981. Prevention of Transgene Expression in Antigen Presenting Cells Correlate with Modulated Immune Response after In Vivo Gene Transfer
Authors: Andrea Annoni, Brian Brown, Manuela Battaglia, Angelo Lombardo, Luigi Naldini, Maria Grazia Roncarolo
Journal: Molecular Therapy (2006)

Immune responses against genetically modified cells represents a major obstacle to the success of gene therapy. Cellular and humoral immune responses to vector-associated and transgene-derived proteins lead to prompt elimination of transgene expressing cells abrogating the benefits of gene transfer. Several strategies have been used to limit immune response following gene transfer, including immunosuppression and the use of different routes of administration. As an alternative approach, we and others have shown that restricting transgene expression to hepatocytes, through the use of hepatocyte-specific promoters, can reduce the incidence of anti- transgene immune responses. This phenomenon is believed to occur because of reduced expression of the transgene in the antigen presenting cells (APCs), which results in reduced T-cell priming. However, alternative pathways can not be ruled out. Here we characterized the immunological mechanisms responsible for circumventing the immune response after liver-directed gene therapy. Immunocompetent C57BL/6 mice were systemically injected with lentiviral vectors (LV) encoding GFP under the control of the liver-specific albumin promoter (ALB) or the ubiquitously expressed CMV promoter, as control. To study the anti-transgene cellular response we quantified CD8 T-cells infiltrating the liver and GFP-specific IFN-g producing CD8 T cells at various time points after LV administration. When mice were treated with LV.CMV.GFP, significant lymphocyte infiltration was observed within days after vector administration. Peak infiltration was detected at day 14, and correlated with a reduction in the frequency of GFP positive cells. In mice treated with LV.ALB.GFP, both the CD8 T cells liver infiltration and the frequency of GFP- specific CD8 T cells in the spleen were significantly lower. This led to stable GFP expression in the liver for at least 12 weeks. As expected, mice treated with LV.CMV.GFP had high titers of GFP-specific antibodies, which appeared at day 14 post-treatment, and slowly declined thereafter. Interestingly, a humoral response was also detected in LV.ALB.GFP-treated mice with GFP-specific antibodies detected by day 14 and peaking at day 28. These results indicate that restriction of GFP expression to the liver attenuates the transgene-specific cellular response, but may not prevent the induction of humoral immunity to the intracellular antigen. These findings suggest that CD4 T-cell help is present in LV.ALB.GFP mice, and that restricted transgene expression to hepatocytes may not ensure immunological ignorance. Studies are ongoing to characterize the immune response following gene transfer by our recently described microRNA-regulated LVs (Brown et al. Nature Medicine. in press). These vectors, which selectively de-target expression from hematopoietic cells, may provide an improved means for achieving transgene-specific tolerance. Overall these studies will help us to understand the role of vector design and antigen presentation in the prevention of immune response to the transgene.