January 2020
Cancer therapy with a CRISPR-assisted telomerase-activating gene expression system
Wei Dai, Xinhui Xu, Danyang Wang, Jian Wu, Jinke Wang
Clustered regularly interspaced short palindromic repeats (CRISPR) it is a powerful and versatile genetic engineering tool. This powerful technology can be used to introduced targeted DNA double stranded break (DSB) virtually at any location within the genome. Compared to the other genetic engineering tools – ZFNs and TALENs – it is cheaper and easier to use. CRISPR has opened a new avenue of therapeutic approaches based on gene editing. In particular they are for the most focused on developing treatments for Immune System related disease like Severe Combined Immunodeficiency (SCID) or HIV. CRISPR has found application also in the development of Exon-Skipping based approaches for muscular dystrophies, like in the case of Duchenne Muscular Dystrophy (DMD). However, usage of CRISPR for other severe human disease like Cancer has been limited.
In the following paper, the researchers employ CRISPR as main tool to kill the cancer cells. To achieve this, they develop a two components circuit in which: a catalytically dead-Cas9 (dCas9) linked to a strong transcriptional activator VP64 and a fully active Cas9 (wtCas9) whose expression is controlled by a minimal promoter (MP) flanked by a telomerase-recognizable 3ʹ single-stranded sequence (3ʹ-TTGGGATT GGGATT-5ʹ) . The minimal promoter can´t support on its own the expression of the wtCas9. To express the wtCas9, dCas9-VP64 needs to bind close to the (MP) so that VP64 can sustain the wtCas9 expression. The dCas9 is targeted to the telomerase-recognizable sequence. The latter, to be bound by the dCas9 needs to be extended by the Telomerase. The Telomerase is expressed at high level in cancer cells – to sustain their high rate of cell division – so with this circuit only in the cancer cells – where the telomerase can extend the binding site for the dCas9 – the wt Cas9 will be expressed. The targets of the wtCas9 are the telomeric sequences which allow the cancer cells to continue dividing uncontrolled.
Dai et al, perform a first proof of concept where they use both cancer derived cell lines like HepG2 (Human liver carcinoma) cells and no cancer derived cell line like MRC-5 (Human lung fibroblast). In the experiment they confirm on the one hand that the wtCas9 is expressed only in the cancer-derived cell line and on the other that due to cutting of telomeres sequence by Cas9 the life span of those cells is dramatically reduced. A limitation of application of CRISPR in cancer cells is also the delivery. The researches prove that it is possible to deliver in vivo their constructs by packaging in Adeno-Associated Virus(AAV). In particular they perform an intravenous injection in mouse; previously treated to develop cancer. Intriguingly they confirm that not only the tumor size is reduced but also that the constructs were expressed only at the tumor site, confirming both the efficacy and specificity of their approach. This paper proposes an elegant strategy through which CRISPR may be used to play an active role in cancer therapy.