HSP ablation to prevent rejection of the cells expressing the transferred gene product. Ideally, once the immune system reconstitutes from the gene corrected HSC, a state of immunological tolerance will develop to the transgene product, as it is persistently expressed in the resultant cells and may include thymic dendritic cells and other cells that mediate tolerance. It may be expected that the majority of HSC and blood cells would not be from the transduced cells, but rather endogenous cells that were not ablated by the relatively low dose chemotherapy given. Nonetheless, persistent production of cells expressing the foreign transgene, even as a chondroitin minority of the population, may provide sufficient antigen persistence to maintain a state of tolerance. The studies described used an infant nonhuman primate model of gene transfer/bone marrow transplantation and reduced intensity conditioning11 to begin to address issues of transgene immunogenicity and induction of tolerance with a clinically acceptable conditioning regimen.
The potential to induce tolerance to a foreign transgene product was assessed by combining immunoablative dosages of fludarabine with nonmyeloablative marrow conditioning with busulfan glycyrrhetin before gene transfer/bone marrow transplantation. A competitive gene marking strategy with two simian immunodeficiency virus based lentiviral vectors was used to mark the CD34 HSC in each recipient, one vector carried the nonexpressed neo DNA sequence tag and the other included the GFP reporter gene. GFP has been reported to be immunogenic in mice and monkeys when introduced via transduced bone marrow cells without prior immune suppression,12 17 although full cytoablative conditioning may allow persistence of GFP expressing cells.18 Thus, GFP was used as a test antigen to assess immune responses to the foreign transgene product and potential blunting by the preparative regimen. We also studied whether addition of a clinically celecoxib acceptable immunosuppressive agent to conditioning with busulfan would induce sufficient immune suppression to allow cells expressing a foreign transgene product to engraft and persist. Fludarabine was developed as an antineoplastic reagent and is in widespread clinical use for the treatment of leukemia.
Fludarabine has very potent anti lymphocyte activity, providing efficacy in eradicating lymphocytic leukemia cells, but also results in significant lymphopeniaand immune suppression. Fludarabine has been adopted for use in HSC transplant preconditioning regimens for its immune suppressive activity, and is often combined with busulfan, which is myeloablative, but not particularly immune suppressive, to allow engraftment of allogeneic HSC.22 Because of the absence of published data on the pharmacokinetics of fludarabine in infant rhesus monkeys, animals were treated in three successive cohorts where the fludarabine dosages were successively increased. The findings provide new information on the immunological responses to the GFP transgene product and may provide a platform for additional studies of immune responses and tolerance to novel transgene products in the context of gene therapy using HSC. Results Clinical observations Infant rhesus monkeys were transplanted in three series with escalating intensity of conditioning.