May 07, 2020 Targeted, homology-driven gene insertion in stem cells by ZFN-loaded

Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper ( Cai et al., 2014a ), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZFN-loaded IDLVs 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. We 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. Our findings demonstrate the applicability of nuclease-loaded ‘all-in-one’ IDLVs for site-directed gene insertion in stem cell-based gene therapies. attached to poly-L-lysine coated coverslips by spinoculation at 1200 g for In all the cells or lentiviral particles were washed three times with PBS before fixation with 4% paraformaldehyde. Coverslips were stored in 70% ethanol for at least 15 min. To visualize HA-tagged ZFNs, fixed cells or lentiviral particles were incubated with mouse-derived primary HA mono-clonal antibody (Covance, Princeton, NJ) and secondary Alexa Fluor 488 Donkey anti-Mouse IgG (LifeTechnologies). To visualize p24, the lentiviral particles were stained with rabbit-derived primary p24 polyclonal antibody (ThermoFisher Scientific, Waltman, MA) and secondary Alexa Fluor 568 Donkey-anti-Rabbit IgG (LifeTechnologies). The nuclei were stained by DAPI. Sequential imaging was done by using a 488 nm line of a multiline argon laser (detection of Alexa-488), the 405 nm diode laser (detection of DAPI) and 561 nm DPSS laser (detection of Alexa-568) on a confocal microscope (LSM 710, ZEISS) using 63 (cid:2) oil immersion objective with a numerical aperture of 1.4. transduced with LP-ZFN(CCR5), LP-ZFN(AAVS1), IDLV-ZFN(AAVS1)/ puro and IDLV-ZFN(CCR5)/ puro at a dose of 500 ng p24 per 0.5 (cid:2) 10 5 cells. Five days after transduction, iPSCs treated with IDLV-ZFN(AAVS1)/ puro and IDLV-ZFN(CCR5)/ puro were selected with puromycin (0.5 m g/ml). Representative clones were further stained using alkaline phosphatase and TRA-1-60 live stain dyes (Life Technologies). For colony-form-ing unit assays, after transduction with and incubation for 4 days, 1200 CD34 + cells were mixed with Human Methylcellulose Complete Media (R&D Systems Inc., Minneapolis, MN) and transferred to a 35-mm plate. The plates were incubated in a humidified atmosphere with 5% (vol/vol) CO 2 at 37 ˚ C for two weeks. the case of the AAVS1 locus, primer sets YJ706F-0279 and YJ707F-YJ738R were used as first round and second round PCRs, respectively, to detect 5’ junctions. YJ251F-YJ205R and YJ252F-YJ666R primer sets were used as first round and second round primer set, respectively, to detect 3’ junctions. For insertion into the CCR5 locus, YJ825F-0279 and YJ833F-YJ738R were used for amplification of 5’ junctions. Primer sets YJ251F-YJ253R and YJ252F-YJ208R were used to detect 3’ junctions. The CCR5 locus was amplified using primer set YJ224F-YJ225R, whereas the AAVS1 locus was amplified using primer set YJ222F-YJ223R.