Introduction
Solid tumors, unlike blood cancers, present a unique challenge for drug delivery and efficacy, making them notoriously difficult to treat (Jain 2012). Some key reasons for the challenge include anatomical barriers (e.g., surrounding connective tissue and abnormal blood flow), biological complexity (e.g., cellular heterogeneity and presence of drug suppressors), and delivery system limitations (e.g., lack of drug targeting and penetration).
Ongoing research is aimed at finding novel solutions to overcome these hurdles to develop more effective therapies for solid tumors. Among such approaches, harnessing naturally occurring toxins, as alternatives to classic cytotoxic drugs, and advanced nanoparticle delivery systems are of particular interest.
This blog outlines one such approach recently reported by an interdisciplinary team of researchers (Granot-Matok et al. 2023) at Tel Aviv University in Israel. They used CleanCap® 5-methoxyuridine (5moU)-modified mRNA to encode a naturally occurring bacterial toxin, obtained from TriLink’s custom mRNA synthesis service. The mRNA was encapsulation in a lipid nanoparticle (LNP) for direct injection into a solid tumor. Optimization of the LNP formulation for functional delivery of mRNA employed TriLink reporter genes 5moU-modified Firefly Luciferase (FLuc) mRNA and Enhanced Green Fluorescent Protein (EGFP) mRNA.
The following sections outline how Granot-Matok et al. progressed from the initial design and characterization of PE toxin mRNA, measurement of its toxic effect on cancer cells in vitro, and demonstration of in vivo efficacy in a solid-tumor model.
PE-III mRNA-LNP Design and Characterization
Pseudomonas exotoxin A (PE) induces cell death by apoptosis. During apoptosis, which is a process of programmed cell destruction, there are changes in cellular morphology, plasma membrane blebbing, and cell shrinkage. The cytoskeleton collapses, the nuclear envelope disassembles, and the nuclear DNA breaks up into fragments.
PE is comprised of three functional domains for receptor binding (domain I), internalization and intracellular processing (domain II), and toxic ADP-ribosyltransferase activity (domain III), as reviewed elsewhere. Granot-Matok et al. used mRNA encoding only toxic domain-III (PE-III mRNA) to advantageously:
- Bypass resistance associated with either receptor binding, internalization, or intracellular processing;
- Provide alternative treatment for cancers that do not express an identified unique antigen; and
- Avoid immunogenicity or mutagenicity that can result from administration of the toxin domain-III protein itself or a plasmid DNA vector thereof.
The mRNA obtained from TriLink had been co-transcriptionally capped with a CleanCap® (Cap 1) analog and incorporated 5-moU in place of U. It was comprised of a U-depleted/GC-rich (70%) 654-nt coding sequence for PE-III, flanked by optimized 5’- and 3’-UTRs and a poly(A) tail (TriLink . Cap 1 such as CleanCap analogs and modified nucleotides such as 5-moU provide lower immunogenicity and enhanced translation (Galloway et al. 2019 and Vaidyanathan et al. 2018).
As depicted in Figure 1, the PE-III mRNA was combined with three lipids and cholesterol for self-assembly by turbulent mixing in a serpentine-microchannel device. For reproducibly optimal performance, precisely measured ratios of all these components were required, as well as the inclusion of a novel ionizable lipid that was previously reported by Kampel et al. 2021.
FIGURE 1. Schematic and microscopic image of PE-III mRNA-loaded lipid nanoparticles. (A) Self-assembly of lipids and mRNA to form LNPs via turbulent mixing. (B) Microscopic image of FLuc mRNA-LNPs; 200 nm bar-scale. (C-E) Endosomal uptake, translation into PE-III, and apoptosis. Taken from Granot-Matok et al. (2023) and free to use under CC-BY license.
To evaluate functionality, Granot-Matok et al. studied mRNA-LNPs loaded with either FLuc or EGFP mRNA (5-moU) reporters from TriLink and 4 different cancer cell lines of either human or murine origin. The mRNA-LNPs, which did not exhibit any significant cytotoxicity, demonstrated dose-dependent in vitro expression of both FLuc and EGFP, as reflected by increasing luminescence and fluorescence signal intensities, respectively.
PE-III mRNA-LNPs Induce Cancer Cell Apoptosis In Vitro
It is known that the ADP-ribosyltransferase activity of PE-III kills cells by inactivation of eukaryotic elongation factor 2 that is essential for protein biosynthesis. This induces apoptosis and the cell dies. Granot-Matok et al. hypothesized that LNP-mediated delivery of mRNA encoding only PE-III, without inclusion of the binding and translocation domains of full-length PE, would retain the potential for potent intracellular blockage of protein biosynthesis and lead to apoptosis.
A highly metastatic melanoma cell line (B16F10.9) and 3 other types of cancer cell lines were separately incubated with increasing amounts of PE-III mRNA-LNPs, which in all cases led to >90% reduction in cancer cell viability 48 h post-treatment. It was also confirmed that this cytotoxicity was due to apoptosis, using a conventional staining method and fluorescence-activated cell-sorting analysis. The B16F10.9 cells treated with PE-III mRNA-LNPs had an increasing fraction of apoptotic cells starting from 24 h post-treatment to a highly significant fraction of 95% late-apoptotic/necrotic cells 48 h post-treatment.
To further validate that the PE-III mRNA-LNP mechanism of action correlates with that of full-length PE, and that domain-III triggers inhibition of protein biosynthesis, in vitro protein synthesis was monitored using reporter mRNA. The B16F10.9 cells were incubated with either PE-III mRNA-LNPs or FLuc mRNA-LNPs for 2 h, and then transfected with EGFP mRNA. This led to dose-dependent inhibition of EGFP expression in cells pre-treated with PE-III mRNA-LNPs, but not with FLuc mRNA-LNPs, reaching 100% inhibition at the highest dose tested.
PE-III mRNA-LNPs Cause Apoptosis and Tumor Growth Inhibition In Vivo
To test the efficacy of PE-III mRNA-LNPs in vivo, B16F10.9-melanoma tumor-bearing mice were intratumorally injected with PE-III mRNA-LNPs for comparison to two controls, FLuc mRNA-LNPs and phosphate-buffered saline (PBS). The treatment protocol involved 4 injections, with a gap of 2-3 days between injections, and final analysis 72 h after the last injection.
Mice receiving PE-III mRNA-LNPs had smaller tumor volumes vs. controls at all tested timepoints, and significantly lower tumor volumes at the experiment’s endpoint, as measured in vivo by IVIS imaging and excised-tumor sizes. No significant reduction in the average weight of the treated vs. control mice indicated that there was no substantial systemic toxicity of the treatment.
To validate the mechanism of action of PE-III mRNA, residual tumors were removed on the final day of the experiment and analyzed by immunohistochemical staining. Caspase-3 staining was employed to detect early apoptotic changes, and the TUNEL assay was used to detect late apoptosis reflected by DNA fragmentation. Higher staining for both caspase-3 and TUNEL in the PE-III mRNA-LNP-treated group vs. controls indicated late apoptosis in the treated tumors at the experiment’s endpoint.
Regarding biodistribution, it was found that a single intratumoral injection of FLuc mRNA-LNPs led 24 h later to 100-times more FLuc expression in the tumor vs. intraperitoneal injection of this reporter. Also, there was relatively little FLuc expression in the major blood-filtering organs. Collectively, these observations support tumor-specific biodistribution following intratumoral injection.
Repeated Intratumoral Injection of PE-III mRNA-LNPs Increases Survival
Demonstration of increased survival in an animal model is a critical indicator for clinical development of any type of anticancer drug. Granot-Matok et al. therefore aimed to evaluate the effect of treatment with PE-III mRNA-LNPs on the survival rate of tumor-bearing mice, as well as quantitative measurement of tumor-growth inhibition. To do this using in vivo imaging, mice were first inoculated with B16F10.9 cells that had been stably transduced by a lentivirus to continuously express intratumoral mCherry and FLuc labels.
Treatment started 8-10 days after inoculation when the tumor volumes reached 40-50 mm3 by caliper measurements or, for imaging experiments, when labeled tumors reached or exceeded a threshold luminescence. Groups of mice were then intratumorally injected with either PBS, Fluc mRNA-LNPs, or PE-III mRNA-LNPs every 2-3 days (Figure 2). Each mouse was treated until reaching the ethical sacrificing criterion (tumor volume ≥1,500 mm3) and was then euthanized.
FIGURE 2. Intratumorally injected PE-III mRNA-LNPs led to increased survival rate. (Top) Experimental protocol. (Bottom) Kaplan-Meier plot representing survival rate of all treated groups showing significant positive effect of PE-III mRNA compared to both controls (*p ≤0.05, **p ≤0.01). Taken from Granot-Matok et al. (2023) and free to use under CC-BY license.
The experiment lasted 35 days from tumor inoculation, with 9 injection timepoints and tracking intratumoral mCherry and FLuc intensities by in vivo IVIS imaging every 2-3 days. Treatment with PE-III mRNA-LNPs but not the controls led to significant tumor growth inhibition, as reflected by tumor size independently measured by caliper, mCherry signal, and FLuc signal.
Mice receiving PE-III mRNA-LNPs had reduced tumor volumes at all tested timepoints, and significantly lower tumor volumes at days 20, 22 and 25 post tumor inoculation vs. control groups. There was a highly significant (p ≤0.01) increase in the survival rate of PE-III mRNA-LNPs treated mice vs. controls (Figure 2), with no appreciable average weight loss.
Concluding Comments
The above findings represent the first-ever demonstration of the in vivo efficacy of treating a solid tumor by direct injection of PE-III mRNA-LNPs. This in a mouse model of melanoma, which is 2% of cancer worldwide. PE-III mRNA-LNPs as a possible platform technology could be used to treat other types of solid tumors, such as breast, lung, colon, prostate, and pancreas, which collectively represent an additional 45% of cancer worldwide (World Cancer Research Fund International 2023).
Granot-Matok et al. conclude their report by suggesting that LNPs having surface-exposed moieties for attachment of cancer cell-specific targeting for delivery of PE-III mRNA by simple injection into the blood stream. This would allow clinical applications to either blood cancers or solid tumors, whether accessible by direct injection or not.
Your comments are welcomed, as usual.
Please feel free to share this blog with your colleagues or on social media.
