Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene, predominantly expressed in the liver. Two compounds that knockdown TTR, comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx), are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background, this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown, siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (>80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds

Identification of survival-promoting OSIP108 peptide variants and their internalization in human cells

The plant-derived decapeptide OSIP108 increases tolerance of yeast and human cells to apoptosis-inducing agents, such as copper and cisplatin. We performed a whole amino acid scan of OSIP108 and conducted structure-activity relationship studies on the induction of cisplatin tolerance (CT) in yeast. The use of cisplatin as apoptosis-inducing trigger in this study should be considered as a tool to better understand the survival-promoting nature of OSIP108 and not for purposes related to anti-cancer treatment. We found that charged residues (Arg, His, Lys, Glu or Asp) or a Pro on positions 4–7 improved OSIP108 activity by 10% or more. The variant OSIP108[G7P] induced the most pronounced tolerance to toxic concentrations of copper and cisplatin in yeast and/or HepG2 cells. Both OSIP108 and OSIP108[G7P] were shown to internalize equally into HeLa cells, but at a higher rate than the inactive OSIP108[E10A], suggesting that the peptides can internalize into cells and that OSIP108 activity is dependent on subsequent intracellular interactions. In conclusion, our studies demonstrated that tolerance/survival-promoting properties of OSIP108 can be significantly improved by single amino acid substitutions, and that these properties are dependent on (an) intracellular target(s), yet to be determined

Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis

The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR), expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP). Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO) and small interfering RNA (siRNA) designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease

Portal Hypertensive Polyposis in Advanced Liver Cirrhosis: The Unknown Entity?

Background. Portal hypertension is a serious complication of liver cirrhosis. Objective. To identify relevant endoscopic findings in patients with advanced cirrhosis and consecutive portal hypertension. Methods. This was a retrospective study of liver transplant candidates who underwent upper gastrointestinal endoscopy between April 2011 and November 2015. Results. A total of 1,045 upper endoscopies were analyzed. Portal hypertensive gastric and duodenal polyps were frequently observed and were associated with thrombocytopenia (p = 0.040; OR: 2.4, 95% CI 1.04–5.50), Child-Pugh score > 6 (p = 0.033; OR: 2.3, 95% CI 1.07–4.92), Model for End Stage Liver Disease score > 16 (p = 0.030; OR: 4.1, 95% CI 1.14–15.00), and previous rubber band ligation (p < 0.001; OR = 5.2, 95% CI 2.5–10.7). These polyps often recurred after polypectomy; however, no malignant transformation occurred during the observational time until October 2017. The most common endoscopic finding was esophageal varices, observed in more than 90% of patients. Conclusion. Portal hypertensive polyposis is common in patients with advanced cirrhosis. Our data suggest that these polyps have benign characteristics

Angiotensin II type 1 receptor blockers increase tolerance of cells to copper and cisplatin

The human pathology Wilson disease (WD) is characterized by toxic copper (Cu) accumulation in brain and liver, resulting in, among other indications, mitochondrial dysfunction and apoptosis of hepatocytes. In an effort to identify novel compounds that can alleviate Cu-induced toxicity, we screened the Pharmakon 1600 repositioning library using a Cu-toxicity yeast screen. We identified 2 members of the drug class of Angiotensin II Type 1 receptor blockers (ARBs) that could increase yeast tolerance to Cu, namely Candesartan and Losartan. Subsequently, we show that specific ARBs can increase yeast tolerance to Cu and/or the chemotherapeutic agent cisplatin (Cp). The latter also induces mitochondrial dysfunction and apoptosis in mammalian cells. We further demonstrate that specific ARBs can prevent the prevalence of Cu-induced apoptotic markers in yeast, with Candesartan Cilexetil being the ARB which demonstrated most pronounced reduction of apoptosis-related markers. Next, we tested the sensitivity of a selection of yeast knockout mutants affected in detoxification of reactive oxygen species (ROS) and Cu for Candesartan Cilexetil rescue in presence of Cu. These data indicate that Candesartan Cilexetil increases yeast tolerance to Cu irrespectively of major ROS-detoxifying proteins. Finally, we show that specific ARBs can increase mammalian cell tolerance to Cu, as well as decrease the prevalence of Cu-induced apoptotic markers. All the above point to the potential of ARBs in preventing Cu-induced toxicity in yeast and mammalian cells.status: publishe

Drug induced tolerance of copper treated KO cells.

<p>Cells were treated with Zn, DPA and Zn+DPA and viability was determined by MTT assay. Zn pretreatment was for 2 h. DPA and Cu exposure was for 48 h. The gain of viability following treatment (black) is given relative to the viability of untreated KO (open) and HepG2 cells (shadow). Significance (p<0.05) is indicated: *treated vs untreated; ^†gain by treatment in KO vs. gain in HepG2 cells; ^aZn+DPA treatment vs. Zn; ^bZn+DPA treatment vs. DPA.</p

Angiotensin II type 1 receptor blockers increase tolerance of cells to copper and cisplatin

The human pathology Wilson disease (WD) is characterized by toxic copper (Cu) accumulation in brain and liver, resulting in, among other indications, mitochondrial dysfunction and apoptosis of hepatocytes. In an effort to identify novel compounds that can alleviate Cu-induced toxicity, we screened the Pharmakon 1600 repositioning library using a Cu-toxicity yeast screen. We identified 2 members of the drug class of Angiotensin II Type 1 receptor blockers (ARBs) that could increase yeast tolerance to Cu, namely Candesartan and Losartan. Subsequently, we show that specific ARBs can increase yeast tolerance to Cu and/or the chemotherapeutic agent cisplatin (Cp). The latter also induces mitochondrial dysfunction and apoptosis in mammalian cells. We further demonstrate that specific ARBs can prevent the prevalence of Cu-induced apoptotic markers in yeast, with Candesartan Cilexetil being the ARB which demonstrated most pronounced reduction of apoptosis-related markers. Next, we tested the sensitivity of a selection of yeast knockout mutants affected in detoxification of reactive oxygen species (ROS) and Cu for Candesartan Cilexetil rescue in presence of Cu. These data indicate that Candesartan Cilexetil increases yeast tolerance to Cu irrespectively of major ROS-detoxifying proteins. Finally, we show that specific ARBs can increase mammalian cell tolerance to Cu, as well as decrease the prevalence of Cu-induced apoptotic markers. All the above point to the potential of ARBs in preventing Cu-induced toxicity in yeast and mammalian cells

Oxidative stress and viability of KO cell line.

<p>(A) Oxidative stress following Cu exposure at 0.1 mM Cu for 1 h. The four histograms represent fluorescence of viable cells as obtained by flow cytometry after staining with H₂DCFDA dye. A shift of the two histograms appears in KO cells after Cu exposure (shaded) relative to untreated control (open) indicating induction of OS. (B) Viability of KO (circles) and HepG2 cells (triangle) relative to untreated control was determined by MTT assay after 48 h of Cu exposure. (C) Induction of apoptosis was determined after 24 h of 0.1 mM Cu exposure using Annexin-V staining followed by flow cytometry analysis. (D) Cell viability of knockin versus KO cell is shown. Viability was determined at 0.5 mM Cu by MTT assay after 48 h. Data is represented as mean±SE of three independent experiments. Asterisks indicate significance (p<0.05).</p