- Email: [email protected]
714. Combined Preconditioning and In Vivo Chemoselection With 6-Thioguanine for Selection of Genetically Modified Hematopoietic Stem Cells
SOMATIC STEM CELLS 712. Human Skin Regeneration From Single Gene-Edited Epidermal Stem Cell Clones
Blanca Duarte,1 Francesca Miselli,2 Rodolfo Murillas,1 Alessandra Recchia,2 Marcela Del Río,1,3 Fernando Larcher.1,3 1 Epithelial Biomedicine, CIEMAT-CIBERER, Madrid, Spain; 2 Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 3Department of Bioengineering, University Carlos III de Madrid (UC3M), Madrid, Spain. Ex vivo gene therapy represents one of the various current strategies suitable to treat genodermatoses such as Epidermolysis Bullosa (EB).Efforts are being put on the development of safer and efficient approaches minimizing genotoxic effects due to transgene-induced insertional mutagenesis. Gene-editing through nuclease-facilitated homologous recombination (HR) has been recently proven feasible on skin cells. We have recently reported the ZFN-facilitated, HRmediated insertion of a marker gene into the AAVS1 locus in a limited number of human epidermal cells that, upon grafting, persisted as small foci in the skin regenerated in immunodeficient mice consistent with a stem cell behavior (Coluccio et al., 2013). However, since these foci comprised less than 1% of the total graft surface, the actual capacity of regeneration of a whole epidermal surface has not been proven. In this study we report the engraftment and persistent skin regeneration achieved with a single stem cells clone isolated from HR-mediated gene targeted human keratinocytes. GFP positive keratinocytes (less than 1%) present in culture after co-infection with IDLV-GFP and Ad-ZFN (designed for the AAVS1 locus) were sorted and expanded in culture. Although previous attempts of FACS-selecting viable cells containing less than 5% of GFP positive human keratinocytes failed, in this case supplementation of the culture medium with a ROCK inhibitor allowed the expansion of sorted keratinocytes displaying stem features. Only one of the very few GFP-positive keratinocyte colonies maintained an undifferentiated phenotype and grew larger than two-centimeters in diameter, consisting with the characteristics of a holoclone. The expanded population displayed a highly homogeneous morphology and a sharp peak of fluorescence. PCR analysis of DNA isolated from the clone showed bands consistent with “on target” integration (of the HR-cassette containing the GFP gene) on the AAVS1 locus. Expanded keratinocytes were used to prepare bioengineered skin equivalents for grafting to immunodeficient mice. Four weeks after grafting, human skin regeneration was confirmed through GFP epifluorescence. Histological analysis revealed the regeneration of a human skin with normal architecture and epidermal differentiation markers. Molecular analysis of regenerated skin biopsies is ongoing to ruling out selection of putative off-target or rearrangement events. Our results demonstrate that gene-edited epidermal stem cells preserve their plentiful regenerative capacity without harm and confirm that a strategy based on skin regeneration from very limited number of gene corrected stem cell clones ex vivo is achievable.
713. Fetal Renal Stem Cells Attenuate Ischemic Injury in the Rat Kidney Marina G. M. C. M. da Cunha,1 Silvia Zia,1 Fanny O. Arcolino,1 Elena Levtchenko,1 Jan Deprest,1 Jaan Toelen.1 1 Development and Regeneration, KULeuven, Leuven, Belgium.
Introduction: Despite extensive research into an effective treatment for acute renal injury (AKI), the mortality rate still remains high. Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, as a subtype of these cells is derived from the fetal renal tract. We hypothesize that hAFSCs delivered in rats with renal ischemia and reperfusion (I/R) injury, have a nephroprotective effect due to their immunomodulatory and anti-inflammatory effect, preventing extensive fibrosis as a late complication. Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Materials and Methods: In the first phase of this study, we isolated hAFSCs from human amniotic fluid samples and characterized their immunophenotypic and differentiation properties. Out of 120 clones a cell line with a high expression of CD24 and CD117 and the renal progenitor markers Six2 and Lin1 was selected. In the second phase, I/R injury was performed in thirty male 8week-old Wistar rats and treatment via direct arterial injection was performed 6 hours later. The control group (n=10) received 800ml of vehicle, the treated group (n =10) received 1x106 hAFSCs in 800ml of vehicle. Serum creatinine and proteinuria levels were measured at 24 hours, 48 hours and 2 months after I\R injury. Histological analysis was performed to quantify tubular necrosis and hyaline cast formation at 48 hours, and interstitial fibrosis by Masson’s Trichrome and PAS staining at 2 months. Additionally, the expression of Ki-67, ED-1 and α-SMA was assessed using immunohistochemistry at 48 hours. Results: A subtype of hAFSCs with renal marker expression has been isolated and characterized. These cells showed a nephroprotective effect in the rat ischemia model. There was a significant lower creatinine and proteinuria level at 24 and48 hours. We observed significantly less tubulointerstitial injury, which was characterized by a higher tubular proliferation, lower inflammatory response and an inhibition of myofibroblast expression. The treated group also showed less fibrosis at 2 months. Conclusion: We conclude a subset of cells from hAFSCs with renal phenotype is nephroprotective when delivered in rats with renal ischemia and reperfusion injury.
714. Combined Preconditioning and In Vivo Chemoselection With 6-Thioguanine for Selection of Genetically Modified Hematopoietic Stem Cells
Katrin Hacke,1 Janet A. Treger,1 Brooke T. Bogan,1 Valerie Rezek,1 Munetoshi Narukawa,2 Saki Shimizu,2 Pei-Qi Liu,3 Andreas Reik,3 André M. Lieber,4 Gay M. Crooks,5 Donald B. Kohn,6 Dong Sung An,2 Scott G. Kitchen,1 Philip D. Gregory,3 Gregory J. Cost,3 Michael C. Holmes,3 Noriyuki Kasahara.1 1 Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles; 2School of Nursing, University of California, Los Angeles; 3Sangamo BioSciences, Richmond; 4Division of Medical Genetics, Department of Medicine, University of Washington, Seattle; 5Department of Pathology & Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles; 6Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles. We have recently developed a novel and highly efficient strategy that uses the purine analog 6-thioguanine (6TG) as a single agent for both preconditioning and in vivo chemoselection of hypoxanthine/ guanine-phosphoribosyltransferase (HPRT)-deficient hematopoietic stem and progenitor cells (HSPC). In a murine model of bone marrow (BM) transplantation, combined 6TG conditioning and post-transplantation chemoselection consistently achieved ~95% engraftment of HPRT-deficient donor BM in the absence of any other cytotoxic conditioning regimen. Moreover, long-term reconstitution of histologically and immunophenotypically normal hematopoiesis was observed in both primary and secondary recipients, without significant toxicity. To translate this strategy for combined 6TG preconditioning and chemoselection into a clinically feasible approach, we are testing two different approaches for genetic modification of normal HSPC to render them HPRT-deficient and thus 6TG-resistant: (1) We have constructed and validated a lentiviral vector expressing short-hairpin RNA (shRNA) that targets a consensus sequence present in both human and murine HPRT genes, and which achieves effective longterm reduction of HPRT expression in cells from either species in vitro. In both primary murine BM cells and human CD34+ HSPC S275
SOMATIC STEM CELLS lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Transplantation of vector-transduced HPRT-knockdown CD34+ HSPC into irradiated NOD scid gamma (NSG) mice resulted in stable and long-term reconstitution with genetically modified cells indicating no adverse effects of the HPRT knockdown on CD34+ HSPC engraftment. (2) We have further employed engineered zinc finger nucleases (ZFNs) to target the human HPRT locus for gene disruption. Protocols for HPRT-ZFN delivery into CD34+ HSPC by electroporation or Ad5/ F35 adenoviral transduction were established, and enhanced gene modification rates of up to >80% were observed after HPRT disruption and 6TG selection in vitro. In on-going studies, the efficiency and safety of combined 6TG preconditioning and in vivo chemoselection are currently being tested for enhancing engraftment of genetically modified HPRT-deficient CD34+ HSPC in humanized mouse models.
715. KEL-Null Red Blood Cells Derived From In Vitro Differentiation of Zinc-Finger Nuclease-Edited HSCs
David F. Hui,1 Allen Zhu,1 Rajarajeswari Sivalenka,2 Nithianandan Selliah,2 Kristina Tess,2 Chang Li,2 Matt Mendel,1 Philip D. Gregory,1 Stewart Abbot,2 Xiaokui Zhang,2 Gregory J. Cost.1 1 Sangamo BioSciences, Richmond, CA; 2Celgene Cellular Therapeutics, Warren, NJ.
Transfusion of in vitro-differentiated red blood cells (RBC) manufactured from CD34-positive hematopoietic stem cells (HSC) promises to alleviate the immunological and logistical complications inherent to transfusion of donated whole blood. Adverse clinical outcomes associated with immune reactions to donor alloantigens (such as Kell) outside of the traditional ABO/Rhesus matching criteria are well documented. Manufacture of RBC ex vivo gives the opportunity to use HSC populations that have been selected or engineered to eliminate such antigens. We thus sought to eliminate the Kell antigen by disrupting the KEL gene in CD34+ HSCs to be used for ex vivo RBC manufacture. We first generated a zinc-finger nuclease (ZFN) pair that cleaved ~45% of KEL alleles in CD34+ HSCs. Treatment with these KEL-specific ZFNs did not alter the spectrum of HSC differentiation. We readily isolated KEL-null BFU-E cell clones from HSC pools differentiated in vitro on methylcellulose containing-plates. Finally, we transfected HSCs with the KEL ZFNs at large-scale and used the resulting cells in a scaled-down RBC manufacturing system in suspension flask cultures using protocols established as part of a DARPA-funded* Blood Pharming program. Evaluation of expansion rates indicated that KEL-null cells expanded as well as the mock transfected control population. Expression of the erythroid differentiation markers CD235a and CD71 as well as enucleation were evaluated by flow cytometry and revealed no significant differences between the mock transfected and Kellknockout cultures. We conclude that ZFN-mediated KEL inactivation in HSCs is compatible with subsequent RBC differentiation and manufacture and highlights the potential to further modify protein expression patterns in HSC-derived RBC populations. *Awards: FA9550-08-1-03-0392 & HR0011-12-2-0015.
716. Large-Scale Efficacy, Pharmacology and Toxicology Study of Human Parthenogenetic Stem Cell Derived Neural Stem Cells in Non-Human Primate Model of Parkinson’s Disease
Ruslan Semechkin,1 Rodolfo Gonzalez,1 Ibon Garitaonandia,1 Alina Ostrowska,1 Tatiana Abramihina,1 Gerald Wambua,1 Maxim Poustovoitov,1 Alexander Noskov,1 Andrew Crain,2 Caleb McEntire,3 Tiffany Chu,1 Louise Laurent,4 John Elsworth,3 Evan Snyder,2 D. Eugene Redmond.3 1 International Stem Cell Corporation, Carlsbad, CA; 2SanfordBurnham Medical Research Institute, La Jolla, CA; 3Yale University School of Medicine, New Haven, CT; 4University of California San Diego, San Diego, CA.
Cell based therapies hold great promise in the treatment of Parkinson’s disease. Clinical studies have shown that implantation of fetal neural tissue provides very long-term symptomatic relief in patients with PD without pharmacological dopaminergic therapy. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells (hpSCs) offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos, can be expanded indefinitely, and be made in an HLA homozygous manner, decreasing the chances of immune rejection. We have previously reported that human parthenogenetic stem cell derived neural stem cells (hpNSCs) survive long term, successfully engraft and increase brain dopamine levels in rodent and non-human primate models of PD. In the present comprehensive large-scale pharmacology, toxicology and efficacy study, hpNSCs were manufactured under cGMP conditions and injected bilaterally into the striatum and substantia nigra of immunosuppressed MPTP-lesioned African green monkeys with moderate to severe clinical Parkinsonian symptoms. Behavioral changes and motor movements were evaluated against sham vehicle control based on a Parkinsonian summary score. Additionally necropsy, histopathological analysis of multiple organs, and biodistribution of hpNSCs was performed to determine the safety profile of the implanted cells. Interim results show a reduction in Parkinson’s scores after implantation of hpNSCs and demonstrate the safety of the therapy and its potential clinical application in Parkinson’s disease.
717. Characterization and Enhancement of Transgene Expression From Mitotically Stable Non-Integrating Lentiviral Vector Episomes in Dividing Cells
Santhosh Chakkaramakkil Verghese,1,2 Natalya Goloviznina,1,2 Anupama Narla,3 Hans-Joachim Lipps,4 Peter Kurre.1,2 1 Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR; 2Department of Pediatrics, Oregon Health & Science University, Portland, OR; 3Department of Pediatrics, Stanford University School of Medicine, Stanford, CA; 4Department of Cell Biology, University of Witten/Herdecke, Witten, Germany. Non-integrating lentiviral vectors provide safety towards insertional mutagenesis and effectively mediate the gene delivery into non-dividing tissue targets. In dividing targets like hematopoietic stem cells (HSCs), the non-replicating vector genomes are successively lost with each cell division. We developed a mitotically stable (“anchored”) niLV by incorporating human ß-interferon derived Scaffold/Matrix Associated Regions (S/MARs) sequence in a 3rd generation lentivector (termed ‘aniLV’). Unique properties of lentivectors including active nuclear transport and circularization of vector DNA during abortive integration were harnessed in the aniLV vector design. The aniLV transduced actively dividing targets including human derived cell lines (293T and HS-5 cells) and murine
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Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Blanca Duarte,1 Francesca Miselli,2 Rodolfo Murillas,1 Alessandra Recchia,2 Marcela Del Río,1,3 Fernando Larcher.1,3 1 Epithelial Biomedicine, CIEMAT-CIBERER, Madrid, Spain; 2 Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 3Department of Bioengineering, University Carlos III de Madrid (UC3M), Madrid, Spain. Ex vivo gene therapy represents one of the various current strategies suitable to treat genodermatoses such as Epidermolysis Bullosa (EB).Efforts are being put on the development of safer and efficient approaches minimizing genotoxic effects due to transgene-induced insertional mutagenesis. Gene-editing through nuclease-facilitated homologous recombination (HR) has been recently proven feasible on skin cells. We have recently reported the ZFN-facilitated, HRmediated insertion of a marker gene into the AAVS1 locus in a limited number of human epidermal cells that, upon grafting, persisted as small foci in the skin regenerated in immunodeficient mice consistent with a stem cell behavior (Coluccio et al., 2013). However, since these foci comprised less than 1% of the total graft surface, the actual capacity of regeneration of a whole epidermal surface has not been proven. In this study we report the engraftment and persistent skin regeneration achieved with a single stem cells clone isolated from HR-mediated gene targeted human keratinocytes. GFP positive keratinocytes (less than 1%) present in culture after co-infection with IDLV-GFP and Ad-ZFN (designed for the AAVS1 locus) were sorted and expanded in culture. Although previous attempts of FACS-selecting viable cells containing less than 5% of GFP positive human keratinocytes failed, in this case supplementation of the culture medium with a ROCK inhibitor allowed the expansion of sorted keratinocytes displaying stem features. Only one of the very few GFP-positive keratinocyte colonies maintained an undifferentiated phenotype and grew larger than two-centimeters in diameter, consisting with the characteristics of a holoclone. The expanded population displayed a highly homogeneous morphology and a sharp peak of fluorescence. PCR analysis of DNA isolated from the clone showed bands consistent with “on target” integration (of the HR-cassette containing the GFP gene) on the AAVS1 locus. Expanded keratinocytes were used to prepare bioengineered skin equivalents for grafting to immunodeficient mice. Four weeks after grafting, human skin regeneration was confirmed through GFP epifluorescence. Histological analysis revealed the regeneration of a human skin with normal architecture and epidermal differentiation markers. Molecular analysis of regenerated skin biopsies is ongoing to ruling out selection of putative off-target or rearrangement events. Our results demonstrate that gene-edited epidermal stem cells preserve their plentiful regenerative capacity without harm and confirm that a strategy based on skin regeneration from very limited number of gene corrected stem cell clones ex vivo is achievable.
713. Fetal Renal Stem Cells Attenuate Ischemic Injury in the Rat Kidney Marina G. M. C. M. da Cunha,1 Silvia Zia,1 Fanny O. Arcolino,1 Elena Levtchenko,1 Jan Deprest,1 Jaan Toelen.1 1 Development and Regeneration, KULeuven, Leuven, Belgium.
Introduction: Despite extensive research into an effective treatment for acute renal injury (AKI), the mortality rate still remains high. Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, as a subtype of these cells is derived from the fetal renal tract. We hypothesize that hAFSCs delivered in rats with renal ischemia and reperfusion (I/R) injury, have a nephroprotective effect due to their immunomodulatory and anti-inflammatory effect, preventing extensive fibrosis as a late complication. Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Materials and Methods: In the first phase of this study, we isolated hAFSCs from human amniotic fluid samples and characterized their immunophenotypic and differentiation properties. Out of 120 clones a cell line with a high expression of CD24 and CD117 and the renal progenitor markers Six2 and Lin1 was selected. In the second phase, I/R injury was performed in thirty male 8week-old Wistar rats and treatment via direct arterial injection was performed 6 hours later. The control group (n=10) received 800ml of vehicle, the treated group (n =10) received 1x106 hAFSCs in 800ml of vehicle. Serum creatinine and proteinuria levels were measured at 24 hours, 48 hours and 2 months after I\R injury. Histological analysis was performed to quantify tubular necrosis and hyaline cast formation at 48 hours, and interstitial fibrosis by Masson’s Trichrome and PAS staining at 2 months. Additionally, the expression of Ki-67, ED-1 and α-SMA was assessed using immunohistochemistry at 48 hours. Results: A subtype of hAFSCs with renal marker expression has been isolated and characterized. These cells showed a nephroprotective effect in the rat ischemia model. There was a significant lower creatinine and proteinuria level at 24 and48 hours. We observed significantly less tubulointerstitial injury, which was characterized by a higher tubular proliferation, lower inflammatory response and an inhibition of myofibroblast expression. The treated group also showed less fibrosis at 2 months. Conclusion: We conclude a subset of cells from hAFSCs with renal phenotype is nephroprotective when delivered in rats with renal ischemia and reperfusion injury.
714. Combined Preconditioning and In Vivo Chemoselection With 6-Thioguanine for Selection of Genetically Modified Hematopoietic Stem Cells
Katrin Hacke,1 Janet A. Treger,1 Brooke T. Bogan,1 Valerie Rezek,1 Munetoshi Narukawa,2 Saki Shimizu,2 Pei-Qi Liu,3 Andreas Reik,3 André M. Lieber,4 Gay M. Crooks,5 Donald B. Kohn,6 Dong Sung An,2 Scott G. Kitchen,1 Philip D. Gregory,3 Gregory J. Cost,3 Michael C. Holmes,3 Noriyuki Kasahara.1 1 Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles; 2School of Nursing, University of California, Los Angeles; 3Sangamo BioSciences, Richmond; 4Division of Medical Genetics, Department of Medicine, University of Washington, Seattle; 5Department of Pathology & Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles; 6Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles. We have recently developed a novel and highly efficient strategy that uses the purine analog 6-thioguanine (6TG) as a single agent for both preconditioning and in vivo chemoselection of hypoxanthine/ guanine-phosphoribosyltransferase (HPRT)-deficient hematopoietic stem and progenitor cells (HSPC). In a murine model of bone marrow (BM) transplantation, combined 6TG conditioning and post-transplantation chemoselection consistently achieved ~95% engraftment of HPRT-deficient donor BM in the absence of any other cytotoxic conditioning regimen. Moreover, long-term reconstitution of histologically and immunophenotypically normal hematopoiesis was observed in both primary and secondary recipients, without significant toxicity. To translate this strategy for combined 6TG preconditioning and chemoselection into a clinically feasible approach, we are testing two different approaches for genetic modification of normal HSPC to render them HPRT-deficient and thus 6TG-resistant: (1) We have constructed and validated a lentiviral vector expressing short-hairpin RNA (shRNA) that targets a consensus sequence present in both human and murine HPRT genes, and which achieves effective longterm reduction of HPRT expression in cells from either species in vitro. In both primary murine BM cells and human CD34+ HSPC S275
SOMATIC STEM CELLS lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Transplantation of vector-transduced HPRT-knockdown CD34+ HSPC into irradiated NOD scid gamma (NSG) mice resulted in stable and long-term reconstitution with genetically modified cells indicating no adverse effects of the HPRT knockdown on CD34+ HSPC engraftment. (2) We have further employed engineered zinc finger nucleases (ZFNs) to target the human HPRT locus for gene disruption. Protocols for HPRT-ZFN delivery into CD34+ HSPC by electroporation or Ad5/ F35 adenoviral transduction were established, and enhanced gene modification rates of up to >80% were observed after HPRT disruption and 6TG selection in vitro. In on-going studies, the efficiency and safety of combined 6TG preconditioning and in vivo chemoselection are currently being tested for enhancing engraftment of genetically modified HPRT-deficient CD34+ HSPC in humanized mouse models.
715. KEL-Null Red Blood Cells Derived From In Vitro Differentiation of Zinc-Finger Nuclease-Edited HSCs
David F. Hui,1 Allen Zhu,1 Rajarajeswari Sivalenka,2 Nithianandan Selliah,2 Kristina Tess,2 Chang Li,2 Matt Mendel,1 Philip D. Gregory,1 Stewart Abbot,2 Xiaokui Zhang,2 Gregory J. Cost.1 1 Sangamo BioSciences, Richmond, CA; 2Celgene Cellular Therapeutics, Warren, NJ.
Transfusion of in vitro-differentiated red blood cells (RBC) manufactured from CD34-positive hematopoietic stem cells (HSC) promises to alleviate the immunological and logistical complications inherent to transfusion of donated whole blood. Adverse clinical outcomes associated with immune reactions to donor alloantigens (such as Kell) outside of the traditional ABO/Rhesus matching criteria are well documented. Manufacture of RBC ex vivo gives the opportunity to use HSC populations that have been selected or engineered to eliminate such antigens. We thus sought to eliminate the Kell antigen by disrupting the KEL gene in CD34+ HSCs to be used for ex vivo RBC manufacture. We first generated a zinc-finger nuclease (ZFN) pair that cleaved ~45% of KEL alleles in CD34+ HSCs. Treatment with these KEL-specific ZFNs did not alter the spectrum of HSC differentiation. We readily isolated KEL-null BFU-E cell clones from HSC pools differentiated in vitro on methylcellulose containing-plates. Finally, we transfected HSCs with the KEL ZFNs at large-scale and used the resulting cells in a scaled-down RBC manufacturing system in suspension flask cultures using protocols established as part of a DARPA-funded* Blood Pharming program. Evaluation of expansion rates indicated that KEL-null cells expanded as well as the mock transfected control population. Expression of the erythroid differentiation markers CD235a and CD71 as well as enucleation were evaluated by flow cytometry and revealed no significant differences between the mock transfected and Kellknockout cultures. We conclude that ZFN-mediated KEL inactivation in HSCs is compatible with subsequent RBC differentiation and manufacture and highlights the potential to further modify protein expression patterns in HSC-derived RBC populations. *Awards: FA9550-08-1-03-0392 & HR0011-12-2-0015.
716. Large-Scale Efficacy, Pharmacology and Toxicology Study of Human Parthenogenetic Stem Cell Derived Neural Stem Cells in Non-Human Primate Model of Parkinson’s Disease
Ruslan Semechkin,1 Rodolfo Gonzalez,1 Ibon Garitaonandia,1 Alina Ostrowska,1 Tatiana Abramihina,1 Gerald Wambua,1 Maxim Poustovoitov,1 Alexander Noskov,1 Andrew Crain,2 Caleb McEntire,3 Tiffany Chu,1 Louise Laurent,4 John Elsworth,3 Evan Snyder,2 D. Eugene Redmond.3 1 International Stem Cell Corporation, Carlsbad, CA; 2SanfordBurnham Medical Research Institute, La Jolla, CA; 3Yale University School of Medicine, New Haven, CT; 4University of California San Diego, San Diego, CA.
Cell based therapies hold great promise in the treatment of Parkinson’s disease. Clinical studies have shown that implantation of fetal neural tissue provides very long-term symptomatic relief in patients with PD without pharmacological dopaminergic therapy. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells (hpSCs) offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos, can be expanded indefinitely, and be made in an HLA homozygous manner, decreasing the chances of immune rejection. We have previously reported that human parthenogenetic stem cell derived neural stem cells (hpNSCs) survive long term, successfully engraft and increase brain dopamine levels in rodent and non-human primate models of PD. In the present comprehensive large-scale pharmacology, toxicology and efficacy study, hpNSCs were manufactured under cGMP conditions and injected bilaterally into the striatum and substantia nigra of immunosuppressed MPTP-lesioned African green monkeys with moderate to severe clinical Parkinsonian symptoms. Behavioral changes and motor movements were evaluated against sham vehicle control based on a Parkinsonian summary score. Additionally necropsy, histopathological analysis of multiple organs, and biodistribution of hpNSCs was performed to determine the safety profile of the implanted cells. Interim results show a reduction in Parkinson’s scores after implantation of hpNSCs and demonstrate the safety of the therapy and its potential clinical application in Parkinson’s disease.
717. Characterization and Enhancement of Transgene Expression From Mitotically Stable Non-Integrating Lentiviral Vector Episomes in Dividing Cells
Santhosh Chakkaramakkil Verghese,1,2 Natalya Goloviznina,1,2 Anupama Narla,3 Hans-Joachim Lipps,4 Peter Kurre.1,2 1 Papé Family Pediatric Research Institute, Oregon Health & Science University, Portland, OR; 2Department of Pediatrics, Oregon Health & Science University, Portland, OR; 3Department of Pediatrics, Stanford University School of Medicine, Stanford, CA; 4Department of Cell Biology, University of Witten/Herdecke, Witten, Germany. Non-integrating lentiviral vectors provide safety towards insertional mutagenesis and effectively mediate the gene delivery into non-dividing tissue targets. In dividing targets like hematopoietic stem cells (HSCs), the non-replicating vector genomes are successively lost with each cell division. We developed a mitotically stable (“anchored”) niLV by incorporating human ß-interferon derived Scaffold/Matrix Associated Regions (S/MARs) sequence in a 3rd generation lentivector (termed ‘aniLV’). Unique properties of lentivectors including active nuclear transport and circularization of vector DNA during abortive integration were harnessed in the aniLV vector design. The aniLV transduced actively dividing targets including human derived cell lines (293T and HS-5 cells) and murine
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Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy