Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85

Abstract

The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force. 3 Highlights•Transgene integration to human AAVS1 locus decreases MBS85 expression.•Decrease in MBS85 expression increases cellular contractile force.•MBS85 is involved in myosin regulatory light chain phosphorylation.

Data set for comparison of cellular dynamics between human AAVS1 locus-modified and wild-type cells

“…Insertion of exogenous gene into the AAVS1 locus has been conducted in recent biological researches. Previously, our data showed that the AAVS1 -modification changes cellular contractile force (Mizutani et al, 2015 [1] ). To assess if this AAVS1 -modification affects cell migration, we compared cellular migration speed and turnover of cytoskeletal protein in human fibroblasts and fibroblasts with a green fluorescent protein gene knocked-in at the AAVS1 locus in this data article.…”

“…Fluorescence recovery was observed after photobleaching for the fluorescent protein-tagged myosin regulatory light chain. Data here are related to the research article “Transgene Integration into the Human AAVS1 Locus Enhances Myosin II-Dependent Contractile Force by Reducing Expression of Myosin Binding Subunit 85” [1] .…”

TALEN mediated gene editing in a mouse model of Fanconi anemia

“…The AAVS1 locus has been defined as a bona-fide safe harbor locus in humans 34 , in which an exogenous gene could be efficiently expressed in various cell lines and iPSCs [28][29][30][31][32][33][34][48][49][50][51][52] . Therefore, in this work, we have decided to carry out a targeted integration strategy into 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).…”

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

“…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).…”