Abstract 1453: MicroRNA replacement and RNAi-mediated silencing of ALK as combined targeted therapies for neuroblastoma
Authors: Perri Patrizia , Paolo Daniela D. , Priddy Leslie , Fiore Annarita D. , Brignole Chiara , Pastorino Fabio , Brown David , Ponzoni Mirco 
Published: May 2015

Abstract
The effective treatment of advanced Neuroblastoma (NB) is still a challenge in pediatric oncology, because the clinical use of most therapeutics is limited by insufficient drug delivery to the tumor and high systemic toxicity. The discovery of the RNAi has great promise for anti-cancer therapeutics but, as in high-grade solid tumors a single ‘oncogene addiction’ is rare, multi-'gene' target combinations are required and a targeted delivery system is mandatory to successfully translate RNAi-based therapeutics into the clinics.
It is now ascertained the master role of ALK and related genes such as PHOX2B, able to promote its transcription, in NB growth and survival, opening new perspectives for RNAi-mediated therapies for NB. Nevertheless, even if the counterpart of tumor suppressor (TS) regulators in NB is not completely known, a number of miRNAs with TS functions have been found aberrantly under-expressed in NB and associated with aggressive phenotypes.
To develop a new generation of targeted and multi-RNAi-directed therapeutics for NB, we are exploiting our well-established NB-specific delivery system, the GD2-Targeted Liposomes entrapping specific siRNAs, TL[siRNA], or miRNAs mimics, TL[miRNA]. The final purpose is to get simultaneously the silencing of the oncogenic functions of ALK and related genes and the replacement of miRNAs with TS functions in clinically relevant mouse models of NB. We have planned to test different combinations of individually entrapped TL[siRNA] or TL[miRNA] for in vivo therapeutic efficacy.
In a pilot study, NB-bearing mice were treated with different TL[siRNA]. After 65 days from i.v. injection of HTLA-230 (WT ALK) cells, we observed: about 50% of TL[siALK]-treated mice still disease-free, marginal effects with TL[siPHOX2A] and moderate effects with TL[siPHOX2B] formulations alone and a significant increased mice survival with an equimolar combination of TL[siRNA] of each gene. The enhanced efficacy of ALK and PHOX2 genes knockdown combination highlights a powerful therapeutic potential.
After pilot transfection studies of SH-SY5Y (F1174L mut ALK) and GI-LI-N (WT ALK) cell lines, TS miRNAs mimics were screened and selected. Cell proliferation was evaluated at different days post-transfection. miR-96, miR-34a and let7b replacement resulted the most effective in inhibiting NB cell growth. Liposomal preparation of miRNA-NC and the above mimics for in vivo studies was tuned up according to the number of negative charges of the stem-loop sequence with an entrapment efficiency of 80-90%. Currently, an ongoing in vivo trial indicates promising results after replacement of miR-34a and let7b.
We believe that the use of tumor-targeted biocompatible carriers of RNAi molecules able to replace loss-of-functions and to knockdown gain-of-functions in cancer cells at the same time is likely to represent an innovative modality for a more effective gene therapy.
Citation Format: Patrizia Perri, Daniela Di Paolo, Leslie Priddy, Annarita Di Fiore, Chiara Brignole, Fabio Pastorino, David Brown, Mirco Ponzoni. MicroRNA replacement and RNAi-mediated silencing of ALK as combined targeted therapies for neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1453. doi:10.1158/1538-7445.AM2014-1453