There is currently a very large research activity in developing lipid- based vectors for therapeutic applications because of their nonimmunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. Indeed cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids currently used in human clinical gene therapy trials worldwide. The vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short-interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. The low efficiencies are the result of poorly understood transfection-related mechanisms at the molecular and self-assembled levels, and a general lack of knowledge about interactions between membranes and double stranded nucleic acids resulting in stable complex formation, and between membrane-nucleic acid complexes and cellular components.
The aims of this research application are (1) to use custom synthesized degradable and PEGylated lipids, and biophysical characterization, in order to clarify the interactions between lipid-nucleic acid complexes and cellular components for improved understanding of structure-function properties, and (2) to clarify structures and interactions between cationic membranes and siRNA in CL-siRNA complexes used in gene silencing. Modern methods of organic and solid phase chemistry will be employed to synthesize multivalent degradable lipids, peptide-PEG-lipids, and acid labile PEG-lipids. The structure of the lipid-nucleic acid complexes will be solved by using synchrotron x-ray diffraction techniques at the Stanford Synchrotron Radiation Laboratory and cryo-electron microscopy at UCSB and the Scripps Research Institute. Confocal microscopy will enable us to track the lipid-nucleic acid complexes and observe their interactions with cells. The structures will be correlated to the biological activity of complexes interacting with cells by quantitative measurements of transfection efficiency and silencing efficiency both in DMEM and in high-serum for in vivo applications. The broad long-range goal of the research is to develop a mechanistic understanding of the biophysical interactions between cationic membranes and biologically active double stranded nucleic acids and between CL-nucleic acid complexes and cells, which will generate custom lipid-carriers of nucleic acids ultimately for use in gene therapeutics and disease control.
The project proposes to use a mechanistic approach to further the understanding of lipid carriers of therapeutic DNA and RNA. This, in turn, will lead to new materials and methods and the development of efficient lipidic DNA and RNA carriers for disease control. The goals will be accomplished by applying biophysical scientific methods to custom designed lipids and therapeutic molecules, made available by advanced synthetic methods.
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