Gutierrez-Guerrero Alejandra, Périan Séverine, Leray Aurélien, Martinello Chiara, Abrey Recalde Maria Jimena, Costa Caroline, Herrero Cecilia Iglesias, Bouzelha Mohammed, Alvarez-Dorta Dimitri, Gouin Sébastien G, Ayuso Eduard, Adjali Oumeya, Büning Hildegard, Deniaud David, Mével Mathieu, Verhoeyen Els
CIRI - International Center for Infectiology Research, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Université Lyon, 69007 Lyon, France.
Nantes Université, CNRS, CEISAM UMR 6230, 44000 Nantes, France.
Mol Ther Nucleic Acids. 2025 Feb 22;36(2):102495. doi: 10.1016/j.omtn.2025.102495. eCollection 2025 Jun 10.
Nanoblades are viral particles loaded with the Cas9 protein complexed with gRNA, which allowed efficient gene editing in hematopoietic stem and progenitor cells (HSPCs). Combined with recombinant adeno-associated vector (rAAV) 6 containing two homologous arms to a gene locus resulted in 50% of expression cassette knockin into HSPCs. However, high effective doses of rAAV6 induced HSPC cell death. Here, we demonstrated that, at high doses, rAAV2 was much less toxic for template DNA delivery and allowed transduction levels in HSPCs equivalent to rAAV6. To improve donor template delivery, rAAV2 and rAAV6 were chemically bio-conjugated with a mannose ligand, via the lysine or tyrosine amino acid residues exposed at the adeno-associated vector (AAV) capsid surface. High-level transduction of HSPCs with mannose-coupled rAAV6 vectors accompanied by a remarkable lower toxicity was achieved as compared to control rAAV6 in correlation with highly reduced p53 pathway activation. Mannose-conjugated rAAV6 combined with nanoblades allowed efficient gene knockin and increased survival of HSPCs from 10% to 80% as compared to the unmodified rAAV6 even in the most immature CD34CD38lowCD90 hematopoietic stem cell (HSC) population. Summarizing, mannose-conjugated rAAV6 maintained high-level donor mediated gene knockin when combined with nanoblades without inducing significant toxicity for the HSPCs, an important feature for clinical translation of HSPC gene-editing strategies.
纳米刀片是装载有与gRNA复合的Cas9蛋白的病毒颗粒，其能够在造血干细胞和祖细胞（HSPCs）中进行高效基因编辑。与含有针对某一基因座的两个同源臂的重组腺相关病毒载体（rAAV）6相结合，可使50%的表达盒敲入HSPCs。然而，高剂量的rAAV6会诱导HSPC细胞死亡。在此，我们证明，在高剂量下，rAAV2对模板DNA递送的毒性要小得多，并且能使HSPCs中的转导水平与rAAV6相当。为了改善供体模板递送，rAAV2和rAAV6通过腺相关病毒（AAV）衣壳表面暴露的赖氨酸或酪氨酸氨基酸残基与甘露糖配体进行化学生物偶联。与对照rAAV6相比，甘露糖偶联的rAAV6载体对HSPCs的高水平转导伴随着显著更低的毒性，这与p53通路激活的高度降低相关。与未修饰的rAAV6相比，甘露糖偶联的rAAV6与纳米刀片相结合，即使在最不成熟的CD34CD38lowCD90造血干细胞（HSC）群体中，也能实现高效的基因敲入，并使HSPCs的存活率从10%提高到80%。总之，甘露糖偶联的rAAV6与纳米刀片结合时，能维持高水平的供体介导的基因敲入，且不会对HSPCs诱导显著毒性，这是HSPC基因编辑策略临床转化的一个重要特征。
