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CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo. Cas9 base editors are promising tools for correcting pathogenic single nucleotide mutations. Here the authors chemically modify mRNA encoding the editor and the gRNA to enhance editing and broaden its application.

Cystic fibrosis is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of CFTR using established animal and cell-based models led to the recent discovery of effective modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we generate a panel of airway epithelial cells using induced pluripotent stem cells from individuals with common or rare CFTR variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function we adapt two established in vitro assays for use in induced pluripotent stem cell-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we detect genotype-specific differences in CFTR baseline function and response to CFTR modulators. These results demonstrate the potential of the human induced pluripotent stem cell platform as a research tool to study CF and in particular accelerate therapeutic development for CF caused by rare variants. Hundreds of mutations in the gene CFTR lead to cystic fibrosis and represent a challenge to developing therapeutics. Here, authors demonstrate the ability of airway cells derived from human iPSCs to model genotype-specific CFTR function as well as pharmacologic rescue of disease causing mutations.

Base editing (BE) can permanently correct over half of known human pathogenic genetic variants without requiring a repair template, thus serving as a promising therapeutic tool to treat a broad spectrum of genetic diseases. However, the broad activity windows of current base editors pose a major challenge to their therapeutic application. Here, we show that integrating a naturally occurring oligonucleotide binding module into the deaminase active center of TadA-8e, a highly active deoxyadenosine deaminase, enhances its editing specificity. When conjugated with a Cas9 nickase or alternative PAM Cas9 variants, the engineered TadA variant—TadA-NW1—consistently achieves robust A-to-G editing efficiencies within an editing window consisting of four nucleotides, substantially narrower than the 10-bp editing window of the TadA-8e-derived ABEs. Moreover, compared to ABE8e, ABE-NW1 shows significantly decreased Cas9-dependent and -independent off-target activity while maintaining similar on-target editing efficiency. Further, TadA-NW1 can be reprogrammed to perform desired cytidine deamination and adenine transversion within a restricted editing window. Finally, in a cystic fibrosis (CF) cell model, ABE-NW1 outperforms existing ABEs in accurately and efficiently correcting the CFTR W1282X variant, one of the most common CF-causing mutations. In all, we engineered a suite of base editors with refined activity windows, enabling more precise base editing. Importantly, this study presents a streamlined genome editor re-engineering strategy to accelerate the development of therapeutic base editing. The broad activity windows of current base editors pose a major challenge to their therapeutic application. Here, the authors established a generalizable re-engineering framework to narrow the activity windows of diverse base editors, streamlining the development of therapeutic base editing.

Nonsense mutations arise from single nucleotide substitutions that result in premature termination codons (PTCs). PTCs result in little to no full-length protein production and loss of mRNA expression through the nonsense-mediated mRNA decay (NMD) pathway. We demonstrate that anticodon engineered (ACE-) tRNAs efficiently suppress the most prevalent cystic fibrosis (CF) causing PTCs, promoting significant rescue of endogenous cystic fibrosis transmembrane conductance regulator (CFTR) transcript abundance and channel function in different model systems. We demonstrate that our best-performing ACE-tRNA, that decodes all UGA PTCs to a leucine amino acid, markedly rescues CFTR channel function from the most prevalent CF causing PTCs that arise from non-leucine encoding codons. Using this single ACE-tRNA variant, we demonstrate significant rescue of CFTR channel function in an immortalized airway cell line and two different primary CF patient-derived intestinal cell models with CFTR nonsense mutations. Thus, ACE-tRNAs have promise as a platform therapeutic for CF and other nonsense-associated diseases.

We report the engineering of lipid nanoparticles (LNPs) to transport CRISPR/Cas9 payloads, including double-stranded DNA (dsDNA) donor templates, designed for homology directed repair (HDR)-mediated site-specific insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to correct cystic fibrosis (CF) in diseased airway epithelium. We screened various nanoparticle formulations, adjusting ratios of Cas9-encoding mRNA, single guide RNAs (sgRNAs), and dsDNA donor templates to optimize gene editing using human bronchial epithelial cells (16HBE14o-) harboring a CF-causing mutation (G542X). Populations of G542X cells edited via LNP delivery of CFTR donors achieved 3 - 3.5% gene integration and yielded comparable CFTR protein expression compared to normal 16HBE14o- controls. These edited populations exhibit restoration of CFTR-dependent Cl- current to ca. 80% of values measured in normal 16HBE14o- cell monolayers. This LNP platform adds capabilities for transporting large gene editing machinery to airway epithelial cells for genomic integration of entire genes, enabling therapeutic solutions that achieve correction of any CF-causing mutation.Competing Interest StatementRAF, PGA, BG, DK, and SJJ are inventors on US patent applications filed by the Regents of the University of California relating to the design of gene editing machinery and lipid nanoparticle formulations targeting epithelial diseases. The other authors declare no competing interests.

Cystic fibrosis (CF) is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane regulator (CFTR) anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of the CFTR channel using established animal and cell-based models led to the recent discovery of effective CFTR modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we assembled a panel of iPSCs derived from individuals with common or rare variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function in patient-specific iPSCs we adapted two established in vitro assays of CFTR function to iPSC-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we quantified CFTR baseline function and response to CFTR modulators and detected genotype-specific differences. Our results demonstrate the potential of the human iPSC platform as a research tool to study cystic fibrosis and in particular accelerate therapeutic development for CF caused by rare mutations. Competing Interest Statement The authors have declared no competing interest.

Inhibitors of the PAD4 enzyme that bind the inactive enzyme link this protein deiminase and the resultant arginine-to-citrulline modification to formation of neutrophil extracellular traps, highly decondensed chromatin structures with both host-defense and pathological roles. PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.