In Vitro ADMET Laboratories Inc., Columbia, MD (QY, APL) and Amgen Inc., San Francisco, CA (SCH, JML), United States.
In Vitro ADMET Laboratories Inc., Columbia, MD (QY, APL) and Amgen Inc., San Francisco, CA (SCH, JML), United States.
Biochem Pharmacol. 2021 Jul;189:114374. doi: 10.1016/j.bcp.2020.114374. Epub 2020 Dec 30.
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.
我们在此报告了一种新型体外实验模型——长期培养的人肝细胞（PCHC）的评估，该模型可用于评估寡核苷酸治疗药物的效力和作用持续时间。我们在 PCHC 的长期培养过程中观察到了一个新的现象，即基于白蛋白、转铁蛋白和转甲状腺素蛋白等特征性肝分化标记基因的 mRNA 谱，观察到 PCHC 的再分化。与已知的培养人肝细胞去分化一致，在培养肝细胞 2 天时观察到标记基因表达下降。在第 7 天观察到了一个新的再分化现象，标记基因的表达增加到与培养的第 1 天相似的水平。分化标记基因的表达在第 7 天最高，随后逐渐下降，但在最长培养时间（41 天）内仍高于第 2 天。这种再分化现象表明，PCHC 可能有助于评估寡核苷酸治疗药物对人肝细胞基因表达的作用持续时间。因此，我们进行了一项使用 PCHC 的概念验证研究，该研究使用了一种 GalNAc 缀合的靶向人次黄嘌呤磷酸核糖转移酶 1（HPRT1）的 siRNA。在第 1 天，用 siRNA 处理的培养物中 HPRT1 mRNA 表达下降至未处理肝细胞的 21%，从第 2 天到第 12 天下降至<10%，从第 16 天到第 33 天下降至<20%，最终在第 41 天恢复至 64%。我们的结果表明，PCHC 代表了一种具有临床相关性的、成本效益高且耗时高效的实验工具，可用于评估 GalNAc-siRNA 的沉默活性，提供关于疗效和疗效持续时间的信息。PCHC 可作为一种物种和细胞类型相关的实验工具，适用于药物开发环境，以辅助寡核苷酸治疗药物的开发。
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