Discovery and delivery strategies for engineered live biotherapeutic products

Abstract

Genetically engineered microbes that secrete therapeutics, sense and respond to external environments, and/or target specific sites in the gut fall under an emergent class of therapeutics, called live biotherapeutic products (LBPs). As live organisms that require symbiotic host interactions, LBPs offer unique therapeutic opportunities, but also face distinct challenges in the gut microenvironment. In this review, we describe recent approaches (often demonstrated using traditional probiotic microorganisms) to discover LBP chassis and genetic parts utilizing omics-based methods and highlight LBP delivery strategies, with a focus on addressing physiological challenges that LBPs encounter after oral administration. Finally, we share our perspective on the opportunity to apply an integrated approach, wherein discovery and delivery strategies are utilized synergistically, towards tailoring and optimizing LBP efficacy.

Keywords: drug delivery; drug discovery; gastrointestinal physiology; live biotherapeutic products; multi-omics.

Figure 1, Key Figure.. Physiological Challenges in the Human Gut.

Upon oral administration, LBPs will encounter various physiological challenges during passage from the stomach (blue), through the small intestine (yellow), and to the colon/large intestine (purple). There are a variety of chemical challenges secreted in the gut, such as (a) acid (stomach), (b) digestive enzymes (stomach and small intestine), and (c) bile salts (small intestine) which either disrupt essential LBP components (e.g., cell wall) or cause internal stresses that lead to LBP death. (d) Immune cells in the gut (small intestine and colon) can actively sense, interact with, and clear exogeneous LBPs. Competition, arising from the existing microbiota (large intestine), can limit LBPs ability to access sufficient (e) nutrients, for growth and metabolism, or (f) space, for adherence, growth, and colonization. Physiological challenges can also be ubiquitously encountered through the gut such as chemical gradients (g) (pH and oxygen) or physical phenomena including (h) peristalsis and (i) epithelial/mucosal turnover. These physiological aspects, and how they serve as challenges to LBPs upon oral administration, are discussed in more detail in Box 1.

Figure 2.. Discovery Approaches to Overcome Physiological Challenges and Control Microenvironment Interactions.

(a) Discovery of LBPs can be guided by investigating chassis in in vitro and in vivo settings, using a variety of ‘omics pipelines. (b) Genomics measures DNA composition at both the organism and community levels and can be used to determine the presence and abundance of LBPs in various locations in the gut and under various microenvironment conditions. These insights identify factors that drive the LBP’s adaptation to the gut and control interactions with other microbiota members. (c) Transcriptomics can identify genes that are differentially regulated in response to physiological challenges in the gut. These differentially regulated genes illuminate in vivo adaptation mechanisms of LBPs. (d) Proteomics can identify proteins in LBPs (as well as their subcellular localization) that facilitate in vivo adaptation. Since proteins on LBP surfaces mediate extracellular interactions, proteomics can provide insight into how LBPs interact and communicate with the dynamic gut microenvironment. (e) Metabolomics can identify the metabolic activity of LBPs in a multi-species community through identification of metabolites and their fluxes, which in turn illuminates how an LBP chassis adapts to gut microenvironments. (f) Functional genomics can be used to achieve pathway optimization and high-throughput strain screening for engineered LBP applications. Pathway optimization enables high activity of the engineered function even under the burden of physiological challenges. High-throughput strain screening can identify candidate chassis that are already gut colonizers and that can receive genetic payloads in situ through horizontal gene transfer.

Figure 3.. Formulation and Genetic Engineering Strategies to Improve LBP Delivery.

Pharmaceutical formulations and genetic engineering strategies can be leveraged to both overcome physiological challenges and utilize physiological microenvironments to improve LBP delivery and LBP function. Examples of pharmaceutical formulation approaches include (a) encapsulation via phospholipid bi-layer membrane LBP coatings, (b) targeting via synthetic adhesin-surface modification of the LBP, and (c) endowing stimuli-responsive functions by using external magnets to direct the movement of magnetized LBPs. Examples of genetic engineering approaches include (d) engineered LBPs which secrete an encapsulating protective and mucoadhesive hydrogel with conjugated therapeutic modalities, (e) LBPs engineered to express adhesins with high binding affinities on the microbial surface for targeting, and (f) the expression of several enzymes for controlled metabolism in response to anoxic conditions for stimuli-responsive therapeutic function. Recently, pharmaceutical formulations and genetic engineering strategies have been combined to (g) encapsulate genetically engineered LBPs in a hydrogel bead which allows diffusion of nutrients into the bead to maintain the engineered function while achieving biocontainment, and (h) enable co-culture of a bacterial cellulose-secreting probiotic organism with engineered LBPs to enable protective encapsulation of the LBP while maintaining its engineered function.

Figure 4.. Integrating Discovery and Delivery for Future LBP Development.

(a) Recent trends in the discovery of LBPs utilize multi-omics (e.g., genomics, transcriptomics, proteomics, metabolomics) strategies to uncover potential new microbial chassis, colonization factors, and adaptation mechanisms to overcome the physiological challenges of the gut. (b) Recent trends in the delivery of LBPs utilize both pharmaceutical formulation and genetic engineering strategies to encapsulate, target, and/or generate stimuli-responsive functions to overcome physiological challenges. (c) The current strategies can be integrated wherein discovery and delivery occur simultaneously for synergistic LBP design (e.g., chassis selection, genetic engineering, formulation approaches). (d) Synergistic LBP discovery and delivery has the potential to inform more sophisticated modeling approaches wherein micro-scale approaches (e.g., metabolic flux analysis, network modeling) are combined with macro-scale approaches (e.g., physiologically-based pharmacokinetic modeling) to enable better prediction for LBP efficacy. Synergistic LBP discovery and delivery also has the potential to pave the way for next-generation LBPs which have both improved survival and site-specific action through genetic engineering and pharmaceutical formulations approaches, ultimately leading to more efficacious LBPs with increased therapeutic response and decreased off-target toxicities.