(615) Antisense Oligonucleotide-Based Drug Development for Cystic Fibrosis Patients Carrying the 2789+5 G-to-A Splicing Mutation

Background: 12% of CFTR disease causing variants affect the correct splicing of the transcripts. 2789+5 G-to-A (2789) is a splicing mutation at the +5 position of the splice donor site of exon 16, the mutation leads to generation of transcripts lacking exon 16 and WT transcripts. In transcripts lacking exon 16 a premature stop codon is generated, leading to degradation of these transcripts by the nonsense mediated mRNA decay mechanism.

The CF 2789 patients are mainly characterized by mild clinical CF manifestations in childhood. Diagnosis is carried out in adulthood due to the development of lung disease and infections by P. aeruginosa. Most of the patients have pancreatic sufficiency. The current approved Vertex modulators do not provide a sufficient therapeutic benefit to 2789 patients. Therefore, further strategies of drug development are essential to address the unmet needs of these patients.

Previous study by Igreja et al. showed that RNA-based antisense oligonucleotides were able to modulate splicing and rescue normal full-length CFTR transcripts. This antisense oligonucleotide led to increase of full-length CFTR protein levels and restore CFTR function in cells expressing the 2789 CFTR minigene. SpliSense aims to develop an inhaled ASO-based therapy for 2789 CF patients.

Methods: Using an in-house algorithm we designed and optimized ASOs, targeted to enhance exon 16 inclusion. These ASOs were screened for their effect on CFTR RNA in a cellular screening system based on 293 cells transfected with a minigene CFTR plasmid. Rectal organoids were established from a heterozygous patient carrying the 2789 mutation (2789/Q359KT360K). The organoids were grown in monolayers on porous filters. RNA was extracted and the level of exon 16 skipping was evaluated using RT-qPCR. Mature CFTR levels were evaluated using western blot and CFTR function was analyzed by the Ussing Chambers.

Results: Screening of candidate ASOs led to the identification of several lead ASOs that significantly decrease the level of aberrantly spliced CFTR mRNA. A functional screening system based on organoids derived from a patient carrying the 2789 mutation was established. Organoids-derived monolayer cells show low levels of full length CFTR and protein as well as low levels of CFTR function. Free uptake of ASO shows a significant reduction of transcripts levels without Ex16 and increment of CFTR activity in a dose dependent manner.

Conclusions: These results together with the know-how of Splisense in delivering ASO drugs to the lungs highlight the potential of our lead ASOs as a therapeutic approach for CF patients carrying the 2789 mutation.

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