- Email: [email protected]
631. Nonhuman Primate Cord Blood Expansion and Transplantation
STEM CELL THERAPIES ber size in progeroid ERCC1-decient mice. These ndings are similar to our previous observations that regenerative potential of MDSCs in skeletal muscle, heart and bone correlates with the ability of the cells to induce angiogenesis. Collectively, these data suggest that the therapeutic effect of adult MDSCs in this mouse model of progeria is due to a paracrine effect, mediated by a soluble factor that promotes angiogenesis and growth.
630. AAV-2 Delivered Factor IX Is Secreted with Full Clotting Activity from Human Adipose Stromal Cells Differentiated towards Hepatocytes
Katherine T. Marcucci,1 Katie Bisordi,2 Shangzhen Zhou,1 Keith L. March,3 Elliot D. Rosen,2,3 Katherine A. High.1,4 1 Department of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Department of Medical and Molecular Genetics, Indiana University Medical Schoool, Indianapolis, IN; 3 Indiana Center for Vasular Biology, Indiana University Medical Schoool, Indianapolis, IN; 4Howard Hughes Medical Institute, Philadelphia, PA. Human Adipose Stromal Cells (hASCs) are an adult stem cell population that can be differentiated towards several lineages, including hepatocytes. The hepatocyte-like hASCs are a potential population for ex vivo gene therapy for autologous cell-based treatment of human Factor IX (hFIX) deciency in Hemophilia B. We differentiated the hASCs towards hepatocytes using a previously published protocol (Talens-Visconti R, et al. 2006. World J Gastroenterol. 12: 5834-45). We monitored hepatocyte differentiation by RT-PCR for liver-specic markers, α-fetoprotein, albumin, HNF4α and CYP3A. By the end of the 21-day protocol, the differentiated hASCs expressed all of the liver-specic markers. We also determined the relative expression levels of γ-glutamyl carboxylase, Vitamin K epoxide reductase, quinone reductase and PACE/furin by RT-qPCR. These genes are required for the Vitamin K-dependent modications of hFIX responsible for full clotting activity. We found that they are expressed in the hASC population prior to and throughout the differentiation protocol. Therefore, we transduced the hepatocyte-like hASCs at day 7 with an AAV-2 vector delivering hFIX under control of the CMV promoter at two MOIs, 5e5 and 1e6 vg/cell. We measured supernatant hFIX levels by ELISA every 3 days for 15 days posttransduction. hFIX in the supernatant of untransduced differentiated cells was below the lower limit of detection (20ng/mL) of the ELISA. However, hFIX from transduced cells was detected in the supernatant as early as 3 days post-transduction and peak levels at day 15 posttransduction were 3.5 and 3.7 µg/106 cells/24 hours for MOIs 5e5 and 1e6vg/cell, respectively. These levels are 3-10 fold higher than those found in the literature for hFIX secretion from a primary or stem cell population transduced by a viral vector. We also measured the clotting activity of the secreted hFIX by activated partial thromboplastin time (aPTT) on days 9 and 15 post-transduction. Secreted hFIX specic activity corresponded with that of normal human plasma on both days for both MOIs. Thus, we have identied an adult stem cell population that secretes fully functional hFIX and can be considered as a source for an autologous cell-based treatment following ex vivo gene therapy for the treatment of Hemophilia.
631. Nonhuman Primate Cord Blood Expansion and Transplantation
Korashon L. Watts,1 R. Keith Humphries,2 Hans-Peter Kiem.1 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Terry Fox Laboratory, BC Cancer Agency Research Centre, Vancouver, BC, Canada.
Umbilical cord blood (CB) has become an attractive source of stem cells for hematopoietic cell transplantation. Signicant research efforts have focused on the development of methods to expand CB Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
cells ex vivo. We have developed a nonhuman primate cord blood transplantation model that can be used to test the efcacy of various expansion conditions in a clinically relevant setting in a large animal. Our rst step was to optimize the procedure for cord blood collection; this proved to be a complex, iterative process involving frequent adjustments to the protocol. Once we were condent that we had established an optimal protocol for cord blood collection, we investigated whether overexpression of the HOXB4 gene can expand nonhuman primate CB cells. A competitive repopulation analysis was used. Autologous CB CD34+ cells from a Macaca nemestrina subject were divided into 2 equal fractions: half were transduced with a gammaretroviral HOXB4-GFP vector, expanded for 9 days, and cryopreserved. The second half were transduced with a YFP control vector and immediately cryopreserved. Both fractions of cells were then intravenously infused into the myeloablated recipient. Hematopoietic recovery was measured by daily complete blood counts; gene marking was analyzed by bi-weekly ow cytometry. LAM-PCR was used to conrm polyclonal repopulation and to analyze retroviral integration sites. Gene transfer efciency was 46% for both HOXB4-GFP and YFP cells. After 9 days of ex vivo expansion, HOXB4-GFP cells showed an overall fold expansion of 78-fold. Hematopoietic recovery, measured as ANC >1000, occurred on Day 19. The animal became transfusion-independent around 3 weeks post-transplant. Marking in the GFP arm peaked at around 28% in granulocytes at Day 15 and then stabilized at around 10% from Day 40 onwards. Marking in the YFP arm was present at <0.5% for the duration of the study; therefore, we observed ∼20-fold expansion of HOXB4-GFP+ repopulating cells relative to control. At 3 months post-transplant, HOXB4-GFP+ cells were present in all lineages. Within the lymphoid lineages, GFP+ cells were present at 3.4% among CD3+ T cells, 9.2% among CD4+ T cells, 3.9% among CD8+ T cells, and 6.0% among CD20+ B cells. Additionally, GFP+ cells were present at a level of 8.7% among CD13+ cells and 9.2% among CD14+ cells. Analysis of platelets at 3 months post-transplant showed that 12% were GFP+. LAM-PCR was performed on GFP-sorted cells 3 months post-transplant, revealing polyclonal repopulation. In summary, we have established and tested a large-animal cord blood transplantation model. Our ndings indicate that HOXB4 transduction facilitates expansion of CB cells to clinically relevant doses and aids in engraftment. This nonhuman primate cord blood transplantation model will allow us to study other expansion techniques as well, such as co-culture with mesenchymal stem cells, transduction using adenoviral vectors, and HOXB4 protein-mediated expansion.
632. Stealth Delivery: Combining Site-Specic Integration and Cassette Design To Achieve Robust Expression without Impacting Endogenous Gene Expression Angelo Lombardo,1,2 Daniela Cesana,1,2 Pietro Genovese,1,2 Elena Provasi,2,3 Bruno Di Stefano,3 Margherita Neri,1 Vania Broccoli,3 Angela Gritti,1 Michael C. Holmes,4 Philip D. Gregory,4 Chiara Bonini,3 Luigi Naldini.1,2 1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2 San Raffaele University, Milan, Italy; 3San Raffaele Scientic Institute, Milan, Italy; 4Sangamo BioSciences Inc., Richmond, CA.
Site-specic integration holds great promise for gene therapy as it may overcome the genotoxic risks of conventional integrating gene transfer vectors. We previously described the use of Zinc Finger Nucleases (ZFN) and Integrase Defective Lentiviral Vectors to insert transgenes into a preselected human genomic site. However, little is known of suitable “safe harbor” loci, and about the impact of transgene insertion on the targeted locus and vice versa. Here we address this issue by targeting two putative safe harbor sites, the CCR5 gene and the AAVS1 locus, and assessing: i) their permissiveness to transgene expression; ii) transcriptional perturbation of the locus and S245
630. AAV-2 Delivered Factor IX Is Secreted with Full Clotting Activity from Human Adipose Stromal Cells Differentiated towards Hepatocytes
Katherine T. Marcucci,1 Katie Bisordi,2 Shangzhen Zhou,1 Keith L. March,3 Elliot D. Rosen,2,3 Katherine A. High.1,4 1 Department of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2Department of Medical and Molecular Genetics, Indiana University Medical Schoool, Indianapolis, IN; 3 Indiana Center for Vasular Biology, Indiana University Medical Schoool, Indianapolis, IN; 4Howard Hughes Medical Institute, Philadelphia, PA. Human Adipose Stromal Cells (hASCs) are an adult stem cell population that can be differentiated towards several lineages, including hepatocytes. The hepatocyte-like hASCs are a potential population for ex vivo gene therapy for autologous cell-based treatment of human Factor IX (hFIX) deciency in Hemophilia B. We differentiated the hASCs towards hepatocytes using a previously published protocol (Talens-Visconti R, et al. 2006. World J Gastroenterol. 12: 5834-45). We monitored hepatocyte differentiation by RT-PCR for liver-specic markers, α-fetoprotein, albumin, HNF4α and CYP3A. By the end of the 21-day protocol, the differentiated hASCs expressed all of the liver-specic markers. We also determined the relative expression levels of γ-glutamyl carboxylase, Vitamin K epoxide reductase, quinone reductase and PACE/furin by RT-qPCR. These genes are required for the Vitamin K-dependent modications of hFIX responsible for full clotting activity. We found that they are expressed in the hASC population prior to and throughout the differentiation protocol. Therefore, we transduced the hepatocyte-like hASCs at day 7 with an AAV-2 vector delivering hFIX under control of the CMV promoter at two MOIs, 5e5 and 1e6 vg/cell. We measured supernatant hFIX levels by ELISA every 3 days for 15 days posttransduction. hFIX in the supernatant of untransduced differentiated cells was below the lower limit of detection (20ng/mL) of the ELISA. However, hFIX from transduced cells was detected in the supernatant as early as 3 days post-transduction and peak levels at day 15 posttransduction were 3.5 and 3.7 µg/106 cells/24 hours for MOIs 5e5 and 1e6vg/cell, respectively. These levels are 3-10 fold higher than those found in the literature for hFIX secretion from a primary or stem cell population transduced by a viral vector. We also measured the clotting activity of the secreted hFIX by activated partial thromboplastin time (aPTT) on days 9 and 15 post-transduction. Secreted hFIX specic activity corresponded with that of normal human plasma on both days for both MOIs. Thus, we have identied an adult stem cell population that secretes fully functional hFIX and can be considered as a source for an autologous cell-based treatment following ex vivo gene therapy for the treatment of Hemophilia.
631. Nonhuman Primate Cord Blood Expansion and Transplantation
Korashon L. Watts,1 R. Keith Humphries,2 Hans-Peter Kiem.1 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Terry Fox Laboratory, BC Cancer Agency Research Centre, Vancouver, BC, Canada.
Umbilical cord blood (CB) has become an attractive source of stem cells for hematopoietic cell transplantation. Signicant research efforts have focused on the development of methods to expand CB Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
cells ex vivo. We have developed a nonhuman primate cord blood transplantation model that can be used to test the efcacy of various expansion conditions in a clinically relevant setting in a large animal. Our rst step was to optimize the procedure for cord blood collection; this proved to be a complex, iterative process involving frequent adjustments to the protocol. Once we were condent that we had established an optimal protocol for cord blood collection, we investigated whether overexpression of the HOXB4 gene can expand nonhuman primate CB cells. A competitive repopulation analysis was used. Autologous CB CD34+ cells from a Macaca nemestrina subject were divided into 2 equal fractions: half were transduced with a gammaretroviral HOXB4-GFP vector, expanded for 9 days, and cryopreserved. The second half were transduced with a YFP control vector and immediately cryopreserved. Both fractions of cells were then intravenously infused into the myeloablated recipient. Hematopoietic recovery was measured by daily complete blood counts; gene marking was analyzed by bi-weekly ow cytometry. LAM-PCR was used to conrm polyclonal repopulation and to analyze retroviral integration sites. Gene transfer efciency was 46% for both HOXB4-GFP and YFP cells. After 9 days of ex vivo expansion, HOXB4-GFP cells showed an overall fold expansion of 78-fold. Hematopoietic recovery, measured as ANC >1000, occurred on Day 19. The animal became transfusion-independent around 3 weeks post-transplant. Marking in the GFP arm peaked at around 28% in granulocytes at Day 15 and then stabilized at around 10% from Day 40 onwards. Marking in the YFP arm was present at <0.5% for the duration of the study; therefore, we observed ∼20-fold expansion of HOXB4-GFP+ repopulating cells relative to control. At 3 months post-transplant, HOXB4-GFP+ cells were present in all lineages. Within the lymphoid lineages, GFP+ cells were present at 3.4% among CD3+ T cells, 9.2% among CD4+ T cells, 3.9% among CD8+ T cells, and 6.0% among CD20+ B cells. Additionally, GFP+ cells were present at a level of 8.7% among CD13+ cells and 9.2% among CD14+ cells. Analysis of platelets at 3 months post-transplant showed that 12% were GFP+. LAM-PCR was performed on GFP-sorted cells 3 months post-transplant, revealing polyclonal repopulation. In summary, we have established and tested a large-animal cord blood transplantation model. Our ndings indicate that HOXB4 transduction facilitates expansion of CB cells to clinically relevant doses and aids in engraftment. This nonhuman primate cord blood transplantation model will allow us to study other expansion techniques as well, such as co-culture with mesenchymal stem cells, transduction using adenoviral vectors, and HOXB4 protein-mediated expansion.
632. Stealth Delivery: Combining Site-Specic Integration and Cassette Design To Achieve Robust Expression without Impacting Endogenous Gene Expression Angelo Lombardo,1,2 Daniela Cesana,1,2 Pietro Genovese,1,2 Elena Provasi,2,3 Bruno Di Stefano,3 Margherita Neri,1 Vania Broccoli,3 Angela Gritti,1 Michael C. Holmes,4 Philip D. Gregory,4 Chiara Bonini,3 Luigi Naldini.1,2 1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2 San Raffaele University, Milan, Italy; 3San Raffaele Scientic Institute, Milan, Italy; 4Sangamo BioSciences Inc., Richmond, CA.
Site-specic integration holds great promise for gene therapy as it may overcome the genotoxic risks of conventional integrating gene transfer vectors. We previously described the use of Zinc Finger Nucleases (ZFN) and Integrase Defective Lentiviral Vectors to insert transgenes into a preselected human genomic site. However, little is known of suitable “safe harbor” loci, and about the impact of transgene insertion on the targeted locus and vice versa. Here we address this issue by targeting two putative safe harbor sites, the CCR5 gene and the AAVS1 locus, and assessing: i) their permissiveness to transgene expression; ii) transcriptional perturbation of the locus and S245