- Email: [email protected]
428. Evaluation of Engraftment and Tolerance Induction after Reduced Intensity Conditioning for Hematopoietic Stem Cell Transplantation in the Rhesus Gene Therapy Model
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I quantitative and qualitative superiority over single-agent-mobilized cells. These results are of obvious relevance to all other genetic disorders in which autotransplantation of genetically manipulated HSCs is attempted.
428. Evaluation of Engraftment and Tolerance Induction after Reduced Intensity Conditioning for Hematopoietic Stem Cell Transplantation in the Rhesus Gene Therapy Model
Naoya Uchida,1 Molly Evans,1 Richard Weitzel,1 Aylin Bonifacino,2 Sandra Price,2 Allen Krouse,2 Mark Metzger,2 Matthew Hsieh,1 Robert Donahue,2 John Tisdale.1 1 NHLBI/NIDDK,NIH, Bethesda, MD; 2Hematology Branch,NHLBI,NIH, Rockville, MD.
Reduced intensity conditioning (RIC) regimens are desirable for hematopoietic stem cell (HSC) transplantation after genetic modification; however, may be insufficient for opening niches for efficient engraftment and inducing immunological tolerance to transgenes. Therefore, we sought to evaluate both engraftment and tolerance for genetically modified cells using our rhesus gene therapy model following RIC. We previously established long-term gene marking in our rhesus model in which transduced CD34+ cells were transplanted following 10Gy total body irradiation (TBI)(J Virol. 2009). In this study, a RIC regimen of 4Gy TBI was utilized for two animals who received CD34+ cells split for transduction with a GFP-expressing chimeric HIV1 vector or a mock vector not expressing a transgene. In vitro GFP+ rates (%GFP) evaluated by flow cytometry were 36-86%, and vector copy number per cell (VCN) evaluated by real time PCR was GFP: 7.5-9.0, mock: 3.54.5. At 2 weeks post-transplant, both animals demonstrated 1.6-2.1 %GFP in red blood cells (RBCs), but undetectable in granulocytes, lymphocytes, or platelets. %GFP in RBCs gradually decreased and was undetectable by 3 months. Interestingly, granulocytes showed 97-99% GFP positivity starting at 1-2 months post-transplant, and declined to undetectable levels by 3-4 months. Similar gene marking levels were observed between GFP and mock vectors until 6 months. These data suggest that 4Gy TBI is insufficient for efficient engraftment of genetically modified hematopoietic repopulating cells. To evaluate whether introduction of transduced HSCs after RIC could establish immunological tolerance for GFP transgene, we performed autologous lymphocyte infusion with GFP marking in these animals. As a control animal for GFP tolerance, we utilized a rhesus who had 5-10 %GFP in peripheral blood cells for 2 years post HSC transplant. Leukapheresis cells were transduced with a WPRE-containing GFP-expressing vector, and the cells were infused into the animals, which mainly contained lymphocytes with efficient GFP/WPRE marking (VCN 0.39-0.61). In the control animal, lymphocyte GFP/ WPRE marking levels rose for 12 weeks post-infusion compared to granulocytes (p<0.01), while no difference in GFP/WPRE signals between lymphocytes and granulocytes was observed in the two RIC transplanted animals. Additionally, we performed mixed lymphocyte culture, in which the two RIC transplanted animals showed in vitro proliferation of T cells after stimulation with GFP+ autologous lymphocytes (p<0.01), while no significant proliferation was observed in the control animal. These data suggest that 4Gy TBI alone is insufficient for inducing tolerance for transgenes and the two RIC transplanted animals have established immunological memory for GFP. In summary, 4Gy TBI was insufficient for efficient engraftment of genetically modified hematopoietic repopulating cells and inducing immunological tolerance. Our findings should be helpful for the design of conditioning regimens in gene therapy trials.
429. Myeloprotective Properties of Inducible (shRNA-Mediated) Knock-Down of DeoxycytidineKinase (dCK) in Human Hematopoietic In Vitro Models
Nico Lachmann,1 Kevin Czarnecki,1 Miriam Hetzel,1 Sebastian Brennig,1 Niels Heinz,2 Bernd Schiedlmeier,2 Thomas Moritz.1 1 RG Reprogramming, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany; 2Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
Drug resistance (CTX-R) gene transfer has been advocated to protect the lymphohematopoietic system from the side effects of anti-cancer chemotherapy. In the context of leukemias cytidine deaminase (CDD), multi-drug-resistance protein 1 (MDR1), and dihydrofolate reductase (DHFR) -mutants protecting from cytidine analogs, anthracyclines and antifolates, respectively, appear of interest. However, resistance genes for other clinically relevant nucleotide analogs with anti-leukemic properties such as fludarabine, clofarabine or cladribine have been missing, so far. As in clinical studies reduced expression of deoxycytidine kinase (dCK) has been linked to nucleotide analog resistance, we here introduce a novel Doxregulated lentiviral vector harbouring a minimal pTET-T11 Pol(II) promoter and an exchangeable shRNA (embedded in a miRNA-30 backbone) to mediate specific knock-down of dCK and nucleotide analog resistance in hematopoietic cells. Robust and Dox-dependent knock-down of 80% of dCK expression analysis within 2-3 days of Dox administration was demonstrated in human promyelocytic HL60 cells by western blot and qRT-PCR analysis. In addition, dCK knock-down in these cells conferred more than 10-fold increased Ara-C resistance in comparison to control cells with LD50 values of 700nM versus 60nM, respectively. Similar protection capabilities were observed also in primary CD34+ hematopoietic stem/progenitor cells. Dox-titration assays revealed Dox concentrations > 0,2μg/ml as sufficient for this effect. Furthermore, dCK knock-down allowed for highly efficient selection of transduced cells by 200nM Ara-C (from 10 to > 95% within 7 days). Most importantly, dCK knock-down also protected HL-60 cells effectively from clofarabine and fludarabine as evident by 6-10-fold increased LD50 concentrations as well as cladribine (5-fold increase). Thus, we here describe a novel Doxinducible, lentiviral “knock-down” vector expressing a dCK-specific shRNA in a miRNA-30 backbone from a minimal pTET-T11 Pol(II) promoter. This construct not only induces resistance to Ara-C in hematopoietic cell lines and efficient in vitro selection of transduced cells, but also provides profound resistance to clofarabine, fludarabine, and cladribine, thus introducing a novel strategy to CTX-R-mediated myeloprotection and calling for further in vivo evaluation.
430. Comparision of In-Utero Gene Therapy and In-Utero Hemopoietic Stem Cell Therapy Approaches in Severe Models of Murine beta Thalassemia
Niraja Dighe,1 Citra Mattar,1 Lay Geok Tan,1 Daren Sandikin,1 Simon Waddington,4 Michael Antoniou,5 Mahesh Choolani,1 Jerry Chan.1,2,3 1 Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, National University of Singapore, Singapore, Singapore; 2Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore, Singapore; 3Cancer and Stem Cell Program,, Duke-NUS Graduate Medical School, Singapore, Singapore; 4Institute for Women’s Health, Gene Transfer Technology Group, University College London, London, United Kingdom; 5Department of Medical and Molecular Genetics, King’s College London School of Medicine, London., United Kingdom. Aim Ex vivo gene therapy using lentiviral vectors has shown phenotypic correction in adult murine models of thalassaemia major.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
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GENE AND CELL THERAPY FOR PULMONARY DISEASES Intra-uterine transfer has potential of averting irreversible organ damage leading to severe disability or premature death. Rescue can be achieved by intrauterine delivery of lentiviral vectors expressing the human beta globin transgene or alternatively with haemopoietic stem cell transplantation. We investigated the dual approaches of intrauterine gene therapy (IUGT) and haemopoietic stem cell therapy (IUHSCT) to correct a murine model of beta thalassaemia major. Methods Hbb/Th3+/- males were backcrossed with C57BL/6 females. In the IUGT arm F1 fetuses were given 5x106 TU/fetus of LV-LCR--globin intravenously. In the IUHSCT arm fetuses were given 2x105-1x106 cells/fetus of either murine liver-derived CD45.1+ mononuclear cells, or human fetal liver-derived haemopoietic stem cells (hflHSC) intraperitoneally at E15-18. Pups were screened by genotype. Vector Copy Number (VCN) and chimerism was analysed at monthly intervals in peripheral blood. Animals were harvested to determine chimerism in the liver, spleen and bone marrow by quantitative PCR and FACS up to 6 months. Phenotype was monitored with haematological indices and blood smears.Uninjected F1 progeny from the same breeding system served as controls. Results Four fetuses received 5x106 TU of LV-LCR--globin. By 20 postnatal weeks hematological indices in treated heterozygote pups showed marked improvement compared with uninjected controls, particularly RBC counts (average 8.25 vs 5.1 E+106) and Hb levels (average 12 vs 9.6g/dL) . Quantitative PCR showed a median VCN of 2.4 copies per cell (range 0.23-4.64) in peripheral blood. However peripheral blood films showed persistent anisocytosis and poikilocytosis. Analysis of CD45.1/GFP donor cell transfer demonstrated peripheral blood chimerism at a median engraftment level of 0.32% (range 0.1-0.6%). Analysis of human cells demonstrated peripheral blood chimerism at a median engraftment level of 0.33% (range 0.13.4%). The median xenogeneic chimerism in the bone marrow, liver and spleen was 0.82% (range 0.05-3.01%) in mice sacrificed at 6 months. DNA chimerism was confirmed by qPCR. There were no differences in haematological parameters between untreated and injected heterozygotes. Conclusion Data from IUGT suggests use of lentivirus LCR--globin can lead to hematological improvement in the thalassemic mouse. Various strategies for IUHSCT including the utility of higher cell doses, manipulation of donor cells by CD26 inhibition and intravenous delivery to improve engraftment are underway.
431. Regulatory T Cells Suitable for Clinical Use Can Be Propagated on Artificial Antigen Presenting Cells
Pappanaicken Kumaresan,1 Chetan Dhamne,1 Sourindra Maiti,1 Janani Krishnamoorthy,1 Radhika Thokala,1 Helen Huls,1 Champlin Richard,1 Dean Lee,1 Dat Tran,2 Laurence Cooper.1 1 Division of Pediatrics, Unit 907, MD Anderson Medical Center, Houston, TX; 2Division of Pediatric Research Center, University of Texas-Houston Medical School, Houston, TX. Regulatory T cells (Treg) are an attractive approach to prevent and/ or control graft-versus-host-disease (GvHD) in patients undergoing allogeneic hematopoietic stem-cell transplantation (HSCT). Since Treg constitute only a small portion of the donor T-cell population, strategies are needed to propagate clinically-sufficient numbers of Treg cells to impact the incidence and severity of GvHD. We have developed a protocol to numerically expand Treg cells by recursive stimulation with --irradiated K562-derived artificial antigen presenting cells (aAPC) in the presence of rapamycin and interleukin (IL)-2. All three of these components to the manufacturing process are available as clinical-grade reagents suitable for use in compliance with current good manufacturing practice. The aAPC were genetically modified to co-express CD64, CD86, CD137L and a membrane-bound variant of IL-15. Our data demonstrates that Treg can sustain proliferation upon cross-linking CD3 by OKT3-loaded S166
aAPC resulting in 500 to 1,000-fold expansion (n=6) after 28 days of culture. The propagated Treg were highly enriched for expression of FoxP3+ (88%) and as anticipated produced minimal amounts of IL2, IFN-, and IL-17 in response to activation. These Treg, suppressed (60-80%) proliferation of allogeneic conventional T cells at a ratio of 1:2 (Treg:Tcon). Multiplexed gene expression analysis was used to quantify mRNA molecules of interest based on the direct digital detection of color-coded target-specific probes. These data revealed that of aAPC-propagated Treg up-regulated expression of FoxP3, CD25, and TGFR1 (2, 5 and 7-fold respectively), while expression of CCR7, CTLA-4, and IL-10 were decreased (2, 6, and 8 fold) as compared to non-expanded freshly-isolated Treg cells. Expression levels for TGF1, IRF4, GATA-3 were unaltered. In summary, our approach to manufacturing supports the outgrowth of Treg cells with desired phenotype and genotype in numbers suitable for their human application in patients at risk for GvHD after allogeneic HSCT.
Gene and Cell Therapy for Pulmonary Diseases 432. A Novel Cancer Cell Vaccine Using Induced Pluripotent Stem Cells Genetically Engineered To Produce GM-CSF Elicits Substantial Antitumor Immunity in a Syngeneic Mouse Model
Hiroyuki Inoue,1,2 Ayumi Wanatabe,1 Chika Sakamoto,1 Megumi Narusawa,1 Shohei Miyamoto,1 Makoto Inoue,3 Koichi Takayama,2 Mamoru Hasegawa,3 Yoichi Nakanishi,2 Kenzaburo Tani.1 1 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 3DNAVEC Corporation, Tsukuba, Jamaica.
Development of a novel therapeutic modality targeting cancer stem cells (CSCs) holds great promise for the eventual eradication of cancer. It was demonstrated that CSCs shared antigenic similarities with embryonic stem cells (ESCs) and the vaccination using ESCs could generate antitumor immunity. However, the use of ESCs raises potential immunological and ethical problematic issues. Recently, by the forced ectopic expression of defined transcription factors, autologous somatic cells were successfully reprogrammed into induced pluripotent stem cells (iPSCs) that closely resemble ESCs. We hypothesized that novel cell vaccines using mouse iPSCs genetically engineered to express the immunostimulatory cytokine of GM-CSF would cross-react CSC cells to induce antitumor immunity against poorly immunogenic syngeneic LLC mouse lung cancer cells which would resolve such problematic issues . Our results of in vitro assays demonstrated that non-transmissible recombinant Sendai virus-mediated mouse GM-CSF gene transfer to iPSCs (iPS/ GM) was effective to produce abundant GM-CSF in vitro and iPS/ GM cells maintained their stemness in terms of morphology and antigenicity as evidenced by the expression of SSEA-1,Oct3/4 and alkaline phosphatase compared with unmodified iPS cells. In vivo antitumor effects of iPS/GM cells were observed in immunocompetent mice subcutaneously treated with irradiated iPS/GM cells in the left flank followed by subsequent challenge with parental LLC cells in the right flank. Mice treated with irradiated iPS/GM cells significantly suppressed the tumor growth of subcutaneous LLC tumors compared with control mice. Of note, no serious adverse events were observed with lack of liver and kidney dysfunctions as evidenced by biochemical analysis. In conclusion, to our knowledge, this is the first report that demonstrated a therapeutic antitumor efficacy of iPSCs vaccine, suggesting that iPSCs-based vaccine strategy could induce CSCs associated antigens-specific antitumor immunity and be a promising modality for cancer immunotherapy. Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
428. Evaluation of Engraftment and Tolerance Induction after Reduced Intensity Conditioning for Hematopoietic Stem Cell Transplantation in the Rhesus Gene Therapy Model
Naoya Uchida,1 Molly Evans,1 Richard Weitzel,1 Aylin Bonifacino,2 Sandra Price,2 Allen Krouse,2 Mark Metzger,2 Matthew Hsieh,1 Robert Donahue,2 John Tisdale.1 1 NHLBI/NIDDK,NIH, Bethesda, MD; 2Hematology Branch,NHLBI,NIH, Rockville, MD.
Reduced intensity conditioning (RIC) regimens are desirable for hematopoietic stem cell (HSC) transplantation after genetic modification; however, may be insufficient for opening niches for efficient engraftment and inducing immunological tolerance to transgenes. Therefore, we sought to evaluate both engraftment and tolerance for genetically modified cells using our rhesus gene therapy model following RIC. We previously established long-term gene marking in our rhesus model in which transduced CD34+ cells were transplanted following 10Gy total body irradiation (TBI)(J Virol. 2009). In this study, a RIC regimen of 4Gy TBI was utilized for two animals who received CD34+ cells split for transduction with a GFP-expressing chimeric HIV1 vector or a mock vector not expressing a transgene. In vitro GFP+ rates (%GFP) evaluated by flow cytometry were 36-86%, and vector copy number per cell (VCN) evaluated by real time PCR was GFP: 7.5-9.0, mock: 3.54.5. At 2 weeks post-transplant, both animals demonstrated 1.6-2.1 %GFP in red blood cells (RBCs), but undetectable in granulocytes, lymphocytes, or platelets. %GFP in RBCs gradually decreased and was undetectable by 3 months. Interestingly, granulocytes showed 97-99% GFP positivity starting at 1-2 months post-transplant, and declined to undetectable levels by 3-4 months. Similar gene marking levels were observed between GFP and mock vectors until 6 months. These data suggest that 4Gy TBI is insufficient for efficient engraftment of genetically modified hematopoietic repopulating cells. To evaluate whether introduction of transduced HSCs after RIC could establish immunological tolerance for GFP transgene, we performed autologous lymphocyte infusion with GFP marking in these animals. As a control animal for GFP tolerance, we utilized a rhesus who had 5-10 %GFP in peripheral blood cells for 2 years post HSC transplant. Leukapheresis cells were transduced with a WPRE-containing GFP-expressing vector, and the cells were infused into the animals, which mainly contained lymphocytes with efficient GFP/WPRE marking (VCN 0.39-0.61). In the control animal, lymphocyte GFP/ WPRE marking levels rose for 12 weeks post-infusion compared to granulocytes (p<0.01), while no difference in GFP/WPRE signals between lymphocytes and granulocytes was observed in the two RIC transplanted animals. Additionally, we performed mixed lymphocyte culture, in which the two RIC transplanted animals showed in vitro proliferation of T cells after stimulation with GFP+ autologous lymphocytes (p<0.01), while no significant proliferation was observed in the control animal. These data suggest that 4Gy TBI alone is insufficient for inducing tolerance for transgenes and the two RIC transplanted animals have established immunological memory for GFP. In summary, 4Gy TBI was insufficient for efficient engraftment of genetically modified hematopoietic repopulating cells and inducing immunological tolerance. Our findings should be helpful for the design of conditioning regimens in gene therapy trials.
429. Myeloprotective Properties of Inducible (shRNA-Mediated) Knock-Down of DeoxycytidineKinase (dCK) in Human Hematopoietic In Vitro Models
Nico Lachmann,1 Kevin Czarnecki,1 Miriam Hetzel,1 Sebastian Brennig,1 Niels Heinz,2 Bernd Schiedlmeier,2 Thomas Moritz.1 1 RG Reprogramming, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany; 2Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
Drug resistance (CTX-R) gene transfer has been advocated to protect the lymphohematopoietic system from the side effects of anti-cancer chemotherapy. In the context of leukemias cytidine deaminase (CDD), multi-drug-resistance protein 1 (MDR1), and dihydrofolate reductase (DHFR) -mutants protecting from cytidine analogs, anthracyclines and antifolates, respectively, appear of interest. However, resistance genes for other clinically relevant nucleotide analogs with anti-leukemic properties such as fludarabine, clofarabine or cladribine have been missing, so far. As in clinical studies reduced expression of deoxycytidine kinase (dCK) has been linked to nucleotide analog resistance, we here introduce a novel Doxregulated lentiviral vector harbouring a minimal pTET-T11 Pol(II) promoter and an exchangeable shRNA (embedded in a miRNA-30 backbone) to mediate specific knock-down of dCK and nucleotide analog resistance in hematopoietic cells. Robust and Dox-dependent knock-down of 80% of dCK expression analysis within 2-3 days of Dox administration was demonstrated in human promyelocytic HL60 cells by western blot and qRT-PCR analysis. In addition, dCK knock-down in these cells conferred more than 10-fold increased Ara-C resistance in comparison to control cells with LD50 values of 700nM versus 60nM, respectively. Similar protection capabilities were observed also in primary CD34+ hematopoietic stem/progenitor cells. Dox-titration assays revealed Dox concentrations > 0,2μg/ml as sufficient for this effect. Furthermore, dCK knock-down allowed for highly efficient selection of transduced cells by 200nM Ara-C (from 10 to > 95% within 7 days). Most importantly, dCK knock-down also protected HL-60 cells effectively from clofarabine and fludarabine as evident by 6-10-fold increased LD50 concentrations as well as cladribine (5-fold increase). Thus, we here describe a novel Doxinducible, lentiviral “knock-down” vector expressing a dCK-specific shRNA in a miRNA-30 backbone from a minimal pTET-T11 Pol(II) promoter. This construct not only induces resistance to Ara-C in hematopoietic cell lines and efficient in vitro selection of transduced cells, but also provides profound resistance to clofarabine, fludarabine, and cladribine, thus introducing a novel strategy to CTX-R-mediated myeloprotection and calling for further in vivo evaluation.
430. Comparision of In-Utero Gene Therapy and In-Utero Hemopoietic Stem Cell Therapy Approaches in Severe Models of Murine beta Thalassemia
Niraja Dighe,1 Citra Mattar,1 Lay Geok Tan,1 Daren Sandikin,1 Simon Waddington,4 Michael Antoniou,5 Mahesh Choolani,1 Jerry Chan.1,2,3 1 Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, National University of Singapore, Singapore, Singapore; 2Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore, Singapore; 3Cancer and Stem Cell Program,, Duke-NUS Graduate Medical School, Singapore, Singapore; 4Institute for Women’s Health, Gene Transfer Technology Group, University College London, London, United Kingdom; 5Department of Medical and Molecular Genetics, King’s College London School of Medicine, London., United Kingdom. Aim Ex vivo gene therapy using lentiviral vectors has shown phenotypic correction in adult murine models of thalassaemia major.
Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
S165
GENE AND CELL THERAPY FOR PULMONARY DISEASES Intra-uterine transfer has potential of averting irreversible organ damage leading to severe disability or premature death. Rescue can be achieved by intrauterine delivery of lentiviral vectors expressing the human beta globin transgene or alternatively with haemopoietic stem cell transplantation. We investigated the dual approaches of intrauterine gene therapy (IUGT) and haemopoietic stem cell therapy (IUHSCT) to correct a murine model of beta thalassaemia major. Methods Hbb/Th3+/- males were backcrossed with C57BL/6 females. In the IUGT arm F1 fetuses were given 5x106 TU/fetus of LV-LCR--globin intravenously. In the IUHSCT arm fetuses were given 2x105-1x106 cells/fetus of either murine liver-derived CD45.1+ mononuclear cells, or human fetal liver-derived haemopoietic stem cells (hflHSC) intraperitoneally at E15-18. Pups were screened by genotype. Vector Copy Number (VCN) and chimerism was analysed at monthly intervals in peripheral blood. Animals were harvested to determine chimerism in the liver, spleen and bone marrow by quantitative PCR and FACS up to 6 months. Phenotype was monitored with haematological indices and blood smears.Uninjected F1 progeny from the same breeding system served as controls. Results Four fetuses received 5x106 TU of LV-LCR--globin. By 20 postnatal weeks hematological indices in treated heterozygote pups showed marked improvement compared with uninjected controls, particularly RBC counts (average 8.25 vs 5.1 E+106) and Hb levels (average 12 vs 9.6g/dL) . Quantitative PCR showed a median VCN of 2.4 copies per cell (range 0.23-4.64) in peripheral blood. However peripheral blood films showed persistent anisocytosis and poikilocytosis. Analysis of CD45.1/GFP donor cell transfer demonstrated peripheral blood chimerism at a median engraftment level of 0.32% (range 0.1-0.6%). Analysis of human cells demonstrated peripheral blood chimerism at a median engraftment level of 0.33% (range 0.13.4%). The median xenogeneic chimerism in the bone marrow, liver and spleen was 0.82% (range 0.05-3.01%) in mice sacrificed at 6 months. DNA chimerism was confirmed by qPCR. There were no differences in haematological parameters between untreated and injected heterozygotes. Conclusion Data from IUGT suggests use of lentivirus LCR--globin can lead to hematological improvement in the thalassemic mouse. Various strategies for IUHSCT including the utility of higher cell doses, manipulation of donor cells by CD26 inhibition and intravenous delivery to improve engraftment are underway.
431. Regulatory T Cells Suitable for Clinical Use Can Be Propagated on Artificial Antigen Presenting Cells
Pappanaicken Kumaresan,1 Chetan Dhamne,1 Sourindra Maiti,1 Janani Krishnamoorthy,1 Radhika Thokala,1 Helen Huls,1 Champlin Richard,1 Dean Lee,1 Dat Tran,2 Laurence Cooper.1 1 Division of Pediatrics, Unit 907, MD Anderson Medical Center, Houston, TX; 2Division of Pediatric Research Center, University of Texas-Houston Medical School, Houston, TX. Regulatory T cells (Treg) are an attractive approach to prevent and/ or control graft-versus-host-disease (GvHD) in patients undergoing allogeneic hematopoietic stem-cell transplantation (HSCT). Since Treg constitute only a small portion of the donor T-cell population, strategies are needed to propagate clinically-sufficient numbers of Treg cells to impact the incidence and severity of GvHD. We have developed a protocol to numerically expand Treg cells by recursive stimulation with --irradiated K562-derived artificial antigen presenting cells (aAPC) in the presence of rapamycin and interleukin (IL)-2. All three of these components to the manufacturing process are available as clinical-grade reagents suitable for use in compliance with current good manufacturing practice. The aAPC were genetically modified to co-express CD64, CD86, CD137L and a membrane-bound variant of IL-15. Our data demonstrates that Treg can sustain proliferation upon cross-linking CD3 by OKT3-loaded S166
aAPC resulting in 500 to 1,000-fold expansion (n=6) after 28 days of culture. The propagated Treg were highly enriched for expression of FoxP3+ (88%) and as anticipated produced minimal amounts of IL2, IFN-, and IL-17 in response to activation. These Treg, suppressed (60-80%) proliferation of allogeneic conventional T cells at a ratio of 1:2 (Treg:Tcon). Multiplexed gene expression analysis was used to quantify mRNA molecules of interest based on the direct digital detection of color-coded target-specific probes. These data revealed that of aAPC-propagated Treg up-regulated expression of FoxP3, CD25, and TGFR1 (2, 5 and 7-fold respectively), while expression of CCR7, CTLA-4, and IL-10 were decreased (2, 6, and 8 fold) as compared to non-expanded freshly-isolated Treg cells. Expression levels for TGF1, IRF4, GATA-3 were unaltered. In summary, our approach to manufacturing supports the outgrowth of Treg cells with desired phenotype and genotype in numbers suitable for their human application in patients at risk for GvHD after allogeneic HSCT.
Gene and Cell Therapy for Pulmonary Diseases 432. A Novel Cancer Cell Vaccine Using Induced Pluripotent Stem Cells Genetically Engineered To Produce GM-CSF Elicits Substantial Antitumor Immunity in a Syngeneic Mouse Model
Hiroyuki Inoue,1,2 Ayumi Wanatabe,1 Chika Sakamoto,1 Megumi Narusawa,1 Shohei Miyamoto,1 Makoto Inoue,3 Koichi Takayama,2 Mamoru Hasegawa,3 Yoichi Nakanishi,2 Kenzaburo Tani.1 1 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 3DNAVEC Corporation, Tsukuba, Jamaica.
Development of a novel therapeutic modality targeting cancer stem cells (CSCs) holds great promise for the eventual eradication of cancer. It was demonstrated that CSCs shared antigenic similarities with embryonic stem cells (ESCs) and the vaccination using ESCs could generate antitumor immunity. However, the use of ESCs raises potential immunological and ethical problematic issues. Recently, by the forced ectopic expression of defined transcription factors, autologous somatic cells were successfully reprogrammed into induced pluripotent stem cells (iPSCs) that closely resemble ESCs. We hypothesized that novel cell vaccines using mouse iPSCs genetically engineered to express the immunostimulatory cytokine of GM-CSF would cross-react CSC cells to induce antitumor immunity against poorly immunogenic syngeneic LLC mouse lung cancer cells which would resolve such problematic issues . Our results of in vitro assays demonstrated that non-transmissible recombinant Sendai virus-mediated mouse GM-CSF gene transfer to iPSCs (iPS/ GM) was effective to produce abundant GM-CSF in vitro and iPS/ GM cells maintained their stemness in terms of morphology and antigenicity as evidenced by the expression of SSEA-1,Oct3/4 and alkaline phosphatase compared with unmodified iPS cells. In vivo antitumor effects of iPS/GM cells were observed in immunocompetent mice subcutaneously treated with irradiated iPS/GM cells in the left flank followed by subsequent challenge with parental LLC cells in the right flank. Mice treated with irradiated iPS/GM cells significantly suppressed the tumor growth of subcutaneous LLC tumors compared with control mice. Of note, no serious adverse events were observed with lack of liver and kidney dysfunctions as evidenced by biochemical analysis. In conclusion, to our knowledge, this is the first report that demonstrated a therapeutic antitumor efficacy of iPSCs vaccine, suggesting that iPSCs-based vaccine strategy could induce CSCs associated antigens-specific antitumor immunity and be a promising modality for cancer immunotherapy. Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy