Knowledge of CFTR function and cell type expression has advanced greatly since its discovery in 1989.
Indeed, drug therapies such as Ivacafter and Trikafta restore function for most CFTR mutation classes;
however, ~10% of people with CF cannot benefit from these drugs because their CFTR proteins are only
partially produced and there is little the drugs can do to help. Here we use an adenosine deaminase fused to a
CRISPR-Cas9 nickase (termed an Adenine Base Editor (ABE)) that converts an Adenine to a Guanine which
is critical for full length protein production for certain mutations. ABEs do not create double stranded DNA
breaks, do not require homologous recombination templates, and are effective in quiescent cells of the
airways. We recently reported correction of CFTR nonsense mutations using electroporated ABEs in human
airway epithelial cells in vitro as determined by next generation sequencing and correction of chloride current.
However, the lung is a challenging organ to correct using gene editing due to an enormous surface area and
multiple mechanisms to resist vector uptake. Delivery of the ABE to enough of the appropriate airway cells to
be therapeutic is the problem we address in this proposal. Our research group has demonstrated success
using many categories of reagents for modifying the genomes of airway cells. In this proposal, we compare two
ABE delivery tools: adeno-associated viruses (AAVs) and viral-like particles (VLPs). They each have their pros
and cons. We will compare AAV-ABEs and VLP-ABEs in multiple in vitro and in vivo models. We will identify
reagents with improved airway progenitor cell targeting. In vivo editing efficiency will be examined in a new
reporter mouse model termed Gene Editing Reporter 14 (GER14) and an established GFP transgenic pig
model. In addition, in vivo CFTR function will be examined in genetically modified mice with the endogenous
mouse Cftr exon 12 replaced with the human CFTR exon 12 with the R553X mutation (termed hEx12R553X).
Lastly, we ask if secreted mucins inhibit AAV or eVLP delivery or editing efficiency in models of advanced lung
disease. Our goal is to provide a life-long gene repair strategy that could be adapted for a great number of CF
causing mutations. The reagents, methods, and data generated by these experiments could be applied to base
editing for other monogenic disorders, thereby significantly advancing the gene therapy field.