Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

Ordovás, Laura; Boon, Ruben; Pistoni, Mariaelena; Chen, Yemiao; Wolfs, Esther; Guo, Wenting; Sambathkumar, Rangarajan; Bobis‐Wozowicz, Sylwia; Helsen, Nicky; Vanhove, Jolien; Berckmans, Pieter; Cai, Qiuyin; Vanuytsel, Kim; Eggermont, Kristel; Vanslembrouck, Veerle; Schmidt, Béla Z.; Raitano, Susanna; Bosch, Ludo Van Den; Nahmias, Yaakov; Cathomen, Toni; Struys, Tom; Verfaillie, Catherine M.

doi:10.1016/j.stemcr.2018.01.034

Cited by 50 publications

(78 citation statements)

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“…Despite the absence of EGFP expression in Mbs85-edited WT HSPCs, targeted integration in up to 20.98 ± 9.4% of clones were observed using 2.5 µg of each TALEN monomer together with 4 µg of the EGFP reporter donor (Fig 4E). This observation and the very low proportion of EGFPexpressing cells observed in liquid cultures suggests a restricted expression of transgenes in the Mbs85 locus of mHSPCs as has previously been reported in the human AAVS1 locus of hESCs (Ordovas et al, 2015).…”

Section: Discussionsupporting

confidence: 79%

“…The AAVS1 locus has been defined as a bona-fide safe harbor locus in humans (Lombardo et al, 2011), in which an exogenous gene could be efficiently expressed in various cell lines and iPSCs (DeKelver et al, 2010;Dreyer et al, 2015;Hockemeyer et al, 2009;Li et al, 2017;Lombardo et al, 2011;Mizutani et al, 2016;Mizutani et al, 2015;Oceguera-Yanez et al;Ordovas et al, 2015;Ramachandra et al, 2011;Smith et al, 2008;Zou et al, 2011). Therefore, in this work, we have decided to carry out a targeted integration strategy in the mouse Mbs85 orthologous locus in order to asses if the corresponding mouse locus may serve as a new site where gene editing can be performed in mouse disease models such as Fanconi anemia (FA).…”

Section: Discussionmentioning

confidence: 99%

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Therapeutic gene editing in hematopoietic progenitor cells from a mouse model of Fanconi anemia

Pino-Barrio

Giménez

Villanueva

et al. 2018

Preprint

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The promising ability to genetically modify hematopoietic stem and progenitor cells (HSPCs) by precise gene editing remains challenging due to their sensitivity and poor permissiveness. This represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus that phenotypically corrects primary cells derived from a mouse model of Fanconi anemia (FA).By co-delivering TALENs and a donor therapeutic FANCA cassette template to the Mbs85 locus (ortholog of the hAAVS1 safe harbor locus), we achieved efficient gene targeting (23%) in FA mouse embryonic fibroblasts (MEFs). This resulted in the phenotypic correction of these cells, as revealed by the improvement of their hypersensitivity to mitomycinC. Moreover, robust evidence of targeted integration was observed in murine WT and FA-A hematopoietic progenitor cells (HPC) reaching mean targeted integration values of 20.98% and 16.33% respectively, with phenotypic correction of FA HPCs. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy in a murine safe harbor locus, such as the Mbs85 gene, of MEFs and murine HPC from a FA mouse model. Supplementary Table 3. Primers and probes used in this study. Forward and reverse primers as well as probes, their sequence, their melting temperature (Tm) and the product size is indicated. In the case of probes, the fluorochrome to which each probe was conjugated is also indicated.

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Therapeutic gene editing in hematopoietic progenitor cells from a mouse model of Fanconi anemia

Pino-Barrio

Giménez

Villanueva

et al. 2018

Preprint

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“…Ordovás et al recently reported that transgene expression inhibition at the AAVS1 site was observed in a lineage-dependent manner in human iPSCs due to DNA methylation and other unknown mechanisms. 32 We did not observe transgene silencing in GFP clones that showed robust GFP expression not only in undifferentiated status but also throughout the differentiation toward all three germ layers. This result suggests that the AAVS1 site can indeed support stable transgene expression in rhesus macaque.…”

Section: Discussionmentioning

confidence: 58%

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

et al. 2017

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“…[23][24][25] For initial validation of gRNAs designed for Cpf1, we used the reporter plasmid pCAG-EGxxFP-AAVS1, which was previously constructed to contain an approximately 500 bp genomic region of AAVS1 14) integrated into the enhanced green fluorescent protein (EGFP) coding region (Supplementary Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver

Tsukamoto

Sakai

Iizuka

et al. 2018

Biological & Pharmaceutical Bulletin

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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 system is now widely used as a genome editing tool. CRISPR-associated endonuclease in Prevotella and Francisella 1 (Cpf1) is a recently discovered Cas endonuclease that is designable and highly specific with efficiencies comparable to those of Cas9. Here we generated the adenovirus (Ad) vector carrying an Acidaminococcus sp. Cpf1 (AsCpf1) expression cassette (Ad-AsCpf1) for the first time. Ad-AsCpf1 was applied to primary human hepatocytes prepared from humanized mice with chimeric liver in combination with the Ad vector expressing the guide RNA (gRNA) directed to the Adeno-associated virus integration site 1 (AAVS1) region. The mutation rates were estimated by T7 endonuclease I assay around 12% of insertion/ deletion (indel). Furthermore, the transduced human hepatocytes were viable (ca. 60%) at two weeks post transduction. These observations suggest that the Ad vector-mediated delivery of the CRISPR/AsCpf1 system provides a useful tool for genome manipulation of human hepatocytes.

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Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

Cited by 50 publications

References 0 publications

Therapeutic gene editing in hematopoietic progenitor cells from a mouse model of Fanconi anemia

Therapeutic gene editing in hematopoietic progenitor cells from a mouse model of Fanconi anemia

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver

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