Abstract
BackgroundForkhead box P3+ (FOXP3+) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the FOXP3 gene in animal models and humans results in loss of differentiation potential into Treg cells and are responsible for several immune-mediated inflammatory diseases. Strategies of increasing FOXP3 expression represent a potential approach to increase the pool of Tregs within the lymphocyte population and may be employed in therapies of diverse autoimmune conditions. In the present study, a dCas9 CRISPR-based method was systematically employed to achieve upregulation and sustained high expression of endogenous FOXP3 in HEK293 and human Jurkat T cell lines through targeting of the core promotor, three known regulatory regions of the FOXP3 gene (CNS1–3), and two additional regions selected through extensive bioinformatics analysis (Cage1 and Cage2).ResultsUsing an activator-domain fusion based dCas9 transcription activator, robust upregulation of FOXP3 was achieved, and an optimal combination of single guide RNAs was selected, which exerted an additive effect on FOXP3 gene upregulation. Simultaneous targeting of FOXP3 and EOS, a transcription factor known to act in concert with FOXP3 in initiating a Treg phenotype, resulted in upregulation of FOXP3 downstream genes CD25 and TNFR2. When compared to ectopic expression of FOXP3 via plasmid electroporation, upregulation of endogenous FOXP3 via the Cas9-based method resulted in prolonged expression of FOXP3 in Jurkat cells.ConclusionsTransfection of both HEK293 and Jurkat cells with dCas9-activators showed that regulatory regions downstream and upstream of FOXP3 promoter can be very potent transcription inducers in comparison to targeting the core promoter. While introduction of genes by conventional methods of gene therapy may involve a risk of insertional mutagenesis due to viral integration into the genome, transient up- or down-regulation of transcription by a CRISPR–dCas9 approach may resolve this safety concern. dCas9-based systems provide great promise in DNA footprint-free phenotype perturbations (perturbation without the risk of DNA damage) to drive development of transcription modulation-based therapies.
Highlights
Forkhead box P3+ (FOXP3+) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis
Ectopic expression of FOXP3 in naïve T cells and T cell lines via viral transduction has been shown to confer in vivo and in vitro suppressive activity towards Treg cells, demonstrating that Tconv may be reprogrammed into immunosuppressive Treg-like cells [6, 12,13,14]
The core promoter region encompassed 49,264,651–49,265,336 bp region on human chromosome X (ChrX), three regions downstream of core promoter were CNS1 (ChrX: 49,262,712– 49,263,103 bp), CNS2 (ChrX: 49,260,567–49,261,013 bp) and CNS3 (ChrX: 49,258,069–49,258,320 bp) and two regions upstream of core promoter entailed Cage1 (ChrX: 49,266,272–49,266,819 bp) and Cage2 (ChrX: 49,266,819–49,267,319 bp; all above mentioned coordinates pertain to the human genome assembly hGRC37/ hg19)
Summary
Forkhead box P3+ (FOXP3+) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the FOXP3 gene in animal models and humans results in loss of differentiation potential into Treg cells and are responsible for several immune-mediated inflammatory diseases. Loss-of-function mutations of the FOXP3 gene in animal models and humans result in loss of differentiation potential into Treg cells and is responsible for highly aggressive, fatal, systemic immune-mediated inflammatory disease [5]. Many autoimmune conditions, such as type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis and others are characterized by an imbalance between the pools of immune-suppressing Tregs and pro-inflammatory CD4+ conventional T cells [7]. Transdifferentiation of conventional T cells into immunosuppressive Treg-like cells using non-insertional methods via FOXP3 upregulation could provide an alternative approach to increase the pool of therapeutic Treg-like cells
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