A molecular toolkit for heterologous protein secretion across Bacteroides species.

Bacteroides species are abundant, prevalent, and stable members of the human gut microbiota, making them a promising chassis for developing long-term interventions for chronic diseases. Engineering Bacteroides as in situ bio-factories, however, requires efficient protein secretion tools, which are currently lacking. Here, we systematically investigate methods to enable heterologous protein secretion in Bacteroides.

A molecular toolkit for heterologous protein secretion across species.

species are abundant and prevalent stably colonizing members of the human gut microbiota, making them a promising chassis for developing long-term interventions for chronic diseases. Engineering these bacteria as on-site production and delivery vehicles for biologic drugs or diagnostics, however, requires efficient heterologous protein secretion tools, which are currently lacking. To address this limitation, we systematically investigated methods to enable heterologous protein secretion in using both endogenous and exogenous secretion systems.

Short FcRn-Binding Peptides Enable Salvage and Transcytosis of scFv Antibody Fragments.

Therapeutic antibodies have become one of the most widely used classes of biotherapeutics due to their unique antigen specificity and their ability to be engineered against diverse disease targets. There is significant interest in utilizing truncated antibody fragments as therapeutics, as their small size affords favorable properties such as increased tumor penetration as well as the ability to utilize lower-cost prokaryotic production methods. Their small size and simple architecture, however, also lead to rapid blood clearance, limiting the efficacy of these potentially powerful therapeutics.

Living Therapeutics: The Next Frontier of Precision Medicine.

Modern medicine has long studied the mechanism and impact of pathogenic microbes on human hosts, but has only recently shifted attention toward the complex and vital roles that commensal and probiotic microbes play in both health and dysbiosis. Fueled by an enhanced appreciation of the human-microbe holobiont, the past decade has yielded countless insights and established many new avenues of investigation in this area. In this review, we discuss advances, limitations, and emerging frontiers for microbes as agents of health maintenance, disease prevention, and cure.

A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont.

Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species.

Directed Evolution of Targeted Recombinases for Genome Engineering.

Over the past several years, genome engineering has become an established component of basic research endeavors, and is emerging as a vital element of clinical research applications. Site-specific recombinases are one of the several tools that can facilitate genome modification by catalyzing rearrangements between specific DNA targets. Of particular interest are the small serine recombinases, which are modular in both form and function.

Genome-Editing Technologies: Principles and Applications.

Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools.

Protein delivery using Cys2-His2 zinc-finger domains.

The development of new methods for delivering proteins into cells is a central challenge for advancing both basic research and therapeutic applications. We previously reported that zinc-finger nuclease proteins are intrinsically cell-permeable due to the cell-penetrating activity of the Cys2-His2 zinc-finger domain. Here, we demonstrate that genetically fused zinc-finger motifs can transport proteins and enzymes into a wide range of primary and transformed mammalian cell types.

Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign.

Despite recent advances in genome engineering made possible by the emergence of site-specific endonucleases, there remains a need for tools capable of specifically delivering genetic payloads into the human genome. Hybrid recombinases based on activated catalytic domains derived from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are a class of reagents capable of achieving this. The utility of these enzymes, however, has been constrained by their low overall targeting specificity, largely due to the formation of side-product homodimers capable of inducing off-target modifications.

Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering.

Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors.

Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants.

The serine recombinases are a diverse family of modular enzymes that promote high-fidelity DNA rearrangements between specific target sites. Replacement of their native DNA-binding domains with custom-designed Cys₂-His₂ zinc-finger proteins results in the creation of engineered zinc-finger recombinases (ZFRs) capable of achieving targeted genetic modifications. The flexibility afforded by zinc-finger domains enables the design of hybrid recombinases that recognize a wide variety of potential target sites; however, this technology remains constrained by the strict recognition specificities imposed by the ZFR catalytic domains.

Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains.

Expanding the scope of site-specific recombinases for genetic and metabolic engineering.

Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities.

Antibody conjugation approach enhances breadth and potency of neutralization of anti-HIV-1 antibodies and CD4-IgG.

Broadly neutralizing antibodies PG9 and PG16 effectively neutralize 70 to 80% of circulating HIV-1 isolates. In this study, the neutralization abilities of PG9 and PG16 were further enhanced by bioconjugation with aplaviroc, a small-molecule inhibitor of virus entry into host cells. A novel air-stable diazonium hexafluorophosphate reagent that allows for rapid, tyrosine-selective functionalization of proteins and antibodies under mild conditions was used to prepare a series of aplaviroc-conjugated antibodies, including b12, 2G12, PG9, PG16, and CD4-IgG.

A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells.

Zinc-finger recombinases (ZFRs) represent a potentially powerful class of tools for targeted genetic engineering. These chimeric enzymes are composed of an activated catalytic domain derived from the resolvase/invertase family of serine recombinases and a custom-designed zinc-finger DNA-binding domain. The use of ZFRs, however, has been restricted by sequence requirements imposed by the recombinase catalytic domain.

Targeted gene knockout by direct delivery of zinc-finger nuclease proteins.

Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in various mammalian cell types with minimal off-target effects.

ImmunoPET using engineered antibody fragments: fluorine-18 labeled diabodies for same-day imaging.

Combining the specificity of tumor-targeting antibodies with the sensitivity and quantification offered by positron emission tomography (PET) provides tremendous opportunities for molecular characterization of tumors in vivo. Until recently, significant challenges have been faced when attempting to combine antibodies which show long biological half-lives and positron-emitting radionuclides with comparably short physical half-lives, in particular (18)F (half-life, 109 min). A fast and simple microwave-assisted method of generating N-succinimidyl-4-[(18)F]fluorobenzoate has been developed and employed for radiolabeling a small, rapidly targeting HER2-specific engineered antibody fragment, the cys-diabody.

Microfluidic-based 18F-labeling of biomolecules for immuno-positron emission tomography.

Methods for tagging biomolecules with fluorine 18 as immuno-positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.

ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv dimers (diabodies).

Rapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time points. Here, tumor targeting of anti-CD20 diabodies (scFv dimers) for detection of low-grade B-cell lymphomas were evaluated. In addition, the effect of linker length on oligomerization of the diabody was investigated.

Cys-diabody quantum dot conjugates (immunoQdots) for cancer marker detection.

The present work demonstrates the use of small bivalent engineered antibody fragments, cys-diabodies, for biological modification of nanoscale particles such as quantum dots (Qdots) for detection of target antigens. Novel bioconjugated quantum dots known as immunoQdots (iQdots) were developed by thiol-specific oriented coupling of tumor specific cys-diabodies, at a position away from the antigen binding site to amino PEG CdSe/ZnS Qdots. Initially, amino PEG Qdot 655 were coupled with reduced anti-HER2 cys-diabody by amine-sulfhydryl-reactive linker [N-ε-maleimidocaproyloxy] succinimide ester (EMCS) to produce anti-HER2 iQdot 655.

Site-specific, thiol-mediated conjugation of fluorescent probes to cysteine-modified diabodies targeting CD20 or HER2.

Small, engineered antibody fragments such as diabodies (50 kDa noncovalent dimers of single-chain Fv fragments) are useful alternatives to their larger antibody counterparts. However, due to their size, they are more susceptible to disruption of their antigen binding sites when modified using random conjugation techniques. Previous work has demonstrated the utility of a C-terminal cysteine modification for site-specific radiolabeling of an anti-CEA diabody, resulting in the creation of a cys-diabody (CysDb).

Measurement of amphotericin B concentration by resonant Raman spectroscopy--a novel technique that may be useful for non-invasive monitoring.

We wished to determine whether Resonant Raman Spectroscopy (RRS) could be used to measure Amphotericin B (AmB) at therapeutic and subtherapeutic concentrations in a model system mimicking the anterior chamber of the eye. The goal was to develop a technique for non-invasive measurement of AmB levels in the aqueous humor (AH) of the eye. A krypton-ion laser source (406.