PDF file - 687K, Figure S1. dsP21-322 triggers changes in cell morphology associated with growth inhibition. Figure S2. Anti-growth activities of LNP-dsP21-322-2'F and its non-formulated derivatives. Figure S3. Duplex stability in water and detection of nucleic acid background signal in urine. Figure S4. Uptake of LNP-formulated dsP21-322-2'F-FITC by bladder urothelium. Figure S5. GFP signature of orthotopic tumors in excised mouse bladders. Figure S6. Muscle invasive bladder tumor and metastatic bladder tumor in the kidney
ARTICLE ABSTRACT
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.
