Establishment of a conditionally reprogrammed primary eccrine sweat gland culture for evaluation of tissue-specific CFTR function.

BACKGROUND: Sweat chloride concentration is used both for CF diagnosis and for tracking CFTR modulator efficacy over time, but the relationship between sweat chloride and lung health is heterogeneous and informed by CFTR genotype. Here, we endeavored to characterize ion transport in eccrine sweat glands (ESGs). METHODS: First, ESGs were microdissected from a non-CF skin donor to analyze individual glands. We established primary cultures of ESG cells via conditional reprogramming for functional testing of ion transport by short circuit current measurement and examined cell composition by single-cell RNA-sequencing (scRNA-seq) comparing with whole dissociated ESGs. Secondly, we cultured nasal epithelial (NE) cells and ESGs from two people with CF (pwCF) to assess modulator efficacy. Finally, NEs and ESGs were grown from one person with the CFTR genotype F312del/F508del to explore genotype-phenotype heterogeneity. RESULTS: ESG primary cells from individuals without CF demonstrated robust ENaC and CFTR function. scRNA-seq demonstrated both secretory and ductal ESG markers in cultured ESG cells. In both NEs and ESGs from pwCF homozygous for F508del, minimal baseline CFTR function was observed, and treatment with CFTR modulators significantly enhanced function. Notably, NEs from an individual bearing F312del/F508del exhibited significant baseline CFTR function, whereas ESGs from the same person displayed minimal CFTR function, consistent with observed phenotype. CONCLUSIONS: This study has established a novel primary culture technique for ESGs that allows for functional ion transport measurement to assess modulator efficacy and evaluate genotype-phenoytpe heterogeneity. To our knowledge, this is the first reported application of conditional reprogramming and scRNA-seq of microdissected ESGs.

Use of adenine base editing and homology-independent targeted integration strategies to correct the cystic fibrosis causing variant, W1282X.

Small molecule drugs known as modulators can treat ~90% of people with cystic fibrosis (CF), but do not work for premature termination codon variants such as W1282X (c.3846G>A). Here we evaluated two gene editing strategies, Adenine Base Editing (ABE) to correct W1282X, and Homology-Independent Targeted Integration (HITI) of a CFTR superexon comprising exons 23-27 (SE23-27) to enable expression of a CFTR mRNA without W1282X. In Flp-In-293 cells stably expressing a CFTR expression minigene bearing W1282X, ABE corrected 24% of W1282X alleles, rescued CFTR mRNA from nonsense mediated decay and restored protein expression. However, bystander editing at the adjacent adenine (c.3847A>G), caused an amino acid change (R1283G) that affects CFTR maturation and ablates ion channel activity. In primary human nasal epithelial cells homozygous for W1282X, ABE corrected 27% of alleles, but with a notably lower level of bystander editing, and CFTR channel function was restored to 16% of wild-type levels. Using the HITI approach, correct integration of a SE23-27 in intron 22 of the CFTR locus in 16HBEge W1282X cells was detected in 5.8% of alleles, resulting in 7.8% of CFTR transcripts containing the SE23-27 sequence. Analysis of a clonal line homozygous for the HITI-SE23-27 produced full-length mature protein and restored CFTR anion channel activity to 10% of wild-type levels, which could be increased three-fold upon treatment with the triple combination of CF modulators. Overall, these data demonstrate two different editing strategies can successfully correct W1282X, the second most common class I variant, with a concomitant restoration of CFTR function.

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.

SLC26A9 SNP rs7512462 is not associated with lung disease severity or lung function response to ivacaftor in cystic fibrosis patients with G551D-CFTR.

BACKGROUND: The CFTR modulator ivacaftor has been variably effective in treating individuals with cystic fibrosis (CF) who harbor CFTR gating variants such as G551D, as well as other classes of CFTR variants when used with other modulators. Because CFTR genotype does not fully explain this variability, defining genetic modifiers of response to modulator therapy is of particular interest to the field of individualized CF drug therapy. Previous studies have proposed that a variant in SLC26A9 (rs7512462) is associated with lung disease severity and with response to treatment with ivacaftor in individuals with CF who carry G551D or gating variants. METHODS: Given the implications for CF treatment, we re-examined the reported associations in three cohorts; patients enrolled in the Twin and Siblings study at Johns Hopkins University, the CF modifier study at the University of North Carolina at Chapel Hill, and the prospective G551D Observational (GOAL) study. The GOAL study was specifically designed to measure lung function response to ivacaftor. RESULTS: We find no association between SLC26A9 (rs7512462) genotype and lung disease severity (n = 272) or change in lung function at one-, three-, and six-month intervals following ivacaftor treatment(n = 141) in individuals with CF who carry at least one G551D variant. CONCLUSIONS: Our inability to replicate this association indicates that rs7512462 genotype should not be used in treatment decisions.

Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies.

Elucidating the functional consequence of molecular defects underlying genetic diseases enables appropriate design of therapeutic options. Treatment of cystic fibrosis (CF) is an exemplar of this paradigm as the development of CFTR modulator therapies has allowed for targeted and effective treatment of individuals harboring specific genetic variants. However, the mechanism of these drugs limits effectiveness to particular classes of variants that allow production of CFTR protein. Thus, assessment of the molecular mechanism of individual variants is imperative for proper assignment of these precision therapies. This is particularly important when considering variants that affect pre-mRNA splicing, thus limiting success of the existing protein-targeted therapies. Variants affecting splicing can occur throughout exons and introns and the complexity of the process of splicing lends itself to a variety of outcomes, both at the RNA and protein levels, further complicating assessment of disease liability and modulator response. To investigate the scope of this challenge, we evaluated splicing and downstream effects of 52 naturally occurring CFTR variants (exonic = 15, intronic = 37). Expression of constructs containing select CFTR intronic sequences and complete CFTR exonic sequences in cell line models allowed for assessment of RNA and protein-level effects on an allele by allele basis. Characterization of primary nasal epithelial cells obtained from individuals harboring splice variants corroborated in vitro data. Notably, we identified exonic variants that result in complete missplicing and thus a lack of modulator response (e.g. c.2908G>A, c.523A>G), as well as intronic variants that respond to modulators due to the presence of residual normally spliced transcript (e.g. c.4242+2T>C, c.3717+40A>G). Overall, our data reveals diverse molecular outcomes amongst both exonic and intronic variants emphasizing the need to delineate RNA, protein, and functional effects of each variant in order to accurately assign precision therapies.