- Email: [email protected]
716. Optimisation of Lentiviral Transduction Conditions of Haematopoietic Stem Cells for Treatment of Mucopolysaccharidosis Type IIIA (MPSIIIA)
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY III may require only a low level of normal or gene-corrected cells for a permanent and therapeutic outcome.
715. Lentiviral Transduction of MPS1 Bone Marrow Cells Results in High Production of alphaL-Iduronidase In Vivo
Henk Rozemuller,1 Petra Moerer,2 Niek P. van Til,3 JaapJan Boelens,1 Tom J. de Koning,4 Anton C. Martens,2 Gerard Wagemaker,3 Nico M. Wulffraat.1 1 Pediatrics, University Medical Center, Utrecht, Netherlands; 2 Immunology, University Medical Center, Utrecht, Netherlands; 3 Hematology, Erasmus University Medical Center, Utrecht, Netherlands; 4Metabolic Diseases, University Medical Center, Utrecht, Netherlands.
Mucopolysaccharidosis type I (MPS1, Hurler syndrome) is caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA) and is characterized by glycosaminoglycan (GAG) accumulation causing multi-organ failure. Although enzyme replacement therapy or hematopoietic stem cell (HSC) transplantation constitutes significant improvement of disease phenotype, there is still a lack of effect of those therapies on the central nervous system and the skeleton. Aim of this study is to obtain improved correction of disease in MPS1 mice, including central nervous system and skeletal defects, by overexpression of IDUA in hematopoietic cells. Lentiviral vectors with the spleen focus forming virus- (SF) and phosphoglycerate kinase- (PGK) promoters driving codon optimized human IDUA cDNA (IDUAco) expression were constructed. These vectors were used to transduce male lineage-negative (Lin-) MPS1 bone marrow cells with an average of 1 to 2 vector copies per cell, which was confirmed by quantitative PCR (Q-PCR). Transduced bone marrow cells were subsequently transplanted in 3-week-old 6 Gy irradiated MPS1 female mice. Since matched donor HSC transplantation in MPSI patients is currently the preferred treatment, control MPSI mice were transplanted with syngeneic wildtype Lin- cells. Blood and urine samples were collected monthly. On average 80% male cell chimerism was observed in peripheral blood containing 30% IDUA positive cells up to 7 months after transplantation. IDUA levels in blood were 500- and 80-fold of normal wild type levels for the SF-IDUAco and PGK-IDUAco, respectively, or normalised in wildtype bone marrow transplanted MPS1 mice. Analysis of GAG secretion in urine revealed normalization to wild type levels for all transplantation groups. At month 7, the mice were sacrificed and assessment of GAG levels in spleen, liver, lung, heart, and kidney demonstrated significant reduction of GAGs to normal levels. Most importantly, GAG accumulation was significantly decreased in brain in accordance with high levels of IDUA. CT analysis of the skeleton revealed significant improvement of the bone thickness. We conclude that the IDUAco vectors provide long-term expression of high levels of IDUA, resulting in concomitant reduction in GAG secretion in urine, reduction of GAG in other organs, including brain, and in a significant improvement of the skeletal deformations. This warrants further development towards clinical application.
716. Optimisation of Lentiviral Transduction Conditions of Haematopoietic Stem Cells for Treatment of Mucopolysaccharidosis Type IIIA (MPSIIIA)
Ana Sergijenko,1 Alex Langford-Smith,1 Kia Langford-Smith,1 Rob Wynn,2 J. Ed Wraith,3 Simon Jones,2 Fiona Wilkinson,1 Brian Bigger.1 1 University of Manchester, Manchester, United Kingdom; 2Royal Manchester Children’s Hospital, Manchester, United Kingdom; 3St. Mary’s Hospital, Manchester, United Kingdom. MPSIIIA is caused by a mutation in the SGSH gene, leading to disruption of the substrate degradation pathway in lysosomes and a phenotype of severe neurodegeneration. We have shown that lentiviral gene therapy using transplanted haematopoietic stem cells (HSCs) corrects the neurological phenotype in a mouse model of MPSIIIA. The aims of this project are to generate a clinically applicable vector, develop a more efficient transduction protocol for HSCs, and assess vector safety. Lentivirus production was optimised to reduce production cost by using less DNA (10.5 ug/ plate) and a PEI transfection reagent. Transduction of HSCs was also optimised. The addition of MG132 with cytokines and BSA improved transduction by 10%, and addition of valproic acid (VA) increased stem cell numbers by 35%. High titres (2x108 IU/ml) of three lentiviral vectors encoding GFP gene driven by a ubiquitous (PGK) or monocyte specific (CD11b and CD18) human promoters were made and tested in vitro in a number of cell lines, and then in vivo, by transplanting transduced lineage depleted cells into wild type mice. Flow cytometry showed that over 80% of donor cells were transduced. Blood and bone marrow cells were further stained for flow cytometry to assess promoter specificity. Improved SGSH encoding cDNAs were made. It was shown that HSCs transduced with GFP encoding virus had 3 fold more copy numbers than cells transduced with SGSH-long (cDNA + 3’ and 5’ UTRs). The copy number was increased when using a codon-optimised SGSH (SGSH-CO) (cDNA only) and remained high when a stop mutated SGSH-CO-X was used, suggesting that vector size, not SGSH toxicity is important in HSC transduction. SGSH-CO produced more SGSH activity than the non-codon-optimised SGSH. We are currently assessing PGK-SGSH-CO, CD11b-SGSH-CO, and CD18-SGSH-CO in a clinically relevant lentiviral backbone in MPSIIIA mice, with 6 and 12 month follow up to assess therapy and safety. We have shown that addition of MG132 and VA to HSCs increased stem cell numbers and percentage of transduced HSCs. We have also shown that vector size is important in HSC transduction, that SGSH activity is increased by codon optimisation, and therapy is more specific when using cell lineage promoters. This is a promising clinical therapy for MPSIIIA.
717. Disease Improvement with Neonatal Intracranial AAV and Systemic Lentiviral Gene Therapy in Sanfilippo Syndrome Type B Mice
Coy D. Heldermon,1 Elizabeth Qin,2 Kevin K. Ohlemiller,3 Erik D. Herzog,4 Carole Vogler,5 Mark S. Sands.2 1 Medicine, University of Florida, Gainesville, FL; 2Internal Medicine, Washington University in St. Louis, St. Louis, MO; 3 Otolaryngology, Washington University in St. Louis, St. Louis, MO; 4Biology, Washington University in St. Louis, St. Louis, MO; 5 Pathology, St. Louis University, St. Louis, MO. Sanfilippo Syndrome type B (MPS IIIB) is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU). We have previously shown that intracranial AAVbased gene therapy improves several aspects of the disease such as lifespan. In an attempt to correct the disease in the mouse model, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (AAV), intravenous lentiviral-NAGLU (MND)
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
S273
715. Lentiviral Transduction of MPS1 Bone Marrow Cells Results in High Production of alphaL-Iduronidase In Vivo
Henk Rozemuller,1 Petra Moerer,2 Niek P. van Til,3 JaapJan Boelens,1 Tom J. de Koning,4 Anton C. Martens,2 Gerard Wagemaker,3 Nico M. Wulffraat.1 1 Pediatrics, University Medical Center, Utrecht, Netherlands; 2 Immunology, University Medical Center, Utrecht, Netherlands; 3 Hematology, Erasmus University Medical Center, Utrecht, Netherlands; 4Metabolic Diseases, University Medical Center, Utrecht, Netherlands.
Mucopolysaccharidosis type I (MPS1, Hurler syndrome) is caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA) and is characterized by glycosaminoglycan (GAG) accumulation causing multi-organ failure. Although enzyme replacement therapy or hematopoietic stem cell (HSC) transplantation constitutes significant improvement of disease phenotype, there is still a lack of effect of those therapies on the central nervous system and the skeleton. Aim of this study is to obtain improved correction of disease in MPS1 mice, including central nervous system and skeletal defects, by overexpression of IDUA in hematopoietic cells. Lentiviral vectors with the spleen focus forming virus- (SF) and phosphoglycerate kinase- (PGK) promoters driving codon optimized human IDUA cDNA (IDUAco) expression were constructed. These vectors were used to transduce male lineage-negative (Lin-) MPS1 bone marrow cells with an average of 1 to 2 vector copies per cell, which was confirmed by quantitative PCR (Q-PCR). Transduced bone marrow cells were subsequently transplanted in 3-week-old 6 Gy irradiated MPS1 female mice. Since matched donor HSC transplantation in MPSI patients is currently the preferred treatment, control MPSI mice were transplanted with syngeneic wildtype Lin- cells. Blood and urine samples were collected monthly. On average 80% male cell chimerism was observed in peripheral blood containing 30% IDUA positive cells up to 7 months after transplantation. IDUA levels in blood were 500- and 80-fold of normal wild type levels for the SF-IDUAco and PGK-IDUAco, respectively, or normalised in wildtype bone marrow transplanted MPS1 mice. Analysis of GAG secretion in urine revealed normalization to wild type levels for all transplantation groups. At month 7, the mice were sacrificed and assessment of GAG levels in spleen, liver, lung, heart, and kidney demonstrated significant reduction of GAGs to normal levels. Most importantly, GAG accumulation was significantly decreased in brain in accordance with high levels of IDUA. CT analysis of the skeleton revealed significant improvement of the bone thickness. We conclude that the IDUAco vectors provide long-term expression of high levels of IDUA, resulting in concomitant reduction in GAG secretion in urine, reduction of GAG in other organs, including brain, and in a significant improvement of the skeletal deformations. This warrants further development towards clinical application.
716. Optimisation of Lentiviral Transduction Conditions of Haematopoietic Stem Cells for Treatment of Mucopolysaccharidosis Type IIIA (MPSIIIA)
Ana Sergijenko,1 Alex Langford-Smith,1 Kia Langford-Smith,1 Rob Wynn,2 J. Ed Wraith,3 Simon Jones,2 Fiona Wilkinson,1 Brian Bigger.1 1 University of Manchester, Manchester, United Kingdom; 2Royal Manchester Children’s Hospital, Manchester, United Kingdom; 3St. Mary’s Hospital, Manchester, United Kingdom. MPSIIIA is caused by a mutation in the SGSH gene, leading to disruption of the substrate degradation pathway in lysosomes and a phenotype of severe neurodegeneration. We have shown that lentiviral gene therapy using transplanted haematopoietic stem cells (HSCs) corrects the neurological phenotype in a mouse model of MPSIIIA. The aims of this project are to generate a clinically applicable vector, develop a more efficient transduction protocol for HSCs, and assess vector safety. Lentivirus production was optimised to reduce production cost by using less DNA (10.5 ug/ plate) and a PEI transfection reagent. Transduction of HSCs was also optimised. The addition of MG132 with cytokines and BSA improved transduction by 10%, and addition of valproic acid (VA) increased stem cell numbers by 35%. High titres (2x108 IU/ml) of three lentiviral vectors encoding GFP gene driven by a ubiquitous (PGK) or monocyte specific (CD11b and CD18) human promoters were made and tested in vitro in a number of cell lines, and then in vivo, by transplanting transduced lineage depleted cells into wild type mice. Flow cytometry showed that over 80% of donor cells were transduced. Blood and bone marrow cells were further stained for flow cytometry to assess promoter specificity. Improved SGSH encoding cDNAs were made. It was shown that HSCs transduced with GFP encoding virus had 3 fold more copy numbers than cells transduced with SGSH-long (cDNA + 3’ and 5’ UTRs). The copy number was increased when using a codon-optimised SGSH (SGSH-CO) (cDNA only) and remained high when a stop mutated SGSH-CO-X was used, suggesting that vector size, not SGSH toxicity is important in HSC transduction. SGSH-CO produced more SGSH activity than the non-codon-optimised SGSH. We are currently assessing PGK-SGSH-CO, CD11b-SGSH-CO, and CD18-SGSH-CO in a clinically relevant lentiviral backbone in MPSIIIA mice, with 6 and 12 month follow up to assess therapy and safety. We have shown that addition of MG132 and VA to HSCs increased stem cell numbers and percentage of transduced HSCs. We have also shown that vector size is important in HSC transduction, that SGSH activity is increased by codon optimisation, and therapy is more specific when using cell lineage promoters. This is a promising clinical therapy for MPSIIIA.
717. Disease Improvement with Neonatal Intracranial AAV and Systemic Lentiviral Gene Therapy in Sanfilippo Syndrome Type B Mice
Coy D. Heldermon,1 Elizabeth Qin,2 Kevin K. Ohlemiller,3 Erik D. Herzog,4 Carole Vogler,5 Mark S. Sands.2 1 Medicine, University of Florida, Gainesville, FL; 2Internal Medicine, Washington University in St. Louis, St. Louis, MO; 3 Otolaryngology, Washington University in St. Louis, St. Louis, MO; 4Biology, Washington University in St. Louis, St. Louis, MO; 5 Pathology, St. Louis University, St. Louis, MO. Sanfilippo Syndrome type B (MPS IIIB) is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU). We have previously shown that intracranial AAVbased gene therapy improves several aspects of the disease such as lifespan. In an attempt to correct the disease in the mouse model, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (AAV), intravenous lentiviral-NAGLU (MND)
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
S273