Validation of hTERT and Survivin promoters as key components of Boomerang system

In Boomerang system, we utilize two cancer-specific promoters for the activation of the system. For a proof-of-concept we selected human hTERT and Survivin promoters. For assessing their expression levels in cancer cells vs. “healthy" cells we performed gene expression analysis using quantitative real-time PCR (qPCR), where the expression of human hTERT and Survivin was evaluated in several human cancer cell lines compared to healthy cells (fibroblasts) (Fig.1).

Hyperactivation of the selected promoters was validated by using vectors expressing GFP (link to vector) and mCherry (link to vector) under the control of hTERT and Survivin promoters, respectively (Fig. 2). For this study we used both calcium phosphate transfection method (Fig. 2A) and AAV transduction (Fig. 2B).

Results:

The results show higher expression levels of survivin and hTERT in several human cancer cell lines compared to human fibrobalsts (healthy cells) (Fig.1). These results suggest marked promoter hyperactivation in cancer cells, confirming the eligibility for use of these two promoters in our studies.

Fig. 1. hTERT and Survivin expression levels 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.

Following simultaneous transfection with phTERT -GFP and pSurvivin-mCherry using calcium phosphate, GFP and mCherry expression was evident only in human cancer cells (HT1080 fibrosarcoma), compared to undetected levels in healthy fibroblasts (Fig. 2A). In merged image, colocalization of GFP and mCherry is evident in some of the cells (yellow).

Similar results were obtained with AAV transduction, with more cells expressing GFP and mCherry, suggesting that AAV transduction is more efficient.

A
B
Fig. 2. eGFP and mCherry expression, under human TERT and survivin promoters respectively in cancer cells (fibrosarcoma) and healthy fibroblasts, after 48 h (A) calcium phosphate plasmid transfection; and AAV transduction (B). Bar: 200 micron.

Similar expression of GFP was obtained in cancer and healthy cells when we used GFP under the control of constitutive promoter, CMV (link to vector), confirming that the presence of GFP/mCherry expression in cancer cells is not due the differences in transduction efficiency, but solely stems from promoter hyperactivaiton in these cells (Fig. 3).

Fig. 3. eGFP expression under constitutive promoter CMV in HT1080 cells (fibrosarcoma) and healthy fibroblasts, after AAV transduction. Bar: 200 micron.

Boomerang activation system – functional prototype in action

In our project, we designed cancer-specific CRISPR/Cas9-mediated activation of a gene of interest (modeled by GFP).

We tested our three component-based activation system by transfection or transduction of human cancer cells (HT1080 fibrosarcoma) and healthy cells with three vectors:

1) phTERT dCas9-VP64-pAAV (link to vector)

2) pSurvivin-gMLP-pAAV (link to vector)

3) pMLPm-eGFP-pAAV (link to vector)

Results:

Following simultaneous CaP transfection of three plasmids, eGFP expression was detected only in cancer cells, compared to undetected levels in healthy cells (Fig. 4).

These results show that the expression of dCas9-VP64 and gRNA under the control of cancer-specific promoters (TERT and survivin) drives the activation of the system only in cancer cells (Fig. 4). As a control group we included pMLPm-eGFP-pAAV alone to exclude GFP expression due to the "leakage" of the synthetic promoter (minimal promoter).

In our experiments we did not obtained GFP expression following AAV vectors transduction of the activation system three components. This might occur due to poor packaging efficacy of the virus (due to much larger than recommended size of phTERT-dCas9-VP64 vector – 8.1 kb).

Fig.4. eGFP expression from synthetic activation promoter exclusively in cancer cells after successful activation of CRISPR-based activation core driven by dCas9-VP64 - under the control of hTERT promoter, and guide RNA (targeting the synthetic activation promoter)- under the control of human survivin promoter. Bar:100 micron.

Complete design of Boomerang knockout system - ready for testing

We also engineered AAV construct in which the human TERT promoter controls the expression of "classical" Cas9 (SaCas9) - phTERT-SaCas9. The construct, together with gRNA under the control of human survivin promoter, was designed to drive knockout of Ubiquitin B (Ubb) gene.

Results:

We performed transfection and transduction experiments in cancer cells (HT1080) and healthy cells with the two components of the knockout system:

1) phTERT-SaCas9-pAAV (link to vector)

2) pSurvivin-gUBB -pAAV (link to vector)

Due to lack of time, we did not perform mutation and sequencing analysis to detect the effect of our system on the Ubb gene.

Since Ubb expression is essential for the growth of cancer cells, we expected to observe a decrease in proliferation rate and cell growth. At the time of writing, no effect of the Boomerang knockout system on cell growth was observed both on cancer and healthy cells.

Further studies are required to more extensively evaluate the performance of the designed constructs and confirm SaCas9-introduced mutations.

Conclusion and future aspects

In our project we designed cancer-specific promoter-driven CRISPR/Cas9 activation system that can be applied to cancer therapy.

The design allows an expression of any desired protein for applications such as induction of cancer cell apoptosis or cell death (e.g. diphteria toxin A) or tumor labeling for complete surgical removal.

We showed a functional prototype of the “Boomerang activation system” with GFP expression, showing a potential of this platform. Future attempts should be invested in increasing the efficiency of the transfection/transduction, and performing extended studies in additional cancer cell types.

Future studies should also include the validation of “Boomerang Knockout system”

In our studies we selected AAV for the delivery of “Boomerang system” due to several important advantages, such as low pathogenicity and mild immune response. AAV is used today in advanced clinical trials for gene therapy. One AAV-mediated gene therapy has already been approved for marketing at 2012 by the European Commission (Glybera - for treatment of LPLD - lipoprotein lipase deficiency). More information available here. Definitely, the efficacy of our system will be dependent on the development of effective delivery approaches in the future.

Promoter-based Boomerang system design avoids several major drawbacks in cancer therapy, such as reliance on cell proliferation or the existence of specific mutations. Various 'omics' analyses that are being developed or already available today allow dissection of each patient's tumor genetic and expression patterns based on simple biopsy. The Boomerang system could be easily personalized to specific patient and tumor profile by selecting hyperactivated promoter sets based on the screening results. With DNA technologies becoming cheaper and more advanced, the Boomerang platform could be readily a part of personalized medicine approaches developed for efficient and highly specific cancer therapy.