Lactobacillus mediated delivery of therapeutic peptides in the gastrointestinal tract

Abstract: Lactobacilli are normal residents of the human gastrointestinal (GI) tract. Many species belonging to the genera Lactobacillus with health beneficial properties have been introduced as probiotics. Genetic engineering of Lactobacillus could potentially combine the colonizing ability and probiotic effect with an engineered therapeutic function. The aim of this thesis was to explore the possibility of using lactobacilli as vectors for delivery of peptides in the GI tract for therapy against type 2 diabetes and other immune-related diseases. GLP-1, an incretin hormone that can stimulate insulin secretion, is used in the treatment of type 2 diabetes. In paper I, we designed and expressed the pentameric GLP-1 (trypsin cleavable oligomers) both in a secreted form and anchored on the surface of L. paracasei BL23. The pentameric GLP-1 retains its bioactivity both in vitro and in the intestine of diabetic rat following digestion by intestinal trypsin. When given by gavage to diabetic rats, the non-expressor Lactobacillus showed significant anti-diabetic effect but GLP-1 expression did not provide an additional insulinotropic effect possibly due to the low levels produced. These results indicate that L. paracasei BL23 itself might be used as an alternative treatment method for type 2 diabetes, but further work is needed to increase the expression level of GLP-1 by Lactobacillus. Interleukin-22 (IL-22) plays a prominent role in epithelial regeneration and protecting intestinal stem cells from immune-mediated tissue damage. In paper II, Lactobacillus strains were constructed for delivery of IL-22 as a new therapeutic strategy for acute graft-versushost disease (GVHD) in the GI tract. The secretion and surface anchoring of mouse IL-22 by L. paracasei BL23 was demonstrated and the biological activity of IL-22 produced by Lactobacillus was shown in vitro. In paper III, we developed a co-expression vector to produce two rotavirus-specific VHH antibody fragments in Lactobacillus. Both antibody fragments (ARP1 and ARP3) were expressed in secreted and surface anchored forms and their ability to bind to various rotavirus serotypes was demonstrated in vitro. This vector was then used in paper IV, to simultaneously produce both secreted Interleukin-21 (IL-21) and anchored CD40 ligand (CD40L) on the surface of lactobacilli. IL-21/CD40L based stimulation may be a potential therapy for IgA deficient patients. In this paper, IL-21(or hybrid IL-21/4) and CD40L were expressed individually, or co-expressed in L. paracasei BL23. We showed that Lactobacillus expressing IL-21, IL-21/4 and CD40L individually can induce IgA secretion in PBMCs from healthy donors. This work provides the basis for the use of genetically modified lactobacilli for delivery of therapeutic peptides in the GI tact. The delivery strategies could be further developed to facilitate easy and inexpensive access to other peptide drugs.

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