In situ reprogramming of gut bacteria by oral delivery

Abstract

Abundant links between the gut microbiota and human health indicate that modification of bacterial function could be a powerful therapeutic strategy. The inaccessibility of the gut and inter-connections between gut bacteria and the host make it difficult to precisely target bacterial functions without disrupting the microbiota and/or host physiology. Herein we describe a multidisciplinary approach to modulate the expression of a specific bacterial gene within the gut by oral administration. We demonstrate that an engineered temperate phage λ expressing a programmable dCas9 represses a targeted E. coli gene in the mammalian gut. To facilitate phage administration while minimizing disruption to host processes, we develop an aqueous-based encapsulation formulation with a microbiota-based release mechanism and show that it facilitates oral delivery of phage in vivo. Finally we combine these technologies and show that bacterial gene expression in the mammalian gut can be precisely modified in situ with a single oral dose.

Microencapsulation of Bacteriophages Using Membrane Emulsification in Different pH-Triggered Controlled Release Formulations for Oral Administration

“…Capsules made from L-55 and L100 may be suitable for in vivo studies involving small animal models, e.g., mice and rats; the intestinal physiological pH values in these animals is well suited to these polymer dissolution profiles. L-55 and L100 capsules would also allow delivery of therapeutic cargo to the different parts of the small intestine in humans ((proximal pH 5.8-6.5); distal (pH 6.6-7.5)) [28]. S100 capsules provide the possibility of delivering payloads further down the GI tract in the large intestine where the pH is~7, allowing site specific delivery strategies to be considered.…”

New technologies for developing phage-based tools to manipulate the human microbiome

“…This strategy is most relevant in cases where only one bacterial taxon is responsible for the disease, for example, C. difficile-associated diarrhoea. (ii) Targeting the function by using phages as vectors to deliver a variety of modulatory interventionssuch as specific genes to introduce a new function to the community (which is beneficial to the host), such as genes which encode essential metabolites; the CRISPR/Cas nuclease system, which can selectively eliminate bacterial taxa based on their genetic content [56], which is useful when there is functional redundancy in the population, as is the case with TMAO-producing bacteria; or programmable nuclease deactivated Cas9 to repress virulence genes, for example, suppressing shiga toxin expression in E. coli [57]. This is an interesting approach as its lower impact on bacterial fitness might protect against selection for resistance.…”

“…(iv) Phages are sensitive to low pHthus, their oral delivery is challenging. To protect phages against gastric acid and proteases in the GI tract they can be encapsulated using a layer-by-layer assembly approach that triggers phage release upon entry into the large intestinethat part of the GI tract which contains the highest number of bacteria [57]. (v) The host immune system can neutralise phages.…”

The Many Applications of Engineered Bacteriophages—An Overview

“…Rather than using CRISPR for cutting target DNA, researchers can target a nuclease-deactivated Cas9 (dCas9) to a specific gene to alter its expression. As a proof of principle for in vivo alteration of gene expression of microbes in the gut, Hsu and colleagues engineered phage λ to express dCas9 and a guide RNA targeting the gene for red fluorescent protein (λ::dCas9 rfp ) [ 53 ]. When the engineered phage was added to a culture of a reporter strain of E. coli containing the genomically integrated RFP gene, RFP fluorescence was significantly reduced relative to the control lacking the targeting crRNA.…”