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208. Hematopoetic Transduction In Vivo by Intraveneous Retroviral Gene Therapy in Neonatal MPS VII Dogs
GENETIC AND METABOLIC DISEASES: PART ONE and Flt-3L was used. Thirteen NOD/SCID/MPSVII mice were transplanted with CD34+ cells transduced with GUSB vector, 4 mice received cells transduced with an analogous EGFP-expressing lentiviral vector, and 5 mice were transplanted with mock transduced cells. After 6 days in liquid culture, 22% of cells exposed to GUSB vector expressed enzyme. This bulk culture had 1204 ± 16 U of GUSB specific activity (nmol substrate/hour/mg protein) compared to 16 ± 0.2 U in the mock transduced liquid culture. Treated NOD/ SCID/MPSVII mice will be analyzed at twelve weeks following transplantation. A fluorogenic substrate specific for GUSB will be used to measure the percentage of corrected cells in multiple hematopoietic lineages by flow cytometry. Based on previous data from our laboratory, stable in vivo marking at a level comparable to that seen in vitro is anticipated. In addition, a broad distribution of GUSB-expressing cells and widespread reversal of disease pathology is expected. Results from this study will provide critical pre-clinical data concerning the correction of diseased human HPCs and their therapeutic effect in vivo.
WBCs difficult to evaluate by this method. However, WBC GUSB activity was not proportional to serum activity. The decrease in typical severe cytoplasmic granulation, seen in 87-95% of WBCs in untreated MPS VII-affected dogs, to 25-52% in the 3 non-HGFtreated dogs, and to 0% in the HGF-treated dog was likely to cross correction from serum GUSB. Current efforts include evaluating in vivo expression and the identification of integration sites by linear amplification-mediated PCR to compare to ex vivo, cytokine-stimulated CD34+ protocols that have recently shown insertional mutatgenesis in children with X-SCID. Supported by NIH grants DK54481, RR02512, DK48028, RR T32-07063
208. Hematopoetic Transduction In Vivo by Intraveneous Retroviral Gene Therapy in Neonatal MPS VII Dogs
The long-term goal of this study is to determine if transduction of CD34+ canine marrow cells with FVIII encoding HIV-1 vector will result in the appearance of therapeutic levels of FVIII in the plasma of normal and hemophilic dogs. As a first step towards this goal, we have created HIV-1 vectors that encode the B-domain-deleted human or canine FVIII under control of mouse phosphoglycerate kinase (PGK) or elongation factor 1 alpha (EF1a ) promoter-enhancer elements. The woodchuck post-transcriptional regulatory element (WPRE) was inserted downstream of the transgene to enhance FVIII production. High-titered (2 x 107 IU/ml), vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped virus stocks were generated by multi-plasmid transient transfection of 293T cells and used for infection of naïve 293T cells. The supernatants from the transduced cells were tested for secretion of FVIII by ELISA (for human FVIII) and COATest Factor VIII chromogenic assay (for functional canine and human FVIII). The results indicated that high levels, up to 234,000 milliUnits/ml of functional FVIII were produced by both canine and human FVIII encoding vectors. The human FVIII encoding vectors were also tested in primary canine marrow mononuclear cells (MNC). The supernatants of transduced cultures showed detectable (~28 milliUnits/ml) accumulation of human FVIII by ELISA. Canine FVIII encoding vectors were used for transduction of CD34+ canine marrow cells. The cells were prestimulated with a combination of human IL-1, IL-3, IL-6, FLT3-ligand and canine stem cell factor for 48 h, and then transduced with canine FVIII expressing HIV-1 vector by spinoculation on fibronectin fragment (CH296)-coated plates. Transductions were repeated three times, approximately every 12 hours. The transduced cells were transplanted into the marrow donor preconditioned by total body irradiation with 200 cGy. Two dogs were entered into this study. Genomic DNA from peripheral blood granulocytes and lymphocytes as well as marrow MNC were tested for the presence of HIV-1 vector sequences. In the first animal, HIV-1 vector sequences were detected in both granulocytes and lymphocytes as well as marrow MNC for 5 weeks. The results of further follow up of the transplanted animals will be presented.
Thomas O’Malley,1 Paula Henthorn,1 Lingfei Xu,2 N. Matthew Ellinwood,1 Hamutal Mazier,1 Katherine Parker Ponder,2 Mark Haskins.1 1 Pathobiology and Clinical Studies, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, United States; 2 Internal Medicine, Washington University School of Medicine, St. Louis, MO, United States. Mucopolysaccharidosis (MPS) VII is caused by deficient activity of beta-glucuronidase (GUSB). This enzyme is responsible for the catabolism of glycosaminoglycans and its deficiency leads to storage of these substrates. In a liver-targeted therapy, three dogs were treated on day 2-3 of life with 3.1x10E9 infectious units (IU)/kg of a Moloney murine leukemia based vector containing the human alpha-1-antitrypsin promoter, canine GUSB cDNA, and woodchuck post transcriptional regulatory element. Another neonatal MPS VII dog received 2.5mg/kg of human hepatocyte growth factor (HGF) prior to the injection of 12x10E9 IU/kg of RV. The dogs have been followed for 2+ years. The serum GUSB activity has been constant at 29-131% (74-335 nmole (nm)/h/ml) of normal (255 nm/h/ml) for the non-HGF dogs and 5200% (13,321 nm/h/ml) for the HGF dog. The purpose of this study was to evaluate peripheral blood for proviral sequence by PCR. This was done at 6 months posttreatment for the non-HGF dogs and 1 year for the HGF dog. All animals were positive for proviral sequence, which has remained stable throughout the study. The copy number is estimated to be 0.5% and appears uniform in all four dogs. A fifth dog, recently treated with14X10E9 IU/kg of vector had provirus detected in the blood at 23 days. At the age of treatment, the liver is still a hematopoetic organ in the dog. Therefore, it is possible that transduction of hematopoetic cells occurred in the liver rather than the bone marrow, although the latter cannot be discounted. The vector has been shown in vitro to over-express GUSB in an early monocytic canine cell line despite carrying a liver-specific promoter. Thus, it is possible that hematopoietic cells overexpress GUSB in vivo, and by migrating from the circulation into hard to reach target tissues such as joints and the brain, may facilitate clinical improvement by cross-correction of non-transduced cells through uptake of secreted enzyme by the mannos-6-phosphate receptor. GUSB activity in white blood cells (WBCs) was 2.3-4.1% (15-26 nm/h/mg) of normal (648 nm/h/mg) in the non-HGF dogs and 7.2% (47 nm/h/mg) of normal in the HGF-treated dog. Cross-correction by uptake of GUSB from the serum by mannose-6-phosphate receptors in the plasma membrane of cells makes expression within S82
209. Marrow Stem Cell Gene Therapy for Canine Hemophilia A Narasimhachar Srinivasakumar,1 Michail M. Zaboikin,1 Tatiana N. Zaboikina,1 Friedrich G. Schuening.1 1 Department of Medicine, Vanderbilt University, Nashville, TN.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
WBCs difficult to evaluate by this method. However, WBC GUSB activity was not proportional to serum activity. The decrease in typical severe cytoplasmic granulation, seen in 87-95% of WBCs in untreated MPS VII-affected dogs, to 25-52% in the 3 non-HGFtreated dogs, and to 0% in the HGF-treated dog was likely to cross correction from serum GUSB. Current efforts include evaluating in vivo expression and the identification of integration sites by linear amplification-mediated PCR to compare to ex vivo, cytokine-stimulated CD34+ protocols that have recently shown insertional mutatgenesis in children with X-SCID. Supported by NIH grants DK54481, RR02512, DK48028, RR T32-07063
208. Hematopoetic Transduction In Vivo by Intraveneous Retroviral Gene Therapy in Neonatal MPS VII Dogs
The long-term goal of this study is to determine if transduction of CD34+ canine marrow cells with FVIII encoding HIV-1 vector will result in the appearance of therapeutic levels of FVIII in the plasma of normal and hemophilic dogs. As a first step towards this goal, we have created HIV-1 vectors that encode the B-domain-deleted human or canine FVIII under control of mouse phosphoglycerate kinase (PGK) or elongation factor 1 alpha (EF1a ) promoter-enhancer elements. The woodchuck post-transcriptional regulatory element (WPRE) was inserted downstream of the transgene to enhance FVIII production. High-titered (2 x 107 IU/ml), vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped virus stocks were generated by multi-plasmid transient transfection of 293T cells and used for infection of naïve 293T cells. The supernatants from the transduced cells were tested for secretion of FVIII by ELISA (for human FVIII) and COATest Factor VIII chromogenic assay (for functional canine and human FVIII). The results indicated that high levels, up to 234,000 milliUnits/ml of functional FVIII were produced by both canine and human FVIII encoding vectors. The human FVIII encoding vectors were also tested in primary canine marrow mononuclear cells (MNC). The supernatants of transduced cultures showed detectable (~28 milliUnits/ml) accumulation of human FVIII by ELISA. Canine FVIII encoding vectors were used for transduction of CD34+ canine marrow cells. The cells were prestimulated with a combination of human IL-1, IL-3, IL-6, FLT3-ligand and canine stem cell factor for 48 h, and then transduced with canine FVIII expressing HIV-1 vector by spinoculation on fibronectin fragment (CH296)-coated plates. Transductions were repeated three times, approximately every 12 hours. The transduced cells were transplanted into the marrow donor preconditioned by total body irradiation with 200 cGy. Two dogs were entered into this study. Genomic DNA from peripheral blood granulocytes and lymphocytes as well as marrow MNC were tested for the presence of HIV-1 vector sequences. In the first animal, HIV-1 vector sequences were detected in both granulocytes and lymphocytes as well as marrow MNC for 5 weeks. The results of further follow up of the transplanted animals will be presented.
Thomas O’Malley,1 Paula Henthorn,1 Lingfei Xu,2 N. Matthew Ellinwood,1 Hamutal Mazier,1 Katherine Parker Ponder,2 Mark Haskins.1 1 Pathobiology and Clinical Studies, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, United States; 2 Internal Medicine, Washington University School of Medicine, St. Louis, MO, United States. Mucopolysaccharidosis (MPS) VII is caused by deficient activity of beta-glucuronidase (GUSB). This enzyme is responsible for the catabolism of glycosaminoglycans and its deficiency leads to storage of these substrates. In a liver-targeted therapy, three dogs were treated on day 2-3 of life with 3.1x10E9 infectious units (IU)/kg of a Moloney murine leukemia based vector containing the human alpha-1-antitrypsin promoter, canine GUSB cDNA, and woodchuck post transcriptional regulatory element. Another neonatal MPS VII dog received 2.5mg/kg of human hepatocyte growth factor (HGF) prior to the injection of 12x10E9 IU/kg of RV. The dogs have been followed for 2+ years. The serum GUSB activity has been constant at 29-131% (74-335 nmole (nm)/h/ml) of normal (255 nm/h/ml) for the non-HGF dogs and 5200% (13,321 nm/h/ml) for the HGF dog. The purpose of this study was to evaluate peripheral blood for proviral sequence by PCR. This was done at 6 months posttreatment for the non-HGF dogs and 1 year for the HGF dog. All animals were positive for proviral sequence, which has remained stable throughout the study. The copy number is estimated to be 0.5% and appears uniform in all four dogs. A fifth dog, recently treated with14X10E9 IU/kg of vector had provirus detected in the blood at 23 days. At the age of treatment, the liver is still a hematopoetic organ in the dog. Therefore, it is possible that transduction of hematopoetic cells occurred in the liver rather than the bone marrow, although the latter cannot be discounted. The vector has been shown in vitro to over-express GUSB in an early monocytic canine cell line despite carrying a liver-specific promoter. Thus, it is possible that hematopoietic cells overexpress GUSB in vivo, and by migrating from the circulation into hard to reach target tissues such as joints and the brain, may facilitate clinical improvement by cross-correction of non-transduced cells through uptake of secreted enzyme by the mannos-6-phosphate receptor. GUSB activity in white blood cells (WBCs) was 2.3-4.1% (15-26 nm/h/mg) of normal (648 nm/h/mg) in the non-HGF dogs and 7.2% (47 nm/h/mg) of normal in the HGF-treated dog. Cross-correction by uptake of GUSB from the serum by mannose-6-phosphate receptors in the plasma membrane of cells makes expression within S82
209. Marrow Stem Cell Gene Therapy for Canine Hemophilia A Narasimhachar Srinivasakumar,1 Michail M. Zaboikin,1 Tatiana N. Zaboikina,1 Friedrich G. Schuening.1 1 Department of Medicine, Vanderbilt University, Nashville, TN.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy