Combined transgene immortalized urothelial cells capable of reprogramming and hepatic differentiation.

Human primary cells, including urine-derived cells (UCs), are an excellent source for generation of pluripotent stem cells (iPSCs) to model disease. However, replicative senescence starts early and shortens the time window for generation of iPSCs. We addressed the question whether combinations of transgenes allows efficient immortalization of UCs, iPSC generation, and differentiation into hepatocyte-like cells (HLCs). Retroviral transfer of three gene cassettes HPVE6E7 (H), hTERT/p53DD (T), cyclinD1/CDK4R24C (C) encoding five genes was established in primary UCs. Long-term cell proliferation was observed in cells carrying transgenes H, HT, HC, and HCT, whereas cells carrying transgenes C, T and CT showed early senescence similar to UCs. iPSCs could be exclusively generated from immortalized UCs transduced with transgenes HCT and HC. iPSC colonies appeared however later and in smaller number as compared to UCs. Using an established hepatic differentiation protocol, HLCs were obtained with high efficacy. Of note, a high expression of individual transgenes was observed in immortalized UCs, which was down-regulated after reprogramming in four out of five genes. One transgene was re-expressed in HLCs as compared to iPSCs. Our data suggest that individual transgene combinations result in advanced growth rates of immortalized cells and do not prevent iPSC formation and HLC differentiation. Retroviral transgene expression is mostly silenced in iPSCs but can be rarely re-expressed after hepatic differentiation. An extended time window for iPSC establishment can be proposed that allows straightforward functional analyses of differentiated cells.

In Vitro and In Vivo Effects of SerpinA1 on the Modulation of Transthyretin Proteolysis.

Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.

SERPINA1 modulates expression of amyloidogenic transthyretin.

Hereditary transthyretin amyloidosis (ATTR) is caused by amyloid deposition of misfolded transthyretin (TTR) in various tissues. Recently, reduction of circulating serum TTR, achieved via silencing oligonucleotides, was introduced as therapy of ATTR amyloidosis. We explored the impact of Serpin Family A Member 1 (SERPINA1) on TTR mRNA and protein expression. Oncostatin M (OSM) induced SERPINA1 in hepatoma cells and mice, while concomitantly TTR expression was significantly reduced. SERPINA1 knockdown resulted in specific elevated TTR expression in hepatoma cells; however other genes belonging to the group of acute phase proteins were unaffected. In mice, serum TTR was elevated after mSERPINA1 knockdown throughout antisense treatment. Following SERPINA1 knockdown, TTR deposition in several tissues, including dorsal root ganglia and intestine, was found to be increased, however numbers did not exceed significance levels. The data suggest that SERPINA1 is a co-factor of TTR expression. Our findings provide novel insight in the regulation of TTR and reveal a role of SERPINA1 in the pathogenesis of ATTR amyloidosis.

APOE polymorphism in ATTR amyloidosis patients treated with lipid nanoparticle siRNA.

The novel class of compounds represented by lipid nanoparticle (LNP)-encapsulated siRNA formulations has an enormous potential to target disease, notably of the liver. Endocytosis of LNPs is believed to be mediated by APOE, an important serum protein of lipoprotein homeostasis. APOE polymorphisms affect binding to hepatic receptors and have been associated with development of specific disease. Here, the role of APOE was investigated with regard to the efficacy of Patisiran, the first LNP-siRNA recently approved for clinical use in patients having transthyretin amyloidosis (ATTR amyloidosis). Patisiran was evaluated in the human hepatoma cell line HepG2 after knockdown of APOE. The APOE genotype was determined in ATTR amyloidosis patients treated with Patisiran. TTR knockdown was monitored in consecutive plasma up to week 12. Downregulation of APOE suppressed efficacy of Patisiran in HepG2 cells. TTR levels were found to be robustly reduced (84.7% ± 1%) following Patisiran treatment in patients plasma. Analysis of APOE polymorphisms in ATTR amyloidosis patients revealed three most frequent genotypes E3/3, E3/4 and E3/2. APOE stratification of patients did not show significant differences of TTR plasma concentrations following treatment. Our results suggest that APOE is an important mediator of TTR silencing by Patisiran, however efficacy is independent of the APOE genotype.

Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy.

BACKGROUND: Patients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD. METHODS: Since current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy. RESULTS: Under treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells. CONCLUSION: We conclude that repeated AGAL activity measurements in patients' white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.

Hepatocyte-like cells reveal novel role of SERPINA1 in transthyretin amyloidosis.

Transthyretin (TTR)-related familial amyloid polyneuropathy (ATTR) results from aggregation and extracellular disposition of misfolded TTR mutants. Growing evidence suggests the importance of hepatic chaperones for the modulation of pathogenesis. We took advantage of induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) from ATTR patients (ATTR-HLCs) to compare chaperone gene expression to that in HLCs from healthy individuals (H-HLCs). From the set of genes analyzed, chaperones that are predominantly located extracellularly were differently expressed. Expression of the chaperones showed a high correlation with TTR in both ATTR-HLCs and H-HLCs. In contrast, after TTR knockdown, the correlation was mainly affected in ATTR-HLCs suggesting that differences in TTR expression triggers aberrant chaperone expression. Serpin family A member 1 (SERPINA1) was the only extracellular chaperone that was markedly upregulated after TTR knockdown in ATTR-HLCs. Co-immunoprecipitation revealed that SERPINA1 physically interacts with TTR. In vitro assays indicated that SERPINA1 can interfere with TTR aggregation. Taken together, our results suggest that extracellular chaperones play a crucial role in ATTR pathogenesis, in particular SERPINA1, which may affect amyloid formation.

[Transthyretin Familial Amyloid Polyneuropathy - Disease Profile of a Multisystem Disorder]. / Transthyretin-assoziierte familiäre Amyloidpolyneuropathie.

Transthyretin-related Familial Amyloid Polyneuropathy (ATTR Amyloidosis, former FAP, here called TTR-FAP) is a rare, progressive autosomal dominant inherited amyloid disease ending fatal within 5 - 15 years after final diagnosis. TTR-FAP is caused by mutations of transthyretin (TTR), which forms amyloid fibrils affecting peripheral and autonomic nerves, the heart and other organs. Due to the phenotypic heterogeneity and partly not specific enough clinical symptoms, diagnosis of TTR-FAP can be complicated. False diagnoses can include idiopathic polyneuropathy, chronic inflammatory demyelinating polyneuropathy, diabetic neuropathy as well as paraneoplastic syndrome. Hence, it is assumed that many cases remain unreported. Early and correct diagnosis of TTR-FAP is crucial, since appropriate therapeutic options exist. TTR-FAP should always be differentially diagnosed, when apart from a progressive peripheral polyneuropathy, additional symptoms as autonomic dysfunction, cardiomyopathy, gastrointestinal disorders, unexpected loss of weight, carpal tunnel syndrome, restrictions of renal function, epileptic fits, and corneal and vitreous body clouding occur. Histological evidence of amyloid and successive immunohistochemical evidence of transthyretin as well as genetic testing for transthyretin mutations, lead to an accurate diagnosis.

Organic cation transporter 3 mediates cisplatin and copper cross-resistance in hepatoma cells.

Platinum-based drugs are first-line compounds in the treatment of many solid cancers. Major obstacles are tumors that become resistant and toxic side effects, both largely due to the expression of transporters that mediate the cellular processing of platinum. In this study, we addressed the establishment of cisplatin resistance in the absence of copper transporter ATP7B that has been previously found to be overexpressed in various resistant cells. Cisplatin sensitivity, induction of apoptosis, drug accumulation, and transporter gene expression were determined in hepatoma cell lines. Knockout or overexpression of copper transporter ATP7B did not affect cisplatin sensitivity. Cisplatin resistant cells showed a stably reduced cisplatin accumulation and a downregulation of organic cation transporter 3 (OCT3). In contrast, OCT3 overexpression could reverse resistance. Reduced MT1 expression was detected in the resistant cell line, however transient and highly dependent on the presence of cisplatin. Cross-resistance to copper was also associated with OCT3 downregulation. Our results suggest that a decreased level of OCT3 expression results in resistance to cisplatin and copper. OCT3 may represent a novel target for improved prognosis and anticancer therapy, including HCC.

Downregulation of hepatic multi-drug resistance protein 1 (MDR1) after copper exposure.

Copper homeostasis is strictly regulated in mammalian cells. We investigated the adaptation of hepatocytes after long-term copper exposure. Copper-resistant hepatoma HepG2 cell lines lacking ATP7B were generated. Growth, copper accumulation, gene expression, and transport were determined. Hepatocyte-like cells derived from a Wilson disease (WD) patient and the liver of a WD animal model were also studied. The rapidly gained copper resistance was found to be stable, as subculturing of cells in the absence of added copper (weaning) did not restore copper sensitivity. Intracellular copper levels and the expression of MT1 and HSP70 were increased, whereas the expression of CTR1 was reduced. However, the values normalized after weaning. In contrast, downregulation of multi-drug resistance protein 1 (MDR1), encoding P-glycoprotein (P-gp), was shown to be permanent. Calcein assays confirmed the downregulation of MDR1 in the resistant cell lines. MDR1 knockdown by siRNA resulted in increased copper resistance and decreased intracellular copper. Treatment of the resistant cells with verapamil, a known inducer of MDR1, was followed by increased copper-induced toxicity. Downregulation of MDR1 was also observed in hepatocyte-like cells derived from a WD patient after copper exposure. In addition, MDR1 was downregulated in Long-Evans Cinnamon rats when the liver copper was elevated. The results indicate that downregulation of MDR1 is an adaptation of hepatic cells after sustained copper exposure when ATP7B is non-functional. Our data add to the versatile functions of MDR1 in the hepatocyte and may have an impact on the treatment of copper-related diseases, prominently WD.

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.

Methanobactin reverses acute liver failure in a rat model of Wilson disease.

In Wilson disease (WD), functional loss of ATPase copper-transporting ß (ATP7B) impairs biliary copper excretion, leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration- and European Medicines Agency-approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure, leaving liver transplantation as the only viable treatment option. Here, we investigated the therapeutic utility of methanobactin (MB), a peptide produced by Methylosinus trichosporium OB3b, which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes, including hepatic copper accumulation, liver damage, and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover, MB treatment prevented hepatocyte death, subsequent liver failure, and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD.

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.