137 Development of a Safe Hematopoietic Stem Cell (HSC) Mobilization Strategy for Gene Therapy of Thalassemia Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Ge[.]
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 GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY I CD34+cells HU as pretreatment is an effective strategy to overcome this limitation, however, it requires a rather prolonged treatment with a critical washout period for a sufcient CD34+cell yield in time Furthermore, the small effect on correction implies that after a year a signicant percentage of the therapeutic gene expression in liver is provided by integrated scAAV vector genomes 138 Long Term Expression in Liver from scAAV Seems Derived from Integrated Vector Genomes 139 Human Amnion Epithelial (AE) Stem Cell Transplant Improves Disease Phenotype and Survival in Mouse Models of Intermediate Maple Syrup Urine Disease (MSUD) Paula S M Miranda,1 Maude Flageul,2 Christine Kaeppel,3 Lysbeth ten Bloemendaal,1 Dominique Aubert,2 Nowrouzi Ali,3 Manfred Schmidt,3 Nicolas Ferry,2 Piter J Bosma.1 Tytgat Lab for Liver and Intestinal Research, Academic Medical Center, Amsterdam, Netherlands; 2Biotherapies Hepatiques, INSERM UMR 948, Nantes, France; 3Nationales Zentrum für Tumorerkrankungen, DKFZ, Heidelberg, Germany A single portal vein injection of our double-stranded scAAV8LP1-UGT1A1 vector provides life-long correction of inherited unconjugated hyperbilirubinemia in the Gunn rat, the relevant rat model for Crigler-Najjar syndrome type (CN-1) Patients with CN-1 lack hepatic UGT1A1 activity resulting in accumulation of unconjugated bilirubin, a neurotoxic compound in serum and tissues, which if left untreated causes bilirubin encephalopathy The life-long AAV mediated correction obtained in Gunn rats, suggest that gene therapy for CN-1 is feasible Recombinant AAV vectors have been reported to persist in episomal form in post-mitotic tissues This episomal persistence explains the loss efcacy of single stranded AAV liver directed gene therapy caused by hepatocyte proliferation occurring in neonatal animals and upon liver damage However, the effect of hepatocyte proliferation on long-term efcacy of double stranded scAAV vectors has not been reported Therefore we decided to evaluate the effect of liver damage on scAAV mediated correction We performed 2/3 partial hepatectomy in Gunn rats at 12 weeks (n=8) or at 52 weeks (n=6) after vector injection Just prior to the hepatectomy serum bilirubin levels in both groups were 35 +/- 11 uM In rats receiving a partial hepatectomy at 12 weeks after vector injection, the correction in serum bilirubin was totally lost This loss of correction is comparable to that reported for single stranded AAV previously However, in contrast to this, in rats hepatectomized at 52 weeks serum bilirubin levels increases only to 60uM and remain signicantly below that of non treated animals The effects on serum were subsequently conrmed by a substantial reduction (>80%) of vector genome copies in liver upon hepatectomy performed at 12 weeks while upon hepatectomy performed at 52 weeks no signicant decrease was observed Our data suggest that in non proliferating hepatocytes, as is the case in Gunn rats, scAAV genomes gradually integrate into the host genome The observation that a 2/3 hepatectomy at 52 weeks after scAAV injection does not abolish correction suggests that at that time a signicant part of the UGT1A1 expression is derived from integrated genomes To identify the vector integration sites we are performing LAM-PCR using genomic DNA obtained from liver before and after partial hepatectomy In conclusion, our data seem to indicate that integration of scAAV in host genomes gradually increases Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy K J Skvorak,1 K Dorko,1 M Hansel,1 F Marongiu,1 V Tahan,1 K M Gibson,2 Q Sun,3 T Bottiglieri,3 E Arning,3 J Davila,4 S Strom.1 Pathology, Univ Pittsburgh, Pittsburgh, PA; 2Biol Sciences, Michigan Tech Univ., Houghton, MI; 3Inst Metab Dis., Baylor Univ Med Ctr., Dallas, TX; 4Pzer Global Research, St Louis, MO MSUD (OMIM 248600) is a disorder of branched chain amino acid (BCAA; leucine, isoleucine, valine) catabolism MSUD is treated primarily by dietary manipulation, though compliance is variable often resulting in catabolic crisis Orthotopic liver transplantation greatly improves outcomes, but cost is signicant and donor organs are scarce To explore novel preclinical treatment options, a viable transgenic mouse model of intermediate MSUD (iMSUD) was developed (Homanics, et al., BMC Med Gen 2006, 7:33) Using this model, proof of principle studies determined that mouse hepatocyte transplantation significantly improved disease phenotype (i.e., corrected or partially corrected key amino acids in the brain and blood, enzyme activity, and monoamines) and lengthened survival (Mol Ther 2009, 17(7):126; Biochim Acta 2009, 1792(10):1004) Applying this rationale, human-derived stem cells were explored as a possible alternative to hepatocytes for use in cell transplant studies Human AE (hAE) cells are derived from human amnion and share many characteristics with pluripotent embryonic stem cells hAE cells (1x10^6/100µL) were directly injected into the livers of neonatal iMSUD pups Two injections were given during the rst 10 days of life After weaning (21 days of age), hAE cell “booster shots” (2x10^6 cells) were administered bi-weekly until sacrice (∼35 days of age) The lifespan of iMSUD mice was signicantly extended by hAE transplant [hAE Tx: 100% survival at 35 days (n=7) vs untreated iMSUD: 0% survival at 35 days (n=7); p30% At the time of abstract submission, brain amino acid data are pending completion We propose that hAE cells may be an alternate source for cell transplantation as a therapeutic intervention for MSUD and other liver-based inborn errors of metabolism S53 ... values and homovanillic acid was decreased by >30% At the time of abstract submission, brain amino acid data are pending completion We propose that hAE cells may be an alternate source for cell transplantation... this rationale, human-derived stem cells were explored as a possible alternative to hepatocytes for use in cell transplant studies Human AE (hAE) cells are derived from human amnion and share many... lysine, and proline were all normalized to control levels in hAE transplanted animals Other serum amino acids were partially corrected BCAA/alanine ratio, a more powerful indicator of disease, was