Safety in our Project

From the beginning of the brainstorming, the main idea involved the expression of therapeutic proteins that would act as toxins for tumor cells. At this point, we realized that we needed more specificity if healthy tissue was to be unharmed. In the hypothetical case that our bacteria were liberated into the environment and some way it survived and maintained its plasmid without a selectivity marker, it would be a catastrophe. This imaginary scenario led us to the idea of implementing safety locks in the design of our project.

The first issue we needed to address was the potential harm of the bacteria triggered by the immune response against the lipid A and the Braun lipoprotein components of the Lipopolysaccharide (LPS) membrane of the Escherichia coli. Thus, we devoted our <a href="module1.html">Module I</a> to develop a strategy to avoid this problem. Literature showed that the deletion of the msbB and the lpp genes can produce viable strains; furthermore, according to studies performed by he mutants of this strain by Stritzker et, al., the bacteria with the msbB deletion could still be able to reach and colonize tumours. This action also reduced the side effects in mice.

We wanted that the action of our therapy was site-directed; as a result, we designed our phages to internalize in the mammalian cells. The infection in bacteria is initiated when P3 binds to the F pilus on the surface of an E. coli which facilitates the translocation of the viral DNA into the cytoplasm. In our case the P3 will be binded with the peptide CTVALPGGYVRVC, that specifically binds to glucose-regulated protein 78 (GRP78). Normally, GRP78 expression is maintained at low levels but is upregulated in stress environments and induced in tumor environments. Thus, the phages will only bind to this cancer-specific receptor that express this receptor protein, ignoring the healthy cells. For more information, you can check <a href="module3.html">Module III</a>.

Also, we needed to design our genes that we wanted to insert in a way their products wouldn’t harm humans, and it was necessary to decide which therapeutic proteins we were going to produce as tumor toxins. From our past project and bibliographical research, we found out that the soluble part of the protein TAT-Apoptin (<a href="http://parts.igem.org/Part:BBa_K1166005">BBa_K1166005</a>)was a protein that has an antitumoral activity with high specificity leaving healthy tissue in its majority unharmed. Nonetheless, the protein’s activity could be enhanced by using a survivin siRNA, which overexpressed by tumoral cells; thus, we needed to regulate the expression of it. This was accomplished by using a hTERT (human telomerase reverse transcriptase) promoter (<a href=" http://parts.igem.org/Part:BBa_K404106">BBa_K404106</a>), which is induced by several transcription factors found in cancerous cells, which will stop the siRNa from activating in a healthy cell. For more information, you can check <a href="module4.html">Module IV</a>.

Then, the bacteria was designed to produce its recombinant bacteriophage under a quorum sensing system, which is described in <a href="module2.html">Module II</a>. Also, the strains that are being used in the project cannot survive outside laboratory conditions for more than two weeks because they are derived from E.coli K12. Therefore, the probability of producing bacteriophages outside the laboratory conditions is very low.

Finally, its important to recall that the project is not ready to be used as an industrial product, as more research is needed. This knowledge will be generated after all the experimental tests on this strain are finished, which must include in vivo and clinical trials, outside the reach of a one-summer test. Also, a construct without antibiotic resistance is yet to be developed and the use of an auxotrophic dependence construct needs to be developed previous to its release.

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References

Stritzker, J., Hill, P. J., Gentsche, I., & Szalay, A. A. (2010). Myristoylation negative msbB-mutants of probiotic E. coli Nissle 1917 retain tumor specific colonization properties but show less side effects in immunocompetent mice. Bioengineered, 1(2), 139-145.

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Safety in our Project

References