Lipid nanoparticles-loaded with toxin mRNA represents a new strategy for the treatment of solid tumors

Abstract

Background and rationale: Cancer therapy have evolved remarkably over the past decade, providing new strategies to inhibit cancer cell growth using immune modulation, with or without gene therapy. Specifically, suicide gene therapies and immunotoxins have been investigated for the treatment of tumors by direct cancer cell cytotoxicity. Recent advances in mRNA delivery also demonstrated the potential of mRNA-based vaccines and immune-modulators for cancer therapeutics by utilizing nanocarriers for mRNA delivery. Methods: We designed a bacterial toxin-encoding modified mRNA, delivered by lipid nanoparticles into a B16-melanoma mouse model. Results: We showed that local administration of LNPs entrapping a modified mRNA that encodes for a bacterial toxin, induced significant anti-tumor effects and improved overall survival of treated mice. Conclusions: We propose mmRNA-loaded LNPs as a new class of anti-tumoral, toxin-based therapy.

Keywords: cancer therapy; gene therapy; immunotoxins; lipid nanoparticles; mRNA; suicide-gene therapy.

Figure 1

Schematic and microscopic representation of toxin encoding mmRNA-loaded lipid nanoparticles. A. Self-assembly of lipid mixture and mRNA molecules in acidic buffer composes mRNA-LNPs. mRNA is encoding for the pseudomonas exotoxin A (PE) toxin, which is delivered by the LNPs to cancer cells (C). The delivered mmPE is then translated by target cells into PE toxin (D) that induces apoptosis (E). B. Representative TEM image of Firefly Luciferase mmRNA-loaded LNPs. Bar scale - 200 nm.

Figure 2

Physicochemical characterization and in-vitro expression of MC3-mmRNA-LNPs and EA-PIP-mmRNA-LNPs. A-B. Chemical structures of MC3 (A) and EA-PIP (B) ionizable lipids. C-D. LNPs' size and zeta potential measurements by dynamic light scattering (DLS). E. mmRNA encapsulation efficiency as reflected in a RiboGreen-based assay, allowing mmRNA concentration calculation, according to the absorbance of an RNA-binding dye. F. Firefly luciferase expression in B16F10.9 cells 48 h post incubation with mmFluc-LNPs composed of either MC3 or EA-PIP lipids. H-G. EGFP expression level in B16F10.9 cells 48 h post incubation with mmEGFP-LNPs composed of either MC3 (H) or EA-PIP (I) lipids. Multiple paired t-tests statistical analysis was performed by Prism GraphPad, *p ≤ 0.05, ****P < 0.0001.

Figure 3

mmPE-LNPs therapeutic effect in-vitro: cancer cell viability reduction via apoptosis and protein translation inhibition. A. B16F10.9 cell viability rate 48 h post treatment with mmPE-LNPs. Cells were incubated with increasing doses of mmPE-LNPs, composed of either MC3 or EA-PIP lipids. 48 h post treatment, the viability rate of treated cells was exceptionally low (~10%) in all tested conditions. B. B16F10.9 cells pre-treated with mmPE-LNPs, had lower expression levels of transfected EGFP mmRNA compared to cells pre-treated with mmFluc LNPs, suggesting that mmPE LNPs inhibit protein translation. C. PI-Annexin assay for determination of necrosis and apoptosis rates 3 h, 24 h and 48 h post treatment. D. Graphical representation of PI-Annexin-V-stained cells according to their viability state: live, necrotic, early apoptotic or late apoptotic at all tested timepoints, indicating mmPE-LNPs caused significant apoptosis 48 h post treatment.

Figure 4

Intratumorally-injected, repeated doses of mmPE-LNPs caused in-vivo apoptosis in tumor cells. Representative images of mice tumors IHC stained for either caspase-3 (A-C) or TUNEL (E-G), yellow arrows mark stained cells. Left panel (A, E) represents PBS-treated mice, middle panel (B, E) represents mmFluc-LNPs treated mice and right panel (C, G) represents mmPE-LNPs treated mice (0.15 mg/Kg, 50 µl, four doses). All specimens (N = 3) were classified by a veterinary pathologist according to the following: Grade 0 = no positive reaction at all, Grade 1= Only few cells are positive (< 5 cells per a X20 field), Grade 2 = Very mild positive stain (5-15 cells per a X20 field), Grade 3 = Mild positive stain (15-25 cells per a X20 field), Grade 4= Moderate positive stain (25-50 cells per a X20 field), Grade 5 = Marked positive stain (> 50 cells per a X20 field). D&H are graphical representations of the average scoring of caspase-3 and TUNEL positive cells, respectively.

Figure 5

Intratumorally-injected mmPE-LNPs effect on tumor growth in a B16F10.9 mouse model. A. Experiment settings and timeline. Mice were subcutaneously inoculated with B16F10.9 melanoma cells and were treated with four intratumoral, repeated doses of either PBS, mmFluc LNPs or mmPE LNPs (0.15 mg/Kg, 50 µl), starting 12 days post tumor inoculation. B. Average percentage of mice's weight compared to first day of treatment, representing weight loss rate. C. Ex-vivo tumors at experiment endpoint, visually demonstrating tumor sizes. D. Average tumor volume of mice from first day of treatments [n = 6 mice / group. Student's t-test statistical analysis of every pair of groups was performed using Prism GraphPad, *p ≤0.05.

Figure 6

Safety profile analysis of mmPE-LNPs. A-H. Histology of liver (A-D) and spleen (E-H) samples (hematoxylin and eosin staining, representative images) of either healthy (A,E), PBS-injected (B,F), mmFluc-LNPs-injected (C,G) or mmPE-LNPs-injected mice (D,H), after four intratumoral injections, 0.15 mg/Kg, 50 µl. I. Liver enzymes of mice intratumorally injected with either mmFluc LNPs or mmPE LNPs, compared to untreated mice, 24 h post injection (0.15 mg/Kg, 50 µl), indicating no significant increase has occurred in the treatment group.

Figure 7

Intratumorally injected mmPE-LNPs caused tumor volume reduction, cancer-cell specific expression and better survival rate, with minimal systemic toxicity. A. Experiment timeline and settings. B. Average percentage of mice's weight compared to first day of treatment, representing weight loss rate. No significant weight loss in the treated group (mmPE, repeated doses, 0.15 mg/Kg, 50 µl) compared to the control groups (PBS and mmFluc) was observed. C. Average tumor volume of all groups. [n = 6 mice / group. Statistical analysis was done using student's t-test for every pair of groups, performed by Prism GraphPad, *p ≤0.05]. D. Tumor volume of each individual mouse until reaching sacrificing criteria (tumor volume ≥ 1500 mm³). E. Kaplan-Meier plot representing survival rate of all groups., showing significant positive effect of mmPE-LNPs on mice's survival rate. [Log-rank (Mantel-Cox) test was used for curve comparison using Prism GraphPad, *p ≤0.05, **p ≤0.01.]. F. Representative FACS analysis of mCherry-labeled tumor cells of mice receiving a single IT dose of mmEGFP-LNPs, 24 h post injection. A substantial fraction of almost 60% out of the mCherry-labeled cells also co-expressed EGFP (FITC), demonstrating a high delivery rate. Additionally, nearly all EGFP expressing cells were also mCherry positive, representing extremely high specificity.

Figure 8

Intratumorally-injected mmPE-LNPs inhibited B16-melanoma growth, as reflected by lower luminescence and fluorescence signals of labelled tumors. A&C. Representative IVIS images showing mCherry-luc labelled B16F10.9 tumor progression as reflected by either mCherry (A) or Firefly-luciferase (C) signals starting 48 h post the first injection (PBS, mmFluc LNPs or mmPE LNPs, 0.15 mg/Kg, 50 µl). Mice reaching a threshold tumor size of 1500 mm³ were sacrificed and therefore had no imaging in later timepoints. Mice have reached larger tumors sooner in the control groups compared to the mmPE-LNPs treated groups. B&D. Average flux of mCherry (B) or Firefly Luciferase (D) signals from tumors of all groups over time showing that tumor signals were significantly lower in the treatment group for both Firefly luciferase and mCherry reporters at days 20, 22 and 25 post tumor inoculation, compared to the control groups. [Statistical analysis was done using student's t-test for every pair of groups, performed by Prism GraphPad, *p ≤0.05.]