Overview




Motivation




Although it is one of the most researched and funded fields in medicine, cancer is still a major cause of morbidity and mortality worldwide, with 14 million new cases and over 8 million deaths per year. It is the second cause of death worldwide, and it’s responsible for quarter of the death cases among developed countries. If current trends continue, cancer will soon surpass heart disease as the leading cause of death in the U.S
The failure of current therapies to cure cancer is due to a few reasons:
1. Most treatments cannot distinguish precisely enough between cancer and healthy cells. Low specificity means higher toxicity and high rate of adverse effects.

2. Cancer cells have an extremely complex pathophysiology with multiple biological pathways allowing their infinite growth and resistance to treatment. Thus, intervening with only one of this pathways, as most current therapies do, is doomed to fail.

3. Cancer is not a single disease, but a collection of diseases arising from different genetic mutations, involving abnormal cell growth.

Our aim , therefore, is to develop cancer therapy that is both highly specific for cancer cells, efficient, and personalized for each tumor and patient genetics.


Boomerang




This summer we have set our goal to design and test a synthetic machine which could distinguish individual cancer cells from healthy tissue. Our design makes sure that the function of our machine will be limited exclusively to cancer cells. Our machine does so by being operated by 2 separate cancer-specific promoters, which are highly and predominantly activated in cancer cells (1, 2) (link to Results figure of TERT and survivin). By using two separate promoters we ensure that our system will be exclusively activated only in cancer cells, with minimal, if any, expression in healthy cells. Simply by changing the promoters that control the system parts, our modular system makes it easy to design the system to fit the genetic profile of each individual malignancy.

There were several ways in which we can deliver our system in the body, and we chose AAV (Adeno Associated Virus) because of its many advantages, including low pathogenicity and mild immune response. AAV is used today in advanced clinical trials for gene therapy, and 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 (3, 4).

In our specific design for the prototype/proof-of-concept studies we use promoters which are linked to tumor proliferation (human telomerase-reverse transcriptase (hTERT) promoter) and enhanced survival (human survivin promoter), both known to be highly active in multiple cancer cell types (1, 2).

The Design

We have constructed two separate designs, both utilizing different versions of CRISPR/Cas9 system:


Knock-out of genes essential for cancer cell survival (e.g., to inhibit tumor proliferation and induce apoptosis)	Expression of exogenous proteins which could: 1) label the tumor in a way which would enable surgeons to identify its edges for its complete removal (e.g., a chromophore) 2) lead to cancer cell death (e.g., by expression of an apoptotic protein); 3) to produce a biomarker detectable in blood and/or urine for cancer diagnosis

Why Boomerang?

	Like a boomerang thrown by a person which flies back instantly, our synthetic machine uses cancer cells' own genetic alterations against them.

Part improvement

Our project led us to improving on an existing biobrick phTERT-K404106.
Our part: BBa_K1699001 is shorter, and we have characterized and validated it experimentally. We have also added data which shows that our part is working well in driving transcription of downstream genes specifically human cancer cells.
For more information please visit our Part improvement page.

References

(1) The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters

(2) Applications of the CRISPR–Cas9 system in cancer biology

(3) Oncolytic viruses: a new class of immunotherapy drugs

(4) Targeting of tumor radioiodine therapy by expression of the sodium iodide symporter under control of the survivin promoter

(5) In vivo genome editing using Staphylococcus aureus Cas9.

(6) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing

(7) DDownregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention

(8) RNA-guided gene activation by CRISPR-Cas9-based transcription factors.

(9) Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas

(10) Generation of Constitutively Active Recombinant Caspases-3 and -6 by Rearrangement of Their Subunits

(11) One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell

(12) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture