Team:Penn/Barriers

Currently, in the field of medicine, groundbreaking approaches to therapeutics are constantly being developed. With the advent of synthetic biology, bacterial therapy is rapidly emerging as a promising source of therapeutic potential for many diseases, especially cancer. Consequently, bacteria can actually be seen as the ideal vector for the production and delivery of drugs for many diseases.

One reason bacterial therapies have been proven to be better than many existing therapies is due to the fact that many bacteria already exist in a commensal or even mutualistic relationship with the human body. The reason this serves as an important advantage over other options is that it lessens the chance of a harmful response from the immune system. Introducing foreign entities into the body reduces the chance that the therapy would continue to work after a long period of time since the body's defense mechanisms would kick in. This is exactly why working with or engineering strains that are part of the various microbiomes of the human body would be ideal. Escherichia coli is an example of a commensal bacteria in the gut that has been engineered to treat cholera. Scientists have managed to program E. coli to express cholera autoinducer 2 (CAI-2), inhabit the gut, and effectively block the quorum sensing that cholera use to propagate disease in the gut [1]. Another example of this has been through engineering Lactobacillus L. jensenii, a vaginal strain to produce anti-HIV proteins to serve as a preventive measure again HIV [2].

Another feature that makes bacteria optimal is that they already contain compounds and metabolic pathways that can release or produce drugs. As a result, this helps reduce costs for extra materials by utilizing natural processes. Complete chemical synthesis is very costly so any efforts that can harness the biosynthetic processes are greatly preferred. However, using a purely biosynthetic pathway leads to low yields. This is where synthetic biology techniques come into play where low prices and high yields can become a reality. A case where synthetic processes have been utilized effectively is in the case of the anti-malarial drug Artemisinin. Scientists have successfully managed to engineer E. coli to produce a precursor of Artemisinin by altering some pathways that can purified in a cost-effective manner [3]. Another great bacterium to engineer is Lactobacillus, due to the fact that it is part of pathway of fermenting milk in to yogurt. Using this strategy, in 2008, MIT's iGEM team attempted to manipulate Lactobacillus bulgaricus to produce an antimicrobial peptide that would inhibit the binding of Streptococcus mutans. This would eventually prevent the formation of cavities. Moreover, this method effectively creates a cheaper, sustainable way to keep the therapy functional since small amounts of the bacteria are required to re-ferment milk and keep producing this antimicrobial yogurt for one's teeth [4].

In addition, a large proportion of diseases are bacterial. This allows one to consider the possible interplay between different bacteria. One way in which this interaction has been embraced is in the earlier cholera therapeutic with E. coli. Here we have two bacteria, engineered E. coli Nissle 1917 and Vibrio cholera, of which both have the ability to quorum sense or communicate with other cells. However, the latter is pathogenic and the former is not. As a result, since bacteria only have the ability to communicate with other bacteria that produce the specific CAI-2 it allows the E. coli to trick the Vibrio cholera to believe that the they have already inhabited that part of the gut and prevent the formation of the cholera biofilm [1]. These types of bacterial interactions are an interesting consideration to make when creating therapeutics where other non-pathogenic bacteria can beat pathogenic bacteria at their own game by emulating their behaviour.

Many bacteria are also facultative or obligate anaerobes. This is incredibly relevant for many diseases where hypoxic areas need to be targeted, the most obvious one being cancer. Initial attempts were done with facultative anaerobes (although considered to be an aerobe at the time) such as Listeria monocytogenes because they are noninvasive and are easy to control through antibiotics. However although they did cause some tumors to regress, ultimately they preferred aerobic environments [5]. Then they tried Salmonella which was another facultative anaerobe, but thought to favor anaerobic environments more. Due to this preference they were predicted to accumulate around the necrotic centers of cancerous tumors, and especially after modifications made to amino acid machinery [6]. Nevertheless, it remained flawed due to the fact that it was invasive and the safety and viability of the option in the long-term remained unpredictable. Finally, more recently, an attempt was made with Clostridium, an obligate anaerobe. This was a great candidate since it would only target oxygen-depleted areas of tumors, and thus not be able to cause any systemic disease. In addition, Clostridium are sporulating bacteria which enable them to be metabolically active only in areas of necrotic tissue. As a result, the Clostridium would be inert everywhere else and thus unlikely to cause an immune response [7]. When this was tried out in practice the localization of Clostridium tetani turned out to be highly concentrated in hypoxic areas and therefore made it a suitable target for cancer therapies [8]. Clostridium was eventually tried out years later as a vector and proved to have sustained anti-tumor effects [9].

Bacteria are also advantageous by the fact that they motile due to their flagella. This is especially useful in diseases where the area of infection is large and deep. The bacterial flagellum is useful in these situations because they have the ability to penetrate tissues [10]. Moreover, this also illustrates the point that bacteria will not be entropically limited, unlike passive molecules, and can thus acquire energy to reach areas that are not as easily accessible. This results in allowing the bacterial density to be higher in areas away from the vascular source from which they arrived. Once again this sort of advantage primes bacterial therapies well for areas of tumors that are quite large and deep. Furthermore, the flagella allow bacteria to exhibit chemotaxis which is once again beneficial for cancer therapies. Several bacteria have shown to accumulate preferentially in tumorous regions due to the nutrients provided from dying cells [11].

Additional points can be made to show why bacterial therapeutics are just plain convenient. First of all, bacterial genetics are very well-understood. This makes them easy to manipulate and build circuits in, similar to electrical engineering. In addition, bacteria are responsive to their environment which enables the potential to create "smarter" therapeutics that would enable precise controls even after delivery, and eventually ensure long-term success of the treatment. These controls can be achieved through creating circuits that are responsive to their environment using various promoter strategies. Finally, bacteria are very easily externally detectable through various techniques that include the use of light, magnetic resonance imaging, and positron emission tomography. This will enable scientists to measure the state of the tumor and therefore the success and efficacy of treatment.

However despite all these advantages, bacterial therapies have a low presence in the market, and engineered bacterial therapies are practically non-existent. One reason is that bacterial therapies often have trouble obtaining venture capital since many people still remain uncomfortable with putting genetically-modified organisms in their bodies. As a result, this essentially inhibits the commercialization of these products. But even before that, these therapies encounter additional resistance from the governmental agencies like the FDA and they can't even enter the market to begin with. Just this year, 2012, has Artemisinin, which comes from both genetically engineered bacteria and yeast become FDA-approved and ready to come in to the market soon. What's interesting is that these types of bacterial therapeutics, or at least probiotics widely used in Europe and Asia, but only recently has the market been rising in the United States [12]. Several companies such as Osel and Oragenics are companies that exclusively focus on developing bacterial therapeutics. Osel focuses primarily on harnessing the microbiomes already present in the human body. Although they have several probiotics in clinical trials none of their actual engineered probiotics have been able to go past the pre-clinical stage [13]. Oragenics focuses more on oral delivery of probiotics and has managed to bring a few products into the market.

Thus, by understanding these the various advantages bacterial therapies have, we hope to create two systems: a light-activated bacterial therapeutic and antimicrobial biofilm. However, also being aware that many of these bacterial therapeutic never make it into the clinic we hope to analyze why from a scientific, governmental, and social perspective to better understand the issue. Then based on this analysis offer some solutions that can streamline the process of eventually bringing these engineered bacterial systems into the clinic.

Although bacterial present themselves as ideal vehicles for drug delivery for various types of diseases there are several hurdles that need to be overcome before they become clinically viable. One major issue is the control of drug delivery. It is essential that the drug be delivered at high enough concentrations to sufficiently induce the appropriate therapeutic effects, yet low enough as to not be toxic to the body. [need to find existing solutions] The inherent toxicity of bacteria also presents an additional challenge. Although these engineered bacteria may appear to non-pathogenic in various healthy animal and human trials, their remaining virulence may still be able to affect patients who are immunocompromised, such as those with HIV and cancer. Scientists have attempted to engineer pathogenic strains bacteria such as Salmonella so that they may have decreased virulence. However, these efforts have not been successful in clinical trials [1].The bacteria remained too virulent to be used inside the human body due to their immunogenicity and pathogenicity outweighing any therapeutic effects[1]. As a result, scientists have been experimenting with nonpathogenic bacteria like Escherichia coli Nissle 1917. This strain that has been shown to be completely non-pathogenic not exporting any toxins. Scientists have been trying to engineer their various systems into this strain, however it has only been shown to be successful in situations where they behave commensally in the gut flora [2].

In addition, since bacterial therapeutics can be variable in their targeting efficiency they pose additional challenges. The problem is that cancer is unique in two ways. First, there are many types of cancer, each of which may have several subtypes. Each cancer subtype has a unique profile, meaning that a single bacterial therapeutic will interact differently with what may at first glance seem like the same type of disease. Secondly, each person's body will interact with the drug differently. Due to this uncertainty, the ideal dosage of the bacterial therapeutic varies between patients, making dosage decisions more difficult and less accurate. These ideas are what Moreover, this lack of consistent targeting efficiency will pose a larger problem for diseases that have metastatic potential. Not only won't the drug be able to spread uniformly, in the case of cancer, throughout the tumor, but also it won't be able to target more distal areas easily.

An additional issue is that the engineered bacteria may be genetically unstable, which may create mutations that alter the pathogenicity or growth potential of the cell itself.

Another problem is the method of delivery itself. Oral delivery of bacterial therapeutics forces bacteria to experience the harsh environment of the GI tract which causes low survival rates. This can be alleviated through higher dosages, but that can then cause an immunogenic response.

[1] Duan, F., & March, J. C. (2010). Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model. Proceedings of the National Academy of Sciences of the United States of America, 107(25), 11260–11264. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/20534565

[2] Liu, X., Lagenaur, L. A., Lee, P. P., & Xu, Q. (2008). Engineering of a human vaginal Lactobacillus strain for surface expression of two-domain CD4 molecules. Applied and Environmental Microbiology, 74(15), 4626–4635. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2504410&tool=pmcentrez&rendertype=abstract

[3] Tsuruta, H., Paddon, C. J., Eng, D., Lenihan, J. R., Horning, T., Anthony, L. C., Regentin, R., et al. (2009). High-Level Production of Amorpha-4,11-Diene, a Precursor of the Antimalarial Agent Artemisinin, in Escherichia coli. (A. Gregson, Ed.)PLoS ONE, 4(2), 12. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2637983&tool=pmcentrez&rendertype=abstract

[4] "Biogurt: A Sustainable and Savory Drug Delivery System." Main Page. MIT, n.d. Web. 02 Aug. 2012. .

[5] Pan, Z. K., Ikonomidis, G., Pardoll, D. & Paterson, Y. (1995). Regression of established tumors in mice mediated by the oral administration of a recombinant Listeria monocytogenes vaccine. Cancer Res 55, 4776-4779.

[6] Pawelek, J. M., Low, K. B. & Bermudes, D. (1997). Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 57, 4537-4544.

[7] Van Mellaert, L., Barbe, S. & Anne, J. (2006). Clostridium spores as anti-tumour agents. Trends Microbiol 14, 190-196.

[8] Lambin, P., Theys, J., Landuyt, W., Rijken, P., Van Der Kogel, A., Van Der Schueren, E., Hodgkiss, R., et al. (1998). Colonisation of Clostridium in the body is restricted to hypoxic and necrotic areas of tumours. Anaerobe, 4(4), 183–188. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16887640

[9] Theys, J., Pennington, O., Dubois, L., Anlezark, G., Vaughan, T., Mengesha, A., Landuyt, W., et al. (2006). Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo. British Journal of Cancer, 95(9), 1212–1219. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2360559&tool=pmcentrez&rendertype=abstract

[10] Toley, B. J., & Forbes, N. S. (2012). Motility is critical for effective distribution and accumulation of bacteria in tumor tissue. Integrative biology : quantitative biosciences from nano to macro, 4(2), 165–76. Retrieved from http://pubs.rsc.org/en/content/articlehtml/2012/ib/c2ib00091a

[11] . Kasinskas, R. W. & Forbes, N. S. Salmonella typhimurium specifically chemotax and proliferate in heterogeneous tumor tissue in vitro. Biotechnol. Bioeng. 94, 710–721 (2006).

[12] Starling, Shane. "Global Probiotics Market Approaching $30bn by 2015: Report."NutraIngredients.com. N.p., 15 Sept. 2010. Web. 03 Aug. 2012. .

[13] "Clinical Development." Osel: Harnessing the Microbiome. N.p., n.d. Web. 03 Aug. 2012. .