Li Shuojun, Du Moqing, Deng Jiamin, Deng Guiyun, Li Jiaying, Song Zhiyong, Han Heyou
State Key Laboratory of Agriculture Microbiology, College of life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
State Key Laboratory of Agriculture Microbiology, College of Science, Huazhong Agricultural University, Wuhan 430070, China.
Acta Biomater. 2022 Dec;154:597-607. doi: 10.1016/j.actbio.2022.10.015. Epub 2022 Oct 13.
The CRISPR/Cas9 mediated genome editing have provided a promising strategy to correct multiple mutations of Duchenne muscular dystrophy (DMD). However, the delivery of CRISPR/Cas9 system into mammalian cell for DMD gene editing mainly relies on adeno associated virus (AAV)-mediated transport. Meanwhile, the protospacer adjacent motif (PAM) requirement of wild-typed Cas9 protein causing the target sites for exon splice acceptor site are restricted to limited regions. Here, we developed a biomineralized PAMLess Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This method described a facile synthesis of biomineralized NPs with high SpRY pDNA encapsulation efficiency. In vitro results show that the Bm-SpRY NPs have the obvious advantages of well biocompatibility and protecting SpRY pDNA from enzyme degradation and efficient delivery under high serum condition. Cell studies demonstrated that Bm-SpRY NPs enable rapid cellular uptake, endo-lysosomes escape and nucleus transport. Meanwhiles, the DMD gene editing via Bm-SpRY NPs pathway is transient process without genomic integration. We evaluated multiple target regions with different PAMs for the DMD exon 51 splice acceptor site through Bm-SpRY NPs method and found that the target region with TAG PAM has the highest editing efficiency and significant preferential mutation. In vivo results show that intramuscular injection of Bm-SpRY NPs enable DMD gene mutation in muscle tissue without tissue damage. This study may extend the advanced application of CRISPR system for DMD therapy. STATEMENT OF SIGNIFICANCE: The gene editing technology of CRISPR/Cas9 provides an effective treatment strategy for the Duchenne muscular dystrophy (DMD) therapy. However, the delivery of CRISPR system in mammalian cell mainly relies on viral mediated transport and the NGG or NAG requirement of wild-typed Cas9 protein limits the target region in DMD gene. Here, the present study provides a biomineralized PAM Less Cas9 (SpRY) variant nanoparticles (Bm-SpRY NPs) for DMD gene editing in vitro and in vivo. This study may extend the application of CRISPR system for DMD gene therapy.
CRISPR/Cas9介导的基因组编辑为纠正杜氏肌营养不良症（DMD）的多种突变提供了一种很有前景的策略。然而，将CRISPR/Cas9系统导入哺乳动物细胞进行DMD基因编辑主要依赖腺相关病毒（AAV）介导的转运。同时，野生型Cas9蛋白对原间隔相邻基序（PAM）的要求使得外显子剪接受体位点的靶位点局限于有限区域。在此，我们开发了一种生物矿化的无PAM Cas9（SpRY）变体纳米颗粒（Bm-SpRY NPs）用于体外和体内的DMD基因编辑。该方法描述了一种简便的合成具有高SpRY pDNA包封效率的生物矿化纳米颗粒的方法。体外结果表明，Bm-SpRY NPs具有良好的生物相容性、能保护SpRY pDNA免受酶降解以及在高血清条件下高效递送等明显优势。细胞研究表明，Bm-SpRY NPs能够实现快速的细胞摄取、从内溶酶体逃逸并进入细胞核转运。同时，通过Bm-SpRY NPs途径进行的DMD基因编辑是一个无基因组整合的瞬时过程。我们通过Bm-SpRY NPs方法评估了DMD外显子51剪接受体位点具有不同PAM的多个靶区域，发现具有TAG PAM的靶区域具有最高的编辑效率和明显的优先突变。体内结果表明，肌肉注射Bm-SpRY NPs能够在肌肉组织中实现DMD基因突变且不造成组织损伤。本研究可能会拓展CRISPR系统在DMD治疗中的先进应用。重要性声明：CRISPR/Cas9基因编辑技术为杜氏肌营养不良症（DMD）治疗提供了一种有效的治疗策略。然而，CRISPR系统在哺乳动物细胞中的递送主要依赖病毒介导的转运，并且野生型Cas9蛋白对NGG或NAG的要求限制了DMD基因中的靶区域。在此，本研究提供了一种生物矿化的无PAM Cas9（SpRY）变体纳米颗粒（Bm-SpRY NPs）用于体外和体内的DMD基因编辑。本研究可能会拓展CRISPR系统在DMD基因治疗中的应用。
