Lipid-encapsulated siRNA for hepatocyte-directed treatment of advanced liver disease

Abstract

Lipid-based RNA nanocarriers have been recently accepted as a novel therapeutic option in humans, thus increasing the therapeutic options for patients. Tailored nanomedicines will enable to treat chronic liver disease (CLD) and end-stage liver cancer, disorders with high mortality and few treatment options. Here, we investigated the curative potential of gene therapy of a key molecule in CLD, the c-Jun N-terminal kinase-2 (Jnk2). Delivery to hepatocytes was achieved using a lipid-based clinically employable siRNA formulation that includes a cationic aminolipid to knockdown Jnk2 (named siJnk2). After assessing the therapeutic potential of siJnk2 treatment, non-invasive imaging demonstrated reduced apoptotic cell death and improved hepatocarcinogenesis was evidenced by improved liver parenchyma as well as ameliorated markers of hepatic damage, reduced fibrogenesis in 1-year-old mice. Strikingly, chronic siJnk2 treatment reduced premalignant nodules, indicative of tumor initiation. Furthermore, siJnk2 treatment led to a significant activation of the immune cell compartment. In conclusion, Jnk2 knockdown in hepatocytes ameliorated hepatitis, fibrogenesis, and initiation of hepatocellular carcinoma (HCC), and hence might be a suitable therapeutic option, to define novel molecular targets for precision medicine in CLD.

Fig. 1. Design and validation of siRNA sequence for Jnk2 inhibition.

a Bioinformatics-based sequence design for the inhibiting Jnk2 mRNA resulted in 12 different siRNA sets. The different binding regions of each siRNA set is shown. b Selection of the most efficient siRNA sets targeting Jnk2 in cell-culture experiments. Hepa 1–6 were treated with either medium only, or transfected with Lipofectamine or one out of 12 different computer-defined Jnk2-siRNAs [1 nmol] for 24 h and the Jnk2 mRNA expression was quantified by using qRT-PCR. Results are expressed as fold increase. c Hepa 1–6 cells were treated with medium only or transfected with Lipofectamine or siJnk2 (which corresponded to siRNA sets 3 or 4) in a concentration ranging from 0.125–10 nmol for 24 h and subsequently Jnk2 mRNA levels were determined by using qRT-PCR. Results are expressed as fold increase. d Primary murine WT hepatocytes received either medium only, or were transfected with Lipofectamine or siJnk2 sets 3 or 4 in a concentration ranging from 0.125–5 nmol for 24 h. The mRNA levels of Jnk2 were quantified by using qRT-PCR and results are shown as fold increase. Results are expressed as mean ± S.E.M. ***P < 0.001.

Fig. 2. Genetic deletion of Jnk2 reduces liver cancer at advanced stages.

NEMO^ΔHepa, JNK2^ΔHepa, and NEMO/JNK2^ΔHepa mice and their respective control animals (NEMO^Control, JNK2^Control, and NEMO/JNK2^Control were generated. Mice were sacrificed at the age of 52 weeks. Livers were extracted and analyzed macros- and microscopically for phenotypic characterization. a Macroscopic images of livers of 1-year-old control mice (upper images) and their corresponding knockout mice NEMO^ΔHepa, JNK2^ΔHepa, and NEMO/JNK2^ΔHepa (lower images), scale bars: 1 cm. Number of tumor nodules ≥0.5 cm in diameter were quantified in livers of NEMO^ΔHepa and NEMO/JNK2^ΔHepa animals at the age of 52 weeks (right). b Representative histological liver sections of the identical control animals (upper images) and the knockout mice (upper images) at the age of 52 weeks were stained with hematoxylin and eosin (left). Serum liver ALT (right) of 52-week-old animals is displayed in U/l (n = 11–13). (Scale bars: 100 μm).

Fig. 3. Lipid-based nanoparticles for organ and cell-type-specific delivery of siRNA molecules.

a Scheme of T-junction-based production of lipid-based nanoparticles for siRNA delivery, which are composed of a lipid mixture containing the aminolipid KL-52, 1,2-distearoyl-3-phosphatidylcholine (DSPC), α-[3’-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-ω-methoxy-polyoxyethylene (PEG-c-DOMG), and cholesterol dissolved in ethanol and of a therapeutic siRNA in aqueous buffer. b C57BL6J mice aged 7–8 weeks were treated with a single dose of luciferase (siLuc) or siJnk2-LNP in vivo with a concentration of 0.2 mg/kg BW for 6 h (n = 3). Jnk2 mRNA expression was determined in isolated liver, spleen, kidney, muscle, heart, and fat tissue. c Jnk2 mRNA levels were analyzed in hepatocytes, KC and HSC isolated from livers of WT mice that were treated with either siLuc or siJnk2-LNP. d Jnk2 mRNA expression in whole liver extracts in doses ranging from 0.05 to 0.2 mg/kg after 6 or 168 h at the indicated doses. Quantitative RT-PCR results were displayed as fold induction. e Protein levels of JNK2, pJNK, and JNK were analyzed by immunoblotting of whole liver extracts at siRNA doses ranging from 0.05 to 0.2 mg/kg for 6 or 168 h. GAPDH was used as loading control. Ratio: Normalization of each protein level relative to GAPDH was determined by densitometry. Results are expressed as means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001; ns no significance.

Fig. 4. Hepatocyte-specific siRNA-dependent Jnk2 deletion in an early phase of chronic liver injury.

Male 8-week-old NEMO^Control and NEMO^ΔHepa animals were injected on a weekly basis with a single dose of 0.2 mg/kg siLuc or siJnk2-LNP, over a period of 4 weeks and sacrificed at 12 weeks of age (n = 12). a Schematic cartoon of the siJnk2-LNP injection protocol. b Accumulation of Duramycin-NIR790 in the liver, a quantitative biomarker of cell death. c Representative H&E-stained liver sections prepared from 12-week-old NEMO^Control (upper panels) and NEMO^ΔHepa (lower panels) mice receiving either siLuc-LNP or siJnk2-LNP injections. (Scale bars: 100 μm). d Liver injury reflected by alanine aminotransferase (ALT) enzyme activity. e Immunoblot analysis of whole liver extracts detecting cleaved caspase-3 and PCNA. GAPDH was used as loading control. f Caspase-3 activity in the livers from 12-week-old NEMO^Control mice compared to NEMO^ΔHepa mice receiving siLuc-LNP or siJnk2-LNP injections. The activity was represented as relative fluorescence units (RFU). g Hepatic mRNA levels of PCNA were quantified and displayed as fold increase for the identical treatment groups. Data are presented as means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. Treatment of advanced stage liver tumors with lipid nanoparticles targeting Jnk2 in Hepatocytes.

Male 44-week-old NEMO^ΔHepa mice were injected weekly with a single dose of 0.2 mg/kg luciferase (siLuc) or siJnk2-LNP beginning at 44 weeks of age until reaching 52 weeks of age. a Experimental setup of the weekly injections of the LNP. b Representative macroscopic images (left) of the livers of NEMO^ΔHepa mice at an age of 44 weeks (control) and with 52 weeks. Dashed circles indicate tumoral lesions (scale bars: 1 cm). Number of tumor nodules ≥0.5 cm in diameter (right) were quantified. c Representative H&E staining of liver sections (left) of untreated 44-week-old mice and the two treatment groups after siRNA-LNP treatment. Liver injury reflected by serum ALT (right, n = 12–14). d Expression of JNK2 and pJNK2 was analyzed by immunoblotting of liver samples of one-year-old NEMO^ΔHepa mice treated with either siLuc-LNP or siJnk2-LNP. e Quantitative RT-PCR analysis of the mRNA levels of Jnk2 and survivin for the same NEMO^ΔHepa treatment groups were determined and are represented as fold increase. Data are presented as means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6. Effects of Jnk2-directed siRNA lipid nanoparticles on cell death in mice with advanced liver cancer.

Mice aging 44 weeks were treated with either siLuc or siJnk2-LNP for another 8 weeks. a 52-week-old NEMO^ΔHepa mice were injected with Duramycin-NIR790 and non-invasive µ-CT-FMT-based whole body imaging was performed. Representative FMT/µCT images showing the accumulation of Duramycin-NIR790 in the liver. b µCT-FMT-based quantification of Duramycin in livers. c Liver tissue extracts were subjected to immunoblotting for PCNA and cleaved Caspase-3. The same membrane was then stripped and reblotted for GAPDH, used as loading control. d Activity of cleaved caspase 3 and PCNA e mRNA expression. f Flow-cytometry analysis demonstrated changes in immune cell populations. Cells were counted and quantified and given as cell numbers for the whole liver. Data analysis is expressed as means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.