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Vectorology - GEG Tech top picks
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Targeted, homology-driven gene insertion in stem cells by ZFN-loaded ‘all-in-one’ lentiviral vectors
Targeted, homology-driven gene insertion in stem cells by ZFN-loaded ‘all-in-one’ lentiviral vectors | Building on previous work (Cai et al., 2014), HIV-derived lentiviral vectors are harnessed to insert user-defined genes into a safe locus in the genome of human stem cells.
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Here, the scientists design non integrating lentiviral vectors (NILVs) "all in one" which contains ZFN that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. They show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Their findings demonstrate the applicability of nuclease-loaded ‘all-in-one’ NILVs for site-directed gene insertion in stem cell-based gene therapies. 
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Scientists recently developed a ZF transcription repressor (ZF-KOX1) able to bind to expanded DNA CAG-repeats in the huntingtin (HTT) gene, which are found in Huntington’s disease (HD). In the present work, they sought to develop a long-term single-injection gene therapy approach in the brain. Nonetheless, they found that using rAAV vector pCAG promoter (CMV early enhancer element and the chicken β-actin promoter) led to a strong reduction in ZF expression by 6 weeks after injection. They therefore tested a new non-viral promoter to see whether the host-adapted ZF expression could be sustained for a longer time. Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat) resulted in up to 77 % repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 1010 virions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that longer term effects are possible.