An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts.

In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines.

Impact of mRNA chemistry and manufacturing process on innate immune activation.

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation, here, we compared, in multiple cell and in vivo models, mRNA that encodes human erythropoietin incorporating either canonical uridine or 1-methyl-pseudouridine (1mΨ), synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1mΨ made by the modified process, while mRNA containing canonical uridine was immunostimulatory regardless of process.

Targeting Cancer Gene Dependencies with Anthrax-Mediated Delivery of Peptide Nucleic Acids.

Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the gene, rendering these cancers vulnerable after further suppression.

Characterization of Novel Piperidine-Based Inhibitor of Cathepsin B-Dependent Bacterial Toxins and Viruses.

Exploiting the host endocytic trafficking pathway is a common mechanism by which bacterial exotoxins gain entry to exert virulent effects upon the host cells. A previous study identified a small-molecule, 1-(2,6-dimethyl-1-piperidinyl)-3-[(2-isopropyl-5-methylcyclohexyl)oxy]-2-propanol, that blocks the process of anthrax lethal toxin (LT) cytotoxicity. Here, we report the characterization of the bioactivity of this compound, which we named RC1.

Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase.

Ras/Rap1-specific endopeptidase (RRSP) is a cytotoxic effector domain of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of highly virulent strains of Vibrio vulnificus. RRSP blocks RAS-MAPK kinase signaling by cleaving Ras and Rap1 within the switch I region between Y32 and D33. Although the RRSP processing site is highly conserved among small GTPases, only Ras and Rap1 have been identified as proteolytic substrates.

Delivery of Non-Native Cargo into Mammalian Cells Using Anthrax Lethal Toxin.

The intracellular delivery of peptide and protein therapeutics is a major challenge due to the plasma membrane, which acts as a barrier between the extracellular environment and the intracellular milieu. Over the past two decades, a nontoxic PA/LFN delivery platform derived from anthrax lethal toxin has been developed for the transport of non-native cargo into the cytosol of cells in order to understand the translocation process through a protective antigen (PA) pore and to probe intracellular biological functions. Enzyme-mediated ligation using sortase A and native chemical ligation are two facile methods used to synthesize these non-native conjugates, inaccessible by recombinant technology.

A d-Amino Acid at the N-Terminus of a Protein Abrogates Its Degradation by the N-End Rule Pathway.

Eukaryotes have evolved the ubiquitin (Ub)/proteasome system to degrade polypeptides. The Ub/proteasome system is one way that cells regulate cytosolic protein and amino acids levels through the recognition and ubiquitination of a protein's N-terminus via E1, E2, and E3 enzymes. The process by which the N-terminus stimulates intracellular protein degradation is referred to as the N-end rule.

Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen.

A variety of pathogenic bacteria infect host eukaryotic cells using protein toxins, which enter the cytosol and exert their cytotoxic effects. Anthrax lethal toxin, for example, utilizes the membrane-spanning translocase, protective antigen (PA) pore, to deliver the protein toxin lethal factor (LF) from the endosome into the cytosol of cells. Previous work has investigated the delivery of natural peptides and enzymatic domains appended to the C-terminus of the PA-binding domain of lethal factor (LFN) into the cytosol via PA pore.

Delivery of mirror image polypeptides into cells.

Mirror image peptides have unique stability and immunogenic properties in mammals, making them attractive agents to investigate. Their properties inside cells have been mostly unexplored because biopolymers are difficult to transport across cellular membranes. Here, we used protective antigen (PA) from anthrax toxin to deliver mirror image polypeptide cargo into the cytosol of mammalian cells when conjugated to the C-terminus of the PA-binding domain of lethal factor, LF.

Delivery of antibody mimics into mammalian cells via anthrax toxin protective antigen.

Antibody mimics have significant scientific and therapeutic utility for the disruption of protein-protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol.

Flow-based enzymatic ligation by sortase A.

Sortase-mediated ligation (sortagging) is a versatile, powerful strategy for protein modification. Because the sortase reaction reaches equilibrium, a large excess of polyglycine nucleophile is often employed to drive the reaction forward and suppress sortase-mediated side reactions. A flow-based sortagging platform employing immobilized sortase A within a microreactor was developed that permits efficient sortagging at low nucleophile concentrations.

Protein thioester synthesis enabled by sortase.

Proteins containing a C-terminal thioester are important intermediates in semisynthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. Here we report a simple strategy, utilizing sortase A, for routine preparation of recombinant proteins containing a C-terminal (α)thioester.

A water soluble vitamin B12-ReI fluorescent conjugate for cell uptake screens: use in the confirmation of cubilin in the lung cancer line A549.

A water soluble vitamin B(12)-rhenium conjugate was synthesized and used in concert with intrinsic factor to screen for cubilin receptor-mediated uptake in lung cancer cells. Internalization of the conjugate demonstrated that it could be used to rapidly screen for the cubilin receptor in living cells, subsequently confirmed with Western blotting and RT-PCR.

Trifluoracetic acid-assisted crystallization of vitamin B12 results in protonation of the phosphate group of the nucleotide loop: insight into the influence of crystal packing forces on vitamin B12 structures.

In the course of experiments concerning our ongoing project on the synthesis of vitamin B(12) (cyanocobalamin, CNCbl) bioconjugates for drug-delivery purposes, we observed the formation of well-shaped red parallelepipeds from a concentrated aqueous solution of the HPLC-purified vitamin. The X-ray structural investigation (MoK(α)) at 98 K on these crystals revealed a CNCbl-TFA salt of formula [CNCbl(H)](TFAc)·14H(2)O (1, where TFA = trifluoracetic acid; TFAc(-) = trifluoracetate anion), in which a proton transfer from the trifluoracetic acid to the phosphate-O4P oxygen atoms is observed. 1 crystallizes in the standard orthorhombic P2(1)2(1)2(1) space group, a = 16.

Targeting the cubilin receptor through the vitamin B(12) uptake pathway: cytotoxicity and mechanistic insight through fluorescent Re(I) delivery.

The intrinsic factor (IF) vitamin B(12) ileum anchored receptor, cubilin, mediates endocytotic uptake of the IF complex of vitamin B(12) to the blood serum. This receptor was targeted for the selective delivery and accumulation of a new bioprobe, a B(12) conjugate of rhenium 2, in the cubilin expressing placental choriocarcinoma BeWo cell line. Competitive uptake and cytotoxicity assays of 2 were investigated and interactions with nuclear DNA explored.

Hepatoma cell uptake of cationic multifluorescent quantum dot liposomes.

Cationic multifluorescent quantum dot liposomes (QD-Ls) have been prepared with both hydrophobic and hydrophilic CdSe/ZnS quantum dots by reverse phase evaporation. QD incorporation was confirmed by fluorescence and confocal microscopy. Incorporation did not affect QD photoactivity or damage bilayer or liposome structure.

Targeting the folate receptor (FR): imaging and cytotoxicity of ReI conjugates in FR-overexpressing cancer cells.

The synthesis, characterization, in vitro imaging, and cytotoxic properties of a new folate conjugate of rhenium(I) are reported. The conjugate [FA-PEG-BQAV-Re(CO)3]+ (gamma-4) was screened against an adriamycin- and cisplatin-resistant human ovarian cancer cell line (A2780/AD) that overexpresses the folate receptor (FR). Compound gamma-4 was internalized by a folate-receptor-mediated endocytotic pathway, which results in internal accumulation of gamma-4.