Medical Applications of Synthetic Biology - Samantha Simpson

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Project Proposal

My project will be on medical applications of synthetic biology. I will reference Anderson's paper on utilizing quorum-sensing and hypoxia-responsive genes coupled with invasin from Yersinia tuberculosis to invade cancer-causing cells. Coupled with Critchley's work, which describes a bacterial system that invades eukaryotic cells and delivers proteins coded for in the bacteria's genome, one could possibly create 'search and destroy' E.coli that can locate, invade, and kill tumor cells. Garmory's paper also highlights the possibility of using bacteria as a drug-delivering system, specifically vaccine vectors. Kobayashi describes cells that produce a protective biofilm layer after exposure to DNA-damaging agents. Ro engineers yeast to create an anti-malarial drug that can be created at a lower cost than the previous standard production process. These papers, and others that have a focus on direct medical applications such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines, will be referenced to create a cohesive overview of applications of synthetic biology to the medical field.


Paper

Synthetic biology is a rapidly emerging field that strives to re-design existing biological systems and components or fabricate novel biological systems and components. The end result of experimentation in synthetic biology is to help scientists understand a naturally occurring genetic pathway more fully or to create a new, useful pathway. Recent research efforts have focused on engineering bacteria to create a new form of biofuel or to make promoters of varying efficiencies for more exact gene expression. Other research efforts focus on engineering yeast to make antimalarial drugs or engineering E. coli to recognize and invade tumor-like cells, both of which are medical applications of synthetic biology. Advancements such as novel cancer therapies, vaccination technology, biological protection, and the creation of new medicines highlight synthetic biology’s potential to create breakthroughs in both the prevention and treatment side of medical science. This paper will examine various medical applications of synthetic biology and further development that needs to be done before current research can be used in hospitals and doctors’ offices.


Prevention

Engineering live bacteria to act as a vaccine vector is an area of interest in synthetic biology. Bacteria were first thought to be good vectors because they would not degrade at mucosal surface and would survive the low pH in the gastrointestinal tract like most other orally administered antigens (Garmory, 2003). A particular strain of Salmonella with one or more deletions in the shikimate pathway, which creates a precursor for phenylalanine and tyrosine, is a promising vector because it gets into the body easily but does not harmfully effect the tissue and is gone within a week or two of introduction. Scientists engineered this strain to carry the Yersinia pestis V antigen, and studied its ability to protect mice from Y. pestis. The V antigen is generally injected via an intramuscular route, however, in this study, 20 mice were given the V antigen via the intragastric route so scientists could test the effectiveness of an oral administration. Unfortunately, the inoculation was not extremely successful and only 6 of 20 mice infected with Y. pestis showed a strong response (Garmory, 2003). Garmory and colleagues cited several reasons for this: the copy number of the plasmid containing the V antigen might not have been high enough; the attenuated Salmonella may not have been able to reach cells that would be targeted by Y. pestis; and the inability of the cell to secrete the antigen. The authors remain hopeful that attenuated bacteria vectors may someday produce an orally administered long-lasting response for protection against bubonic and pneumonic plague (Garmory, 2003). Many changes need to be made to live vector vaccine design before it is an effective method of treating or preventing disease, such as solving the issue of specifity of a vaccine administered orally, and creating a bacterial chassey that can secrete an antigen at an appropriate time.


Another method of disease prevention with a synthetic biology approach is related to skin cancer. E. coli was engineered to sense single-stranded DNA, and this DNA damage was coupled with a mechanism to produce a biofilm around the cell. The biofilm output module was tested with DNA damage done by mitomycin C, a DNA crosslinking agent, or UV irradiation, a known cause of skin cancer. Single-stranded DNA activates RecA, which in turn represses the C1 repressor protein, thus allowing the transcription of the PL promoter (Kobayashi, 2004). The PL promoter is on the biofilm-forming output plasmid (pBFR), which controls biofilm formation (Fig. 1). The biofilm protection, when part of a toggle switch mechanism, lasted indefinitely when pulsed with UV light at 8 J/m2 for only 2 seconds (Kobayashi, 2004) (Fig. 2). The biofilm-production mechanism has not been engineered in eukaryotic cells, but a prokaryote-based system that could aid with human skin disease is imaginable. For example, a sunscreen that changes colors when the DNA damage may be too intense for a sunbather as a warning, or a sunscreen that becomes more protective as the possibility of genetic damage increased. Further research should focus on how to best implement the biofilm producing output module to protect human DNA.

Toggleswitch.JPG UVinducedbiofilm.JPG
Fig. 1. The RecA / traA toggleswitch mechanism. (Kobayashi, 2004 - Permission Pending) Fig. 2. Crystal violet absorbance measures biofilm formation. (Kobayashi, 2004 - Permission Pending)


Treatment

Synthetic biologists consider finding a cure for cancer a reachable goal. Previous to the development of synthetic biology as a field, it was known that three types of bacteria, Bifodobacterium, Clostridium, and Salmonella all preferentially infect the dense cells of tumors (Pawelek, 2003). All three types of bacteria are associated with reducing tumor size in patients who are infected with them, and it was noted that all three bacteria act anaerobically. Capitalizing on the idea that tumors create an unusually dense, anaerobic mass of cells, Christopher Voigt’s lab engineered bacteria to identify and invade cancer cells. They utilized the lux quorum sensing from Vibrio fischeri to identify cells growing at high densities (Fig. 3) and the fdhF promoter to identify cells growing in anaerobic conditions in vitro (Fig. 4) (Anderson, 2006). They coupled this identification mechanism with the invasin output module from Yersinia pseudotuberculosis so the E. coli could invade specific cells.

InvasinDensity.JPG InvasinAnaerobic2.JPG
Fig. 3. The red line represents when the invasin output module was paired with the inducible promoter lux which senses high cell density, the black line is constitutively on invasin. (Anderson, 2006 - Permission Pending) Fig. 4. The invasin output module was paired with the inducible promoter fdhF which senses anaerobic conditions. (Anderson, 2006 - Permission Pending)

Next, scientists hope to insert a gene that would destroy the invaded cells, making the complete cycle a search, invade, and destroy loop that would only be sensitive to cancer cells. One promising destroy mechanism has been implemented in humans via intratumoral injection, based on Salmonella that contains the E. coli cytosine deaminase gene that converts 5-fluorocytosine to 5-fluorouracil, a chemotherapy drug (Nemunaitis, 2003). Unfortunately, while the tumors did produce 5-fluorouracil (5-FU), they did not produce it in high enough yields to cause regression of the cancer. Another experiment flooded mouse cells via intratumoral injection with 6-methylpurine-2’deoxyribose (6-MPDR), which was converted to the toxin 6-methyl purine (MeP) by the enzyme purine nucleoside phosphorylase (PNP), which is naturally found in E. coli (Critchley, 2004) (Fig. 5).

Tumor.JPG

Fig. 5. Tumor size after injections. Cancer cells were injected into mice, and 5 days later injections with PBS, 6-MPDR, and invasin-enhanced E-coli began. Tumor cells recieving injections of invasin-enhanced E.coli and 6-MPDR grew the slowest, but did not get smaller. (Critchley, 2004 - Permission Pending)


The problem with this is that even though MeP was injected into the tumor, all cells in the body might be exposed to the toxin. In addition to the search and destroy mechanism, scientists hope to increase selectivity by combining the hypoxia-sensing and density-sensing units so that E. coli will only enter eukaryotic cells that exhibit both characteristics. This would make it more likely that invasin-enhanced E. coli would only invade cancer cells rather than muscle tissue that had low oxygen due to exercise (Anderson, 2006). To create a fully functional, synthetic biology-based approach to cancer therapy, scientists need to develop bacteria that discriminately invades cancer cells and destroys them. Most of the pieces are there – scientists have engineered E. coli that selectively invades hypoxic cells or dense cells and Salmonella that can destroy tumors in vivo – they just need to be put together effectively.


Finally, synthetic biology has made its mark on the field of medicine by creating drugs that are less expensive to produce than those made by pharmaceutical companies. Jay Keasling’s project involving the antimalarial drug artemisinin is the best example. Yeast was engineered to increase normal farnesyl pyrophosphate (FPP) production, convert FPP to amorphadiene, and oxidizing amorphadiene to artemisinic acid, which can then easily be separated from the yeast cells and converted to artemisinin in the laboratory (Fig. 6). This innovation allows the production of artemisinin at lower prices than those currently on the market, and without regard to environmental constraints (Ro, 2006). On a completely different tangent, Collins’s lab engineered T7 bacteriophages to deliver biofilm-degrading enzymes such as dispersin B after infecting and replicating in an E. coli biofilm. Dispersin B works to degrade an adhesin that is critical for biofilm formation. Phages that released dispersin B were found to be 99.997% effective at removing biofilm, which is 4.5 orders of magnitude higher than non-enzymatic phages, and caused a 3.65 log10 reduction in bacterial cells recovered from biofilmwhen compared to untreated biofilms (Lu, 2007) (Fig. 7). The specificity of phages for a certain type of bacteria make them a viable vector to be used in human treatments, which may range from removing dental plaques to cleaning inserted medical devices. Before phages are ready for use in medicine, however, a well-characterized phage library must be created so that way any specific biofilm can be targeted using the dispersin B method. This also requires scientists being able to identify the type of bacteria in the biofilm, which may be growing inside the body. Using synthetic biology to engineer an antimalarial drug in yeast or a biofilm-destroying enzyme in bacteriophages greatly increases the possible contributions to the field of medicine.

Artemesinin.JPG BiofilmDegradation.JPG
Fig. 6. All the steps in the box indicate steps that were optimized in yeast for a maximum artemisinic acid output. (Ro, 2006 - Permission Pending) Fig. 7. The amount of biofilm was decreased when the dispersin B enzymes were incorporated into T7 bacteriophages. (Lu, 2007 - Permission Pending)


Conclusion

Medical applications of synthetic biology are wide-ranging and eminently applicable to daily life. Synthetic biology based cancer treatments that utilize E.coli engineered to sense and attack tissues with high cell density and low oxygen availability are promising and need to be tested in vivo. Designing vectors out of attenuated bacteria may be a safe, effective method for disease treatment, but need to be able to reach a target tissue if administered orally. Using biofilms to sense sun damage potential may decrease the likelihood of skin cancer. Finally, using engineered cells to produce medicines in a cost-effective, environmentally-friendly way may revolutionize the pharmaceutical industry. Investing more research efforts in the field of synthetic biology may turn out to be an investment in medical technology.

Works Cited

-Anderson JC, Clarke EJ, Arkin, AP, and Voigt CA (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of Molecular Biology 355:619-27.

-Critchley RJ, Jezzard S, Radford KJ, Goussard S, Lemoine NR, et al. (2004). Potential therapeutic applications of recombinant, invasive E. coli. Gene Therapy 11:1224-33.

-Garmory HS, Leary SEC, Griffin, KF, Williamson D, Brown, KA, and Titball RW (2003). The use of live attenuated bacteria as a delivery system for heterologous antigens. Journal of Drug Targeting 11:471-79.

-Kobayashi H, et al. (2004). Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-19.

-Lu, TK, and Collins JJ (2007). Dispersing biofilms with engineered enzymatic bacteriophage. PNAS 104: 11197-11202.

-Nemunaitis J, Cunningham C, Senzer N, Kuhn J, Cramm J, Litz C, et al. (2003). Pilot trial of genetically modified, attenuated Salmonella expressing cytosine deaminase gene in refractory cancer patients. Cancer Gene Therapy 10:737-744.

-Pawelek JM, Low KB, and Bermudes D (2003). Bacteria as tumour-targeting vectors. Lancet Oncology 4:548-56.

-Ro D, et al. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940-43.

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