A. T. Tsang1,2, X. Yu1, R. Goraczniak5, M. Brenneman1,5, S. Gunderson3,5, D. R. Carpizo1,2,4 4Rutgers University,Department Of Pharmacology,Piscataway, NJ, USA 5Silagene Inc.,Hillsborough, NJ, USA 1Cancer Institute Of New Jersey,Division Of Surgical Oncology,New Brunswick, NJ, USA 2Robert Wood Johnson – Rutgers,Department Of Surgery,New Brunswick, NJ, USA 3Rutgers University,Department Of Molecular Biology And Biochemistry,Piscataway, NJ, USA
Introduction:
Activating mutations of the KRAS gene are key drivers of pancreatic cancer. However, despite decades of effort, the KRAS protein has proved refractory to small-molecule targeting. Many RNA interference-based studies targeting RAS have demonstrated therapeutic effects, but technical difficulties of delivery in vivo have impeded translation of this approach to the clinic. U1 Adaptors are synthetic oligonucleotides that enable the U1 small nuclear ribonucleoprotein complex to stably bind within the terminal exon of any chosen pre-mRNA target. This interferes with the obligatory polyadenosine tail addition step in mRNA maturation, resulting in selective destruction of the mRNA in the nucleus. The U1 Adaptor gene silencing mechanism is thus distinct from those of siRNA or antisense oligonucleotides, and offers important advantages for their use as therapeutic agents. In a proof-of-concept study, U1 adaptors targeting BCL2 and GRM-1 oncogenes effectively suppressed growth of human melanoma xenograft tumors in mice.
Methods:
We sought to translate this technology to target human KRAS in pancreatic cancer. We first screened a set of U1 Adaptors targeting human KRAS pre-mRNA at eight different positions within the terminal exon coding sequence and untranslated region. Adaptors were evaluated in vitro using the human pancreatic cancer cell line MiaPaca-2 (KRASG12C). KRAS mRNA was measured by quantitative PCR, and KRAS protein expression was determined using standard Western blot techniques. Cell growth inhibition assays were performed and viable cells were counted using flow cytometry over 12 days. Adaptors that gave the best in vitro results were then evaluated in MiaPaca-2 xenografts. For in-vivo delivery, Adaptors were complexed with nanoparticles linked to a tumor-targeting peptide, cRGD, and administered by tail vein injection twice weekly. Tumors were then evaluated for KRAS gene and protein expression.
Results:
U1 Adaptor screening revealed a range of KRAS gene silencing as measured by quantitative PCR. Candidate Adaptors 1, 2 and 3 reduced KRAS mRNA expression by 65%, 73% and 76% respectively; as effectively as an siRNA control. We demonstrated potent inhibition of MiaPaca-2 cell growth and knockdown of KRAS protein expression, using Adaptors modified with locked nucleic acid (LNA). We then evaluated Adaptors 2 and 3 in mice bearing MiaPaca-2 xenografts. We observed significant tumor growth inhibition, by as much as 68% using Adaptor 3 (p=0.0002), compared to the vehicle control by day 34. Tumors were then analyzed and found to have KRAS protein reduction.
Conclusion:
We have demonstrated that the U1 Adaptor method of gene silencing can be successfully applied to target human KRAS both in vitro and in vivo. These results support the continued investigation of U1 Adaptor technology as a strategy for therapeutic targeting of RAS oncogenes.