135 Cell Autonomy in Methylmalonic Acidemia (MMA) Implications for Gene and Cell Therapy Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S50[.]
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY I of mutation on actual cAMP concentrations Importantly, mutationtransduced SSCs were altered in their ability to differentiate in vitro and in vivo (altered ectopic bone formation in mice) LV-GNASR201C injection into mouse zygotes generated pups that were positive for the transgene (up to 66% of the injected embryos) The mutation was inherited for at least four generations 100% of the transgenic animals tested displayed increased cAMP levels when measured in the presence of IBMX Following repeated backcrossing, up to 100% of the F4 transgenic animals displayed transgene expression and 65% an FD characteristic skeletal phenotype The cell and animal models were then used for testing a gene therapy approach for FD The LV encoded GNASR201C allele specic interfering sequence was delivered in vitro (in WT or LVGNASR201C-SSCs) and in vivo by intravenous injection in FD transgenic animals In SSCs, we could correct the FD phenotype both in molecular terms (selective know down of the mutated protein), and functionally (cAMP reduction and recovery of SSC differentiation capacities) In vivo, we are currently testing the ability of the same vector of correcting the FD skeletal phenotype Taken together, our data demonstrate that FD phenotpye can be transferred in vivo and in vitro, and specically silenced, at least in vitro, using lentiviral vectors These models provide important opportunities to dissect the disease mechanisms, and to design and validate in vitro and in vivo, preclinical approaches for gene correction 134 Bone Marrow Transplantation Cures Hemophilia A Antonia Follenzi,1,2 Sanj Raut,3 Simone Merlin,2 Rita Sarkar,4 Sanjeev Gupta.1 Pathology, Liver Research Center, Einstein College of Medicine, Bronx; 2Medical Sciences, University of Piemonte Orientale, Novara, Italy; 3Haemostasis Section, National Institute for Biological Standards and Controls, Hertfordshire, United Kingdom; 4Genetics, University of Pennsylvania, Philadelphia, PA Identication of cells capable of synthesizing and releasing FVIII will be critical for therapeutic development in hemophilia A Recent studies indicated that endothelial cells, particularly liver sinusoidal endothelial cells, are major source of FVIII, although the origin of endothelial cells is incompletely dened and FVIII could potentially be expressed in additional cell types To determine whether donor bone marrow (BM) could produce cells able to synthesize and release FVIII, we transplanted genetically marked healthy BM cells in hemophilia A mice We analyzed FVIII production and correction of hemophilia by several assays Extensive BM chimerism was observed after transplantation of 2x106, as well as 10x106 of total BM cells Remarkably, we found therapeutic correction was lower in hemophilia mice receiving 2x106 BM cells rather than 10x106 BM cells (55%, n 42, versus 100%, n 44, respectively, p5% to 22% of normal levels, with correction of hemophilia over at least 12 mo We measured plasma Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy FVIII antigen in several mice 2, and mo after transplantation of 10x106 BM cells and found that plasma FVIII antigen and activity levels were in agreement with one another, increasing condence in our ndings We did not detect neutralizing antibodies against FVIII in hemophilia A mice after BM transplants, including mice with or without detectable FVIII antigen and plasma FVIII activity Moreover, hemophilia A mice with correction of plasma FVIII activity survived tail clip-induced bleeding, again in agreement with therapeutic cure We found that induction of parenchymal and/or endothelial injuries with J02 antibody, monocrotaline or CCl4 did not improve therapeutic correction in hemophilia A mice Also, despite these hepatic and endothelial injuries to recruit transplanted cells, donor BM-derived hepatocytes or endothelial cells were extremely rare, and did not account for therapeutic benets By contrast, we discovered FVIII was produced in donor BM-derived mononuclear cells and mesenchymal stromal cells (MSC), which expressed FVIII mRNA and protein Also, we found FVIII mRNA expression in total human cord blood, BM and BM cell fractions, i.e., CD34+ and CD133+ progenitor cells, CD33+ myeloid cells, and CD105+ mesenchymal and MSC Immunostaining of MSC, veried to be vimentin-positive, showed FVIII protein Moreover, injection of healthy Kupffer cells (liver macrophage/mononuclear cells), which predominantly originate from BM, or of healthy BM -derived MSC protected hemophilia A mice from bleeding challenge Conclusions: BM transplantation cured hemophilia A through reconstitution of mononuclear cells and MSC These insights in additional cellular origins of FVIII offer new mechanisms for understanding pathophysiological alterations in FVIII synthesis and release and for developing therapies in hemophilia A 135 Cell Autonomy in Methylmalonic Acidemia (MMA): Implications for Gene and Cell Therapy Irini Manoli,1 Justin R Sysol,1 Randy J Chandler,1 Jennifer Sloan,1 Patricia Zerfas,2 Victoria Hoffmann,2 Mones Abu-Asab,3 Maria Tsokos,3 Charles P Venditti.1 GMBB/NIH, Bethesda, MD; 2ORS/NIH, Bethesda, MD; 3CCR/ NIH, Bethesda, MD Introduction: A severe mitochondriopathy affecting selected cells, particularly hepatocytes, in mice and patients with methylmalonic acidemia (MMA) has been rmly established [Chandler et al., FASEB J 23:1252] Whether mitochondrial dysfunction is cell autonomous and/or dependent upon circulating metabolites is unknown but critical to investigate If high methylmalonic acid levels are toxic to cells that express the enzyme, successful gene and cell therapy for MMA may require a degree of correction not achievable with current delivery methods To examine the consequences of liver-specic correction in MMA and to study disease pathophysiology in extra-hepatic organs, we generated transgenic Mut-/- mice that express the Mut cDNA in a liver-specic fashion Methods: An insulated construct, TgINS-Alb-Mut, expresses the Mut gene under the control of the murine albumin promoter/α-fetoprotein enhancer Mut-/-;TgINS-Alb-Mut mice were characterized with matched control littermates Liver and kidney from transgenic mice or from MMA patients who underwent transplantation (n=4) were studied by electron microscopy Results: Mut-/-;TgINS-Alb-Mut animals were present in expected Mendelian proportions and fully viable post-weaning RNA from different tissues of the Mut-/-;TgINS-Alb-Mut mice at weeks, and months revealed stable liver-specic transgene expression between 8.2-18±1% of the Mut+/- levels 13C-propionate oxidation in the Mut-/-;TgINS-Alb-Mut was 54.7±9.2%, compared to 76.5±4.5 in the Mut+/- and 10±2 in the Mut-/- mice after 25 minutes Challenge with a high-protein diet resulted in weight loss versus baseline and treated controls Plasma methylmalonic acid levels (µM) in the Mut-/-;TgINSAlb-Mut mice were 500±177 at baseline, 1500±620 after months and 1983±418 after months on high-protein diet, while they remained S51 GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY I unchanged in the treated controls, 4.8.±1.1 Despite the massive elevations of plasma metabolites on a high protein diet, liver function tests and hepatic ultrastructure remained normal in the Mut-/-;TgINSAlb-Mut mice However, histological examination of their kidneys revealed tubulointerstitial nephritis Proximal tubular cells contained megamitochondria with short or missing cristae and proteinaceous matter Similar changes were observed in native tissues obtained from MMA patients Summary: Low-level hepatic expression of the Mut enzyme uniformly rescued Mut-/- mice from neonatal lethality, restored growth, increased 13C-propionate oxidative capacity and prevented characteristic hepatic ultrastructural mitochondrial changes from developing The maintenance of normal hepatic ultrastructure in the presence of massive elevations of circulating metabolites establishes that cell-autonomous effects likely underlie hepatic megamitochondria formation Since Mut-/-;TgINS-Alb-Mut mice can be induced to develop the kidney disease of MMA, this model will be useful to study gene and/ or cell therapies directed toward the proximal tubule, another cell type that would be benecial to correct in the treatment of MMA 136 Efcacious Gene Therapy in Pompe Disease Requires Mannose-6-Phosphate Receptor Expression Baodong Sun,1 Deeksha Bali,1 Y.-T Chen,1 Dwight D Koeberl.1 Pediatrics/Division of Medical Genetics, Duke University Medical Center, Durham, NC The curative potential of gene therapy in lysosomal storage diseases has been increasingly recognized, as the limitations of enzyme replacement therapy (ERT) have become evident, especially in Pompe disease The enzyme dosages required for ERT in Pompe disease range up to 100-fold greater than those in other lysosomal disorders This high enzyme requirement can be partially attributed to the need to treat the muscle mass, which comprises approximately 40% of body weight; however, poor uptake of acid α-glucosidase (GAA) by skeletal muscle has been linked to the low abundance of the cation-independent mannose-6-phosphate receptor (MPR) in skeletal muscle compared to heart ERT in MPR knockout/GAA knockout mice (either 20 mg/kg and 100 mg/kg every other week) suggested that skeletal muscle (quadriceps, triceps, gastrocnemius) was largely dependent on MPR for rhGAA uptake and glycogen clearance in vivo as compared to GAA knockout (80, 76 and 72% reduced uptake respectively) Antibody responses against GAA also reduced efcacy from ERT Enhanced efcacy through the evasion of antibody responses against GAA in Pompe disease has been achieved through two gene therapy strategies, either by the induction of immune tolerance with hepatic-restricted expression from AAV-LSPhGAA, or by the correction of accumulated intracellular glycogen with musclerestricted GAA expression from AAV-MHCK7hGAA We evaluated the impact of MPR-mediated uptake of GAA by evaluating gene therapy in MPR knockout/GAA knockout mice The essential role of MPR was emphasized by the lack of efcacy for either strategy, as demonstrated by markedly reduced biochemical correction of GAA deciency and of glycogen accumulations in MPR knockout/ GAA knockout mice, in comparison with administration of the same vectors in GAA knockout mice that expressed MPR Somewhat surprisingly, high-level muscle-restricted GAA expression with AAVMHCK7hGAA also failed to reduce glycogen storage in absence of MPR expression The latter result indicated that MPR expression was critical not only to receptor-mediated uptake of GAA, but also to the intracellular trafcking of GAA to lysosomes Administration of AAV-MHCK7hGAA achieved supraphysiologic GAA activity, but failed to signicantly reduce glycogen accumulations in the skeletal muscle of MPR knockout/GAA knockout mice Specically, the glycogen content of hind-limb muscles and the diaphragm was reduced signicantly more in the GAA knockout mice that expressed S52 MPR (30-90%) following AAV-MHCK7hGAA administration, whereas the same vector treatment failed to reduce the glycogen content of these muscles in MPR knockout/GAA knockout mice In summary, MPR expression was critical to gene therapy in Pompe disease mice for both receptor-mediated uptake and intracellular targeting of GAA An optimized gene therapy strategy in lysosomal storage disorders should emphasize both tissue-restricted expression to evade antibody responses and the expression of MPR in the target tissues of experimental subjects 137 Development of a Safe Hematopoietic Stem Cell (HSC) Mobilization Strategy for Gene Therapy of Thalassemia Evangelia Yannaki,1 Thalia Papayannopoulou,2 Erica Jonlin,2 Ioannis Batsis,1 Pamela Becker,2 Fani Zervou,1 Angeliki Xagorari,1 Garyfalia Karponi,1 Varnavas Constantinou,1 Achilles Anagnostopoulos,1 Athanasios Fassas,1 George Stamatoyannopoulos.2 Gene and Cell Therapy Center, Hematology-BMT Unit, George Papanicolaou Hospital, Thessaloniki, Greece; 2Medical Genetics, Hematology, University of Washington, Seattle, WA Effective gene therapy for thalassemia, requires high numbers of transduced HSCs to be reinfused to patients and mobilized peripheral blood will be probably the primary source of HSCs There is limited information on mobilization of adults with β-thalassemia using GCSF Rare events of splenic rupture or thrombosis with GCSF in normal donors raise safety concerns because of splenomegaly and hypercoagulability which are characteristic of thalassemic patients Pretreatment with Hydroxyurea (HU) could reduce risks by decreasing the spleen size in non-splenectomized and the high numbers of circulating cells in splenectomized patients We assessed the safety and efcacy of GCSF mobilization, with or without HU pretreatment, in 12 splenectomized and 12 non-splenectomized adults with β-thalassemia major Nine splenectomized and nonsplenectomized patients received HU for month with a 1-2 week interval between HU cessation and GCSF initiation Two aphereses were conducted with a target CD34+cell dose ≥2X106/kg No severe adverse events were observed In non-splenectomized patients, HU decreased the spleen size over baseline and mobilization resulted in lower maximum spleen size increase compared to no-HU group HU negatively affected the CD34+cell yield, when the washout period before GCSF was week, however, a 2-week interval between HU discontinuation and GCSF administration markedly improved the CD34+yield Non-splenectomized patients w/o HU mobilized successfully Splenectomized patients, were a priori considered effective mobilizers due to the absence of trapping HSCs by the spleen; however, they mobilized in most cases poorly They responded excessively to GCSF, despite the reduced and adjusted to the WBC level GCSF doses, by developing hyperleukocytosis which necessitated, in all but one patient, early therapeutic leukapheresis HU negatively affected the yield when the washout period before GCSF was ≤10days; however a 2-week washout period, which allowed bone marrow to recover after the myelosuppressive effect of HU, impressively improved mobilization Importantly, those patients could tolerate almost 2fold higher GCSF doses w/o producing excessive leukocytosis In all splenectomized patients, HU decreased the high platelets and WBCs before GCSF, potentially reducing the risk of thrombosis and partially preventing hyperleukocytosis during mobilization HU did not affect the clonogenic capacity of HSCs in terms of CFU-GM/BFU-E in splenectomized or not subjects We conclude that mobilization of splenectomized thalassemia patients is challenging but not inherently inefcient The mandatory GCSF-dose modications to avoid hyperleukocytosis crucially affect the yield of Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... expression was critical to gene therapy in Pompe disease mice for both receptor-mediated uptake and intracellular targeting of GAA An optimized gene therapy strategy in lysosomal storage disorders... Stamatoyannopoulos.2 Gene and Cell Therapy Center, Hematology-BMT Unit, George Papanicolaou Hospital, Thessaloniki, Greece; 2Medical Genetics, Hematology, University of Washington, Seattle, WA Effective gene therapy. .. hepatic megamitochondria formation Since Mut-/-;TgINS-Alb-Mut mice can be induced to develop the kidney disease of MMA, this model will be useful to study gene and/ or cell therapies directed