Enhancement of Polymer-mediated Gene Expression by Hyperosmotic Polymannitol and Polyxylitol based Gene Transporters

Abstract

Successful treatment of genetic diseases by gene therapy requires efficient delivery of therapeutic DNA into specific cells, followed by safe and long-term expression of the encoded gene product at physiologically relevant levels. Efficient translocation of transgene faces physical and metabolic barriers throughout its voyage to the nucleus of targeted cells. Crossing the biological barriers such as cell membrane, tumor mass and blood brain barrier (BBB) are challenging for effective and efficient gene therapy. Novel strategies and rational designing of polymeric vector can lead to the evolution of highly efficient and safe gene delivery carrier. This study uncovers the implications and applications of polyhydroxyl containing hyperosmotic polymers in crossing biological barriers for enhanced gene delivery. This dissertation aims to: (i) The synthesis of polymannitol- and polydixylitol-based vectors to utilize the osmotic activity due to hydroxyl groups for accelerated cellular uptake and enhanced gene transfection. Various endocytosis inhibition studies verify the stimulation of caveolae and cyclooxygenase-2 (COX-2) - dependent endocytosis to be the main route of cellular internalization to account for enhanced transgene expression. (ii) The research introduces the application of polydixylitol based hyperosmotic polymer (PdXYP) in transmigrating BBB by intra-arterial infusion of osmotic polyol by triggering cellular uptake via selective stimulation of caveolae-mediated endocytosis. In vitro BBB model and in vivo transfection results showed higher transfection efficiency in brain astrocytes. In vivo bioimaging and mechanistic investigation proved the potential of the system reaching deeper in to the tumor mass. (iii) A combination treatment that uses hyperosmotic non-viral vectors and nanopatterned matrix to promote gene delivery into cells indicates the importance of synergistic cues in designing non-viral gene delivery platforms and strategies for gene therapy. (iv) The delivery of therapeutic siRNA in cancer cells using polymannitol-based vector (PMGT) exemplifies the vector as a strategy to inhibit tumor progression. c-Jun N-terminal kinase 2 (JNK2) is primarily responsible for the oncogenic transformation of the transcription factor c-Jun. Expression of the proto-oncogene c-Jun progresses the cell cycle from G1 to S phase, but when its expression becomes awry it leads to uncontrolled proliferation and angiogenesis. Delivering a JNK2 siRNA in tumor tissue was anticipated to reverse the condition with subsequent onset of apoptosis which predominantly requires an efficient delivering system capable of penetrating through the compact tumor mass. In the present study, it was demonstrated that PMGT with inherent hyperosmotic properties was able to penetrate through and deliver the siJNK2 in the subcutaneous tumor of xenograft mice. Hyperosmotic activity of polymannitol was shown to account for the enhanced therapeutic delivery both in vitro and in vivo because of the induction of COX-2 which firstly stimulates caveolin-1 for caveolae-mediated endocytosis of the polyplexes and secondly activates caspase-9 to induce apoptosis in cancer cells. Further suppression of JNK2 and hence c-Jun expression led to the inhibition of tumor growth in xenograft mice model. Thus, the synthesis and application of hyperosmotic polymers demonstrated their facilitation in crossing the biological barriers by elevating the osmolarity of the extracellular matrix.