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Practical methods for enhancing protein production in vivo remain a challenge. RNA activation (RNAa) is emerging as one potential solution by using double-stranded RNA (dsRNA) to increase endogenous gene expression. This approach, although related to RNA interference (RNAi), facilitates a response opposite to gene silencing. Duplex dsP21-322 and its chemically modified variants are examples of RNAa-based drugs that inhibit cancer cell growth by inducing expression of tumor suppressor p21WAF1/CIP1 (p21). In this study, we investigate the therapeutic potential of dsP21-322 in an orthotopic model of bladder cancer by formulating a 2′-fluoro-modified derivative (dsP21-322-2′F) into lipid nanoparticles (LNP) for intravesical delivery. LNP composition is based upon clinically relevant formulations used in RNAi-based therapies consisting of PEG-stabilized unilamellar liposomes built with lipid DLin-KC2-DMA. We confirm p21 induction, cell-cycle arrest, and apoptosis in vitro following treatment with LNP-formulated dsP21-322-2′F (LNP-dsP21-322-2′F) or one of its nonformulated variants. Both 2′-fluoro modification and LNP formulation also improve duplex stability in urine. Intravesical delivery of LNP-dsP21-322-2′F into mouse bladder results in urothelium uptake and extends survival of mice with established orthotopic human bladder cancer. LNP-dsP21-322-2′F treatment also facilitates p21 activation in vivo leading to regression/disappearance of tumors in 40% of the treated mice. Our results provide preclinical proof-of-concept for a novel method to treat bladder cancer by intravesical administration of LNP-formulated RNA duplexes. Cancer Res; 72(19); 5069–79. ©2012 AACR.

Small RNA molecules are a promising new class of drugs by offering expanded targets not druggable by conventional therapies with high target specificity and low toxicity. However, their clinical use is significantly hindered by the lack of vehicles that deliver the molecules efficiently to target tissues and cells. The bladder is an easily accessible hollow organ and is ideal for local delivery of drugs or molecules such as small RNA. RNA activation (RNAa) is a newly discovered mechanism of gene induction triggered by promoter targeted double-stranded RNA, also known as small activating RNA (saRNA). In this study, we investigate, in an orthotopic bladder cancer model, antitumor effects of a saRNA (dsP21) targeting the promoter of the p21WAF1/CIP1 (p21) gene, a key negative regulator of the cell cycle rarely mutated or deleted in bladder cancer. Introducing dsP21 into bladder cancer cells activated p21 expression with subsequent inhibition of cell proliferation, arrest of the cell cycle and induction of apoptosis accompanied by the activation of caspase 3 and PARP. Chemical modification (2′-Fluoro) and subsequent formulation of dsP21 in lipid nanoparticles (LNPs) retained its RNAa activity with minimal immunostimulatory effect and extended its stability in urine, and when delivered intravesically it could well diffuse into the bladder wall. Delivery of LNP-formulated dsP21 (LNP-dsP21) into mouse bladder with established human bladder cancer significantly inhibited tumor growth and extended animal survival with demonstrated p21 activation in vivo. Of particular significance, LNP-dsP21 treatment caused the regression or disappearance of established tumors in 30% of the treated mice. Our results provide proof-of-principle that targeted activation of p21 can be applied to the treatment of bladder cancer and LNP-formulated small RNA can be successfully delivered to the bladder by intravesical instillation. Further clinical development of RNAa-based intravesical therapy is warranted for the treatment of residual and recurring bladder cancer in humans. Acknowledgement: financial support from the AACR Henry Shepard Bladder Cancer Research Grants (09-60-30-LI). 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 LB-363. doi:1538-7445.AM2012-LB-363