B. A. Krasnick1, M. S. Strand1, N. Sankpal1, Y. Bi1, P. Goedegebuure1, S. Wickline2, H. Pan2, R. C. Fields1 1Washington University,Surgery,St. Louis, MO, USA 2University Of South Florida College Of Medicine,Cardiology,Tampa, FL, USA
Introduction: For patients with metastatic colorectal cancer (CRC), 5 year survival is ~10%. Unlike conventional chemotherapy, where treatment is not targeted and often inefficient, nanoparticles (NPs) have the potential to allow precise delivery of cargo (such as siRNA or other small molecule inhibitors) directly to sites of disease. The cargo being delivered can theoretically target multiple different pathways simultaneously, including those not “druggable” by other means (such as KRAS). Here, we describe our peptide based, endosomolytic NP system designed to deliver siRNA against KRAS to several model CRC tumor systems.
Methods: Quasar 705 (Q705) tagged siRNA NPs (Fluorescent NP) were given via IV injection to mice, and in vivo uptake was assessed. To assess Fluorescent NP uptake we utilized confocal microscopy, in vivo and ex vivo imaging systems, as well as multi-color flow cytometry. CT26 murine CRC cells, which harbor a KRAS mutation are used throughout. For patient derived xenografts (PDXs), a unique KRAS mutant CRC cell line, 322, was derived. Subcutaneous (SQ) CT26 ± GFP/Luciferase labelled murine CRC tumors were created by injecting tumor cells into the right flank of BALB/C mice. For liver tumors, a hemi-splenectomy liver metastasis model was used, with CT26 GFP/luciferase labelled CRC cells injected into the BALB/C hemispleen. A KRAS specific siRNA was used create our KRAS siRNA NP (KRAS NP) for IV treatment, and KRAS RNA level was assessed via RT-PCR.
Results: Our Fluorescent NP localizes to SQ murine CRC tumors (Figure Part A, N=5 per group—Fluorescent NP or Control NP treated) with minimal non-tumor uptake, as well as 322 PDX CRC tumors (not shown). Our Fluorescent NP localizes specifically to GFP positive murine CRC tumor cells and not to non-tumor cells in a SQ tumor model (Figure Part B, N=3 per group—Fluorescent NP and Control NP treated). In our liver metastasis model, Fluorescent NP is delivered specifically to GFP+ tumor cells in the liver (Figure Part C). Mice harboring CT26 SQ tumors were then treated with KRAS NP, which leads to a 50% knockdown in tumor KRAS RNA expression as compared to Control NP treated mice (p<0.0001).
Conclusion: Our peptide based nanoparticle localizes to in vivo CRC cells in metastatic and heterotopic model systems. This system can be used to specifically target KRAS expression. We are currently exploring the therapeutic potential of this system in multiple pre-clinical systems as a novel platform for targeted delivery of precision therapeutics.
