Using CRISPR/Cas9 to insert an ancestral uricase gene

The metabolic product of purine metabolism in human is uric acid. In most other mammals, uric acid is further oxidize to 5-hydroxyisourate by the enzyme uricase. High concentration of uric acid is associated with human diseases like gout because uric acid is highly insoluble. The human uricase gene is not functional and therefore referred to as a pseudogene. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein 9 (CRISPR/Cas9) system has recently emerged as an efficient methodology for genome editing to repair or reverse genetic mutations. We exploited the CRISPR/Cas9 system to knock-in a functional ancestral uricase into the genome of Huh7 cells, a hepatocyte-derived cellular carcinoma human cell line. Three-dimensional (3D) models, such as organoids and spheroids, present a paradigm for research since these structures present a complex model closer to the architecture and environment of tissues when compare to 2D models. In addition, 3D models show better cell-cell interactions and cellular response. Western blot analyses confirmed the expression of ancestral uricase with the expected protein size. Next, we analyzed the ability of spheroids to metabolize uric acid. Ancestral uricase-positive spheroids were able to metabolize uric acid at different concentrations (range from 2.5 mg/dL to 7.5 mg/dL of uric acid). Spheroids were subjected to starvation, absence of both glucose and fetal bovine serum, and the intracellular uric acid and ATP levels were determined. Uricase-positive spheroids showed lower intracellular uric acid and high ATP levels. To induce triglycerides and uric acid production, spheroids were grown in the presence of 20 mM fructose. Uricase-positive spheroids showed a reduction in both triglycerides and uric acid levels. Our results suggest that these human spheroids expressing ancestral uricase will help prevent and/or treat hyperuricemia (gout) and other fatty-liver diseases through gene therapy.

人类嘌呤代谢（purine metabolism）的终产物为尿酸。在绝大多数其他哺乳动物体内，尿酸可被尿酸酶（uricase）进一步氧化为5-羟异尿酸。由于尿酸溶解度极低，高浓度尿酸与痛风等人类疾病密切相关。人类的尿酸酶基因已丧失功能，因此被称为假基因。成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR）相关蛋白9（CRISPR/Cas9）系统近年来已成为高效的基因组编辑技术，可用于修复或逆转遗传突变。本研究利用CRISPR/Cas9系统，将具有功能的祖先型尿酸酶敲入人肝细胞源性肝癌细胞系Huh7的基因组中。三维（3D）模型（如类器官（organoids）与球状体（spheroids））为相关研究提供了理想范式：相较于二维（2D）模型，这类结构能够模拟更贴近体内组织的架构与微环境，同时可更好地重现细胞间相互作用与细胞应答反应。蛋白质免疫印迹（Western blot）分析证实，祖先型尿酸酶以预期的分子量成功表达。随后，我们检测了球状体对尿酸的代谢能力：表达祖先型尿酸酶的球状体可代谢浓度范围为2.5 mg/dL至7.5 mg/dL的尿酸。将球状体置于无糖且不含胎牛血清的环境中进行饥饿处理后，检测细胞内尿酸与三磷酸腺苷（adenosine triphosphate, ATP）水平，结果显示，表达尿酸酶的球状体胞内尿酸水平更低，且ATP水平更高。为诱导甘油三酯与尿酸的生成，我们将球状体置于含20 mM果糖的培养基中培养，此时表达尿酸酶的球状体的甘油三酯与尿酸水平均出现显著下降。本研究结果表明，这类表达祖先型尿酸酶的人源球状体可通过基因治疗手段，助力预防和/或治疗高尿酸血症（痛风）及其他脂肪性肝病。