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

<div><p>Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (<i>TTR</i>) 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-TTR_Rx), 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. <i>TTR</i> 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 <i>TTR</i> 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.</p></div

Characteristics of FAP patients.

<p>Characteristics of FAP patients.</p

TTR knockdown in FAP patient derived hepatocyte-like cells.

<p>(A) qRT-PCR analysis of <i>TTR</i> mRNA expression in FAP HLCs treated with compounds (n = 5). Mean±SE of FAP1 to FAP5 are shown. (B) Immunocytochemistry stainings of TTR in FAP HLCs 24 hours after treatment with compounds. FAP4 derived cells are shown as one typical example. Exposure time was consistently adjusted to 1/6 second. Scale bars, 50 μM. (C) TTR Western blot analysis of cell culture supernatants derived from HLCs. FAP4 derived cells are shown as one typical example. (D) Quantification of TTR expression as obtained by Western blots. Mean±SE of FAP1, FAP4 and FAP5 are shown. (E) qPCR analysis of different hepatic and housekeeping marker genes in FAP HLCs after treatment with siTTR1 (triangle) and TTR-ASO (squares). Mean of FAP1 to FAP5 are shown. Data were normalized and set relative to untreated control cells. Absolute ΔΔCt-values are given. Dotted line indicates significance level. ** p< 0.01; ns, not significant.</p

Characterization of urine cells.

<p>(A) Brightfield image of UCs at p2 after isolation. FAP3 derived cells are shown as one typical example. Scale bar, 100 μM. (B) Flow cytometry analysis of UCs at p2 (n = 4). Total expression of markers used for characterization of mesenchymal stem cells is given. (C) Relative mRNA expression of epithelial, fibroblast and renal marker genes in UCs (n = 6). Data were normalized to housekeeping gene.</p

Suppressing transthyretin production in mice, monkeys and humans using 2nd-Generation antisense oligonucleotides

<p>Transthyretin amyloidosis (ATTR amyloidosis) is a rare disease that results from the deposition of misfolded transthyretin (TTR) protein from the plasma into tissues as amyloid fibrils, leading to polyneuropathy and cardiomyopathy. IONIS-TTR_Rx (ISIS 420915) is a 2nd-Generation 2′-<i>O</i>-(2-methoxyethyl) modified “2′-MOE” antisense oligonucleotide (ASO) that targets the TTR RNA transcript and reduces the levels of the TTR transcript through an RNaseH1 mechanism of action, leading to reductions in both mutant and wild-type TTR protein. The activity of IONIS-TTR_Rx to decrease TTR protein levels was studied in transgenic mice bearing the Ile84Ser human TTR mutant, in cynomolgus monkeys and in healthy human volunteers. Robust (>80%) reductions of plasma TTR protein were obtained in all three species treated with IONIS-TTR_Rx, which in mice and monkeys was associated with substantial reductions in hepatic TTR RNA levels. These effects were dose-dependent and lasted for weeks post-dosing. In a Phase 1 healthy volunteer study, treatment with IONIS-TTR_Rx for four weeks was well tolerated without any remarkable safety issues. TTR protein reductions up to 96% in plasma were observed. These nonclinical and clinical results support the ongoing Phase 3 development of IONIS-TTR_Rx in patients with ATTR amyloidosis.</p

Reprogramming of urine cells obtained from FAP patients.

<p>(A) Typical brightfield images of UCs from a FAP patient at different times after reprogramming. FAP2 derived cells are shown. Scale bars, 100 μM. (B) Immunofluorescence stainings of cells after reprogramming. DAPI was used for nuclear counterstaining. FAP5 derived cells are shown as one typical example. Scale bars, 50 μM. (C) Gene expression analysis of reprogrammed cells. Relative expression to housekeeping gene is shown. Mean±SE of FAP1 to FAP5 are shown. (D) EB expression profile of pluripotency- and lineage-specific markers derived from FAP iPSCs. Dots indicate sample scores of cells derived from patients FAP1 (black), FAP2 (red) and FAP4 (green). Human embryonic stem (hES) and iPS cell lines served as control (box plot). (E) DNA sequencing chromatograms of the <i>TTR</i> gene derived from FAP iPSCs. Heterozygotic variant nucleotides are shown by arrow.</p