Part:BBa_K1699001

Human short TERT promoter

Human short TERT promoter.

hTERT (human Telomerase Reverse Transcriptase) is a human promoter which controls the transcription of Telomerase, a gene highly expressed in cancer cells, and not in healthy ones (1).

Usage and Biology

hTERT promotes the transcription of the catalytic subunit of telomerase. Telomerase elongates the ends of chromosomes, regions called telomers. hTERT is not active in somatic cells and is highly active in most if not all cancer cell types. Therefore, the expression of an exogenous gene under the control of hTERT promoter will likely occur in cancer cells only. We have used this promoter as part of a two promoter system (the other one being survivin promoter, also highly active in cancer cells), in order drive the expression, and subsequently activate the core of our system (based on CRISPR?Cas9 technology) exclusively in cancer cells.

Characterization

The validity of hTERT promoter for the application of cancer-specific gene expression was performed using quantitative real-time PCR (qPCR), where the expression of hTERT was evaluated in several human cancer cell lines comprared to healthy cells (fibroblasts) (Fig. 1). The results show thousand-fold and higher TERT expression levels in cancer cells, suggesting marked promoter hyperactivation.

Fig. 1. hTERT epxression level in several human cancer cell types, evaluated by qPCR. HF-human fibroblasts (healthy cells); cancer cell lines: HepG2 - hepatocarcinoma, A549 - lung adenocarcinoma, MDA-MB-231 - breast adenocarcinoma, HT1080 - fibrosarcoma.

This part was used and validated by BGU 2015 team in a following constructs.
1. phTERT-GFP: AAV (adeno-acociated virus)- vector expressing GFP under the control of human short TERT promoter was constructed (Fig. 2).

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Fig. 2. Plasmid map of AAV vector expressing eGFP under the control of human short TERT promoter.

Following calcium phoshpate plasnid transfection or AAV transduction, robust GFP expression was evident only in cancer cells, compared to undetected levels in healthy cells (Fig. 3).

Fig. 3.

2. phTERT-dCas9-VP64: AAV vector expressing dCas9-VP64 (engineered version of "classical" Cas9 for transcriptional activation of any gene of interest) under the control of human short TERT promoter was constructed as a part of functional prototype of two cancer-specific promoter-driven CRISPR/Cas9 activation system of BGU 2015 Boomerang team (Fig. 4).

Fig. 4.

Following calcium phoshpate plasnid transfection or AAV transduction, GFP expression was detected only in cancer cells, compared to undetected levels in healthy cells (Fig. 5).

Fig. 5.

Here, the GFP expression was driven by minimal synthetic promoter, after successul expression of dCas9-VP64 - under the control of hTERT, and guide RNA trageting the synthetic promoter - under the control of human survivin promoter. The unique ribozyme design was used to drive gRNA synthesis under "unusual" control of RNA polymerase II (and not III) promoter.

References

1. The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters http://www.nature.com/gt/journal/v8/n7/pdf/3301421a.pdf

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]