- Email: [email protected]
316. Therapeutic Potential of Genetically Modified Neural Stem Cells (NSCs) in a Mouse Model of Globoid Cells Leukodystrophy (GLD)
GENE THERAPY FOR LYSOSOMAL STORAGE DISEASES AND OTHER GENETIC DISORDERS functional lysosomal enzyme galactocerebrosidase (GALC). This is an autosomal recessive neurodegenerative disease affecting both the central and peripheral nervous systems that in its most severe form results in death before the age of 2 years old for humans and approximately 35-40 days in the mouse model. To date there is no cure for this disease, though bone marrow transplants have been helpful with prolonging the onset of the disease if admnistered before symptoms appear. However, bone marrow transplants often are complicated by graft vs. host disease and do not actually cure the disease. This study attempts to evaluate the effect of intraventricular administration of mesenchymal stem cells on the pathology of Krabbe’s disease in the twitcher mouse model using adult murine mesenchymal stem cells isolated from the bone marrow or adipose tissue. Pups were injected bilaterally on post-natal day (PND) 3-4 with either adipose derived or bone marrow derived mesenchymal stem cells (ASCs or BMSCs respectively) at a dose of 20,000 cells/ ul and 1 ul per hemisphere for a total of 40,000 cells. Body weights, lifespan, and motor function were evaluated beginning with PND15. Tissues were also harvested when the mice were euthanized for immunohistochemistry, GALC enzymatic assays, cytokine analysis, cell tracking, and Western blot analysis. Survival analysis curves indicate a signicant difference in lifespan between cell treated and control twitcher mice. Body weights for treated twitchers were also higher than those of controls, and motor function analyzed by the wire hang maneuver and gait analysis was also improved in cell treated twitchers. These injected cells also seem to exert potent antiinammatory effects in the central nervous system. iNOS levels in the brains of treated mice were reduced and expression of several inammatory cytokines appears to be suppressed in the treated mice. Western blot analysis for CD163 also revealed decreased macrophage inltration in the brains of twitchers treated with ASCs and BMSCs.While these improvements are promising, the twitcher mice still exhibited symptoms associated with Krabbe’s disease. This approach only targets the central nervous system, therefore a combination therapy approach to treating this disease will most likely be more benecial.
315. Therapeutic Efcacy of Hematopoietic Stem Cell Gene Therapy for Hurler Type 1 Mucopolysaccharidosis
Stefania Delai,1,2 Ilaria Visigalli,1 Letterio S. Politi,3 Carmela Di Domenico,4 Emanuela Mrak,5 Riccardo Brambilla,6 Federica Cerri,7 Daniela Ungaro,8 Merel Stok,9 Francesca Sanvito,10 Ubaldo Del Carro,8 Alessandro Rubinacci,5 Katherine P. Ponder,11 Angelo Quattrini,7 Paola Di Natale,4 Luigi Naldini,1,12 Alessandra Bif.1 1 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientic Institute, Milan, Italy; 2University of Perugia, Perugia, Italy; 3Neuroradiology, San Raffaele Scientic Institute, Milan, Italy; 4University of Naples, Naples, Italy; 5Bone metabolism, San Raffaele Scientic Institute, Milan, Italy; 6Genetic of Behavior, San Raffaele Scientic Institute, Milan, Italy; 7Experimental Neurology, San Raffaele Scientic Institute, Milan, Italy; 8Experimental Neurophysiology, San Raffaele Scientic Institute, Milan, Italy; 9 Erasmus MC, Rotterdam, Netherlands; 10Anatomopathology, San Raffaele Scientic Institute, Milan, Italy; 11Division of Hematology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO; 12Vita Salute University, Milan, Italy. Type 1 Mucopolysaccharidosis (MPS I) is an inherited lysosomal storage disorder caused by the deciency of α-L-iduronidase (IDUA), which is responsible for systemic glycosamminoglycans (GAGs) accumulation, leading to skeletal and joint disease, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly and mental retardation. The available treatment options (enzyme replacement therapy – ERT – and hematopoietic cell transplantation – HCT) are poorly effective on skeletal and brain disease manifestations, leaving Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
treated patients with substantial disabilities. In order to improve the therapeutic efcacy of HCT and target the skeletal and neurological disease manifestations, we are developing a gene therapy strategy for MPS I based on IDUA supra-normal expression in hematopoietic stem cells (HSC) and their progeny by means of lentiviral vector (LV)-mediated gene transfer. Transduction of murine HSC with a LV encoding the human IDUA cDNA led to enzyme over-expression up to 150 fold above normal donors’ levels in their differentiated progeny, which could thus more abundantly correct affected tissues upon inltration. Indeed, when these cells were transplanted into Idua-/- mice, supra-normal enzyme expression was observed in circulating hematopoietic cells and in all tested diseased tissues, including the brain, in which transplantation of wild type HSC failed to deliver the functional enzyme. Importantly, gene therapy corrected all major disease manifestations, decreasing GAGs storage in tissues and urines and improving auditory impairment and retinal structure. Moreover, gene therapy demonstrated a greater efcacy than wild type HSC transplantation in correcting the MPS I-related skeletal and behavioral defects. Interestingly, the extent of therapeutic efcacy is solely dependent from the enzyme activity measured in vivo in the differentiated progeny of the transplanted stem cells. Thus, these results demonstrate that enzyme over-expression into hematopoietic cells renders HSC gene therapy effective in tissues refractory to correction following transplantation of normal donors’ HSC, paving the way for future clinical testing. *Equel contribution
316. Therapeutic Potential of Genetically Modied Neural Stem Cells (NSCs) in a Mouse Model of Globoid Cells Leukodystrophy (GLD)
Margherita Neri,1 Alessandra Ricca,2 Silvia Ungari,1 Ilaria Visigalli,1 Alessandra Bif,1 Ilaria Di Girolamo,3 Sabata Martino,3 Lucia Sergi Sergi,1 Luigi Naldini,1,2 Angela Gritti.1 1 Telethon Inst. for Gene Therapy (HSR-TIGET), San Raffaele Scientic Institute, Milano, Italy; 2Vita-Salute San Raffaele University Medical School, Milano, Italy; 3Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia, Italy. Globoid Cell Leukodystrophy (GLD) is a rare genetic disorder caused by the deciency of the lysosomal enzyme β-galactocerebrosidase (GALC), characterized by progressive central and peripheral demyelination. We evaluated the potential of Neural Stem Cell (NSC)based approach to correct the metabolic defect and to ameliorate pathology in Twitcher (Twi) mice, a true model of GLD. We derived NSC lines from the subventricular zone of neonatal Twi mice and of wt littermates. Using bidirectional lentiviral vectors (bdLV) encoding for GALC and GFP, we achieved high efciency of transduction (>80%) and supraphysiological GALC levels (2-3 fold the wt levels) in mutant NSC, with no toxicity or functional impairment due to transgene over-expression. Similar supraphysiological GALC levels were obtained following bdLV.GALC transduction of wt NSC. Of importance, gene-corrected cells allowed more efcient metabolic cross-correction of GALC-decient NSC then wt cells in vitro, due to more efcient enzyme secretion in the extracellular milieau. In order to obtain a stable source of GALC-secreting cells in the brain we transplanted wt or GALC over-expressing NSCs into the telencephalic lateral ventricles of neonatal Twi mice (1x10^6 total cells, bilateral injection). Forty days after transplant we found NSC (identied by GFP expression) widely distributed into the brain parenchyma, from the olfactory bulb to the hippocampus. Also, many cells were found lining the ventricles, along the corpus callosum and in the external capsula. Engrafted NSC (1-3% of the total injected cells) either expressed glial cell markers or retained antigenic features of immature neural cells (i.e. expression of the neuroepithelial S121
GENE THERAPY FOR LYSOSOMAL STORAGE DISEASES AND OTHER GENETIC DISORDERS marker nestin) and did not show proliferative activity. Transplanted NSC produced and secreted the GALC protein, as demonstrated by immunohistochemistry using anti-GALC antibodies. Most important, GALC activity was restored to 50% of wt levels in brain and spinal cord tissues of NSC-transplanted Twi mice, indicating widespread and efcient transport of the bioactive enzyme in CNS tissues (likely through CSF ow) coupled to active cross-correction. We are currently investigating whether this metabolic correction correlates with amelioration of pathology, clearance of tissue storage and rescue of the phenotype, also evaluating the potential therapeutic advantage of GALC-overexpressing NSC as compared to the wt counterpart. These results, together with our preliminary data indicating the feasibility of safe GALC-overexpression in human fetal NSC (a therapeutically relevant cell type), warrant further consideration of NSC gene therapy for the treatment of GLD, likely in combination with other approaches ensuring enzymatic reconstitution in visceral organs and in the PNS.
317. Adeno-Associated Virus and Tetanus Toxin Fragment C Based Vectors for Gene Therapy in the CNS Carlos Gay-Antaki,1 John H. Wolfe.1,2 University of Pennsylvania, Philadelphia, PA; 2Children’s Hospital of Philadelphia, Philadelphia, PA.
1
Neurogenetic diseases have widespread lesions in the CNS, this presents a problem for treatment because it requires methods that can treat widespread areas. The tetanus toxin fragment C (TTC) the neurotropic atoxic fragment of the tetanus holotoxin, is a potential neurotropic carrier that has been fused to reporter proteins such as beta galactosidase and green uorescent protein, and to therapeutic proteins such as superoxide dismutase and glial derived neurotrophic factor. However, the methods using TTC have been limited to protein delivery models. Our lab has previously shown that a fusion protein between TTC and the lysosomal enzyme beta glucuronidase (GUSB) has biochemical properties similar to the wild-type enzyme but different secretion proles, demonstrating the bifunctionality of the chimeric molecule. Furthermore we have shown that this fusion protein can be synthesized in mammalian cells in vivo. In this study we have successfully introduced TTC fused to the lysosomal enzyme GUSB into a functional adeno-associated virus (AAV) vector. Our results show unprecedented levels of enzymatic activity 1 month post-injection in the CNS of mice after a single injection of 1ul of vector into the dentate gyrus, compared to experiments using the wild-type enzyme. We also explored the ability of the fusion protein to enter the CNS after an intramuscular injection of the vector using the hypoglossal pathway as a model. Our results show that the fusion protein is able to bypass the blood brain barrier and deliver the therapeutic enzyme into the expected nuclei in the CNS. This study shows that inserting a TTC fusion construct into an AAV vector has potential for use in gene therapy for diseases that affect the CNS by delivering the vector directly in the brain, in which case TTC is distributed widely via neurons, or by injecting muscle and allowing for the protein to bypass the BBB through the axonal transport of TTC in motor neurons. This is an important step in the development of TTC mediated delivery as a therapeutic method because it eliminates the need for repeated delivery of fused protein, as in previous models, as it is being continuously synthesized in vivo.
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318. Lineage Specic Lentiviral Mpl Expression Rescues Hematopoietic Stem Cell Defects in Mpl-/Mice
Dirk Heckl,1 Daniel C. Wicke,1 Johann Meyer,1 Axel Schambach,1 Johanna Krause,1 Matthias Ballmaier,2 Christopher H. Baum,1 Ute Modlich.1 1 Department of Experimental Hematology, Hannover Medical School, Hannover, Germany; 2Department of Pediatric Hematology, Hannover Medical School, Hannover, Germany.
Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare genetic disorder that is caused by inactivating mutations in the thrombopoietin receptor MPL. Children with CAMT develop severe thrombocytopenia and aplastic anemia. With the aim to develop a gene therapy for CAMT and to avoid severe adverse reactions induced by ectopic Mpl expression (uncontrolled expansion and exhaustion of hematopoietic cells and increased frequency of leukemia), we developed self-inactivating lentiviral vectors that express eGFP or Mpl by different lineage-specic promoters derived from human MPL, murine Mpl and human GPIba. For comparison we used the ubiquitously active PGK promoter. In vitro experiments conrmed lineage-specic expression from the specic promoters in the megakaryocytic cell line HEL, with no expression in other tested hematopoietic cell lines. In vivo expression was restricted to megakaryocytes (CD41+) and hematopoietic stem cells (LSK CD34- Flt3-), with minor activity in other lineages and progenitor populations. All vectors corrected defective megakaryopoiesis of Mpl/- cells in vitro, proving the vectors’ functionality. As the next step, we established a murine model for CAMT gene therapy based on the transplantation of in vitro corrected Mpl-/- hematopoietic cells into Mpl-/- mice. Mpl expressed from the PGK promoter caused death 6 days post transplantation (6/7 mice) due to rupture of enlarged spleens (359-667mg) with erythroid hyperproliferation. This underlines the extreme sensitivity of Mpl-negative hematopoiesis to ectopic Mpl expression. Mice transplanted with bone marrow cells expressing Mpl from the lineage specic promoters survived long term, whereas mice that were transplanted with eGFP transduced Mpl-/- bone marrow cells showed high lethality during establishment of long term hematopoiesis (>12 weeks) revealing Mpl-/- HSC defects. Both early and late post-transplant complications were prevented when using the lineage-specic promoters. Furthermore, platelet production was restored (platelet counts >400x103/µl PB) in approx. 50% of transplanted mice (n=23). Multinucleated megakaryocytes were found in the bone marrow 6-7 months after BMT indicating improved megakaryocytic maturation after gene therapy. In addition, the number of HSCs as identied as LSK population by FACS was signicantly increased in Mpl-corrected animals (GPIbaP.Mpl: 0.031%, GFP: 0.014, p=0.038). q-PCR analysis detected physiological expression levels of Mpl mRNA in LSK cells of mice expressing Mpl from lineage specic promoters. The engraftment of secondary Mpl/- recipients with Mpl corrected BM from the primary mice gave further evidence for the correction of long term HSC numbers. Here we present for the rst time, the correction of a stem cell signalling defect by gene therapy. Our results highlight the toxicity of ectopic overexpression of Mpl but also show the potential for transcriptionally regulated lentiviral vectors to circumvent genotoxic and transgene related, potentially lethal adverse reactions.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
315. Therapeutic Efcacy of Hematopoietic Stem Cell Gene Therapy for Hurler Type 1 Mucopolysaccharidosis
Stefania Delai,1,2 Ilaria Visigalli,1 Letterio S. Politi,3 Carmela Di Domenico,4 Emanuela Mrak,5 Riccardo Brambilla,6 Federica Cerri,7 Daniela Ungaro,8 Merel Stok,9 Francesca Sanvito,10 Ubaldo Del Carro,8 Alessandro Rubinacci,5 Katherine P. Ponder,11 Angelo Quattrini,7 Paola Di Natale,4 Luigi Naldini,1,12 Alessandra Bif.1 1 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientic Institute, Milan, Italy; 2University of Perugia, Perugia, Italy; 3Neuroradiology, San Raffaele Scientic Institute, Milan, Italy; 4University of Naples, Naples, Italy; 5Bone metabolism, San Raffaele Scientic Institute, Milan, Italy; 6Genetic of Behavior, San Raffaele Scientic Institute, Milan, Italy; 7Experimental Neurology, San Raffaele Scientic Institute, Milan, Italy; 8Experimental Neurophysiology, San Raffaele Scientic Institute, Milan, Italy; 9 Erasmus MC, Rotterdam, Netherlands; 10Anatomopathology, San Raffaele Scientic Institute, Milan, Italy; 11Division of Hematology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO; 12Vita Salute University, Milan, Italy. Type 1 Mucopolysaccharidosis (MPS I) is an inherited lysosomal storage disorder caused by the deciency of α-L-iduronidase (IDUA), which is responsible for systemic glycosamminoglycans (GAGs) accumulation, leading to skeletal and joint disease, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly and mental retardation. The available treatment options (enzyme replacement therapy – ERT – and hematopoietic cell transplantation – HCT) are poorly effective on skeletal and brain disease manifestations, leaving Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
treated patients with substantial disabilities. In order to improve the therapeutic efcacy of HCT and target the skeletal and neurological disease manifestations, we are developing a gene therapy strategy for MPS I based on IDUA supra-normal expression in hematopoietic stem cells (HSC) and their progeny by means of lentiviral vector (LV)-mediated gene transfer. Transduction of murine HSC with a LV encoding the human IDUA cDNA led to enzyme over-expression up to 150 fold above normal donors’ levels in their differentiated progeny, which could thus more abundantly correct affected tissues upon inltration. Indeed, when these cells were transplanted into Idua-/- mice, supra-normal enzyme expression was observed in circulating hematopoietic cells and in all tested diseased tissues, including the brain, in which transplantation of wild type HSC failed to deliver the functional enzyme. Importantly, gene therapy corrected all major disease manifestations, decreasing GAGs storage in tissues and urines and improving auditory impairment and retinal structure. Moreover, gene therapy demonstrated a greater efcacy than wild type HSC transplantation in correcting the MPS I-related skeletal and behavioral defects. Interestingly, the extent of therapeutic efcacy is solely dependent from the enzyme activity measured in vivo in the differentiated progeny of the transplanted stem cells. Thus, these results demonstrate that enzyme over-expression into hematopoietic cells renders HSC gene therapy effective in tissues refractory to correction following transplantation of normal donors’ HSC, paving the way for future clinical testing. *Equel contribution
316. Therapeutic Potential of Genetically Modied Neural Stem Cells (NSCs) in a Mouse Model of Globoid Cells Leukodystrophy (GLD)
Margherita Neri,1 Alessandra Ricca,2 Silvia Ungari,1 Ilaria Visigalli,1 Alessandra Bif,1 Ilaria Di Girolamo,3 Sabata Martino,3 Lucia Sergi Sergi,1 Luigi Naldini,1,2 Angela Gritti.1 1 Telethon Inst. for Gene Therapy (HSR-TIGET), San Raffaele Scientic Institute, Milano, Italy; 2Vita-Salute San Raffaele University Medical School, Milano, Italy; 3Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia, Italy. Globoid Cell Leukodystrophy (GLD) is a rare genetic disorder caused by the deciency of the lysosomal enzyme β-galactocerebrosidase (GALC), characterized by progressive central and peripheral demyelination. We evaluated the potential of Neural Stem Cell (NSC)based approach to correct the metabolic defect and to ameliorate pathology in Twitcher (Twi) mice, a true model of GLD. We derived NSC lines from the subventricular zone of neonatal Twi mice and of wt littermates. Using bidirectional lentiviral vectors (bdLV) encoding for GALC and GFP, we achieved high efciency of transduction (>80%) and supraphysiological GALC levels (2-3 fold the wt levels) in mutant NSC, with no toxicity or functional impairment due to transgene over-expression. Similar supraphysiological GALC levels were obtained following bdLV.GALC transduction of wt NSC. Of importance, gene-corrected cells allowed more efcient metabolic cross-correction of GALC-decient NSC then wt cells in vitro, due to more efcient enzyme secretion in the extracellular milieau. In order to obtain a stable source of GALC-secreting cells in the brain we transplanted wt or GALC over-expressing NSCs into the telencephalic lateral ventricles of neonatal Twi mice (1x10^6 total cells, bilateral injection). Forty days after transplant we found NSC (identied by GFP expression) widely distributed into the brain parenchyma, from the olfactory bulb to the hippocampus. Also, many cells were found lining the ventricles, along the corpus callosum and in the external capsula. Engrafted NSC (1-3% of the total injected cells) either expressed glial cell markers or retained antigenic features of immature neural cells (i.e. expression of the neuroepithelial S121
GENE THERAPY FOR LYSOSOMAL STORAGE DISEASES AND OTHER GENETIC DISORDERS marker nestin) and did not show proliferative activity. Transplanted NSC produced and secreted the GALC protein, as demonstrated by immunohistochemistry using anti-GALC antibodies. Most important, GALC activity was restored to 50% of wt levels in brain and spinal cord tissues of NSC-transplanted Twi mice, indicating widespread and efcient transport of the bioactive enzyme in CNS tissues (likely through CSF ow) coupled to active cross-correction. We are currently investigating whether this metabolic correction correlates with amelioration of pathology, clearance of tissue storage and rescue of the phenotype, also evaluating the potential therapeutic advantage of GALC-overexpressing NSC as compared to the wt counterpart. These results, together with our preliminary data indicating the feasibility of safe GALC-overexpression in human fetal NSC (a therapeutically relevant cell type), warrant further consideration of NSC gene therapy for the treatment of GLD, likely in combination with other approaches ensuring enzymatic reconstitution in visceral organs and in the PNS.
317. Adeno-Associated Virus and Tetanus Toxin Fragment C Based Vectors for Gene Therapy in the CNS Carlos Gay-Antaki,1 John H. Wolfe.1,2 University of Pennsylvania, Philadelphia, PA; 2Children’s Hospital of Philadelphia, Philadelphia, PA.
1
Neurogenetic diseases have widespread lesions in the CNS, this presents a problem for treatment because it requires methods that can treat widespread areas. The tetanus toxin fragment C (TTC) the neurotropic atoxic fragment of the tetanus holotoxin, is a potential neurotropic carrier that has been fused to reporter proteins such as beta galactosidase and green uorescent protein, and to therapeutic proteins such as superoxide dismutase and glial derived neurotrophic factor. However, the methods using TTC have been limited to protein delivery models. Our lab has previously shown that a fusion protein between TTC and the lysosomal enzyme beta glucuronidase (GUSB) has biochemical properties similar to the wild-type enzyme but different secretion proles, demonstrating the bifunctionality of the chimeric molecule. Furthermore we have shown that this fusion protein can be synthesized in mammalian cells in vivo. In this study we have successfully introduced TTC fused to the lysosomal enzyme GUSB into a functional adeno-associated virus (AAV) vector. Our results show unprecedented levels of enzymatic activity 1 month post-injection in the CNS of mice after a single injection of 1ul of vector into the dentate gyrus, compared to experiments using the wild-type enzyme. We also explored the ability of the fusion protein to enter the CNS after an intramuscular injection of the vector using the hypoglossal pathway as a model. Our results show that the fusion protein is able to bypass the blood brain barrier and deliver the therapeutic enzyme into the expected nuclei in the CNS. This study shows that inserting a TTC fusion construct into an AAV vector has potential for use in gene therapy for diseases that affect the CNS by delivering the vector directly in the brain, in which case TTC is distributed widely via neurons, or by injecting muscle and allowing for the protein to bypass the BBB through the axonal transport of TTC in motor neurons. This is an important step in the development of TTC mediated delivery as a therapeutic method because it eliminates the need for repeated delivery of fused protein, as in previous models, as it is being continuously synthesized in vivo.
S122
318. Lineage Specic Lentiviral Mpl Expression Rescues Hematopoietic Stem Cell Defects in Mpl-/Mice
Dirk Heckl,1 Daniel C. Wicke,1 Johann Meyer,1 Axel Schambach,1 Johanna Krause,1 Matthias Ballmaier,2 Christopher H. Baum,1 Ute Modlich.1 1 Department of Experimental Hematology, Hannover Medical School, Hannover, Germany; 2Department of Pediatric Hematology, Hannover Medical School, Hannover, Germany.
Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare genetic disorder that is caused by inactivating mutations in the thrombopoietin receptor MPL. Children with CAMT develop severe thrombocytopenia and aplastic anemia. With the aim to develop a gene therapy for CAMT and to avoid severe adverse reactions induced by ectopic Mpl expression (uncontrolled expansion and exhaustion of hematopoietic cells and increased frequency of leukemia), we developed self-inactivating lentiviral vectors that express eGFP or Mpl by different lineage-specic promoters derived from human MPL, murine Mpl and human GPIba. For comparison we used the ubiquitously active PGK promoter. In vitro experiments conrmed lineage-specic expression from the specic promoters in the megakaryocytic cell line HEL, with no expression in other tested hematopoietic cell lines. In vivo expression was restricted to megakaryocytes (CD41+) and hematopoietic stem cells (LSK CD34- Flt3-), with minor activity in other lineages and progenitor populations. All vectors corrected defective megakaryopoiesis of Mpl/- cells in vitro, proving the vectors’ functionality. As the next step, we established a murine model for CAMT gene therapy based on the transplantation of in vitro corrected Mpl-/- hematopoietic cells into Mpl-/- mice. Mpl expressed from the PGK promoter caused death 6 days post transplantation (6/7 mice) due to rupture of enlarged spleens (359-667mg) with erythroid hyperproliferation. This underlines the extreme sensitivity of Mpl-negative hematopoiesis to ectopic Mpl expression. Mice transplanted with bone marrow cells expressing Mpl from the lineage specic promoters survived long term, whereas mice that were transplanted with eGFP transduced Mpl-/- bone marrow cells showed high lethality during establishment of long term hematopoiesis (>12 weeks) revealing Mpl-/- HSC defects. Both early and late post-transplant complications were prevented when using the lineage-specic promoters. Furthermore, platelet production was restored (platelet counts >400x103/µl PB) in approx. 50% of transplanted mice (n=23). Multinucleated megakaryocytes were found in the bone marrow 6-7 months after BMT indicating improved megakaryocytic maturation after gene therapy. In addition, the number of HSCs as identied as LSK population by FACS was signicantly increased in Mpl-corrected animals (GPIbaP.Mpl: 0.031%, GFP: 0.014, p=0.038). q-PCR analysis detected physiological expression levels of Mpl mRNA in LSK cells of mice expressing Mpl from lineage specic promoters. The engraftment of secondary Mpl/- recipients with Mpl corrected BM from the primary mice gave further evidence for the correction of long term HSC numbers. Here we present for the rst time, the correction of a stem cell signalling defect by gene therapy. Our results highlight the toxicity of ectopic overexpression of Mpl but also show the potential for transcriptionally regulated lentiviral vectors to circumvent genotoxic and transgene related, potentially lethal adverse reactions.
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy