Data are presented in total flux (p/s) ± SEM; n = 15 animals per treatment group and n = 8 animals in the PBS group; ****P < 0.0001. One-way ANOVA was used to assess the significance at day 41. (H) Survival curves of 005 GBM–bearing mice. n = 30 animals per treatment group and n = 8 animals in the PBS group. ****P < 0.0001. Log-rank (Mantel-Cox) test was used for curve comparison.

Fig. 5. Therapeutic genome editing in OV8-bearing mice.

(A) Schematic illustration of targeted cLNP production using ASSET (23). (B and C) Tumor targeting and accumulation of Cy5.5-cLNPs in OV8 tumor–bearing mice as analyzed by the IVIS in vivo imaging system, 4 hours after injection. (B) Representative fluorescence imaging of tumors extracted from mCherry-OV8–bearing mice. Top, mCherry OV8 tumors; bottom, Cy5.5-cLNP signal accumulation. (C) Quantification of mean fluorescence intensity of Cy5.5-cLNP accumulation in mCherry-OV8 tumors. Data are means ± SD of three independent experiments. One-way ANOVA was used to assess the significance, *P < 0.05. (D) Percentage of gene editing events in the PLK1 locus as determined by NGS analysis, 48 hours after injection of I-sgGFP, T-sgGFP, I-sgPLK1, or T-sgPLK1 cLNPs (0.75 mg/kg). (E) Experimental design. Ten and 17 days after tumor inoculation, I-sgGFP, T-sgGFP, I-sgPLK1, or T-sgPLK1 cLNPs (0.75 mg/kg) were injected intraperitoneally. Tumor growth was monitored using mCherry fluorescence of OV8-mCherry cells by the IVIS in vivo imaging system. (F and G) Tumor growth inhibition by dual-dose treatment with cLNPs. (F) Representative fluorescence imaging of OV8-bearing mice. (G) OV8 tumor growth curve quantification. Data are presented in total flux (p/s) ± SEM; n = 10 per group. One-way ANOVA was used to assess the significance at day 49; ****P < 0.0001. (H) Survival curves of OV8-bearing mice. n = 10 animals per treatment group. ***P < 0.0001. Log-rank (Mantel-Cox) test was used for curve comparison.