(692) The importance of CFTR mRNA testing to uncover other variants in CF genotype that affect CFTR expression

Background: To date, more than 2000 CFTR variants have been described in CFTR1 database, of which 466 have been assessed in the CFTR2 project. Seven mutation classes have been proposed, based on the impact of each mutation on processing, trafficking and function of CFTR. Many mutations however cause multiple defects at the molecular level, blurring the strict distinction between the ‘theratypes’. Determine the full spectrum of mutations/variants present in CFTR gene may help understand better the disfunction at the protein level. Moreover, unravelling the cellular impairments caused by rare mutations provided the needed input to design better CFTR-directed treatments. Using patient derived models improves the accuracy of the findings, especially when splicing is affected. This study is focused on the L927P rare mutation that has a higher incidence (2.4%) in Belgian people with CF (pWCF) and no available treatment yet.

Methods: Rescue of CFTR function with tezacaftor/ivacaftor (teza/iva) and elexacaftor/tezacaftor/ivacaftor (elexa/teza/iva) in organoids from 12 patients carrying the L927P mutation in one allele was assessed by FIS assay. To test the effect of CFTR mutations present on these organoids at the transcript level, we isolated RNA from the organoids, produced the cDNA by retro-transcription and amplified 8 different fragments that cover the complete coding region of CFTR. Sanger sequencing was done when alternative transcripts were obtained.

Results: The FIS assay results showed no rescue when teza/iva is used on organoids from patients with L927P in trans with non F508del minimal function mutation (n=4) or L927P/L927P ( (n=2) (AUC at 0.8µMFsk=114.3±39.6), and low rescue for L927P/F508del (n=7) organoids (AUC at 0.8µMFsk=964.7±103.0). With elexa/teza/iva we observed high rescue with L927P/F508del (AUC at 0.8µMFsk=3869.8±139.0) but low rescue was observed for the other patients (AUC at 0.8µMFsk=746.3±122.3). mRNA testing showed an alternative transcript in all samples, that by sequencing was proved to result from inclusion of 97nts from IVS7 between Ex7 and Ex8. After DNA sequencing, the mutation c.870-1113_1110delGAAT was found in IVS7 for all samples. The L927P is thus in complex allele with this intronic mutation. For L927P/L927P organoids the alternative transcript band is more intense than the normal transcript band obtained by the RT-PCR amplification. Quantitative analysis of the amplified transcripts will be performed. Treatment of the organoids with oligo nucleotides to revert the abnormal transcripts is under evaluation to determine if increase of correct size transcripts can be obtained and if that will help to increase the rescue of L927P mutant protein by modulators.

Conclusions: In patient-derived rectal organoids, L927P is not efficiently rescued by the CFTR modulators currently available. L927P is found in cis with c.870-1113_1110delGAAT, an intronic mutation causing abnormal splicing and thus reduced levels of full-length L927P transcripts, maybe masking a possible response of L927P to modulators. A therapeutic strategy combining an increase in normally spliced L927P transcripts by oligo nucleotides and modulators may reach higher levels of CFTR rescue.

Knowing the complete CFTR genetic background is important to design the best therapeutic solutions for each patient with CF.

Acknowledgements: Acknowledgements to all the patients that participate in this study. This work was funded by the Belgian CF patient association and by the PTAC Research Grant n°001713G220 from CFF.

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