Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA
Autor: | Qian Yang, Albert P. Li, Julie M. Lade, Sara C. Humphreys |
---|---|
Rok vydání: | 2020 |
Předmět: |
0301 basic medicine
Hypoxanthine Phosphoribosyltransferase Acetylgalactosamine Cell Culture Techniques Drug Evaluation Preclinical Biochemistry Marker gene 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Gene expression Gene silencing Humans Gene Silencing RNA Small Interfering Gene Hypoxanthine Cells Cultured Pharmacology chemistry.chemical_classification RNA Molecular biology In vitro 030104 developmental biology chemistry Transferrin 030220 oncology & carcinogenesis Hepatocytes |
Zdroj: | Biochemical pharmacology. 189 |
ISSN: | 1873-2968 |
Popis: | We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1,10% from days 2 to 12,20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics. |
Databáze: | OpenAIRE |
Externí odkaz: |