studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 905A
Disclosures:
Claire Emson - Employment: KineMed
Martin Decaris - Employment: KineMed Inc.
Scott M. Turner - Employment: KineMed, Inc.
Carine Beysen - Employment: KineMed, Inc; Stock Shareholder: KineMed, Inc
Kelvin Li - Employment: KineMed, Inc; Stock Shareholder: KineMed, Inc
Rohit Loomba - Advisory Committees or Review Panels: Galmed Inc, Tobira Inc,
Arrowhead Research Inc; Consulting: Gilead Inc, Corgenix Inc, Janssen and
Janssen Inc, Zafgen Inc, Celgene Inc, Alnylam Inc, Inanta Inc, Deutrx Inc; Grant/
Research Support: Daiichi Sankyo Inc, AGA, Merck Inc, Promedior Inc, Kinemed
Inc, Immuron Inc, Adheron Inc
The following authors have nothing to disclose: Carolyn Hernandez, Jeffrey Y.
Cui, Leander Lazaro, David A. Brenner, Marc Hellerstein
1426
In vivo cell specific gene silencing in the liver using
novel siRNA-loaded nanohydrogel particles
Leonard Kaps; Institute of Translational Immunology and Research
Center for Immunotherapy (FZI), Mainz, Germany
Background: Efficient in vivo transport of active siRNA to specific
liver cells remains challenging. Compared to lipoplex or
polyplex formulations, cationic nanohydrogel particles (NHP)
can serve as superior oligonucleotide carriers [Siegwart DJ et
al, PNAS 2011]. We have designed NHP [Nuhn L et al, ACS
Nano 2012] that serve as stable carriers for in vivo siRNA
delivery [Nuhn L et al, Biomacromolecules 2014]. Results: NHP
composed of block copolymers loaded with negative control
siRNA up to 600 nM siRNA and of a size of 30 to 40 nm did
not show cytotoxicity for murine 3T3 fibroblasts, Raw or MHS
macrophages, HepG2 human liver cells or AML-12 murine
benign hepatocytes. In full media FITC-labelled NHP were
taken up efficiently reaching up to 90% after 1 h incubation as
determined by FACS analysis. NHP loaded with procollagen
a1(I) or CD68 targeted siRNA yielded a robust knockdown
in 3T3 and Raw cells, respectively, after 48 h of incubation
as determined by qPCR. In vivo studies using near infrared
labelled NHP loaded with Cy5 labelled procollagen a1(I)
siRNA were performed in lCCl4 treated mice. After i.v. injection
NHP accumulated in the liver (80%), as determined by in
vivo and ex vivo near infrared imaging. IHC and ex vivo FACS
analysis revealed a preferential colocalization with myofibroblasts
(α-SMA+) and macrophages (CD45+ F4/80+ CD11b+).
NHP loaded with procollagen a1(I) siRNA generated an up to
80% in vivo knockdown in CCl4-induced liver fibrosis. Efficient
knockdown was further confirmed by decreased liver collagen
(Sirius red staining and hydroxyproline content). Conclusions:
siRNA loaded NHP accumulate in the liver and especially in
(myo-) fibroblasts and macrophages/Kupffer cells. Due to their
intrinsic specificity for nonparenchymal cells, an apparent lack
of toxicity and a high loading capacity, they are attractive
carriers for siRNA-based antifibrotic or immune modulatory
therapies.
Disclosures:
The following authors have nothing to disclose: Leonard Kaps
1427
Host microbiota dictates the profibrotic impact of PAMPs
in the liver
Suriguga Suriguga 1 , Floris Fransen 2 , Dorenda Oosterhuis 1 , Geny
M. Groothuis 3 , Paul D. Vos 2 , Henricus A. Mutsaers 1 , Peter Olinga 1 ;
1 Division of Pharmaceutical Technology and Biopharmacy, Department
of Pharmacy, University of Groningen, Groningen, Netherlands;
2 Division of Medical Biology, Department of Pathology and
Medical Biology, University Medical Center Groningen, University
of Groningen, Groningen, Netherlands; 3 Division of Pharmacokinetics,
Toxicology and Targeting, Department of Pharmacy, University
of Groningen, Groningen, Netherlands
Background Pathogen-associated molecular patterns (PAMPs),
e.g. lipopolysaccharide (LPS) and flagellin, might promote liver
fibrosis via the gut-liver axis. However, how these bacterial
components initiate fibrogenesis needs further clarification.
In germ-free (GF) mice, the liver has no history of interaction
with PAMPs, while in colonized (specific pathogen free, SPF)
mice the liver is exposed to PAMPs. Therefore, this study was
designed to investigate the possible fibrogenic effect of PAMPs
in SPF and GF mice, using precision-cut liver slices (PCLS) as
fibrosis model. Methods PCLS from SPF and GF mice were
incubated with or without 10 mg/ml LPS or 50 ng/ml flagellin
for 48h. Viability of PCLS was assessed by measuring their
ATP content. Gene expression of key fibrosis markers Procollagen1α1
(Col1α1), α-Smooth Muscle Actin (α-SMA), Heat
Shock Protein 47 (Hsp47), and Plasminogen Activator Inhibitor-1
(PAI-1) were determined by real-time qPCR. Results Both
SPF and GF mice liver slices were viable up to 48h. Solely in
SPF PCLS, we observed spontaneous onset of fibrosis during
culture up to 48h. Yet, treatment with LPS or flagellin did not
augment the fibrotic response. In contrast, in GF PCLS, Hsp47
expression was significantly up-regulated in the presence of
LPS: 1.4-fold, while Col1α1 and α-SMA tended to increase
compared to control slices incubated for 48h. PAI-1, a marker
for transforming growth factor-β (TGF-β) pathway activation,
was significantly elevated up to 81.8-fold in SPF and 37.2-fold
in GF PCLS after culture (48h) compared to directly after slicing.
Exposure to LPS further increased PAI-1 gene expression in
GF PCLS (2.9-fold), but not in SPF mice. Moreover, in GF liver
slices treatment with flagellin significantly increased expression
of Col1α1 (1.8-fold), α-SMA (1.8-fold), and Hsp47 (1.2-fold),
while it did not effect PAI-1. Conclusion Both LPS and flagellin
could exacerbate fibrogenesis in GF mouse liver slices but not