The primary goal of this research project is to develop techniques of direct in vivo gene transfer into muscle and liver cells, important target cells for the treatment of inborn errors of metabolism (IEM). Preliminary results indicate the RNA and DNA expression vectors can be directly injected into mouse muscle and express their respective reporter genes. The direct transfer of polynucleotides into muscle will be further developed and will be used as a model system for the development of expression vectors that have increased expression and stability in non- dividing cells, such as muscle cells. Cationic lipids have recently been shown to efficiently transfect RNA and DNA into a wide variety of cells in culture and possibly into muscle cell in vivo. However, the cationic lipid formulations that have been developed to date (commercially available as LipofectinTM) may not be able to transfect genes in vivo because they are inactivated by specific components of serum that may also be present in muscle or liver. A variety of formulation containing a previously developed cationic lipid (DOTMA) and four new cationic lipids (DOTAP, DPRI, DPTMA, and DORI) will be screened in vitro for their ability to mediate the transfection of polynucleotides into fibroblasts in the presence of calf serum. It is encouraging that cationic lipid vesicles containing phosphatidylcholine instead of DOPE or vesicles containing more cationic lipid are not inhibited by serum in vitro. The cationic lipid formulations that can deliver and express polynucleotides in vitro in the presence of calf serum will be tested in vivo by injecting them intramuscularly and intrahepatically into mice and rats and into the portal circulation of rats. The clinical relevance of these techniques will be assessed by determining whether they can modify or correct the biochemical phenotype of the phenylalanine hydroxylase (PAH) deficient mouse, a model for inborn Errors of Metabolism characterized by a circulating toxic metabolite.
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