750. Expression Levels of the Amphotropic Receptor May Explain Gestational Age-Dependence of Tissue Transduction in Fetal Sheep

Abstract

We have recently reported that the gestational age of the recipient determines the pattern and level of transgene expression following in utero retroviral gene transfer by direct injection into pre-immune fetal sheep (Molecular Therapy 11(2):284-293, 2005). These studies suggest that the developmental stage of each organ at the time of injection may determine its susceptibility to in utero gene transfer, and that each organ possesses its own unique temporal window during which optimal transduction can be achieved. In the present studies, we sought to determine the mechanism by which the recipient's age was affecting transduction efficiency and subsequent transgene expression. One can envision two mechanisms whereby the age of the tissue could affect its susceptibility to transduction with murine retroviral vectors. The first of these is the proliferative state of the tissue, since these vectors require cell division and nuclear membrane breakdown for genomic integration. To investigate this possibility, we have begun performing analysis of the fetal liver and lung at various gestational ages using BrdU labeling and staining of tissue sections with antibodies to Ki-67 and PH3, both markers of mitosis. Thus far, our results provide evidence that the proliferative state of the various tissues is not the primary mechanism by which transduction efficiency is being limited, since no significant difference was observed in the percentage of cells labeled in each tissue at the various ages studied. The other primary mechanism whereby the age of the tissue could alter its susceptibility to transduction is through age-dependent alterations in the levels of the receptor used for viral attachment and entry, namely, the amphotropic receptor, PiT2. To test this hypothesis, we custom synthesized an antibody to the ovine PiT2 receptor and developed an ELISA to evaluate the various fetal tissues at different gestational ages to determine whether the age of each tissue impacts upon its levels of PiT2 expression. Thus far, our results suggest that peaks in the levels of PiT2 expression occur at different stages of development in different tissues, thereby providing a possible explanation for the observed alterations in transduction efficiency. We are currently conducting real-time RT-PCR assays to confirm these alterations in the levels of PiT2 expression as a function of gestational age. It is hoped that these ongoing studies will enable us to determine the optimal stages for gene delivery to each of the major organs of the developing fetus and delineate the mechanisms that govern the levels of gene transfer that can be obtained.