(604) SpliSense’s ASO SPL84-23-1 Properly Distributes and is Retained in CF-like Mice Lungs

Background: SpliSense is developing inhaled antisense oligonucleotide (ASO) drugs for the treatment of CF patients carrying mutations with an unmet need. SpliSense’s most advanced program, SPL84-23-1, is targeted to produce normal CFTR protein in patients carrying the 3849+10kb C-to-T mutation. SPL84-23-1 (SPL ASO) was shown to restore CFTR activity in HNE and HBE cells from 3849+10kb C-to-T homozygous and heterozygous patients. As part of phase 1-2 study preparation, this study assessed the ability of SPL84-23-1 to diffuse and distribute in CF lungs, which are characterized by thick, viscous hyper-concentrated mucus.

Methods: SPL ASO stability was measured following incubation in a pool of sputum from 10 CF patients or in Rat lung lysosomal lysate (RLuLL). Following incubation, SPL ASO was tested for its integrity using Northern blot with a specific probe. Migration of the Cy3 and Cy5 Labeled ASO through mucus was analyzed by a 1-dimensional diffusion assay through mucus harvested from human bronchial epithelium cell culture and prepared to 2, 4, and 8% solids. Lung distribution of SPL ASO was analyzed in WT, betaENaC mice and monkeys. Mice were treated with the ASO via intratracheal administration, at different times, while monkeys were treated with SPL ASO by inhalation. following the administration, the lungs were fixed with formalin and processed to slides for histopathologic evaluation. ASO was detected on the lung sections using in-situ hybridization with a labeled probe.

Results: SPL84-23-1 was found to be stable in mucus from CF patients for at least 48 hrs. SPL84-23-1 was shown to diffuse through concentrated mucus, demonstrating a viscosity ~100-fold lower than macroscopic particles, indicating its ability to permeate hyper-concentrated mucus. Additionally, SPL84-23-1 appeared to be nuclease-resistant in RLuLL, supporting its ability to stably enter the cell via lysosomes. SPL84-23-1 comparably distributed in both WT and CF-like lung phenotype (β-ENaC-overexpressing) mice following intratracheal administration, specifically in bronchial and bronchiolar epithelium, at an estimated coverage of 90%. Furthermore, SPL84-23-1 remained detectable in the cytoplasm of respiratory epithelial cells at all parts of the airways in WT mice for up to 4 weeks post-dose. Inhaled SPL84-23-1 distribution in lungs of NHPs will also be discussed.

Conclusions: SPL84-23-1 was stable in CF mucus and Lung Lysosomal Lysate, was able to properly migrate through CF mucus, properly distributed in CF-like mouse lungs, properly detected in the target regions and cells, and remained in WT lungs for up to 4 weeks. Altogether the stability, diffusion, and distribution studies in CF mucus and lungs support the feasibility of weekly/bi-weekly inhaled delivery of SPL84-23-1 to CF patients in the clinic.

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