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

PLoS One. 2016 Sep 1;11(9):e0161455. doi: 10.1371/journal.pone.0161455. eCollection 2016.

Abstract

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.

MeSH terms

  • Adult
  • Aged
  • Amyloid Neuropathies, Familial / drug therapy*
  • Amyloid Neuropathies, Familial / genetics
  • Amyloid Neuropathies, Familial / urine
  • Cell Differentiation
  • Drug Evaluation, Preclinical
  • Female
  • Gene Knockdown Techniques
  • Hepatocytes / drug effects*
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Male
  • Middle Aged
  • Oligonucleotides, Antisense / pharmacology
  • Oligonucleotides, Antisense / therapeutic use*
  • Prealbumin / genetics
  • RNA, Messenger / genetics
  • RNA, Small Interfering / genetics

Substances

  • Oligonucleotides, Antisense
  • Prealbumin
  • RNA, Messenger
  • RNA, Small Interfering

Grants and funding

Part of the work was supported by the “Förderwettbewerb Translationale Stammzellforschung des Landes NRW (w1403ts011a)” and a grant of the “Innovative Medizinische Forschung" (IMF) dedicated to Y. Avsar. In addition, Ionis Pharmaceuticals and Alnylam Pharmaceuticals provided support in the form of salaries for authors S. Guo, EA, BM (Ionis Pharmaceuticals,Inc.) and JG (Alnylam Pharmaceuticals, Inc.), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Both funders supported research materials. The specific roles of these authors are articulated in the ‘author contributions’ section.