Lysosomal Acid Lipase Deficiency Lipid Insights December 9, 2013

Lysosomal Acid Lipase Deficiency

Lipid Insights December 9, 2013

Daniel J. Rader, MD LAL Deficiency

. History . Pathophysiology and genetic epidemiology . Liver disease . Dyslipidemia and atherosclerosis . Diagnostic considerations . Therapeutic considerations . Summary Biology of Lysosomal Acid Lipase

LDL particle lysosome

Free cholesterol and free fatty acids

nucleus

Hepatocyte Lysosomal Acid Lipase (LAL) Deficiency: A single disease, with marked clinical heterogeneity

LAL Deficiency is sometimes called: . Wolman disease (infantile presentation with growth failure) . Cholesteryl Ester Storage Disease or CESD (LAL deficiency presentation in children and adults) . Acid Cholesteryl Ester Hydrolase Deficiency, Type 2 . Acid Lipase Disease . Cholesteryl Ester Hydrolase Deficiency Storage Disease . LIPA Deficiency LAL Deficiency

. History . Pathophysiology and genetic epidemiology . Liver disease . Dyslipidemia and atherosclerosis . Diagnostic considerations . Therapeutic considerations . Summary A brief history: The first phenotypic descriptions

. Abramov, Shorr, and Wolman, 1956: “Generalized xanthomatosis with calcified adrenals” (“Wolman” disease) . Fredrickson, 1963: “Cholesterol ester storage disease” – “…likely a specific hepatic defect in cholesterol ester metabolism.” . Patrick and Lake : “Deficiency of an acid lipase in Wolman’s disease”, Nature, 1969 . Burke and Schubert : “Deficient activity of hepatic acid lipase in cholesterol ester storage disease”, Science, 1972 . Cortner et al: “Genetic variation of lysosomal acid lipase”, Pediatric Research, 1976 . Goldstein, et al: “Role of lysosomal acid lipase in the metabolism of plasma Low density lipoprotein,” JBC, 1975 . Anderson, et al: Cloning of the lysosomal acid lipase cDNA, JBC, 1991 . Anderson, et al: Mutations at the lysosomal acid cholesterol esterase gene locus in Wolman disease. Proc Natl Acad Sci USA, 1994 . Klima H, et al: “A splice junction mutation causes deletion of a 72-base exon from the mRNA for lysosomal acid lipase in a patient with cholesteryl ester storage disease.” J Clin Invest, 1993. A brief history: Unraveling the molecular basis (1) the protein

Healthy Individuals LAL Deficient Patients

LDL particle LDL particle lysosome lysosome

Free cholesterol and free fatty acids

nucleus nucleus Hepatocyte

• Accumulation of abnormal lipid in lysosome AND • Disruption of normal lipid homeostasis LAL Deficiency: One Disease Presenting Across a Clinical Continuum

Adults

Children o Minimal residual LAL enzyme activity o Minimal residual LAL enzyme activity o Advanced liver disease o Premature liver o Premature fibrosis/cirrhosis cardiovascular events o Accelerated Infants atherosclerosis o No LAL enzyme o Profound growth failure o Persistent vomitting /diarrhea o Median age of death (3.4 months) LAL Deficiency presenting in infancy (historically also called Wolman Disease)

. Marked accumulation of cholesteryl esters and Weight-for-age percentiles: triglycerides in many tissues Boys, birth to 12 months 33.1 . Prominent hepatic and GI manifestations 30.9 28.7 – Persistent vomiting, diarrhea 26.5 97th 24.3 85th 22.1 – Hepatomegaly and liver failure 50th 19.8 15th 17.6 3rd – Splenomegaly WeightPounds in 15.4 – Abdominal distension 13.2 11 – Profound growth failure 8.8 6.6 LAL Deficient 4.4 . Adrenal calcification frequently present 1 2 3 4 5 6 7 8 9 10 1112 Age (months) . Rapidly progressive and fatal within 1st year of life . No approved treatments; only palliative care . Incidence: 1:300,000 to 1,000,000 – Increased with consanguinity (e.g., Persian Jews)

12 Molecular Epidemiology of LAL Deficiency Presenting in Children and Adults

. Many different mutations have been described . A single mutation has been described in 50-60% of the cases of LAL deficiency presenting in children and adults: – A point mutation at Exon 8 Splice Junction (E8SJM) which leads to ~3-5% normally spliced mRNA . Estimated prevalence of 1:40,000 to 1:300,000 – Similar to other lysosomal storage disorders (e.g., Gaucher, Fabry, Pompe). LAL Deficiency: Genetic Epidemiology

Author Journal Carriers of E8SJM/ Estimated Sample Size Prevalence*

Muntoni et al Arterioscler 10/2023 1:43,000 to 1:78,000 Thromb Vasc Biol ; (German population) 2007

Grabowski et Scriver’s OMMBID; 9/7011 1:159,000 to 1:294,000 al 2012 (European Americans)

Scott et al Hepatology; 2013 14/4569 1:111,000 to 1:204,000 (Caucasian + Hispanic)

Stitziel et al Arterioscler 88/27,472 1:102,000 to 1:189,000 Thromb Vasc Biol; (European ancestry) 2013

*Range based upon assumption of the “common” E8SJM representing 51 to 69% of all disease causing mutations LAL Deficiency

. History . Pathophysiology and genetic epidemiology . Clinical manifestations including liver disease . Dyslipidemia and atherosclerosis . Diagnostic considerations . Therapeutic considerations . Summary LAL Deficiency presenting in childhood or adulthood: a rare disease with a common phenotype

. Shortened lifespan and morbidity . Prominent hepatic manifestations – Fatty liver (microvesicular steatosis) – Elevated transaminases – Fibrosis and cirrhosis – Liver failure (often early in life)

. Cardiovascular involvement Affected liver removed during transplant – Elevated LDL cholesterol surgery age 9 – Low HDL frequently observed – Variably elevated triglycerides – Accelerated atherosclerosis . Other manifestations: – Splenomegaly – GI manifestations – Lymphadenopathy Clinical Summary (Bernstein et al.; review of 135 cases)

Age of onset Male (birth – 44) Female (1 month-68)

Distribution of 27% between birth and 1 years, Age of Onset 62% between age 3 and 12 years, 11% during adolescence or as adults. 4% patients whose diagnoses were made at autopsy

Hepatomegaly Presented in 99% of patients

Splenomegaly Presented in 74% of patients

Transaminase Elevated AST and/or ALT activities were present in all cases reporting Levels serum transaminase activities, with significant fluctuations at different time points

Bernstein et al. Cholesteryl Ester Storage Disease: Review of the Findings in 135 Reported Patients with an Under-Diagnosed Disease. Journal of Hepatology (2013). Epub Clinical Summary (Bernstein et al.)

Liver • Occurred in all pts Dysfunction • Of the 11 reported deaths, the majority (73%) were due to liver and/or Liver failure Failure • Progression to esophageal varices was reported in 12 cases. 4 additional deaths from liver were reported after publication • Death due to liver disease progression occurred at 7 to 56 years old, • 50% of deaths were in patients under 21 years of age.

Liver Biopsy • Findings were consistent among patients, and appeared (83%) independent of age, genotype, or other factors • A striking orange-yellow in color and diffuse, uniform microvesicular steatosis with minimal zonal differences within the hepatic lobule. • 72 (64%) had fibrosis and/or cirrhosis • 17 patients (15%) who had both fibrosis and cirrhosis in initial or subsequent biopsies

Bernstein et al. Cholesteryl Ester Storage Disease: Review of the Findings in 135 Reported Patients with an Under-Diagnosed Disease. Journal of Hepatology (2013). Epub ALT Abnormal In A Broad Spectrum Of Patients With Late Onset LAL Deficiency

ALT Highest recorded Female Male

Avg 108 93

Std Dev 74 70

Published Median 102 70 literature ULN Q1 48 43

Q3 143 110

Count 17 11

Source: Synageva data on file

LAL Deficiency

. History . Pathophysiology and genetic epidemiology . Liver disease . Dyslipidemia and atherosclerosis . Diagnostic considerations . Therapeutic considerations . Summary Role of LAL In Hepatic Cholesterol And Lipid Homeostasis

Decreased hepatic LAL activity would be predicted to o Decrease cellular free cholesterol o Increase activation of SREBPs, leading to increased cholesterol synthesis, lipogenesis and VLDL production o Reduce expression of LXRs, leading to reduced cholesterol efflux and HDL biogenesis Review of dyslipidemia in published LAL deficiency cases

• 69 publications describing 111 cases: 94 cases had sufficient data to analyze • The median highest recorded LDL-C in the cases where LDL-C was reported: –215 mg/dL in females (n=16) –241 mg/dL in males (n=16) • There was marked variability in triglycerides; the median highest recorded triglyceride was 215 mg/dL • The median lowest recorded HDL-C values were 24 mg/dL in females (n=21) and 22 mg/dL in males (n=21)

In a recent literature review of LAL deficiency involving 135 patients (Bernstein, et al 2013), only 43 pts had reported LDL-C values and 79% of these had LDL-C levels >200 mg/L)

Bernstein et al, Hepatology Feb 2013 [epub] Elevated LDL-C is common in patients with

documented LAL deficiency

C value mg/ C value

- LDL Combined hyperlipidemia is a common

feature of LAL deficiency

) )

Cholesterol (mg/ Cholesterol Highest Recorded Total Total Recorded Highest

Highest Recorded Triglyceride (mg/dL) Low HDL-C is a characteristic feature of

LAL deficiency

) )

Cholesterol (mg/ Cholesterol Highest Recorded Total Total Recorded Highest

Lowest Recorded HDL-Cholesterol (mg/dL) Low HDL-C in LAL deficiency is mechanistically linked to ABCA1 (Bowden KL et al, 2011)

Increase in ABCA1 mRNA with LDL . Recent data indicates that cholesterol flux out of loading lysosomes is a key regulator of ABCA1 Normal CESD expression (Bowden KL et al, 2011) . Fibroblasts from LAL deficient patients – Decreased basal and LDL stimulated ABCA1 and ABCG1 expression – Decreased apoA-I mediated efflux of phosphatidylcholine, sphingomyelin and Decreased cholesterol efflux in unesterified cholesterol (UC) CESD fibroblasts – LXR agonists correct ABCA1 expression but not efflux Normal – Decreased generation endogenous

oxysterols including 27 hydrocholesterol CESD

29 Circulation The Role of Macrophage LAL In Atherosclerosis

. Macrophages endocytose cholesteryl esters by receptor mediated uptake of modified and aggregated LDL

. Macrophages express LAL which is required for effective hydrolysis of CE in the lysosome

. The LIPA gene has been identified by GWAS as significantly associated with coronary artery disease

http://lancastria.net/blog/wp-content/uploads/2011/07/atherosclerosis_8_lancastria.jpg

30 Early Onset Cerebrovascular Disease in LAL Deficiency

Haller W et al, JPGN 50 (5) . Cerebrovascular 555 2010 Disease described as incidental finding in other case reports

vom Dahl S et . No systematic al; J. Hepatol. 31 (1999) 741- analysis of 746. prevalence of LAL Deficiency in “young” stroke patients LAL Deficiency

. History . Pathophysiology and genetic epidemiology . Liver disease . Dyslipidemia and atherosclerosis . Diagnostic considerations . Therapeutic considerations . Summary LAL Deficiency Presents Across the Age Continuum with Significant Morbidity

33 Case

47 y/o woman referred for management of dyslipidemia Medications: – HCTZ 25 mg – atorvastatin 20 mg PMH: HTN SH: alcohol 1-2 drinks/week, non-smoker Labs: untreated atorvastatin 20 mg – TG 203 mg/dL 174 mg/dL – Total chol 270 mg/dL 215 mg/dL – LDL 195 mg/dL 144 mg/dL – HDL 34 mg/dL 36 mg/dL – AST 45 U/L 56 U/L – ALT 72 U/L 80 U/L PE: height 5’10”, weight 195 lbs, BMI 28, BP 132/76 mild splenomegaly Which children/adults should ideally be tested for LAL Deficiency? . Patients with: – Unexplained (e.g., not viral or alcoholic) persistently elevated serum transaminases, i.e., ALT and/or AST (may be modest) with or without hepatomegaly, and – Elevation in LDL cholesterol (~ >160 mg/dl), and – Decreased HDL cholesterol (~ < 50 mg/dL)

– Patients with biopsy proven microvesicular steatosis without known etiology (e.g., Reye syndrome or drug toxicity) Which enriched population of children/adults should be tested for LAL Deficiency? . Non-obese patients with: – Unexplained (e.g., not viral or alcoholic) persistently elevated serum transaminases, i.e., ALT and/or AST (may be modest) with or without hepatomegaly, and – Elevation in LDL cholesterol (~ >160 mg/dl), and – Decreased HDL cholesterol (~ < 50 mg/dL) – Age < 60 years old

– Patients with biopsy proven microvesicular steatosis without known etiology (e.g., Reye syndrome or drug toxicity) Why the focus on non-obese patients?

[not drawn to scale]

obese

Number General of People Population with with Metabolic Hepatomegaly, Syndrome ALT & AST, or LDL & HDL LAL Deficiency

weight Dried Blood Spot (DBS) Enzyme Assay

. LAL activity is determined by measurement of total lipase activity and lipase activity in the presence of a LAL specific inhibitor . Allows for the possibility of testing high risk populations

Hamilton J, et al. Clin Chim Acta. 2012;413:1207-10. Available Labs to Perform DBS for LALD

US UK LabCorp Yorkhill Hospital Integrated Genetics Specialty Testing Group The Biochemistry Department Center for Molecular Biology and Pathology Yorkhill, Glasgow, United Kingdom LAL Assay Information Yorkhill Biochemistry Website Test code: 402300

Massachusetts General Hospital (MGH) Brazil Neurogenetics DNA/Biochemical Diagnostics Lab Rede DLD Brasil Boston, MA Medical Genetics Service MGH Lab Form Hospital de Clinicas de Porto Alegre Tel + 55 51 33598011 Seattle Children's Hospital [email protected] Department of Laboratories www.dld.ufrgs.br (in development) Seattle, WA Seattle Children's DBS Lab Form Japan Jikei University School of Medicine HIBM Research Group Department of Genetics & Genome Science, Minato-ku, Reseda, CA Tokyo, Japan HIBM Requisition Forms Genetics & Genome Science Website

Additional Labs : www.laldeficiencysource.com Where are these patients: Strategies to Finding Patients

Simple blood tests Right Right Right Test . Dried blood spot Practice Patient . Enzyme assay . DNA sequencing

Geneticists Suspected Enzyme DNA Patients Dried Blood Spot Hepatologists; Liver Gastroenterologists Manifestations

Lipid Lipidologists Manifestations

Liver Pathologists Pathology LAL Deficiency

. Statins Statins in LAL Deficiency

In a recent literature review of LAL deficiency involving 135 patients (Bernstein, et al 2013), 35 patients were reported to be taking statins: – 8 had no liver biopsy findings reported – 15 had biopsy findings with fibrosis, cirrhosis or LALD- associated hepatopathology, but no long-term or sequential follow-up data reported – 12 patients on statins had sequential data available and all demonstrated progressive liver disease that was more advanced in subsequent biopsies – Six patients treated with statins required transplantation or died from liver failure

Bernstein et al, Hepatology Feb 2013 [epub] 43 LAL Deficiency: Therapeutic considerations

. Statins . Other lipid-lowering drugs LAL Deficiency: Therapeutic considerations

. Statins . Other lipid-lowering drugs . New therapeutic approaches (ie enzyme replacement therapy) Sebelipase alfa Targeting and Activity

Terminal mannose/GlcNac and mannose-6-phosphate (M6P)

for targeted delivery Oregon green- Lysotracker red Overlap images labeled sebelipase alfa

6 Uptake into key cells 5 Normal Human Fibroblasts

4 LD Fibroblasts Lysosomal localization 3 demonstrated 2

Corrects enzyme deficiency 1 0 Cellular LAL Activity (nUnits/cell)Activity CellularLAL 0 0.160.16 0.50.5 1.61.6 55.0 sebelipase alfa (ug/ml) Source: Synageva data on file LAL Deficiency Rat Model Reproduces Key Aspects of Human Disease

Accumulation Lipid Growth failure Increased Mortality Substrate in Liver, Spleen and Gut

Rat model 1st described Japan 1990 Maximal life span in LAL-/- rats is (Yoshida H, Kuriyama M. Lab Animal Sci. approximately 14 weeks 1990;40(5):486-9. Source: Synageva data on file sebelipase alfa Restores Normal Growth & Increases Survival in Rats

Source: Synageva data on file

48 Sebelipase alfa In Vivo Activity

Source: Synageva data on file * sebelipase alfa 5mg*kg-1 once weekly for 4 weeks Improvements In Growth And Organ Size Is Associated With Correction Of Underlying Pathology

Liver Sebelipase alfa

Placebo

SebelipaseSBC-102 alfa

Source: Synageva data on file LAL-CL01 & LAL-CL04 Trial Design Improvement in Transaminases

n=9 n=8

Mean percent change in ALT & AST is 54% and 30% respectively at week 78. Source: Whitley et al. NASPGHAN 2013 Improvement in Dyslipidemia Profile

n=9 n=8

Source: Whitley et al. NASPGHAN 2013 Rapid, Sustained Reduction in Liver Volume and Fat Fraction (MRI)

10 n=8 n=8 n=7 n=7 n=7 n=6 0 -10% -9-%9% -14% -10 -12%

-20 Week 10 Week 24 -35%-35% -30 Week 52 -42%-42%

Percent Change Percent -40

From CL04 FromCL04 Baseline -55% -50 -55%

-60 Liver Volume Liver Fat Fraction

Source: Whitley et al. NASPGHAN 2013 ARISE Trial

Study Design Endpoints • Randomized, double-blind, • Normalization in ALT* placebo controlled • Change in LDL, non-HDL, TG, HDL • Multi-center global trial • Normalization in AST • N = 50 • Change in liver volume and liver fat content • Improvement in liver histopathology

Key Inclusion Criteria Key Exclusion Criteria • Confirmed Diagnosis of LALD • Severe hepatic dysfunction (Child-Pugh Class C) • Subject is ≥4 years of age • Previous stem cell or liver transplant • ALT ≥1.5x ULN

* Age- and gender-specific ULN provided by the central laboratory performing the assay http://www.clinicaltrials.gov/ct2/show/NCT01757184

Announced on Dec 3rd – Completes Target Enrollment & Data in H2:2014 LAL Deficiency

. History . Pathophysiology and genetic epidemiology . Liver disease . Dyslipidemia and atherosclerosis . Diagnostic considerations . Therapeutic considerations . Summary Summary

 LAL Deficiency is due to mutations in the LIPA gene resulting in a marked decrease in the activity of the enzyme lysosomal acid lipase  The major clinical manifestations are hepatosteatosis leading to fibrosis, and dyslipidemia characterized by elevated LDL-C, variable TG, and low HDL-C  LAL Deficiency should be considered in patients with elevated LDL- C and/or low HDL-C together wit hepatosplenomegaly and/or elevated live transaminases; or metabolic syndrome without overt obesity  The diagnosis of LAL deficiency can be made via dry blood spot for LAL activity  Sebelipase alfa is being developed as enzyme replacement therapy for LAL deficiency