Preprint Review Version 1 Preserved in Portico This version is not peer-reviewed

Gene Therapy Approach for Treating DeltaF508, G542X and R553X Cystic Fibrosis Mutations Through the Usage Of CRISPR Prime Editing

Version 1 : Received: 27 October 2021 / Approved: 12 November 2021 / Online: 12 November 2021 (15:34:03 CET)

How to cite: Tafa, M.; Karışık, S.N.; Aksoy, E.B.; Aslan, R. Gene Therapy Approach for Treating DeltaF508, G542X and R553X Cystic Fibrosis Mutations Through the Usage Of CRISPR Prime Editing. Preprints 2021, 2021110235. https://doi.org/10.20944/preprints202111.0235.v1 Tafa, M.; Karışık, S.N.; Aksoy, E.B.; Aslan, R. Gene Therapy Approach for Treating DeltaF508, G542X and R553X Cystic Fibrosis Mutations Through the Usage Of CRISPR Prime Editing. Preprints 2021, 2021110235. https://doi.org/10.20944/preprints202111.0235.v1

Abstract

Cystic Fibrosis is a rare genetic disease that affects the transmission of chloride ions due to mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Even though there are nearly 2000 mutations identified to be related to the condition, the most common mutation is F508del; deletion of a phenylalanine residue at 508. On the other hand, G542X which is a Class I mutation is also found very commonly and there are no modulator treatments available for it. Furthermore, it was investigated that R553X mutation can as well be corrected simultaneously with G542X mutation. Therefore, the main focus is on designing a gene therapy project that can correct all these three mutations at once by utilizing the prime editing technique via lipid-based delivery. In this way, the mutations can be edited through plasmids that were designed containing 2 pegRNAs and the Cas enzyme. To implement such an approach efficiently, both ex vivo, an animal model, and in vivo steps are to be designed. For the cell line, fibroblasts are selected due to their simplicity and low cost. The animal model of the experiment is determined to be a ferret concerning the high similarity to the human's CFTR protein and finally, the procedure will follow on a direct application in human Cystic Fibrosis patients. The plasmids are thought to be delivered through a cationic liposome that will reach the lungs with the aid of a nebulizer. At the last stage of the experimental procedure, Sanger Sequencing will be done to see if the desired edit within the CFTR has been performed successfully, and Next Generation Sequencing will be executed to see if there has been an off-target mutation in the remainder of the genome. Whereas for detecting the presence and expression of CFTR protein in humans, immunodetection with flow cytometry will be conducted.

Keywords

Cystic Fibrosis; CFTR gene; CRISPR/Cas; pegRNA; Cationic Liposome

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Comments (0)

We encourage comments and feedback from a broad range of readers. See criteria for comments and our Diversity statement.

Leave a public comment
Send a private comment to the author(s)
* All users must log in before leaving a comment
Views 0
Downloads 0
Comments 0
Metrics 0


×
Alerts
Notify me about updates to this article or when a peer-reviewed version is published.
We use cookies on our website to ensure you get the best experience.
Read more about our cookies here.