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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 the ideal 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)+ (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. The efficacy of our system will be dependent on the development of effective delivery approaches. (3).

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.

The Design

The design includes two parts: one with cas9 gene, and the other with a gRNA compatible to three sequences in the second axon of Ubb. Cas9 – the cas9 endonuclease was put under control of h-tert promoter, thus should be expressed predominantly in cells in which it is highly active, namely – cancer cells. When guided with gRNA, cas9 cuts both strands of DNA at the target sites, which leads to the cell trying to fix the double strand break, adding mutations which damage the activity of the mature protein. gRNA – the guide RNA is a hundred bases long molecule with a unique two dimensional structure which binds cas9 and guides it to a dsDNA sequence complementary to 22 base pairs on the 5' end of the molecule.

The second design includes the gRNA and two different parts. dCas9-V64 – dCas9-V64 was engineered so that it lacks endonuclease activity and has 4 V16 activation domains attached. When guided to a specific promoter, dCas9-V64 promotes transcription of genes downstream of binding site. The third part is synthetic promoter regulating GFP. The synthetic promoter has 3 complementary sites for the gRNA from the second part, which, upon binding, should paint cancer cells fluorescent green. **This construct was made as a proof of concept. By utilizing a synthetic promoter we could, in theory, express an apoptotic protein, a toxin and pretty much everything with a promoter.
• A detailed explanation of why your team chose to work on this particular project.
Cancer is the number one cause of mortality in developed countries
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References

(1) Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention Choongseob Oh , Soonyong Park , Eun Kyung Lee2 & Yung Joon Yoo

Inspiration

See how other teams have described and presented their projects:

• Imperial
• UC Davis
• SYSU Software