610. Evaluation of Three Reticuloendothelial System Blocking Modalities on the Outcome of Adenoviral Mediated Gene Therapy in Hemophilic Mice
Authors: Maha Othman, Andrea Labelle, Christine Hough, Alan H. Lazarus, David Lillicrap
Journal: Molecular Therapy (2005)

Gene therapy is an attractive potential treatment for hemophilia A and replication deficient adenovirus has been a commonly used vector for hepatic transgene delivery. However, a major obstacle has been the activation the innate and adaptive immunity when applied in vivo. The virus localizes in the liver within one hour after IV administration and is taken up by liver macrophages. This induces inflammatory cytokines that limit the efficiency of transduction and causes substantial loss of the vector genome. We aimed to evaluate the influence of three modalities of blocking the reticuloendothelial system on the outcome of Ad mediated gene delivery in FVIII KO mi Transfusion of chilled platelets (1 |[times]| 108 platelets) either 1h before (group I) or simultaneously with (group II) Ad. Immunoglobulin preparation (IVIG-50 mg) and the macrophage depleting drug Clodronate (200 uL clodronate liposome). In all mice, we used an E1/E3 deleted with canine FVIII. HNF (1 |[times]| 1011 viral particles). Assessment of the outcome of gene therapy was done by FVIII assay, liver transaminase (ALT) levels, TNF alpha, FVIII inhibitors and platelet count. Our study showed that FVIII expression levels that peaked at 1 week post Ad injection was significantly higher with the clodronate group (n=5, p=0.005) and significantly lower in mice receiving IVIG (n=5, p=0.0001) when compared to the group that received Ad only. In both groups, the FVIII antibodies were significantly lower when compared to the group that received Ad alone. The two chilled platelet groups did not show significant effects on FVIII levels or FVIII inhibitor incidence. The ALT significantly elevated at day 8 in the two groups receiving chilled platelets (group I; n= 5 p=0.01) (group II; n= 3, p=0.002).There was a significant reduction of ALT in the IVIG group (n=5, p= 0.002) when compared to the Ad only group. TNF alpha showed a significant elevation 3 hours post injection in the group receiving IVIG (n=5, p=0.04) and a significant reduction in the Chilled/Ad simultaneously group (n=3, p=0.01). At 1W, a significant reduction was obtained in the clodronate group only (n=5, p= 0.02) and no effect among the other groups. Thrombocytopenia was least evident with the clodronate and IVIG groups and most evident with the two chilled platelet groups (group I: n=5, p= 0.00001)(group II: n=3, 0.002) with longer duration of thrombocytopenia in group II. These data show that there is a potential for using the clodronate in improving the outcome of gene therapy since it not only enhances the expression of FVIII but also significantly reduces the adaptive immune response to FVIII. The IVIG preparation may have a positive influence in modulating the immune response to Ad, however a major obstacle is the limitation of the gene expression presumably due to neutralizing anti- adenovirus antibodies. Although the chilled platelets may play a minor role in blocking liver macrophages, this strategy does not improve the FVIII expression levels and has shown adverse effects in terms of liver injury as well as thrombocytopenia.