Chew Wei Leong, Tabebordbar Mohammadsharif, Cheng Jason K W, Mali Prashant, Wu Elizabeth Y, Ng Alex H M, Zhu Kexian, Wagers Amy J, Church George M
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA.
Nat Methods. 2016 Oct;13(10):868-74. doi: 10.1038/nmeth.3993. Epub 2016 Sep 5.
CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.
腺相关病毒（AAV）介导的CRISPR-Cas9递送在基因治疗方面具有前景，但由于其潜在的免疫原性和有限的有效载荷能力而面临关键障碍。在此，我们展示了使用AAV-拆分Cas9在出生后小鼠中进行基因组工程，这是一个可定制用于基因组编辑、转录调控以及AAV-CRISPR-Cas9其他以前不可行应用的多功能平台。我们确定了影响我们平台功效和临床转化的关键参数，包括病毒生物分布、各器官的编辑效率、抗原性、免疫反应和生理结果。这些结果表明，AAV-CRISPR-Cas9引发具有独特细胞和分子特征的宿主反应，但与其他递送方法不同，它在体内不会诱导广泛的细胞损伤。我们的研究为开发有效的基因组治疗方法奠定了基础。
