Design and application of the transformer base editor in mammalian cells and mice.

Fusing apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like cytidine deaminase with catalytically impaired Cas proteins (e.g., nCas9 or dCas9) provides a novel gene-editing technology, base editing, that grants targeted base substitutions with high efficiency. However, genome-wide and transcriptome-wide off-target mutations are observed in base editing, which raises safety concerns regarding therapeutic applications. Previously, we developed a new base editing system, the transformer base editor (tBE), to induce efficient editing with no observable genome-wide or transcriptome-wide off-target mutations both in mammalian cells and in mice. Here we describe a detailed protocol for the design and application of the tBE. Steps for designing single-guide RNA (sgRNA) and helper sgRNA pairs, making constructs, determining the genome-wide and transcriptome-wide off-target mutations, producing the tBE-containing adeno-associated viruses, delivering adeno-associated viruses into mice and examining the in vivo editing effects are included in this protocol. High-precision base editing by the tBE can be completed within 2-3 weeks (in mammalian cells) or within 6-8 weeks (in mice), with sgRNA-helper sgRNA pairs. The whole process can be collaboratively accomplished by researchers using standard techniques from molecular biology, bioinformatics and mouse husbandry.

Helper-Polymer Based Five-Element Nanoparticles (FNPs) for Lung-Specific mRNA Delivery with Long-Term Stability after Lyophilization.

Lipid nanoparticles (LNPs) carrying therapeutic mRNAs hold great promise in treating lung-associated diseases like viral infections, tumors, and genetic disorders. However, because of their thermodynamically unstable nature, traditional LNPs carrying mRNAs need to be stored at low temperatures, which hinders their prevalence. Herein, an efficient lung-specific mRNA delivery platform named five-element nanoparticles (FNPs) is developed in which helper-polymer poly(ß-amino esters) (PBAEs) and DOTAP are used in combination. The new strategy endows FNPs with high stability by increasing the charge repulsion between nanoparticles and the binding force of the aliphatic chains within the nanoparticles. The structure-activity relationship (SAR) shows that PBAEs with E1 end-caps, higher degrees of polymerization, and longer alkyl side chains exhibit higher hit rates. Lyophilized FNP formulations can be stably stored at 4 °C for at least 6 months. Overall, a novel delivery platform with high efficiency, specificity, and stability was developed for advancing mRNA-based therapies for lung-associated diseases.

Poly(beta-amino ester)-Based Nanoparticles Enable Nonviral Delivery of Base Editors for Targeted Tumor Gene Editing.

Base editing is an emerging genome editing technology with the advantages of precise base corrections, no double-strand DNA breaks, and no need for templates, which provides an alternative treatment option for tumors with point mutations. However, effective nonviral delivery systems for base editors (BEs) are still limited. Herein, a series of poly(beta-amino esters) (PBAEs) with varying backbones, side chains, and end caps were synthesized to deliver plasmids of BEs and sgRNA. Efficient transfection and base editing were achieved in HEK-293T-sEGFP and U87-MG-sEGFP reporter cell lines by using lead PBAEs, which were superior to PEI and lipo3k. A single intratumor injection of PBAE/pDNA nanoparticles induced the robust conversion of stopped-EGFP into EGFP in mice bearing xenograft glioma tumors, indicating successful gene editing by ABEmax-NG. Overall, these results demonstrated that PBAEs can efficiently deliver BEs for tumor gene editing both in vitro and in vivo.