Introduction: Whole human genome short hairpin RNA (shRNA) screen on human squamous cell carcinoma (Hep3) cells identified a panel of novel functional genes that are required for productive cell motility and successful metastatic dissemination in vivo. One such novel protein target encodes for nuclear protein C14orf142, which is up regulated in metastatic clear cell RCC (cRCC). Short interfering RNA (siRNA) are capable of binding to and inducing the degradation of complementary messenger RNAs leading to targeted silencing of functional proteins. The Fusogenic liposome platform is formulated with a fusion associated small transmembrane protein, P14, which ensures highly efficient siRNA delivery into the cellular cytoplasm. Our objective is to investigate whether P14 armored LNPs encapsulating custom siRNA against C14orf142 could interfere with clear cell RCC experimental metastasis in vivo.
Methods: The Precision Nanosystems Nanoassemblr TM benchtop and Spark systems were utilized for precise control of fusosome formulation to generate the P14 armored Entos™ lipid nanoparticle encapsulating 150µg of custom C14orf142 siRNA per injection. One week after wild type 786-0 cell implantation into murine flank, a treatment regimen with C14orf142 siRNA and scramble siRNA was initiated by encapsulating siRNA in Entos™ LNP-P14 as a delivery platform. NSG mice were injected IV via the tail vein with 150µg siRNA twice a week. Once scramble mice reached tumor volumes of 1cm3, all mice were euthanized, distant organs and primary tumors were harvested. Human Alu qPCR was performed to assess 786-0 metastatic dissemination and RT-qPCR was used to analyze C14orf142 knockdown within the primary tumor.
Results: Tumor growth was significantly slower in mice treated with C14orf142 siRNA and resulted in lower tumor weights (0.40±0.10g) at the studies endpoint compared to scramble (0.80±0.16g). Quantitative PCR analysis of primary tumors confirmed decreased C14orf142 mRNA levels measured in tumors derived from mice treated with C14orf142 siRNA (59.2±6.5%) relative to scramble (100%). 786-0 metastasis to the lungs (16.8±3.4%), brain (46.5±12.1%) and liver (36.8±12.9%) was markedly reduced in mice treated with C14orf142 siRNA as compared to scramble siRNA (100%).
Conclusions: P14 armored fusogenic lipid nanoparticles encapsulating siRNA against C14orf142 could potentially pave the way for novel therapeutic approaches in management of metastatic clear cell RCC.
Source of Funding: Kidney Cancer Research Network and Kidney Cancer Canada Research Grant
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