(634) Generation of a CFTR R1162X “TAG” model and comparison of readthrough to the naturally occurring R1162X “TGA”

Background: Premature termination codon (PTC) mutations in the CFTR coding sequence affect ~10% of patients with Cystic Fibrosis (CF) and effective therapies for these patients remain a serious medical need. The four most common CF-causing CFTR PTC mutations are G542X, W1282X, R553X, and R1162X, all of which are “TGA”/ (opal) stop codons. Drug-induced readthrough is a promising therapeutic approach that can restore expression of full length CFTR and consequently suppress Nonsense-Mediated mRNA Decay (NMD). R1162X is thought to be more promiscuous than other PTC mutations to current readthrough molecules and is being investigated in high-throughput screening campaigns to identify novel readthrough molecules. It is well established that nucleotide sequence surrounding a PTC can affect readthrough; however, it is largely unexplored how different stop codons in the same sequence context can affect readthrough. We created a 16HBEge cell model of CFTR with a R1162X “TAG” (amber) PTC and compared it to the previously established 16HBEge cell model with the patient relevant R1162X “TGA” (opal) mutation. These two cell models express different stop codons at the same position in CFTR and can be used to further explore PTC biology.

Methods: The R1162X “TAG” model was created from the 16HBE14o- cell line (WT CFTR) using prime editing. First, 144 prime editing combinations were tested in HEK-293 cells. The top 8 combinations from HEK-293 cells were tested in 16HBE14o- cells in pools of 2-3. The top performing combination resulted in ~5.5% editing efficiency in bulk and was used to generate a R1162X “TAG” expressing heterozygous clonal cell line with high transepithelial resistance in CFTR functional assays.

Results: Preliminary studies comparing R1162X “TGA” and R1162X “TAG” showed that both had little to no function without treatment, as expected. The quadruple combination treatment of tezacaftor, elexacaftor, the aminoglycoside ELX-02, and the cereblon E3 ligase inhibitor CC-90009 [1], in addition to acute potentiation with ivacaftor in the assay, resulted in an increase in CFTR function to ~3% of WT function and that was not different between the R1162X “TGA” and RR162X “TAG” stop codons.

Conclusions: In summary, prime editing resulted in low efficiency editing of the CFTR R1162 region using the 144 prime editing guide RNA and nicking RNA combinations tested; however, a usable, heterozygous cell model expressing the R1162X “TAG” mutation was identified. Preliminary studies showed no significant difference in CFTR function between the R1162X “TAG” and “TGA” models when treated with the quadruple cocktail listed above, suggesting that the sequence context surrounding R1162X is an important component of R1162X readthrough. We expect to use the R1162X “TAG” specificity model to characterize and compare current and future readthrough agents.

Acknowledgements:

References: [1] Surka C, Jin L, Mbong N, Lu CC, Jang IS, Rychak E, Mendy D, Clayton T, Tindall E, Hsu C, Fontanillo C, Tran E, Contreras A, Ng SWK, Matyskiela M, Wang K, Chamberlain P, Cathers B, Carmichael J, Hansen J, Wang JCY, Minden MD, Fan J, Pierce DW, Pourdehnad M, Rolfe M, Lopez-Girona A, Dick JE, Lu G. CC-90009, a novel cereblon E3 ligase modulator targets acute myeloid leukemia blasts and leukemia stem cells. Blood (2021)137(5):661-677. https://doi.org/10.1182/blood.2020008676