1) The outermost protein of the retrovirus, the envelope, carries the primary determinant of viral tropism by virtue of its specific interaction with a cell surface receptor. Retroviral vectors used in human gene therapy clinical trials have the envelope of either amphotropic murine leukemia virus (A-MuLV) or gibbon ape leukemia virus (GaLV). The receptors for A-MuLV and GaLV are closely related, but distinct, multiple-membrane spanning phosphate symporters. Site-directed mutagenesis demonstrated that interactions with a conformational epitope were involved in A-MuLV receptor recognition. These on-going studies reveal the structural requirements for how retroviral vectors interact with their receptors and enter cells. 2) We have developed a method for expressing a genetically-modified retroviral vector which contains chimeric envelope glycoproteins. These types of retroviral vectors will be useful in targeting gene delivery to specific cell types (i.e., tumor cells) in vivo. 3) Experimental evaluation of the assays used for detection of replication-competent retrovirus (RCR) in retroviral vector production material, have demonstrated that presence of replication-defective retroviral vector in clinical grade material can inhibit significantly the ability to detect RCR. A standardized stock of RCR is being developed to allow for calibration of RCR detection assay sensitivity. 4) Phase I clinical trials involving the use of swine tissues have begun. However, pigs carry endogenous retroviruses. We have shown that activation of this normally latent retrovirus occurs in the course of in vitro immune activation of pig peripheral blood lymphocytes. We now have pig cell lines productively infected with retrovirus from two different pig breeds. Preliminary molecular characterization of these swine retrovirus(es) suggest that they may differ from a previously characterized pig retrovirus. These studies will help to elucidate the safety concerns regarding clinical use of porcine cells, tissues, or organs and allow for development of diagnostic assays.
