WS05.06Design of an adenine base editor for temporospatial control of editing

IF 5.4 2区医学 Q1 RESPIRATORY SYSTEM

Journal of Cystic Fibrosis Pub Date : 2025-06-01 DOI:10.1016/j.jcf.2025.03.521

I. Zollo , L. Santos , J. Alves , A.S. Coroadinha , P.T. Harrison , C.M. Farinha

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引用次数: 0

Abstract

Background and Aims

Despite advances with modulators, 10-15% of people with cystic fibrosis, particularly those with nonsense variants, still lack effective treatments. For these cases, gene-based therapies offer the most promising path to a cure. Among gene editing tools, Adenine Base Editor (ABE) is particularly suitable for correcting W1282X, the second most common CF-causing variant without a therapy. However, it remains unclear which cell types in the lung epithelium, and how many of them, need editing to restore CFTR function to therapeutic levels.

Methods and Results

We developed a basal cell line bearing W1282X (BCi W1282X-CFTR) able of differentiating into airway epithelial cell types (1). Additionally, we engineered a split version of ABE (SplitABE) that allows temporal control of editing. Preliminary data show SplitABE reverts the premature stop codon to WT while minimizing bystander edits.

Here, we present a strategy integrating temporal and spatial control of ABE by placing the SplitABE cassette under five cell-type-specific promoters, targeting basal, ciliated, secretory (type 1 and 2), and ionocyte cells (2). These plasmids were used to transfect 293T cells for large-scale lentiviral particle production. Infectious titers, determined via ddPCR after transducing BCi W1282X-CFTR cells, ranged between 7E+06 and 1.5E+07 IU/mL. These titers are now being used to transduce BCi W1282X-CFTR cells at varying MOIs to establish cell lines with cell-type-specific SplitABE expression.

Conclusion

This advanced gene-editing platform, combined with a robust cellular model, will help pinpoint critical cellular targets for CFTR correction and optimize strategies for potential clinical application.

1. Santos L et al (2023) J Cyst Fibr 22 (S2), S32.

2. Zollo I et al (2024) J Cyst Fibr 23 (S2), S162.

Acknowledgements: Work supported by grants HARRIS21G0 and FARINH24G0 from CFF and centre grants UIDP/04046/2020 and UIDB/04046/2020 from FCT, Portugal (to BioISI).

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ws05.06用于编辑时空控制的腺嘌呤碱基编辑器的设计

背景和目的尽管调节剂取得了进展，但仍有10-15%的囊性纤维化患者，特别是无意义变异患者，缺乏有效的治疗方法。对于这些病例，基于基因的治疗提供了最有希望的治愈途径。在基因编辑工具中，腺嘌呤碱基编辑器（Adenine Base Editor， ABE）特别适合在没有治疗的情况下纠正W1282X，这是第二常见的cf致病变异。然而，目前尚不清楚肺上皮中的哪些细胞类型，以及其中有多少细胞需要编辑才能将CFTR功能恢复到治疗水平。方法和结果我们建立了一个携带W1282X （BCi W1282X- cftr）的基底细胞系，该细胞系能够分化为气道上皮细胞类型(1)。此外，我们设计了一个分裂版本的ABE (SplitABE)，允许编辑的时间控制。初步数据显示SplitABE将过早停止密码子还原为WT，同时最小化旁观者编辑。在这里，我们提出了一种整合ABE时间和空间控制的策略，通过将SplitABE盒放置在五种细胞类型特异性启动子下，针对基底细胞、纤毛细胞、分泌细胞（1型和2型）和离子细胞(2)。这些质粒被用于转染293T细胞以大规模生产慢病毒颗粒。BCi W1282X-CFTR细胞转导后，通过ddPCR测定感染滴度，范围为7E+06至1.5E+07 IU/mL。这些滴度现在被用于在不同moi下转导BCi W1282X-CFTR细胞，以建立具有细胞类型特异性SplitABE表达的细胞系。这一先进的基因编辑平台，结合强大的细胞模型，将有助于确定CFTR校正的关键细胞靶点，并优化潜在的临床应用策略。Santos L等（2023）[J]囊肿纤维22 (S2), S32.2。Zollo等（2024）[J]囊肿纤维23 (S2), S162。致谢：这项工作得到了CFF资助HARRIS21G0和FARINH24G0以及葡萄牙FCT资助UIDP/04046/2020和UIDB/04046/2020（给BioISI）的中心资助。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Cystic Fibrosis 医学-呼吸系统

CiteScore

10.10

自引率

13.50%

发文量

1361

审稿时长

50 days

期刊介绍： The Journal of Cystic Fibrosis is the official journal of the European Cystic Fibrosis Society. The journal is devoted to promoting the research and treatment of cystic fibrosis. To this end the journal publishes original scientific articles, editorials, case reports, short communications and other information relevant to cystic fibrosis. The journal also publishes news and articles concerning the activities and policies of the ECFS as well as those of other societies related the ECFS.