Yifat S Oren1, Ofra Avizur-Barchad1, Efrat Ozeri-Galai1, Renana Elgrabli1, Meital R Schirelman1, Tehilla Blinder1, Chava D Stampfer1, Merav Ordan1, Onofrio Laselva2, Malena Cohen-Cymberknoh3, Eitan Kerem4, Christine E Bear5, Batsheva Kerem6. 1. SpliSense Biohouse Labs, Hadassah Ein Kerem, Jerusalem, Israel. 2. Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy; Division of Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada. 3. Pediatric Pulmonology Unit and CF Center, Hadassah Medical Center and Faculty of Medicine, Hebrew University of Jerusalem, Israel. 4. CF Center, Hadassah Hebrew University Medical Center, Jerusalem, Israel. 5. Division of Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada. 6. SpliSense Biohouse Labs, Hadassah Ein Kerem, Jerusalem, Israel; Department of Genetics, The Hebrew University, Jerusalem, Israel. Electronic address: batshevak@savion.huji.ac.il.
Abstract
BACKGROUND: Antisense oligonucleotide- based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to generate skipping over exon 23 of the CFTR transcript, to eliminate the W1282X nonsense mutation and avoid RNA degradation induced by the nonsense mediated mRNA decay mechanism, allowing production of partially active CFTR proteins lacking exon 23. METHODS: ∼80 ASOs were screened in 16HBEge W1282X cells. ASO candidates showing significant exon skipping were assessed for their W1282X allele selectivity and the increase of CFTR protein maturation and function. The effect of a highly potent ASO candidates was further analyzed in well differentiated primary human nasal epithelial cells, derived from a W1282X homozygous patient. RESULTS: ASO screening led to identification of several ASOs that significantly decrease the level of CFTR transcripts including exon 23. These ASOs resulted in significant levels of mature CFTR protein and together with modulators restore the channel function following free uptake into these cells. Importantly, a highly potent lead ASOs, efficiently delivered by free uptake, was able to increase the level of transcripts lacking exon 23 and restore the CFTR function in cells from a W1282X homozygote patient. CONCLUSION: The highly efficient exon 23 skipping induced by free uptake of the lead ASO and the resulting levels of mature CFTR protein exhibiting channel function in the presence of modulators, demonstrate the ASO therapeutic potential benefit for CF patients carrying the W1282X mutation with the objective to advance the lead candidate SPL23-2 to proof-of-concept clinical study.
BACKGROUND: Antisense oligonucleotide- based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to generate skipping over exon 23 of the CFTR transcript, to eliminate the W1282X nonsense mutation and avoid RNA degradation induced by the nonsense mediated mRNA decay mechanism, allowing production of partially active CFTR proteins lacking exon 23. METHODS: ∼80 ASOs were screened in 16HBEge W1282X cells. ASO candidates showing significant exon skipping were assessed for their W1282X allele selectivity and the increase of CFTR protein maturation and function. The effect of a highly potent ASO candidates was further analyzed in well differentiated primary human nasal epithelial cells, derived from a W1282X homozygous patient. RESULTS: ASO screening led to identification of several ASOs that significantly decrease the level of CFTR transcripts including exon 23. These ASOs resulted in significant levels of mature CFTR protein and together with modulators restore the channel function following free uptake into these cells. Importantly, a highly potent lead ASOs, efficiently delivered by free uptake, was able to increase the level of transcripts lacking exon 23 and restore the CFTR function in cells from a W1282X homozygote patient. CONCLUSION: The highly efficient exon 23 skipping induced by free uptake of the lead ASO and the resulting levels of mature CFTR protein exhibiting channel function in the presence of modulators, demonstrate the ASO therapeutic potential benefit for CF patients carrying the W1282X mutation with the objective to advance the lead candidate SPL23-2 to proof-of-concept clinical study.
Authors: Kelly M Martinovich; Anthony Kicic; Stephen M Stick; Russell D Johnsen; Sue Fletcher; Steve D Wilton Journal: Front Pharmacol Date: 2022-03-22 Impact factor: 5.810