137 development of a safe hematopoietic stem cell (HSC) mobilization strategy for gene therapy of thalassemia

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[.]

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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-/-;Tg

INS-Alb-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 bene cial to correct in the treatment of MMA

136 Ef cacious 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

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 ef cacy from ERT Enhanced ef cacy 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

muscle-restricted 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 ef cacy for either strategy,

as demonstrated by markedly reduced biochemical correction of

GAA de ciency 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

AAV-MHCK7hGAA 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 traf cking of GAA to lysosomes Administration

of AAV-MHCK7hGAA achieved supraphysiologic GAA activity,

but failed to signi cantly reduce glycogen accumulations in the

skeletal muscle of MPR knockout/GAA knockout mice Speci cally,

the glycogen content of hind-limb muscles and the diaphragm was

reduced signi cantly more in the GAA knockout mice that expressed

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

1 Gene and Cell Therapy Center, Hematology-BMT Unit, George Papanicolaou Hospital, Thessaloniki, Greece; 2 Medical 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 ef cacy of GCSF mobilization, with or without HU pretreatment, in 12 splenectomized and 12 non-splenectomized adults with β-thalassemia major Nine splenectomized and 6 non-splenectomized patients received HU for 1 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 1 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 inef cient The mandatory GCSF-dose modi cations to avoid hyperleukocytosis crucially affect the yield of

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 suf cient CD34+cell yield

138 Long Term Expression in Liver from scAAV Seems Derived from Integrated Vector Genomes

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

1 Tytgat Lab for Liver and Intestinal Research, Academic Medical Center, Amsterdam, Netherlands; 2 Biotherapies Hepatiques, INSERM UMR 948, Nantes, France; 3 Nationales Zentrum für Tumorerkrankungen, DKFZ, Heidelberg, Germany.

A single portal vein injection of our double-stranded scAAV8-LP1-UGT1A1 vector provides life-long correction of inherited unconjugated hyperbilirubinemia in the Gunn rat, the relevant rat model for Crigler-Najjar syndrome type 1 (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 ef cacy 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 ef cacy 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 signi cantly below that of non treated animals The effects on serum were subsequently con rmed 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 signi cant 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 signi cant 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

in time Furthermore, the small effect on correction implies that after

a year a signi cant percentage of the therapeutic gene expression in liver is provided by integrated scAAV 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)

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

1 Pathology, Univ Pittsburgh, Pittsburgh, PA; 2 Biol Sciences, Michigan Tech Univ., Houghton, MI; 3 Inst Metab Dis., Baylor Univ Med Ctr., Dallas, TX; 4 P zer 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 signi cant 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 sacri ce (∼35 days of age) The lifespan of iMSUD mice was signi cantly extended by hAE transplant [hAE Tx: 100% survival at 35 days (n=7)

vs untreated iMSUD: 0% survival at 35 days (n=7); p<0.0001] Serum amino acids were also signi cantly improved Glutamate, alanine, aspartate, 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 decreased by 70% Alloisoleucine was decreased by 60% and was not signi cantly different from control values BCAAs leucine and valine were also signi cantly improved (50% and 40% decrease, respectively) Isoleucine and citrulline (both elevated in iMSUD animals) were unaffected by cell transplant Other amino acids important to the urea cycle (i.e., arginine, argininosuccinase) were unchanged in all groups Neurotransmitter alterations and brain injury

is characteristic of MSUD Importantly, brain monoamines showed improvements in hAE transplanted animals Dopamine turnover was normalized to control 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 as a therapeutic intervention for MSUD and other liver-based inborn errors of metabolism

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