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Developing an RNAi Therapeutic for Liver Disease Associated with Alpha-1 -Antitrypsin Deficiency

Alfica Sehgal1, Keith S. Blomenkamp2, Stuart Milstein1, Brian R. Bettencourt1, Satya Kuchimanchi1, Klaus B. Charisse1, Amy Simon1, Jeffrey Teckman2, Kevin Fitzgerald1, David Bumcrot1;
1Alnylam Pharmaceuticals, Cambridge, MA; 2Saint Louis University, St Louis, MT

AIM: Liver disease associated with Alpha-1-antitrypsin (AAT) deficiency is a common cause of both morbidity and mortality in patients. We propose that down regulating the mutant allele (Z-AAT) at the mRNA level will prevent the progression and development of liver disease. METHODS: A set of chemically modified siRNAs designed using bioinformatic algorithms was synthesized and screened by transfection in Hep3B cells for activity leading to selection of a highly potent siRNA for further studies. The siRNA was tested in transgenic mice expressing human Z-AAT. These mice develop liver disease and show protein accumulation similar to the human patients. RESULTS: Intravenous administration of theAAT siRNA, formulated in a lipid nanoparticle (LNP-AAT), led to dose-dependent inhibition of human AAT-Z mRNA in livers, with maximum inhibition of >95% observed at a dose of 1 mg/kg, and approximately 85% inhibition at a ten-fold lower dose. Similar reductions in circulating hAAT-Z protein levels were also detected (90% at 1 mg/kg, approximately 50% at 0.1 mg/kg). Additional disease-related endpoints were evaluated in hAAT-Z transgenic mice after seven bi-weekly doses of 0.3 mg/kg LNP-AAT siRNA. Immunoblot analysis of liver lysates demonstrated a 45% reduction in AAT-Z polymers; the precursors of the pathogenic aggregates. A decrease in the extent of liver injury as measured by markers of fibrosis (75% decrease in collagen 1a1 expression) and hepatocyte proliferation (91% reduction in BrdU incorporation) was also observed. Similar physiological benefit as measured by decreased proliferative index was obtained with long-term dosing in aged animals with advanced liver disease. In parallel to the LNP-AAT, we have also developed a N-acetylgalactosamine (GalNAc) -conjugated siRNA (ALN-AAT). This allows for subcutaneous administration and is taken up by the hepatocytes via asialoglycoprotein receptor. ALN-AAT was found to efficiently reduce the levels of mutant circulating protein in these mice in a dose-dependent manner. CONCLUSION: The efficacy achieved by administration of RNA therapeutics targeting AAT in a well-established mouse model of AAT-deficiency associated liver disease supports further development of this novel therapeutic strategy.

Disclosures:

Alfica Sehgal - Employment: Alnylam Pharmaceuticals

Stuart Milstein - Employment: Alnylam

Brian R. Bettencourt - Employment: Alnylam Pharmaceuticals

Satya Kuchimanchi - Employment: Alnylam Pharmaceuticals

Klaus B. Charisse - Employment: Alnylam Pharmaceuticals

Amy Simon - Employment: Alnylam

Jeffrey Teckman - Advisory Committees or Review Panels: The Alpha-1 Project; Consulting: Isis Pharmaceuticals, Arrowhead, Agios; Grant/Research Support: Alnylam, Alpha-1 Foundation; Independent Contractor: Vertex; Speaking and Teaching: Alpha-1 Association

Kevin Fitzgerald - Employment: Alnylam Pharmaceuticals; Patent Held/Filed: Alnylam Pharmaceuticals; Stock Shareholder: Alnylam Pharmaceuticals

The following people have nothing to disclose: Keith S. Blomenkamp, David Bumcrot

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Mutation Analysis of 42 Chinese Patients with Wilson Disease

Yang Wang1,2, Mingran Sun2,3, Shibo Li2, Feng Wang1;
1Department of Infectious Diseases, 1st Hospital of Jilin University, Changchun, China; 2Pediatrics Genetics Laboratory, Oklahoma University Health Sciences Center, Oklahoma City, OK; 3Key Laboratory for Molecular Enzymology and Engineering, College of Life Sciences, Jilin University, Changchun City, China

Background: Mutation of the ATP7B gene is the cause of Wilson's disease(WD), which is an autosomal recessive disorder. The missense mutation Arg778Leu is the most common mutation found in patients ascertained from the southern part of China. However, data regarding the ATP7B mutation in the northern part, especially in Jilin Province, is unknown. Objective: To determine mutation frequency of ATP7B gene in WD patients from Jilin Province of China. Methods: 42 Chinese patients clinically diagnosed with WD from 40 unrelated families from Jilin Province were enrolled in this study. Family members from 19 families were available for the family screening study. 60 healthy Chinese people participated in the control group. DNA was extracted from peripheral blood and whole coding region of the 21 exons of the ATP7B gene were sequenced by Sanger method and analyzed by a commercial mutation software. Results: Mutation analysis revealed 26 identified pathogenic mutations. The frequency of Arg778Leu(c.2333G>T) and Pro992Leu(c.2975C>T) was 28.3% and 14.1% respectively. A novel mutation, Val659Glu (c.1 976C>A) was also identified in one patient. 11 identified mutations haven't been reported in Chinese populations. Family members from 19 family were available for family screening studies. Two family members were found to have compound heterozygous mutations and were diagnosed with WD based on molecular testing results. Conclusions: Arg778Leu and Pro992Leu are most common mutations of ATP7B in Jilin Province. A novel mutation was detected in this study. Molecular assays should be offered for the individual members who are at risk of being affected or carriers of WD.

The novel mutations of Val659Glu (c. 1 976C>A)

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Disclosures:

The following people have nothing to disclose: Yang Wang, Mingran Sun, Shibo Li, Feng Wang

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Hemochromatosis and Arterial Stiffness

Jorge Daruich, Esteban González Ballerga, Ines Alonso, Mariano Duarte, Florencia Yamasato, Sara Schaab, Juan A. Sorda;
Hospital de Clinicas Jose de San Martín, Caba, Argentina

Introduction. Hereditary Hemochromatosis (HH) is a genetically determined disease of iron overload and excessive tissue stored. Recently, an increase of cardiovascular (CV) risk has been reported in this group with controversial data. Pulse wave velocity (PWV) is the simplest way to asses arterial stiffness (AS), and has been used in epidemiological studies demonstrating the predictive value of aortic stiffness for CV events. PWV variation pre and post induced ischemia of brachial artery has been validated for evaluate endothelial dysfunction (ED). We hypothesized that iron-induced vascular fibrosis, and diminish the nitric oxide (NO) vasodilatation that could lead to AS and ED. Material and methods. In this prospective study we tested carotid-femoral PWV for AS (structural impact evaluation) and carotid-braquial PWV variation pre and post induced ischemia for ED (functional component evaluation) in 22 Hereditary Hemochromatosis (HH) patients with diagnostic liver biopsies without diabetes, 48,2 ±15,7 years old compared with healthy patients. Age, sex, weight, height, hips, BMI, blood pressure and heart rate were also recorded. PWV and PWV variation we asses by Complior System. Statistical Methods. Chi-square and the Fisher exact tests. A value of p <0.05 indicated statistical significance. Results. In HH was observed a CF PWV 8,3 ± 1,6 m/s vs. 6,4±0,8 m/s in control group (CG) (p<0.001) and 8,2% PWV reduction in CG and only 0,3 % in HH (p<0.001). Conclusions. These results show a arterial structural and functional impact and they could be considered as a target organ in HH. Our findings may support features research including more patients with HH and others iron overload disorders.

PWV and PWV variation

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Disclosures:

The following people have nothing to disclose: Jorge Daruich, Esteban González Ballerga, Ines Alonso, Mariano Duarte, Florencia Yamasato, Sara Schaab, Juan A. Sorda

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Predicting C282Y homozygote genotype for hemochromatosis using serum ferritin and transferrin saturation in 44,809 participants from the HEIRS Study

Andrew Lim1, Mark Speechley2, Paul Adams1;
1Gastroenterology, University Hospital, London, ON, Canada; 2Epidemiology and Bio-statistics, Western University, London, ON, Canada

Background: The simultaneous interpretation of serum ferritin and transferrin saturation has been used as a clinical guide to diagnose genetic hemochromatosis. Previous studies have used a nomogram and Bayes' theorem in referred patients (McGrath et al, J Lab Clin Med 2002,140-6-8). The HEIRS study screened 101,168 North American participants with serum ferritin, transferrin saturation and C282Y genotyping for the HFE gene. Methods: A subsample of Caucasians (n = 44,809) and logistic regression (Bernoulli regression with logit link) were used to predict individual probabilities of HFE C282Y homozygosity using serum ferritin and transferrin saturation levels. Men (n = 17,323) and women (n = 27,486) were analyzed separately. Regression equations were evaluated using Area Under the Curve (AUC) from receiver operating curve (ROC) analysis and variance explained by Nagelkerke's pseudo R squared. In addition, an Android smartphone App and website application were developed to provide physicians with easy access to predicting C282Y homozygotes of the HFE gene. Results: The logistic regression equation for men had an area under the ROC curve of 0.91 for men and 0.89 for women. The pseudo R square for men was 0.44 and for women was 0.34. There were 137 male (ferritin range 7.5 - 5,200 μg/L) and 177 female C282Y homozygotes (ferritin range 7.5 - 2,960 μg/L). An example of the analysis is a Caucasian man with a transferrin saturation of 50% and a ferritin of 500 μg/L has a 1.3 % [95% Confidence Interval 1.1 - 8.8 %] probability of being a C282Y homozygote. Website model was presented at lim.brichacek.ca. Conclusions: A large primary care based sample of 44,809 participants has led to the development of a new computer/smartphone tool to predict the probability of being a C282Y homozygote of the hemochromatosis gene from serum ferritin and transferrin saturation.

Disclosures:

The following people have nothing to disclose: Andrew Lim, Mark Speechley, Paul Adams

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Prevalence and Risk factors of Iron Overload in Korean Population : Analysis of the Fifth Korea National Health and Nutrition Examination Survey (KNHANES V-1)

Seong Min Chung, Sukho Hong, Eun Sun Jang, Sang Soo Lee, Jung Wha Chung, Sung Wook Yang, Jin-Wook Kim, Sook-Hyang Jeong;
Department of Internal Medicine, Seoul National University Bun-dang Hosipital, Seongnam, Republic of Korea

Background: Excessive accumulation of iron can result in systemic damage and organ dysfunction. However, the prevalence and risk factors of iron overload in Korea population has never been studied. Methods: The results of serum iron-related laboratory tests obtained from 6,007 subjects (≥ 18 years old) who participated in the blood examination as a part of the Fifth Korea National Health and Nutrition Examination Survey (KNHANES V-1) in 2010 were analysed. Case weights that account for the unequal probabilities of selection were applied for all analyses due to the complex sample design of the survey. Results: Among the study population, mean age of 44.19 years, mean serum ferritin levels were 124.7 ng/mL (95% CI, 117.77-131.69) in males, 46.4 ng/mL (95% CI, 44.07-48.65) in females, respectively. Mean transferrin saturation (TS) levels were 41.5% (95% CI, 40.47-42.49) in males, 31.8% (95% CI, 31.07-32.48) in females. The prevalence of iron overload (defined as TS>55% or ferritin >300 for males and TS>50% or ferritin >200 for female) were 21.5% in males, 10.2% in females, respectively. In the iron overload group, both frequencies of current smoking and alcohol drinking were significantly higher than those with normal iron load. The proportion of subjects having liver disease (according to the medical history or abnormal laboratory finding) was significantly higher in iron overload group (36.8%) than in normal iron load group (22.8%, p<0.001). Interestingly, body mass index (BMI) and an index of insulin resistance indicated as HOMA-IR showed lower levels in iron overload group than normal iron load group (BMI 23.6 vs. 23.4 kg/m2, HOMA-IR 2.49 vs. 2.60, p<0.001). In multivariable analysis, iron overload was independent predictive factor of liver disease (OR=1.47; 95% CI, 1.470-1.476) after adjustment for age, gender, anemia, HOMA-IR and current drinking amounts. Conclusions: Iron overload in general Korean population was not associated with insulin resistance, but related with presence of liver disease. Therefore, the prognostic role of iron overload in Korean patients with liver disease should be documented in the future research.

Disclosures:

The following people have nothing to disclose: Seong Min Chung, Sukho Hong, Eun Sun Jang, Sang Soo Lee, Jung Wha Chung, Sung Wook Yang, Jin-Wook Kim, Sook-Hyang Jeong

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Prevalence and Related Factors of Iron Overload in Patients with Hepatocellular Carcinoma in a Hepatitis B Prevalent Region

Sukho Hong1, Eun Shin2, Haeryoung Kim2, Ho Seong Han3, Jai Young Cho3, Yoo-Seok Yoon3, Sang Soo Lee1, Jung Wha Chung1, Sung Wook Yang1, Seong Min Chung1, Eun Sun Jang1, Jin-Wook Kim1, Sook-Hyang Jeong1;
1Internal medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea; 2Pathology, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea; 3Surgery, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea

Background: Excessive accumulation of iron can result in hepatic dysfunction. However, the prevalence and related factors of iron overload in hepatocellular carcinoma (HCC) patients in hepatitis B virus prevalent region was limitedly studied. We analyzed the prevalence, cellular localization and related factors of liver iron overload using liver tissue samples obtained from HCC patients. Methods: We retrospectively enrolled 221 HCC patients who underwent surgical resection from May 2003 to Dec 2011 in Seoul National University Bundang Hospital. Hepatic iron content was measured semi-quan-titatively using adjusted Deugnier's method and simplified Scheuer method in non-tumorous liver samples. The cellular localization of iron deposit was assessed by calculation of hepatocyte iron score (HIS), sinusoidal iron score (SIS) and portal iron score (PIS), and summation of these 3 scores presented as total iron score (TIS) according to adjusted Deugnier's method. Iron distribution at hepatocytes and Kupffer cells according to Scheuer's method was also investigated. The clinical variables related to hepatic iron overload were assessed. Results: Hepatic iron deposition was found in 43% of HCC patients. The mean HIS, SIS, PIS, TIS and corrected TIS (cTIS) by Deugnier's criteria was 2.5, 0.3, 0.1, 2.9 and 1.7, respectively. Hepatocyte and Kupffer cell iron deposition by Scheuer's criteria was 38% and 24%, respectively. The iron overload group (cTIS ≥ 2) showed significantly higher male proportion, higher hemoglobin level, higher mean corpuscular volume (MCV), and higher MELD score than the group showing no iron overload (cTIS < 2). Iron overload at Kupffer cells but not at hepatocytes, was significantly associated with higher mortality. Conclusions: The prevalence of iron overload (cTIS ≥ 2) in HCC was 34%, and the factors related to hepatic iron overload were male, higher levels of MCV, hemoglobin, and MELD score. Iron overload at Kupffer cells rather than at hepatocyte suggests poor prognosis, which warrants further study.

Disclosures:

The following people have nothing to disclose: Sukho Hong, Eun Shin, Haeryoung Kim, Ho Seong Han, Jai Young Cho, Yoo-Seok Yoon, Sang Soo Lee, Jung Wha Chung, Sung Wook Yang, Seong Min Chung, Eun Sun Jang, Jin-Wook Kim, Sook-Hyang Jeong