Esposito Federica, Dell'Aquila Fabio, Rhiel Manuel, Auricchio Stefano, Chmielewski Kay Ole, Andrieux Geoffroy, Ferla Rita, Horrach Paula Sureda, Padmanabhan Arjun, Di Cunto Roberto, Notaro Simone, Santeularia Manel Llado, Boerries Melanie, Dell'Anno Margherita, Nusco Edoardo, Padula Agnese, Nutarelli Sofia, Cornu Tatjana I, Sorrentino Nicolina Cristina, Piccolo Pasquale, Trapani Ivana, Cathomen Toni, Auricchio Alberto
Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.
Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy; Medical Genetics, Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy.
Cell Rep Med. 2024 Jul 16;5(7):101619. doi: 10.1016/j.xcrm.2024.101619. Epub 2024 Jun 18.
Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3' end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.
通过CRISPR-Cas9在高度转录的白蛋白基因座进行肝脏定向腺相关病毒（AAV）载体介导的非同源靶向整合（AAV-HITI）正在研究中，以在新生儿治疗后提供持续的转基因表达。我们表明，在两个遗传性疾病小鼠模型中，靶向白蛋白基因座的3'端可导致约15%的小鼠肝细胞发生有效整合，从而达到全身蛋白质的治疗水平。我们证明，全长HITI供体DNA在核酸酶切割后优先整合，并且尽管在靶基因座中有部分AAV基因组整合，但在脱靶位点未发现明显的染色体重排或插入/缺失。与此一致的是，在1年的随访中未观察到肝细胞癌的证据。最后，如果直接转化为人类，AAV-HITI在被认为安全的载体剂量下是有效的，除了在新生儿中有效外，在成年肝脏中也具有治疗效果。总体而言，我们的数据支持这种基于肝脏定向AAV的敲入策略的开发。
