There are many nations today facing an aging population. Old age brings about numerous ailments, leading to a decline in the quality of life for those affected. Our project aims to prevent the occurrence of a specific illness, gout, by utilizing a biologically engineered medicine in the form of an easy-to-use bottle of liquid medication that is suitable for all age groups. Gout is a metabolic disease characterized by acute arthritis attacks and chronic arthritis symptoms. In addition to arthritis attacks, gout may be accompanied by other symptoms such as fatigue, skin redness, and the formation of nodules[ T. Neogi, Clinical practice. Gout. N Engl J Med 364, 443-452 (2011).]. Gout symptoms usually appear at night or early in the morning. Patients may experience severe joint pain and swelling. These symptoms usually affect the big toe, but can also affect other joints, such as knees, wrists, fingers and elbows. Severe gout can cause patients to lose mobility, while complications can lead to permanent impairment and disability.
Gout is primarily caused by high levels of uric acid in the body, namely by the condition known as hyperuricemia[ Y. Zhu, B. J. Pandya, H. K. Choi, Prevalence of gout and hyperuricemia in the US general population: the National Health and Nutrition Examination Survey 2007-2008. Arthritis Rheum 63, 3136-3141 (2011).]. Uric acid is a waste product produced by the metabolism of purines in food. Most of the uric acid is excreted through the kidneys. However, if the body cannot effectively dispose of uric acid, uric acid levels will rise, forming urate crystals which are deposited in the joints and the surrounding soft tissues, causing pain and an inflammatory response, which then leads to joint pain and swelling. According to research reports, in areas with higher living standards, about 15% of the population have hyperuricemia, and they are all at risk of gout[ G. Singh, B. Lingala, A. Mithal, Gout and hyperuricaemia in the USA: prevalence and trends. Rheumatology (Oxford) 58, 2177-2180 (2019).]. There are two main reasons for increased uric acid levels: First, dietary factors. Intake of purine-rich foods (such as offal, seafood, red meat, etc.) or high-sugar diets will increase the production of purines in the body, which in turn increases the production of uric acid. ; The second is genetic factors. Gout is often related to family inheritance. Some gene mutations can affect the metabolism and excretion of uric acid, causing uric acid to accumulate in the body[ T. J. Major, N. Dalbeth, E. A. Stahl, T. R. Merriman, An update on the genetics of hyperuricaemia and gout. Nat Rev Rheumatol 14, 341-353 (2018).],[ Z. W. Zhou et al., Polymorphisms in GCKR, SLC17A1 and SLC22A12 were associated with phenotype gout in Han Chinese males: a case-control study. BMC Med Genet 16, 66 (2015).]. In addition, some diseases and drugs may also cause gout, such as kidney disease, metabolic disease, hypertension, diabetes, cardiovascular disease, etc. The use of certain medications, such as diuretics and aspirin, can also increase the risk of gout.
The goals of gout treatment are to relieve pain, prevent joint damage, control uric acid levels, and prevent recurrence. At present, there are mainly four types of methods used for the treatment of gout: First, drug treatment, including non-steroidal anti-inflammatory drugs, steroids, indomethacin and other analgesics, as well as diuretics, uric acid synthesis inhibitors and uric acid excretion promoters and other drugs; the second is lifestyle intervention, including weight control, diet adjustment, and increasing exercise. In particular, gout patients should control their intake of meat, alcohol, and high-purine foods, and increase their intake of low-fat dairy products, fruits, and vegetables. The third option is non-drug treatment, including applying ice to affected areas, elevating the affected limb, resting and protecting joints. The fourth choice is surgical treatment, such as joint puncture , fluid extraction and surgical removal of urate deposits in the joints. Although there are many options for treating gout, there are still some noticeable drawbacks. For example, many medications have side effects. NSAIDs can cause gastrointestinal bleeding and kidney problems, while diuretics can cause electrolyte imbalances and dehydration. Long-term use of certain drugs may lead to drug tolerance and increase the risk of adverse reactions. For some people, changing eating habits can be difficult and difficult for patients to adhere to. In addition, surgery itself has risks, such as infection and post-operative pain.
Currently, research on gout is very active worldwide, involving etiology, prevention, treatment, and complications. Etiological research mainly focuses on uric acid metabolism and inflammation. Studies have found that the onset of gout is related to many factors such as heredity, environment, and lifestyle. Research on prevention shows that controlling diet and lifestyle habits, reducing body weight, and limiting alcohol intake can reduce the risk of disease. In addition, preventive drug research for high-risk groups is also underway. Studies have shown that hyperuricemia is closely related to the incidence of gout, and reducing uric acid levels can prevent and treat gout[ T. Bardin, P. Richette, Impact of comorbidities on gout and hyperuricaemia: an update on prevalence and treatment options. BMC Med 7 T. J. Major, N. Dalbeth, E. A. Stahl, T. R. Merriman, An update on the genetics of hyperuricaemia and gout. Nat Rev Rheumatol 14, 341-353 (2018). 8Rebai Y,Wagner L,Gnaien M, et al. Escherichia coli Nissle 1917 Antagonizes Candida albicans Growth and Protects Intestinal Cells from C. albicans -Mediated Damage[J]. Microorganisms,2023,11(8). 9Kleta, S., et al. (2016). “Clinical practice: the probiotic Escherichia coli strain Nissle 1917 (EcN)-from bench to bedside.” European journal of pediatrics, 175(2), 151-162. 10Henker, J., et al. (2007). “Multicenter, double-blind, placebo-controlled trial comparing 2 different formulations of heat-killed Escherichia coli in patients with ulcerative colitis.” American Journal of Gastroenterology, 102(10), 2236-2249. 11Sonnenborn, U. (2016). “Escherichia coli strain Nissle 1917–a valuable resource in basic and applied science and therapeutics.” Microbial ecology in health and disease, 27(sup1), 29758. 12Tursi, A., et al. (2007). “Effects of mesalazine, antibiotics and probiotics on pro-inflammatory cytokines, nitrites and myeloperoxidase in ulcerative colitis.” World Journal of Gastroenterology, 13(23), 3168-3172. ]. Research on treatment mainly focuses on drug treatment and non-drug treatment. Medication treatments include uric acid-lowering drugs, nonsteroidal anti-inflammatory drugs, and steroids. Non-drug treatments include changes in diet and lifestyle. Research on complications focuses more on the relationship between gout and its complications, and how to reduce the risk of complications. Overall, current research on gout is very extensive and active worldwide.
Nissle 1917 is a specific strain of Escherichia coli (E. coli) bacteria that has been extensively researched for its beneficial effects on the gastrointestinal system9. It was first isolated in 1917 by Dr. Alfred Nissle and has since been used as a probiotic to promote gut health10. Nissle 1917 has been shown to have antimicrobial properties, help restore the balance of gut flora, and support the immune system11. It is considered safe for human consumption and has been used in various medical and research applications12.
Though developments for better treatment are already underway, people still suffer from gout. When we learned about the suffering that is inflicted upon individuals by this disease on such a large scale, we made it our goal to develop treatments for it.
The aim is to reduce the concentration of uric acid without causing significant negative side effects to the patient. Our product utilizes a natural method of treatment by targeting the metabolism process of uric acid in the human body. Studies have shown that some animals have the ability to use uric acid oxidase to convert uric acid into allantoin, helping to reduce the accumulation of uric acid in their bodies (Figure 1). Therefore, this study aims to express enzymes such as uric acid oxidase using probiotics to degrade excessive uric acid in the body, thereby reducing the risk of developing gout, utilizing three enzymes: uric acid oxidase, allantoinase, and allantoicase. The feasibility of simultaneously transferring three key enzymes into Nissle 1917 for expression. This approach helps to remove uric acid and prevent the formation of crystals, addressing the root cause of gout7.
To verify the feasibility of this idea, we first constructed the plasmids pET28a-UAO (Uric Acid Oxidase), pET28a-allantoinase, and pET28a-allantoicase. Subsequently, we switched to a different vector in order to transfer them into Nissle 1917. Our experiments produced two end product plasmids that can be inserted into bacteria: pGEX-4T-1-UAO and pGEX-4T-1-UAA. pGEX-4T-1-UAO produces uric acid oxidase while pGEX-4T-1-UAA produces all 3 enzymes. We theorized that we would expect the product to produce UAO, as that would already eliminate the uric acid. The UAA was an experimental attempt as a test to see if the results would be better if all 3 enzymes were produced together (Figure 2).
Figure 2. The outline of subject construction
Our goal is to alleviate the lack of healthy treatments for gout, which is an especially common disease as humanity today continues to improve living standards that could lead to uric acid increases. The world only gives us so much time to be alive, and we wish that more people could spend that time without having to suffer from pain again and again. Though there are still many diseases that continue to plague mankind, we still wish to fight against them, one treatment at a time. The final product that we wish to develop would ideally be easy to use, available to the masses at a reasonable and affordable price, and safe.
T. Neogi, Clinical practice. Gout. N Engl J Med 364, 443-452 (2011).
Y. Zhu, B. J. Pandya, H. K. Choi, Prevalence of gout and hyperuricemia in the US general population: the National Health and Nutrition Examination Survey 2007-2008. Arthritis Rheum 63, 3136-3141 (2011).
G. Singh, B. Lingala, A. Mithal, Gout and hyperuricaemia in the USA: prevalence and trends. Rheumatology (Oxford) 58, 2177-2180 (2019).
T. J. Major, N. Dalbeth, E. A. Stahl, T. R. Merriman, An update on the genetics of hyperuricaemia and gout. Nat Rev Rheumatol 14, 341-353 (2018).
Z. W. Zhou et al., Polymorphisms in GCKR, SLC17A1 and SLC22A12 were associated with phenotype gout in Han Chinese males: a case-control study. BMC Med Genet 16, 66 (2015).
T. Bardin, P. Richette, Impact of comorbidities on gout and hyperuricaemia: an update on prevalence and treatment options. BMC Med 7 T. J. Major, N. Dalbeth, E. A. Stahl, T. R. Merriman, An update on the genetics of hyperuricaemia and gout. Nat Rev Rheumatol 14, 341-353 (2018).
8Rebai Y,Wagner L,Gnaien M, et al. Escherichia coli Nissle 1917 Antagonizes Candida albicans Growth and Protects Intestinal Cells from C. albicans -Mediated Damage[J]. Microorganisms,2023,11(8).
9Kleta, S., et al. (2016). “Clinical practice: the probiotic Escherichia coli strain Nissle 1917 (EcN)-from bench to bedside.” European journal of pediatrics, 175(2), 151-162.
10Henker, J., et al. (2007). “Multicenter, double-blind, placebo-controlled trial comparing 2 different formulations of heat-killed Escherichia coli in patients with ulcerative colitis.” American Journal of Gastroenterology, 102(10), 2236-2249.
11Sonnenborn, U. (2016). “Escherichia coli strain Nissle 1917–a valuable resource in basic and applied science and therapeutics.” Microbial ecology in health and disease, 27(sup1), 29758.
12Tursi, A., et al. (2007). “Effects of mesalazine, antibiotics and probiotics on pro-inflammatory cytokines, nitrites and myeloperoxidase in ulcerative colitis.” World Journal of Gastroenterology, 13(23), 3168-3172.