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464. Modeling Potential HMGA2-Induced Clonal Expansion in a Non-Human Primate Stem Cell Transplant Model
HEMATOLOGIC & IMMUNOLOGIC DISEASES I 464. Modeling Potential HMGA2-Induced Clonal Expansion in a Non-Human Primate Stem Cell Transplant Model
Melissa A. Bonner,1 Sheng Zhou,1 Zhijun Ma,1 Brian P. Sorrentino.1 1 Hematology, St. Jude Children’s Research Hospital, Memphis, TN. A recent clinical trial for β-thalassemia utilizing a β-globin lentiviral vector resulted in a case of potential clonal expansion of cells containing a viral insertion in the HMGA2 gene, resulting in loss of Let-7 regulation and enhancer-driven overexpression of HMGA2. Now 6.5 years post-transplant, this case has not progressed to malignancy nor show signs of continuing clonal expansion. Overexpression of HMGA2 is known to cause HSC expansion in mice, leaving open the question as to whether HMGA2 de-regulation can lead to clonal expansion. Therefore, we have tested whether enforced expression of HMGA2 in autologous HSCs from Macaca nemestrina would result in clonal expansion or leukemia. The human HMGA2 cDNA was cloned from 293T cells, without the 3’ Let-7 sites, into a cl20c MSCV-IRES-GFP plasmid. Lentiviral HMGA2-GFP was produced with a typical unconcentrated titer between 5e7 and 1e8 tu/mL. HMGA2 expression was verified with flow cytometry and western blot analyses in primate CD34+ cells and multiple human cell lines, respectively. For transplant studies, GCSF-mobilized bone marrow was harvested from two macaques, A10W027 and A10W016. Bone marrow was enriched for CD34+ cells and stimulated overnight on RetroNectin-coated plates in XVivo10 media containing HSA, SCF, Flt3-L, and TPO. The CD34+ cells were then split in half and each received two vector exposures (HMGA2-GFP MOI=50, mCherry MOI=100) with a 12 hour washout between applications. After transduction, autologous cells were mixed together in a 50:50 ratio to form a split graft of HMGA2-GFP and mCherry cells and transfused into lethally irradiated recipients. A10W027 and A10W016 are 6 and 2.5 months post-transplant, respectively. Individual colonies from the grafts were analyzed by flow and revealed the transduction efficiencies of the HMGA2-GFP and mCherry vectors to be 6% and 8%, respectively, for A10W027 and 42% and 19%, respectively, for A10W016. The ratio of HMGA2GFP cells to mCherry cells has gone from 0.7 (Graft) to 2.4 in A10W027 with a steady increase in HMGA2-GFP cells starting around 2.5 months post-transplant from 0.8% to 4.0%. Multilineage reconstitution is seen in the HMGA2-GFP and mCherry compartments with CD3, CD14, CD16, and CD20 cells all comprised of a higher level of HMGA2-GFP than mCherry. A10W016 is still early posttransplant and exhibiting flux with transduced cell populations with no obvious early expansion. Monitoring of gene marking continues and deep sequencing analysis is underway to determine definitively if HMGA2 can lead to clonal expansion. Measurements over the next several months should conclusively address whether or not HMGA2 leads to clonal expansion or malignancies in transplanted NHPs.
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465. Gene Therapy for Hereditary Anemia: Preclinical Insertion Site Analysis of a New Lentiviral Vector
Annette Deichmann,1 Olivier Negre,2 Cynthia Bartholomae,1 Raffaele Fronza,1 Christof von Kalle,1 Mitch Finer,3 Gabor Veres,3 Emmanuel Payen,2 Manfred Schmidt.1 1 DKFZ/NCT, German Cancer Research Center/ National Center for Tumor Diseases, Heidelberg, Germany; 2CEA, Institut of Emerging Diseases and Innovative Therapies (iMETI), Fontenay aux Roses, France; 3bluebird bio, Cambridge, MA. Gene therapy using the lentiviral vector HPV569 that carries the ß-globin gene has been proven a valuable therapeutic option in a patient with ß-thalassemia. Recently a new lentiviral vector BB305 has been designed in order to increase the efficiency of gene transfer. A preclinical trial in mice has been set up to investigate the efficacy and safety using BB305. We present the results of the analysis of 10,835 unique integration sites (IS) as an important marker for the safety. 58 mice were transplanted with vector HPV569, BB305 or mock-transduced syngeneic ß-thalassemic bone marrow cells. With regard to the efficacy, complete correction of the phenotype and an oligo/polyclonal hematopoietic reconstitution was observed in all mice. 108 mice received a secondary transplant to evaluate long-term toxicity. Malignant T cell lymphomas were observed in transplanted animals with both vectors but as well in the control group. However, histology, immunohistochemistry and quantitative PCR revealed that the occurrence of these tumors did not derive from transduced donor cells. In pre-transplantation samples we detected 2605 IS in cells transduced with HPV569 and 1372 with BB305, in primary mice 1699 unique IS with vector HPV569 and 2143 IS with vector BB305 and in secondary mice 1353 with HPV569 and 1715 with BB305. Integration of both vectors occurred in genes with about 70% in primary animals and about 54% in secondary animals. The oncogenic potential of the affected genes was looked up in different databases. No significant difference could be detected between preand post-transplantation samples and no preferred integration in Mecom, Lmo2 or Hmga2. Approximately 30% of IS are clustered as common integration sites (CIS). Comparison with a random in silico dataset and another lentiviral preclinical dataset showed that CIS clustering occured in the same genomic areas. In secondary animals we found an oligo/polyclonal hematopoietic reconstitution, but the clonal diversity was reduced as expected and was equivalent for the two vectors. Long-term safety without any signs of clonal outgrowth or in vivo selection has been demonstrated. This complete preclinical insertion site analysis paved the way for the use of this new vector in a clinical setting.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Melissa A. Bonner,1 Sheng Zhou,1 Zhijun Ma,1 Brian P. Sorrentino.1 1 Hematology, St. Jude Children’s Research Hospital, Memphis, TN. A recent clinical trial for β-thalassemia utilizing a β-globin lentiviral vector resulted in a case of potential clonal expansion of cells containing a viral insertion in the HMGA2 gene, resulting in loss of Let-7 regulation and enhancer-driven overexpression of HMGA2. Now 6.5 years post-transplant, this case has not progressed to malignancy nor show signs of continuing clonal expansion. Overexpression of HMGA2 is known to cause HSC expansion in mice, leaving open the question as to whether HMGA2 de-regulation can lead to clonal expansion. Therefore, we have tested whether enforced expression of HMGA2 in autologous HSCs from Macaca nemestrina would result in clonal expansion or leukemia. The human HMGA2 cDNA was cloned from 293T cells, without the 3’ Let-7 sites, into a cl20c MSCV-IRES-GFP plasmid. Lentiviral HMGA2-GFP was produced with a typical unconcentrated titer between 5e7 and 1e8 tu/mL. HMGA2 expression was verified with flow cytometry and western blot analyses in primate CD34+ cells and multiple human cell lines, respectively. For transplant studies, GCSF-mobilized bone marrow was harvested from two macaques, A10W027 and A10W016. Bone marrow was enriched for CD34+ cells and stimulated overnight on RetroNectin-coated plates in XVivo10 media containing HSA, SCF, Flt3-L, and TPO. The CD34+ cells were then split in half and each received two vector exposures (HMGA2-GFP MOI=50, mCherry MOI=100) with a 12 hour washout between applications. After transduction, autologous cells were mixed together in a 50:50 ratio to form a split graft of HMGA2-GFP and mCherry cells and transfused into lethally irradiated recipients. A10W027 and A10W016 are 6 and 2.5 months post-transplant, respectively. Individual colonies from the grafts were analyzed by flow and revealed the transduction efficiencies of the HMGA2-GFP and mCherry vectors to be 6% and 8%, respectively, for A10W027 and 42% and 19%, respectively, for A10W016. The ratio of HMGA2GFP cells to mCherry cells has gone from 0.7 (Graft) to 2.4 in A10W027 with a steady increase in HMGA2-GFP cells starting around 2.5 months post-transplant from 0.8% to 4.0%. Multilineage reconstitution is seen in the HMGA2-GFP and mCherry compartments with CD3, CD14, CD16, and CD20 cells all comprised of a higher level of HMGA2-GFP than mCherry. A10W016 is still early posttransplant and exhibiting flux with transduced cell populations with no obvious early expansion. Monitoring of gene marking continues and deep sequencing analysis is underway to determine definitively if HMGA2 can lead to clonal expansion. Measurements over the next several months should conclusively address whether or not HMGA2 leads to clonal expansion or malignancies in transplanted NHPs.
S178
465. Gene Therapy for Hereditary Anemia: Preclinical Insertion Site Analysis of a New Lentiviral Vector
Annette Deichmann,1 Olivier Negre,2 Cynthia Bartholomae,1 Raffaele Fronza,1 Christof von Kalle,1 Mitch Finer,3 Gabor Veres,3 Emmanuel Payen,2 Manfred Schmidt.1 1 DKFZ/NCT, German Cancer Research Center/ National Center for Tumor Diseases, Heidelberg, Germany; 2CEA, Institut of Emerging Diseases and Innovative Therapies (iMETI), Fontenay aux Roses, France; 3bluebird bio, Cambridge, MA. Gene therapy using the lentiviral vector HPV569 that carries the ß-globin gene has been proven a valuable therapeutic option in a patient with ß-thalassemia. Recently a new lentiviral vector BB305 has been designed in order to increase the efficiency of gene transfer. A preclinical trial in mice has been set up to investigate the efficacy and safety using BB305. We present the results of the analysis of 10,835 unique integration sites (IS) as an important marker for the safety. 58 mice were transplanted with vector HPV569, BB305 or mock-transduced syngeneic ß-thalassemic bone marrow cells. With regard to the efficacy, complete correction of the phenotype and an oligo/polyclonal hematopoietic reconstitution was observed in all mice. 108 mice received a secondary transplant to evaluate long-term toxicity. Malignant T cell lymphomas were observed in transplanted animals with both vectors but as well in the control group. However, histology, immunohistochemistry and quantitative PCR revealed that the occurrence of these tumors did not derive from transduced donor cells. In pre-transplantation samples we detected 2605 IS in cells transduced with HPV569 and 1372 with BB305, in primary mice 1699 unique IS with vector HPV569 and 2143 IS with vector BB305 and in secondary mice 1353 with HPV569 and 1715 with BB305. Integration of both vectors occurred in genes with about 70% in primary animals and about 54% in secondary animals. The oncogenic potential of the affected genes was looked up in different databases. No significant difference could be detected between preand post-transplantation samples and no preferred integration in Mecom, Lmo2 or Hmga2. Approximately 30% of IS are clustered as common integration sites (CIS). Comparison with a random in silico dataset and another lentiviral preclinical dataset showed that CIS clustering occured in the same genomic areas. In secondary animals we found an oligo/polyclonal hematopoietic reconstitution, but the clonal diversity was reduced as expected and was equivalent for the two vectors. Long-term safety without any signs of clonal outgrowth or in vivo selection has been demonstrated. This complete preclinical insertion site analysis paved the way for the use of this new vector in a clinical setting.
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy