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781. Successful Non-Ablative Allogeneic Transplantation after Hematopoietic Stem Cell Transduction with MGMTP140K Lentivirus and In Vivo Chemoselection
TISSUE ENGINEERING & OTHER CELL THERAPIES variety of other tissue types. We have recently shown that expression of OPG by the MSC and conditioned media from MSC subcultures could potentially inhibit osteoclastogenesis in vitro. The present study examined the potential of unmodified MSC in a preclinical mouse model of osteolytic bone metastasis. Six-week-old male SCID mice were injected intra-tibially with the osteolytic prostate cancer cell line, PC3, expressing firefly luciferase, followed by injection of 5x105 unmodified adult mouse bone marrow-derived MSC. Mice were evaluated for tumor growth 4 weeks after the administration of MSC by bioluminescence imaging. Three dimensional µCT and histomorphometry were performed to determine restoration of bone loss by MSC treatment. Results indicated a 90% inhibition of tumor growth and complete protection of tibial bone in the treated mice. To elucidate the mechanism of inhibition of tumor growth, SCID mice were implanted with PC3 cells followed by treatment with MSC, derived from GFP transgenic mouse. Mice were sacrificed seven days after MSC treatment, tibiae harvested and analyzed by histomorphometry. Results indicated formation of woven bone around the cancer cells. Immunohistochemistry with GFP antibody revealed that the new bone was derived mainly from the input MSC. No increase in bone growth was observed in control mice that received only MSC intra-tibially but not cancer cells. Overall, the data signify that relatively abundant amounts of MSC in the tumor microenvironment can provide therapy effects by producing OPG and/or other factor(s) upon interaction with cancer cells. Since the amount of MSC in the bone microenvironment is extremely low, strategies to endogenously mobilize MSC upon bone metastasis of osteolytic cancers may provide significant therapy and reduce morbidity and mortality.
781. Successful Non-Ablative Allogeneic Transplantation after Hematopoietic Stem Cell Transduction with MGMTP140K Lentivirus and In Vivo Chemoselection
Rustom Falahati,1 Linda Flebbe-Rehwaldt,1 Jianqing Zhang,1 Yimin Shi,1 Karin M. L. Gaensler.1 1 Medicine, University of California, San Francisco, CA.
Current allogeneic pre-transplant preparative regimens and posttransplantation immunosuppressive procedures are associated with considerable morbidity. Genetic modification of the stem cells to enable in vivo amplification with successive cycles of low-dose chemotherapy after engraftment, may reduce the intensity of preparative regimens and immunosuppression required, and thereby reduce the risks of allogeneic transplantation. We have developed a murine model in which host vs. graft and graft vs. host immune responses may be separately addressed. To reduce the impact of hostmediated immune responses and graft rejection, we are 1) combining allogeneic transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor hematopoietic stem cells (HSC) and 2) transplanting parental HSC into F1 hybrid recipients. This model system enables the studies on tolerance induction mechanisms to neo-antigens, and allogeneic stem cell engraftment during immune ontogeny. Bone marrow-derived HSC were transduced ex vivo by lentivirus-mediated gene transfer of P140KO6-methylguanine- methyltransferase (MGMTP140K) and GFP (MAG vector). MGMTP140K confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU and enables donor stem cell enrichment using successive cycles of in vivo chemoselection. Our hypothesis is that in vivo administration of BG/BCNU may partially deplete allo-reactive cells of donor and host origin, reducing the need for toxic ablative or immunosuppressive treatment. The feasibility of this model was first tested using 5 x 105 syngeneic whole bone marrow cells (WBM) transduced with MAG and transplanted in neonates. Five weeks after transplantation, 0.5% Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
of cells expressed GFP in peripheral blood prior to chemoselection. After two cycles of BG/BCNU, 39.5% of peripheral blood cells were GFP positive. To evaluate in vivo selection in an allogeneic setting, 2 x 106 MAG-transduced WBM from BALB/c (H-2d) adult mice were transplanted into C57BL/6 x BALB/c F1 (H-2b/d) neonatal mice. Despite having a graft vs. host MHC disparity, we observed an increase from 4.7% (5 weeks after transplant) to 37.4% GFP expression following two cycles of in vivo chemoselection without evidence of GVHD. We have thus demonstrated successful engraftment and enrichment of both syngenic and allogenic HSC expressing MGMTP140K following neonatal transplantation and in vivo selection. Stable engraftment was achieved without myeloablation or post-transplant immunosuppression. These results hold promise for the development of non-myeloablative allogenic transplant approaches using this in vivo selection strategy.
782. Lentivirally Transduced Dendritic Cells Secreting VIP Differentiate into TolerogenicLike DC and Suppress Both Innate and Adaptive Immune Responses
Miguel G. Toscano,1 Francisco Martin,2 Mario Delgado,2 Doina Ganea.1 1 Microbiology and Immunology, Temple University, Philadelphia, PA; 2Immunology and Cell Biology Department, Institute of Parasitology and Biomedicine López Neyra, CSIC, Granada, Spain. It is of increased interest to generate DC with efficient and stable genetic modifications, i.e. expressing a protein with therapeutic effects, to be used as therapeutic tools. Previous results from our laboratory indicated that the neuropeptide vasoactive intestinal peptide (VIP) has both preventive and therapeutic effects in models of autoimmune diseases such as collagen-induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE). We had previously shown that the therapeutic effects of VIP are mediated largely through effects on DC, specifically on the generation of tolerogenic DC. However, direct treatment of human subjects with VIP could lead to significant side effects due to its action on both the cardiovascular system and gastrointestinal tract. Therefore, we show here the use of lentivirally transduced-DC to deliver VIP in order to obtain a local rather than a systemic effect. Mouse bone marrow DC were efficiently transduced by lentiviral vectors carrying eGFP (up to 65% of cells with an MOI of 10). Use of the same vectors carrying human-preproVIP led to a DC population expressing close to 450 pg/ml of VIP. The VIP expressed by transduced DC acted in an autocrine manner; leading to the differentiation of tolerogenic-like DC, characterized by a reduction in the costimulatory molecules CD40 and CD86 following LPS stimulation. In addition, we observed a downregulation in the production in the pro-inflammatory cytokines IL-6 and TNF-α and an increase in IL-10 (anti-inflammatory cytokine). Finally a single injection of 3x106 DC secreting VIP was able to ameliorate significantly clinical EAE and reduce mortality in the ceccal ligation and puncture (CLP) septic peritonitis model.
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781. Successful Non-Ablative Allogeneic Transplantation after Hematopoietic Stem Cell Transduction with MGMTP140K Lentivirus and In Vivo Chemoselection
Rustom Falahati,1 Linda Flebbe-Rehwaldt,1 Jianqing Zhang,1 Yimin Shi,1 Karin M. L. Gaensler.1 1 Medicine, University of California, San Francisco, CA.
Current allogeneic pre-transplant preparative regimens and posttransplantation immunosuppressive procedures are associated with considerable morbidity. Genetic modification of the stem cells to enable in vivo amplification with successive cycles of low-dose chemotherapy after engraftment, may reduce the intensity of preparative regimens and immunosuppression required, and thereby reduce the risks of allogeneic transplantation. We have developed a murine model in which host vs. graft and graft vs. host immune responses may be separately addressed. To reduce the impact of hostmediated immune responses and graft rejection, we are 1) combining allogeneic transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor hematopoietic stem cells (HSC) and 2) transplanting parental HSC into F1 hybrid recipients. This model system enables the studies on tolerance induction mechanisms to neo-antigens, and allogeneic stem cell engraftment during immune ontogeny. Bone marrow-derived HSC were transduced ex vivo by lentivirus-mediated gene transfer of P140KO6-methylguanine- methyltransferase (MGMTP140K) and GFP (MAG vector). MGMTP140K confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU and enables donor stem cell enrichment using successive cycles of in vivo chemoselection. Our hypothesis is that in vivo administration of BG/BCNU may partially deplete allo-reactive cells of donor and host origin, reducing the need for toxic ablative or immunosuppressive treatment. The feasibility of this model was first tested using 5 x 105 syngeneic whole bone marrow cells (WBM) transduced with MAG and transplanted in neonates. Five weeks after transplantation, 0.5% Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
of cells expressed GFP in peripheral blood prior to chemoselection. After two cycles of BG/BCNU, 39.5% of peripheral blood cells were GFP positive. To evaluate in vivo selection in an allogeneic setting, 2 x 106 MAG-transduced WBM from BALB/c (H-2d) adult mice were transplanted into C57BL/6 x BALB/c F1 (H-2b/d) neonatal mice. Despite having a graft vs. host MHC disparity, we observed an increase from 4.7% (5 weeks after transplant) to 37.4% GFP expression following two cycles of in vivo chemoselection without evidence of GVHD. We have thus demonstrated successful engraftment and enrichment of both syngenic and allogenic HSC expressing MGMTP140K following neonatal transplantation and in vivo selection. Stable engraftment was achieved without myeloablation or post-transplant immunosuppression. These results hold promise for the development of non-myeloablative allogenic transplant approaches using this in vivo selection strategy.
782. Lentivirally Transduced Dendritic Cells Secreting VIP Differentiate into TolerogenicLike DC and Suppress Both Innate and Adaptive Immune Responses
Miguel G. Toscano,1 Francisco Martin,2 Mario Delgado,2 Doina Ganea.1 1 Microbiology and Immunology, Temple University, Philadelphia, PA; 2Immunology and Cell Biology Department, Institute of Parasitology and Biomedicine López Neyra, CSIC, Granada, Spain. It is of increased interest to generate DC with efficient and stable genetic modifications, i.e. expressing a protein with therapeutic effects, to be used as therapeutic tools. Previous results from our laboratory indicated that the neuropeptide vasoactive intestinal peptide (VIP) has both preventive and therapeutic effects in models of autoimmune diseases such as collagen-induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE). We had previously shown that the therapeutic effects of VIP are mediated largely through effects on DC, specifically on the generation of tolerogenic DC. However, direct treatment of human subjects with VIP could lead to significant side effects due to its action on both the cardiovascular system and gastrointestinal tract. Therefore, we show here the use of lentivirally transduced-DC to deliver VIP in order to obtain a local rather than a systemic effect. Mouse bone marrow DC were efficiently transduced by lentiviral vectors carrying eGFP (up to 65% of cells with an MOI of 10). Use of the same vectors carrying human-preproVIP led to a DC population expressing close to 450 pg/ml of VIP. The VIP expressed by transduced DC acted in an autocrine manner; leading to the differentiation of tolerogenic-like DC, characterized by a reduction in the costimulatory molecules CD40 and CD86 following LPS stimulation. In addition, we observed a downregulation in the production in the pro-inflammatory cytokines IL-6 and TNF-α and an increase in IL-10 (anti-inflammatory cytokine). Finally a single injection of 3x106 DC secreting VIP was able to ameliorate significantly clinical EAE and reduce mortality in the ceccal ligation and puncture (CLP) septic peritonitis model.
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