CRISPRa-mediated FOXP3 gene upregulation in mammalian cells

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.

IPEX as a Consequence of Alternatively Spliced FOXP3

“…CNS3 located at FOXP3 intron 1 (i.e., between exon 1 and 2) acts as a genetic switch that initiates gene expression through interactions with c-rel (15,16) and atypical NF-κB inhibitor (17). Accordingly, targeting CNS3 chromatin accessibility with a specific ribonucleoprotein (dCas9-catalytically inactive variant of clustered regularly interspaced short palindromic repeats-associated protein 9) has been shown to promote FOXP3 expression in Jurkat cells (18), whereas endogenous long non-coding RNA (FLICR-Foxp3 long intergenic non-coding RNA) induces a FOXP3 low T-cell subset (19). Although high FOXP3 expression levels are commonly attributed to CD4 + CD25 high Treg cells, FOXP3 is also induced by TCR stimulation in naive CD4 + CD25 − T cells and persists for several days in activated CD4 + T cells (20).…”

Super-Treg: Toward a New Era of Adoptive Treg Therapy Enabled by Genetic Modifications

“…Overexpression of the transcription factor HELIOS cooperates with FOXP3 to generate both CD4 + and CD8 + Treg from human Tconv, but particularly CD8 + T cells ( 20 ). Similarly, delivery of dCas9 fused to a transcriptional activator and guides recognizing FOXP3 promoter sequences increases FOXP3 expression ( 104 ).…”

Reprogramming the anti-tumor immune response via CRISPR genetic and epigenetic editing

“…Although not applied in pre-clinical studies, a number of early works also suggest that dCas9-based methods can help circumvent other major tumor immunoevasion mechanisms. For instance, impairment of Treg immunosuppressive function by dCas9-KRAB-mediated forkhead box P3 (FoxP3) transcriptional repression, 139 , 140 dCas9-SAM-mediated enhancement of neoplastic transformation markers, such as MICA, 141 , 142 and dCas9-KRAB-directed repression of immunosuppressive cytokine receptors 143 have demonstrated that dCas9-mediated epigenetic engineering has potential in multiple facets of cancer immunotherapy. Ultimately, dCas9-mediated epigenetic editing to improve tumor immunogenicity and/or boost the host’s anti-tumor immune response opens new therapeutic avenues, particularly in combination with ICI or ACT therapies ( Figure 2 ).…”