The investigator writes that most applications of gene therapy will depend on prolonged expression and readministration of the transgene. Although design of viral vectors with extensive deletions of the genome has resulted in prolonged expression, this approach does not completely abrogate the immune response. It is their contention that a combined strategy that includes tolerization to the vectors, as well as low immunogenicity vectors, will be absolutely necessary for successful long-term gene therapy in humans. He proposes a tolerization strategy that will be applicable clinically and is supported by preliminary data indicating prolonged expression of the transgene and the absence of an immune response. The investigators have shown that antigen presenting cells expressing adenovirus/Fas ligand (Ad/Fas L) and antigen induces systemic tolerance to the Ad antigens without toxicity. Here, they will use APCs that express Fas ligand and have been infected with low-immunogenicity adenovirus-LacZ and other transgenes to induce systemic tolerance to the gene therapy.
In Aim 1, the investigators will determine if the mouse CD11b promoter can specifically up-regulate Fas L in APCs infected with adenovirus with extensive DNA deletions. Expression of Fas L specifically in macrophages using the CD11b promoter has been demonstrated after transient transfection and in transgenic mice.
In Aim II, they will determine if ex-vivo infected APC with adenovirus with extensive DNA deletions can be used to induce T-cell tolerance to adenovirus vector and transgene antigens. APCs will be infected ex-vivo and analyzed for tolerance induction in vivo. The tolerance to transgenes of low and high immunogenicity will be determined.
In Aim III, they will determine if survival of normal APCs will be prolonged using the Fas apoptoss inhibitor CrmA gene and a soluble TNF-receptor to enhance APC and adenovirus survival and tolerization after infection with adenovirus.
In Aim I V, the investigators will develop adenovirus with modified tropism to target Fas L expression to antigen presenting cells for in vivo tolerance induction. This will be accomplished by enhanced targeting to APC using the mannose receptor and by pre-infusion of Ad fiber knob protein to inhibit endogenous tropism of Ad/Fas L to the liver. These experiments should accomplish the objective of devising adenovirus-transgene tolerance methods that are safe and efficiently deliver Fas L to APCs, either by in vivo transfer with relatively high targeting to APCs or in vitro transfer into cells and then transfer of cells that express Fas L. Infection of APCs ex-vivo with Ad/FasL has now been demonstrated to be safe and feasible, and does not induce toxicity upon transfer of these macrophages in vivo. Dr. Mountz and his colleagues believe that the studies will provide a feasible and safe method to enable prolonged and repeated administration of gene therapy that will be required for successful treatment of human diseases.
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