Difference between revisions of "Team:UPF CRG Barcelona/Human Practices"

HUMAN PRACTICES

We believe that bidirectional interaction between society and scientists is crucial for the development of a successful project. In this way, our team has strongly considered expert advice as well as general public opinion, which had directly influenced the direction of our project.

In this part, we have explored the existing market and alternative therapies, enriching our knowledge with advices both from clinical and research areas. We integrated the information given adapting the design of our future probiotic and made it suitable for the actual sanitary system. Even though in this project we are only presenting a proof of concept, we wanted to address all the possible issues when developing a probiotic that is therapy ready and respond to current society needs.

Exploring existing therapies

We first discussed our project with Dr. Salvador Aznar-Benitah, as he has explored the implications of palmitic acid intake in metastasis development. Recently, his team found that overexpression of CD36 allows cells to take up lipids from their environment, positively correlating it with metastatic potential. In fact, the association of high CD36 expression has been associated with poor prognostic in bladder, lung, and breast cancer among others [1].

Benitah’s lab is currently working on CD36 antibodies for therapeutic purposes. Antibodies that block CD36 and therefore its interaction with fatty acids, decrease the ability of cancer cells to metastasize [2]. In preclinical studies with mice, they observed that the administration of CD36-blocking antibodies reduced the number of metastatic tumors and their sizes by 80%. Its administration had also effects even in cases were metastasis had already been established, with a total removal of the metastases in 20% of the animals [2].

“Considering the high cost of antibody-based therapies and the infrastructures necessary to administer them, we propose a low-cost and universally accessible approach”

Solving dietetic needs

The relationship between long chain fatty acid (LCFA) intake and disease has been well described. It has been proved that a diet rich in LCFA can increase the chances of developing a wide range of metabolic diseases such as diabetes or obesity [3] and cancer [4]. Interestingly, and considering recent evidence already mentioned, it has been described that increasing PA consumption might increase the probabilities of developing metastasis.

Dietary restriction of LCFA intake may seem the best approach to this issue. However, as several professionals from the Catalan College of Nutritionists (CoDiNuCat: www.codinucat.cat) pointed out, PA-restriction from diet is not easy. In fact, LCFA are one of the main components of our food intake. In Western diets dietary lipids account for 42% of total ingested calories which makes it infeasible to follow a PA-restricted diet [5]. Information given from Dolors Borau confirmed the lack of an standardized and suitable low LCFA diet for cancer patients and brought up the lack of a proper follow-up when applying this diets. She believes that this is attributed to i) the difficulty to eliminate dietary fats, which are widely used in the elaboration of multiple products offered by the food industry and ii) the low adherence that cancer patients might have in such strict diets.

“Up to date, no standardized and suitable low LCFA diet for cancer patients exists. Low-fat and hypocaloric diets exist but normally lack a proper professional follow-up”

Making our probiotic ready for therapeutics

In the course of our project, we tried to have a system as ready to work in its real environment as possible. In this context, Bernhard Paetzold suggested us to explore and analyse the environment in which our genetically modified bacteria would work. In fact, in the intestine oxygen concentrations are very low, close to anoxia. For this reason, in order to work properly, our product would need to work under anaerobic conditions. However, LCFA are degraded via the beta-oxidation pathway, an aerobic pathway for excellence. In order to tackle this problem, we used flux balance analysis (FBA) to mathematically analyze the flow of metabolites through a metabolic network and predict the cellular growth rate under different environmental conditions or genetic perturbations. This would allow us to to find new strategies to engineer E. coli for achieving a bacterial system capable of efficiently uptaking LCFA in human gut-like conditions (see more).

“Considering the environment in which our probiotic would have to work lead us to explore through flux balance analysis the cellular growth under different environment and genetic perturbations”

Safety concerns

The increasing use of engineered organisms exposes a growing risk of spreading hazardous biological entities into the environment. This leads to a concern in the society, as the general public has pointed out during many of the public engagement activities we have developed (see more go to Education and Public engagement). Therefore, we decided to include key safeguard requirements in our project to prevent the release and proliferation of GMOs in natural ecosystems. In this way, extremely robust biocontainment systems must be established in engineered organisms, especially those within a therapeutic context [9].

After having read and consult with experts in synthetic biology we considered the genomic integration of our design using Multiplex Automated Genome Engineering (MAGE) (go to integration) [10]. This would help us to have stable and robust expression of our system, consequently preventing horizontal gene transfer and reducing the biosafety concerns that could arise from plasmid exchange [11].

“Genomic integration represents a robust biocontainment system, as it prevents horizontal gene transfer”

Integrating our product in a clinical framework

In order to obtain a product suitable for the existing market and sanitary system we developed a theoretical framework in which all the target public needs were included. In this way, information regarding cancer patients and cancer therapy was collected and integrated in our probiotic design.

Maria del Carmen Domínguez Ferreiro and Ana Marina Tarrazo Antelo pointed out that a system to prevent metastasis should be used already from the moment in which an infiltrating neoplasm is diagnosed, of any degree of invasion. However, it would even be from more benefit to those patients in which lymph node involvement had been observed. Thus, our target population will consist on stage 3 and 4 cancer patients, as these two groups are the ones with a higher risk to develop metastasis [6].

Cancer patients in the mentioned stages usually receive chemotherapy by cycles, which means there is rest periods between chemotherapy treatments. Our probiotic would be ingested by the patient in these rest periods, as the probability of developing metastasis is higher. Thus, in the lack of treatment, survival probability of metastatic cells in blood increases [7]. Secondly, it’s important to consider that the chemotherapy damages the microbiome.This could also affect our probiotic, and could therefore produce unbalances in the gut ecosystem (dysbiosis) [7].

“A system able to prevent the distant spread of cancer should be used already from the moment in which an infiltrating neoplasm is diagnosed, of any degree of invasion” - Ana Marina Tarrazo Antelo

Exploring other therapeutic approaches

During the development of our product, we discussed other applications and its ethical considerations. LCFA are involved in many pathologies, among them some of the most common in developed countries. Thus, when trying to develop a system which absorbs LCFA efficiently, some questions about its applicability in other diseases arose.

Cardiovascular disease:

Cardiovascular disease (CVD) is the leading cause of mortality in the Western world. It contains a broad range of pathologies with varied etiologies. LCFA have been described to contribute to the pathogenesis of CVD in many different ways: (1) increase of low-density lipoprotein (LDL) cholesterol, a strong risk factor for CVD (2) atherosclerosis through accumulation of lipids (both saturated and unsaturated fatty acids) and cholesterol in the subendothelial space (3) increase in insulin resistance due to impaired insulin secretion, resulting in hyperinsulinemia and higher glucose levels in blood (4) increase in proinflammatory molecules, such as endotoxines and cytokines [14].

Diabetes:

We also explored its applications in diabetes, as it is one of the most common pathologies in developed countries. In the setting of diabetes, a myocardial use of free fatty acids (FFAs) as fuel at the expense of glucose occurs due to peripheral insulin resistance and the inability of insulin to suppress lipolysis. As a result, an increased energy cost coupled with lipotoxicity and oxidative stress leads to a decrease in cardiac efficiency [17].

Conversely, ketones are the most energetically efficient fuel for the myocardium. Under conditions of mild, persistent hyperketonemia, a shift in fuel metabolism towards a more energy efficient fuel (such as ketone bodies) has been hypothesised to be helpful. This shift improves oxygen consumption at the mitochondrial level, which could improve myocardial work efficiency, enhance myocardial energetics, reduce oxidative stress, improve cardiac systolic function, and ameliorate heart failure [18].

“If your product efficiently diminished this free fatty acids source, could potentially bring to a beneficial metabolic switch avoiding a decrease in the cardiovascular function of diabetes” - Oriol Iborra

Obesity:

Current scientific evidence indicates that dietary fat plays a role in weight loss. Meta-analyses of intervention trials find that fat-reduced diets cause a 3-4 kg larger weight loss than normal-fat diets [19]. However, in some cases dietary fat restrictions can be an issue. Thus, our product could be approached from a merely dietetic point of view. If it would be the case, our probiotic could be administered in situations in which LCFA-restricted diets had low adherency and the patient’s health was strongly impaired. This point raised a lot of ethical considerations, as illicit use of the probiotic for not therapeutic purposes could happen.

Chronic intestinal inflammation pathologies

Lipids, beyond their energetic role have also been considered important components of cellular signal transduction pathways. Their roles in modulating host inflammatory responses are of clinical interest as agents of both promotion and resolution [20]. In this way, dietary long chain fatty acids are also implicated in the pathogenesis of chronic intestinal inflammation, such as inflammatory bowel disease (IBD) or Crohn’s disease.

The conversion of dietary lipids to proinflammatory molecules is well defined. Higher intake of long chain fatty acids such as PA, has been proposed to increase the amount of eicosanoids in the intestine, leading to a growing proportion of potentially inflammatory molecules and thus to an increase the risk of developing IBD [21]. Doctors and experts in the field believe that the contribution of SFAs is minor compared to one of the polyunsaturated fatty acids (PUFAs) which can be metabolized into the proinflammatory prostaglandins, thromboxanes, and leukotrienes. In this way, our product could serve as an efficient potential therapy for this type of pathologies only when coupled to a decrease of PUFAs. Thus, further selectivity issues of our system should be explored.

“Decrease of long chain dietary fats could diminish the amount of potentially inflammatory molecules in the intestine and thus the risk of developing intestinal inflammation pathologies”

Product development:

In order to properly develop our product, we need to make it suitable for the current sanitary system and ready to be introduced to the market. Thus, we explored the requirements and the needs that our probiotic would need to comply to reach that milestone.

Carlota Bernal, a pharmaceutical with experience in the hospital pharmacy, pointed out that as our product is a GMO for therapeutic purposes, we would have to apply to the EMA (European Medicines Agency) to get the commercialization authorization (according to directive 2001/83/CE that establishes a community code on medicines for human use and regulation 1234/2008 among others). However, in case our product was launched for dietetic purposes (as a dietary supplement) it would need fewer controls and easier to commercialize.

References

[1] Enciu, A. M., Radu, E., Popescu, I. D., Hinescu, M. E., & Ceafalan, L. C. (2018). Targeting CD36 as Biomarker for Metastasis Prognostic: How Far from Translation into Clinical Practice?. BioMed research international, 2018.

[2] Pascual, Gloria, et al. "Targeting metastasis-initiating cells through the fatty acid receptor CD36." Nature 541.7635 (2017): 41.

[3] Cani, Patrice D., et al. "Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice." Diabetes (2008).

[4] Nkondjock, A., et al. "Specific fatty acids and human colorectal cancer: an overview." Cancer detection and prevention 27.1 (2003): 55-66.

[5] Aranceta, J., & Pérez-Rodrigo, C. (2012). Recommended dietary reference intakes, nutritional goals and dietary guidelines for fat and fatty acids: a systematic review. British Journal of Nutrition, 107(S2), S8-S22

[6]Metastasis Research. (2018). Patients Perspective. [online] Available at: https://www.metastasis-research.org/patients-perspective [Accessed 16 Oct. 2018].

[7] Montassier, E., Gastinne, T., Vangay, P., Al‐Ghalith, G. A., Bruley des Varannes, S., Massart, S., ... & Knights, D. (2015). Chemotherapy‐driven dysbiosis in the intestinal microbiome. Alimentary pharmacology & therapeutics, 42(5), 515-528.

[8] Iqbal, J., & Hussain, M. M. (2009). Intestinal lipid absorption. American Journal of Physiology-Endocrinology and Metabolism, 296(6), E1183-E1194.

[9] Lee, J. W., Chan, C. T., Slomovic, S., & Collins, J. J. (2018). Next-generation biocontainment systems for engineered organisms. Nature chemical biology, 1.

[10] Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., & Church, G. M. (2009). Programming cells by multiplex genome engineering and accelerated evolution. Nature, 460(7257), 894.

[11] Torres, L., Krüger, A., Csibra, E., Gianni, E., & Pinheiro, V. B. (2016). Synthetic biology approaches to biological containment: pre-emptively tackling potential risks. Essays in biochemistry, 60(4), 393-410.

[12] Feng Y, Cronan JE (2009) Escherichia coli unsaturated fatty acid synthesis: complex transcription of the fabA gene and in vivo identification of the essential reaction catalyzed by FabB. J Biol Chem 284: 29526–29535.

[13] Mansilla, M. C., Cybulski, L. E., Albanesi, D., & de Mendoza, D. (2004). Control of membrane lipid fluidity by molecular thermosensors. Journal of bacteriology, 186(20), 6681-6688.

[14]Briggs, M. A., Petersen, K. S., & Kris-Etherton, P. M. (2017, June). Saturated fatty acids and cardiovascular disease: replacements for saturated fat to reduce cardiovascular risk. In Healthcare (Vol. 5, No. 2, p. 29). Multidisciplinary Digital Publishing Institute.

[15] Clifton, P. M., & Keogh, J. B. (2017). A systematic review of the effect of dietary saturated and polyunsaturated fat on heart disease. Nutrition, Metabolism and Cardiovascular Diseases, 27(12), 1060-1080.

[16] Wang, D. D., & Hu, F. B. (2017). Dietary fat and risk of cardiovascular disease: recent controversies and advances. Annual review of nutrition, 37, 423-446.

[17] Jaswal, J. S., Keung, W., Wang, W., Ussher, J. R., & Lopaschuk, G. D. (2011). Targeting fatty acid and carbohydrate oxidation—a novel therapeutic intervention in the ischemic and failing heart. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1813(7), 1333-1350.

[18] Karpe, F., Dickmann, J. R., & Frayn, K. N. (2011). Fatty acids, obesity, and insulin resistance: time for a reevaluation. Diabetes, 60(10), 2441-2449.

[19] Astrup, A., Ryan, L., Grunwald, G. K., Storgaard, M., Saris, W., Melanson, E., & Hill, J. O. (2000). The role of dietary fat in body fatness: evidence from a preliminary meta-analysis of ad libitum low-fat dietary intervention studies. British Journal of Nutrition, 83(S1), S25-S32.

[20] Shores, D. R., Binion, D. G., Freeman, B. A., & Baker, P. R. (2010). New insights into the role of fatty acids in the pathogenesis and resolution of inflammatory bowel disease. Inflammatory bowel diseases, 17(10), 2192-2204.

[21] Belluzzi, A., Boschi, S., Brignola, C., Munarini, A., Cariani, G., & Miglio, F. (2000). Polyunsaturated fatty acids and inflammatory bowel disease. The American journal of clinical nutrition, 71(1), 339s-342s.