Z Gastroenterol 2016; 54 - KV277
DOI: 10.1055/s-0036-1587053

Evaluierung therapeutischer Oligonukleotide zur Therapie der familiären Amyloidose in einem Stammzell-abgeleiteten Zellmodell

C Niemietz 1, V Sauer 1, J Stella 1, G Chandhok 1, S Guttmann 1, Y Avsar 1, S Guo 2, E Ackermann 2, J Gollob 3, B Monia 2, A Zibert 1, H Schmidt 1
  • 1Klinik für Transplantationsmedizin, Universitätsklinikum Münster, Münster, Deutschland
  • 2Ionis Pharmaceuticals, Carlsbad, CA, USA
  • 3Alnylam Pharmaceuticals, Cambridge, MA, USA

Einleitung: Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene. TTR is secreted into the blood, predominantly by the liver. The tetramer can undergo dissociation resulting in extracellular tissue deposition of TTR, followed by dysfunctions in target tissues. The effects of an antisense oligonucleotide (ASO; IONIS-TTRRx) and small interfering RNA (siRNA; ALN-TTR-02) on TTR synthesis are currently being evaluated in phase II/III clinical trials in patients with FAP. Primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells do not have the genetic background of individual patients.

Ziele: To evaluate IONIS-TTRRx and ALN-TTR-02 in hepatic cells derived from FAP patients.

Methodik: Hepatocyte like cells (HLCs) were generated from five FAP patients using induced pluripotent stem cells (iPSC). Patients had different TTR mutations (Val30Met, Gly47Ala, Arg34Thr) and showed different phenotypes. Differentiation toward HLCs was achieved using a 3-step in-house protocol with a total cultivation time of 14 days. HLCs were characterized by analysis of typical hepatic markers via RT-PCR, immunocytochemistry, flow cytometry, and functional activity. To assess TTR gene silencing, IONIS- TTRRx and ALN-TTR-02 were introduced into HLCs via transfection.

Ergebnis: HLCs of FAP patients could be obtained that showed typical markers of human hepatocytes. TTR mRNA expression was almost equal to human hepatic cells. A significant downregulation (> 80%) of TTR mRNA was induced in HLCs by both therapeutic oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated by both oligonucleotides as determined by ELISA and Western blot analysis. Gene expression of other hepatic markers were not affected by the therapeutic oligonucleotides.

Schlussfolgerung: IONIS- TTRRx and ALN-TTR-02 were shown to be highly efficient in downregulation of TTR in human hepatic like cells obtained from FAP patients. The use of patient-specific cells derived from induced pluripotent stem cells represents an excellent approach to molecularly assess the efficacy and specificity of novel compounds in target cells that have the individual genetic background of patients.