利用肝靶向多肽纳米载体高效递送CRISPR-Cas9治疗苯丙酮尿症模型小鼠的研究

Phenylketonuria(PKU) is a severe chronic hereditary amino acid metabolism disorder disease caused by the defect of liver phenylalanine hydroxylase (PAH). 98% ~ 99% of the disease are caused by PAH gene mutations, among which the mutation in exon 7 (Exon7) is most common. At present, neither low phenylalanine diet therapy, replenish tetrahydrobiopterin, nor conventional gene therapy based on adenovirus carriers, can hardly obtain a satisfactory curative effect. Gene editing technique represented by CRISPR-Cas9 can fundamentally correct gene mutantion, restore the metabolic enzymatic activity, and cure genetic diseases. Therefore, to explore the therapeutic effects of CRISPR-Cas9 technology on PKU is of great importance. Design of a delivery system with high targeting, security and efficiency has been a profound challenge that CRISPR-Cas9 technology should conquer. In this project, a liver specific peptide ligand CSR2a was used as a targeting peptide, gene editing tool enzyme Cas9 was introduced, and a new liver targeting polypeptide nano-vector (L-TPV) based on CRISPR-Cas9 were built. On both sides of PAH gene Exon7, a single guided RNA (sgRNA) will be designed respectively. L-TPV and sgRNA will be assembled into nanoparticles in vitro, which further are injected into PKU model mice through the tail vein. The nano-particles can replace the variation Exon7 using the homologous arm of a normal Exon7 as a template. The therapeutic effect and editing property in vivo will be evaluated. The study can provide supporting evidence and theoretical basis for the treatment of PKU with gene editing.

苯丙酮尿症（PKU）是由肝脏苯丙氨酸羟化酶基因突变所致的严重遗传代谢性疾病，以第七外显子（Exon7）突变最为常见。以CRISPR-Cas9为代表的基因编辑技术能从根本上纠正变异基因，达到治愈疾病的目的。目前报道的CRISPR-Cas9技术递送载体中，病毒载体的运载量小且存在生物风险，非病毒载体转运和编辑效率均较低，并且两类载体均存在靶向性差、脱靶率高等问题。因此，为设计更具靶向性、安全性以及高效性的基因编辑递送系统，本项目拟以肝特异性配体CSR2a作为靶向肽，构建一种具有基因编辑工具酶Cas9活性的新型肝靶向多肽纳米载体（L-TPV），分别与针对Exon7两侧序列设计的单导向RNA，及正常Exon7同源臂供体DNA模板在体外组装成纳米颗粒，通过尾静脉注入并靶向至PKU模型小鼠肝细胞内，修复Exon7，研究体内疗效和编辑性能，为基因编辑用于PKU等遗传病治疗提供实验基础及理论依据。

苯丙酮尿症（PKU）是一种常染色体隐性遗传性疾病，患儿出生后如不能得到及时有效治疗，将因高苯丙氨酸血症以及中间代谢产物对中枢神经系统的毒性作用，导致严重的智能发育障碍。低苯丙氨酸饮食是一种有效的治疗方法，但存在诸多问题，患者生活质量低下，即使严格遵循饮食要求，某些患者仍会出现认知和情感异常。补充苯丙氨酸解氨酶或BH4只适用于部分患者，长期效果不佳且副作用严重；基因治疗研究虽然已取得一些成效，但并未修复PAH基因缺陷，无法实现PAH的长期、高效表达。基因编辑能从根本上纠正变异基因，恢复苯丙氨酸羟化酶活性，达到治愈苯丙酮尿症的目的。因此，本项目将CSR2a小肽作为特异性肝靶向配体，并引入基因编辑工具酶Cas9，成功设计并构建一种基于CRISPR-Cas9的新型肝靶向TPV（L-TPV）用于PKU的治疗。实验证明L-TPV可与识别PAH基因Exon 7两端序列的sgRNA体外组装成纳米颗粒，保护核酸不被降解，并通过CSR2a与肝细胞上受体结合而被内吞入细胞，特异性进入肝细胞内，在PAH基因Exon 7两侧形成DSB，敲除变异Exon 7，并可以借助含有Exon 7同源臂序列的单链供体DNA(ssODN)为模板敲入纠正的Exon 7序列。经验证，该方案不仅可以实现对PKU模型小鼠PAH基因的高效编辑，而且大大降低了脱靶效率，显著提高了小鼠的生存率。该研究项目将为基因编辑技术用于PKU等遗传病治疗提供理论及实验依据，并为基因治疗药物的研制和临床应用奠定基础。