Engineered LBP for the Treatment of Metabolic Diseases

With the continuous development of synthetic biology, researchers have used synthetic biology technology to design different gene circuits in various microbial host cells to create strains with specific functions required by humans. Engineered microbial drugs have been reported in clinical trials for various diseases.

Some metabolic diseases, such as phenylketonuria (PKU), are caused by a deficiency in the enzyme required for specific metabolism or defects in the enzyme activity, which blocks normal metabolic pathways in cells and causes toxic substances to accumulate in the cells. Studies have shown that specific enzymes or engineered probiotics containing specific pathways can alleviate metabolic disorders in organisms, as shown in the figure. Engineered bacteria can synthesize the required enzymes for metabolism, leading to the restoration of metabolic pathways. They can inhibit the synthesis of toxic products or metabolize them into non-toxic products, thereby treating such metabolic diseases.

Phenylketonuria (PKU) is a rare autosomal recessive genetic disease caused by a deficiency in phenylalanine hydroxylase, which is essential for converting the essential amino acid phenylalanine (Phe) into tyrosine. An alternate enzyme, phenylalanine ammonia lyase (PAL), which is present in microorganisms or plants, can potentially be used for PKU treatment. Engineered bacteria LBPs, ingested orally, can target and convert Phe produced by food metabolism in the intestines, reduce its entry into the bloodstream and alleviate the condition. Researchers inserted genes encoding PAL and l-amino acid deaminase into the genome of Escherichia coli Nissle 1917 to create an engineered bacterium named SYNB1618. This bacterium was engineered using two Phe transporter genes (pheP) and three PAL genes (stlA) integrated into the chromosome and regulated by the anaerobic inducible promoter P_fnrs. To reduce the impact of anaerobic processes on bacterial density, the P_tac promoter was used to control two additional stlA genes. In animal experiments, SYNB1618 significantly reduced plasma Phe levels in Pah^enu2/enu2 mice and prevented a surge in Phe levels in the blood plasma of healthy non-human primates (NHPs) after ingestion. The results of phase Ⅰ/Ⅱa clinical trials showed that SYNB1618 was safe and well-tolerated, and all participants cleared the bacteria within 4 days after the last injection. Moreover, SYNB1618 could consume Phe and convert it into non-toxic metabolites in the gastrointestinal tract of healthy volunteers and PKU patients. SYNB1934, an improved strain evolved from SYNB1618, had a stronger ability to degrade Phe. The results of phase Ⅰ clinical trials showed that SYNB1934 can dose-dependently increase the level of the degradation product, trans-cinnamic acid, in plasma. According to measurements of Phe degradation biomarkers, the activity of SYNB1934 was twice that of SYNB1618.

Hyperammonemia is a metabolic disorder characterized by abnormally high ammonia levels in the blood due to a lack of enzymes and transporters involved in the urea cycle, which metabolize free ammonia into urea. The existing treatments include lactulose and antibiotics, but they are ineffective and have many side effects. Researchers found that oral administration of a probiotic Lactobacillus helveticus NS8 strain can alleviate cognitive decline and anxiety behavior in rats with hyperammonemia, indicating that reducing ammonia levels in the gut using gut microbiota may be an effective treatment for hyperammonemia. In addition, an engineered bacterium SYNB1020, constructed from ECN as the host organism, can effectively convert ammonia into l-arginine. Genes encoding the negative regulatory factor ThyA and ArgR were deleted in this strain to activate the transcription of several genes involved in arginine biosynthesis and transport, and the gene encoding the feedback-resistant N-acetylglutamate synthase ArgA215 was integrated into the genome to increase arginine synthesis. The use of SYNB1020 in the spf^ash model of hyperammonemia in mice could reduce blood ammonia levels and increase survival rates to 50%. Unfortunately, the effect of reducing blood ammonia levels in clinical trials of phase Ⅰb/Ⅱa was not ideal, mainly because the indication chosen by the researchers was acute hyperammonemia, and engineered bacteria LBPs could not exert their effects quickly enough within 0.5 hours to achieve the expected results. However, the therapeutic mechanism of this engineered bacterium is rational. This also gives LBP developers a hint that selecting appropriate indications is crucial in clinical trials of drugs.

Diabetes is a group of metabolic diseases characterized by high blood sugar levels due to defects in pancreatic insulin secretion or impaired biological effects of insulin, leading to various complications such as cardiovascular disease, Alzheimer's disease, stroke, and nerve damage. It is divided into type 1 diabetes (T1D) and type 2 diabetes (T2D). Currently, insulin and anti-diabetic drugs as well as cytokine-based therapies are the main treatments for diabetes. Compared with these treatments, engineered bacterial drugs have fewer side effects and can relieve pain caused by injections. Scientists have designed a human gut microbiome strain, Lactococcus lactis, to treat T1D. The modified Lactococcus lactis can secrete complete insulin precursor and biologically active immunoregulatory cytokine interleukin-10 (IL-10). Researchers treated non-obese diabetic (NOD) mice with this recombinant Lactococcus lactis combined with low-dose non-specific immunoregulatory agent anti-CD3. The results showed that 59% of experimentally treated NOD mice had normal blood sugar levels compared with the control group. In addition, researchers designed a recombinant strain Lactobacillus gasseri that secretes GLP-1-(1-37), which can lower blood sugar levels. Although engineered bacteria have made significant progress in treating diabetes in animal models, there have been few clinical trial reports so far.

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For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.