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513. The Immunosuppressant Drug Rapamycin Relieves Lentiviral Transduction Resistance in Human Hematopoietic Stem Cells
ADULT STEM CELLS NON-MESENCHYMAL 512. Comparison of Commercially Available Furin ELISA Kits To Assess Furin Knockdown in cGMP Manufactured FANG™ Vaccines
Connor Phalon,1 Padmasini Kumar,1 Yang Yu,1 Fabienne Norvell,1 John Nemunaitis,1,2,3,4 Neil Senzer,1,2,3,4 Phillip B. Maples.1 1 Gradalis, Inc., Dallas; 2Mary Crowley Cancer Research Centers, Dallas; 3Medical City HCA, Dallas; 4Texas Oncology, P.A., Dallas. We developed a cGMP manufactured autologous tumor-based vaccine FANGTM, incorporating a plasmid encoding GMCSF and a proprietary bifunctional shRNA targeting Furin. Furin, also known as furin/PACE/SPC1, is an enzyme which belongs to the subtilisin-like proprotein convertase family. The proprotein convertases process latent precursor proteins into their biologically active products. Furin’s many substrates include the inactive prepro precursors of TGFβ1 and TGFβ2. Both TGFβ1 and TGFβ2 have been shown to play a significant role in tumor progression, metastasis, and tumor-induced immunosuppression. During the manufacture of the autologous vaccine an aliquot of cells from before and after transfection with FANGTM plasmid are placed in culture. Cells and supernatants are collected at Day 0, 1, 2, 3, 4, 7, 10 and 14 days. Furin protein was quantified by two different ELISA kits; R&D Systems (Minneapolis, MN) and USCN (Wuhan, China). Twenty pairs of culture supernatants (before and after transfection) were tested using both kits. An increase in the Furin levels were detected in the pre transfected supernatants with increasing time in the 14 day culture using either kits, but with very different quantities detected. Average Values R&D PRE (pg) R&D POST (pg) USCN PRE (ng) USCN POST (ng)
Day 3 64.5 ± 54.2 50.9 ± 33.4 34.6 ± 40.1 3.4 ± 8.0
Day 4 69.5 ± 61.6 51.8 ± 31.1 37.6 ± 42.0 3.5 ± 5.9
Day 7 95.7 ± 80.4 49.8 ± 37.6 35.3 ± 41.4 4.1 ± 9.5
Day 10 110.3 ± 91.4 56.7 ± 45.2 39.8 ± 39.9 4.8 ± 10.3
The R&D Systems kit increased from an average of 22.5 pg/106 cells at day 0 to 150.7 pg/106 cells at day 14 and the USCN kit increased from 22.3 ng/106 cells at day 0 to 44.7 ng/106 cells at day 14. The percentage of Furin knockdown also differed between the kits. We observed 25.5% knockdown from day 4 pre and post samples with the R&D Systems kit, and a 90.7% knockdown from the USCN kit. Each kit was tested with both kit’s standards and commercially available Furin proteins from R&D Systems, Abcam, and Santa Cruz Biotechnology at the same concentrations. The USCN kit was unable to detect any of the proteins other than its own standard. The R&D Systems kit detected its own standard and the Furin protein ordered from R&D systems was detected at 10% of expected concentrations, but was unable to detect furin from any other commercial source. In addition, we have recently discovered a kit made by Abcam. This kit will be tested and compared to the USCN and R&D systems kits. The knockdown of TGFβ1 and TGFβ2 (determined by ELISA) were significant and consistent, averaging 93.5% and 92.5% respectively, indicating a high percentage of Furin knockdown with the FANGTM vaccine, corresponding to data obtained from the USCN kit. Based on these results, the USCN ELISA will be used for future samples. Other Furin protein quantitation methods will be explored to verify the USCN ELISA accuracy and reproducibility.
Adult Stem Cells Non-Mesenchymal 513. The Immunosuppressant Drug Rapamycin Relieves Lentiviral Transduction Resistance in Human Hematopoietic Stem Cells Cathy X. Wang,1 Bruce E. Torbett.1,2 Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA; 2Molecular Pathology, University of California, San Diego, La Jolla, CA.
1
Hematopoietic stem cells (HSCs) in the native, non-cycling state are extremely resistant to transduction by HIV-1 vectors, representing S198
a major hurdle to lentiviral-mediated gene delivery; promoting cell cycling to achieve high-level transduction may result in the loss of stem cell potency. Here we demonstrate dramatically improved transduction efficiency of human CD34+ HSCs by HIV-1 vectors in the presence of the immunosuppressant drug rapamycin. In ex vivo cultures, the presence of rapamycin (10-20 ng/ul) during transduction increases EGFP marker expression two-fold from 40% to 80% in human cord blood CD34+ cells that have undergone 24h of prestimulation but do not demonstrate cell cycle activation, at an MOI of 50. The transduction rate of adult bone marrow CD34+ cells, which are more therapeutically relevant and extremely refractory to lentiviral transduction, is also increased two-fold from 20% to 40% with rapamycin treatment. Moreover, increased transduction efficiency is observed in freshly isolated, quiescent CD34+ cells of both cord blood and adult bone marrow origin, with magnitudes ranging from 1.3 to 2-fold. Importantly, rapamycin-treated CD34+ cells maintain equivalent colony forming potential to control cells in semi-solid methylcellulose medium, and engraft irradiated NSG mice up to time of sacrifice at 19 weeks post injection with equivalent levels of chimerism (40% human cells in the bone marrow) to control-cell engrafted mice, indicating that rapamycin treatment does not compromise development potential of progenitor cells. The rapamycin treatment group of mice shows stable and dramatically higher percentages of EGFP expressing cells in peripheral blood and all tissues examined, across all hematopoietic lineages, with a striking four-fold difference of 80% vs. 20% EGFP expression in bone marrow human CD45+ cells, at a modest MOI of 25. The high levels of EGFP expression are preserved in secondary transplant recipients, indicating that rapamycin-treated cells are capable of longterm engraftment in the NSG mouse model. Quantitative PCR for HIV-1 DNA shows accelerated lentiviral vector replication kinetics post cell entry in rapamycin-treated cord blood CD34+ cells. Given that rapamycin is a well-known inducer of autophagy, we speculate that HIV-1 may be utilizing the autophagic machinery to facilitate early stages of infection, leading to more efficient integration into the host cell genome. Accordingly, 3-methyladenine, an autophagyinhibiting molecule, reduces transduction efficiency by half in both quiescent and short-term pre-stimulated cord blood CD34+ cells in ex vivo cultures. However, we cannot currently rule out involvement of non-autophagic mechanisms. The phenomenon is HSC specific, as we do not observe rapamycin-mediated increase in transduction in more differentiated primary myeloid cells. Taken together, our findings provide mechanistic insights into the regulation of HIV pre-integration steps in HSCs, and implicate a significant role for the immunosuppressant drug rapamycin in the field of HSC gene delivery.
514. FVIII Expression and Secretion in Circulating Blood Cell Types Capable of Correcting Bleeding in Hemophilia A Mice
Simone Merlin,1 Diego Zanolini,1 Gabriella Ranaldo,1 Maria Feola,1 Elvira S. Cannizzo,1 Guido Valente,1 Maria Prat,1 Sanjeev Gupta,2 Antonia Follenzi.1 1 University of Piemonte Orientale A. Avogadro, Novara, Italy; 2 Medicine, Albert Einstein College of Medicine, New York.
Identification of cells capable of synthesizing and releasing factor VIII (FVIII) is critical for insights into pathophysiological mechanisms and for developing therapeutic approaches in hemophilia A. Endothelial cells, particularly liver sinusoidal endothelial cells (LSEC), express FVIII most in the body. However, recent studies of bone marrow (BM) transplantation suggested additional cell types could synthesize and release FVIII, and also correct bleeding in hemophilia A mice. Therefore, to establish the ability of circulating blood cells in expressing FVIII, we analyzed several murine and human hematopoietic cell types. First, we generated polyclonal antibody against recombinant human FVIII. The mono-specificity Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
Connor Phalon,1 Padmasini Kumar,1 Yang Yu,1 Fabienne Norvell,1 John Nemunaitis,1,2,3,4 Neil Senzer,1,2,3,4 Phillip B. Maples.1 1 Gradalis, Inc., Dallas; 2Mary Crowley Cancer Research Centers, Dallas; 3Medical City HCA, Dallas; 4Texas Oncology, P.A., Dallas. We developed a cGMP manufactured autologous tumor-based vaccine FANGTM, incorporating a plasmid encoding GMCSF and a proprietary bifunctional shRNA targeting Furin. Furin, also known as furin/PACE/SPC1, is an enzyme which belongs to the subtilisin-like proprotein convertase family. The proprotein convertases process latent precursor proteins into their biologically active products. Furin’s many substrates include the inactive prepro precursors of TGFβ1 and TGFβ2. Both TGFβ1 and TGFβ2 have been shown to play a significant role in tumor progression, metastasis, and tumor-induced immunosuppression. During the manufacture of the autologous vaccine an aliquot of cells from before and after transfection with FANGTM plasmid are placed in culture. Cells and supernatants are collected at Day 0, 1, 2, 3, 4, 7, 10 and 14 days. Furin protein was quantified by two different ELISA kits; R&D Systems (Minneapolis, MN) and USCN (Wuhan, China). Twenty pairs of culture supernatants (before and after transfection) were tested using both kits. An increase in the Furin levels were detected in the pre transfected supernatants with increasing time in the 14 day culture using either kits, but with very different quantities detected. Average Values R&D PRE (pg) R&D POST (pg) USCN PRE (ng) USCN POST (ng)
Day 3 64.5 ± 54.2 50.9 ± 33.4 34.6 ± 40.1 3.4 ± 8.0
Day 4 69.5 ± 61.6 51.8 ± 31.1 37.6 ± 42.0 3.5 ± 5.9
Day 7 95.7 ± 80.4 49.8 ± 37.6 35.3 ± 41.4 4.1 ± 9.5
Day 10 110.3 ± 91.4 56.7 ± 45.2 39.8 ± 39.9 4.8 ± 10.3
The R&D Systems kit increased from an average of 22.5 pg/106 cells at day 0 to 150.7 pg/106 cells at day 14 and the USCN kit increased from 22.3 ng/106 cells at day 0 to 44.7 ng/106 cells at day 14. The percentage of Furin knockdown also differed between the kits. We observed 25.5% knockdown from day 4 pre and post samples with the R&D Systems kit, and a 90.7% knockdown from the USCN kit. Each kit was tested with both kit’s standards and commercially available Furin proteins from R&D Systems, Abcam, and Santa Cruz Biotechnology at the same concentrations. The USCN kit was unable to detect any of the proteins other than its own standard. The R&D Systems kit detected its own standard and the Furin protein ordered from R&D systems was detected at 10% of expected concentrations, but was unable to detect furin from any other commercial source. In addition, we have recently discovered a kit made by Abcam. This kit will be tested and compared to the USCN and R&D systems kits. The knockdown of TGFβ1 and TGFβ2 (determined by ELISA) were significant and consistent, averaging 93.5% and 92.5% respectively, indicating a high percentage of Furin knockdown with the FANGTM vaccine, corresponding to data obtained from the USCN kit. Based on these results, the USCN ELISA will be used for future samples. Other Furin protein quantitation methods will be explored to verify the USCN ELISA accuracy and reproducibility.
Adult Stem Cells Non-Mesenchymal 513. The Immunosuppressant Drug Rapamycin Relieves Lentiviral Transduction Resistance in Human Hematopoietic Stem Cells Cathy X. Wang,1 Bruce E. Torbett.1,2 Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA; 2Molecular Pathology, University of California, San Diego, La Jolla, CA.
1
Hematopoietic stem cells (HSCs) in the native, non-cycling state are extremely resistant to transduction by HIV-1 vectors, representing S198
a major hurdle to lentiviral-mediated gene delivery; promoting cell cycling to achieve high-level transduction may result in the loss of stem cell potency. Here we demonstrate dramatically improved transduction efficiency of human CD34+ HSCs by HIV-1 vectors in the presence of the immunosuppressant drug rapamycin. In ex vivo cultures, the presence of rapamycin (10-20 ng/ul) during transduction increases EGFP marker expression two-fold from 40% to 80% in human cord blood CD34+ cells that have undergone 24h of prestimulation but do not demonstrate cell cycle activation, at an MOI of 50. The transduction rate of adult bone marrow CD34+ cells, which are more therapeutically relevant and extremely refractory to lentiviral transduction, is also increased two-fold from 20% to 40% with rapamycin treatment. Moreover, increased transduction efficiency is observed in freshly isolated, quiescent CD34+ cells of both cord blood and adult bone marrow origin, with magnitudes ranging from 1.3 to 2-fold. Importantly, rapamycin-treated CD34+ cells maintain equivalent colony forming potential to control cells in semi-solid methylcellulose medium, and engraft irradiated NSG mice up to time of sacrifice at 19 weeks post injection with equivalent levels of chimerism (40% human cells in the bone marrow) to control-cell engrafted mice, indicating that rapamycin treatment does not compromise development potential of progenitor cells. The rapamycin treatment group of mice shows stable and dramatically higher percentages of EGFP expressing cells in peripheral blood and all tissues examined, across all hematopoietic lineages, with a striking four-fold difference of 80% vs. 20% EGFP expression in bone marrow human CD45+ cells, at a modest MOI of 25. The high levels of EGFP expression are preserved in secondary transplant recipients, indicating that rapamycin-treated cells are capable of longterm engraftment in the NSG mouse model. Quantitative PCR for HIV-1 DNA shows accelerated lentiviral vector replication kinetics post cell entry in rapamycin-treated cord blood CD34+ cells. Given that rapamycin is a well-known inducer of autophagy, we speculate that HIV-1 may be utilizing the autophagic machinery to facilitate early stages of infection, leading to more efficient integration into the host cell genome. Accordingly, 3-methyladenine, an autophagyinhibiting molecule, reduces transduction efficiency by half in both quiescent and short-term pre-stimulated cord blood CD34+ cells in ex vivo cultures. However, we cannot currently rule out involvement of non-autophagic mechanisms. The phenomenon is HSC specific, as we do not observe rapamycin-mediated increase in transduction in more differentiated primary myeloid cells. Taken together, our findings provide mechanistic insights into the regulation of HIV pre-integration steps in HSCs, and implicate a significant role for the immunosuppressant drug rapamycin in the field of HSC gene delivery.
514. FVIII Expression and Secretion in Circulating Blood Cell Types Capable of Correcting Bleeding in Hemophilia A Mice
Simone Merlin,1 Diego Zanolini,1 Gabriella Ranaldo,1 Maria Feola,1 Elvira S. Cannizzo,1 Guido Valente,1 Maria Prat,1 Sanjeev Gupta,2 Antonia Follenzi.1 1 University of Piemonte Orientale A. Avogadro, Novara, Italy; 2 Medicine, Albert Einstein College of Medicine, New York.
Identification of cells capable of synthesizing and releasing factor VIII (FVIII) is critical for insights into pathophysiological mechanisms and for developing therapeutic approaches in hemophilia A. Endothelial cells, particularly liver sinusoidal endothelial cells (LSEC), express FVIII most in the body. However, recent studies of bone marrow (BM) transplantation suggested additional cell types could synthesize and release FVIII, and also correct bleeding in hemophilia A mice. Therefore, to establish the ability of circulating blood cells in expressing FVIII, we analyzed several murine and human hematopoietic cell types. First, we generated polyclonal antibody against recombinant human FVIII. The mono-specificity Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy