Engineered Telomerase RNA Component (eTERC), a Potential Lncrna Medicine to Extend Replicative Lifespan and Telomere Length in Dyskeratosis Congenita Patient Stem Cells

Telomere length governs cellular replicative capacity and decreases in hematopoietic cells as a function of age. Accelerated telomere attrition due to extrinsic pressures or rare genetic disorders of impaired telomere maintenance such as dyskeratosis congenita (DC) is associated with bone marrow failure (BMF) and evolution to myelodysplastic syndrome and leukemia. For DC and other telomere biology disorders (TBDs), there are no targeted curative therapies and new approaches are needed. Manipulating telomerase, the ribonucleoprotein complex that extends telomeres, could be useful to extend cellular self-renewal in these settings. However, ectopic expression of the telomerase reverse transcriptase (TERT) raises safety concerns about conferring immortalization and cancer risk. Here, we describe the systematic engineering of a synthetic form of the long non-coding RNA (lncRNA) component of telomerase, TERC, that robustly restores telomere length and extends replicative lifespan when introduced transiently into human cells. We found that unmodified full-length in vitro transcribed (IVT) TERC RNA showed no telomere lengthening despite TERT overexpression when introduced into TERC-null 293T human cell lines. This could be explained by nucleolytic degradation, improper folding or trafficking of an IVT lncRNA, and/or lack of association of exogenous TERC with TERT. To address the first possibility, we innovated and applied a novel method for 3’ end stabilization of RNAs, namely self-limiting 2’-O-methyladenosine (2’OMeA) tailing by the non-canonical polymerase TENT4B. 2’OMeA modification by TENT4B conferred 3’ exonuclease resistance to oligonucleotides in vitro and increased the function of an enhanced green fluorescent protein mRNA in cells. Upon introduction of 2’OMeA 3’ end-modified IVT TERC into TERC-null 293T cells, TERT-dependent telomere elongation and reconstitution of telomerase activity could be observed, albeit at modest levels. Next, considering the distinct 2,2,7-trimethylguanosine (tmg) cap structure of TERC may be important not only for nuclease resistance but RNA trafficking, we replaced the standard 5’ cap used in IVT for mRNAs with tmg followed by 2’OMeA tailing by TENT4B (hereafter called “engineered TERC” or eTERC). When co-transfected with TERT mRNA in TERC-null 293T cells, eTERC RNA showed optimal telomerase reconstitution. A single transient exposure to eTERC yielded rapid telomere elongation >1 kilobase within three days and forestalled telomere-induced senescence for 65 population doublings. We next asked whether eTERC could increase telomere length in the setting of various genetic lesions that cause TBDs and introduced eTERC into induced pluripotent stem cells (iPSCs) derived from DC patients. In this case without exogenous TERT, eTERC was capable of robust telomere elongation within three days in iPSCs from nine different DC patients carrying mutations in the genes most commonly associated with TBDs (TERT, TERC, RTEL1, DKC1, TINF2, and PARN). Telomere length gradually returned to baseline 69 days following a single electroporation, showing a significant boost in replicative capacity but with intact homeostatic mechanisms after “hit-and-run” telomere elongation by eTERC. Collectively, our results provide a novel method for enzymatic 3’ end stabilization of RNAs of any size that could be useful in therapeutic RNA engineering applications. We demonstrate a rapid, robust way to elongate telomeres and propose eTERC as a potential first synthetic lncRNA medicine with the means to extend cellular replicative lifespan. eTERC is being advanced for investigation in human hematopoietic stem and progenitor cells from patients, which could be useful for TBD-associated BMF and a broader span of ex vivo gene and cell therapy applications.