Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10 kb C-to-T splicing mutation,Journal of Cystic Fibrosis

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Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10 kb C-to-T splicing mutation
Journal of Cystic Fibrosis ( IF 5.2 ) Pub Date : 2021-07-02 , DOI: 10.1016/j.jcf.2021.06.003
Yifat S Oren ₁ , Michal Irony-Tur Sinai ₂ , Anita Golec ₃ , Ofra Barchad-Avitzur ₄ , Venkateshwar Mutyam ₅ , Yao Li ₅ , Jeong Hong ₆ , Efrat Ozeri-Galai ₄ , Aurélie Hatton ₃ , Chen Leibson ₂ , Liran Carmel ₂ , Joel Reiter ₇ , Eric J Sorscher ₆ , Steve D Wilton ₈ , Eitan Kerem ₉ , Steven M Rowe ₅ , Isabelle Sermet-Gaudelus ₁₀ , Batsheva Kerem ₂

Affiliation

Department of Genetics, The Life Sciences Institute, The Hebrew University, Jerusalem, Israel; SpliSense Therapeutics, Jerusalem, Givat Ram, Israel.
Department of Genetics, The Life Sciences Institute, The Hebrew University, Jerusalem, Israel.
Institut Necker Enfants Malades, INSERM U1151 Université de Paris, Faculté de Médecine Necker, Paris, France.
SpliSense Therapeutics, Jerusalem, Givat Ram, Israel.
Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, United States of America.
Department of Pediatrics, Emory University, Atlanta, GA, United States of America.
Pediatric Pulmonary and Sleep Unit, Department of Pediatrics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Murdoch, Western Australia, Australia; Perron Institute for Neurological and Translational Science, University of Western Australia, Nedlands, Western Australia, Australia.
Hadassah-Hebrew University Medical Center, Department of Pediatrics and Cystic Fibrosis Center, Jerusalem, Israel.
Institut Necker Enfants Malades, INSERM U1151 Université de Paris, Faculté de Médecine Necker, Paris, France; Cystic Fibrosis Center, Hôpital Necker Enfants Malades, Assistance Publique Hôpitaux de Paris, Paris, France; Université de Paris, France; European Reference Network Lung.

Background

Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10 kb C-to-T splicing mutation in the CFTR gene.

Methods

We have screened, in FRT cells expressing the 3849+10 kb C-to-T splicing mutation, ~30 2′-O-Methyl-modified phosphorothioate ASOs, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The effect of highly potent ASO candidates on the splicing pattern, protein maturation and CFTR function was further analyzed in well differentiated primary human nasal and bronchial epithelial cells, derived from patients carrying at least one 3849+10 kb C-to-T allele.

Results

A highly potent lead ASO, efficiently delivered by free uptake, was able to significantly increase the level of correctly spliced mRNA and completely restore the CFTR function to wild type levels in cells from a homozygote patient. This ASO led to CFTR function with an average of 43% of wild type levels in cells from various heterozygote patients. Optimized efficiency of the lead ASO was further obtained with 2′-Methoxy Ethyl modification (2′MOE).

Conclusion

The highly efficient splicing modulation and functional correction, achieved by free uptake of the selected lead ASO in various patients, demonstrate the ASO therapeutic potential benefit for CF patients carrying splicing mutations and is aimed to serve as the basis for our current clinical development.

中文翻译：

为携带 3849+10 kb C-to-T 剪接突变的囊性纤维化患者开发基于反义寡核苷酸的药物

背景

用于剪接调节的基于反义寡核苷酸 (ASO) 的药物最近被批准用于各种未满足需求的遗传疾病。在这里，我们旨在开发一种基于 ASO 的剪接调节疗法，用于在 CFTR 基因中携带 3849+10 kb C-to-T 剪接突变的囊性纤维化 (CF) 患者。

方法

我们筛选了在表达 3849+10 kb C-to-T 剪接突变的 FRT 细胞中，约 30 个 2'-O-甲基修饰的硫代磷酸酯 ASO，旨在防止识别和包含由于突变而产生的神秘外显子. 在分化良好的原代人鼻和支气管上皮细胞中进一步分析了高效 ASO 候选物对剪接模式、蛋白质成熟和 CFTR 功能的影响，这些细胞来源于携带至少一个 3849+10 kb C-to-T 等位基因的患者。

结果

通过自由摄取有效递送的高效先导 ASO 能够显着增加正确剪接的 mRNA 水平，并将 CFTR 功能完全恢复到纯合子患者细胞中的野生型水平。这种 ASO 导致 CFTR 功能在来自各种杂合子患者的细胞中平均达到 43% 的野生型水平。通过 2'-甲氧基乙基修饰 (2'MOE) 进一步获得了铅 ASO 的优化效率。