- Email: [email protected]
629. Adult Muscle-Derived Stem Cells Rescue Progeria Stem Cell Dysfunction and Promote Angiogenesis
STEM CELL THERAPIES Next, we investigated kinetics and strength of these promoters as compared to a strong constitutive promoter (PGK) in experimental arthritis with two relapsing ares of inammation. Surprisingly, only Saa3 and Cxcl1 were strongly upregulated (10-50 fold) during the rst challenge, while a second challenge induced strong luciferase expression for all constructs (10-200 fold). The Saa3 promoter activity showed a clear correlation with joint edema as measured by 99m Tc uptake. Interestingly, in the absence of a second are the Cxcl1 promoter activity still increased whereas the Saa3 promoter activity returned to basal levels. Promoter analysis suggested that hypoxiaresponse elements in the Cxcl1 promoter are likely causative for this difference in expression prole with Saa3. Finally, we characterized the response of computationally-dened promoters in RA synovial tissue of varying inammatory subtypes. Saa3, Cxcl1, Cxcl5 and Il1b promoters could be induced by stimulation of RA synovial broblasts with pro-inammatory stimuli. Uniquely, relative Saa3 promoter responses to cytokines and a TLR4 agonist were signicantly higher in broblasts with an inammatory genetic imprint both as a group as in individual samples. Relative Cxcl1 promoter responses did not discriminate between inammatory synovial broblast subtypes. These data demonstrate that computational design of promoters is of great value for the development of disease-regulated gene therapy, Towards personalized arthritis gene therapy, the Saa3 promoter appears an excellent candidate for sensing the extent of joint inammation and responding differentially in human arthritic synovium according to their inammatory phenotype.
Stem Cell Therapies 628. Contribution of PDGFRĮ-Positive Bone Marrow Cells for Epithelial Regeneration in Genetic Blistering Skin Disease, RDEB
Katsuto Tamai,1 Takenao Chino,1 Takehiko Yamazaki,1 Yasufumi Kaneda.1 1 Gene Therapy Science, Osaka University Gradutate School of Medicine, Suita, Osaka, Japan. Physiologic homeostasis of epithelial structures is maintained by resident stem cells residing in the epithelia themselves. In the skin of patients with recessive dystrophic epidermolysis bullosa (RDEB), the epidermal stem cell pool is continually depleted as a result of detachment of full-thickness epidermis from dermis due to genetic dysfunction of the dermo-epidermal basement membrane zone. Nevertheless, the epidermal regeneration mechanisms in RDEB still adequately maintain functional epithelium in the skin, suggesting epithelial stem/progenitor cell-supplementation from extracutaneous sources. In this study, we demonstrate that detached RDEB epithelia recruit platelet-derived growth factor receptor alpha (PDGFRα)positive bone marrow cells (PDGFRα+ BMCs) via circulation. To elucidate the role of the mobilized PDGFRα-positive bone marrow cells in the regeneration of RDEB skin in vivo, we combined PDGFRα-positive/GFP-positive bone marrow cells (P/G-BMCs) with wild-type bone marrow cells, and transplanted those cells to the lethally irradiated mice to generate P/G-BMT mouse, followed by engraftment of RDEB mouse skin on the back of the P/G-BMT mouse. In 4 weeks after the RDEB skin engraftment, signicant numbers of GFP-positive cells were observed in both mesenchymal and epithelial tissues of the engrafted RDEB skin. The GFP-positive epithelial cells were then shown to express of K5 in the regenerating RDEB epithelia, clearly demonstrating that the PDGFRα+ BMCs contain particular population to provide epithelial cells in the RDEB skin. To further investigate PDGFRα+ BMCs, we observed PDGFRα+ BMCs in vivo by intravital two-photon imaging of the knock-in mice, in which histon H2B-EGFP fusion gene is inserted in the PDGFRα gene locus to express H2B-EGFP fusion protein in the nucleus of the PDGFRα-expressing cells. PDGFRα+ BMCs were shown to reside S244
particularly on and around surface of the bone and trabeculae in bone marrow. Flow cytometry analysis of the knock-in mouse BMCs showed that the PDGFRα-positive BMCs are all lineage-negative and c-kit-negative, suggesting undifferentiated non-hematopoietic cell populations. The PDGFRα+ BMCs were then shown to differentiate to be K5-positive keratinocytes in culture. Finally, we succeeded to observe intravital imaging of the PDGFRα+ BMCs mobilizing around blood vessels to egress into the systemic circulation by stimulation with skin injury-inducing danger signaling. Collectively, our data suggest novel supplementation mechanism of Lin-/PDGFRα+/c-kit- BMCs to the injured skin via circulation to accelerate wound healing. Particularly for RDEB, PDGFRα+ BMCs of the patients may be an ideal target for cell and gene therapy to provide bone marrow-derived epithelial cells harboring therapeutic gene products in the regenerating skin.
629. Adult Muscle-Derived Stem Cells Rescue Progeria Stem Cell Dysfunction and Promote Angiogenesis
Mitra Lavasani,1 Andria R. Robinson,2 Aiping Lu,1 Joseph M. Feduska,1 Jeremy S. Tilstra,3 Paul D. Robbins,3 Laura J. Niedernhofer,2 Johnny Huard.1 1 Department of Orthopaedic Surgery, Stem Cell Research Center, University of Pittsburgh, Pittsburgh, PA; 2Department of Microbiology and Molecular Genetics, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 3Department of Microbiology and Molecular Genetics, Unviersity of Pittsburgh, School of Medicine, Pittsburgh, PA. Severely reduced expression of the DNA repair endonuclease ERCC1-XPF leads to a dramatic progeria (disease of accelerated aging) in humans. Genetic deletions of either the excision repair cross-complementation group 1 (Ercc1) gene or the xeroderma pigmentosum, complementation group F (Xpf) gene in mice causes reduced lifespan with premature onset of severe aging that mimics human progeria. Ercc1-/- mice die in the 4th week of life with multiple symptoms associated with advanced age. We previously showed that muscle-derived stem cells (MDSCs) isolated from ERCC1-decient mice are defective in proliferation and myogenic differentiation. Also, we also showed that intraperitoneal (IP) and intramuscular (IM) injection of wild type (WT) MDSCs into Ercc1-/- mice resulted in engraftment in skeletal muscle and non-muscle tissues. Remarkably, intraperitoneal delivery of WT-MDSCs led to a significant improvement in the healthspan and lifespan of ERCC1-decent mice. In the current study, we hypothesized that a paracrine factor(s) secreted by donor cells is responsible for rescuing the ERCC1-decient mice post-transplantation. We tested this hypothesis in vitro by coculturing WT- with ERCC1-decient-MDSCs and measuring if the proliferation and differentiation defects of ERCC1-decient MDSCs are rescued. Our preliminary data show that the proliferation and myogenic differentiation of progeria ERCC1-decient MDSCs was signicantly improved when co-cultured in a transwell system with WT-MDSCs. These results support the conclusion that adult MDSCs release soluble factors that are growth promoting. We also further characterized how WT-MDSCs could rescue age-related degenerative changes. We found that Ercc1-/- mice display signicantly reduced CD31+ blood vessels per muscle ber compared to their wild-type littermates. The average muscle ber size (cross-sectional area) was also signicantly reduced in the Ercc1-/- mice compared to WT littermates. Injection of WT-MDSCs into Ercc1-/- mice IP resulted in a signicant increase in both vascular supply and muscle ber size compared to the non-injected mutant animals. The Ercc1-/- mice receiving IM injection of WT-MDSCs also showed an improvement in these parameters compared to non-injected controls. Here we demonstrate that injection of the WT-MDSCs promotes angiogenesis and leads to improved vascularization of the muscle as well as muscle Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
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
Stem Cell Therapies 628. Contribution of PDGFRĮ-Positive Bone Marrow Cells for Epithelial Regeneration in Genetic Blistering Skin Disease, RDEB
Katsuto Tamai,1 Takenao Chino,1 Takehiko Yamazaki,1 Yasufumi Kaneda.1 1 Gene Therapy Science, Osaka University Gradutate School of Medicine, Suita, Osaka, Japan. Physiologic homeostasis of epithelial structures is maintained by resident stem cells residing in the epithelia themselves. In the skin of patients with recessive dystrophic epidermolysis bullosa (RDEB), the epidermal stem cell pool is continually depleted as a result of detachment of full-thickness epidermis from dermis due to genetic dysfunction of the dermo-epidermal basement membrane zone. Nevertheless, the epidermal regeneration mechanisms in RDEB still adequately maintain functional epithelium in the skin, suggesting epithelial stem/progenitor cell-supplementation from extracutaneous sources. In this study, we demonstrate that detached RDEB epithelia recruit platelet-derived growth factor receptor alpha (PDGFRα)positive bone marrow cells (PDGFRα+ BMCs) via circulation. To elucidate the role of the mobilized PDGFRα-positive bone marrow cells in the regeneration of RDEB skin in vivo, we combined PDGFRα-positive/GFP-positive bone marrow cells (P/G-BMCs) with wild-type bone marrow cells, and transplanted those cells to the lethally irradiated mice to generate P/G-BMT mouse, followed by engraftment of RDEB mouse skin on the back of the P/G-BMT mouse. In 4 weeks after the RDEB skin engraftment, signicant numbers of GFP-positive cells were observed in both mesenchymal and epithelial tissues of the engrafted RDEB skin. The GFP-positive epithelial cells were then shown to express of K5 in the regenerating RDEB epithelia, clearly demonstrating that the PDGFRα+ BMCs contain particular population to provide epithelial cells in the RDEB skin. To further investigate PDGFRα+ BMCs, we observed PDGFRα+ BMCs in vivo by intravital two-photon imaging of the knock-in mice, in which histon H2B-EGFP fusion gene is inserted in the PDGFRα gene locus to express H2B-EGFP fusion protein in the nucleus of the PDGFRα-expressing cells. PDGFRα+ BMCs were shown to reside S244
particularly on and around surface of the bone and trabeculae in bone marrow. Flow cytometry analysis of the knock-in mouse BMCs showed that the PDGFRα-positive BMCs are all lineage-negative and c-kit-negative, suggesting undifferentiated non-hematopoietic cell populations. The PDGFRα+ BMCs were then shown to differentiate to be K5-positive keratinocytes in culture. Finally, we succeeded to observe intravital imaging of the PDGFRα+ BMCs mobilizing around blood vessels to egress into the systemic circulation by stimulation with skin injury-inducing danger signaling. Collectively, our data suggest novel supplementation mechanism of Lin-/PDGFRα+/c-kit- BMCs to the injured skin via circulation to accelerate wound healing. Particularly for RDEB, PDGFRα+ BMCs of the patients may be an ideal target for cell and gene therapy to provide bone marrow-derived epithelial cells harboring therapeutic gene products in the regenerating skin.
629. Adult Muscle-Derived Stem Cells Rescue Progeria Stem Cell Dysfunction and Promote Angiogenesis
Mitra Lavasani,1 Andria R. Robinson,2 Aiping Lu,1 Joseph M. Feduska,1 Jeremy S. Tilstra,3 Paul D. Robbins,3 Laura J. Niedernhofer,2 Johnny Huard.1 1 Department of Orthopaedic Surgery, Stem Cell Research Center, University of Pittsburgh, Pittsburgh, PA; 2Department of Microbiology and Molecular Genetics, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 3Department of Microbiology and Molecular Genetics, Unviersity of Pittsburgh, School of Medicine, Pittsburgh, PA. Severely reduced expression of the DNA repair endonuclease ERCC1-XPF leads to a dramatic progeria (disease of accelerated aging) in humans. Genetic deletions of either the excision repair cross-complementation group 1 (Ercc1) gene or the xeroderma pigmentosum, complementation group F (Xpf) gene in mice causes reduced lifespan with premature onset of severe aging that mimics human progeria. Ercc1-/- mice die in the 4th week of life with multiple symptoms associated with advanced age. We previously showed that muscle-derived stem cells (MDSCs) isolated from ERCC1-decient mice are defective in proliferation and myogenic differentiation. Also, we also showed that intraperitoneal (IP) and intramuscular (IM) injection of wild type (WT) MDSCs into Ercc1-/- mice resulted in engraftment in skeletal muscle and non-muscle tissues. Remarkably, intraperitoneal delivery of WT-MDSCs led to a significant improvement in the healthspan and lifespan of ERCC1-decent mice. In the current study, we hypothesized that a paracrine factor(s) secreted by donor cells is responsible for rescuing the ERCC1-decient mice post-transplantation. We tested this hypothesis in vitro by coculturing WT- with ERCC1-decient-MDSCs and measuring if the proliferation and differentiation defects of ERCC1-decient MDSCs are rescued. Our preliminary data show that the proliferation and myogenic differentiation of progeria ERCC1-decient MDSCs was signicantly improved when co-cultured in a transwell system with WT-MDSCs. These results support the conclusion that adult MDSCs release soluble factors that are growth promoting. We also further characterized how WT-MDSCs could rescue age-related degenerative changes. We found that Ercc1-/- mice display signicantly reduced CD31+ blood vessels per muscle ber compared to their wild-type littermates. The average muscle ber size (cross-sectional area) was also signicantly reduced in the Ercc1-/- mice compared to WT littermates. Injection of WT-MDSCs into Ercc1-/- mice IP resulted in a signicant increase in both vascular supply and muscle ber size compared to the non-injected mutant animals. The Ercc1-/- mice receiving IM injection of WT-MDSCs also showed an improvement in these parameters compared to non-injected controls. Here we demonstrate that injection of the WT-MDSCs promotes angiogenesis and leads to improved vascularization of the muscle as well as muscle Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
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