Reversible hTERT Modulation in Human T Cell: Lifespan Extension, Functional Maintenance and Applications in Immunotherapy

Abstract

hTERT gene as the main regulator of telomerase activity has been extensively studied in terms of cell proliferation and senescence. However, its research in immune application was long stagnated by the concerns of oncogenic risk and genomic instability. This this study, we intended to prolong the lifespan of the immune cell and restored it back to relative normal state before the manifestation of any malignant transformation. Meanwhile, we took advantage of the cells’ extended longevity to enhance their immune functions.

To alleviate the doubts, we first established temporal control of hTERT gene through CRISPRa and CRISPRi epigenetic modification. Comparing to the conventional RNAi and overexpression system, the dCas9 is capable of control the endogenous gene expression by only transiently binding to DNA instead of altering DNA sequence. In our study, four CRISPRa tool: p300 histone acetyltransferase, TET1 DNA demethylase, VPH and VPR were used to activate hTERT while the CRISPRi KRAB-MeCP2 was used to downregulate the hTERT. The KRAB-MeCP2 can provide stable, long-term silencing even with transient expression that ensure the persistent downregulation of hTERT. The delay of senescence through this activation was proved in our previous study. In this work, we observed a significant reduction in both mRNA and protein level after CRISPRi induction. Additionally, we detected significant changes in the expression of key genes associated with cellular senescence. Specifically, we observed an upregulation of TP53 and CDKN2A, both of which play pivotal roles in the regulation of the senescence response, alongside a reduction in LAMNB1, a gene involved in nuclear structure that is often dysregulated in immortalized cells. Also, the immortalized cell was observed dead after a week followed the CRISPRi induction. Here, we confirmed the downregulation of hTERT after CRISPRi induction and halt of immortality in the previously hTERT activated cells.

We further investigate the functional characteristics of the engineered immune cells. The hTERT modulated PBMCs exhibit the similar phenotypes and function characteristics as in primary PBMCs. Moreover, the immune response against antigen including activation, proliferation and cytokine production were all intact.

Based on prior results, we trained T cells that have specific target for WT-1 tumor associated antigen. WT-1 is a self-antigen so its expression in healthy person is relatively rare. Hence, the starting material for culturing WT-1 specific T cells is generally very low and hard to sort out. In this work, we post an alternative strategy that instead of searching the ocean for a grain of sand, just simply prolong the culture and add a few more rounds of WT-1 antigen peptide stimulation for the T cell have been previously hTERT activated. The replicative senescence escaped by the hTERT activation allowed enough time for WT-1-specific cell to expend through multiple rounds of stimulation and naturally grow into the dominate population without initial sorting. We have co-cultured the trained T cell with A549, MDA-MB-231 and MCF-7 those with different levels of WT-1 overexpression confirmed the enhanced cytotoxicity. Also, we downregulated hTERT level of T cell after prolonged expansion and subsequently co-culture with those tumor cells. The result shows similar cytotoxicity level suggested the downregulation of hTERT does not immediate shut down the telomerase activity but left a window for the cells to exert their full function before death.

In conclusion, this study successfully manipulated hTERT by upregulating the expression for extension of lifespan with subsequent downregulation for restoring its fate to death. The T cells’ main characteristics, phenotypes and functions remain mostly unchanged during the changes. More importantly, we presented a simple, cost-effective and safe approach for the expansion of WT-1-specific T cells that are otherwise difficult to expand due to the low frequency.

Date of Award	30 Apr 2026
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Hiu Wai Raymond LAM (Supervisor) & Dong SUN (Supervisor)