Journal Pre-proof

Evaluation of Antisense Oligonucleotides Therapy Targeting Hsd17b13 in a Fibrosis

Mice Model

Yanling Ma, Hong Cai, Julia Smith, Ching-Hsuen Chu, Stephen E. Mercer, Stephanie
Boehm, Ivar Mcdonald, Bradley Zinker, Dong Cheng
PII: S0022-2275(24)00019-1
DOI: https://doi.org/10.1016/j.jlr.2024.100514
Reference: JLR 100514

To appear in: Journal of Lipid Research

Received Date: 20 June 2023

Revised Date: 29 January 2024
Accepted Date: 30 January 2024

Please cite this article as: Ma Y, Cai H, Smith J, Chu CH, Mercer SE, Boehm S, Mcdonald I, Zinker B,
Cheng D, Evaluation of Antisense Oligonucleotides Therapy Targeting Hsd17b13 in a Fibrosis Mice
Model, Journal of Lipid Research (2024), doi: https://doi.org/10.1016/j.jlr.2024.100514.

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© 2024 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and
Molecular Biology.
 Evaluation of Antisense Oligonucleotides Therapy Targeting Hsd17b13 in a

Fibrosis Mice Model

Yanling Ma 1,2, Hong Cai 1, Julia Smith 1, Ching-Hsuen Chu 1, Stephen E. Mercer 1, Stephanie

Boehm1, Ivar Mcdonald 1, Bradley Zinker 1, Dong Cheng 1,2

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1
Bristol-Myers Squibb Company

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2
Corresponding author
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Correspondence:
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Yanling Ma, PhD., Bristol-Myers Squibb Company

Address: 3401 Princeton Pike, Lawrence Township, NJ, 08648, United States
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Email: [email protected]
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Dong Cheng, PhD., Bristol-Myers Squibb Company

Address: 3401 Princeton Pike, Lawrence Township, NJ, 08648, United States

Email: [email protected]

Author contributions:

Conceptualization: BZ, DC
Data Curation and Investigation: YM, HC, JS, C-H C, SM, SB

Formal Analysis: YM

Writing – original draft: YM

Writing – review & editing: All authors

Supervision: IM, BZ, DC

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Source of funding

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This study was supported by Bristol Myers Squibb
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Abbreviations: 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13); Non-alcoholic fatty liver
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disease (NAFLD); Non-alcoholic steatohepatitis (NASH); Asparate aminotransferase (AST); Alanine

aminotransferase (ALT); Triglycerides (TG); choline-deficient, L-amino acid-defined, high-

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fat diet (CDAHFD)

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Declarations of interest: none.

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Highlights:

Hsd17b13 antisense oligonucleotide (ASO) showed robust gene knock-down efficacy in vitro and in vivo

Hsd17b13 ASO therapy significantly affected hepatic steatosis

Hsd17b13 ASO therapy had no effect on hepatic fibrosis in mice fibrosis model
Abstract

Objective: Human genetic evidence suggests a protective role of loss-of-function variants in 17-beta

hydroxysteroid dehydrogenase 13 (HSD17B13) for liver fibrotic diseases. Although there is limited

preclinical experimental data on Hsd17b13 antisense oligonucleotide (ASO) or siRNA in a fibrosis

model, several ASO and siRNA approaches are being tested clinically as potential therapies for non-

alcoholic steatohepatitis (NASH).. The aim of this study was to assess the therapeutic potential of

Hsd17b13 ASO in a preclinical advanced NASH-like hepatic fibrosis in vivo model.

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Methods: Hsd17b13 ASO was tested in primary mouse hepatocytes for efficacy and specificity. Mice

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were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 4 weeks for disease

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induction and then administered with a scramble oligonucleotide negative control or Hsd17b13 ASO for 8
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weeks. At the time of sacrifice, hepatic histology, gene expression, and serum panels were examined.
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Results: In vitro testing on primary hepatocytes demonstrated that Hsd17b13 ASO exhibited strong

efficacy and specificity for knock-down of the Hsd17b13 gene. In CDAHFD induced steatotic and
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fibrotic mice, therapeutic administration of Hsd17b13 ASO resulted in a significant and dose-dependent
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reduction of hepatic Hsd17b13 gene expression. The CDAHFD group exhibited considerably elevated
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liver enzyme levels, hepatic steatosis score, hepatic fibrosis, and increased expression of fibrotic and

inflammatory gene expression, indicating an advanced NASH-like hepatic fibrosis phenotype. While

Hsd17b13 ASO therapy significantly affected hepatic steatosis, it had no effect on hepatic fibrosis.

Conclusion: Our findings demonstrate, for the first time, that Hsd17b13 ASO effectively suppressed

Hsd17b13 gene expression both in vitro and in vivo, and had a modulatory effect on hepatic steatosis in

mice, but did not affect fibrosis in the CDAHFD mouse model of NASH.

Key Words:

Hsd17b13, antisense oligonucleotide (ASO), fibrosis, steatosis, CDAHFD

Introduction

Non-alcoholic steatohepatitis (NASH), a leading cause of liver disease in the general population, is

characterized by excessive hepatic fat accumulation with complications of hepatic inflammation, fibrosis,

and liver cell damage. Without intervention, NASH could progress into advanced liver diseases, such as

cirrhosis and liver cancer, and may even necessitate liver transplantation. Unfortunately, there are

currently no FDA approved drugs for the treatment of NASH; the main disease management strategies

rely on lifestyle modifications and the treatment of comorbidities (1).

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Accumulating evidence suggests NASH have a heritable component (2, 3), with genetic variants in

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several genes such as PNPLA3 (4), TM6SF2 (5), MBOAT7 (6) associated with the disease severity. More

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recently, SNPs within or near the HSD17B13 gene, which encodes a lipid droplet associated protein (7-9)
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known as hydroxysteroid 17-beta dehydrogenase 13, has also been linked with NASH (10-14).
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Specifically, a splice variant (rs72613567) and rs62305723 encoding for a P260S mutation in HSD17B13

are reported to be associated with lower liver enzyme levels and decreased severity of NASH (10). Both
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splice variants and P260S mutants are enzymatically inactive and/or unstable, leading to the hypothesis
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that inhibiting HSD17B13 through small molecule inhibitors or interfering its gene expression through
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siRNA or ASO might mimic the beneficial effect observed for these variants. In line with this hypothesis,

HSD17B13 ASO and siRNA therapy have been developed and are currently undergoing clinical testing in

phase I trials for the treatment of NASH.

Despite the enthusiasm for clinical development of HSD17B13 based therapy, preclinical data regarding

the pathophysiology of this protein is still limited and no study has reported the preclinical use of

HSD17B13 ASO or siRNA to date. One recent study has evaluated AAV mediated Hsd17b13 gene

knock-down using shRNA; however, this approach would not be directly applicable for human use (15).

Studies performed in HSD17B13 knock-out mice has shown interesting phenotypes that are not entirely

consistent with human genetic findings (16). To investigate the therapeutic efficacy of Hsd17b13 ASO on

preclinical models, we conducted studies using primary hepatocytes and an in vivo diet-induced NASH-
like hepatic fibrosis mouse model. We found that Hsd17b13 ASO robustly inhibited Hsd17b13 gene

expression in vitro and in vivo, and therapeutic treatment of Hsd17b13 ASO modulated hepatic steatosis

but did not decrease hepatic fibrosis in the rodent NASH model.

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Materials and Methods

Antisense Oligonucleotides (A2B5D-038 and A2B5D-039).

Oligonucleotides were synthesized using standard phosphoramidite chemistry on an Äkta

Oligopilot 150 oligonucleotide synthesizer. Hsd17b13 ASO had a 5’ to 3’ sequence of

CGCttttaaggcaCGC while the scramble ASO had a sequence of GGCcaatacgccgTCA where

capital letters designate locked nucleic acid (LNA) nucleobases and lower-case letters designate

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unmodified DNA. Cytosines in bold indicate 5-methylcytosines. The ASOs have full

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phosphorothioate backbone linkages.

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Animals re
Six-week-old C57B6/J male mice were obtained from Jackson Labs (Bar harbor, ME) and were
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housed two per cage in a temperature- and humidity-controlled environment with free access to
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food and water. A week later, CDAHFD diet (L-Amino Acid Deficient Rodent Diet with 60

kcal% Fat with 0.1% Methionine and no added choline, Research Diet, #A06071302) was given
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to 60 mice, while 12 mice continue feeding on a control diet (Control Rodent Diet 10 kcal% Fat,
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Research Diet, #A13012807) during the full experiment period. Mice on CDAHFD were

randomly divided into 5 groups after the disease induction 4-weeks of diet treatment (n=12)

based on body weight and were given saline (vehicle), scramble ASO (50 mpk), or Hsd17b13

ASO at 10, 25, or 50 mpk subcutaneously once a week for 8 consecutive weeks. The mice on

control diet were also given saline subcutaneously once a week. Body weight was taken at the

start of diet treatment, and once a week during drug treatment period. Food intake was measured

twice before drug treatment and the last week of drug treatment. All mice were sacrificed one

week after the last dose and plasma and tissue sample were collected for further analysis. All
experiments were approved by the BMS Animal Care and Use Committee (ACUC),

Lawrenceville, NJ.

Primary Hepatocytes Culture and Treatment

Mouse primary hepatocytes (Lonza, Cat: MBCP01) were thawed and cultured according to the

manufacture’s instruction. Briefly, cells were plated in Collagen I-coated 96-well plate (Corning,

Cat: 356649) at the density of 1.8X104 cells/well for 6 hours Lonza maintenance media

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(MM250-1 and MM250-2). ASOs with concentration ranging from 8.23 nM to 6,000 nM were

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then added into the wells and incubated with primary hepatocytes for 24 to 72 hours.

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RNA Extraction and qPCR re
RNA was extracted from mouse liver tissue or primary hepatocytes using MagMAXTM-96Total
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RNA isolation kit (Life Technologies, Cat: AM1830M) and processed on AB Applied Biosystem
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MagMax express 96 system. Superscript Vilo (Invitrogen, Cat: 100011932) was used for cDNA

synthesis using MJ Research thermal cycler. Real time qPCR was conducted and analyzed in
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Biorad CFX 384 Real Time System. qPCR probes were purchased from Life Technologies with
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Assay IDs listed in table 1.

Plasma Marker Measurement

Blood samples were collected into EDTA-coated Microvette CB300 tubes, and plasma samples

were diluted 2-fold with diH2O before measurement. Diluted samples were analyzed using the

AU680 Clinical Chemistry Analyzer for ALT (Beckman Coulter, #OSR6107), AST (Beckman

Coulter, #OSR6109), ALP (Beckman Coulter, #OSR6004), glucose (Beckman Coulter, #OSR),

triglyceride (Beckman Coulter, #OSR6118), total bile acids (Genway BQ, GWB-BQK086), and

NEFA (Wako Diagnostics, #NEFA-HR).

Liver Triglyceride Measurement

Liver tissue (approximately 100 mg) was homogenized in DPBS (10 μL/mg tissue) buffer on

Precellys Homogenizer. The homogenates were diluted 4-fold in DPBS and analyzed

immediately using the AU680 Clinical Chemistry Analyzer for triglyceride (Beckman Coulter,

#OSR6118).

Liver Histology

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The medial and left lateral lobes of the liver were collected, fixed in 10% neutral buffered

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formalin and then embedded in paraffin for histology analysis. Tissue sections were cut onto

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Superfrost charged slides and stained with H&E for quantification of liver steatosis (steatosis
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area %) and stained with picrosirius red (PSR) for quantification of liver fibrosis content (area
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%) using the HALO software (version 2.3, Indica Labs, Inc.).

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Liver Hydroxyproline Measurement

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Liver tissue was homogenized in 1 ml DPBS buffer on Precellys Homogenizer and dried in a
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speed vac for 4 hours after adding Glacial Acetic Acid. Samples were hydrolyzed in 1 ml of 6 M

HCl solution for 20 hours at 110 ºC and cooled down to room temperature. Filtered samples were

measured for hydroxyproline using Quickzyme Hydroxyproline Kit (Quickzyme,

QZBHYPRO5) according to the manufacture’s instruction.

Statistical Analysis

Nonlinear fit with four parameters variable slope analysis was used for calculation of IC50 of

Hsd17b13 ASO in primary hepatocytes. One-way AVOVA with Tukey multiple comparisons

test was used for difference between treatment groups in vivo.

Results

Hsd17b13 ASO effectively inhibited Hsd17b13 gene expression in primary hepatocytes

To determine the knock-down potency, Hsd17b13 ASO was tested in mouse primary

hepatocytes for 24, 48, and 72 hrs. Negative control scramble ASO had no effect on the gene

expression of Hsd17b13, while Hsd17b13 ASO effectively eliminated Hsd17b13 gene

expression with an IC50 value of 83, 76, and 29 nM, respectively at 24, 48, and 72 hrs (Figure 1),

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suggesting this ASO could efficiently inhibit Hsd17b13 gene expression in vitro. To further

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elucidate the specificity of this ASO, gene expression of Hsd17b11, Hsd17b6, Rdh10, and Dhrs3

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were tested in the same experiment setting and none of these genes were affected by Hsd17b13
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ASO.
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Hsd17b13 ASO effectively inhibited Hsd17b13 gene expression in vivo

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To study the therapeutic potential of Hsd17b13 ASO, we designed an in vivo study on Hsd17b13

ASO using a CDAHFD-induced fibrosis mouse model (Figure 2A). To mimic human NASH
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therapy in the clinical setting, the therapeutic treatment, in contrast to preventative treatment was
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applied to mice with established fibrosis induced by 4-week CDAHFD treatment. Consistent

with previous report (17) mice on CDAHFD maintained their body weight, while mice on

regular chow diet keep gaining weight steadily during the study (Figure 2B). There is no

significant difference in body weight among groups under CDAHFD diet receiving vehicle,

control ASO or Hsd17b13 ASO at different doses. Food intake was measured before and after

ASO treatment and no difference was found among groups (Figure 2C). Interestingly, we

observed that CDAHFD diet treatment significantly induced Hsd17b13 gene expression (Figure

2D), which is consistent with the clinical observation that HSD17B13 gene expression is
elevated in NASH patients compared with healthy control population (10). Control ASO didn’t

change Hsd17b13 expression as expected. Importantly, as shown in the primary hepatocytes,

Hsd17b13 ASO significantly eliminated Hsd17b13 gene expression by 80%, 94%, and 98%

respectively at 10, 25, and 50 mpk (Figure 2D) after 8-week treatment.

Hsd17b13 ASO on plasma markers, liver weight and liver TG

CDAHFD significantly increased plasma liver enzyme levels (AST, ALT, and ALP, Figure 3A-

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C), but decreased blood glucose level (Figure 3D). As previously described, plasma bile acid

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level is induced by CDAHFD treatment (18) (Figure 3E). While Control ASO did not change

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any of the plasma markers compared with vehicle group, Hsd17b13 ASO at higher doses
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significantly increased plasma level of AST (Figure 3A) and ALT (Figure 3B). The highest dose,
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50 mpk group slightly but significantly decreased ALP level (Figure 3C). No changes were noted

in glucose, bile acids, or NEFA by Hsd17b13 ASO treatment compared with control ASO
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(Figure 3D-F). A low dose of Hsd17b13 ASO at 10 mpk, however, showed similar plasma
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markers with control ASO in all respects despite of an 80% knock-down rate on the Hsd17b13
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gene.

After 12 weeks of CDAHFD diet, mice showed a significantly increased liver weight (Figure

4A), decreased body weight (Figure 4B), and an increased liver weight to body weight ratio

(Figure 4C). The hepatic triglyceride (TG) content was significantly elevated in CDAHFD

treated animals compared to the regular chow group (Figure 4D). Consistently, the mice liver

histology and steatosis score evaluation suggested a severe steatosis phenotype under CDAHFD

compared to regular chow (Figure 4E-G). Accordingly, the lipid droplets diameter significantly

increased in the CDAHFD group as well (Figure 4F). Control ASO, again did not change any of

these parameters significantly compared with the vehicle only, while Hsd17b13 ASO decreased
liver weight, liver weight to body weight ratio, liver TG, liver steatosis, and LDs diameter dose-

dependently (Figure 4).

Therapeutic Hsd17b13 ASO intervention does not affect hepatic fibrosis in mice

We next investigated whether hepatic fibrosis was regulated by Hsd17b13 ASO using PSR

staining and gene expression panel analysis. As expected, CDAHFD diet largely induced hepatic

fibrosis in mice (Figure 5). However, therapeutic intervention of Hsd17b13 ASO did not

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improve hepatic fibrosis. Consistent with this observation, hepatic fibrosis related genes (Col1a1,

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Col1a2, Col3a1, Col4a1, Etgf, Pai-1, Acta1, Tgfb, Mmp7, and Mmp12) showed no difference

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between the vehicle and Hsd17b13 ASO treated groups (Figure 6). Furthermore, there was no
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difference in inflammatory-related genes (Cxcl10, Tnfα, IL1b, Il16, Ccl2, Ym1, and Gal3) among
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the vehicle and Hsd17b13 ASO treated groups (Figure 7), indicating that therapeutic intervention

with Hsd17b13 ASO does not improve liver fibrosis in mice.

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Discussion

Recent human genetic findings that loss of function variants in HSD17B13 protect patients from

developing severe chronic liver disease have generated enthusiasm of pharmaceutical industry on

HSD17B13 based therapy. At least two phase I clinical trials have been conducted or are

ongoing to evaluate the safety and tolerability of HSD17B13 ASO or siRNA in humans

(19)(https://beta.clinicaltrials.gov/study/NCT05560607;

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https://beta.clinicaltrials.gov/study/NCT04565717; https://beta.clinicaltrials.gov/study/NCT05519475).

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However, no experimental data evaluating Hsd17b13 ASO in preclinical fibrosis model has been

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reported. Data from current study showed that Hsd17b13 ASO effectively and specifically

inhibited Hsd17b13 gene expression in primary hepatocytes and further in vivo testing confirmed
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the dose-dependent gene inhibition in fibrotic mice liver. However, therapeutic treatment of
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Hsd17b13 ASO decreased hepatic steatosis but did not alter the hepatic fibrosis in CDAHFD
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mice model.
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Animal models play a pivotal role in understanding pathophysiology of human diseases and
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evaluating therapeutic options for human diseases with unmet medical needs (20, 21). However,

there are currently no ideal animal models that fully resemble all aspects of NASH

characteristics (22). High fat diet or Western diet induced obesity models are often used as

hepatic steatosis model for NASH, with limited hepatic fibrosis components (16). The CDAHFD

model has been well established to represent the fibrosis aspect of human NASH leading to its

wide use in academic research and pharmaceutical studies for drug discovery in the NASH field

(23). Furthermore, data from previous publications suggest that Hsd17b13 knock-out animals

showed no protective role in obesogenic diet or alcohol-induced hepatic fibrosis (16). Thus, we

decided to use CDAHFD model to better evaluate from hepatic fibrosis perspective. Our data
showed that mice developed hepatic steatosis and fibrosis after 12-week of CDAHFD diet, as

evidenced by histology, biochemical assays, serum parameters, and hepatic gene expression,

suggesting of successful development of NASH-like fibrotic liver diseases in mice. It is

important to acknowledge that while the CDAHFD model successfully resembles the hepatic

steatosis and fibrosis characteristics of human NASH, like all other diet-induced NASH animal

models, it also has its limitations. The CDAHFD mice model doesn’t develop metabolic

syndromes that are commonly associated with most human NASH patients. Therefore, this

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model cannot be used to study the relationship between NASH with metabolic syndromes.

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Additionally, although this model can develop bridging fibrosis and fibrotic nodules if the mice

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are on the CDAHFD diet for 26 or more weeks, that was not the case in the current study, as our
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model was under the CDAHFD diet for 12 weeks total.
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As expected, Hsd17b13 ASO showed robust potency in vitro and in vivo, with knock-down rate
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of 80%, 94%, and 98% at 10, 25, and 50 mpk, respectively. Surprisingly, two higher doses
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showed modulation of liver enzymes levels, suggesting potential safety concerns for use of this

ASO in preclinically and clinically settings. It is possible that over-inhibiting Hsd17b13 is

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harmful to the mice liver. We tested specificity of the Hsd17b13 ASO against several Hsd family

members (Hsd17b11, Rdh10, Dhrs3, and Hsd17b6), none of them were affected by Hsd17b13

ASO. However, we cannot exclude the possibility that this effect is mediated by off-target effects

mediated through other unintended RNA targets that were not included in our study or

hybridization-independent toxicity, which requires further investigation as described by Kamola

et al (24). Further studies evaluating different Hsd17b13 ASO sequences in a similar

experimental setting are also warranted. Besides liver enzymes, all doses of Hsd17b13 ASO had

no effect on other serum parameters measured in this study.

It is intriguing that mice treated with Hsd17b13 ASO showed decreased liver weight and hepatic

steatosis, consistent with previous reports that Hsd17b13 might regulate hepatic lipid metabolism

using genetic modified mouse models. Adam et al. reported that Hsd17b13 knockout mice

showed increased hepatic steatosis compared with control mice (25). However, another study

demonstrated no overall steatosis but changes in lipid droplet morphology in Hsd17b13 knockout

mice under an obesogenic diet (16). Since HSD17B13 has been demonstrated as lipid droplet

associated protein (7, 8), it is plausible that manipulating Hsd17b13 gene expression by ASO or

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knockout may play a role in lipid metabolism. Further studies investigating the mechanism

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between Hsd17b13 and lipid modulation are of great importance to understand the

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pathophysiology of HSD17B13 and its therapeutic use in humans.
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Another interesting finding is that therapeutic treatment with Hsd17b13 ASO did not decrease
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hepatic fibrosis in CDAHFD mice model at any given dose despite of robust gene inhibition.
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One might argue that two groups of mice might have been over-dosed with Hsd17b13 ASO since
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they showed increased liver enzyme levels. However, when considering the lowest dose of 10

mpk only, no abnormal liver enzyme level was observed in this group. Nonetheless, there was
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still no trend of fibrosis improvement measured by PSR staining, hydroxyproline, and fibrotic

gene expression, despite of 80% of gene knockdown, supporting the notion that Hsd17b13 gene

modulation may not affect fibrosis in mice. A previous study discussed the potential inter-species

difference between mouse and human HSD17b13 protein, which might explain this unique

finding (16). A recent study reported that Hsd17b13 gene silencing by AAV-shRNA induced

minor decrease of hepatic fibrosis in mice (15). There are several potential reasons that could

explain the differences between our studies. Firstly, Luukkonen et al. assessed the preventive

effects of Hsd17b13 KD by administering the fibrosis inducing diet at the same time as AAV
injection, while we used a more stringent method of inducing disease development in mice for 4

weeks before Hsd17b13 ASO treatment to better mimic the therapeutic process in human NASH.

Secondly, the duration of the treatment may also be accountable for the difference; mice in

Luukkonen’s study received AAV-shRNA treatment for 14 weeks, while in our study it was 8

weeks of ASO treatment. Thirdly, although statistically significant, the decrease in fibrosis

caused by AAV-shRNA against Hsd17b13 was minor; only a 20% decrease in fibrosis score and

~10% decrease in liver hydroxyproline was observed in Luukkonen’s study. Notably, we

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adjusted our unit (to µg/g tissue) to allow for better comparation with Luukkonen’s study data.

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The Ctrl AAV and Con ASO groups in two studies showed similar level of hepatic

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hydroxyproline (1300-1400 µg/g). In our study, compared with Con ASO, the Hsd17b13 ASO
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groups showed numerically lower value of hydroxyproline, but without statistical significance.
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Lastly, although less likely, we cannot exclude the possibility that the differences caused by diet
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sources, or the two gene silencing methods of AAV-shRNA and ASO, which warrants further

investigations on those interesting findings.

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In conclusion, we have tested Hsd7b13 ASO therapy for the first time in a preclinical disease
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model that parallels that of human NASH and demonstrated that Hsd17b13 intervention

disturbed hepatic steatosis but not fibrosis, raising the concern of developing HSD17B13 based

therapy for a NASH indication. Further studies should focus on using humanized animal model

or alternate ASOs against Hsd17b13 to better shed light on the topic of HSD17B13’s

pathophysiological roles in NASH.

Data Availability

All data are contained within the manuscript.

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Figure Legend

Figure 1. Hsd17b13 ASO effectively inhibited Hsd17b13 gene expression in primary

hepatocytes. Primary mouse hepatocytes were treated with Hsd17b13 ASO (10 nM to 6 μM) for

(A) 24 hrs, (B) 48 hrs, or (C)72 hrs. Cellular RNA was extracted and quantified for Hsd17b13

gene expression. Scramble ASO was used as negative control. The IC50 of Hsd17b13 ASO was

calculated to be 83 nM, 76 nM, and 29 nM at 24 hrs, 48 hrs, and 72 hrs, respectively. Data are

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presented as Mean±SEM.

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Figure 2. Study design, body weight, food intake and in vivo knock down of Hsd17b13 by

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Hsd17b13 ASO. (A) Study design. Mice were given control diet or CDAHFD for 4 weeks prior
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to control or ASO treatment initiation for disease induction. Mice in the control chow diet group
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were given vehicle; mice in the CDAHFD group were randomly divided into 5 groups and were

given either: vehicle, control ASO at 50 mpk, or Hsd17b13 ASO at 10 mpk, 25 mpk, or 50 mpk,
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respectively for 8 weeks post disease induction. Vehicle and Hsd17b13 ASOs were administered
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through subcutaneous injection, once per week. Mice were sacrificed at the end of the
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experiment and evaluated on NASH markers. (B) Body weight was measured once a week

during the experiment. (C) Food intake prior to respective treatment experiment and at the last

week during the experiment. (D) Hsd17b13 gene expression of each group at end of the

experiment showing 80%, 94%, and 98% knock-down of Hsd17b13 gene by Hsd17b13 ASO at

10, 25, or 50 mpk, respectively. ** P<0.01; ****P<0.0001.

Figure 3. Plasma markers in mice with 8 weeks of vehicle, control ASO, or Hsd17b13 ASO

treatment. (A) Plasma ALT (B) Plasma AST (C) Plasma ALP (D) Plasma glucose (E) Plasma

bile acids (F) Plasma NEFA after 8 weeks of vehicle, control ASO at 50 mpk, or Hsd17b13 ASO

at 10 mpk, 25 mpk, or 50 mpk, respectively. *P<0.05; ** P<0.01; ****P<0.0001.

Figure 4. Liver weight, liver histology, and liver TG in mice with 8 weeks of vehicle, control

ASO, or Hsd17b13 ASO treatment. (A) Mice liver weight after 8 weeks of vehicle, control

ASO at 50 mpk, or Hsd17b13 ASO at 10 mpk, 25 mpk, or 50 mpk treatment, (B) Mice body

weight at the end of the experiment (C) calculated liver weight to body weight ratio (D) Liver

TG content after 8 weeks of vehicle or ASOs treatment (E) Representative liver hematoxylin and

eosin (H&E) stain images of mice in each treatment groups. (F) Steatosis area of mice liver after

8 weeks of vehicle or ASOs treatment. (J) Lipid vacuole size of mice liver after 8 weeks of

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vehicle or ASOs treatment. (G) Representative H&E-stained mice liver tissue after 8 weeks of

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vehicle or ASOs treatment. *P<0.05, ** P<0.01, ***P<0.001, ****P<0.0001.

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Figure 5. Liver fibrosis evaluation in mice after 8 weeks of vehicle or ASOs treatment. (A)
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Quantified PSR positive staining area. (B) Liver hydroxyproline content in mice of each
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treatment groups (vehicle, control ASO at 50 mpk, or Hsd17b13 ASO at 10 mpk, 25 mpk, or 50
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mpk). (C) Representative Picrosirius Red stained mice liver tissue after 8 weeks of vehicle or
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ASOs treatment. ****P<0.0001.

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Figure 6. Hepatic fibrotic genes expression in mice with 8 weeks of vehicle or ASOs

treatment. mRNA was extracted from animals from all groups (vehicle, control ASO at 50 mpk,

or Hsd17b13 ASO at 10 mpk, 25 mpk, or 50 mpk) and qPCR was used to determine gene

expression of (A) Col1a1 (B) Col1a2 (C) Col3a1 (D) Col4a1 (E) Etgf (F) Pai-1 (G) Acta1 (H)

Tgfb (I) Mmp7 and (J) Mmp12. *P<0.05, **P<0.01, ****P<0.0001.

Figure 7. Hepatic inflammatory genes expression in mice with 8 weeks of vehicle or ASOs

treatment. mRNA was extracted from animals from all groups (vehicle, control ASO at 50 mpk,

or Hsd17b13 ASO at 10 mpk, 25 mpk, or 50 mpk) and qPCR was used to determine gene
expression of (A) Cxcl10 (B) Tnfα (C) IL1b (D) Il16 (E) Ccl2 (F) Ym1 and (G) Gal3. *P<0.05,

**P<0.01, *** P<0.001, ****P<0.0001.

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 Table 1. qPCR probes

Gene Name qPCR Probe Assay ID

Ubc Mm02525934_g1
B2m Mm00437762_m1
Pum1 Mm01180596_m1
Rpl13a Mm05910660_g1
Hsd17b13 Mm01203271_m1
Cxcl10 Mm00445235_m1
Tnfα Mm00443258_m1
Il1b Mm00434228_m1
Il6 Mm00446190_m1

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Ccl2 Mm00441244_m1

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Ym1 Mm00657889_m1
Gal3 Mm00802901_m1

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Col1a1 Mm00801666_g1
Col1a2 Mm00483888_m1
Col3a1 Mm00802330_m1
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Col4a1 Mm01210125_m1
Ctgf Mm01192933_g1
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Pai-1(sepine1) Mm00435858_m1
Asma Mm007255412_S1
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Mmp7 Mm00487724_m1
Mmp12 Mm00500554_m1
Tgfb1 Mm01178820_m1
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Rdh10 Mm.PT.58.9541215
Hsd17b11 Mm.PT.58.42741584
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Dhrs3 Mm.PT.58.29788869
Hsd17b6 Mm.PT.58.43950773
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Author contributions:

Conceptualization: BZ, DC

Data Curation and Investigation: YM, HC, JS, C-H C, SM, SB

Formal Analysis: YM

Writing – original draft: YM

Writing – review & editing: All authors

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Supervision: IM, BZ, DC

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We declare that this manuscript has not been published before or under consideration for publication
elsewhere. All authors have approved the manuscript and agree with its submission to Journal of Lipid
Research. Conflict of interest: none

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