A novel single step double positive double negative selection strategy for β-globin gene replacement

Towards β-globin gene-targeting with integrase-defective lentiviral vectors

“…cells transduced with IN M eGFP-lentivirus significantly decreased after 21 days due to the dilution of the non-integrated eGFP gene (Yáñez-Muñoz et al 2006;Engelman et al 1995;Wiskerchen and Muesing 1995). Our results also showed that the number of selected clones in both cell lines (transduced with IN M or IN N pLGT-lentivirus) is greater than in our previous study, which used naked DNA because LV vectors facilitate delivery of the transgene to the nucleus (Khanahmad et al 2006;Sadelain et al 2004). More colonies were attained from transduction of cells with the IN M LV vector than with the IN N LV vector because integrase-defective LV vectors are less able to promote integration than normal LV vectors.…”

“…Gene therapy is an ideal strategy for the treatment of monogenic disorders and many previous studies have aimed to develop an effective gene therapy for b-thalassemia (Malik and Arumugam 2005;Nathwani et al 2005). To this end, several different techniques have been applied, including viral vectors, gene-targeting, antisense snRNA, and RNAi (Khanahmad et al 2006). Among these approaches, gene-targeting and viral vectors are considered the most promising strategies for gene therapy for b-thalassemia (Buchschacher and Wong-Staal 2000).…”

“…In gene-targeting, replacement vectors are used commonly to create subtle mutations. In a previous study, we showed that a 'double positive double negative' strategy ensures the complete replacement of the b-globin gene in one step of HR and using a single construct (Khanahmad et al 2006). Despite the numerous advantages of gene-targeting, it is not sufficient for clinical gene therapy of b-thalassemia because of the low frequency of homologous recombination events.…”

“…The HBG fragment amplified for this construct is a complete b-globin expression cassette, containing its endogenous promoter, exons, introns, terminator, and a 3 0 -enhancer that lies at the end of HBG (Trudel and Costantini 1987). The mechanism of gene-targeting and PNS strategy were described previously (Khanahmad et al 2006). Briefly, homologous recombination is facilitated by two homologous stems (USBHG and DSBHG); cells containing the two homologous stems and the complete b-globin gene were selected with hygromycin B and G-418 in the medium.…”