Gel-based application of siRNA to human epithelial cancer cells induces RNAi-dependent apoptosis.

Gene silencing by RNA interference (RNAi) operates at the level of mRNA that is targeted for destruction with exquisite sequence specificity. In principle, any disease-related mRNA sequence is a putative target for RNAi-based therapeutics. To develop this therapeutic potential, it is necessary to develop ways of inducing RNAi by clinically acceptable delivery procedures. Here, we ask if inducers of RNAi can be delivered to human cells via a gel-based medium. RNAi was induced using synthetic small interfering RNAs (siRNAs), which bypass the need for expression vectors and carry the added bonus of high potency and immediate efficacy. Established cultures of human cells of normal and tumor origin were overlaid with an agarose/liposome/siRNA gel formulation without adverse effects on cell viability or proliferation. Epithelial cancer cells (but not normal human fibroblasts) proved vulnerable to specific siRNAs delivered via the agarose/liposome/siRNA formulation. Moreover, proapoptotic siRNAs induced apoptosis of cervical carcinoma cells (treated with human papillomavirus [HPV] E7 siRNA) and of colorectal carcinoma cells (treated with Bcl-2 siRNA). Thus, we demonstrate successful topical gel-based delivery of inducers of RNAi to human epithelial cancer cells. Topical induction of RNAi opens an important new therapeutic approach for treatment of human diseases, including cervical cancer and other accessible disorders.

[1]  Elisabeth G E de Vries,et al.  Clinical potential of inhibitors of survival pathways and activators of apoptotic pathways in treatment of cervical cancer: changing the apoptotic balance. , 2005, The Lancet. Oncology.

[2]  P. Sharp,et al.  RNAi Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals , 2000, Cell.

[3]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[4]  B. Clotet,et al.  Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication , 2002, AIDS.

[5]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[6]  A. Ganser,et al.  Specific inhibition of bcr-abl gene expression by small interfering RNA. , 2003, Blood.

[7]  David S. Jones,et al.  Rheological and mucoadhesive characterization of polymeric systems composed of poly(methylvinylether-co-maleic anhydride) and poly(vinylpyrrolidone), designed as platforms for topical drug delivery. , 2003, Journal of pharmaceutical sciences.

[8]  D. Weissman,et al.  Inhibition of HIV-1 Infection by Small Interfering RNA-Mediated RNA Interference1 , 2002, The Journal of Immunology.

[9]  B. Cullen,et al.  Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference , 2002, Journal of Virology.

[10]  D. Dykxhoorn,et al.  Killing the messenger: short RNAs that silence gene expression , 2003, Nature Reviews Molecular Cell Biology.

[11]  P. Whittaker,et al.  RNA interference: From gene silencing to gene-specific therapeutics , 2005, Pharmacology & Therapeutics.

[12]  J. Robinson,et al.  Oral cavity as a site for bioadhesive drug delivery , 1994 .

[13]  J. Lieberman,et al.  siRNA-directed inhibition of HIV-1 infection , 2002, Nature Medicine.

[14]  Ali Ehsani,et al.  Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells , 2002, Nature Biotechnology.

[15]  R. Bernards,et al.  Stable suppression of tumorigenicity by virus-mediated RNA interference. , 2002, Cancer cell.

[16]  L. Nielsen,et al.  Bioadhesive drug delivery systems: I. Characterisation of mucoadhesive properties of systems based on glyceryl mono-oleate and glyceryl monolinoleate , 1998 .

[17]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[18]  W. Coulter,et al.  Efficacy of bioadhesive patches in the treatment of recurrent aphthous stomatitis. , 1996, Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology.

[19]  J. Milner,et al.  Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference , 2002, Oncogene.

[20]  Ming Jiang,et al.  Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cells. , 2003, Genes & development.

[21]  Douglas S. Conklin,et al.  Gene expression: RNA interference in adult mice , 2002, Nature.

[22]  David L. Lewis,et al.  Efficient delivery of siRNA for inhibition of gene expression in postnatal mice , 2002, Nature Genetics.

[23]  John Rossi,et al.  Inhibition of HIV-1 by lentiviral vector-transduced siRNAs in T lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell-derived macrophages. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.

[24]  Michael T. McManus,et al.  Gene silencing in mammals by small interfering RNAs , 2002, Nature Reviews Genetics.

[25]  J. H. Price,et al.  A randomised controlled trial evaluating a novel cytotoxic drug delivery system for the treatment of cervical intraepithelial neoplasia , 1997 .

[26]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[27]  P. McCarron,et al.  Sustained Release of 5‐Fluorouracil from Polymeric Nanoparticles , 2000, The Journal of pharmacy and pharmacology.

[28]  D. Baltimore,et al.  Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. V. D. Van Der Zee,et al.  Sensitivity to Fas-mediated apoptosis in high-risk HPV-positive human cervical cancer cells: relationship with Fas, caspase-8, and Bid. , 2005, Gynecologic oncology.