Complete TCR-α Gene Locus Control Region Activity in T Cells Derived In Vitro from Embryonic Stem Cells

Abstract

Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin’s gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein we report manifestation of all key features of mouse T cell receptor (TCR)-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC). High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and upregulated during the expected stage transition of T cell development. We further report that de novo introduction of TCRα LCR linked transgenes into existing T cell lines yields incomplete LCR activity. Together, these data indicate that establishing full TCRα LCR activity requires critical molecular events occurring prior to final T-lineage determination. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.

Adapting in vitro embryonic stem cell differentiation to the study of locus control regions

“…Analogous to the results seen for the β-globin LCR, we have reported that the TCRα LCR cannot drive transgene expression in a copy number-dependent manner after its direct introduction into differentiated T cells (Lahiji et al, 2013). This is in stark contrast to the activity of identical TCRα LCR-driven reporter constructs in transgenic mice, within which all the hallmarks of TCRα LCR activity are consistently observed (Harrow and Ortiz, 2005; Knirr et al, 2010).…”

“…Since the estimated number of integrated transgene copies in these clones (3 and 5) are within a range that yields copy number-dependent expression (Lahiji et al, 2013), this apparent silencing is not due to excessive transgene copy number. Thus, it seems that transfected ESCs will be less hospitable to the function to YFP, and perhaps any non-mammalian reporter cDNA, during differentiation than are standard cell lines.…”

“…The protocol for in vitro derivation of T cells, and other hematopoietic cell types, from mouse ESC was carried out as described previously (Holmes and Zuniga-Pflucker, 2009; Lahiji et al, 2013). Emerging hematopoietic stem cells from day eight co-cultures were harvested and transferred onto OP9-DL1 cell monolayers (to derive T cells) (Schmitt et al, 2004) or OP9 cell monolayers (to derive monocytic and erythroid cells) (Nakano et al, 1994).…”

“…These results led us to use the TCRα LCR to test the ability of the aforementioned in vitro ESC differentiation model to support full LCR activity. Indeed, we found that this system enables full recapitulation of all aspects of TCRα LCR activity seen in vivo (Lahiji et al, 2013). This work has validated in vitro ESC differentiation, as a much needed alternative approach to transgenic mice for the study of LCRs.…”

T-cell receptor α enhancer is inactivated in αβ T lymphocytes

“…ES cells (34). Hence, features of the hCD2 reporter dictate the developmental profile of Tcra LCR-hCD2 activity.…”