Associate Professor The Chinese University of Hong Kong Hong Kong SAR, China (People's Republic)
Abstract: AAV-CRISPR-mediated liver-specific knock-in restored hemostasis in neonatal hemophilia B mice
Zhenjie ZHANG1,2,3, Siqi ZHANG1, Xiangjun HE1, BO FENG1,2 1The Chinse University of Hong Kong, School of Biomedical Sciences , Hong Kong SAR, China, 2Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Centre for Regenerative Medicine and Health, Hong Kong SAR, China; 3 GEINQure Biotech Co. Ltd., Hong Kong SAR, China
AAV-delivered CRISPR/Cas9 (AAV-CRISPR) has shown promising potentials in preclinical models to efficiently insert therapeutic gene sequences in somatic tissues. However, the AAV input doses required were prohibitively high and posed serious risk of toxicity. Here, we performed AAV-CRISPR mediated homology-independent knock-in at a new target site in mAlb 3'UTR and demonstrated that single dose of AAVs enabled long-term integration and expression of hF9 transgene in both adult and neonatal hemophilia B mice (mF9 -/-), yielding high levels of circulating human Factor IX (hFIX) and stable hemostasis restoration during entire 48-week observation period. Furthermore, we achieved hemostasis correction with a significantly lower AAV dose through liver-specific gene knock-in using hyperactive hF9R338L variant. The plasma antibodies against Cas9 and AAV in the neonatal mice receiving low-dose AAV-CRISPR were negligible, which lent support to the development of AAV-CRISPR mediated somatic knock-in for treating inherited diseases.
Funding Source: This study was supported by funds provided by the Research Grants Council of Hong Kong [14116719, 14115520, 14106024 to B.F.], and the Health@InnoHK Program launched by Innovation Technology Commission of the Hong Kong SAR, China.
