Abstract 5647: Marked sensitivity to mRNA targeting by LNA antisense in tumor cells without a delivery system: Lessons learned and potential translation to the clinic

Background: Unlike siRNAs, single-stranded locked nucleic acid-based antisense oligonucleotides (LNA-ONs) have shown the ability to down regulate mRNAs in vitro and in vivo without any delivery systems such as transfection reagents or liposomes. Hence, LNA-ONs may have significant advantages as a therapeutic compared to siRNAs. Investigation of LNA-ONs that target HIF-1α, survivin, or androgen receptor in cancer patients is ongoing. EZN-3920, a LNA-ON to HER3, down regulates HER3 mRNA over a large concentration range (IC50 = 0.1-10 μM) when multiple cancer cell lines are evaluated (Zhang et al, 2011 Gene Therapy. 18:326). We assess here 1) the generality of down-regulation efficiency using two additional LNA-ONs in multiple cell lines and in cells prepared directly from patient tumors, and 2) the correlation of target down-regulation, growth inhibition, and LNA-ON concentration observed in vitro with in vivo xenograft models. Methods: LNA-ONs were either added to tissue culture media (i.e. no transfection) in vitro or prepared in saline and given IV in vivo. Endpoints were measured by qRT-PCR, MTT, Western Blot analysis, and tumor size. Gene Expression Profiling was performed by Asuragen, Inc. Concentration of LNA-ONs in tissues was measured by LC/MS/MS. Results: LNA-ONs targeting PI3KCA p110α (EZN-4150) or β-catenin (EZN-3892) potently down regulated the target mRNA in 50% of tumor cell lines, thereby confirming the observations made with EZN-3920. Diverse sensitivity of the HER3-directed LNA-ON was also observed in cells derived from over 40 primary tumors. Cell lines that were highly sensitive to LNA-ONs in proliferation assay were also responsive in xenograft tumor models. When anti-tumor efficacy of the LNA-ONs was achieved, the concentration of the drug within the tumor matched the concentration required to effectively down regulate the target in vitro. To understand the mechanisms underlying the resistance to LNA-ONs-mediated down-modulation, several cell lines were made highly resistant to LNA-ONs in vitro by chronic exposure to the agent over several months. Tumors from such cell lines were also resistant to LNA-ONs in xenograft models and can be cross-resistant to multiple LNA-ONs. We are currently identifying common genetic differences in the sensitive and resistant cell. Conclusion: We have shown that LNA-ONs, given without any delivery systems, have broad utility in many cancer cell lines including primary tumor cells in which the intended target is a driver for growth. Furthermore, those cell lines sensitive to LNA-ONs in vitro or ex vivo may help identify tumors in animal models that will be responsive to LNA-ONs. Lastly, mechanistic studies on resistant cells may provide an understanding of the diverse response of tumors to LNA-ONs that could ultimately be used to select patients who might preferentially respond to therapy with LNA-ONs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5647. doi:1538-7445.AM2012-5647