Introduction:: One of the most popular immortalization approaches is ectopic overexpression of human telomerase reverse transcriptase (hTERT) to maintain sufficient telomere lengths for avoiding replicative senescence. Compared to other immortalization methods such as overexpressing SV40T antigens or suppressing tumor suppressor genes, hTERT prolongs replicative lifespans without the loss of primary cell functions or malignant transformation. However, randomly integrate the hTERT transgene into the genome via viral vectors can lead to insertional mutagenesis and genome instability. Moreover, only brief reconstitution of telomerase activity is achieved when overexpressed with episomal plasmids. To circumvent these issues, we utilized CRISPR/dCas9-based epigenetic modifiers (p300 histone acetyltransferase and TET1 DNA demethylase) and transcriptional activators (VPH and VPR) to reactivate the endogenous TERT gene in unstimulated T-cells derived from the peripheral blood mononuclear cell (PBMC) by rewiring the epigenetic marks of the TERT promoter. We also have validated the effectiveness of these CRISPR tools in HEK293FT and THP-1 cells by upregulating their endogenous TERT expressions. Importantly, we have successfully delayed cellular senescence of resting T-cells for at least two months without affecting T-cell marker expression by reactivating endogenous TERT expressions. The strategy of cell immortalization described here can be potentially adopted and generalized to delay cell death or even immortalize any other cell types.
Materials and Methods:: We envision to extend replicative lifespan of T cells derived from primary PBMCs by activating its endogenous TERT expression using CRISPR/dCas9-based epigenetic modifiers dCas9-p300 (Addgene# 61357) and dCas9-TET1 (Addgene# 167983) and transcriptional activators dCas9-VPH (Addgene# 158091) and dCas9-VPR(Addgene# 63798) . To construct the required sgRNA guide sequence, a pair of annealed oligonucleotides was cloned into a pU6-sgRNA expression cassette (Addgeen# 53188) bearing the sgRNA scaffold backbone and tracrRNA using BbsI. The oligonucleotides were designed based on the target site sequence (20 bp), and they were flanked on the 3’ end by a 3-bp 5’-NGG-3’ PAM sequence. The CRISPR RGEN Tool, Cas-Designer, was used to identify the target sequence of sgRNAs. The first nucleotide of the transcribed gRNA was a guanine nucleotide (G) to maximize the U6 promoter activity. The selected sgRNA target sequences had no potential off-target sites of RNA-guided endonucleases within 2-nt mismatches. The pU6-seq primer was used for DNA sequencing to confirm successful guide sequence insertion in the sgRNA. We used the 4D-NucleofectorTM system (Lonza, Basel, Switzerland) and P3 Primary Cell 4D-NucleofectorTM X Kit S (Lonza, Basel, Switzerland) to transfect the plasmid into PBMCs. And then, Analysis of hTERT mRNA and protein expression by qPCR and flow cytometer with FITC-conjugated anti-human hTERT (1µl of 200µg/ml to 100µl PBS) (Santa Cruz, Dallas, Texas, USA). Additionally, this experiment evaluated the characteristics of immortalized T cells, including the cell cycle and proliferation rate.
Results, Conclusions, and Discussions:: we have successfully delayed cellular senescence and extended replicative lifespans of unstimulated T cells derived from human PBMC by reactivating its endogenous TERT expression using a combination of CRISPR/dCas9-based epigenetic modifiers and transcriptional activators. Compared to exogenous telomerase-overexpressing methods, reactivation of endogenous telomerase can alleviate potential host immune response, thereby allowing safer adoptive transfer of TERT-expressing T cells in vivo. Targeted reactivation of endogenous telomerase can also minimize the risk of developing genomic instability and malignant transformation in T cells, as it ensures a functioning physiological level of TERT expression. Although it has been reported that external stimuli (e.g., interleukin-2, monoclonal antibodies CD3 and CD28) are capable of briefly reactivating endogenous telomerase gene in human T lymphocytes, such TERT reactivation is temporary and difficult to support subsequent stimulations for immunotherapy applications. On the contrary, the use of CRISPR/dCas9-based epigenetic modifiers in our study can prolong the TERT reactivation in the T cells by rewiring the epigenetic states that regulate TERT expression. We have observed the TERT-expressing T cell lines for three months of culture after the epigenetic modification and verified that their actual replicative lifespans do not come with accelerated cell division in vitro. Nevertheless, whether these TERT-expressing T cells would eventually become malignantly transformed and would lose their primary T-cell characteristics require further investigations for an even longer period.
In essence, this work exhibits that the transfected PBMCs contain immortalized T-cells, verified by CD3 expression, cell size distribution, as well as the continuous cell proliferation. In addition to PBMC-derived T cells, we have demonstrated the effectiveness of these CRISPR tools in HEK293FT and THP-1-derived macrophages. CRISPR/dCas9-based epigenetic modifiers and transcriptional activators could robustly upregulate the TERT expression in HEK293FT and THP-1-derived macrophages, despite no noticeable expression of hTERT at a baseline level. We envision that the strategy of cell immortalization described here can be applied and generalized to other primary human cells.