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Researchers at our world-class Gene Therapy Center are continuously developing new methods to safely deliver genes to patients with diseases caused by a faulty or missing gene. One such method uses an adeno-associated viral vector (AAV), which can be designed to carry a specific gene and insert it into a specific body tissue. Viral vectors are also being used to deliver and insert various RNA molecules that turn genes off or on in specific tissues. Using these methods allows greater control over gene function in live animals. These techniques have been developed in a close collaboration between the Gene Therapy Center and the RNA Therapeutic Institute under Drs. Terence Flotte, Guangping Gao, and Philip D Zamore. Their data from experiments on mice suggest that the combined use of AAV and RNA that turns off genes (a mechanism called RNA interference) can clarify how one type of gene-silencing RNA, microRNA (miRNA), functions in mammals. This novel and powerful combined method promises to be unique in its potential to clarify how miRNA works, especially in cancer, metabolic and degenerative disorders, and infectious diseases.

A potential application for this combined technology is treating abnormal cholesterol metabolism. High blood cholesterol levels, known as hypercholesterolemia, are a major risk factor for cardiovascular disease, the most common cause of illness and death in the US. One form of hypercholesterolemia is due to inherited mutations in the cell receptor molecule that binds and absorbs low density cholesterol (LDL, bad cholesterol) from the blood under normal conditions. The mutated LDL receptor can malfunction, leading to accumulation of bad cholesterol in the blood and resulting in disease symptoms. The defective gene for the LDL receptor can be replaced with a normal gene in gene-replacement therapy using AAVs, but this treatment may lead the body to generate an immune response against the replaced normal LDL receptor.

To avoid this problem, Drs. Flotte, Gao, and Zamore used an alternative to gene-replacement therapy for the faulty LDL receptor gene, i.e., the body's normal miRNA control of cholesterol levels. Cholesterol metabolism is controlled by the most abundant miRNA in the liver, miR-122, by an unknown mechanism that does not directly affect LDR receptors. Therefore, the researchers made an artificial RNA that inhibits miR122, packaged it in AAV, inserted it into mice, and evaluated its effects on cholesterol metabolism. One injection of this vector reduced mouse miR-122 by 80% and tripled the activity of genes normally inhibited by miR-122. Interestingly, injecting this vector into mice also reduced their cholesterol levels by half. In another set of experiments, the researchers used mice with no LDL receptors. Even when these mice are kept on a normal diet, they have high cholesterol levels as seen in humans with hypercholesterolemia. When the same vector was injected into these diseased mice, their cholesterol and LDL levels fell rapidly by one-third to one-half and these levels remained low. More importantly, these injections had no visible toxic effects on the mice. These data suggest that AAV-based miRNA therapeutics can efficiently and stably inhibit miRNA and that this method can be used to treat hypercholesterolemia and other diseases caused by miRNA deregulation. In addition, studying AAV-mediated miR-122 regulation in laboratory animals with known genetic defects may help scientists to understand the molecular process(s) of miR-122-regulated cholesterol metabolism.