Abstract 1515: Combination siRNA and gene delivery to SKOV-3 cancer cells by a customizable HER2 targeted biopolymeric vector

Abstract

Abstract INTRODUCTION: The major hurdle in successful targeted cancer gene therapy is the lack of a suitable gene delivery vehicle, leaving room for research into innovative delivery vehicles. We have designed and bioengineered a customizeable biopolymeric vector that can overcome intracellular barriers and facilitate gene transfer. The biopolymeric vector contains multiple functional domains including a DNA condensing motif to condense the genetic material into nanosize particles, a targeting peptide to target HER2 positive SKOV-3 cancer cells and internalize, an endosomolytic motif to aid in endosomal escape of the cargo into cytosol, and a nuclear localization (NLS) signal to translocate the genetic material to the cell nucleus. For siRNA delivery, a biopolymer was specifically designed without NLS motif in its structure as the target site for siRNA is cytoplasmic. For pDNA delivery purposes, the biopolymer with NLS in its structure was complexed with either pEGFP (encodes GFP) or therapeutic gene pTKSR39 (encodes mutant thymidine kinase). For siRNA delivery, the biopolymer without NLS was complexed with either GFP siRNA or Bcl-2 siRNA. This is the first report of a gene delivery system that can be customized specifically for pDNA or siRNA delivery to cancer cells. METHODS: Recombinant biopolymers were genetically engineered in E. coli and purified. The biopolymers were complexed with GFP siRNA, Bcl-2 siRNA, pEGFP, and pTKSR39 to form nanoparticles. The size of nanoparticles were characterized by Dynamic Light Scattering. Each biopolymer in complex with siRNA or pDNA was used to transfect to SKOV-3 ovarian cancer cells. Flow cytometry was used to measure the total green fluorescence. The expression of TKSR39 and knockdown of Bcl-2 gene were examined by westernblot analysis. The ability of the biopolymers in complex with Bcl-2 siRNA or pTKSR39 (in combination with Ganciclovir) to kill cancer cells was examined using a WST assay. Stable SKOV-3 cells that express GFP (SKOV-3-GFP) were also prepared for GFP siRNA studies. RESULTS & CONCLUSIONS: The biopolymer without NLS was able to deliver GFP siRNA to the cell cytoplasm and resulted in significant knockdown of GFP in SKOV-3-GFP cells. This biopolymer was able to knockdown the Bcl-2 gene and result in significant cell death. The biopolymer with NLS was unable to reduce the level of GFP expression in SKOV-3-GFP cells and also unable to knockdown Bcl-2 gene significantly. However, the biopolymer with NLS was successful in delivering both pEGFP and pTKSR39 to the SKOV-3 cells efficiently. The biopolymer equipped with NLS and in complex with pTKSR39 was able to kill SKOV-3 cells significantly after Ganciclovir treatment. These and previous studies show that our novel biopolymeric system can be custom designed for various gene therapy needs. Combination of siRNA and gene delivery shows promise as a multi-faceted therapy with numerous potential applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1515.