Lothar Steidler

Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10

Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clinical use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA− hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacteriums capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.

Comparative and Functional Analysis of Sortase-Dependent Proteins in the Predicted Secretome of Lactobacillus salivarius UCC118

ABSTRACT Surface proteins are important factors in the interaction of probiotic and pathogenic bacteria with their environment or host. We performed a comparative bioinformatic analysis of four publicly available Lactobacillus genomes and the genome of Lactobacillus salivarius subsp. salivarius strain UCC118 to identify secreted proteins and those linked to the cell wall. Proteins were identified which were predicted to be anchored by WXL-binding domains, N- or C-terminal anchors, GW repeats, lipoprotein anchors, or LysM-binding domains. We identified 10 sortase-dependent surface proteins in L. salivarius UCC118, including three which are homologous to mucus-binding proteins (LSL_0152, LSL_0311, and LSL_1335), a collagen-binding protein homologue (LSL_2020b), two hypothetical proteins (LSL_1838 and LSL_1902b), an enterococcal surface protein homologue (LSL_1085), a salivary agglutinin-binding homologue (LSL_1832b), an epithelial binding protein homologue (LSL_1319), and a proteinase homologue (LSL_1774b). However, two of the genes are gene fragments and four are pseudogenes, suggesting a lack of selection for their function. Two of the 10 genes were not transcribed in vitro, and 1 gene showed a 10-fold increase in transcript level in stationary phase compared to logarithmic phase. The sortase gene was deleted, and three genes encoding sortase-dependent proteins were disrupted. The sortase mutant and one sortase-dependent protein (mucus-binding homologue) mutant showed a significant reduction in adherence to human epithelial cell lines. The genome-wide investigation of surface proteins can thus help our understanding of their roles in host interaction.

Secretion of biologically active murine interleukin-10 by Lactococcus lactis.

We investigated the ability of Lactococcus lactis to secrete biologically active, murine interleukin-10 (mIL-10). mIL-10 was synthesized as a fusion protein, consisting of the mature part of the eukaryotic protein fused to the secretion signal of the lactococcal Usp45 protein. The secreted protein was analyzed by PAGE, ELISA and bioassay.We show that L. lactis can efficiently secrete biologically active, murine IL-10. Determination of the N-terminal amino acid sequence confirmed correct processing of the fusion polypeptide by the lactococcal signal peptidase. The amount of mIL-10, accumulating in the medium, could be increased by a factor of ten by growing the cells in an optimized medium, buffered at near-neutral pH. Under these conditions, up to 30 mg of mIL-10 was obtained from a 10-litre fermentation.

Genetically engineered probiotics

Probiotic micro-organisms have been used for many years. Originating as food supplements, they are now most often administered orally and offer an attractive alternative for treating of intestinal disorders. A better understanding of the mechanisms by which these micro-organisms act has now opened up possibilities for designing new probiotic strains. Through genetic engineering, it is possible not only to strengthen the effects of existing strains, but also to create completely new probiotics. These need not necessarily be composed only of bacterial products but can also include elements of regulatory systems or enzymes derived from a foreign-human-source. If designed carefully and with absolute attention to biological safety in its broadest sense, the development of genetically modified probiotics has the potential to revolutionize alimentary health.

Characterization of ApuB, an Extracellular Type II Amylopullulanase from Bifidobacterium breve UCC2003

ABSTRACT The apuB gene of Bifidobacterium breve UCC2003 was shown to encode an extracellular amylopullulanase. ApuB is composed of a distinct N-terminally located α-amylase-containing domain which hydrolyzes α-1,4-glucosidic linkages in starch and related polysaccharides and a C-terminally located pullulanase-containing domain which hydrolyzes α-1,6 linkages in pullulan, allowing the classification of this enzyme as a bifunctional class II pullulanase. A knockout mutation of the apuB gene in B. breve UCC2003 rendered the resulting mutant incapable of growth in medium containing starch, amylopectin, glycogen, or pullulan as the sole carbon and energy source, confirming the crucial physiological role of this gene in starch metabolism.

Therapeutic Drug Delivery by Genetically Modified Lactococcus lactis

Abstract:  Food‐grade bacteria have been consumed throughout history without associated pathologies and are, therefore, absolutely safe to ingest. Unexpectedly, Lactococcus lactis (L. lactis), known from cheese production, can be genetically engineered to constantly secrete satisfactory amounts of bioactive cytokines. Both of these features enabled the development of a new kind of topical delivery system: topical and active delivery of therapeutic proteins by genetically modified micro‐organisms. The host organisms record inspired the development of applications that target intestinal diseases. In a variety of mouse models, chronic colon inflammation can be successfully treated with (interleukin) IL‐10–secreting L. lactis. Trefoil factor (TFF) producer strains have also been shown to be very effective in the treatment of acute colitis. Such novel therapeutic strains are textbook examples of genetically modified (GM) organisms. There are legitimate concerns with regard to the deliberate release of GM micro‐organisms. On development of these applications, therefore, we have engineered these bacteria in such a way that biological containment is guaranteed. The essential gene thyA, encoding thymidylate synthase, has been exchanged for IL‐10. This makes the GM strain critically dependent on thymidine. Lack of thymidine, for example, resulting from thymidine consumption by thyA‐deficient strains–will irreversibly lead to induced “thymidine‐less death.” This accomplishment has created the possibility of using this strategy for application in human medicine.

AG013, a mouth rinse formulation of Lactococcus lactis secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis

Non-clinical studies, focusing on the pharmacodynamics (PD), pharmacokinetics (PK) and safety pharmacology of genetically modified Lactococcus lactis (L. lactis) bacteria, engineered to secrete human Trefoil Factor 1 (hTFF1), were performed to provide proof-of-concept for the treatment of oral mucositis (OM) patients. L. lactis strain sAGX0085 was constructed by stably inserting an htff1 expression cassette into the bacterial genome, and clinically formulated as a mouth rinse (coded AG013). PD studies, using different oral dosing regimens, were performed in a clinically relevant hamster model for radiation-induced OM. The PK profile was assessed in healthy hamsters and in hamsters with radiation-induced OM. In addition, in vitro and in vivo safety pharmacology studies were conducted, in pooled, complement-preserved human serum, and in neutropenic hamsters and rats respectively. Topical administration of L. lactis sAGX0085/AG013 to the oral mucosa significantly reduced the severity and course of radiation-induced OM. PK studies demonstrated that both living L. lactis bacteria, as well as the hTFF1 secreted, could be recovered from the administration site for maximum 24h post-dosing, without systemic exposure. The in vitro and in vivo safety pharmacology studies confirmed that L. lactis sAGX0085 could not survive in systemic circulation, not even under neutropenic conditions. The results from the PD, PK and safety pharmacology studies reported here indicate that in situ secretion of hTFF1 by topically administered L. lactis bacteria provides a safe and efficacious therapeutic tool for the prevention and treatment of OM.

Oral Delivery of Glutamic Acid Decarboxylase (GAD)-65 and IL10 by Lactococcus lactis Reverses Diabetes in Recent-Onset NOD Mice

Growing insight into the pathogenesis of type 1 diabetes (T1D) and numerous studies in preclinical models highlight the potential of antigen-specific approaches to restore tolerance efficiently and safely. Oral administration of protein antigens is a preferred method for tolerance induction, but degradation during gastrointestinal passage can impede such protein-based therapies, reducing their efficacy and making them cost-ineffective. To overcome these limitations, we generated a tolerogenic bacterial delivery technology based on live Lactococcus lactis (LL) bacteria for controlled secretion of the T1D autoantigen GAD65370–575 and the anti-inflammatory cytokine interleukin-10 in the gut. In combination with short-course low-dose anti-CD3, this treatment stabilized insulitis, preserved functional β-cell mass, and restored normoglycemia in recent-onset NOD mice, even when hyperglycemia was severe at diagnosis. Combination therapy did not eliminate pathogenic effector T cells, but increased the presence of functional CD4+Foxp3+CD25+ regulatory T cells. These preclinical data indicate a great therapeutic potential of orally administered autoantigen-secreting LL for tolerance induction in T1D.

Intracellular Accumulation of Trehalose Protects Lactococcus lactis from Freeze-Drying Damage and Bile Toxicity and Increases Gastric Acid Resistance

ABSTRACT Interleukin-10 (IL-10) is a promising candidate for the treatment of inflammatory bowel disease. Intragastric administration of Lactococcus lactis genetically modified to secrete IL-10 in situ in the intestine was shown to be effective in healing and preventing chronic colitis in mice. However, its use in humans is hindered by the sensitivity of L. lactis to freeze-drying and its poor survival in the gastrointestinal tract. We expressed the trehalose synthesizing genes from Escherichia coli under control of the nisin-inducible promoter in L. lactis. Induced cells accumulated intracellular trehalose and retained nearly 100% viability after freeze-drying, together with a markedly prolonged shelf life. Remarkably, cells producing trehalose were resistant to bile, and their viability in human gastric juice was enhanced. None of these effects were seen with exogenously added trehalose. Trehalose accumulation did not interfere with IL-10 secretion or with therapeutic efficacy in murine colitis. The newly acquired properties should enable a larger proportion of the administered bacteria to reach the gastrointestinal tract in a bioactive form, providing a means for more effective mucosal delivery of therapeutics.

Actobiotics™ as a Novel Method for Cytokine Delivery

Interleukin‐10 (IL‐10) is central in immune downregulation, but so far its use in inflammatory diseases remains cumbersome. For treatment of inflammatory bowel disease, adequate amounts of IL‐10 must reach the intestinal lining. Systemic injection of a pharmacologically active doses of recombinant human (rh) IL‐10 results in very low mucosal levels of protein and severe toxicity and side effects. In animal models, topical and active delivery of IL‐10 by ingestion of recombinant Lactococcus lactis (L. lactis) was shown to be a valuable alternative. Starting thereof we have developed a novel pharmaceutical platform. Our expertise and TopAct™ (topical and active) delivery technology allows use of recombinant L. lactis– ActoBiotics™– in clinical practice. Here we discuss the development of recombinant L. lactis for intestinal delivery of rhIL‐10 in humans.