Potent and Selective Inhibition of A‑to‑I RNA Editing with 2′‑<i>O</i>‑Methyl/Locked Nucleic Acid-Containing Antisense Oligoribonucleotides

ADARs (adenosine deaminases acting on RNA) are RNA editing enzymes that bind double helical RNAs and deaminate select adenosines (A). The product inosine (I) is read during translation as guanosine (G), so such changes can alter codon meaning. ADAR-catalyzed A to I changes occur in coding sequences for several proteins of importance to the nervous system. However, these sites constitute only a very small fraction of known A to I sites in the human transcriptome, and the significance of editing at the vast majority sites is unknown at this time. Site-selective inhibitors of RNA editing are needed to advance our understanding of the function of editing at specific sites. Here we show that 2′-<i>O</i>-methyl/locked nucleic acid (LNA) mixmer antisense oligonucleotides are potent and selective inhibitors of RNA editing on two different target RNAs. These reagents are capable of binding with high affinity to RNA editing substrates and remodeling the secondary structure by a strand-invasion mechanism. The potency observed here for 2′-<i>O</i>-methyl/LNA mixmers suggests this backbone structure is superior to the morpholino backbone structure for inhibition of RNA editing. Finally, we demonstrate antisense inhibition of editing of the mRNA for the DNA repair glycosylase NEIL1 in cultured human cells, providing a new approach to exploring the link between RNA editing and the cellular response to oxidative DNA damage

The Molecular Basis for Binding of an Electron Transfer Protein to a Metal Oxide Surface

Achieving fast electron transfer between a material and protein is a long-standing challenge confronting applications in bioelectronics, bioelectrocatalysis, and optobioelectronics. Interestingly, naturally occurring extracellular electron transfer proteins bind to and reduce metal oxides fast enough to enable cell growth, and thus could offer insight into solving this coupling problem. While structures of several extracellular electron transfer proteins are known, an understanding of how these proteins bind to their metal oxide substrates has remained elusive because this abiotic–biotic interface is inaccessible to traditional structural methods. Here, we use advanced footprinting techniques to investigate binding between the <i>Shewanella oneidensis</i> MR-1 extracellular electron transfer protein MtrF and one of its substrates, α-Fe₂O₃ nanoparticles, at the molecular level. We find that MtrF binds α-Fe₂O₃ specifically, but not tightly. Nanoparticle binding does not induce significant conformational changes in MtrF, but instead protects specific residues on the face of MtrF likely to be involved in electron transfer. Surprisingly, these residues are separated in primary sequence, but cluster into a small 3D putative binding site. This binding site is located near a local pocket of positive charge that is complementary to the negatively charged α-Fe₂O₃ surface, and mutational analysis indicates that electrostatic interactions in this 3D pocket modulate MtrF–nanoparticle binding. Strikingly, these results show that binding of MtrF to α-Fe₂O₃ follows a strategy to connect proteins to materials that resembles the binding between donor–acceptor electron transfer proteins. Thus, by developing a new methodology to probe protein–nanoparticle binding at the molecular level, this work reveals one of nature’s strategies for achieving fast, efficient electron transfer between proteins and materials

Preferential Identification of Agonistic OX40 Antibodies by Using Cell Lysate to Pan Natively Paired, Humanized Mouse-Derived Yeast Surface Display Libraries

To discover therapeutically relevant antibody candidates, many groups use mouse immunization followed by hybridoma generation or B cell screening. One modern approach is to screen B cells by generating natively paired single chain variable fragment (scFv) display libraries in yeast. Such methods typically rely on soluble antigens for scFv library screening. However, many therapeutically relevant cell-surface targets are difficult to express in a soluble protein format, complicating discovery. In this study, we developed methods to screen humanized mouse-derived yeast scFv libraries using recombinant OX40 protein in cell lysate. We used deep sequencing to compare screening with cell lysate to screening with soluble OX40 protein, in the context of mouse immunizations using either soluble OX40 or OX40-expressing cells and OX40-encoding DNA vector. We found that all tested methods produce a unique diversity of scFv binders. However, when we reformatted forty-one of these scFv as full-length monoclonal antibodies (mAbs), we observed that mAbs identified using soluble antigen immunization with cell lysate sorting always bound cell surface OX40, whereas other methods had significant false positive rates. Antibodies identified using soluble antigen immunization and cell lysate sorting were also significantly more likely to activate OX40 in a cellular assay. Our data suggest that sorting with OX40 protein in cell lysate is more likely than other methods to retain the epitopes required for antibody-mediated OX40 agonism

GMP Manufacturing and IND-Enabling Studies of a Recombinant Hyperimmune Globulin Targeting SARS-CoV-2

Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus