Professor T.I. Tikchonenko is a well-known molecular virologist, experienced in the genetic engineering of animal viruses, construction of antisense RNA (asRNA) genes, and development of transgenic and chimeric animals. Dr. Overbaugh's laboratory has developed several molecularly-defined retroviral model systems for studying the mechanisms of T cell cytopathicity and immunodeficiency disease induced by retroviruses. The collaborative effort of these two teams will focus on applying the expertise of Dr. Tikchonenko in developing antisense antiviral strategies for inhibition of feline leukemia virus (FeLV) infection. These studies will expand currently funded efforts in Dr. Overbaugh's laboratory on the 'Regulation of FeLV expression and pathogenesis' (NIH/NCI R01 CA51080), which is the parent grant for this application, to include antisense strategies for inhibition of FeLV replication and expression. We propose to engineer several different constructs expressing FeLV asRNA genes and test their efficacy in inhibiting FeLV replication, expression and cytopathicity in cell culture systems. The FeLV variants used for virus challenge will include representatives of: mildly lymphomagenic subgroup-A FeLVs, a subgroup-B FeLV isolated from the brain tissue of an infected cat, and FeLV-FAIDS variants that induce cytopathicity in T cells and immunodeficiency disease in cats. The antiviral effects of chemically synthesized asDNA targeted at different parts of the FeLV genome will be tested in cell culture in conjunction with successful asRNA methods. Retroviral vectors will be constructed for expression of asRNAs with maximal activity as determined in the initial studies, and these will be used to introduce exogenous genes expressing asRNA into primary cells. We propose to develop this latter approach with the hope of eventually introducing asRNAS in vivo by transplantation. The long term goal of the studies proposed here will be to develop a FeLV model system to test the feasibility and efficacy of antisense approaches as anti-retroviral therapies.
