ORAL PRESENTATIONS

Table 3 (abstract O134).

Biliary epithelial cell (BEC) activation, % expression (n =5)

CD4+CD28−ve CD4+CD28+ve CD8+CD28−ve CD8+CD28+ve

ICAM-1 33.43±16 9.34±4.2 28.4±13.4 16.17±5.7HLA-DR 52.1±15.6 33.54±6.06 46.59±17.01 49.9±14.2CD40 33.22±18.3 30.1±16.6 46.9±14.3 34.4±16.5

Table 4 (abstract O134).

CD28−ve frequency (after 21 days in culture)a Significance

Untreated 1,25(OH)2D3 TNFa TNFa+1,25(OH)2D3

% CD28*−ve 22.94±6.34 1.96±0.8 37.6±5.2 3.66±1.29 *p < 0.05 (Untreated vs 1,25(OH)2D3

**p < 0.01 (1,25(OH)2D3 vs TNFa)*p < 0.05 (TNFa vs TNFa+1,25(OH)2D3)

a All cells were treated initially with aCD3/aCD28 beads and IL-2 (50U/ml) was supplemented in culture.

Conclusions: A high proportion of CD28−ve T cells are present in

PSC liver. Vitamin D abrogates the pathogenic effects of CD28−ve

cells representing a therapeutic opportunity.

O135

INHIBITION OF INTESTINAL BILE ACID ABSORPTION

BY ASBT INHIBITOR A4250 PROTECTS AGAINST

BILE ACID-MEDIATED CHOLESTATIC LIVER INJURY IN MICE

A. Baghdasaryan1, P. Jha1, M. Muller1, N. Auer1, A. Deutschmann2,

C. Zohrer2, I. Pahlman3, H. Graffner3, P. Fickert2, M. Trauner1.1Hans Popper Laboratory of Molecular Hepatology, Division of

Gastroenterology and Hepatology, Department of Internal Medicine III,

Medical University of Vienna, Vienna, 2Laboratory of Experimental and

Molecular Hepatology, Division of Gastroenterology and Hepatology,

Department of Internal Medicine, Medical University of Graz, Graz,

Austria; 3Albireo, Gothenburg, Sweden

E-mail: anyutabag@gmail.com

Background and Aims: Approximately 95% of biliary bile acids

(BAs) are reabsorbed in the gut and circulate back to the liver for

further secretion into bile. Therefore, pharmacological inhibition of

the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2)

may protect against BA-mediated liver injury in cholestasis.

Methods: 8-week-old Mdr2−/− (Abcb4−/−) mice (model of cholestatic

liver injury and sclerosing cholangitis) received either a diet

supplemented with A4250 (0.01% w/w) – a highly potent and

selective ASBT inhibitor – or a chow diet and liver injury was

assessed after 4 weeks. Bile flow and composition were analyzed

after 1 week. As a complementary model, wild type mice received

A4250-supplemented diet over 1 week before and after common

bile duct ligation (CBDL).

Results: Compared with chow-fed Mdr2−/− mice A4250 significantly

reduced serum ALT (111±22 vs. 484±214U/L), ALP (122±9 vs.

210±47U/L) and serum BAs (25±6 vs. 86±30mmol/L), inhibited

proinflammatory (Tnf-a, Vcam-1, Mcp-1) and profibrogenic (Col1a1,

Col1a2) gene expression and reduced bile duct proliferation

(mRNA and immunohistochemistry for K19). Furthermore, A4250

significantly reduced bile flow (0.9±0.2 vs. 1.7±0.2ml/gLW/min)

and biliary BA output (3.1±1.6 vs. 35.6±13.0mmol/L/gLW/min),

which correlated with Bsep repression, while Ntcp and Cyp7a1

were induced. In CBDL mice, A4250 reduced serum ALP

(711±212 vs. 1278±355U/L, p < 0.05) and BA levels (168.8±57.7

vs. 249.8±141.3mmol/L n.s.).

Conclusions: Pharmacological ASBT inhibition attenuates

cholestatic liver injury by reducing biliary BA output.

O136

A NOVEL, HYBRID RECOMBINANT AAV-piggyBac TRANSPOSON

VECTOR PERMITS ROBUST LONG-TERM PHENOTYPE

CORRECTION OF THE PROGRESSIVE FAMILIAL INTRAHEPATIC

CHOLESTASIS TYPE 3 MOUSE MODEL IN VIVO

S.M. Siew1,2, S.C. Cunningham1, I.E. Alexander1. 1Gene Therapy

Research Unit, Children’s Medical Research Institute, 2Department of

Gastroenterology, The Children’s Hospital at Westmead, Sydney, NSW,

Australia

E-mail: smsiew777@yahoo.com.au

Background and Aims: Efficient liver-targeted gene transfer using

recombinant adeno-associated viral (rAAV) vectors has led to

promising therapeutic efficacy in pre-clinical models and clinical

trials. However, delivery of conventional, predominantly non-

integrating rAAV vectors to juvenile mice results in loss of persistent

transgene expression in the liver due to rapid liver growth and

hepatocyte proliferation. The piggyBac transposon system permits

stable genomic modification of mammalian cells, but targeted

delivery is relatively inefficient in vivo.

Aim: To develop a hybrid vector, utilising high transduction

efficiency of rAAV together with piggyBac transposase-mediated

somatic integration to stably express human ABCB4 under a

hepatocyte-specific promoter/enhancer in vivo, and to correct

chronic liver disease in a murine model of Progressive

Familial Intrahepatic Cholestasis type 3, lacking canalicular

phosphatidylcholine translocation.

Figure: Comparison of biliary phosphatidylcholine concentrations.

Methods: Neonatal Abcb4−/− mice were injected intraperitoneally

with 5×1011 vector genomes of the hybrid vector, co-administered

with a rAAV expressing piggyBac transposase (pBase+), and were

Journal of Hepatology 2014 vol. 60 | S45–S66 S57

ORAL PRESENTATIONS

compared to untreated homozygotes and those that receive hybrid

vector without co-administered transposase vector (pBase−).

Results: The pBase+ cohort had stable, persistent expression

of hABCB4, resulting in phenotype correction, extending into

adulthood. pBase+ homozygotes exhibited no hepatosplenomegaly

with dramatically reduced periportal inflammation and liver

fibrosis, in contrast to untreated and pBase− controls. A single

therapeutic injection at birth led to >8-fold increased mean biliary

phosphatidylcholine concentration in juveniles (4 weeks) and

adults (12 weeks), compared with untreated and pBase− controls.

Conclusions: This novel and powerful hybrid rAAV-piggyBac

transposon vector strategy has potential for treating a wide

array of inherited liver diseases of early onset, and with further

development, may translate into future therapeutic benefit in the

clinic.

O137

4-PHENYLBUTYLATE AMELIORATES LIVER FIBROSIS IN

PATIENTS WITH PROGRESSIVE FAMILIAL INTRAHEPATIC

CHOLESTASIS (PFIC) TYPE 2 AND PRURITUS IN PATIENTS WITH

PFIC TYPE 1

Y. Hasegawa1, H. Kondou1, S. Naoi2, K. Bessho1, M. Ukitsu3,

M. Sasaki3, T. Tsunoda4, A. Inui4, H. Nagasaka5, Y. Miyoshi1,

H. Hayashi2. 1Department of Pediatrics, Graduate School of

Medicine, Osaka University, Suita City, 2Laboratory of Molecular

Pharmacokinetics, Graduate School of Pharmaceutical Sciences,

The University of Tokyo, Tokyo, 3Department of Pediatrics, School

of Medicine, Iwate Medical University, Morioka, 4Department of

Pediatric Hepatology and Gastroenterology, Saiseikai Yokohamashi

Tobu Hospital, Yokohama, 5Department of Pediatrics, Takarazuka City

Hospital, Takarazuka, Japan

E-mail: yhase@ped.med.osaka-u.ac.jp

Background and Aims: Progressive familial intrahepatic cholestasis

type 1 (PFIC1) and 2 (PFIC2) are caused by mutation in FIC1/ATP8B1

and BSEP/ABCB11 genes, and lead to cholestatic cirrhosis with

intractable pruritus during infancy. No medical therapy has

been established for these diseases. We previously reported that

treatment with 4-phenylbutyrate (4PB) increased BSEP expression

on hepatocanalicular membrane (Hayashi H et al. Hepatology 2007),

suggesting its therapeutic potency for patients with PFIC1 and

PFIC2 who retain BSEP function. In this study, we aim to evaluate

therapeutic effects of 4PB in patients with both PFIC1 and PFIC2.

Methods: Patients with PFIC1 (n =5) and PFIC2 (n =2) were enrolled

in this study. 4PB (Swedish Orphan Biovitrum, Sweden) was

administered orally with gradually increasing dosages (200, 350,

and 500mg/kg/day) for 6 months. Biochemical and clinical data

were collected, liver biopsies were performed before and at the

end of the therapy, and severity of pruritus was scored according

to previous reports.

Results: Liver function tests including AST, ALT, total and direct

bilirubin were improved after administration of 4PB in the patients

with PFIC2, but not with PFIC1. Fibrosis and cholestasis were

improved, and partial restoration of BSEP expression at the

canalicular membrane were detected in the liver from PFIC2

patients. In PFIC1 patients, pruritus was markedly attenuated and

they were released from sleep disturbance during the therapy.

Conclusions: 4PB might be a new therapeutic medication for PFIC1

patients who have intractable pruritus and PFIC2 patients who

retain BSEP function, resulting in improvement of their quality of

lives.

O138

PHASE 1 CLINICAL TRIAL OF LIVER DIRECTED GENE THERAPY

WITH rAAV5-PBGD IN ACUTE INTERMITTENT PORPHYRIA:

PRELIMINARY SAFETY DATA

D. D’Avola1,2, E. Lopez-Franco3, P. Harper4, A. Fontanellas3,

N. Grosios5, B. Sangro1,2, A. Henrichson4, F. Salmon5, C. Fuertes1,

S. Abecia6, A. Paneda7, M. Paz7, M.E. Cornet7, M.D.M. Municio7,

I. Troconiz8, C. Kaeppel9, R. Juan7, C. von Kalle9, M. Schmidt9,

H. Petry5, G. Gonzalez-Aseguinolaza3, R. Enriquez de Salamanca6,

J. Prieto1,2,3. 1Liver Unit, Clinica Universidad de Navarra, 2Clinica

Universidad de Navarra, Centro de Investigacion Biomedica

en Red de Enfermedades Hepaticas y Digestivas (CIBEREHD),3Division of Hepatology and Gene Therapy, Centro de Investigacion

Medica Aplicada (CIMA), Pamplona, Spain; 4Porfyricentrum

Sverige, Karolinska Universitetssjukhuset, Solna, Sweden; 5UniQure,

Amsterdam, Netherlands; 6Centro de Investigacion, Hospital 12 de

Octubre, Madrid, 7DIGNA Biotech, 8Department of Pharmacy and

Pharmaceutical Technology, Universidad de Navarra, Pamplona, Spain;9National Center for Tumor Diseases (NCT) and German Cancer

Research Center (DKFZ), Heidelberg, Germany

E-mail: ddavola@unav.es

Background and Aims: Acute intermittent porphyria (AIP) is a

rare genetic disease due to mutations in the gene encoding

porphobilinogen deaminase (PBGD). The aim of this trial is to

explore the safety of liver-directed gene therapy in patients with

severe AIP.

Methods: In this first-in-human phase 1 clinical trial, 4 increasing

doses of a recombinant adeno-associated serotype 5 vector

expressing a codon-optimized human-PBGD transgene under

the control of a liver-specific promoter (rAAV5-PBGD) were

administered to 8 patients with severe AIP by single intravenous

infusion. Besides safety, humoral and T-cell response against capsid

proteins and recombinant protein as well as vector shedding were

analyzed.

Results: rAAV5-PBGD was well tolerated. No treatment-related

serious adverse events were observed. No changes in blood cell

count, liver and renal tests were observed with a median follow-

up of 8 months (range 3–11). One patient at the highest dose

experienced an AIP attack 3 days post-injection. The transient,

slight increase in transaminases detected in this patient was likely

related to the attack and not to the vector. All patients developed

neutralizing antibodies against AAV capsid. No T-cell response was

detected within the 12 weeks post-treatment follow-up. Vector

clearance from all biological fluids was completed after 2 weeks in

the lowest dose and 4 weeks in the highest dose cohorts.

Conclusions: The treatment with rAAV5-PBGD is safe in patients

with AIP. No cellular immune response against the vector or the

transgene was detected.

S58 Journal of Hepatology 2014 vol. 60 | S45–S66