GB2545395A

GB2545395A - Engineered lactococcus lactis producing biologically active GLP-1

Info

Publication number: GB2545395A
Application number: GB1520963.8A
Authority: GB
Inventors: Narbad Arjan; Wegmann Udo; Backhed Fredrik
Original assignee: Institute of Food Research Ltd
Current assignee: Quadram Institute Bioscience
Priority date: 2015-11-27
Filing date: 2015-11-27
Publication date: 2017-06-21
Also published as: GB201520963D0

Abstract

The present invention relates to a recombinant L. lactis strain expressing GLP-1. The present invention further relates to the use of said recombinant L. lactis strain expressing GPL-1 in medicine, in particular in the treatment of diabetes. The present invention further relates to said recombinant L. lactis strain expressing GPL-1 wherein said recombinant L. lactis strain releases GLP-1 in the gut over a period of several days. The present invention further relates to compositions comprising said recombinant L. lactis strain expressing GPL-1 wherein the said composition is encapsulated to be predominantly released in the gut and to increase its shelf life. A food composition comprising a recombinant Lactococcus lactis strain is also claimed. The present invention further discloses a method for the preparation of said recombinant L. lactis strain expressing GLP-1 utilising the GLP-1 expression plasmid vector pUK200_glp1.

Description

Engineered Lactococcus lactis producing biologically active GLP-1

TECHNICAL FIELD OF THE INVENTION

The disclosure herein relates to a recombinant L. lactis strain expressing glucagon like peptide 1 (GLP-1). The present invention further relates to the use of recombinant L. lactis strains in medicine, in particular in the treatment of diabetes. The present invention further relates to said recombinant L. lactis strain wherein the recombinant L. lactis strain releases GLP-1 in the gut over a period of several days. The present invention further relates to compositions comprising the invention. The present invention further discloses a method for producing the recombinant L. lactis strain expressing GLP-1.

BACKGROUND OF THE INVENTION

Glucagon like peptide 1 (GLP-1) is a 30 amino acid peptide hormone produced by enteroendocrine L-cells. GLP-1 modulates a wide range of physiological processes in the host, e.g., it stimulates post-prandial insulin release, reduces appetite and slows down gastric emptying. GLP-1 offers therapeutic advantages over traditional anti-diabetic drugs such as metformin, e. g., weight loss and reduced risk of hypoglycemia. GLP-1 has a short half-life (~2 min) and is rapidly degraded by dipeptidyl peptidase 4 (DPP4). There are two classes of GLP-1 based drugs: GLP-1 mimetics and DPP4 inhibitors. Whereas GLP-1 mimetics are only available as injections, DPP4 inhibitors are available as oral dosage forms. Both of these drugs may have adverse side effects such as the risk of acute pancreatitis and thyroid carcinoma. Thus, there is a need for new oral drugs that could increase systemic levels of active GLP-1.

There is a recent interest in the genetic modification of food-grade bacteria to express eukaryotic peptides. Lactococcus lactis (thereafter referred to as L. lactis) is a homofermentative Gram-positive bacterium used as a starter culture in a wide variety of fermented food products. Its small genome size, the availability of tightly regulated promoter systems and sophisticated genetic tools as well as its mild proteolytic character and GRAS (generally regarded as safe) status make it the ideal host for the production, secretion and delivery of therapeutic eukaryotic proteins. Previously, it has been demonstrated that recombinant L. lactis producing interleukin 10 (IL-10) was protective against colitis in experimental colitis and IllO-l- mouse models. The recombinant strain also showed therapeutic efficacy when administered orally in the form of a capsule to Crohn’s disease patients and is now being tested in a large scale phase 2a clinical trial [Braat H, Rottiers P, Hommes DW, Huyghebaert N, Remaut E, et al. (2006) A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin Gastroenterol Hepatol 4: 754-759], Another study showed that the constitutive secretion of GLP-1 from recombinant Lactobacillus gasseri cells reprogrammed the differentiation of enterocytes into insulin producing cells thus improving glucose tolerance in a streptozotocin induced type 1 diabetes model [Duan FF, Liu JFt, March JC (2015) Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes 64: 1794-1803],

The present invention thus relates to a new recombinant L. lactis strain expressing GLP-1 for use in medicine, in particular for the treatment of diabetes. Additionally, as GLP-1 has a short half-life (~2 min), the ability of the recombinant L. lactis strain to persist in the gastrointestinal tract (GI tract) for several days enables a steady release of GLP-1 in the gut.

SUMMARY OF TUI INVENTION

The present invention relates to a recombinant L. lactis strain expressing GLP-1.

The present invention further relates to the use of said recombinant L. lactis strain expressing GPL-1 in medicine, in particular in the treatment of diabetes.

The present invention further relates to said recombinant L. lactis strain expressing GPL-1 wherein said recombinant L. lactis strain releases GLP-1 in the gut over a period of several days.

The present invention further relates to compositions comprising said recombinant L. lactis strain expressing GPL-1.

The present invention further discloses a method for the preparation of said recombinant L. lactis strain expressing GPL-1.

DESCRIPTION OF THE FIGURES

Figure 1: A) glpl-expressing vector B) Insulin release from pancreatic islets of wild type (WT) and GLP-1 receptor knock out (GLP1R-KO) mice treated with Ex4 (exendin4, positive control), culture supernatant from recombinant uk200 and glpl L. lactis strains. Data is expressed as percent of blank (culture media). C) Copies of total recombinant L. lactis strains (uk200 + glpl) and D) recombinant glpl L. lactis strain in cecal contents of WT and GLP1R-KO mice supplemented with recombinant uk200 and glpl L. lactis strains. E) GLP-1 levels in portal vein blood and F) vena cava blood from WT and GLP1R-KO mice supplemented with recombinant uk200 and glpl L. lactis strains. * p<0.05, *** p<0.001

Figure 2:

Intraperitoneal glucose tolerance with average area under the curve (in inset) of A) wild type (WT) and B) GLP-1 receptor knock out (GLP1R-KO) mice supplemented with recombinant uk200 and glpl L. lactis strains. Insulin levels from C) WT D) GLP1R-KO mice supplemented with recombinant uk200 and glpl L. lactis strains. * p<0.05, * p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus relates to recombinant L. lactis strain expressing GLP-1.

The inventors observed that a recombinant L. lactis strain producing active GLP-1 (strain FI10937) stimulates a significant insulin release (p<0.001) from primary islets isolated from WT mice compared with uk200 L. lactis control strain (FI10936) (Figure IB). The effect was absent in islets isolated from GLP1R-KO mice confirming specificity. Insulin release by recombinant GLP-1 has been shown before in HIT-T15 cells [Agarwal P, Khatri P, BillackB, Low WK, Shao J (2014) Oral delivery of glucagon like peptide-1 by a recombinant Lactococcus lactis. Pharm Res 31: 3404-3414],

To further investigate the in vivo functionality of these strains, WT and GLP1R-KO mice were administered with uk200 (FI10936) and glpl L. lactis (FI10937) strains each for 7 days. By performing qPCR it was confirmed that the abundance of total recombinant L. lactis was similar in mice colonised with either the vector control or the GLP-1 expressing strain independent of mouse genotype (Figure 1C). Furthermore, the glpl L. lactis strain (FI 10937) colonized WT and GLP1R-KO mice at a similar level (Figure ID). There was a significantly higher level of active GLP-1 in the vena porta of mice treated with FI10937 irrespective of the host genotype (FigurelE). A similar trend was observed in vena cava samples but it did not reach statistical significance (FigurelF). These results are in accordance with a previous report that showed the increased circulating levels of IL-12 after administration through genetically engineered bacteria.

To further investigate whether the bacterially produced GLP-1 had physiologic effects, WT mice were treated with the glpl L. lactis strain FI10937. It was observed that WT mice supplemented with GLP-1-expressing L. lactis FI 10937 had an improved glucose tolerance compared with mice treated with the vector only control strain FI10936 (Figure 2A). In contrast, GLP1R-KO mice displayed no improvement in glucose tolerance after treatment with the FI10937 L. lactis strain compared to those treated with the control strain FI10936 (Figure 2B). There was no significant difference in insulin levels of mice supplemented with either FI 10936 or FI 10937 L. lactis strain (Figures 2C and 2D). GLP-1 has an insulinotrophic effect but considering the short half-life of this hormone and the non-proximity with the pancreas, it has been shown that portal GLP-1 also has an insulin independent role in regulating glucose homeostasis through hepato-portal GLPR. With bacterial supplementation and subsequent increase in portal GLP-1, it seems that in this case GLP-1 is acting through hepato-portal sensing in response to intraperitoneal infusion of glucose, which may explain why no differences in insulin levels between the groups were observed.

Taken together, it was demonstrated that the recombinant L. lactis strain producing GLP-1 stimulates insulin secretion in isolated islets and produces better glucose tolerance in supplemented mice. Both of these effects were absent in GLP1R-KO mice thus confirming that they were exclusively mediated by GLP-1 and that GLP1R is required to manifest the beneficial effects of the GLP-1 producing recombinant L.lactis strain FI10936.

In summary these findings provide evidence that recombinant L. lactis strains have potential clinical applications in medicine, in particular for the treatment of diabetes.

Accordingly, the invention further relates to said recombinant L. lactis strain expressing GLP-1 for use in medicine.

In a preferred embodiment of the invention, said recombinant L. lactis strain expressing GLP-1 may be used in diabetes, in particular type 2 diabetes.

The invention further relates to said recombinant L. lactis strain expressing GLP-1 wherein said recombinant L. lactis strain releases GLP-1 in the gut over a period of several days. GLP-1 has a short half-life (~2 min) and is rapidly degraded by DDP4. It is therefore important that GLP-1 is released steadily by said recombinant L. lactis to perpetuate its physiological effect. This is achieved by using the L. lactis strain which is known to persist in the gut for several days. The survival of the probiotic bacteria can be further improved by preparing a freeze-dried preparation of the bacteria in the presence of protectants. Such protectants enable the bacteria to be protected against the stomach acids and bile salts hence improving its transit through the gut and overall survival.

The invention further relates to a pharmaceutical composition comprising said recombinant L. lactis strain expressing GLP-1 and a pharmaceutically acceptable excipient.

The invention further relates to a food composition or food supplement comprising said recombinant L. lactis strain expressing GLP-1 and a suitable carrier.

The pharmaceutical and food compositions may be liquid, comprising said recombinant L. lactis strain expressing GLP-1, or solid, comprising dried said recombinant L. lactis strain expressing GLP-1 that can be reactivated when put in a suitable environment.

Said recombinant L. lactis strain expressing GLP-1 according to the invention may be dried by any system including freeze-dried or spray-dried and can contain suitable known adjuvants such as cryoprotectants. The preparation of the invention may be in the form of a tablet, a powder or similar form, containing a dose of said recombinant L. lactis. The powder may then be mixed with solid food or foods with a high water-content, such as fermented milk products, for example yogurt, or reconstituted using a suitable liquid such as water, milk or similar drinkable liquids.

Alternatively, the dried preparation may be encapsulated in a suitable recipient to protect the recombinant L. lactis during storage or during exposure to stomach acid. In particular, said recombinant L. lactis strain composition may be encapsulated so that it is predominantly released in the gut. The dried preparation may also be encapsulated in a suitable recipient to control the release of said recombinant L. lactis. In particular, encapsulation is desired when the invention is to be used in liquid or moist products to prevent the bacteria from growing and/or fermenting, and therefore from reducing its shelf-life. Suitable compounds for encapsulation for improving the shelf-life, as well as methods for carrying out the encapsulation are known in the art. Examples of encapsulation methods known in the art are presented in W02012/101167 and in a review by Kaila Kailasapathy [Kailasapathy K (2002)

Microencapsulation of Probiotic Bacteria: Technology and Potential Applications. Curr. Issues Intest. Microbiol. 3: 39-48],

The preparations of the invention may further comprise all desired components, and/or additives which are suited for use in food or pharmaceuticals including flavours, colourings, preservatives, sugar, etc., as long as they do not affect the viability of said recombinant L. lactis strain expressing GLP-1 present therein.

In another embodiment, said food composition may be a probiotic formulation comprising said recombinant L. lactis strain expressing GLP-1 and a suitable carrier. The probiotic micro-organisms do not form part of any delivery system of GLP-1. Examples of probiotic organisms that may be used are listed in WO2009/127519 and include yeasts such as Saccharomyces, Debaromyces, Kluyveromyces and Pichia, moulds such as Aspergillus, Rhizopus, Mucor and Penicillium and bacteria such as the genera Bifidobacterium, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Oenococcus and Lactobacillus. Kluyveromyces lactis may also be used.

Suitable carriers may be an immediate-release carrier or a slow-release carrier and may comprise micro-crystalline cellulose (MCC), dextran, com starch, flour, talc, sucrose, mannitol, lactose, calcium carbonate, polyvinylpyrrolidone (PVP), polyethylene oxide, hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), polyvinyl alcohol (PVA) or the like.

The preparation can further contain prebiotic compounds, in particular specific fibres that produce butyrate/butyric acid or propionate/propionic acid upon fermentation; nitrogen donors such as proteins; and specific vitamins, minerals and/or trace elements.

The invention further relates to a method of production of said L. lactis strain expressing GLP-1 by transforming a GLP-1 expression vector into a L. lactis strain.

In a preferred embodiment, said GLP-1 expression vector is a nisin-inducible vector.

In another preferred embodiment, said GLP-1 expression vector is plasmid pUK200 glp l.

Examples

The invention is illustrated by the following non-limiting examples.

Example 1: Construction of recombinant GLP-1 producing L. lactis strain A 206-bp synthetic gene construct encoding the murine GLP-1 peptide (1-37) n-terminally fused to the lactococcal Usp45 signal peptide was created in silico and its codon usage was optimised for lactococcal expression. The resulting gene cassette was obtained through gene synthesis and subsequently cloned into the E. coli plasmid pEX-A (Eurofins, Germany). The cassette contains Ncol and BamHI restriction sites at its 5’ and 3’ends, respectively, allowing for the translational fusion of the gene to the nisA start codon in the lactococcal expression vector pUK200 [Wegmann U, Klein JR, Drumm I, Kuipers OP, Henrich B (1999) Introduction of peptidase genes from Lactobacillus delbrueckii subsp lactis into Lactococcus lactis and controlled expression. Applied and Environmental Microbiology 65: 4729-4733], The gene was excised from pEX-A using Ncol and BamHI and ligated into pUK200, which had been restricted in the same way, resulting in plasmid pUK200_glpl. The empty vector and the nisin-inducible Glpl expression vector were then transformed into the nisin producing strain L. lactis FI5876 [Dodd HM, Horn N, Hao Z, Gasson MJ (1992) A lactococcal expression system for engineering nisins. Applied and Environmental Microbiology 58: 3683-3693] to generate strains FI10936 (uk200) and FI10937 (glpl), respectively. The vector diagram is shown in Figure 1 A.

Example 2: Culture conditions

Both the vector only control and the glpl producing L. lactis strains were grown overnight at 30°C for 16 hours in M17 media (pH 7.0) supplemented with glucose (2% wt/vol final concentration). Thereafter, the cultures were pelleted and subsequently resuspended in fresh M17 media (pH 8.5) supplemented with glucose (2% wt/vol final concentration) and growth was observed for ~3 hours until the pH reached 7.0. There was no difference in the growth rates observed for the two strains. The GLP-1 expressing L. lactis produced ~ 60 pmol/1 of GLP-1 in the supernatant.

Example 3: Ex vivo glucose stimulated insulin release from isolated islets

Islets were isolated from WT and GLP1R-KO mice as previously described [Wichmann A, Allahyar A, Greiner TU, Plovier H, Lunden GO, et al. (2013) Microbial modulation of energy availability in the colon regulates intestinal transit. Cell Host Microbe 14: 582-590], Five islets per group were incubated with either 16 mM glucose-Krebs ringer buffer (blank) or 2μΜ Exendin4 prepared in 16 mM glucose-Krebs ringer buffer (positive control) or cell-free supernatants of culture medium from uk200 and glpl L. lactis diluted 1:4 in 16 mM glucose-Krebs ringer buffer at 37°C for 60 minutes. After incubation, islets were sedimented, supernatants were collected and insulin was measured using the insulin ELISA kit (Crystal Chem). All stimulations were performed in duplicate and repeated three times independently.

Example 4: In vivo experiment WT (n=15) and GLP1R-KO (n=18) mice 10-12 weeks of age were randomized to receive 200μ1 oral daily gavages of either lxlO10 colony forming units (cfu) of the vector only control FI10936 or GLP-1 producer FI10937 L. lactis cultures for 7 days. Fresh cultures were grown every day for gavage as described before. Mice were fed normal chow and water ad libitum and an intraperitoneal glucose tolerance test was performed on day 8-9. Mice were fasted for 4 hours and administered an intraperitoneal injection of 20% glucose solution prepared in PBS (2 mg/g body weight). Blood was drawn from the tail vein at -30, 0, 15, 30, 60, 90 and 120 min after glucose injection and the blood glucose levels were measured using a Bayer glucometer. Additional blood was collected for insulin measurements at 0, 15 and 30 min time points. Blood from the vena porta and the vena cava was also collected; DPP4 inhibitor (5pl) and Aprotinin (2μ1) were added to EDTA tubes and syringes prior to blood collection to prevent degradation of GLP-1. Insulin was measured by low range ELISA (CrytalChem) and an active GLP-1 assay was performed using the Mesoscale discovery kit. Cecal contents were collected and frozen immediately.

Example 5: Quantitative PCR for L. lactis

Bacterial DNA was extracted from cecal contents using the Machery Nagel NucleoSpin® Soil kit according to manufacturer’s instructions. The DNA was diluted to a concentration of lng/μΐ and the total number of recombinant L. lactis cells was determined in a SYBR green based qPCR using forward primer 5’-CCTTCTACCCATTATTACAGCAGG-3’ and reverse primer 5’-ACCACGACCTTTAACAAGCC-3’. GLP-1 L. lactis colonization levels were determined using forward primer 5’-AGCGAAGATGTTGTCTGTTAG-3’ and reverse primer 5’-GGCACTCGGCACTTAATG-3\ Total DNA extracted from glpl L. lactis was used as standard.

Example 6: Statistical analysis

Data are shown as mean±SEM. Unpaired Student’s t test (for 2 groups) or one way of analysis of variance (ANOVA; for 3 or more groups) was performed to determine the significance between groups using GraphPad Prism software. Significance was established at P<0.05.

Claims

1. A recombinant Lactococcus lactis strain expressing GLP-1.

2. A recombinant Lactococcus lactis strain as defined in claim 1 for use in medicine.

3. A recombinant Lactococcus lactis strain as defined in claim 1 for use in the treatment of diabetes.

4. A recombinant Lactococcus lactis strain as defined in claim 1 for use in the treatment of type 2 diabetes.

5. A method of treatment of diabetes using a recombinant Lactococcus lactis strain as defined in claim 1.

6. A method of treatment of type 2 diabetes using a recombinant Lactococcus lactis strain as defined in claim 1.

7. The use of a recombinant Lactococcus lactis strain as defined in claim 1 for the manufacture of a medicament for the treatment of diabetes.

8. The use of a recombinant Lactococcus lactis strain as defined in claim 1 for the manufacture of a medicament for the treatment of type 2 diabetes.

9. A recombinant Lactococcus lactis strain as defined in claim 1 wherein said recombinant Lactococcus lactis strain releases GLP-1 in the gut during several days.

10. A pharmaceutical composition comprising a recombinant Lactococcus lactis strain as defined in claim 1 and a pharmaceutically acceptable excipient.

11. A food composition comprising a recombinant Lactococcus lactis strain as defined in claim 1 and a suitable carrier.

12. A food composition according to claim 11 wherein said food composition is a probiotic formulation comprising a recombinant Lactococcus lactis strain as defined in claim 1 and a suitable carrier.

13. A composition according to any one of claims 10 to 12 wherein said composition is encapsulated.

14. A composition according to claim 13 wherein said composition is encapsulated in such a way that a recombinant Lactococcus lactis as defined in claim 1 is predominantly released in the gut.

15. A composition according to claim 13 wherein said composition is encapsulated in such a way that the shelf-life of a recombinant Lactococcus lactis as defined in claim 1 is increased.

16. A method of producing a recombinant Lactococcus lactis strain as defined in claim 1 wherein a GLP-1 expression vector is transformed into a Lactococcus lactis strain.

17. A recombinant Lactococcus lactis strain as defined in claim 1 produced according to claim 16 wherein said GLP-1 expression vector is nisin-inducible.

18. A recombinant Lactococcus lactis strain as defined in claim 1 produced according to claim 17 wherein said GLP-1 expression vector is plasmid pUK200_glpl.