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687. “Same Day” Ex Vivo Regional Gene Therapy: A Novel Approach To Enhance Bone Repair
RNA VIRUS VECTORS II 686. In Vivo Immunization with Integration Decient Lentiviral Vectors
Biliang Hu,1 Pin Wang.1 1 Chemical Engineering, University of Southern California, Los Angeles, CA. Despite the high efficiency of in vivo immunization with lentiviral vectors as the delivery carrier, safety concern on insertional mutagenesis remains the major obstacle in translating lentiviral vectors to clinical applications. Therefore, it is important to investigate whether an integration decient lentiviral vectors (IDLV) that is defective in vector integration but is competent to mediate transgene expression in certain target cells can be adapted for vaccine applications. Towards this goal, we generated the vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped and HIV-1-based self-inactivating IDLV harboring a decient integrase with a D64V mutation. The resulting IDLV efciently mediated the transient GFP expression in dividing HEK293T cells and maintained the persistent GFP expression in nondividing bone marrow derived dendritic cells (BMDCs). In addition, GFP+CD11c+ dendrtic cells (DCs) were detected in the inguinal lymph node close to the injection site, suggesting that the injected IDLV indeed transduced DCs in vivo and the modied DCs could migrate to the draining lymph node. We then demonstrated that the IDLV was highly potent in delivering the ovalbumin (OVA) antigen to BMDCs to stimulate the OVA-specic OT1 transgenic T cells in vitro. Upon the direct in vivo immunization by the IDLV encoding OVA, we observed a substantial frequency of OVA-specic CD8 T cells (up to 27%) and strong antibody response, which were comparable to that elicited by the integrating lentiviral vectors. Study on the kinetics of the frequency of OVAspecic CD8 T cells indicated that IDLV was as efcacious as its integrating counterpart in the induction of durable antigen-specic T cell response. Moreover, a single immunization with the IDLV could provide protection against the growth of tumor cells bearing the OVA antigen. This work evidently shows that immunization by IDLV can be a promising and safe approach for the development of cancer vaccines.
687. “Same Day” Ex Vivo Regional Gene Therapy: A Novel Approach To Enhance Bone Repair
Mandeep S. Virk, O. Sugiyama, Sang H. Park, Jay R. Lieberman. Orthopaedic Surgery, Univ of Connecticut Health Center, Farmington, CT; Orthopaedic Surgery, JV Luck Research Ctr., Los Angeles, CA. Introduction Ex vivo regional gene therapy using culture expanded bone marrow stromal cells has the potential to be developed as part of a comprehensive tissue engineering strategy to manage large bone defects. However, the traditional 2 step approach of ex vivo regional gene therapy requires harvest and culture expansion of bone marrow cells before they are genetically manipulated and implanted in the donor. The purpose of this study was to assess the efcacy of a “Same Day” strategy for ex vivo gene therapy wherein the harvest and viral transduction of the bone marrow cells was followed by implantation into the target anatomic site without cell expansion. Materials and Methods BM was harvested from the femurs and tibias of male Lewis rats and processed according to a previously published protocol [1]. The buffy coat cells (SD-RBMCs) at the interface were collected and the SD-RBMCs were plated into a 6well plate with 5×106 cells and viral transductions were carried out for 1 hour at MOI of 25 with a modied lentiviral vector (LV) using a two step transcriptional amplication (TSTA) system (TSTALV-BMP-2) [1]. The critical sized femoral defects (8mm) were surgically created in 14-week old male Lewis S268
rats as previously described [2]. The transduced SD-RBMCs were delivered to the femoral defect on a compression resistant matrix. Study Groups Eighteen male Lewis rats underwent femoral defect surgeries and were randomized to two groups: Group I (n=13) animals received 15 x 106 SD-RBMCs + LVTSTA-BMP-2 Group II (control animals; n=5) received 15 x 106 non transduced SD-RBMCs New bone formed in the defect was assessed by plain xrays, histology and histomorphometry. Results The efcacy of the LV-TSTA-BMP-2 vector was tested in the RBMCs. The BMP-2 production in the LV-TSTA-BMP-2 transduced RBMCs was more than 10X higher compared to the LV-RhMLVBMP-2 transduced cells at an MOI of 25. All the defects in Group I animals (SD-RBMCs + TSTA-LV-BMP-2; 13/13) demonstrated complete radiographic healing at 8 weeks. None of the 5 defects in Group II (SD-RBMCs) demonstrated osseous continuity on plain xray. Histological analysis of the defects in Group I demonstrated a continuous cortex with new trabecular bone extending the length of the defect. In contrast, the defects in Group II demonstrated sparse endosteal new bone formation. The bone area/tissue area (BA/ TA) in Group I (0.14±0.02) was signicantly higher than Group II (0.04±0.02). Conclusion A limitation of the adoption of ex vivo gene therapy for clinical use is the cost and inconvenience of a two step cell harvest and expansion approach. The results of this study suggest that a “Same Day” strategy gene therapy is feasible with mononuclear cell fraction of RBMCs transduced with a LV vector that has strong transgene expression. Furthermore, genetically manipulated BM cells using the “Same Day” strategy can effectively heal a critical sized rat femoral defect model. “Same Day” gene therapy has the potential to be a cost effective strategy to treat difcult bone repair problems. References [1] Iyer et al. Proc Natl Acad Sci 2001,98(25)L14595-600 [2] Lieberman et al. J Bone Joint Surg 1999;81:905-17
688. The R Peptide Sequence of XMRV Envelope Glycoprotein Determined the Pseudotyping Specicity with HIV-1 and MLV Vectors
Toshie Sakuma,1 Suk See De Ravin,2 Harry L. Malech,2 Yasuhiro Ikeda.1 1 Mayo Clinic, Rochester, MN; 2National Institute of Health, Bethesda, MD. Retroviral and lentiviral vectors are effective gene delivery vehicles which are currently evaluated in clinical trials. Variations in the viral envelope (Env) glycoproteins, which are used to pseudotype retroviral or lentiviral vectors, can alter the vector performance, including the stability, titers, host range and tissue tropism. Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a novel human retrovirus recently identied in patients with prostate cancer. XMRV targets XPR1 cell surface receptor, which is expressed in a broad range of human tissues including hematopoietic stem cells. Pseudotyping with XMRV Env would allow targeting of XPR1-expressing tissues. Here, we characterized the XMRV Env-pseudotyped retro- and lentiviral vectors. Although the wildtype XMRV Env poorly pseudotyped HIV-1-based lentiviral vectors and Moloney MLV-based retroviral vectors, replacement of the C-terminal 11 amino acid residues of XMRV Env with the corresponding 6 amino acid residues of amphotropic MLV Env signicantly increased the XMRV Envpseudotyped HIV and MLV vector titers. The increased MLV titer with the modied XMRV Env (XMRV/Rampho) correlated with improved Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
Biliang Hu,1 Pin Wang.1 1 Chemical Engineering, University of Southern California, Los Angeles, CA. Despite the high efficiency of in vivo immunization with lentiviral vectors as the delivery carrier, safety concern on insertional mutagenesis remains the major obstacle in translating lentiviral vectors to clinical applications. Therefore, it is important to investigate whether an integration decient lentiviral vectors (IDLV) that is defective in vector integration but is competent to mediate transgene expression in certain target cells can be adapted for vaccine applications. Towards this goal, we generated the vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped and HIV-1-based self-inactivating IDLV harboring a decient integrase with a D64V mutation. The resulting IDLV efciently mediated the transient GFP expression in dividing HEK293T cells and maintained the persistent GFP expression in nondividing bone marrow derived dendritic cells (BMDCs). In addition, GFP+CD11c+ dendrtic cells (DCs) were detected in the inguinal lymph node close to the injection site, suggesting that the injected IDLV indeed transduced DCs in vivo and the modied DCs could migrate to the draining lymph node. We then demonstrated that the IDLV was highly potent in delivering the ovalbumin (OVA) antigen to BMDCs to stimulate the OVA-specic OT1 transgenic T cells in vitro. Upon the direct in vivo immunization by the IDLV encoding OVA, we observed a substantial frequency of OVA-specic CD8 T cells (up to 27%) and strong antibody response, which were comparable to that elicited by the integrating lentiviral vectors. Study on the kinetics of the frequency of OVAspecic CD8 T cells indicated that IDLV was as efcacious as its integrating counterpart in the induction of durable antigen-specic T cell response. Moreover, a single immunization with the IDLV could provide protection against the growth of tumor cells bearing the OVA antigen. This work evidently shows that immunization by IDLV can be a promising and safe approach for the development of cancer vaccines.
687. “Same Day” Ex Vivo Regional Gene Therapy: A Novel Approach To Enhance Bone Repair
Mandeep S. Virk, O. Sugiyama, Sang H. Park, Jay R. Lieberman. Orthopaedic Surgery, Univ of Connecticut Health Center, Farmington, CT; Orthopaedic Surgery, JV Luck Research Ctr., Los Angeles, CA. Introduction Ex vivo regional gene therapy using culture expanded bone marrow stromal cells has the potential to be developed as part of a comprehensive tissue engineering strategy to manage large bone defects. However, the traditional 2 step approach of ex vivo regional gene therapy requires harvest and culture expansion of bone marrow cells before they are genetically manipulated and implanted in the donor. The purpose of this study was to assess the efcacy of a “Same Day” strategy for ex vivo gene therapy wherein the harvest and viral transduction of the bone marrow cells was followed by implantation into the target anatomic site without cell expansion. Materials and Methods BM was harvested from the femurs and tibias of male Lewis rats and processed according to a previously published protocol [1]. The buffy coat cells (SD-RBMCs) at the interface were collected and the SD-RBMCs were plated into a 6well plate with 5×106 cells and viral transductions were carried out for 1 hour at MOI of 25 with a modied lentiviral vector (LV) using a two step transcriptional amplication (TSTA) system (TSTALV-BMP-2) [1]. The critical sized femoral defects (8mm) were surgically created in 14-week old male Lewis S268
rats as previously described [2]. The transduced SD-RBMCs were delivered to the femoral defect on a compression resistant matrix. Study Groups Eighteen male Lewis rats underwent femoral defect surgeries and were randomized to two groups: Group I (n=13) animals received 15 x 106 SD-RBMCs + LVTSTA-BMP-2 Group II (control animals; n=5) received 15 x 106 non transduced SD-RBMCs New bone formed in the defect was assessed by plain xrays, histology and histomorphometry. Results The efcacy of the LV-TSTA-BMP-2 vector was tested in the RBMCs. The BMP-2 production in the LV-TSTA-BMP-2 transduced RBMCs was more than 10X higher compared to the LV-RhMLVBMP-2 transduced cells at an MOI of 25. All the defects in Group I animals (SD-RBMCs + TSTA-LV-BMP-2; 13/13) demonstrated complete radiographic healing at 8 weeks. None of the 5 defects in Group II (SD-RBMCs) demonstrated osseous continuity on plain xray. Histological analysis of the defects in Group I demonstrated a continuous cortex with new trabecular bone extending the length of the defect. In contrast, the defects in Group II demonstrated sparse endosteal new bone formation. The bone area/tissue area (BA/ TA) in Group I (0.14±0.02) was signicantly higher than Group II (0.04±0.02). Conclusion A limitation of the adoption of ex vivo gene therapy for clinical use is the cost and inconvenience of a two step cell harvest and expansion approach. The results of this study suggest that a “Same Day” strategy gene therapy is feasible with mononuclear cell fraction of RBMCs transduced with a LV vector that has strong transgene expression. Furthermore, genetically manipulated BM cells using the “Same Day” strategy can effectively heal a critical sized rat femoral defect model. “Same Day” gene therapy has the potential to be a cost effective strategy to treat difcult bone repair problems. References [1] Iyer et al. Proc Natl Acad Sci 2001,98(25)L14595-600 [2] Lieberman et al. J Bone Joint Surg 1999;81:905-17
688. The R Peptide Sequence of XMRV Envelope Glycoprotein Determined the Pseudotyping Specicity with HIV-1 and MLV Vectors
Toshie Sakuma,1 Suk See De Ravin,2 Harry L. Malech,2 Yasuhiro Ikeda.1 1 Mayo Clinic, Rochester, MN; 2National Institute of Health, Bethesda, MD. Retroviral and lentiviral vectors are effective gene delivery vehicles which are currently evaluated in clinical trials. Variations in the viral envelope (Env) glycoproteins, which are used to pseudotype retroviral or lentiviral vectors, can alter the vector performance, including the stability, titers, host range and tissue tropism. Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a novel human retrovirus recently identied in patients with prostate cancer. XMRV targets XPR1 cell surface receptor, which is expressed in a broad range of human tissues including hematopoietic stem cells. Pseudotyping with XMRV Env would allow targeting of XPR1-expressing tissues. Here, we characterized the XMRV Env-pseudotyped retro- and lentiviral vectors. Although the wildtype XMRV Env poorly pseudotyped HIV-1-based lentiviral vectors and Moloney MLV-based retroviral vectors, replacement of the C-terminal 11 amino acid residues of XMRV Env with the corresponding 6 amino acid residues of amphotropic MLV Env signicantly increased the XMRV Envpseudotyped HIV and MLV vector titers. The increased MLV titer with the modied XMRV Env (XMRV/Rampho) correlated with improved Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy