704 long term correction of crigler najjar syndrome and scale up production of an optimized AAV8 vector expressing the UGT1A1 transgene

704 Long Term Correction of Crigler Najjar Syndrome and Scale Up Production of an Optimized AAV8 Vector Expressing the UGT1A1 Transgene Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright ©[.]

Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy

S280

Genetic DisorDers anD Metabolic liver Disease

702 Long Term Metabolic Correction of Wilson

Disease

Oihana Murillo,1 Daniel Moreno-Luqui,1 Cristina Gazquez,1

Debora Martínez-Espartosa,2 Ignacio Monreal,2 Laura Guembe,1

Armando Moreno,3 Fernando Corrales,3 Jesús Prieto,1,3 Ruben

Hernández-Alcoceba,1 Gloria González-Aseguinolaza.1

1 Gene Therapy and Regulation of Gene Expression, FIMA,

Pamplona, Spain; 2 University Clinic of Navarra, UNAV,

Pamplona, Spain; 3 Hepatology program, FIMA, Pamplona, Spain.

Wilson disease (WD) is an autosomal recessively inherited copper

storage disorder due to mutations in the ATP7B gene resulting in

impaired biliary copper excretion and hepatic copper accumulation

The increased hepatic copper concentration causes hepatocellular

injury of variable intensity from acute liver failure to chronic hepatitis

evolving to cirrhosis Most often the first manifestation of the disorder

is the hepatic disease which may be followed by copper deposition

in the brain with resulting neurological damage Current treatments

are based on copper chelators that promote urinary excretion of the

metal These therapies should be maintained lifelong, may cause

side effects and do not restore normal copper metabolism In this

work we assessed the efficacy of gene therapy to treat this condition

We transduced the liver of Atp7b-/- mice (a model of WD) with an

adenoassociated vector serotype 8 (AAV8) encoding the human

ATP7B cDNA placed under the control of the liver-specific

a1-antitripsin promoter After vector administration we performed serial

determinations of serum transaminases, serum holoceruloplasmin

concentration and ferroxidase activity and urinary copper excretion

In addition we assessed liver cooper concentration, oxidative status

of hepatic proteins and histological liver damage in samples obtained

at 6 months after therapy We observed a dose-dependent therapeutic

effect manifested by the reduction of transaminasemia and urinary

copper excretion and normalization of serum holoceruloplasmin

Furthermore, we documented complete reversal of all hepatic

alterations, including copper content, histopathological changes and

protein oxidation Conclusion: our data demonstrate that gene therapy

provides long term correction of WD in a clinically relevant animal

model of this disorder

703 Liver-Directed Gene Therapy for Primary

Hyperoxaluria Type 1

Raffaele Castello,1 Roberta Borzone,1 Patrizia Annunziata,1

Pasquale Piccolo,1 Nicola Brunetti-Pierri.1,2

1 Telethon Institute of Genetics and Medicine, Pozzuoli, Naples,

Italy; 2 Translational Medicine, Federico II University, Naples,

Italy.

Primary hyperoxaluria type 1 (PH1) is an inborn error of

liver metabolism due to deficiency of peroxisomal enzyme

alanine:glyoxylate-aminotransferase (AGT) which catalyzes the

conversion of glyoxylate to glycine In PH1 patients, glyoxylate

cannot be converted into glycine and is oxidized to oxalate resulting

in hyperoxaluria The excess of oxalate causes deposition of

insoluble calcium oxalate in the kidney and other tissues leading

to nephrolithiasis, nephrocalcinosis, kidney failure, and systemic

tissue damage Combined liver/kidney transplantation is the only

available therapeutic strategy for disease treatment Gene therapy

is an attractive option to provide a definitive cure for PH1 Towards

this goal, we investigated helper-dependent adenoviral (HDAd)

vectors for liver-directed gene therapy of PH1 We injected PH1 mice

with an HDAd encoding AGT under the control of a liver-specific

promoter and observed normalization of urinary oxalate at the doses

of 5x10e12 and 1x10e13 vector particles (vp)/kg and partial correction

with 1x10e12 vp/kg Following challenge with Ethylene Glycol

(EG), a precursor of glyoxylate, we observed reduced elevations of

urinary oxalate in HDAd-injected mice compared to saline controls

Next, we hypothesized that overexpression of glyoxylate reductase/ hydroxypyruvate reductase (GRHPR) or glutamate-pyruvate transaminase (GPT) results in reduction of hyperoxaluria in PH1 by steering glyoxylate towards glycolate synthesis or transamination, respectively To test this hypothesis, we injected PH1 mice with HDAd vectors expressing GRHPR or GPT Both vectors resulted

in significant reduction of hyperoxaluria and co-injection of the two vectors resulted in long-term normalization of oxalate excretion Therefore, metabolic diversion towards non-toxic metabolites has potential for treatment of hyperoxaluria and vector-mediated GRHPR and/or GPT overexpression may be an alternative or adjunctive strategy to enhance efficiency of gene replacement therapy for PH1

We have recently developed a minimally invasive method to improve the therapeutic index of HDAd based on balloon occlusion catheter to achieve preferential delivery of the vector to the liver (Brunetti-Pierri et al., 2009 and 2012) This method may permit in humans correction of PH1 using clinically relevant lower doses of HDAd Based on risk:benefit assessment, PH1 is an attractive disease for clinical application of this method

704 Long-Term Correction of Crigler-Najjar Syndrome and Scale-Up Production of an Optimized AAV8 Vector Expressing the UGT1A1 Transgene

Giulia Bortolussi,1 Fanny Collaud,2 Remco van Dijk,3 Giuseppe Ronzitti,2 Severine Charles,2 Samia Martin,2 Alban Vignaud,2 Florence Lacoste,2 Christine Le Bec,2 Matthias Hebben,2 Fulvio Mavilio,2 Piter Bosma,3 Andres F Muro,1 Federico Mingozzi.2

1 Mouse Molecular Genetics Group, ICGEB, Trieste, Italy;

2 Genethon, Evry, Paris, France; 3 Tytgat Institute for Liver and Intestinal Research, Amsterdam, Netherlands.

Crigler-Najjar syndrome (CN) is an autosomal recessive rare disorder caused by mutations in the UDP-glucuronosyltransferase 1 isotype A1 (UGT1A1) gene In severe CN, lack or reduced activity

of UGT1A1 results in high levels of serum unconjugated bilirubin (UCB), which can lead to brain damage and death Treatment of CN consists of phototherapy for 10-12 hours per day to convert UCB into soluble photoisomers without the need of conjugation, which presents several limitations and has a major impact on life quality of the patients Liver transplantation is the only curative option for CN The limited therapeutic options available prompted us to develop a new therapy for CN based on the transfer of a corrected copy of the UGT1A1 gene to hepatocytes

We developed an AAV8 vector optimized for the liver expression of the hUGT1A1 transgene (AAV8-hUGT1A1) Safety and efficacy of correction of total serum bilirubin (TB) levels with AAV-hUGT1A1 were demonstrated in mouse and rat models of CN at vector doses as low as 5x10 11 vector genomes (vg)/kg, a result confirmed also by the detection of conjugated bilirubin in bile of treated rats In juvenile

CN rats, long-term correction of total bilirubin levels were observed for more than 8 months following AAV8-hUGT1A1 gene transfer

In neonate Ugt1a1-/- mice, intraperitoneal delivery of the vector at doses as low as 1x10 9 vg/mouse resulted in correction of TB at 4 weeks However, vector delivery in P2 and P4 animals resulted in lower efficiency of correction when compared with P11-transduced mice, a finding likely due to the more advanced development of the liver at later time points

Based on these data, dose finding studies were performed in CN rats, which showed a profile of liver transduction with AAV8 vectors similar to humans, i.e lower efficiency of transduction than mice

No differences in efficacy of correction of TB were observed in male

vs female rats

A scalable process for the production of AAV8-hUGT1A1 production was established based on a protocol of triple transfection

Molecular Therapy Volume 23, Supplement 1, May 2015

Genetic DisorDers anD Metabolic liver Disease

of suspension HEK293 cells, leading to high yield vector preparations

with excellent purity profile In vitro transduction assays in human

hepatocytes based on Western blot for hUGT1A1 protein and in

vivo studies in CN rats demonstrated that vectors produced with the

suspension process have potency characteristics undistinguishable

from research grade vectors produced by triple transfection of

adherent cells and purified by cesium chloride gradient centrifugation

In conclusion, our data demonstrate the safety and efficacy of gene

transfer for Crigler-Najjar syndrome in two relevant animal models of

the disease and provide tools and a strong rationale for the translation

of these results in human subjects

705 Selective Advantage of Hepatocytes

Expressing Wild-Type Alpha-1 Antitrypsin (AAT) in

a Novel Human Liver Xenograft Model: A Model for

Correction of a Gain-Of-Function Mutation

Qiushi Tang,1 Michael S BrehmM,2 Terence R Flotte,1 Darcy L

Reil,2 Leonard D Shultz,3 Dale L Greiner,2 Christian Mueller.1

1 Gent Therapy Center, UMass Medical School, Worcester, MA;

2 Diabetes Center of Excellence, UMass Medical School, Worcester,

MA; 3 The Jackson Laboratory, Bar Harbor, ME.

Hepatocytes play a central role in energy metabolism and secretion

of plasma proteins and represent an important target for gene therapy

of single gene disorders Alpha-1 antitrypsin (AAT) deficiency

is one such disorder, in which one common missense mutation

(E342K, known as the PI*Z allele) results in impaired secretion of

AAT In the majority of patients with AAT deficiency, this causes a

lung disease due to a lack of the normal AAT-mediated protection

of lung elastin from neutrophil elastase In a subset of patients,

accumulation of Z-mutant AAT protein triggers hepatocyte injury

leading to inflammation and cirrhosis As vector-mediated methods

for correction of these two defects have been developed for both in

vivo and ex vivo use, we sought to determine whether correction of

the Z-mutant defect in hepatocytes might confer a selective advantage

for repopulation of hepatocytes within an intact liver To test this

concept, a transgenic mouse strain expressing a human PI*Z allele

was crossed with the NOD-SCID-gamma chain knockout (NSG)

strain to create a recipient strain (PI*Z-NSG) for human hepatocyte

xenotransplantation Initial comparisons indicated that PI*Z-NSG

recipients support more efficient engraftment of normal (wt-AAT)

human primary hepatocytes as compared with NSG recipients A

number of procedures to optimize primary hepatocyte engraftment,

and pretreatment of the recipient with monocrotaline further

accentuated the level of engraftment Finally, we propose to test the

paradigm of using the selective advantage of wt-AAT hepatocytes

to model gene and cell therapy of AAT deficiency, through the

introduction of a dual-function lentivirus vector expressing both a

myc-tagged wild-type AAT and a synthetic miRNA to knockdown the

endogenous allele The NSG-PI*Z recipient strain could be used as

a platform for future studies of both cell-based and genetic therapies

of AAT deficiency and further could predict a selective advantage of

corrected cells in AAT deficient patients In addition this new liver

xenograft model provides a setting in which to easily a reproducibly

repopulate a mouse liver with human hepatocytes

*These authors contributed equally to this work

of AAV9-Iduronidase Prevents Emergence

of Neurologic Disease and Neurocognitive Dysfunction in a Murine Model of

Mucopolysaccharidosis Type I

Lalitha Belur,1 Megan Buckvold,1 Kelly Podetz-Pedersen,1 Maureen Riedl,2 Lucy Vulchanova,2 Leah R Hanson,3 Karen Kozarsky,4 William H Frey,3 Walter C Low,5 Carolyn Fairbanks,2

R Scott McIvor.1

1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2 Neuroscience, University of Minnesota, Minneapolis, MN; 3 Alzheimer’s Research Center, Regions Hospital,

St Paul, MN; 4 REGENXBIO Inc., Washington, DC; 5 Neurosurgery, University of MInnesota, Minneapolis, MN.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive storage disease caused by deficiency of alpha-L-iduronidase (IDUA), resulting in accumulation of glycosaminoglycans (GAGs) In the severe form of the disease (Hurler syndrome), death results by age

10 Current treatments for this disease include hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) However, ERT is ineffective in treating CNS disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier, and while there is neurologic benefit to HSCT the procedure is associated with significant morbidity and mortality

We have taken a novel approach to treat neurologic disease associated with Hurler syndrome, using intranasal administration

of an IDUA-encoding AAV9 vector A CAGS regulated AAV9-IDUA vector was infused intranasally into adult mice (2-3 months

of age) that had been immunotolerized at birth with Aldurazyme to prevent anti-IDUA immune response Mice sacrificed at 3 months post-infusion exhibited IDUA enzyme activity levels that were 100-fold that of wild type in the olfactory bulb, with wild type levels of enzyme restored in all other parts of the brain Intranasal treatment with AAV9-IDUA also resulted in clearance of tissue GAG storage materials in all parts of the brain QPCR analysis of vector genomes indicated only background levels in all portions of the brain There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb but there was no evidence for the presence of transduced cells in other portions of the brain This indicates that clearing of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain At 6 months of age, intranasally treated animals along with age-matched heterozygote and IDUA-deficient control animals were subjected to neurocognitive testing using the Barnes maze Unaffected heterozygote animals exhibited improved performance in this test while MPS I mice displayed a deficit in locating the escape Remarkably, MPS I mice treated intranasally with AAV9-IDUA exhibited behavior similar

to the heterozygote controls, demonstrating prevention of the neurocognitive deficit seen in the untreated MPS I animals There was

no significant difference between heterozygote animals and treated animals, while latency to escape was significantly different between these two groups and MPS I deficient animals (P<0.001) This novel, non-invasive strategy for intranasal AAVIDUA administration could potentially be used to treat CNS manifestations of several disorders, including lysosomal storage diseases