690. Gene Delivery Targeting CCR3 Using SV40-Derived Vectors: A Novel Therapeutic Approach for Allergic Diseases
Authors: Lokesh Agrawal, David S. Strayer
Journal: Molecular Therapy (2006)

CCR3 is constitutively expressed on eosinophils, basophils, mast cells and type 2 T lymphocytes. These cells collectively constitute the majority of the inflammatory infiltrates in many diseases caused by immunologic hypersensitivity. CCR3 is known to act as a receptor for several chemokines: eotaxin, RANTES, MIP1-α and MIP1-β. Eotaxin is produced by monocytes and T lymphocytes. It stimulates migration of eosinophils and is involved in maintaining allergic responses. Thus targeting CCR3 for downregulation may be an appealing therapeutic approach in asthma.The present study focuses on the role of CCR3 in asthmatic diseases in the lung and as a target for manipulation by gene transfer. Two strategies were applied to target CCR3. First we used the anti- CCR3 hybridoma clone 5E8-G9-B4 to develop a single chain antibody (SFv) construct containing variable heavy (VH) and variable light (VL) chain, separated by a flexible hinge region. The SFv was made using overlap PCR and cloned into a mammalian expression vector. Second, we tested a panel of small interfering siRNA's targeting a sequence shared by murine and human CCR3 mRNAs. For initial studies U87-CD4-CCR3 cell line was utilized.Initial studies with the siRNA were performed by adding the soluble RNAs to cultured cells. In parallel, the effectiveness of the SFv was studied in transfection experiments. Analysis of CCR3 cell membrane expression was done by cytofluorimetry (FACS) and immunostaining. We identified one anti-CCR3 siRNA, R3-526, that decreased cell membrane CCR3 60–80% when added to U87- CD4-CCR3 cultures. CCR3 expression was most inhibited at siRNA concentrations of 1nM to 10nM. Transfected SFv also reduced CCR3 to a similar extent in the same cells. Comparable results were obtained using monocyte-derived macrophages. Inhibition of CCR3- signaled eotaxin activity was tested by measuring cellular calcium release in U87-CD4-CCR3 cells in response to eotaxin. Both the SFv and R3-526 siRNA largely blocked eotaxin signaling. As well, the SFv and R3-526 siRNA inhibited eotaxin-induced chemotaxis in these cells. Current studies are underway to examine both the effectiveness of expressing these moieties using SV40-derived gene transfer vectors. SV40-derived vectors were used because they provide permanent transgene expression in airway lining cells as well as the bone marrow-derived cells resident in the lungs. Thus, SV(5E8) and SV(siRNA-CCR3) anti-CCR3 vectors were also made to study their effectiveness in CCR3 downregulation. The effectiveness of inhibiting eosinophil and mast cell CCR3 is under study.Thus, modulating CCR3 has potential as a means to control in immune hypersensitivity responses, e.g., in allergic asthma and other diseases. CCR3 is constitutively expressed on eosinophils, basophils, mast cells and type 2 T lymphocytes. These cells collectively constitute the majority of the inflammatory infiltrates in many diseases caused by immunologic hypersensitivity. CCR3 is known to act as a receptor for several chemokines: eotaxin, RANTES, MIP1-α and MIP1-β. Eotaxin is produced by monocytes and T lymphocytes. It stimulates migration of eosinophils and is involved in maintaining allergic responses. Thus targeting CCR3 for downregulation may be an appealing therapeutic approach in asthma. The present study focuses on the role of CCR3 in asthmatic diseases in the lung and as a target for manipulation by gene transfer. Two strategies were applied to target CCR3. First we used the anti- CCR3 hybridoma clone 5E8-G9-B4 to develop a single chain antibody (SFv) construct containing variable heavy (VH) and variable light (VL) chain, separated by a flexible hinge region. The SFv was made using overlap PCR and cloned into a mammalian expression vector. Second, we tested a panel of small interfering siRNA's targeting a sequence shared by murine and human CCR3 mRNAs. For initial studies U87-CD4-CCR3 cell line was utilized. Initial studies with the siRNA were performed by adding the soluble RNAs to cultured cells. In parallel, the effectiveness of the SFv was studied in transfection experiments. Analysis of CCR3 cell membrane expression was done by cytofluorimetry (FACS) and immunostaining. We identified one anti-CCR3 siRNA, R3-526, that decreased cell membrane CCR3 60–80% when added to U87- CD4-CCR3 cultures. CCR3 expression was most inhibited at siRNA concentrations of 1nM to 10nM. Transfected SFv also reduced CCR3 to a similar extent in the same cells. Comparable results were obtained using monocyte-derived macrophages. Inhibition of CCR3- signaled eotaxin activity was tested by measuring cellular calcium release in U87-CD4-CCR3 cells in response to eotaxin. Both the SFv and R3-526 siRNA largely blocked eotaxin signaling. As well, the SFv and R3-526 siRNA inhibited eotaxin-induced chemotaxis in these cells. Current studies are underway to examine both the effectiveness of expressing these moieties using SV40-derived gene transfer vectors. SV40-derived vectors were used because they provide permanent transgene expression in airway lining cells as well as the bone marrow-derived cells resident in the lungs. Thus, SV(5E8) and SV(siRNA-CCR3) anti-CCR3 vectors were also made to study their effectiveness in CCR3 downregulation. The effectiveness of inhibiting eosinophil and mast cell CCR3 is under study. Thus, modulating CCR3 has potential as a means to control in immune hypersensitivity responses, e.g., in allergic asthma and other diseases.