(680) A strategy to edit disease-causing, segmental duplications in CFTR

Background: There are numerous variants of the CFTR gene that cause CF by preventing production of the CFTR protein. These variants are not capable of responding to modulators, and thus alternative strategies are needed to provide CFTR function to individuals carrying these mutations. One such group of mutations is those in which large segments of the CFTR gene are either duplicated or deleted, referred to as segmental duplications and deletions, respectively. A large proportion of individuals with cystic fibrosis, but for whom two pathogenic CFTR variants are not detected by conventional genotyping, are now recognized to carry duplications and/or deletions, undetected previously because the underlying sequence is not altered, but rather differs only in copy number. To date, there are 9 such duplications in CFTR that have been reported in the literature. We have added strategies to our program in gene editing to address these variants by a process to remove duplicated sequences and render the CFTR gene intact, in single copy, capable of producing functional CFTR protein. This strategy requires no template DNA and thus minimizing the number of editing components needed.

Methods: The strategy involves the use of sequence-targeted DNA editors such as CRISPR/Cas9 that induce double-strand DNA breaks at user-selected sites in the CFTR gene. Sites in non-coding portions of the duplicated region (introns) are identified computationally as DNA cleavage sites for which gRNAs with high on-target scores and low off-target scores are selected. Successful gRNAs, in combination with Cas9, will remove the duplicated sequence by recognizing and cleaving a unique sequence that occurs twice because of the duplication. The duplication is excised and non-homologous end-joining repairs the cleaved ends. This project focuses on a single mutation, CFTRdup7-11, as a proof-of-concept study with the ultimate goal of developing a panel of editors that could treat all of the known duplications. A fluorescent and luminescent reporter system was developed in which duplicated gRNA recognition sequences flank transcriptional terminators such that cleavage of the gRNA sequences allow production and detection of the reporter. The modular nature of the reporter allows multiple gRNAs to be compared in parallel. Primary nasal epithelia cells have recently been obtained from an individual with CF who carries the CFTRdup7-11 allele in trans to a frame-shifting deletion.

Results: The reporter construct was used to evaluate gRNAs with cognate sequences in introns 9 and 10, and compared to a reference gRNA in exon 11. We found the gRNAs for both introns 9 and 10 were substantially more effective than the exon 11 gRNA. These gRNAs are now being evaluated in the primary airway cells to determine if they restore detectable CFTR function. Short circuit current measurements show that monolayers of these primary cells fail to respond to CFTR-stimulating compounds, such as forskolin, and experiments to measure currents after editing are underway.

Conclusions: Segmental duplications and deletions are becoming increasingly appreciated as advanced sequencing technologies are applied. We have developed a fluorescence and luminescence-based reporter system on which to evaluate platforms for removing duplications. The reporter system allows rapid screening of candidate gRNAs to empirically test for efficiency of editing.

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