Protospacer modification improves base editing of a canonical splice site variant and recovery of CFTR function in human airway epithelial cells.

Canonical splice site variants affecting the 5' GT and 3' AG nucleotides of introns result in severe missplicing and account for about 10% of disease-causing genomic alterations. Treatment of such variants has proven challenging due to the unstable mRNA or protein isoforms that typically result from disruption of these sites. Here, we investigate CRISPR-Cas9-mediated adenine base editing for such variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We validate a CFTR expression minigene (EMG) system for testing base editing designs for two different targets. We then use the EMG system to test non-standard single-guide RNAs with either shortened or lengthened protospacers to correct the most common cystic fibrosis-causing variant in individuals of African descent (c.2988+1G>A). Varying the spacer region length allowed placement of the editing window in a more efficient context and enabled use of alternate protospacer adjacent motifs. Using these modifications, we restored clinically significant levels of CFTR function to human airway epithelial cells from two donors bearing the c.2988+1G>A variant.

Toward Gene Transfer Nanoparticles as Therapeutics.

Genetic medicine has great potential to treat the underlying causes of many human diseases with exquisite precision, but the field has historically been stymied by delivery as the central challenge. Nanoparticles, engineered constructs the size of natural viruses, are being designed to more closely mimic the delivery efficiency of viruses, while enabling the advantages of increased safety, cargo-carrying flexibility, specific targeting, and ease in manufacturing. The speed in which nonviral gene transfer nanoparticles are making progress in the clinic is accelerating, with clinical validation of multiple nonviral nucleic acid delivery nanoparticle formulations recently FDA approved for both expression and for silencing of genes. While much of this progress has been with lipid nanoparticle formulations, significant development is being made with other nanomaterials for gene transfer as well, with favorable attributes such as biodegradability, scalability, and cell targeting. This review highlights the state of the field, current challenges in delivery, and opportunities for engineered nanomaterials to meet these challenges, including enabling long-term therapeutic gene editing. Delivery technology utilizing different kinds of nanomaterials and varying cargos for gene transfer (DNA, mRNA, and ribonucleoproteins) are discussed. Clinical applications are presented, including for the treatment of genetic diseases such as cystic fibrosis.