Liposome Mediated Delivery of sirn to Hepatic Stellate ells lfica Sehgal, Mohammad Zafari, oris Klebanov, Greg Hinkle, Satya Kuchimanchi, Sarfraz Shaikh, Martin Maier, Jonathan O Shea, Lauren Speciner, kin kinc, Daniel nderson,* Robert Langer,* Tatiana Novobrantseva, Victor Kotelianski, David umcrot* lnylam Pharmaceuticals, ambridge, M 242; *Koch Institute for Integrative ancer Research at MIT, ambridge, M
bstract pproximately 28, mericans die every year from chronic liver diseases (LD) including chronic hepatitis, alcoholism and non-alcoholic fatty liver. While all LDs can lead to hepatic fibrosis, therapeutic interventions generally target the etiological agent of the disease and do not directly address the underlying fibrosis. Hepatic stellate cells (HSs) play a key role in the cause and progression of liver fibrosis. n insult/injury to the liver activates the quiescent stellate cells which then synthesize extracellular matrix (EM) as part of a wound healing response. However, repeated liver injury leads to further proliferation of stellate cells, increased matrix deposition, and ultimately, liver fibrosis or cirrhosis. Therefore, reducing the production of EM by stellate cells would be predicted to inhibit the progression of fibrotic disease. Here we describe lipid nanoparticle (LNP) mediated sirn delivery to quiescent and activated stellate cells in three mouse models of liver injury: induction by carbon tetrachloride, thioacetamide or bile-duct ligation. sirns formulated in LNPs comprising the cationic lipid, 2-2 (PNS, 2: 7(5)-864) were administered via intravenous injection. Silencing of the HS-specific targets, collagena (cola) or rhon demonstrated effective sirn delivery to stellate cells. We achieved durable target knock-down in a dose dependent manner with an I 5 of approximately. mg/kg for cola and.-.3 mg/kg for rhon. Delivery to activated HSs was further confirmed using sirns directed against a second HS target, smooth muscle actin (cta2). We propose that sirn-mediated inhibition of cola, or other EM components, warrants further exploration as a novel therapeutic approach for liver fibrosis. Figure. Stellate cells Stellate cells play key role in liver fibrosis dapted from J. linical Investigation 25 (5:2-29)
Stellate cells play key role in liver fibrosis dapted and modified from lin Sci (27) 2:265; lin Liver Dis. (28) 2: 939 Hepatic stellate cells (HS): Ito cells, fat storing cells, interstitial cells, lipocytes Described by Kupffer (876) as liver sternzellen (star shaped) Present in Space of Disse 5-% of total resident liver cells Major storage sites for retinoids (vit ) in normal livers Injury to liver induces a wound healing response hemokine signaling ctivation of stellate cells ctivated HSs become highly proliferative Synthesize fibrotic matrix rich in Type I ollagen Scarring of tissue hronic insult leads to fibrosis and eventually cirrhosis Targeting stellate cells Stellate cells are the major players in fibrosis hence targeting them can open different avenues for treatment ollagen (ol) is the principal collagen in fibrotic liver and is expressed by stellate cells in the liver Decrease in ollagena (ola) after treatment with sirn indicates delivery to activated stellate cells ola is a heterotrimer: 2 chains of pro-a(i) & chain of pro-a2(i) ola forms homotrimers in absence of ola2 ola2 degrades intra-cellularly in absence of ola Decrease in ollagen should Destabilize the fibrillar architecture Provide negative feedback to the HSs and lead to HS apoptosis/clearing Promote recovery of normal liver function
Figure 2. Silencing of quiescent stellate cells.4.4 Relative RhoN mrn/gpdh.2.8.6.4.2 Relative Levels of RhoN/GPDH.2.8.6.4.2 PS.3..3 Day 2 Day Day 2 Day 7 Day 4 Day 2 Day 2 RhoN Luc Luc RhoN Luc Figure 2. Silencing of quiescent stellate cells.. Dose dependent silencing of RhoN mediated by LNP-RhoN sirn. nimals (n=5) were IV dosed with,.3,.,.3 mpk LNP-RhoN or mpk LNP-Luc or PS and sacrificed 48 hours after the dose.. nimals (n=5) were dosed with.5 mg/kg LNP-RhoN or LNP-Luc and sacrificed on the indicated Day. RhoN is a small GTPase, and its expression in normal liver is limited to stellate cells. Silencing of RhoN in whole livers signifies delivery to quiescent stellate cells. Figure 3. l 4 induced liver injury Liver damage 24 hours after single I 4 dose MO l4 Induction of ola message by I 4 Serum Levels 2,899 29 n=5 LT 2,4 62 n=5 ST Relative ola mrn/gpdh 5 4 3 2. MO 37.2 n=5 l 4 Mineral oil treated animals I 4 treated animals Figure 3. l 4 induced liver injury. Two doses of l 4 were administered 7 days apart, and animals were sacrificed 24 (in ) or 48 hours (, ) after the treatment.. Levels of LT and ST measured in serum of treated mice (n=5). verage of ola mrn measured in livers after treatment (n=5).. Representative liver sections stained with Sirius red.
Figure 4. Knockdown in activated stellate cells Day: 9 2 l 4 gavage (solvent mineral oil) IV dose Sacrifice Relative ola mrn/gpdh.6.4.2.8.6.4.2 Relative SM mrn Levels/GPDH.4.2.8.6.4.2 3.5. 3 PS PS LNP-ola (mg/kg) LNP-Luc 3 3 PS PS LNP-SM LNP-Luc Figure 4. Target knockdown in activated stellate cells.. Dose dependent ola mrn knockdown with LNP formulated sirn in acute injury model. The mrn levels were measured using Taqman-QPR in total livers. The graph represents normalized signal for ola mrn, using GPDH as internal control. Luc: Luciferase, sirn control (n= per l 4 group). a- SM mrn knock-down with LNP-SM.
Figure 5. Delivery to stellate cells in different injury models 6 9 4 8 Relative ola mrn/gpdh 2 8 6 4 2 Ola/GPDH mrn (relative to control) 7 6 5 4 3 2 PS LNP-ola LNP-Luc PS T Saline PS PS LNP-ola LNP-Luc Normal D Ligation Percent Survival 2 8 6 4 2 Luc ola PS 5 7 9 2 Days after DL Figure 5. Delivery to activated stellate cells in different injury models.. LNP mediated delivery to stellate cells in mice treated with thioacetamide (T), an acute injury agent. Mice were given 3 weekly doses of T and cola expression was measured in livers 2 days after sirn dose. (n=). LNP mediated delivery in severe acute injury model of bileduct ligation (DL). D ligation was done on Day. nimals were dosed with sirn on days 4 and 7 and sacrificed on day 8 (n=5).. DL mice were IV dosed on days 5, 7 and with PS, LNP- ola or LNP-Luc (n=5). The number of surviving animals were counted each day and all remaining animals were sacrificed on Day 2. nimals treated with LNP-ola could sustain the injury longer than other two groups. Figure 6. LNP delivery to stellate cells in chronic injury model Day: 8 5 22 23 27 29 3 33 36 37 39 l 4 or MO IV dose.5 mg/kg x Sacrifice 8 7 ola/gpdh mrn (relative to control) 6 5 4 3 2 PS LNP-siLuc LNP-siola PS LNP-siLuc LNP-siola
ollagen protein measured by western blot MO l4 -ola l4 -Luc Pro-olI 3 Relative Signal Intensity 25 2 5 5 2 3 4 5 6 7 8 9 2 3 -ola l4-luc Robust delivery to stellate cells under conditions of chronic liver injury Multi-insult, multi-dose model ~5-fold induction of ola mrn by l 4 ~95% knock-down of ola mrn ~9% reduction in collagen I protein Figure 6. LNP mediated delivery to stellate cells in a chronic injury model.. The experimental plan is depicted in the line diagram. ola mrn knockdown with LNP- sirn in multi-insult, multi-dose model. The mrn levels were measured using Taqman-QPR in total livers. The graph represents normalized signal for ola mrn, using GPDH as internal control. Luc: Luciferase, sirn control (n=). ollagen protein measured by western blot (top). Quantification of ol normalized to b-actin is shown in the graph below. The western blot was scanned using Lior Odyssey.
Figure 7. ola knockdown is mediated by RNi RN sample Ligate adaptor Day: 9 I 4 or MO gavage IV dose LNP-ola Sacrifice cdn synthesis Gene specific primer I 4 - ola I 4 st rd PR Nested PR nticipated band size TOPO cloning Sanger sequencing lones with 5 RE cleavage product I 4 - LNP-ola I 4 8% (8/) % (/) Figure 7. ola knockdown is mediated by RNi. animals were given l 4 by oral gavage.. Scheme for 5 RE assay. Total liver mrn was processed using Generacer kit (Invitrogen) for cdn synthesis and 5 RE to analyze the cleaved cola. The PR products were cloned into T vectors and sequenced.. nimals were injected with LNP-ola and sacrificed after 24 hours.. garose gel showing DN bands for nested PR. 8% of the clones from LNP-ola group showed expected cleavage product.
Figure 8. Delivery to hepatocytes under conditions of liver injury IFN-α TNF-α TGF-β TGF-β R SMD7 P SMD 2/3 SMD 4 SMD-independent pathways p38 JNK -bl P3K TK SMD4 p3 P SMD2/3 EMT and other profibrotic responses ollagen gene expression Figure 8. artoon depiction of TGF-b SMD signaling pathway in liver fibrosis. Different signaling pathways lead to production of extracellular matrix components including ollagen. Relative SMD2 mrn Levels/GPDH.4.2.8.6.4.2 sirn SMD2/3 Luc ola/gpdh mrn (relative to control) 6 4 2 8 6 4 2 sirn PS ola SMD2/3 Luc l 4 D Relative FVII mrn/gpdh.2.8.6.4.2 LNP-FVII LNP-Luc LNP-FVII LNP-Luc SMD2/3 sirn decreases SMD2 mrn levels by ~8% after l 4 injury ola mrn reduced by ~4%, likely downstream effect of SMD2 mrn reduction FVII knock-down demonstrates LNP-siRN delivery under conditions of liver injury Figure 8. Delivery to hepatocytes under conditions of liver injury. set of sirn targeting both SMD2 and SMD3 were screened in NIH 3T3 cells. The lead candidate was formulated for in vivo experiment. Liver injury was induced using l 4. nimals were injected with sirn against SMD2/3, cola or Luc and samples processed for Taqman-QPR.. SMD2/3 mrn levels normalized to GPDH.. ola mrn levels after treating the animals with sirn against ola, SMD2/3 or Luc are shown, normalized to GPDH.. Delivery to hepatocytes was assessed by measuring knock-down of FVII after l 4 treatment. Similar levels of knock-down were observed in animals treated with l 4 or the vehicle, mineral oil. (Luc: sirn against luciferase: as a control, FVII: sirn against Factor VII, a protein specifically synthesized by hepatocytes in the liver.) Summary Potent and reproducible LNP mediated delivery to quiescent and activated stellate cells Tested LNP-siRN delivery to activated stellate cells under different models of liver injury cute and chronic injury model for pericentral injury: l 4 ile-duct ligation injury model for portal fibrosis Thioacetamide induced injury in pericentral and periportal areas Delivery confirmed with silencing of multiple markers Robust knockdown of ola in activated stellate cells Maximum knockdown ~9%, ED 5 around. mg/kg Delivery to hepatocytes in chronically injured liver Supports further development of ola targeting as a therapeutic approach