Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency.

The use of modified nucleotides to suppress the interferon response and maintain translation of self-amplifying RNA (saRNA), which has been achieved for mRNA, has not yet succeeded. We identify modified nucleotides that, when substituted at 100% in saRNA, confer innate immune evasion and robust long-term protein expression, and when formulated as a vaccine, protect against lethal SARS-CoV-2 challenge in mice. This discovery advances saRNA therapeutics by enabling prolonged protein expression at low doses.

Complete substitution with modified nucleotides suppresses the early interferon response and increases the potency of self-amplifying RNA.

Self-amplifying RNA (saRNA) will revolutionize vaccines and in situ therapeutics by enabling protein expression for longer duration at lower doses. However, a major barrier to saRNA efficacy is the potent early interferon response triggered upon cellular entry, resulting in saRNA degradation and translational inhibition. Substitution of mRNA with modified nucleotides (modNTPs), such as N1-methylpseudouridine (N1mΨ), reduce the interferon response and enhance expression levels. Multiple attempts to use modNTPs in saRNA have been unsuccessful, leading to the conclusion that modNTPs are incompatible with saRNA, thus hindering further development. Here, contrary to the common dogma in the field, we identify multiple modNTPs that when incorporated into saRNA at 100% substitution confer immune evasion and enhance expression potency. Transfection efficiency enhances by roughly an order of magnitude in difficult to transfect cell types compared to unmodified saRNA, and interferon production reduces by >8 fold compared to unmodified saRNA in human peripheral blood mononuclear cells (PBMCs). Furthermore, we demonstrate expression of viral antigens in vitro and observe significant protection against lethal challenge with a mouse-adapted SARS-CoV-2 strain in vivo . A modified saRNA vaccine, at 100-fold lower dose than a modified mRNA vaccine, results in a statistically improved performance to unmodified saRNA and statistically equivalent performance to modified mRNA. This discovery considerably broadens the potential scope of self-amplifying RNA, enabling entry into previously impossible cell types, as well as the potential to apply saRNA technology to non-vaccine modalities such as cell therapy and protein replacement.

Review of Tolerability of Fremanezumab for Episodic and Chronic Migraine.

Calcitonin gene-related peptide (CGRP) monoclonal antibodies (mAbs) were the first class of medication specifically developed for the prevention of migraine. Fremanezumab is one of four CGRP mAbs currently available and is approved by the US Food and Drug Administration (FDA) for the preventative treatment of episodic and chronic migraines. This narrative review summarizes the history of fremanezumab development, the trials that led to its approval, and the later studies published evaluating its tolerability and efficacy. Evidence of fremanezumab for clinically significant efficacy and tolerability in patients with chronic migraine is especially important when considering the high level of disability, lower quality of life scores, and higher levels of health-care utilization associated with this condition. Multiple clinical trials demonstrated superiority of fremanezumab over placebo in terms of efficacy while demonstrating good tolerability. Treatment-related adverse reactions did not differ significantly compared to placebo and dropout rates were minimal. The most commonly observed treatment-related adverse reaction was mild-to-moderate injection site reaction, described as erythema, pain, induration, or swelling at the injection site.

Radiolabeled Biodistribution of Expansile Nanoparticles: Intraperitoneal Administration Results in Tumor Specific Accumulation.

Nanoparticle biodistribution in vivo is an essential component to the success of nanoparticle-based drug delivery systems. Previous studies with fluorescently labeled expansile nanoparticles, or "eNPs", demonstrated a high specificity of eNPs to tumors that is achieved through a materials-based targeting strategy. However, fluorescent labeling techniques are primarily qualitative in nature and the gold-standard for quantitative evaluation of biodistribution is through radiolabeling. In this manuscript, we synthesize 14C-labeled eNPs to quantitatively evaluate the biodistribution of these particles in a murine model of intraperitoneal mesothelioma via liquid scintillation counting. The results demonstrate a strong specificity of eNPs for tumors that lasts one to 2 weeks postinjection with an overall delivery efficiency to the tumor tissue of 30% of the injected dose which is congruent with prior reports of preclinical efficacy of the technology. Importantly, the route of administration is essential to the eNP's material-based targeting strategy with intraperitoneal administration leading to tumoral accumulation while, in contrast, intravenous administration leads to rapid clearance via the reticuloendothelial system and low tumoral accumulation. A comparison against nanoparticle delivery systems published over the past decade shows that the 30% tumoral delivery efficiency of the eNP is significantly higher than the 0.7% median delivery efficiency of other systems with sufficient quantitative data to define this metric. These results lay a foundation for targeting intraperitoneal tumors and encourage efforts to explore alternative, nonintravenous routes, of delivery to accelerate the translation of nanoparticle therapies to the clinic.

Minimally invasive, sustained-release relaxin-2 microparticles reverse arthrofibrosis.

Substantial advances in biotherapeutics are distinctly lacking for musculoskeletal diseases. Musculoskeletal diseases are biomechanically complex and localized, highlighting the need for novel therapies capable of addressing these issues. All frontline treatment options for arthrofibrosis, a debilitating musculoskeletal disease, fail to treat the disease etiology-the accumulation of fibrotic tissue within the joint space. For millions of patients each year, the lack of modern and effective treatment options necessitates surgery in an attempt to regain joint range of motion (ROM) and escape prolonged pain. Human relaxin-2 (RLX), an endogenous peptide hormone with antifibrotic and antifibrogenic activity, is a promising biotherapeutic candidate for musculoskeletal fibrosis. However, RLX has previously faltered through multiple clinical programs because of pharmacokinetic barriers. Here, we describe the design and in vitro characterization of a tailored drug delivery system for the sustained release of RLX. Drug-loaded, polymeric microparticles released RLX over a multiweek time frame without altering peptide structure or bioactivity. In vivo, intraarticular administration of microparticles in rats resulted in prolonged, localized concentrations of RLX with reduced systemic drug exposure. Furthermore, a single injection of RLX-loaded microparticles restored joint ROM and architecture in an atraumatic rat model of arthrofibrosis with clinically derived end points. Finally, confirmation of RLX receptor expression, RXFP1, in multiple human tissues relevant to arthrofibrosis suggests the clinical translational potential of RLX when administered in a sustained and targeted manner.

In situ gelling and dissolvable hydrogels for use as on-demand wound dressings for burns.

Currently, no dressings utilized in burn clinics provide adhesion, hydration or mechanical strength on the same order as human skin as well as the ability to be atraumatically removed. We report the synthesis, characterization, and in vivo evaluation of in situ polymerized and subsequent dissolvable hydrogels as burn wound dressings. Hydrogel dressings, from a small library of synthesized materials form in situ, exhibit storage moduli between 100-40 000 Pa, dissolve on-demand within 10 minutes to 90 minutes, swell up to 350%, and adhere to both burned and healthy human skin at 0.2-0.3 N cm-2. Further, results from an in vivo porcine second degree burn model demonstrate functional performance with healing equivalent to conventional treatments with the added benefit of facile, in situ application and subsequent removal via dissolution.

Temporary In Situ Hydrogel Dressings for Colon Polypectomies.

Currently, no dressings are utilized after removal of polyps during a colonoscopy rendering these tissue sites susceptible to bleeding, sepsis, and perfusion. We report the design specifications, synthesis, and ex vivo evaluation of in situ polymerized hydrogels as colon wound dressings post polypectomy. The hydrogels exhibited varied properties to include moduli between 100 and 16 000 Pa, dissolution times between 4 h to 7 days or longer, swelling up to 200%, and adhesion to colon tissue from 0.1 to 0.4 N/cm2. The hydrogels displayed minimal cytotoxicity, prevented the migration/spread of bacteria, and exhibited rapid gelation, a requirement for application to the lumen of the colon via an endoscope. This work highlights the structure-property relationship of hydrogels prepared from N-hydroxysuccinimide functionalized PEG cross-linkers and hyperbranched polyethylenimines or 4-arm PEG-NH2 star polymers, and their potential as colon wound dressings.

The Prognosis of Arthrofibroses: Prevalence, Clinical Shortcomings, and Future Prospects.

Fibrosis is the dysregulated biosynthesis of connective tissue that results from persistent infection, high serum cholesterol, surgery, trauma, or prolonged joint immobilization. As a disease that impacts connective tissue, it is prevalent across the body and disrupts normal extracellular and tissue organization. Ultimately, fibrosis impairs the tissue structural, mechanical, or biochemical function. This review describes the clinical landscape of joint fibrosis, that is, arthrofibrosis, including the risk factors and causes, as well as current clinical treatments and their shortcomings. Because treating arthrofibrosis remains an unmet clinical challenge, we present several animal models used for exploration of the physiopathology of arthrofibrosis and summarize their use for testing novel treatments. We then discuss therapeutics for the prevention or treatment of arthrofibrosis that are in preclinical development and in ongoing clinical trials. We conclude with recent findings from molecular biological studies of arthrofibroses that shed insight on future areas of research for improved treatments.

Deimmunized Lysostaphin Synergizes with Small-Molecule Chemotherapies and Resensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

There is an urgent need for novel agents to treat drug-resistant bacterial infections, such as multidrug-resistant Staphylococcus aureus (MRSA). Desirable properties for new antibiotics include high potency, narrow species selectivity, low propensity to elicit new resistance phenotypes, and synergy with standard-of-care (SOC) chemotherapies. Here, we describe analysis of the antibacterial potential exhibited by F12, an innovative anti-MRSA lysin that has been genetically engineered to evade detrimental antidrug immune responses in human patients. F12 possesses high potency and rapid onset of action, it has narrow selectivity against pathogenic staphylococci, and it manifests synergy with numerous SOC antibiotics. Additionally, resistance to F12 and ß-lactam antibiotics appears mutually exclusive, and, importantly, we provide evidence that F12 resensitizes normally resistant MRSA strains to ß-lactams both in vitro and in vivo These results suggest that combinations of F12 and SOC antibiotics are a promising new approach to treating refractory S. aureus infections.

Globally deimmunized lysostaphin evades human immune surveillance and enables highly efficacious repeat dosing.

There is a critical need for novel therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant pathogens, and lysins are among the vanguard of innovative antibiotics under development. Unfortunately, lysins' own microbial origins can elicit detrimental antidrug antibodies (ADAs) that undermine efficacy and threaten patient safety. To create an enhanced anti-MRSA lysin, a novel variant of lysostaphin was engineered by T cell epitope deletion. This "deimmunized" lysostaphin dampened human T cell activation, mitigated ADA responses in human HLA transgenic mice, and enabled safe and efficacious repeated dosing during a 6-week longitudinal infection study. Furthermore, the deimmunized lysostaphin evaded established anti-wild-type immunity, thereby providing significant anti-MRSA protection for animals that were immune experienced to the wild-type enzyme. Last, the enzyme synergized with daptomycin to clear a stringent model of MRSA endocarditis. By mitigating T cell-driven antidrug immunity, deimmunized lysostaphin may enable safe, repeated dosing to treat refractory MRSA infections.

Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 ß-lactamase (P99ßL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 109 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99ßL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

The evolution of function within the Nudix homology clan.

The Nudix homology clan encompasses over 80,000 protein domains from all three domains of life, defined by homology to each other. Proteins with a domain from this clan fall into four general functional classes: pyrophosphohydrolases, isopentenyl diphosphate isomerases (IDIs), adenine/guanine mismatch-specific adenine glycosylases (A/G-specific adenine glycosylases), and nonenzymatic activities such as protein/protein interaction and transcriptional regulation. The largest group, pyrophosphohydrolases, encompasses more than 100 distinct hydrolase specificities. To understand the evolution of this vast number of activities, we assembled and analyzed experimental and structural data for 205 Nudix proteins collected from the literature. We corrected erroneous functions or provided more appropriate descriptions for 53 annotations described in the Gene Ontology Annotation database in this family, and propose 275 new experimentally-based annotations. We manually constructed a structure-guided sequence alignment of 78 Nudix proteins. Using the structural alignment as a seed, we then made an alignment of 347 "select" Nudix homology domains, curated from structurally determined, functionally characterized, or phylogenetically important Nudix domains. Based on our review of Nudix pyrophosphohydrolase structures and specificities, we further analyzed a loop region downstream of the Nudix hydrolase motif previously shown to contact the substrate molecule and possess known functional motifs. This loop region provides a potential structural basis for the functional radiation and evolution of substrate specificity within the hydrolase family. Finally, phylogenetic analyses of the 347 select protein domains and of the complete Nudix homology clan revealed general monophyly with regard to function and a few instances of probable homoplasy. Proteins 2017; 85:775-811. © 2016 Wiley Periodicals, Inc.

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family.

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74-compound library of known Nudix enzyme substrates. We found substrates for four enzymes with kcat /Km values >10,000 M-1  s-1 : Q92EH0_LISIN of Listeria innocua serovar 6a against ADP-ribose, Q5LBB1_BACFN of Bacillus fragilis against 5-Me-CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8-oxo-dATP and 3'-dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty-two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported kcat /Km values exhibited against these canonical substrates are well under 105 M-1  s-1 . By contrast, several Nudix enzymes show much larger kcat /Km values (in the range of 105 to >107 M-1  s-1 ) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810-1822. © 2016 Wiley Periodicals, Inc.

Molecular function prediction for a family exhibiting evolutionary tendencies toward substrate specificity swapping: recurrence of tyrosine aminotransferase activity in the Iα subfamily.

The subfamily Iα aminotransferases are typically categorized as having narrow specificity toward carboxylic amino acids (AATases), or broad specificity that includes aromatic amino acid substrates (TATases). Because of their general role in central metabolism and, more specifically, their association with liver-related diseases in humans, this subfamily is biologically interesting. The substrate specificities for only a few members of this subfamily have been reported, and the reliable prediction of substrate specificity from protein sequence has remained elusive. In this study, a diverse set of aminotransferases was chosen for characterization based on a scoring system that measures the sequence divergence of the active site. The enzymes that were experimentally characterized include both narrow-specificity AATases and broad-specificity TATases, as well as AATases with broader-specificity and TATases with narrower-specificity than the previously known family members. Molecular function and phylogenetic analyses underscored the complexity of this family's evolution as the TATase function does not follow a single evolutionary thread, but rather appears independently multiple times during the evolution of the subfamily. The additional functional characterizations described in this article, alongside a detailed sequence and phylogenetic analysis, provide some novel clues to understanding the evolutionary mechanisms at work in this family.

A continuous fluorescence assay for the characterization of Nudix hydrolases.

The common substrate structure for the functionally diverse Nudix protein superfamily is nucleotide-diphosphate-X, where X is a large variety of leaving groups. The substrate specificity is known for less than 1% of the 29,400 known members. Most activities result in the release of an inorganic phosphate ion or of a product bearing a terminal phosphate moiety. Reactions have typically been monitored by a modification of the discontinuous Fiske-SubbaRow assay, which is relatively insensitive and slow. We report here the development of a continuous fluorescence assay that enables the rapid and accurate determination of substrate specificities in a 96-well format. We used this novel assay to confirm the reported substrate characterizations of MutT and NudD of Escherichia coli and to characterize DR_1025 of Deinococcus radiodurans and MM_0920 of Methanosarcina mazei. Novel findings enabled by the new assay include the following. First, in addition to the well-characterized hydrolysis of 8-oxo-dGTP at the α-ß position, MutT cleaves at the ß-γ phosphate bond at a rate of 3% of that recorded for hydrolysis at the α-ß position. Second, MutT also catalyzes the hydrolysis of 5-methyl-dCTP. Third, 8-oxo-dGTP was observed to be the best substrate for DR_1025 of the 41 compounds screened.

Probing the informational and regulatory plasticity of a transcription factor DNA-binding domain.

Transcription factors have two functional constraints on their evolution: (1) their binding sites must have enough information to be distinguishable from all other sequences in the genome, and (2) they must bind these sites with an affinity that appropriately modulates the rate of transcription. Since both are determined by the biophysical properties of the DNA-binding domain, selection on one will ultimately affect the other. We were interested in understanding how plastic the informational and regulatory properties of a transcription factor are and how transcription factors evolve to balance these constraints. To study this, we developed an in vivo selection system in Escherichia coli to identify variants of the helix-turn-helix transcription factor MarA that bind different sets of binding sites with varying degrees of degeneracy. Unlike previous in vitro methods used to identify novel DNA binders and to probe the plasticity of the binding domain, our selections were done within the context of the initiation complex, selecting for both specific binding within the genome and for a physiologically significant strength of interaction to maintain function of the factor. Using MITOMI, quantitative PCR, and a binding site fitness assay, we characterized the binding, function, and fitness of some of these variants. We observed that a large range of binding preferences, information contents, and activities could be accessed with a few mutations, suggesting that transcriptional regulatory networks are highly adaptable and expandable.

Engineering homooligomeric proteins to detect weak intersite allosteric communication: aminotransferases, a case study.

The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.

Specificity and cooperativity at ß-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions.

Establishing a quantitative understanding of the determinants of affinity in protein-protein interactions remains challenging. For example, TEM-1/ß-lactamase inhibitor protein (BLIP) and SHV-1/BLIP are homologous ß-lactamase/ß-lactamase inhibitor protein complexes with disparate K(d) values (3 nM and 2 µM, respectively), and a single substitution, D104E in SHV-1, results in a 1000-fold enhancement in binding affinity. In TEM-1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV-1 D104 does not in the wild type SHV-1/BLIP co-structure. Here, we present a 1.6 Å crystal structure of the SHV-1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV-1 D104 and BLIP K74 and a 400-fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network.

The fitness landscapes of cis-acting binding sites in different promoter and environmental contexts.

The biophysical nature of the interaction between a transcription factor and its target sequences in vitro is sufficiently well understood to allow for the effects of DNA sequence alterations on affinity to be predicted. But even in relatively simple in vivo systems, the complexities of promoter organization and activity have made it difficult to predict how altering specific interactions between a transcription factor and DNA will affect promoter output. To better understand this, we measured the relative fitness of nearly all Escherichia coli sigma(70) -35 binding sites in different promoter and environmental contexts by competing four randomized -35 promoter libraries controlling the expression of the tetracycline resistance gene (tet)against each other in increasing concentrations of drug. We sequenced populations after competition to determine the relative enrichment of each -35 sequence. We observed a consistent relationship between the frequency of recovery of each -35 binding site and its predicted affinity for sigma(70) that varied depending on the sequence context of the promoter and drug concentration. Overall the relative fitness of each promoter could be predicted by a simple thermodynamic model of transcriptional regulation, in which the rate of transcriptional initiation (and hence fitness) is dependent upon the overall stability of the initiation complex, which in turn is dependent upon the energetic contributions of all sites within the complex. As implied by this model, a decrease in the free energy of association at one site could be compensated for by an increase in the binding energy at another to produce a similar output. Furthermore, these data show that a large and continuous range of transcriptional outputs can be accessed by merely changing the -35, suggesting that evolved or engineered mutations at this site could allow for subtle and precise control over gene expression.

Active site prediction using evolutionary and structural information.

MOTIVATION: The identification of catalytic residues is a key step in understanding the function of enzymes. While a variety of computational methods have been developed for this task, accuracies have remained fairly low. The best existing method exploits information from sequence and structure to achieve a precision (the fraction of predicted catalytic residues that are catalytic) of 18.5% at a corresponding recall (the fraction of catalytic residues identified) of 57% on a standard benchmark. Here we present a new method, Discern, which provides a significant improvement over the state-of-the-art through the use of statistical techniques to derive a model with a small set of features that are jointly predictive of enzyme active sites. RESULTS: In cross-validation experiments on two benchmark datasets from the Catalytic Site Atlas and CATRES resources containing a total of 437 manually curated enzymes spanning 487 SCOP families, Discern increases catalytic site recall between 12% and 20% over methods that combine information from both sequence and structure, and by >or=50% over methods that make use of sequence conservation signal only. Controlled experiments show that Discern's improvement in catalytic residue prediction is derived from the combination of three ingredients: the use of the INTREPID phylogenomic method to extract conservation information; the use of 3D structure data, including features computed for residues that are proximal in the structure; and a statistical regularization procedure to prevent overfitting.