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222. Lentiviral Vector-Based Ex Vivo Gene Therapy Approach for Metachromatic (MLD) and Globoid (GLD) Leukodystrophies
GENETIC AND METABOLIC DISEASES: PART ONE observed up to day 222. Vectors expressing canine FVIII from the CAG and ET promoters were generated, and are currently being tested in normal and Hemophilia A dogs. I am an employee and stock holder in Cell Genesys.
damage in both mouse models are ongoing. Efficacy evaluations are based on neurophysiological, comportamental and neuromotor studies in MLD mice and on monitoring survival in Twitcher mice.
GENE THERAPY FOR THE NERVOUS SYSTEM I
223. Genetic Interventions To Decrease Neurological Injury In Vivo
222. Lentiviral Vector-Based Ex Vivo Gene Therapy Approach for Metachromatic (MLD) and Globoid (GLD) Leukodystrophies
William O. Ogle,1 Daniela Kaufer,1 Zachary S. Pincus,1 Theodore C. Dumas,1 Klaus M. Dinkel,3 Deveroux Ferguson,2 Kelsey L. Clark,1 Robert M. Sapolsky.1 1 Biological Sciences, Stanford University, Stanford, CA; 2 Psychiatry, Stanford University, Stanford, CA; 3Leibniz Institute for Neurobiology, Magdeburg, Germany.
Alessandra Biffi,1 Michele De Palma,2 Angelo Quattrini,3 Ilaria Visigalli,1 Diego Dolcetta,1 Claudio Bordignon,1 Luigi Naldini.1,2 1 San Raffaele-Telethon Institute for Gene Therapy (H.S.R.TIGET), Scientific Institute H.S. Raffaele, Milano, Italy; 2 Laboratory for Gene Transfer and Therapy, Institute for Cancer Research, University of Torino, Candiolo, Italy; 3Neuropathology Unit, Scientific Institute H.S. Raffaele, Milano, Italy. MLD and GLD are inborn lipidosis caused by the deficiency of the lysosomal enzymes arylsulfatase A (ARSA) and galactocerebrosidase (GALC), which result in storage of undegraded sulphatides in the CNS and PNS, leading to progressive demyelination. BMT provides a clinically feasible method for permanently replacing deficient lysosomal enzyme activity. Donor derived cells migrate into disease target organs and become a local and permanent source of the missing enzyme. We are evaluating an ex vivo gene therapy approach for MLD and GLD based on transplantation of LV-transduced autologous HSC. We first investigated the timing and extent of migration of donor derived cells into the nervous system of wild type mice. We transduced HSC, enriched from the total BM by lineage negative (Lin-) selection, by a short ex vivo incubation with advanced generation LV expressing GFP from the hPGK promoter and injected the cells into lethally irradiated mice. Three months after the transplant FACS analysis of blood samples demonstrated high engraftment of GFP expressing cells (62-95%, n=16) in the different hematopoietic lineages. The BM of primary recipients was used to perform secondary transplants. Multi-lineage transgene expressing cells fully engrafted the secondary hosts, indicating transduction of primitive long-term repopulating cells and stable transgene expression in their progeny for up to 12 months. Transplanted mice were sacrificed at 3, 6 and 9 months after BMT to study the migration of BM-derived transduced cells. We detected a significant migration of transgene expressing cells into CNS and PNS that increased progressively with longer times after BMT. Co-localization studies showed that transduced cells contributed to the turnover of the resident microglial/ macrophage populations. Nine months after BMT, 20-30% of the microglia in randomly collected sections from the neocortex, hippocampus and cerebellum, and 50-70% of the F4/80+ population in sections from peripheral ganglia and sciatic nerve were represented by GFP+ cells. These results demonstrate the efficacy of this approach in targeting transgene expression to the CNS and, for the first time, PNS. We then transduced ARSA deficient (As2-/-) Lincells from MLD mice with PGK-ARSA LV. Transduced cells exhibited ARSA activity levels 10-15 fold higher than wild type cells and secretion of active enzyme in the medium. As2-/- mice examined 810 weeks after transplantation with ARSA LV transduced cells showed multiple vector copies in 65-98% (n=40) of circulating PBMC with ARSA activity levels 8-10 fold higher than those of wild type mice. Similar studies are in progress in Twitcher mice with PGK-GALC LV, which efficiently rescued GALC activity in Twi-/- cells. Preliminary results demonstrated that ARSA overexpression can prevent sulphatide accumulation in MLD target organs. Long-term studies to evaluate the safety and therapeutic impact of this approach on the progression of the hystopathological S88
Neuronal injuries generate elevated glucocorticoid (GC) levels enhancing injury due to increased neurotoxicity. Glucocorticoids are hormones secreted during stress affecting various target tissues. The increase in neurotoxicity is due to the neurons within the hippocampus becoming sensitized by GCs to insults such as hypoxia-ischemia (Cardiac arrest), hypoglycemia, or hyperexcitation (Seizures). The result of this neuroendangerment is an increase in neuronal death as measured by neuron survival in vitro and lesion size in vivo. Previously we have demonstrated that several genes selected to interfere with GC actions can decrease the endangering effects of this hormone during neuronal injury in vitro. These genes which diminish the effects of corticosterone (cort), a glucocorticoid, in primary rat hippocampal cultures are: 1) 11 beta-Hydroxy steroid dehydrogenase type 2 (11bHSD2) which converts the glucocorticoid corticosterone to the inactive cortisone. 11bHSD2 acts unidirectionally diminishing cort’s effective dose. 2) A dominant negative glucocorticoid receptor (dnGR) 3) A chimeric gene combining the estrogen receptor (ER) DNA binding domain and the glucocorticoid receptor ligand binding domain (GR/ER) diverting the glucocorticoid signal into protective, estrogenic actions. 11bHSD2, dnGR, or the GR/ER under the control of a constitutive promoter as is the reporter gene, green flourescent protein(GFP). Glucocorticoid neuroendangerment is modulated by 11bHSD2, dnGR, and GR/ER in vivo. Initial experiments to test the reduction in lesion size due to the effects of the anit-glucocorticoid genes were performed. Adult male Sprague-Dawley rats had Herpes Simplex Virus (HSV) amplicons containing 11bHSD2, dnGR, GR/ER, or Green Flourescent Protein control (GFP) were delivered via stereotaxic injection into the dentate gyrus, one hemisphere receiving the experimental amplicon. The other hemisphere received the control amplicon. Kainic acid, to induce hyperexcitation was delivered in conjunction with the experimental amplicons. Expression was verified by immunoflourescence from hippocampal slices. Total lesion volume was determined from 30um coronal sections. Total lesion size in the CA3 area of he hippocampus was measured using the nonlesioned hemisphere to determine the size of the intact CA3 region. Results will be presented. GR/ER chimeric receptor can activate the estrogen receptor pathway in vitro. We anticipated that this chimeric protein will not only attenuate the endangering effects of GCs, but divert the GC signal into protective, estrogenic actions. Using semi-quantitative and real time PCR the GR/ER chimeric receptor, in the presence of cort., activates brain derived nerve growth factor (BDNF) gene expression and repress serum glucocorticoid kinase (SGK) gene expression at levels comparable to the native estrogen receptor. Our preliminary data suggests that these amplicons can modulate the cellular response to cort. within hippocampal neurons in vivo. Resulting in an attenuation of the endangerment and decreasing neurotoxic enhancement within the hippocampus during neuronal injury.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
damage in both mouse models are ongoing. Efficacy evaluations are based on neurophysiological, comportamental and neuromotor studies in MLD mice and on monitoring survival in Twitcher mice.
GENE THERAPY FOR THE NERVOUS SYSTEM I
223. Genetic Interventions To Decrease Neurological Injury In Vivo
222. Lentiviral Vector-Based Ex Vivo Gene Therapy Approach for Metachromatic (MLD) and Globoid (GLD) Leukodystrophies
William O. Ogle,1 Daniela Kaufer,1 Zachary S. Pincus,1 Theodore C. Dumas,1 Klaus M. Dinkel,3 Deveroux Ferguson,2 Kelsey L. Clark,1 Robert M. Sapolsky.1 1 Biological Sciences, Stanford University, Stanford, CA; 2 Psychiatry, Stanford University, Stanford, CA; 3Leibniz Institute for Neurobiology, Magdeburg, Germany.
Alessandra Biffi,1 Michele De Palma,2 Angelo Quattrini,3 Ilaria Visigalli,1 Diego Dolcetta,1 Claudio Bordignon,1 Luigi Naldini.1,2 1 San Raffaele-Telethon Institute for Gene Therapy (H.S.R.TIGET), Scientific Institute H.S. Raffaele, Milano, Italy; 2 Laboratory for Gene Transfer and Therapy, Institute for Cancer Research, University of Torino, Candiolo, Italy; 3Neuropathology Unit, Scientific Institute H.S. Raffaele, Milano, Italy. MLD and GLD are inborn lipidosis caused by the deficiency of the lysosomal enzymes arylsulfatase A (ARSA) and galactocerebrosidase (GALC), which result in storage of undegraded sulphatides in the CNS and PNS, leading to progressive demyelination. BMT provides a clinically feasible method for permanently replacing deficient lysosomal enzyme activity. Donor derived cells migrate into disease target organs and become a local and permanent source of the missing enzyme. We are evaluating an ex vivo gene therapy approach for MLD and GLD based on transplantation of LV-transduced autologous HSC. We first investigated the timing and extent of migration of donor derived cells into the nervous system of wild type mice. We transduced HSC, enriched from the total BM by lineage negative (Lin-) selection, by a short ex vivo incubation with advanced generation LV expressing GFP from the hPGK promoter and injected the cells into lethally irradiated mice. Three months after the transplant FACS analysis of blood samples demonstrated high engraftment of GFP expressing cells (62-95%, n=16) in the different hematopoietic lineages. The BM of primary recipients was used to perform secondary transplants. Multi-lineage transgene expressing cells fully engrafted the secondary hosts, indicating transduction of primitive long-term repopulating cells and stable transgene expression in their progeny for up to 12 months. Transplanted mice were sacrificed at 3, 6 and 9 months after BMT to study the migration of BM-derived transduced cells. We detected a significant migration of transgene expressing cells into CNS and PNS that increased progressively with longer times after BMT. Co-localization studies showed that transduced cells contributed to the turnover of the resident microglial/ macrophage populations. Nine months after BMT, 20-30% of the microglia in randomly collected sections from the neocortex, hippocampus and cerebellum, and 50-70% of the F4/80+ population in sections from peripheral ganglia and sciatic nerve were represented by GFP+ cells. These results demonstrate the efficacy of this approach in targeting transgene expression to the CNS and, for the first time, PNS. We then transduced ARSA deficient (As2-/-) Lincells from MLD mice with PGK-ARSA LV. Transduced cells exhibited ARSA activity levels 10-15 fold higher than wild type cells and secretion of active enzyme in the medium. As2-/- mice examined 810 weeks after transplantation with ARSA LV transduced cells showed multiple vector copies in 65-98% (n=40) of circulating PBMC with ARSA activity levels 8-10 fold higher than those of wild type mice. Similar studies are in progress in Twitcher mice with PGK-GALC LV, which efficiently rescued GALC activity in Twi-/- cells. Preliminary results demonstrated that ARSA overexpression can prevent sulphatide accumulation in MLD target organs. Long-term studies to evaluate the safety and therapeutic impact of this approach on the progression of the hystopathological S88
Neuronal injuries generate elevated glucocorticoid (GC) levels enhancing injury due to increased neurotoxicity. Glucocorticoids are hormones secreted during stress affecting various target tissues. The increase in neurotoxicity is due to the neurons within the hippocampus becoming sensitized by GCs to insults such as hypoxia-ischemia (Cardiac arrest), hypoglycemia, or hyperexcitation (Seizures). The result of this neuroendangerment is an increase in neuronal death as measured by neuron survival in vitro and lesion size in vivo. Previously we have demonstrated that several genes selected to interfere with GC actions can decrease the endangering effects of this hormone during neuronal injury in vitro. These genes which diminish the effects of corticosterone (cort), a glucocorticoid, in primary rat hippocampal cultures are: 1) 11 beta-Hydroxy steroid dehydrogenase type 2 (11bHSD2) which converts the glucocorticoid corticosterone to the inactive cortisone. 11bHSD2 acts unidirectionally diminishing cort’s effective dose. 2) A dominant negative glucocorticoid receptor (dnGR) 3) A chimeric gene combining the estrogen receptor (ER) DNA binding domain and the glucocorticoid receptor ligand binding domain (GR/ER) diverting the glucocorticoid signal into protective, estrogenic actions. 11bHSD2, dnGR, or the GR/ER under the control of a constitutive promoter as is the reporter gene, green flourescent protein(GFP). Glucocorticoid neuroendangerment is modulated by 11bHSD2, dnGR, and GR/ER in vivo. Initial experiments to test the reduction in lesion size due to the effects of the anit-glucocorticoid genes were performed. Adult male Sprague-Dawley rats had Herpes Simplex Virus (HSV) amplicons containing 11bHSD2, dnGR, GR/ER, or Green Flourescent Protein control (GFP) were delivered via stereotaxic injection into the dentate gyrus, one hemisphere receiving the experimental amplicon. The other hemisphere received the control amplicon. Kainic acid, to induce hyperexcitation was delivered in conjunction with the experimental amplicons. Expression was verified by immunoflourescence from hippocampal slices. Total lesion volume was determined from 30um coronal sections. Total lesion size in the CA3 area of he hippocampus was measured using the nonlesioned hemisphere to determine the size of the intact CA3 region. Results will be presented. GR/ER chimeric receptor can activate the estrogen receptor pathway in vitro. We anticipated that this chimeric protein will not only attenuate the endangering effects of GCs, but divert the GC signal into protective, estrogenic actions. Using semi-quantitative and real time PCR the GR/ER chimeric receptor, in the presence of cort., activates brain derived nerve growth factor (BDNF) gene expression and repress serum glucocorticoid kinase (SGK) gene expression at levels comparable to the native estrogen receptor. Our preliminary data suggests that these amplicons can modulate the cellular response to cort. within hippocampal neurons in vivo. Resulting in an attenuation of the endangerment and decreasing neurotoxic enhancement within the hippocampus during neuronal injury.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy