Telomerase reconstitution immortalizes human hepatocytes

HEPATOLOGYVol. 34, No. 4, Pt. 2, 2 0 0 1

AASLD ABSTRACTS

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TELOMERASE RECONSTITUTION IMMORTALIZES HUMAN HEPATOCYTES. H e n n i n g Wege, Hai T Le, Jian W u , Univ of CA, Davis Med Ctr, Sacramento, CA; Ranjit Girl, Harmeet Malhi, Sanjeev Gupta, Albert Einstein Coll of Medicine, Bronx, NY; Mark A Zern, Univ of CA, Davis Med Ctr, Sacramento, CA

AN INTERIM ANALYSIS OF PERV INFECTIVITY IN 74 PATIENTS TREATED W I T H A BIOARTIFICIAL LIVER IN A PROSPECTIVE, RANDOMIZED, MULTICENTER CONTROLLED TRIAL. Zorina Pitkin, Circe Biomedical, Inc, Lexington, MA; William Switzer, Centers for Disease Control & Prevention, Atlanta, GA; Louisa Chapman, Centers for Disease Control and Prevention, Atlanta, GA; A n t h o n y C Stevens, Barry Solomon, Circe Biomedical, Inc, Lexington, MA; Vedapuri S h a n m u g a m , Vinod Bhullar, A h h a f Hussain, Aprille Matthews, Paul Sandstrom, Walid Heneine, Centers for Disease Control a n d Prevention, Atlanta, GA

Background: The shortage of h u m a n hepatocytes a n d hepatocyte lines with excellent liver-specific functions limits hepatocellular transplantation a n d bioartificial liver support. Therefore, we immortalized h u m a n hepatocytes b y the reconstitution of telomerase, a ribonncleoprotein essential for c h r o m o s o m e stability. Methods: The catalytic telomerase subunit, telomerase reverse transcriptase (TERT), was delivered to cultured h u m a n hepatocytes by retroviral transduction. T r a n s d u c e d cells were evaluated for transgene expression, telomerase activity, g r o w t h ability, a n d hepatocyte-specific functions. Results: W e first transduced isolated fetal h u m a n hepatocytes. Real-time quantitative RT-PCR revealed high levels of TERT expression in transduced cells. After transduction, the relative telomerase activity (RTA), measured by the telomeric repeat amplification protocol, increased from 0.3-+0.2 to 9 8 . 6 + 1 3 . 5 ( p < 0 . 0 0 1 ) . Telomerase-positive cells preserved their m e a n telomere length above 9kb, whereas the m e a n telomere length of u n t r a n s d u c e d control cells decreased below 6kb after 25 population doublings (PD). U n t r a n s d u c e d fetal h u m a n hepatocytes terminated proliferation after approximately 35PD a n d the rate of DNA synthesis, determined by BrdU-incorporation, decreased to 9.6+5.0%. In contrast, telomerase-rescued cells continue to proliferate (70PD to date) a n d sustain high DNA synthesis rates of 35-+3.8% ( p < 0 . 0 0 1 ) . The immortalized fetal h u m a n hepatocytes synthesize albumin, glucose-6-phosphatase, a n d glycogen, d e m o n s t r a t e d b y histochemical a n d immunofluorescent assays. RT-PCR assays revealed that immortalized cells express receptors for a variety of g r o w t h factors, including HGF, EGF, T G F a , TGF]31 and 2, IGF, a n d FGF, similar to primary fetal h u m a n hepatocytes. Moreover, immortalized cells express hepatocyte-enriched transcription factors, s u c h as C/EBPa, w h i c h is also present in p r i m a r y fetal a n d adult h u m a n hepatocytes. W e then transduced hepatocytes isolated from a h u m a n infant liver. Again, transduced cells reached high RTA levels of 56.3 -+4.5. After m o r e than 30 days in culture a n d 5 subpassages post-transduction (to date), the telomerized cells continue to proliferate a n d to express albumin m R N A abundantly, as s h o w n b y quantitative real-time RT-PCR. Conclusions: Telomerase reconstitution immortalizes h u m a n hepatocytes. Immortalized cells expressing TERT retain hepatocyte-specific functions, a n d thus constitute a novel source of cells for liver therapies, including hepatocellular transplantation a n d bioartificial liver support.

Background: The HepatAssistLiver Support system (LAS) is an extracorporeal bioartificial liver containing primary porcine hepatocytes placed in a bioreactor behind a porous membrane. Pig ceils contain endogenous retroviruses capable of infecting human cells in-vitro. However, to date there has been no evidence for porcine endogenous retrovirus (PERV) infection in patients who have been treated with living porcine cells. We previously reported that 28 patients treated with LAS tested negative for PERVinfection. It was also demonstrated that a membrane used in LA5 provides an additional safety barrier by separating patient plasma perfused through LAS from porcine cells. We now present the first report on a prospectively designed study with a comprehensive PERVfollowup in a large (n= 74) patient population treated with LAS.Methods: Patients diagnosed with acute liver failure were prospectively randomized to either standard of care (SOC)(n=56) or LAS treatments (n=74). All patients consented to PERV testing as part of the protocol. LASgroup patients received daily 6-hour LAS treatments. Bloodsamples were collected from both SOC andLAS patients at baseline. Additional blood samples were collected from LAS patients immediately after the Iast LA5 treatment and at 1, 2, 3, 6, 9 and 12 months post randomization. PERVtesting was performed in a blinded fashion by testing (1)peripheral blood lymphocytes (PBLs) for PERVgag and pol sequences and for pig mitochondrial DNA (mtDNA) by PCR, and (2) serum for seroreactivity to PERVantigens by Western blot(Wb). Results: No evidence of PERV infection was identified in any of 74 patients enrolled in 18 centers in the U.S. and Europe and treated with LAS between 1998-2001(for the b'kS study group and the list of participating investigatorsplease see the abstract submitted to AASLD2001 by Stevenset al.). A total of 2341,AS treatments were administered to the 74 LA5group patients; individual patients received frmn 1-9 treatments which averaged 3.2 treatments per patient over a three-day period. Thirty-six patients (49%) received liver transplants and subsequent immunosuppressive therapy for up to 2.5 years. For 34 patients, samples were collected to or beyond 3 months post LAStreatment and 21 patients were at 1-yearpost treatment. Specificantibodies to the diagnostic 30kD PERVgag protein were not detected in any patients. However, 18 patients (14%) in both the SOC (n=5) and LAS (n= 13) groups were found to have pre-existingnon-specific or non-specificseroreativity to only a protein of approximately 55kD also present in the 293 non-infected cell line, but not the major PERVgag protein of 30kD. These were interpreted as non-specific cross reactivity unrelated to the subsequent LAS exposure. These patients were also negative for PERVgag RNA sequences by RT-PCR analysis.No other patients demonstrated any seroreactivityto PERVWb at any point. Pig mtDNA was detected in PBLsfrom 4 patients in samples taken immediately after the last LA5 treatment, indicating presence of pig DNA in 5% of exposed patients. All other samples (including samples collected from these 4 patients at other time points) tested negativefor mtDNAas well as PERVgag and pol. The subsequent samples tested negative for PERV infection. Summary: To date, 74 patients treated with the LAS from 1-9 times, for a total of 234 treatments, have no evidence of PERVinfection at time points ranging from immediatelyafter to 12 months post exposure despite immunosupression in approximately half of the treated patients. Conclusion: This is the first prospectively designed study demonstrating evidence of absence of PERV infection in a large patient population treated multiple times with a membrane based LAS containing porcine hepatocytes.

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I-IEPATOCYTE TRANSPLANTATION AS A TREATMENT FOR GLYCOGEN STORAGE DISEASE TYPE I A. Manrizio Muraca, Alberto Burlina, Daniele Neri, Maria T Vilei, A n n a Granato, Paolo Fehracco, Chiara Ferraresso, Gianpero Giron, Giorgio Gerunda, University of Padova, Padova Italy

INTRASPLENIC TRANSPLANTATION OF ENCAPSULATED CELLS - A NOVEL APPROACH TO CELL THERAPY. Takeshi Aoki, Yutaka Umehara, Chiara Ferraresso, Nozomu Sugiyama, Yvette Middleton, Itzhak Avital, AchiIles A Demetriou, Jacek Rozga, Cedars-Sinai Medical Center, Los Angeles, CA

Hepatocyte transplantation has been successfully used as an alternative to liver transplantation in a few patients with liver-based inherited metabolic disorders. W e treated with this procedure a 47 year-old w o m a n with glycogen storage disease type I A (GSD IA) diagnosed at 3 years of age by clinical presentation a n d enzymatic assay in the liver. Despite of orally administered u n c o o k e d cornstarch every three hours, she was unable to maintain blood glucose constantly above 3.5 mmolA, as required for optimal metabolic control of the disease. Hypertriglyceridemia (20-22 mmol/L; n.v. < 3 . 5 ) , hyperuricemia (0.75 mmol/L; n.y. < 0 . 6 0 ) a n d hyperlactacidemia (4-6.5 mmol/L; n.v. < 2 . 2 ) were also noted. Her liver was enlarged a n d irregular, a n d muhiple adenomas were diagnosed on ultrasound, CT scan a n d NMR. Compliance with dietary treatment was increasingly difficult because of the p o o r quality of life, a n d the patient therefore sought an alternative cure for her hypoglycaemia. Permission to perform allogeneic hepatocyte transplantation was granted b y the Ethics Committee of the University Hospital of Padova a n d both the patient a n d her family gave informed written consent. Two billion viable hepatocytes were infused by gravity at a rate of approximately 25 million cells/min in two separate sessions over a period of 230 m i n via one catheter, while the other catheter was used for separate heparin infusion at a rate of 20 UIfKg/hr. The total n u m b e r of cells infused was limited because of pre-existing portal a n d p u l m o n a r y hypertension. Basal portal pressure was 15 m m Hg a n d it increased u p to 31 m m Hg d u r i n g the second cell infusion, while portal flow decreased in parallel from 14 to 6 cm/s. Basal PAP was 45 m m Hg, a n d increased u p to 50 m m Hg, b u t the procedure was well tolerated. A triple i m m u n o s u p p r e s s i o n regime was initiated immediately with mycophenolate mofetil, tacrolimus a n d steroids. Metabolic stability was achieved in 3 days a n d the patient was placed o n a n o r m a l diet; she was able to tolerate at least 7 hours fasting with n o r m a l blood glucose levels (ranging from 70 to 110 mg/dl), normal lactate a n d free fatty acids. Blood triglycerides d r o p p e d to 7 mmo[/L a n d thereafter showed large fluctuations, but eventually stabilized a r o u n d 10-15 mmol/L. Blood uric acid was not affected. The metabolic i m p r o v e m e n t is persisting six m o n t h s after cell infusion u n d e r tacrolimus immunosuppression. The present observation indicates that hepatocyte transplantation can markedly improve the metabolic defect in GSD IA, with considerable i m p r o v e m e n t in quality of life.

INTRODUCTION: For many diseases, cell therapy may represent a simple, low risk and cost effective alternative to whole organ replacement. Moreover, it could allow efficient use of donor organs, which are in critically short supply. In some instances, whole organ replacement is not even an option. For example, transplantation of pancreatic islets for Type 1 diabetes is presently viewed as the most promising alternative to insulin therapy. Cell therapy is likely to succeed clinically if cells survive at the transplantation site and are protected against rejection. Many techniques have been used over the years for cell transplantation, including subcutaneous, intraperitoneal, intraportal, intrasplenic, and kidney subcapsular injection. However, only with intraportal and intrasplenic injection, are cells placed in the bloodstream and have instant access to oxygen and nutrients. As regards rejection, encapsulation has been shown to allow transplantation of cells without immunosuppression. We hypothesized that transplantation of encapsulated cells into the spleen would offer these two benefits. MATERIALS AND METHODS: Male allogeneic Sprague-Dawley and syngeneic inbred Lewis rats (n=48; 300g) were used. Hepatocytes were harvested by the in situ two-step liver perfusion method. Cells were seeded in 100 kDa and 500 kDa hollow-fibers (polyvinylidene difiuoride PVDF). Rat spleens (n=90) were implanted with 2 fibers, each 2 - cm long. They were either empty or loaded with 5x105 cells. No immunosuppression was used. Rats were killed in batches of 3-5 after 1, 3, 5, 7,14 and 28 days. Additionally, human HepG2 ceils (American Type Culture Collection) were encapsulated using alginate/ poly-L-lysine (ALP) and transplanted into the spleen; control rats were transplanted with free HepG2 cells and with non-encapsulated syngeneic inbred Lewis rat hepatocytes. Again, no immunosuppression was used. Blood human albumin levels were measured using Western blotting; immunostaining for albumin was also performed. RESULTS: Empty fibers induced peri-capsular fibrosis which was slightly greater around the fibers with allogeneic cells. Hepatocytes transplanted in 500-kDa fibers survived for 7 days, while those placed in 100 kDa fibers remained viable for 4 weeks (end of study). Remarkably, ALP capsules, whether with or without HepG2 cells, produced no local fibrotic response. Moreover, encapsulated HepG2 cells remained viable, stained positive for albumin and human albumin was detected in blood samples at all time points studied, i.e., even after 4 weeks; in contrast, non-encapsulated cells lost viability and function after 7 days. In control rats transplanted with syngeneic rat hepatocytes, Western blotting revealed no signal for human albumin. CONCLUSIONS: This study suggests that after intrasplenic transplantation of encapsulated cells, excellent survival of cells, including xenogeneic HepG2 cells, was due to the placement of immunoisolated cells directly into the bloodstream. Further, lack of peri-capsular fibrosis indicates that this novel approach to cell therapy has the potential to allow transplantation of allo- and xenogeneic cells (e.g., pancreatic islets) without immunosuppression.