- Email: [email protected]
780. Therapeutic Potential of Adult Mesenchymal Stem Cells in Osteolytic Bone Metastasis
TISSUE ENGINEERING & OTHER CELL THERAPIES 778. Development of Lentiviral Therapy for Netherton Syndrome
Waseem Qasim,1 Gill Talbot,2 Fernando Larcher,3 John Harper,2 Adrian Thrasher,1 Wei Li DI.1 1 Molecular Immunology Unit, UCL Institute of Child Health, London, United Kingdom; 2Immunobiology Unit, Institute of Child Health, London, United Kingdom; 3Regenerative Medicine Unit, Centro de Investigaciones Energéticas, Madrid, Spain. Mutations in the SPINK5 gene, which encodes the Lymphoepithelial Kazal-type-related inhibitor (LEKTI), give rise to a Netherton syndrome (NS). The disease is characterised by ichthyosiform erythroderma, atopic diathesis and a defective skin barrier. Infants can develop severe dehydration soon after birth and severe forms of NS are associated with 10% mortality in the first year of life. Ex-vivo transduction of keratinocyte stem cells is now feasible, and the corrected epidermal sheets generated could provide valuable source of skin grafts. This approach has been successful for other skin diseases such as the blistering disorder, epidermolysis bullosa, where murine retroviral vectors were used to correct keratinocyte stem cells during ex-vivo culture, and the corrected skin sheets then used as grafts in affected patients. In NS, gene modified autologous skin sheets could provide an important protective barrier against fluid loss as well as serve as a source of systemic LEKTI protein delivery. We generated self-inactivating HIV-1 based lentiviral vectors to encode codon optimised SPINK5, linked to the enhanced green fluorescent reporter protein (eGFP). The vectors encode the Spleen Focus Forming Virus (SFFV) LTR and were pseudotyped with the vesicular stomatitis virus (VSV) envelope. We demonstrate high levels of transduction in both keratinocyte cell lines, and in primary keratinocyte stem cells. The vectors were used to generate gene modified epidermal sheets from healthy donor and Netherton patient cells. Western blot analysis confirmed the reconstitution of LEKTI expression and the presence of correctly processed LEKTI species in the NS cells. Culture on a dermal support matrix was used to generate 3-dimensional organotypic skin cultures, and immunohistochemical staining was used to demonstrate that epidermal eGFP expression co-localised with LEKTI expression. In subsequent long term culture experiments we have detected reductions in gene expression over time for SPINK5/eGFP vectors (but not for eGFP control vectors), and have investigated the underlying mechanism of this phenomenon. Increased methylation of CpG sites was detected within the SFFV promoter of vectors encoding SPINK5/eGFP compared to eGFP alone, suggesting that SPINK5 encoding vectors may be more prone to silencing over time. Culture of cells in the methylation inhibitor Azacytidine resulted in partial reversal of silencing. The issue is being addressed through modification of promoter/enhancer regions and the longer term durability of gene correction of primary cells is being studied in human/murine chimeric skin graft models.
779. Long-Term Safety of Controlled VEGF Expression by Rapid FACS-Purification of Transduced Myoblasts in Chronic Ischemia
Thomas Wolff, Heidi Misteli, Edin Mujagic, Philipp Fueglistaler, Roberto Gianni-Barrera, Uta Helmrich, Lorenz Gurke, Michael Heberer, Andrea Banfi. Departments of Biomedicine and of Surgery, Basel University Hospital, Basel, Switzerland. This study aims to: 1) develop a clinically-applicable method to achieve controlled angiogenesis in vivo by cell-based gene delivery; and 2) test its safety in chronic ischemia. The long-term objective is to develop a safe and efficient therapeutic angiogenesis strategy for the treatment of ischemia. Vascular Endothelial Growth Factor (VEGF) gene delivery is a powerful strategy to revascularize ischemic tissue. However, excessive VEGF induces deleterious effects, such S298
as angioma growth, and we have previously shown that the induction of normal or aberrant angiogenesis by VEGF depends exclusively on the amount secreted in the microenvironment, and not on its total dose, as it remains localized in the matrix around each producing cell (JCI 2004; FASEB J 2006). To achieve clinically applicable, controlled expression in vivo, we developed a FACS-based method to rapidly purify genetically-engineered progenitors producing desired levels of VEGF. Primary mouse myoblasts were transduced with a retroviral construct in which the VEGF164 gene was linked through an Internal Ribosomal Entry Site to a truncated version of CD8a, acting as a reporter gene. Differences in VEGF expression were reflected by the cell-surface amount of CD8a, detected and quantified on live cells by FACS. FACS-purified populations were implanted in nonischemic (mouse) and chronically ischemic (rat) skeletal muscles. The expression of VEGF was found to be linearly correlated (R2=0.897) with that of CD8 at the single cell level analyzing individual clones isolated across the whole range of expression in the heterogeneous primary population (range from 2 to 142 ng/106 cells/day). A set of clones was implanted in the ear muscle of SCID mice to correlate VEGF expression with the 3-dimensional morphology of induced vessels after 4 weeks in whole-mount preparations. The clone expressing the highest VEGF level (34.0±1.7 ng/106 cells/day) which induced normal angiogenesis was selected as a reference. Cells expressing similar CD8a levels (and therefore of VEGF) were rapidly purified from the primary population. VEGF and CD8a expression of the sorted cells was stable during in vitro expansion over 23 doublings. In non-ischemic muscle tissue, the heterogeneous population always caused angioma growth. However, purified cells caused robust normal angiogenesis, which was stable after 4 months, and completely prevented angioma formation. Also after implantation in chronically ischemic tissue, the FACS-purified population induced only normal angiogenesis around the fibers where myoblasts had engrafted, leading to a doubling of vessel length density compared to control cells, and completely avoided aberrant vascular structures, which were caused instead by the unsorted population. Long-term safety and stability of this angiogenic response was verified up to 3 months after implantation. In conclusion, rapid FACS-purification of VEGF-expressing myoblasts induced safe angiogenesis in vivo, both in normal and chronically ischemic muscle. This technology is a promising novel approach to achieve therapeutic angiogenesis.
780. Therapeutic Potential of Adult Mesenchymal Stem Cells in Osteolytic Bone Metastasis
Diptiman Chanda,1 Tatyana Isayeva,1 Sanjay Kumar,1 Jonathan A. Hensel,1 Gene P. Siegal,1 Selvarangan Ponnazhagan.1 1 Pathology, University of Alabama at Birmingham, Birmingham, AL. Bone metastasis is characteristic to many cancers including carcinomas of the breast, prostate, lung, thyroid and kidney, which leads to severe bone pain, susceptibility to fracture, spinal cord compression. A majority of bone metastatic cancers produce osteolytic lesions whereas prostate cancer generates osteoblastic phenotype. However, osteoblastic metastasis in prostate cancer is preceded by osteolytic event and prevention of which has been shown to inhibit prostate cancer progression in the bone. The primary mechanism responsible for bone destruction is receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated osteoclastic bone resorption. Increased expression of RANKL has been observed in osteolytic malignancies. Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL and prevents binding of RANKL to RANK on pre-osteoclasts and subsequent inactivation of the osteoclastic activity. Homing of adult bone marrow-derived mesenchymal stem cells (MSC) to the sites of tumorigenesis is well known besides their ability to self-renew and differentiate into bone, cartilage, fat and a Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
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.
S299
Waseem Qasim,1 Gill Talbot,2 Fernando Larcher,3 John Harper,2 Adrian Thrasher,1 Wei Li DI.1 1 Molecular Immunology Unit, UCL Institute of Child Health, London, United Kingdom; 2Immunobiology Unit, Institute of Child Health, London, United Kingdom; 3Regenerative Medicine Unit, Centro de Investigaciones Energéticas, Madrid, Spain. Mutations in the SPINK5 gene, which encodes the Lymphoepithelial Kazal-type-related inhibitor (LEKTI), give rise to a Netherton syndrome (NS). The disease is characterised by ichthyosiform erythroderma, atopic diathesis and a defective skin barrier. Infants can develop severe dehydration soon after birth and severe forms of NS are associated with 10% mortality in the first year of life. Ex-vivo transduction of keratinocyte stem cells is now feasible, and the corrected epidermal sheets generated could provide valuable source of skin grafts. This approach has been successful for other skin diseases such as the blistering disorder, epidermolysis bullosa, where murine retroviral vectors were used to correct keratinocyte stem cells during ex-vivo culture, and the corrected skin sheets then used as grafts in affected patients. In NS, gene modified autologous skin sheets could provide an important protective barrier against fluid loss as well as serve as a source of systemic LEKTI protein delivery. We generated self-inactivating HIV-1 based lentiviral vectors to encode codon optimised SPINK5, linked to the enhanced green fluorescent reporter protein (eGFP). The vectors encode the Spleen Focus Forming Virus (SFFV) LTR and were pseudotyped with the vesicular stomatitis virus (VSV) envelope. We demonstrate high levels of transduction in both keratinocyte cell lines, and in primary keratinocyte stem cells. The vectors were used to generate gene modified epidermal sheets from healthy donor and Netherton patient cells. Western blot analysis confirmed the reconstitution of LEKTI expression and the presence of correctly processed LEKTI species in the NS cells. Culture on a dermal support matrix was used to generate 3-dimensional organotypic skin cultures, and immunohistochemical staining was used to demonstrate that epidermal eGFP expression co-localised with LEKTI expression. In subsequent long term culture experiments we have detected reductions in gene expression over time for SPINK5/eGFP vectors (but not for eGFP control vectors), and have investigated the underlying mechanism of this phenomenon. Increased methylation of CpG sites was detected within the SFFV promoter of vectors encoding SPINK5/eGFP compared to eGFP alone, suggesting that SPINK5 encoding vectors may be more prone to silencing over time. Culture of cells in the methylation inhibitor Azacytidine resulted in partial reversal of silencing. The issue is being addressed through modification of promoter/enhancer regions and the longer term durability of gene correction of primary cells is being studied in human/murine chimeric skin graft models.
779. Long-Term Safety of Controlled VEGF Expression by Rapid FACS-Purification of Transduced Myoblasts in Chronic Ischemia
Thomas Wolff, Heidi Misteli, Edin Mujagic, Philipp Fueglistaler, Roberto Gianni-Barrera, Uta Helmrich, Lorenz Gurke, Michael Heberer, Andrea Banfi. Departments of Biomedicine and of Surgery, Basel University Hospital, Basel, Switzerland. This study aims to: 1) develop a clinically-applicable method to achieve controlled angiogenesis in vivo by cell-based gene delivery; and 2) test its safety in chronic ischemia. The long-term objective is to develop a safe and efficient therapeutic angiogenesis strategy for the treatment of ischemia. Vascular Endothelial Growth Factor (VEGF) gene delivery is a powerful strategy to revascularize ischemic tissue. However, excessive VEGF induces deleterious effects, such S298
as angioma growth, and we have previously shown that the induction of normal or aberrant angiogenesis by VEGF depends exclusively on the amount secreted in the microenvironment, and not on its total dose, as it remains localized in the matrix around each producing cell (JCI 2004; FASEB J 2006). To achieve clinically applicable, controlled expression in vivo, we developed a FACS-based method to rapidly purify genetically-engineered progenitors producing desired levels of VEGF. Primary mouse myoblasts were transduced with a retroviral construct in which the VEGF164 gene was linked through an Internal Ribosomal Entry Site to a truncated version of CD8a, acting as a reporter gene. Differences in VEGF expression were reflected by the cell-surface amount of CD8a, detected and quantified on live cells by FACS. FACS-purified populations were implanted in nonischemic (mouse) and chronically ischemic (rat) skeletal muscles. The expression of VEGF was found to be linearly correlated (R2=0.897) with that of CD8 at the single cell level analyzing individual clones isolated across the whole range of expression in the heterogeneous primary population (range from 2 to 142 ng/106 cells/day). A set of clones was implanted in the ear muscle of SCID mice to correlate VEGF expression with the 3-dimensional morphology of induced vessels after 4 weeks in whole-mount preparations. The clone expressing the highest VEGF level (34.0±1.7 ng/106 cells/day) which induced normal angiogenesis was selected as a reference. Cells expressing similar CD8a levels (and therefore of VEGF) were rapidly purified from the primary population. VEGF and CD8a expression of the sorted cells was stable during in vitro expansion over 23 doublings. In non-ischemic muscle tissue, the heterogeneous population always caused angioma growth. However, purified cells caused robust normal angiogenesis, which was stable after 4 months, and completely prevented angioma formation. Also after implantation in chronically ischemic tissue, the FACS-purified population induced only normal angiogenesis around the fibers where myoblasts had engrafted, leading to a doubling of vessel length density compared to control cells, and completely avoided aberrant vascular structures, which were caused instead by the unsorted population. Long-term safety and stability of this angiogenic response was verified up to 3 months after implantation. In conclusion, rapid FACS-purification of VEGF-expressing myoblasts induced safe angiogenesis in vivo, both in normal and chronically ischemic muscle. This technology is a promising novel approach to achieve therapeutic angiogenesis.
780. Therapeutic Potential of Adult Mesenchymal Stem Cells in Osteolytic Bone Metastasis
Diptiman Chanda,1 Tatyana Isayeva,1 Sanjay Kumar,1 Jonathan A. Hensel,1 Gene P. Siegal,1 Selvarangan Ponnazhagan.1 1 Pathology, University of Alabama at Birmingham, Birmingham, AL. Bone metastasis is characteristic to many cancers including carcinomas of the breast, prostate, lung, thyroid and kidney, which leads to severe bone pain, susceptibility to fracture, spinal cord compression. A majority of bone metastatic cancers produce osteolytic lesions whereas prostate cancer generates osteoblastic phenotype. However, osteoblastic metastasis in prostate cancer is preceded by osteolytic event and prevention of which has been shown to inhibit prostate cancer progression in the bone. The primary mechanism responsible for bone destruction is receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated osteoclastic bone resorption. Increased expression of RANKL has been observed in osteolytic malignancies. Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL and prevents binding of RANKL to RANK on pre-osteoclasts and subsequent inactivation of the osteoclastic activity. Homing of adult bone marrow-derived mesenchymal stem cells (MSC) to the sites of tumorigenesis is well known besides their ability to self-renew and differentiate into bone, cartilage, fat and a Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
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.
S299