Engineering protein nanoparticles for drug delivery.

Protein nanoparticles offer a highly tunable platform for engineering multifunctional drug delivery vehicles that can improve drug efficacy and reduce off-target effects. While many protein nanoparticles have demonstrated the ability to tolerate genetic and posttranslational modifications for drug delivery applications, this review will focus on three protein nanoparticles of increasing size. Each protein nanoparticle possesses distinct properties such as highly tunable stability, capacity for splitting or fusing subunits for modular surface decoration, and well-characterized conformational changes with impressive capacity for large protein cargos. While many of the genetic and posttranslational modifications leverage these protein nanoparticle's properties, the shared techniques highlight engineering approaches that have been generalized across many protein nanoparticle platforms.

An extracellular glucose sensor for substrate-dependent secretion and display of cellulose-degrading enzymes.

The efficient hydrolysis of lignocellulosic biomass into fermentable sugars is key for viable economic production of biofuels and biorenewable chemicals from second-generation feedstocks. Consolidated bioprocessing (CBP) combines lignocellulose saccharification and chemical production in a single step. To avoid wasting valuable resources during CBP, the selective secretion of enzymes (independent or attached to the surface) based on the carbon source available is advantageous. To enable enzyme expression and secretion based on extracellular glucose levels, we implemented a G-protein-coupled receptor (GPCR)-based extracellular glucose sensor; this allows the secretion and display of cellulases in the presence of the cellulosic fraction of lignocellulose by leveraging cellobiose-dependent signal amplification. We focused on the glucose-responsiveness of the HXT1 promoter and engineered PHXT1 by changing its core to that of the strong promoter PTHD3 , increasing extracellular enzyme activity by 81%. We then demonstrated glucose-mediated expression and cell-surface display of the ß-glucosidase BglI on the surface of Saccharomyces cerevisiae. The display system was further optimized by re-directing fatty acid pools from lipid droplet synthesis toward formation of membrane precursors via knock-out of PAH1. This resulted in an up to 4.2-fold improvement with respect to the baseline strain. Finally, we observed cellobiose-dependent signal amplification of the system with an increase in enzymatic activity of up to 3.1-fold when cellobiose was added.

Highly modular hepatitis B virus-like nanocarriers for therapeutic protein encapsulation and targeted delivery to triple negative breast cancer cells.

Protein therapeutics offer enormous clinical impact in treating a variety of diseases by offering high selectivity with limited off-target effects. However, delivery challenges severely hinder functional proteins from reaching their target cells and necessitate frequent administration. To address these problems, nanocarrier encapsulation can provide protease protection and enhanced targeted transportation of functional proteins to their intended disease site. Inspired by their viral analogues, virus-like particles (VLPs) are non-infectious viral capsids that have potential for drug delivery applications because of their shared structural characteristics, such as high loading capacity, particle stability, and structural uniformity. Here, we describe a modular hepatitis B virus (HBV) VLP delivery platform offering tunable modifications of both the exterior and interior viral capsid surfaces via SpyCatcher-SpyTag bioconjugation and a multi-expression system, respectively. This new platform facilitates modification with epidermal growth factor receptor (EGFR)-targeting proteins and encapsulation with both model green fluorescent protein (GFP) and prodrug-converting yeast cytosine deaminase (yCD) enzyme. The resultant targeted VLPs demonstrated enhanced uptake and toxicity in EGFR-overexpressing triple negative breast cancer (TNBC) cells in contrast to non-malignant breast epithelial cells.

Assembly of Metabolons in Yeast Using Cas6-Mediated RNA Scaffolding.

Cells often localize pathway enzymes in close proximity to reduce substrate loss via diffusion and to ensure that carbon flux is directed toward the desired product. To emulate this strategy for the biosynthesis of heterologous products in yeast, we have taken advantage of the highly specific Cas6-RNA interaction and the predictability of RNA hybridizations to demonstrate Cas6-mediated RNA-guided protein assembly within the yeast cytosol. The feasibility of this synthetic scaffolding technique for protein localization was first demonstrated using a split luciferase reporter system with each part fused to a different Cas6 protein. In Saccharomyces cerevisiae, the luminescence signal increased 3.6- to 20-fold when the functional RNA scaffold was also expressed. Expression of a trigger RNA, designed to prevent the formation of a functional scaffold by strand displacement, decreased the luminescence signal by nearly 2.3-fold. Temporal control was also possible, with induction of scaffold expression resulting in an up to 11.6-fold increase in luminescence after 23 h. Cas6-mediated assembly was applied to create a two-enzyme metabolon to redirect a branch of the violacein biosynthesis pathway. Localizing VioC and VioE together increased the amount of deoxyviolacein (desired) relative to prodeoxyviolacein (undesired) by 2-fold. To assess the generality of this colocalization method in other yeast systems, the split luciferase reporter system was evaluated in Kluyveromyces marxianus; RNA scaffold expression resulted in an increase in the luminescence signal of up to 1.9-fold. The simplicity and flexibility of the design suggest that this strategy can be used to create metabolons in a wide range of recombinant hosts of interest.

Advances in consolidated bioprocessing using synthetic cellulosomes.

The primary obstacle impeding the more widespread use of biomass for energy and chemical production is the absence of a low-cost technology for overcoming their recalcitrant nature. It has been shown that the overall cost can be reduced by using a 'consolidated' bioprocessing (CBP) approach, in which enzyme production, biomass hydrolysis, and sugar fermentation can be combined. Cellulosomes are enzyme complexes found in many anaerobic microorganisms that are highly efficient for biomass depolymerization. While initial efforts to display synthetic cellulosomes have been successful, the overall conversion is still low for practical use. This limitation has been partially alleviated by displaying more complex cellulsome structures either via adaptive assembly or by using synthetic consortia. Since synthetic cellulosome nanostructures have also been created using either protein nanoparticles or DNA as a scaffold, there is the potential to tether these nanostructures onto living cells in order to further enhance the overall efficiency.

Recombinant protein polymer-antibody conjugates for applications in nanotechnology and biomedicine.

Currently, there are over 100 antibody-based therapeutics on the market for the treatment of various diseases. The increasing importance of antibody treatment is further highlighted by the recent FDA emergency use authorization of certain antibody therapies for COVID-19 treatment. Protein-based materials have gained momentum for antibody delivery due to their biocompatibility, tunable chemistry, monodispersity, and straightforward synthesis and purification. In this review, we discuss progress in engineering the molecular features of protein-based biomaterials, in particular recombinant protein polymers, for introducing novel functionalities and enhancing the delivery properties of antibodies and related binding protein domains.

Tunable and Modular miRNA Classifier through Indirect Associative Toehold Strand Displacement.

The programmability of nucleic acids allows detection devices with complex behaviors to be designed de novo. While highly specific, these high-order circuits are usually sequence constrained, making their adaptability toward biological targets challenging. Here, we devise a new strategy called indirect associative strand displacement to decouple sequence constraints between miRNA inputs and de novo strand displacement circuits. By splitting circuit inputs into their toehold and branch migration regions and controlling their association through a docking strand, we demonstrate how any miRNA sequence can be interfaced with synthetic DNA circuits, including catalytic hairpin assembly and a four-input classifier.

Strategies for Multienzyme Assemblies.

Proteins are not designed to be standalone entities and must coordinate their collective action for optimum performance. Nature has developed through evolution the ability to co-localize the functional partners of a cascade enzymatic reaction in order to ensure efficient exchange of intermediates. Inspired by these natural designs, synthetic scaffolds have been created to enhance the overall biological pathway performance. In this chapter, we describe several DNA- and protein-based scaffold approaches to assemble artificial enzyme cascades for a wide range of applications. We highlight the key benefits and drawbacks of these approaches to provide insights on how to choose the appropriate scaffold for different cascade systems.

A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells.

To effectively reprogram cellular regulatory networks towards desired phenotypes, it is critical to have the ability to provide precise gene regulation in a spatiotemporal manner. We have previously engineered toehold-gated guide RNA (thgRNA) to enable conditional activation of dCas9-mediated transcriptional upregulation in mammalian cells using synthetic RNA triggers. Here, we demonstrate that microRNA (miR)-gated thgRNAs can be transcribed by type II RNA polymerase to allow multiplexed transcriptional activation using both mRNA and miR. Activation is achieved only by proper miR-mediated processing of the flanking 5' cap and 3' poly A tail and hairpin unblocking by mRNA via strand displacement. This new AND-gate design is exploited to elicit conditional protein degradation based on induced expression of a specific ubiquibody. This new strategy may find many new applications in an RNA-responsive manner.

Dynamic modulation of enzyme activity by synthetic CRISPR-Cas6 endonucleases.

In nature, dynamic interactions between enzymes play a crucial role in defining cellular metabolism. By controlling the spatial and temporal organization of these supramolecular complexes called metabolons, natural metabolism can be tuned in a highly dynamic manner. Here, we repurpose the CRISPR-Cas6 family proteins as a synthetic strategy to create dynamic metabolons by combining the ease of RNA processing and the predictability of RNA hybridization for protein assembly. By disturbing RNA-RNA networks using toehold-mediated strand displacement reactions, on-demand assembly and disassembly are achieved using both synthetic RNA triggers and mCherry messenger RNA. Both direct and 'Turn-On' assembly of the pathway enzymes tryptophan-2-monooxygenase and indoleacetamide hydrolase can enhance indole-3-acetic acid production by up to ninefold. Even multimeric enzymes can be assembled to improve malate production by threefold. By interfacing with endogenous mRNAs, more complex metabolons may be constructed, resulting in a self-responsive metabolic machinery capable of adapting to changing cellular demand.

EGFR Ligand Clustering on E2 Bionanoparticles for Targeted Delivery of Chemotherapeutics to Breast Cancer Cells.

Naturally occurring protein nanocages are promising drug carriers because of their uniform size and biocompatibility. Engineering efforts have enhanced the delivery properties of nanocages, but cell specificity and high drug loading remain major challenges. Herein, we fused the SpyTag peptide to the surface of engineered E2 nanocages to enable tunable nanocage decoration and effective E2 cell targeting using a variety of SpyCatcher (SC) fusion proteins. Additionally, the core of the E2 nanocage incorporated four phenylalanine mutations previously shown to allow hydrophobic loading of doxorubicin and pH-responsive release in acidic environments. We functionalized the surface of the nanocage with a highly cell-specific epidermal growth factor receptor (EGFR)-targeting protein conjugate, 4GE11-mCherry-SC, developed previously in our laboratories by employing unnatural amino acid (UAA) protein engineering chemistries. Herein, we demonstrated the benefits of this engineered protein nanocage construct for efficient drug loading, with a straightforward method for removal of the unloaded drug through elastin-like polypeptide-mediated inverse transition cycling. Additionally, we demonstrated approximately 3-fold higher doxorubicin internalization in inflammatory breast cancer cells compared to healthy breast epithelial cells, leading to targeted cell death at concentrations below the IC50 of free doxorubicin. Collectively, these results demonstrated the versatility of our UAA-based EGFR-targeting protein construct to deliver a variety of cargoes efficiently, including engineered E2 nanocages capable of site-specific functionalization and doxorubicin loading.

Deciphering the Design Rules of Toehold-Gated sgRNA for Conditional Activation of Gene Expression and Protein Degradation in Mammalian Cells.

A new class of toehold-gated gRNAs (thgRNAs) has been created to provide conditional gene regulation via RNA-mediated activation. However, the detailed design principles remain elusive. Here, we presented an investigation into the design rules for conditional gRNAs by systematically varying the toehold, stem, and flexible loop regions of thgRNA for optimal gene activation in HeLa cells. We determined that nonspecific interactions between the toehold region and the flexible loop are the main driver for the background leak observed in the OFF state. By trimming the toehold length from 15 to 5 nt, the improved thgNT-F design led to a 38-fold increase in the activated ON state with no observable background leak. The same design rule was successfully adapted to target two different regions on the mCherry mRNA with the same impressive fold change. Using the thgRNA to direct conditional protein degradation, we showed up to 8-fold knockdown of a reporter protein through activating expression of a bifunctional ubiquibody GS2-IpaH9.8. This new strategy may find many new applications for cell culture control or cell therapy by removing unwanted proteins in an RNA-responsive manner.

Incorporation of Endosomolytic Peptides with Varying Disruption Mechanisms into EGFR-Targeted Protein Conjugates: The Effect on Intracellular Protein Delivery and EGFR Specificity in Breast Cancer Cells.

Intracellular delivery of protein therapeutics remains a significant challenge limiting the majority of clinically available protein drugs to extracellular targets. Strategies to deliver proteins to subcellular compartments have traditionally relied on cell-penetrating peptides, which can drive enhanced internalization but exhibit unreliable activity and are rarely able to target specific cells, leading to off-target effects. Moreover, few design rules exist regarding the relative efficacy of various endosomal escape strategies in proteins. Accordingly, we developed a simple fusion modification approach to incorporate endosomolytic peptides onto epidermal growth factor receptor (EGFR)-targeted protein conjugates and performed a systematic comparison of the endosomal escape efficacy, mechanism of action, and capacity to maintain EGFR-targeting specificity of conjugates modified with four different endosomolytic sequences of varying modes of action (Aurein 1.2, GALA, HA2, and L17E). Use of the recently developed Gal8-YFP assay indicated that the fusion of each endosomolytic peptide led to enhanced endosomal disruption. Additionally, the incorporation of each endosomolytic peptide increased the half-life of the internalized protein and lowered lysosomal colocalization, further supporting the membrane-disruptive capacity. Despite this, only EGFR-targeted conjugates modified with Aurein 1.2 or GALA maintained EGFR specificity. These results thus demonstrated that the choice of endosomal escape moiety can substantially affect targeting capability, cytotoxicity, and bioactivity and provided important new insights into endosomolytic peptide selection for the design of targeted protein delivery systems.

Outer membrane vesicles (OMVs) enabled bio-applications: A critical review.

Outer membrane vesicles (OMVs) are nanoscale spherical vesicles released from Gram-negative bacteria. The lipid bilayer membrane structure of OMVs consists of similar components as bacterial membrane and thus has attracted more and more attention in exploiting OMVs' bio-applications. Although the endotoxic lipopolysaccharide on natural OMVs may impose potential limits on their clinical applications, genetic modification can reduce their endotoxicity and decorate OMVs with multiple functional proteins. These genetically engineered OMVs have been employed in various fields including vaccination, drug delivery, cancer therapy, bioimaging, biosensing, and enzyme carrier. This review will first briefly introduce the background of OMVs followed by recent advances in functionalization and various applications of engineered OMVs with an emphasis on the working principles and their performance, and then discuss about the future trends of OMVs in biomedical applications.

Engineering a Blue Light Inducible SpyTag System (BLISS).

The SpyCatcher/SpyTag protein conjugation system has recently exploded in popularity due to its fast kinetics and high yield under biologically favorable conditions in both in vitro and intracellular settings. The utility of this system could be expanded by introducing the ability to spatially and temporally control the conjugation event. Taking inspiration from photoreceptor proteins in nature, we designed a method to integrate light dependency into the protein conjugation reaction. The light-oxygen-voltage domain 2 of Avena sativa (AsLOV2) undergoes a dramatic conformational change in its c-terminal Jα-helix in response to blue light. By inserting SpyTag into the different locations of the Jα-helix, we created a blue light inducible SpyTag system (BLISS). In this design, the SpyTag is blocked from reacting with the SpyCatcher in the dark, but upon irradiation with blue light, the Jα-helix of the AsLOV2 undocks to expose the SpyTag. We tested several insertion sites and characterized the kinetics. We found three variants with dynamic ranges over 15, which were active within different concentration ranges. These could be tuned using SpyCatcher variants with different reaction kinetics. Further, the reaction could be instantaneously quenched by removing light. We demonstrated the spatial aspect of this light control mechanism through photopatterning of two fluorescent proteins. This system offers opportunities for many other biofabrication and optogenetics applications.

Engineering bionanoparticles for improved biosensing and bioimaging.

The importance of bioimaging and biosensing has been clear with the onset of the COVID-19 pandemic. In addition to viral detection, detection of tumors, glucose levels, and microbes is necessary for improved disease treatment and prevention. Bionanoparticles, such as extracellular vesicles and protein nanoparticles, are ideal platforms for biosensing and bioimaging applications because of their propensity for high density surface functionalization and large loading capacity. Scaffolding large numbers of sensing modules and detection modules onto bionanoparticles allows for enhanced analyte affinity and specificity as well as signal amplification for highly sensitive detection even at low analyte concentrations. Here we demonstrate the potential of bionanoparticles for bioimaging and biosensing by highlighting recent examples in literature that utilize protein nanoparticles and extracellular vesicles to generate highly sensitive detection devices with impressive signal amplification.

Self-assembling protein nanocages for modular enzyme assembly by orthogonal bioconjugation.

The wide variety of enzymatic pathways that can benefit from enzyme scaffolding is astronomical. While enzyme co-localization based on protein, DNA, and RNA scaffolds has been reported, we still lack scaffolds that offer well-defined and uniform three-dimensional structures for enzyme organization. Here we reported a new approach for protein co-localization using naturally occurring protein nanocages as a scaffold. Two different nanocages, the 25 nm E2 and the 34 nm heptatitis B virus, were used to demonstrate the successfully co-localization of the endoglucanase CelA and cellulose binding domain using the robust SpyTag/SpyCatcher bioconjugation chemistry. Because of the simplicity of the assembly, this strategy is useful not only for in vivo enzyme cascading but also the potential for in vivo applications as well.

Conditional Protein Rescue by Binding-Induced Protective Shielding.

Synthetic protein-level circuits offer an extra layer of cellular control on top of conventional gene-level circuits. Here, we describe a technology that allows conditional protein rescue (CPR) from proteasomal degradation using different protein inputs as masking agents. A target protein is fused to a degron tag and an affinity sensor domain. The use of nanobodies as the sensor domain offers a generalizable strategy to execute a wide range of protein-level circuits with ease. The utility of this new strategy was successfully demonstrated to distinguish cancer cells out of a healthy population using the HPV-specific E7 protein as a cellular marker. Because CPR can be programmed to execute more complex Boolean logic designs using cell-specific proteomes, this platform offers a highly modular and scalable framework for a wide range of applications based on synthetic protein circuits.

Modular Hepatitis B Virus-like Particle Platform for Biosensing and Drug Delivery.

The hepatitis B virus-like particle (HBV VLP) is an attractive protein nanoparticle platform due to the availability of 240 modification sites for engineering purposes. Although direct protein insertion into the surface loop has been demonstrated, this decoration strategy is restricted by the size of the inserted protein moieties. Meanwhile, larger proteins can be decorated using chemical conjugations; yet these approaches perturb the integrity of more delicate proteins and can unfavorably orient the proteins, impairing active surface display. Herein, we aim to create a robust and highly modular method to produce smart HBV-based nanodevices by using the SpyCatcher/SpyTag system, which allows a wide range of peptides and proteins to be conjugated directly and simply onto the modified HBV capsids in a controlled and biocompatible manner. Our technology allows the modular surface modification of HBV VLPs with multiple components, which provides signal amplification, increased targeting avidity, and high therapeutic payload incorporation. We have achieved a yield of over 200 mg/L for these engineered HBV VLPs and demonstrated the flexibility of this platform in both biosensing and drug delivery applications. The ability to decorate over 200 nanoluciferases per VLP improved detection signal by over 1500-fold, such that low nanomolar levels of thrombin could be detected by the naked eye. Meanwhile, a dimeric prodrug-activating enzyme was loaded without cross-linking particles by coexpressing orthogonally labeled monomers. This along with a epidermal growth factor receptor-binding peptide enabled tunable uptake of HBV VLPs into inflammatory breast cancer cells, leading to efficient suicide enzyme delivery and cell killing.