457. The Benefits of CH-296 during Ex Vivo Gene Transfer Go beyond the Ability to Co-Localize the Retroviral Particle and Target Cell

Abstract

Successful gene therapy depends on at least two parameters: introduction of the corrective gene into primitive hematopoietic progenitor cells (HPC) and maintenance of the long-term engraftment potential of these ex vivo manipulated progenitors post-transplantation. The use of the recombinant fibronectin fragment, CH-296, increases retroviral gene transfer by co-localizing the retroviral particle and the target cell which expresses VLA-4 and VLA-5 integrins. In a xenotransplantation model, our lab previously demonstrated the maintenance of long-term reconstituting hematopoietic progenitors during ex vivo gene transfer either on a pre-established irradiated bone marrow stromal monolayer or CH-296-coated plates. In contrast, HPC cultured on BSA-coated plates in the presence of IL-3, IL-6, and SCF maintained short- but not long-term engraftment potential. Here, we compared the molecular changes that occur in bone marrow and cord blood HPC cultured on CH-296 vs. BSA-coated plates in the presence of cytokines. Bone marrow HPC were cultured in the presence of IL-3, IL-6, and SCF for 48 hours. Subcellular fractionation followed by immunoblotting showed an induction of p21Cip1 protein in the cytoplasmic compartment of HPC cultured on BSA-coated plates. Since p21Cip1 is upregulated during monocytic differentiation, we hypothesized that lack of integrin engagement in the presence of cytokines might induce the loss of HPC primitive characteristics. Confirming our hypothesis, we observed a loss of c-myb, CD34, and GATA-2 expression in HPC maintained in suspension. In addition, we observed changes in MAPK phosphorylation and localization in HPC cultured on fibronectin, as compared to BSA-coated plates. Extrapolating the same hypothesis to cordblood derived HPCs, we observed consistently higher levels of reactive oxygen species (ROS) in cordblood HPC on BSA-coated plates, as compared to cells on CH-296. Aside from regulating cell differentiation, high levels of ROS have been shown to induce apoptosis. Consistent with this, we documented lower levels of phosphorylated cytoplasmic AKT in HPC on BSA-coated plates, as compared to cells on CH-296 plates. Based on these preliminary observations, we hypothesize that the suppression of differentiation markers such as p21Cip1 and apoptotic inducers such as intracellular reactive oxygen species, the maintenance of transcription factors such as GATA-2 and c-Myb, and the enhanced phosphorylation of the pro-survival regulator AKT in HPCs during ex vivo culture on CH-296 may all contribute to the ability of these cells to achieve long term engraftment following transplantation into sublethally irradiated immunodeficient mice. We therefore propose that inclusion of CH-296 during ex vivo culture of HPC may be important even during non-retroviral mediated gene transfer in order to maintain HPC engraftment potential.