Christian M. Brommel

Base editors can correct disease-causing genetic variants. After a neonate had received a diagnosis of severe carbamoyl-phosphate synthetase 1 deficiency, a disease with an estimated 50% mortality in early infancy, we immediately began to develop a customized lipid nanoparticle-delivered base-editing therapy. After regulatory approval had been obtained for the therapy, the patient received two infusions at approximately 7 and 8 months of age. In the 7 weeks after the initial infusion, the patient was able to receive an increased amount of dietary protein and a reduced dose of a nitrogen-scavenger medication to half the starting dose, without unacceptable adverse events and despite viral illnesses. No serious adverse events occurred. Longer follow-up is warranted to assess safety and efficacy. (Funded by the National Institutes of Health and others.).

Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38-82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.

The list of successful gene therapy trials using adeno-associated virus (AAV)-based vectors continues to grow and includes a wide range of monogenic diseases. Replication incompetent AAV genomes typically remain episomal and expression dilutes as cells divide and die. Consequently, long-term transgene expression from AAV is best suited for quiescent cell types, such as retinal cells, myocytes, or neurons. For genetic diseases that involve cells with steady turnover, AAV-conferred correction may require routine readministration, where every dose carries the risk of developing an adaptive immune response that renders treatment ineffective. Here, we discuss innovative approaches to permanently modify the host genome using AAV-based platforms, thus potentially requiring only a single dose. Such approaches include using AAV delivery of DNA transposons, homologous recombination templates into safe harbors, and nucleases for targeting integration. In tissues with continual cell turnover, genetic modification of progenitor cell populations will help ensure persistent therapeutic outcomes. Combining the safety profile of AAV-based gene therapy vectors with the ability to integrate a therapeutic transgene creates novel solutions to the challenge of lifelong curative treatments for human genetic diseases.

Abstract In a newborn pig cystic fibrosis (CF) model, the ability of gland-containing airways to fight infection was affected by at least two major host-defense defects: impaired mucociliary transport and a lower airway surface liquid (ASL) pH. In the gland-containing airways, the ASL pH is balanced by CFTR (CF transmembrane conductance regulator) and ATP12A, which, respectively, control HCO3 − transport and proton secretion. We found that, although porcine small airway tissue expressed lower amounts of ATP12A, the ASL of epithelial cultures from CF distal small airways (diameter < 200 μm) were nevertheless more acidic (compared with non-CF airways). Therefore, we hypothesized that gland-containing airways and small airways control acidification using distinct mechanisms. Our microarray data suggested that small airway epithelia mediate proton secretion via ATP6V0D2, an isoform of the V0 d subunit of the H+-translocating plasma membrane V-type ATPase. Immunofluorescence of small airways verified the expression of the V0 d2 subunit isoform at the apical surface of Muc5B+ secretory cells, but not ciliated cells. Inhibiting the V-type ATPase with bafilomycin A1 elevated the ASL pH of small airway cultures, in the presence or absence of HCO3 −, and decreased ASL viscosity. These data suggest that, unlike large airways, which are acidified by ATP12A activity, small airways are acidified by V-type ATPase, thus identifying V-type ATPase as a novel therapeutic target for small airway diseases.

The airway-surface liquid pH (pHASL) is slightly acidic relative to the plasma and becomes more acidic in airway diseases, leading to impaired host defense. CO2 in the large airways decreases during inspiration (0.04% CO2) and increases during expiration (5% CO2). Thus, we hypothesized that pHASL would fluctuate during the respiratory cycle. We measured pHASL on cultures of airway epithelia while changing apical CO2 concentrations. Changing apical CO2 produced only very slow pHASL changes, occurring in minutes, inconsistent with respiratory phases that occur in a few seconds. We hypothesized that pH changes were slow because airway-surface liquid has little carbonic anhydrase activity. To test this hypothesis, we applied the carbonic anhydrase inhibitor acetazolamide and found minimal effects on CO2-induced pHASL changes. In contrast, adding carbonic anhydrase significantly increased the rate of change in pHASL. Using pH-dependent rates obtained from these experiments, we modeled the pHASL during respiration to further understand how pH changes with physiologic and pathophysiologic respiratory cycles. Modeled pHASL oscillations were small and affected by the respiration rate, but not the inspiratory:expiratory ratio. Modeled equilibrium pHASL was affected by the inspiratory:expiratory ratio, but not the respiration rate. The airway epithelium is the only tissue that is exposed to large and rapid CO2 fluctuations. We speculate that the airways may have evolved minimal carbonic anhydrase activity to mitigate large changes in the pHASL during breathing that could potentially affect pH-sensitive components of ASL.