Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

Abstract

Stable expression of transgenes is essential in both therapeutic and research applications. Traditionally, transgene integration has been accomplished via viral vectors in a semi-random fashion, but with inherent integration site biases linked to the type of virus used. The randomly integrated transgenes may undergo silencing and more concerningly, can also lead to dysregulation of endogenous genes. Gene dysregulation can lead to malignant transformation of cells and has unfortunately given rise to cases of leukaemia in gene therapy trials. Genomic safe harbour (GSH) loci have been proposed as safe sites for transgene integration. To date, a number of sites in the human genome have been used for directed integration; however none of these pass scrutiny as bona fide GSH. Here, we conducted a computational analysis to identify 25 putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage

“…to knock in the mCerulean3 transgene at the 3’ end of the MYH6 endogenous locus using a modified homology-directed repair donor cassette as previously described. 14,15 CRISPR-mediated NF2 knockout (KO) hESC cell lines were derived from the H1 MYH6 -mCerulean3 reporter line. iPSC and embryonic stem cell H1 lines were cultured on Geltrex-coated 6-well plates (Geltrex diluted at 1:200 Dulbecco's Modified Eagle Medium F12 medium) and maintained in mTesR1 medium (No.…”

Manipulating and studying gene function in human pluripotent stem cell models

“…Moreover, as already discussed, the most popular GSH, the AAVS1 locus, does not support homogeneous transgene expression with many promoters and can be methylated. In this light, the recent identification of several novel human GSH sites may pave the way to more predictable hPSC genome editing [258][259][260]. Such GSH candidates were identified through a combination of methods and were rigorously assessed for widely accepted criteria for bona fide GSH status, but their use in hPSCs and their derivatives remains to be demonstrated.…”