Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

Background: Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors.

Sleeping Beauty mRNA-LNP enables stable rAAV transgene expression in mouse and NHP hepatocytes and improves vector potency.

Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene.

Efficient retroelement-mediated DNA writing in bacteria.

The ability to efficiently and dynamically change information stored in genomes would enable powerful strategies for studying cell biology and controlling cellular phenotypes. Current recombineering-mediated DNA writing platforms in bacteria are limited to specific laboratory conditions, often suffer from suboptimal editing efficiencies, and are not suitable for in situ applications. To overcome these limitations, we engineered a retroelement-mediated DNA writing system that enables efficient and precise editing of bacterial genomes without the requirement for target-specific elements or selection.

Bacteriophage Therapy Testing Against Shigella flexneri in a Novel Human Intestinal Organoid-Derived Infection Model.

Objective: Enteric bacterial pathogens cause diarrheal disease and mortality at significant rates throughout the world, particularly in children younger than 5 years. Our ability to combat bacterial pathogens has been hindered by antibiotic resistance, a lack of effective vaccines, and accurate models of infection. With the renewed interest in bacteriophage therapy, we sought to use a novel human intestinal model to investigate the efficacy of a newly isolated bacteriophage against Shigella flexneri.

An ingestible bacterial-electronic system to monitor gastrointestinal health.

Biomolecular monitoring in the gastrointestinal tract could offer rapid, precise disease detection and management but is impeded by access to the remote and complex environment. Here, we present an ingestible micro-bio-electronic device (IMBED) for in situ biomolecular detection based on environmentally resilient biosensor bacteria and miniaturized luminescence readout electronics that wirelessly communicate with an external device. As a proof of concept, we engineer heme-sensitive probiotic biosensors and demonstrate accurate diagnosis of gastrointestinal bleeding in swine.

Synthetic Biogenesis of Bacterial Amyloid Nanomaterials with Tunable Inorganic-Organic Interfaces and Electrical Conductivity.

Amyloids are highly ordered, hierarchal protein nanoassemblies. Functional amyloids in bacterial biofilms, such as Escherichia coli curli fibers, are formed by the polymerization of monomeric proteins secreted into the extracellular space. Curli is synthesized by living cells, is primarily composed of the major curlin subunit CsgA, and forms biological nanofibers with high aspect ratios.

Microbiome therapeutics - Advances and challenges.

The microbial community that lives on and in the human body exerts a major impact on human health, from metabolism to immunity. In order to leverage the close associations between microbes and their host, development of therapeutics targeting the microbiota has surged in recent years. Here, we discuss current additive and subtractive strategies to manipulate the microbiota, focusing on bacteria engineered to produce therapeutic payloads, consortia of natural organisms and selective antimicrobials.

Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation.

Bacteriophage-based synthetic biology for the study of infectious diseases.

Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic engineering and synthetic biology. Here, we discuss bacteriophage-based technologies and their application to the study of infectious diseases.

Synthesis and patterning of tunable multiscale materials with engineered cells.

Many natural biological systems--such as biofilms, shells and skeletal tissues--are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning.