Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function.

The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.

Safety and Biodistribution of Nanoligomers Targeting the SARS-CoV-2 Genome for the Treatment of COVID-19.

As the world braces to enter its fourth year of the coronavirus disease 2019 (COVID-19) pandemic, the need for accessible and effective antiviral therapeutics continues to be felt globally. The recent surge of Omicron variant cases has demonstrated that vaccination and prevention alone cannot quell the spread of highly transmissible variants. A safe and nontoxic therapeutic with an adaptable design to respond to the emergence of new variants is critical for transitioning to the treatment of COVID-19 as an endemic disease. Here, we present a novel compound, called SBCoV202, that specifically and tightly binds the translation initiation site of RNA-dependent RNA polymerase within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, inhibiting viral replication. SBCoV202 is a Nanoligomer, a molecule that includes peptide nucleic acid sequences capable of binding viral RNA with single-base-pair specificity to accurately target the viral genome. The compound has been shown to be safe and nontoxic in mice, with favorable biodistribution, and has shown efficacy against SARS-CoV-2 in vitro. Safety and biodistribution were assessed using three separate administration methods, namely, intranasal, intravenous, and intraperitoneal. Safety studies showed the Nanoligomer caused no outward distress, immunogenicity, or organ tissue damage, measured through observation of behavior and body weight, serum levels of cytokines, and histopathology of fixed tissue, respectively. SBCoV202 was evenly biodistributed throughout the body, with most tissues measuring Nanoligomer concentrations well above the compound KD of 3.37 nM. In addition to favorable availability to organs such as the lungs, lymph nodes, liver, and spleen, the compound circulated through the blood and was rapidly cleared through the renal and urinary systems. The favorable biodistribution and lack of immunogenicity and toxicity set Nanoligomers apart from other antisense therapies, while the adaptability of the nucleic acid sequence of Nanoligomers provides a defense against future emergence of drug resistance, making these molecules an attractive potential treatment for COVID-19.

Reversing radiation-induced immunosuppression using a new therapeutic modality.

Radiation-induced immune suppression poses significant health challenges for millions of patients undergoing cancer chemotherapy and radiotherapy treatment, and astronauts and space tourists travelling to outer space. While a limited number of recombinant protein therapies, such a Sargramostim, are approved for accelerating hematologic recovery, the pronounced role of granulocyte-macrophage colony-stimulating factor (GM-CSF or CSF2) as a proinflammatory cytokine poses additional challenges in creating immune dysfunction towards pathogenic autoimmune diseases. Here we present an approach to high-throughput drug-discovery, target validation, and lead molecule identification using nucleic acid-based molecules. These Nanoligomer™ molecules are rationally designed using a bioinformatics and an artificial intelligence (AI)-based ranking method and synthesized as a single-modality combining 6-different design elements to up- or downregulate gene expression of target gene, resulting in elevated or diminished protein expression of intended target. This method additionally alters related gene network targets ultimately resulting in pathway modulation. This approach was used to perturb and identify the most effective upstream regulators and canonical pathways for therapeutic intervention to reverse radiation-induced immunosuppression. The lead Nanoligomer™ identified in a screen of human donor derived peripheral blood mononuclear cells (PBMCs) upregulated Erythropoietin (EPO) and showed the greatest reversal of radiation induced cytokine changes. It was further tested in vivo in a mouse radiation-model with low-dose (3 mg/kg) intraperitoneal administration and was shown to regulate gene expression of epo in lung tissue as well as counter immune suppression. These results point to the broader applicability of our approach towards drug-discovery, and potential for further investigation of our lead molecule as reversible gene therapy to treat adverse health outcomes induced by radiation exposure.

Nanoligomers Targeting Human miRNA for the Treatment of Severe COVID-19 Are Safe and Nontoxic in Mice.

The devastating effects of the coronavirus disease 2019 (COVID-19) pandemic have made clear a global necessity for antiviral strategies. Most fatalities associated with infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) result at least partially from uncontrolled host immune response. Here, we use an antisense compound targeting a previously identified microRNA (miRNA) linked to severe cases of COVID-19. The compound binds specifically to the miRNA in question, miR-2392, which is produced by human cells in several disease states. The safety and biodistribution of this compound were tested in a mouse model via intranasal, intraperitoneal, and intravenous administration. The compound did not cause any toxic responses in mice based on measured parameters, including body weight, serum biomarkers for inflammation, and organ histopathology. No immunogenicity from the compound was observed with any administration route. Intranasal administration resulted in excellent and rapid biodistribution to the lungs, the main site of infection for SARS-CoV-2. Pharmacokinetic and biodistribution studies reveal delivery to different organs, including lungs, liver, kidneys, and spleen. The compound was largely cleared through the kidneys and excreted via the urine, with no accumulation observed in first-pass organs. The compound is concluded to be a safe potential antiviral treatment for COVID-19.

Development of potent and effective synthetic SARS-CoV-2 neutralizing nanobodies.

The respiratory virus responsible for coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected nearly every aspect of life worldwide, claiming the lives of over 3.9 million people globally, at the time of this publication. Neutralizing humanized nanobody (VHH)-based antibodies (VHH-huFc) represent a promising therapeutic intervention strategy to address the current SARS-CoV-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity VHH bacteriophage library, several potent neutralizing VHH-huFc antibodies were identified and evaluated for their capacity to tightly bind to the SARS-CoV-2 receptor-binding domain, to prevent binding of SARS-CoV-2 spike (S) to the cellular receptor angiotensin-converting enzyme 2, and to neutralize viral infection. Preliminary preclinical evaluation of multiple VHH-huFc antibody candidates demonstrate that they are prophylactically and therapeutically effective in vivo against wildtype SARS-CoV-2. The identified and characterized VHH-huFc antibodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic.

Facile accelerated specific therapeutic (FAST) platform develops antisense therapies to counter multidrug-resistant bacteria.

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase Klebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae, and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST platform offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells.

Design of a De Novo Aggregating Antimicrobial Peptide and a Bacterial Conjugation-Based Delivery System.

Antibacterial resistance necessitates the development of novel treatment methods for infections. Protein aggregates have recently been applied as antimicrobials to disrupt bacterial homeostasis. Past work on protein aggregates has focused on genome mining for aggregation-prone sequences in bacterial genomes rather than on rational design of aggregating antimicrobial peptides. Here, we use a synthetic biology approach to design an artificial gene encoding a de novo aggregating antimicrobial peptide. This artificial gene, opaL (overexpressed protein aggregator lipophilic), disrupts bacterial homeostasis by expressing extremely hydrophobic peptides. When this hydrophobic sequence is disrupted by acidic residues, consequent aggregation and antimicrobial effect decrease. Further, we developed a probiotic delivery system using the broad-host range conjugative plasmid RK2 to transfer the gene from donor to recipient bacteria. We utilize RK2 to mobilize a shuttle plasmid carrying opaL by adding the RK2 origin of transfer. We show that opaL is nontoxic to the donor, allowing for maintenance and transfer since its expression is under control of a promoter with a recipient-specific T7 RNA polymerase. Upon mating of donor and recipient Escherichia coli, we observe selective growth repression in T7 polymerase-expressing recipients. This technique could be used to target desired pathogens by selecting pathogen-specific promoters to control T7 RNA polymerase expression and provides a basis for the design and delivery of aggregating antimicrobial peptides.

Isolating the Escherichia coli Transcriptomic Response to Superoxide Generation from Cadmium Chalcogenide Quantum Dots.

Nanomaterials have been extensively used in the biomedical field and have recently garnered attention as potential antimicrobial agents. Cadmium telluride quantum dots (QDs) with a bandgap of 2.4 eV (CdTe-2.4) were previously shown to inhibit multidrug-resistant clinical isolates of bacterial pathogens via light-activated superoxide generation. Here we investigate the transcriptomic response of Escherichia coli to phototherapeutic CdTe-2.4 QDs both with and without illumination, as well as in comparison with the non-superoxide-generating cadmium selenide QDs (CdSe-2.4) as a negative control. Our analysis sought to separate the transcriptomic response of E. coli to the generation of superoxide by the CdTe-2.4 QDs from the presence of cadmium chalcogenide nanoparticles alone. We used comparisons between illuminated CdTe-2.4 conditions and all others to establish the superoxide generation response and used comparisons between all QD conditions and the no treatment condition to establish the cadmium chalcogenide QD response. In our analysis of the gene expression experiments, we found eight genes to be consistently differentially expressed as a response to superoxide generation, and these genes demonstrate a consistent association with the DNA damage response and deactivation of iron-sulfur clusters. Each of these responses is characteristic of a bacterial superoxide response. We found 18 genes associated with the presence of cadmium chalcogenide QDs but not the generation of superoxide by CdTe-2.4, including several that implicated metabolism of amino acids in the E. coli response. To explore each of these gene sets further, we performed both gene knockout and amino acid supplementation experiments. We identified the importance of leucyl-tRNA downregulation as a cadmium chalcogenide QD response and reinforced the relationship between CdTe-2.4 stress and iron-sulfur clusters through examination of the gene tusA. This study demonstrates the transcriptomic response of E. coli to CdTe-2.4 and CdSe-2.4 QDs and parses the different effects of superoxide versus material effects on the bacteria. Our findings may provide useful information toward the development of QD-based antibacterial therapy in the future.

Designing Superoxide-Generating Quantum Dots for Selective Light-Activated Nanotherapy.

The rapid emergence of superbugs, or multi-drug resistant (MDR) organisms, has prompted a search for novel antibiotics, beyond traditional small-molecule therapies. Nanotherapeutics are being investigated as alternatives, and recently superoxide-generating quantum dots (QDs) have been shown as important candidates for selective light-activated therapy, while also potentiating existing antibiotics against MDR superbugs. Their therapeutic action is selective, can be tailored by simply changing their quantum-confined conduction-valence band (CB-VB) positions and alignment with different redox half-reactions-and hence their ability to generate specific radical species in biological media. Here, we show the design of superoxide-generating QDs using optimal QD material and size well-matched to superoxide redox potential, charged ligands to modulate their uptake in cells and selective redox interventions, and core/shell structures to improve their stability for therapeutic action. We show that cadmium telluride (CdTe) QDs with conduction band (CB) position at -0.5 V with respect to Normal Hydrogen Electron (NHE) and visible 2.4 eV bandgap generate a large flux of selective superoxide radicals, thereby demonstrating the effective light-activated therapy. Although the positively charged QDs demonstrate large cellular uptake, they bind indiscriminately to cell surfaces and cause non-selective cell death, while negatively charged and zwitterionic QD ligands reduce the uptake and allow selective therapeutic action via interaction with redox species. The stability of designed QDs in biologically-relevant media increases with the formation of core-shell QD structures, but an appropriate design of core-shell structures is needed to minimize any reduction in charge injection efficiency to adsorbed oxygen molecules (to form superoxide) and maintain similar quantitative generation of tailored redox species, as measured using electron paramagnetic resonance (EPR) spectroscopy and electrochemical impedance spectroscopy (EIS). Using these findings, we demonstrate the rational design of QDs as selective therapeutic to kill more than 99% of a priority class I pathogen, thus providing an effective therapy against MDR superbugs.

Assessing Different Reactive Oxygen Species as Potential Antibiotics: Selectivity of Intracellular Superoxide Generation Using Quantum Dots.

Reactive oxygen species (ROS) represent a broad range of chemical species including superoxide, hydroxyl, singlet oxygen, and hydrogen peroxide. Each species behaves differently in the cellular environment. Some can play specific roles as intracellular signaling molecules, while others act primarily as indiscriminate oxidants. Several recent reports have promoted the use of exogenous ROS as therapeutic agents with applications from cancer therapies to novel antimicrobials. However, therapeutics, specifically antibiotics, should either kill or inhibit the growth of harmful cells (bacteria here) without harming the host cells, and hence selectivity of action is of vital importance. Here, we show that among different ROS, only superoxide was found to be bactericidal, killing a range of multidrug-resistant (MDR) pathogens without affecting the viability or growth of mammalian cells. Superoxide has a high thermodynamic capacity to be a strong oxidant. However, its lack of reactivity with cellular components at a physiological pH, except for the inactivation of biosynthetic enzymes containing labile iron-sulfur clusters, is key to its selectivity. The role of iron in bacterial pathogenesis also makes superoxide a strong candidate for antimicrobial therapy. Additionally, using a series of selective scavengers, we show that the superoxide radical is therapeutically effective and selective compared to other ROS like hydroxyl radicals, confirming previous results that used Escherichia coli gene knockouts to show that superoxide selectively deactivates some enzymes rather than causing indiscriminate damage of cellular components. In our in vitro studies, intracellular superoxide generation using light-activated quantum dots yielded highly selective and effective antimicrobial action. We screened 45 clinical MDR bacterial isolates and observed inhibition/therapeutic action in all strains, highlighting the applicability of such nanoparticle superoxide therapy. These results can pave the way for rational design of nanoscale therapies as precision medicine.

Potentiating antibiotics in drug-resistant clinical isolates via stimuli-activated superoxide generation.

The rise of multidrug-resistant (MDR) bacteria is a growing concern to global health and is exacerbated by the lack of new antibiotics. To treat already pervasive MDR infections, new classes of antibiotics or antibiotic adjuvants are needed. Reactive oxygen species (ROS) have been shown to play a role during antibacterial action; however, it is not yet understood whether ROS contribute directly to or are an outcome of bacterial lethality caused by antibiotics. We show that a light-activated nanoparticle, designed to produce tunable flux of specific ROS, superoxide, potentiates the activity of antibiotics in clinical MDR isolates of Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Despite the high degree of antibiotic resistance in these isolates, we observed a synergistic interaction between both bactericidal and bacteriostatic antibiotics with varied mechanisms of action and our superoxide-producing nanoparticles in more than 75% of combinations. As a result of this potentiation, the effective antibiotic concentration of the clinical isolates was reduced up to 1000-fold below their respective sensitive/resistant breakpoint. Further, superoxide-generating nanoparticles in combination with ciprofloxacin reduced bacterial load in epithelial cells infected with S. enterica serovar Typhimurium and increased Caenorhabditis elegans survival upon infection with S. enterica serovar Enteriditis, compared to antibiotic alone. This demonstration highlights the ability to engineer superoxide generation to potentiate antibiotic activity and combat highly drug-resistant bacterial pathogens.

ROS mediated selection for increased NADPH availability in Escherichia coli.

The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli.

Transcriptional Interference in Convergent Promoters as a Means for Tunable Gene Expression.

An important goal of synthetic biology involves the extension and standardization of novel biological elements for applications in medicine and biotechnology. Transcriptional interference, occurring in sets of convergent promoters, offers a promising mechanism for building elements for the design of tunable gene regulation. Here, we investigate the transcriptional interference mechanisms of antisense roadblock and RNA polymerase traffic in a set of convergent promoters as novel modules for synthetic biology. We show examples of elements, including antisense roadblock, relative promoter strengths, interpromoter distance, and sequence content that can be tuned to give rise to repressive as well as cooperative behaviors, therefore resulting in distinct gene expression patterns. Our approach will be useful toward engineering new biological devices and will bring new insights to naturally occurring cis-antisense systems. Therefore, we are reporting a new biological tool that can be used for synthetic biology.

Photoexcited quantum dots for killing multidrug-resistant bacteria.

Multidrug-resistant bacterial infections are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics. Metal nanoparticles can induce cell death, yet the toxicity effect is typically nonspecific. Here, we show that photoexcited quantum dots (QDs) can kill a wide range of multidrug-resistant bacterial clinical isolates, including methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and extended-spectrum ß-lactamase-producing Klebsiella pneumoniae and Salmonella typhimurium. The killing effect is independent of material and controlled by the redox potentials of the photogenerated charge carriers, which selectively alter the cellular redox state. We also show that the QDs can be tailored to kill 92% of bacterial cells in a monoculture, and in a co-culture of E. coli and HEK 293T cells, while leaving the mammalian cells intact, or to increase bacterial proliferation. Photoexcited QDs could be used in the study of the effect of redox states on living systems, and lead to clinical phototherapy for the treatment of infections.

Sequence-Specific Peptide Nucleic Acid-Based Antisense Inhibitors of TEM-1 ß-Lactamase and Mechanism of Adaptive Resistance.

The recent surge of drug-resistant superbugs and shrinking antibiotic pipeline are serious challenges to global health. In particular, the emergence of ß-lactamases has caused extensive resistance against the most frequently prescribed class of ß-lactam antibiotics. Here, we develop novel synthetic peptide nucleic acid-based antisense inhibitors that target the start codon and ribosomal binding site of the TEM-1 ß-lactamase transcript and act via translation inhibition mechanism. We show that these antisense inhibitors are capable of resensitizing drug-resistant Escherichia coli to ß-lactam antibiotics exhibiting 10-fold reduction in the minimum inhibitory concentration (MIC). To study the mechanism of resistance, we adapted E. coli at MIC levels of the ß-lactam/antisense inhibitor combination and observed a nonmutational, bet-hedging based adaptive antibiotic resistance response as evidenced by phenotypic heterogeneity as well as heterogeneous expression of key stress response genes. Our data show that both the development of new antimicrobials and an understanding of cellular response during the development of tolerance could aid in mitigating the impending antibiotic crisis.