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 .
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 . 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 .
Even though it is a highly promising strategy, he estimates it would take at least another four years to finish the needed clinical trials. Besides, considering the high cost of antibody-based therapies and the infrastructures necessary to administer them, our team thought about possible alternatives. This way, we propose a low-cost and accessible approach within everyone’s reach based on synthetic biology.
Salvador Aznar Benitah is a group Leader at Institute for Research in Biomedicine at the Barcelona (IRB Barcelona) since September 2013. Leading the research group stem cells and cancer, he has explored the implications of palmitic acid in metastasis development. Institute for Research in Biomedicine at the Barcelona.
“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  and cancer . 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 . 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.
Furthermore, as many experts have noted, even though PA is physiologically essential for obtaining energy, neuronal activity or respiration our body can synthesized it de novo. Therefore, there shouldn’t be any problem in restricting the overload of PA in the diet.
Considering this, our project sheds light on the need of an alternative approach to this absence in cancer patients dietary requirements. Our idea is to target the availability of LCFA directly in the gut with a probiotic that could absorb this harmfuls acids in an efficient way.
Dolors Borau, graduated in Nutritionism and Dietetics and currently working preparing diets either for private or public institutions. CoDiNuCat refers to the school of Dietist and nutritionist of Catalonia, a public corporation who aims to keep and enhance healthcare. Sònia Vallés is the dietist and nutritionist from the school to whom we talked with.
“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).
When considering administration it’s also important to be aware of the location of fatty acid absorption inside the gut. This absorption occurs mainly in the jejunum but also in the ilium (5%) . Knowing this, our probiotic design will need to consider degradable layers to ensure maximal concentration and efficiency of our probiotic in the jejunum.
Bernhard Paetzold is the co-Founder of S-Biomedic, a company focused on developing cosmetics based on naturally enhanced bacteria to address chronic problems with the skin (such as acne).
“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”
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 .
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) . 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 .
We also explored additional mechanisms that could be developed to further enhance biosafety. In this way, we posed and discussed within a theoretical framework the possibility of developing a biocontainment system through different KO. FabA and FabB were proposed as potential targets for developing termosensible strains. These genes are responsible for the synthesis of unsaturated fatty acids (UFAs) essential for maintaining cell membrane fluidity . In this way, if the UFAs synthesis is impaired, bacteria are not able to hold temperature-dependent changes due to instabilities in the membrane. Consequently, KO of either gene leads to UFA auxotrophy , which has also been described as a method for biocontainment.
Considering society concerns and needs remains a key issue for our team. In this way, when participating in many events such as Pint of Science, Week of Science or Youth Mobile Festival, we noticed how society was concerned about genetically modified organisms (GMO's). Therefore, in order to change that and disminishes people worries, we were especially careful and seek advice in experts in order to develope a system as safe as possible.
“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 .
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 . 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) .
Regarding administration, it’s important to consider that cancer patients suffer a lot of digestive problems. Therefore, we consider that the best way to deliver our probiotic could be as a pill or a liquid solution.
We have also contacted Juan M. Vázquez Lago, to see how our probiotic is in concordance with ethic criteria in actual medicine. As Juan explained “considering the different factors that involve the project, our modified probiotic would not be an ethical problem”.
Maria del Carmen Domínguez Ferreiro is a pharmaceutical and biologist, head of section of the colorectalcancer screening program of Galicia.
Ana Marina Tarrazo Antelo is a MD Doctor in breast cancer, working at the galician program for the early detection of breast cancer.
Juan M. Vázquez Lago is a Medical Doctor in preventive medicine and public health. He is also a public Health Technician of the Management of Integrated Management of Santiago as well as the President of the Territorial Research Ethics Committee of Santiago-Lugo, Spain and a member of the Autonomous Committee of Research Ethics of Galicia, Spain.
“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 (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 .
We discussed with cardiovascular research professionals the possibilities of developing our product for cardiovascular approaches. They pointed out the controversial on recent studies about the relationship between CVD prevention and dietary fats. Indeed, some recent meta-analyses of observational studies have shown no association between SFA and CVD risk . However, it must be noted that an important feature still to be considered is the foods and/or nutrient replacements which are associated with diets low in SFA. Usually, these are coupled with higher intake of poliunsaturated fatty acids (PUFAs), which have been also described as harmful for the cardiovascular system . In this way, we propose an improved alternative, as our probiotic would provide a LCFA-restricted diet without the increment of other dietary fats.
Professionals from Catalan Institute of Cardiovascular sciences (ICCC) and Co-founders & CEO at GlyCardial Diagnostics are developing kit to identify a marker protein with which to diagnose infarction in earlier stages.
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 .
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 .
In this way and taking into account all the given information, we hypothesized that if our product efficiently diminished this FFAs source could potentially bring to a beneficial metabolic switch avoiding this decrease in the cardiovascular function. However, as Oriol Iborra and Egea pointed out “in order to successfully work, our product would need to be coupled to an increase in the hiperketonemia, a more efficient energy mean”.
Oriol Iborra and Egea, is currently working at the Fundació Institut Investigació Germans Trias i Pujol (Cardiology Unit) from ICREC research group (cardiac regeneration and Heart Failure). He works in the mechanisms of action of sacubitril/valsartan on cardiac remodeling.
“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
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 . 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 . 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 . 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”
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.
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