(1) We are evaluating an alternative gene therapy method using a new methodology, RNA/DNA hybrids, targeting cultured cells as well as utilizing the hybrids to create animal models of disease. The RNA and DNA portions have different functions, RNA targets the hybrid to a specific region in the genome, the DNA portion is thought to then induce a repair process to modify a single targeted base, resulting in the desired change within the genome. The method has been used to induce targeted single base genomic changes in cultured cells for three targets apolipoprotein B, hemoglobin and alkaline phosphatase. (2) Peptide nucleic acid (PNA) oligonucleotides have a higher affinity for native DNA than normal DNA oligonucleotides. PNA's hybridization to DNA is also more significantly destabilized by a single base mismatch. Utilizing these attributes, we have developed a PCR method to screen for rare single base mutations that constitute as little as 0.25% of the total sequences. Using a PNA that blocks PCR amplification of the normal sequence, the rare sequence is preferentially amplified. (3) We are investigating various gene therapy vector delivery systems in collaboration with Dr. Nancy Templeton Smyth at the NCI. She has developed methods that are reportedly up to 100 times more potent than traditional cationic liposome methods. We are evaluating these methods in the delivery of gene therapy vectors, RNA/DNA hybrids and antisense oligonucleotides to hepatocytes. 4) We have been evaluating the effect of lovastatin, a lipid lowering drug, on the markedly atherogenic lipoprotein Lp(a) in patients with systemic lupus erythematosus (SLE); another study is evaluating thyroid hormone induced changes in Lp(a) in thyroid cancer patients. During treatment for thyroid cancer, thyroid hormone levels are iatrogenically varied, allowing the effect of high and low levels of thyroid hormone on Lp(a) to be determined. We have been evaluating the effect of niacin, another lipid lowering drug, on the markedly atherogenic lipoprotein Lp(a). Other collaborative studies involve determining the lipid effect of IL-6 and IL-10 in patients. Both interleukins significantly reduced cholesterol, triyglcerides and apolipoprotein B. 5)The potential toxicity of gene therapy and/or embryonic stem cell manipulation is being evaluated in a cohort of mice that carry an obesity and hyprlipidemia phenotype. They resulted from experiments that were designed to create a homeobox knockout using embryonic stem cells. An evaluation is being carried out to determine if some portion of the targeting vector used to create the knockout has integrated in the genome disrupting an important gene in weight regulation and hyperlipidemia.
