Assistant Professor of Engineering Brown University, United States
Introduction: Kinase inhibitors are an established cancer therapeutic; 69 of the 80 currently FDA-approved kinase inhibitors are used in cancer treatment. While approved kinase inhibitors target around 24 kinases in various classes: protein-serine/threonine, dual specificity protein kinases (MEK1/2), non-receptor protein-tyrosine kinases, and receptor protein-tyrosine kinases, this represents only a small fraction of the 518-member protein kinase enzyme superfamily. Traditionally, structural-development of these approved small molecule kinase inhibitors (based on protein kinase X-ray crystal structures) has suffered from long development times, off target activity, severe adverse effects, and drug resistance. This project aims to develop kinase-selective siRNA to address the limitations of kinase inhibitors while maintaining therapeutic efficacy. siRNA therapeutics allow the silencing of kinases not readily accessible to other treatment modalities such as small molecule and antibody technologies with sequence-specificity.
Here, a kinase was selected for its dual implication in proliferation, migration, and chemotherapeutic sensitivity in ovarian cancers. siRNA sequence alignment was performed in silico, and custom siRNA sequences were encapsulated into lipid nanoparticles (LNPs) capable of transfecting ovarian cancer cells. Proof-of-concept for therapeutic efficacy in ovarian cancer is currently underway. Differences between the designed siRNA-LNP against a commercially available small molecule kinase inhibitor regarding potency and toxicity will be investigated.
Materials and
Methods: OVCAR3 ovarian cancer cells were cultured in DMEM/F12-media supplemented with 10% FBS and 1% penicillin/streptomycin by volume. Cells were incubated at 37°C with 5% CO2, and passage number kept below 10. Cell viability was monitored through trypan blue staining before and after transfection with siRNA-LNPs.
Lipid nanoparticles (LNPs) were formulated with an ionizable lipid (MC3), a phospholipid (DSPC), cholesterol, and a polyethylene glycol (PEG)-conjugated lipid (DMG-C-PEG2000) according to the Onpattro formulation, molar ratio of 50:10:38.5:1.5, respectively. The lipids were dissolved in ethanol and rapidly hand-mixed with an siRNA-containing aqueous phase (10mM, pH 3, citrate buffer) in a 1:3 volumetric ratio. siRNA was encapsulated in the lipid nanoparticles at a 1:10 mass ratio to the ionizable lipid. The resulting LNPs were dialyzed against PBS using a 20kDA MWCO slide-a-lyzer dialysis unit for a minimum of 6 hours, changing the PBS after 2 hours. The particle z-average size and polydispersity index was measured in triplicate with a Malvern Zetasizer Nano. siRNA encapsulation efficiency was calculated using a Quant-it RiboGreen assay in the presence or absence of triton-X100.
Custom siRNA and the commercial transfection reagent, Dharmafect4, were ordered through Dharmacon. Reverse transfection was performed with antibiotic-free medium according to Dharmafect protocol or with the hand mixed lipid nanoparticles.
RNA was isolated 48 hours post transfection according to the RNeasy Plus Mini Kit. Input RNA was quantified by Nanodrop. Relative quantification of gene expression was quantified using the Luna Universal Probe one-step RT-qPCR kit and Taqman probes with technical triplicates on a Bio-Rad CFX96.
Results, Conclusions, and Discussions: In order to assess the efficacy of our novel kinase-targeted siRNA designed in silico, we transfected OVCAR3 ovarian cancer cells with 5nM and 25nM siRNA using a commercial transfection reagent (Dharmafect 4). RT-qPCR was used to quantify gene silencing, using GAPDH as a reference gene, which we confirmed was stably expressed under experimental conditions. Our siRNA yielded >90% silencing compared to no siRNA controls (treated with Dharmafect4 transfection reagent only, and no treatment). In order to assess the ability of siRNA-LNP formulations to transfect the OVCAR3 cells, our siRNA was encapsulated into MC3 LNPs. The resulting lipid nanoparticles loaded with siRNA had a z-average size of 152.0nm and PDI of 0.097 whereas the empty negative control had a z-average size of 187.8nm and PDI of 0.074. The encapsulation efficiency was 82% by RiboGreen Assay. The amount of siRNA entrapped was 83% of what was input when formulating the lipid nanoparticles. Repeating 5nM and 25nM siRNA doses with siRNA-LNP yielded substantially less silencing than the Dharmafect4 reagent. Previous literature reports that < 2% of siRNA is released from the lipid nanoparticle/endosome, so the dosing was increased for subsequent transfections with lipid nanoparticle formulations. Dosing 100nM and 500nM siRNA-LNP yielded 80-96% silencing, compared to the empty negative control. Statistical significance, in all experiments, was confirmed with a two-sided student t-test.
In conclusion, the designed siRNA showed robust silencing when formulated in MC3 LNPs, dosed at 100nM. Ongoing work includes in vitro proof-of concept experiments to assess the effects of siRNA-LNP kinase silencing on ovarian cancer migration (single cell tracking), viability, and proliferation; as well as direct comparison with FDA-approved small molecule kinase inhibitors. The successful development of siRNA-based kinase inhibitors, will represent a new treatment modality for cancers.
