(618) SPL84-23-1 Antisense Oligonucleotide Modulating the 3849+10kb C-T Splicing Defect, Has an Additive Effect with TRIKAFTA in Primary HBE Cells from 3849 Patients

Background: SpliSense is developing antisense oligonucleotides (ASOs) as inhaled drugs for treatment of Cystic Fibrosis (CF) patients carrying specific mutations. Among the identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, 10-15% affect the correct splicing of the gene transcripts. These include non-canonical splicing mutations that generate both aberrant and correct splicing of the CFTR gene, such as: 3849+10kb C- >T (3849), 2789+5 G- >A, 1811+1.6 kb A- >G, and 3272-26A- >G. These mutations are classified as class V mutations that lead to reduced amounts of normal CFTR proteins at the cell surface. Class V mutations are associated with extensive variation in disease severity, ranging from typical severe CF to a milder disease. However, the lung disease severity and the rate of pseudomonas infection is similar to that of severe CF patients . SPL84-23-1 is an ASO drug developed for treatment of patients carrying the 3849 splicing mutation by decreasing the inclusion of an 84 bp cryptic exon, resulting in the production of normal, fully functional CFTR protein.

Methods: Length optimization and lead ASO selection was performed using free uptake of ASOs at the length of 17 to 20 nt into HNE cells carrying the 3849 mutation. Following treatment, RNA was extracted and analyzed by RT-qPCR. The selected lead ASO was assessed by the Ussing Chamber system in primary respiratory cells derived from patients carrying the 3849 mutation, Human Nasal Epithelial (HNE) and Human Bronchial Epithelial (HBE) cells.

Results: ASO length is an important parameter for efficient delivery. SpliSense aimed to develop an optimal, short ASO drug to avoid toxicity and off target binding but maintain maximal affinity and CFTR activity. After screening process SPL84-23-1, which is a shorter version of our lead ASO SPL84-23, was selected as SpliSense leading drug candidate. The effect of SPL84-23-1 free uptake on both the splicing modulation and CFTR activation was similar to that of SPL84-23. In HBE cells from a homozygous 3849 patient, the effect of SPL84-23-1 was compared to that of SYMDEKO, the approved drug for this patient population. While SYMDEKO had no effect in these cells, SPL84-23-1 led to full restoration of CFTR activity. Moreover, in heterozygous patients (3849/F508del), the effect of SPL84-23-1 was compared to that of TRIKAFTA, the approved drug for this patient population, as well as the combination of SPL84-23-1 with TRIKAFTA. SPL84-23-1 alone had comparable effect to TRIKAFTA. Importantly, the combination of SPL84-23-1 with TRIKAFTA led to a significant additive effect, reaching 66% of WT CFTR activity. These results indicate of the potential of combined treatment in this patient population.

Conclusions: SPL84-23-1 was selected as a lead ASO based on its ability to significantly enhance the production of normal and functional CFTR channels in cultured HNE and HBE cells heterozygous or homozygous for the CFTR 3849 splicing mutation. The full rescue of the 3849 allele and the additive effect with TRIKAFTA highlight the clinical potential of this drug for all patients carrying the 3849 mutation. This project is in advanced IND-enabling studies, aiming to enable the initiation of a clinical study by late 2022.

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