TolerGenics, Inc., an entrepreneurial biotechnology company established in 1997, is developing and commercializing novel therapies for inducing and maintaining epitope-specific immune tolerance using a B-cell delivery strategy. Based on the hypothesis that B-cell antigen presentation of self immunoglobulins would be highly tolerogenic, we employ a patented technology (#5,817,308 """"""""Tolerogenic fusion proteins of immunoglobulins and methods for inducing and maintaining tolerance"""""""") in which we engineer retroviral constructs containing multiple epitopes in frame at the N-terminus of a murine IgG1 heavy (H) chain. This technology has been shown to achieve long-term maintenance of immune tolerance through continuous in vivo presentation of relevant epitopes. Data in three experimental autoimmune models are highly promising in that significant clinical efficacy has been achieved. That is, expression of Ig fusion proteins in B cells can both prevent and reverse autoimmune responsiveness in uveitis and EAE, and significantly delay the onset of diabetes in NOD mice with peri-insulitis. However, safety concerns about the use of retroviral vectors have arisen from recent human clinical trials. Thus, we believe that alternative expression methods for these constructs need to be developed before further trials can ensue. In this application, we wish to test the hypothesis that transient expression of Ig fusion proteins can be tolerogenic, using two new methods for non-insertional expression of genes and/or proteins: Nucleofection[TM] and HIV TAT fusion proteins. Nucleofection[TM] (Nf) is a non-viral transfection methodology for efficient delivery of DNA directly to the nucleus of non-dividing, as well as dividing, cells. We have found that Nf can be used to express GFP constructs at high efficiency in naive human T and B lymphocytes. TAT fusion proteins are rapidly taken up by a variety of cells, including lymphocytes, and can be used to deliver proteins for presentation to the immune system. In our first aim, we will express a tetanus toxin peptide (p947-967)-Ig as a model antigen in human B cells. TT was chosen as a model antigen because the response of uncloned human T-cells from Type I diabetes patients to diabetogenic epitopes is poor. B cells expressing this peptide will be incubated with autologous peripheral T cells in an in vitro readout for tolerance. TAT fusion proteins of p947-967-Ig will be similarly expressed in human B cells for tolerance in vitro. In the second aim, murine B cells from NOD mice will be incubated with TAT-insulin-Ig and then transferred in vivo to test their tolerogenic effects on development of diabetes. As Amaxa technology develops, we will beta test Nf in aim 2 for use with murine B cells as tolerogenic APC in NOD recipients. We expect these novel immune tolerance therapies will ultimately be utilized in the treatment of autoimmune diabetes.