Lactobacillus-vectored Alkaline Phosphatase to Mitigate Alcohol-induced Leaky Gut and LPS Endotoxin Load in the Gut

PROJECT SUMMARY/ ABSTRACT Alcohol misuse is one of the leading causes of mortality, accounting for 178,000 deaths and over 249 billion in medical costs and lost productivity annually in the United States. Chronic alcohol consumption induces gut dysbiosis, increasing intestinal permeability and Gram-negative bacteria, subsequently raising lipopolysaccharide (LPS, endotoxin) levels, and leading to endotoxemia, systemic inflammation, and organ dysfunction. Alkaline phosphatase (AP) is an endogenous enzyme produced in the intestine and plays a key role in detoxifying LPS. Oral administration of Intestinal AP (IAP) attenuated alcohol-induced hepatosteatosis in mice and reduced LPS content in cecal contents and blood and prevented metabolic syndrome in mice. However, oral administration of AP has a challenge of short half-life, poor bioavailability, protease susceptibility, degradation in the acidic and enzyme-rich environment of the stomach, the need for frequent administration at higher concentrations and lack of sustained supply in the intestine. BiomEdit has developed a proprietary Lactobacillus- vectored Drug Delivery Technology (LactoDDT®), which has previously demonstrated its capacity to prevent alcohol-induced leaky gut, partially reduce proinflammatory cytokines and efficiently deliver nanobodies and other targets to gastrointestinal (GIT) tract. BiomEdit also has developed a proprietary AP (InPro®), which neutralizes LPS from different bacteria, reduces LPS-induced cytokine expression in RAW 264.7 macrophages and increases native intestinal AP and improves LPS induced intestinal morphology and restores microbial homeostasis in weaned pigs. Here, we propose to apply a two-pronged approach using: 1) LactoDDT® chassis to prevent alcohol-induced gut leakiness and 2) LactoDDT®-produced AP in the GIT to reduce alcohol-induced increase in endotoxin levels in the gut and its systemic translocation. Our approach will be a paradigm shift in AP delivery overcoming many of the limitations associated with oral administration of AP. SA1: Generation of engineered live biotherapeutics (eLBPs) delivering functional InPro®. In this aim, we propose to generate a total of 20 eLBPs delivering InPro® and evaluate these eLBPs to neutralize LPS in an in vitro assay. SA2: Evaluation of eLBPs for efficacy in a binge-on-chronic alcohol model of leaky gut. In partnership with our collaborator at LSU, we will evaluate the efficacy of final 2 eLBPs with the highest InPro® secretion and activity in a binge-on- chronic alcohol model of leaky gut in mice. A successful outcome of this Phase I work is development of a live bio-preventative or -therapeutic aimed at reducing alcohol-induced leaky gut and increase in LPS load in the gut and its systemic translocation following recent or ongoing alcohol consumption. In Phase II, we will advance the final candidates towards preclinical development in a binge-on-chronic alcohol model of leaky gut and prepare a risk-benefit assessment package for pre-IND meeting with FDA. 1