CFTR Gene Editing via RNA-Guided Endonuclease and Donor Template
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Solution Overview
Problem
Current therapies for cystic fibrosis (CF) do not effectively address the underlying genetic mutations in the CF transmembrane conductance regulator (CFTR) gene, leading to severe organ damage and a lack of a cure for the disease.
Innovation Solution
Development of gene-editing systems that target specific positions in the CFTR gene, specifically using RNA-guided DNA endonucleases and guide RNAs to introduce nucleic acids encoding exons 11 to 27, with homologous arms for efficient insertion and correction of mutations, particularly at positions like 1220, 2068, and 3821 in intron 10, to restore CFTR function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current therapies are used for cystic fibrosis, then treatment is provided, but the underlying genetic mutations in the CFTR gene are not corrected, leading to severe organ damage and no cure
Solution Approach 1:
The therapy is divided into multiple components delivered separately: an RNA-guided endonuclease (e.g., Cas9), a guide RNA (gRNA) targeting specific CFTR mutation sites, and a donor DNA template containing the corrected sequence. This segmentation allows each component to be optimized independently and delivered via appropriate vectors, resolving the contradiction between curative effectiveness and therapeutic complexity.
Solution Approach 2:
The system performs preliminary genetic correction by introducing the RNA-guided endonuclease and guide RNA into cells before administering the donor DNA template. The endonuclease预先 targets and cleaves the mutated CFTR gene at specific sites (e.g., positions 1220, 2068, 3821 in intron 10), creating double-strand breaks that prepare the site for subsequent homology-directed repair with the corrected sequence, thereby achieving curative effectiveness through staged intervention.
2Reliability
If gene-editing systems are developed to correct CFTR mutations, then curative effectiveness is improved, but the complexity of the therapy increases
Solution Approach 1:
The guide RNA serves as an intermediary that bridges the RNA-guided endonuclease and the specific CFTR mutation target sites. The gRNA contains a spacer sequence complementary to the mutated region (e.g., intron 10 positions 1220, 2068, 3821), enabling the endonuclease to locate and cleave the precise mutation site without requiring complex targeting mechanisms, thus improving curative effectiveness while managing system complexity.
Solution Approach 2:
The donor DNA template contains a copy of the corrected CFTR gene sequence flanked by homologous arms that match the regions surrounding the mutation site. This copying approach allows the cell's homology-directed repair mechanism to accurately insert the corrected sequence into the cleaved CFTR gene, achieving high curative effectiveness through simple sequence replication rather than complex editing procedures.
3Manufacturing precision
If RNA-guided endonucleases and guide RNAs are used to target specific positions in the CFTR gene, then editing precision is improved, but the system complexity increases
Solution Approach 1:
The guide RNA is designed with a specific spacer sequence (e.g., 20 nucleotides) that is complementary only to the mutated region of the CFTR gene at specific positions (e.g., intron 10 positions 1220, 2068, 3821). This local specificity ensures that the RNA-guided endonuclease cleaves only at the intended mutation site, achieving high editing precision through localized molecular recognition rather than global system complexity.
Solution Approach 2:
The system achieves editing precision by changing the nucleotide sequence parameter of the guide RNA spacer to match the specific mutation site in the CFTR gene. By adjusting this single parameter (the spacer sequence), the system can precisely target different mutation sites (e.g., positions 1220, 2068, 3821) without requiring changes to the overall system architecture, thereby maintaining simplicity while achieving high precision.
4Productivity
If homologous arms are included in the donor DNA template, then insertion efficiency is improved, but the DNA sequence length increases
Solution Approach 1:
The donor DNA template includes homologous arms that are longer than the minimum required for homology-directed repair (e.g., 50-200 nucleotides on each side of the corrected sequence). This excessive length ensures high insertion efficiency by providing sufficient homology for efficient recombination, while the arms are kept relatively short compared to the entire CFTR gene, thus balancing insertion efficiency with manageable DNA length for delivery.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The gene-editing systems achieve high editing rates and cell survival, correcting 99% of non-responsive CFTR alleles, thereby potentially treating CF by enhancing CFTR activity and improving lung function.
Implementation Method 1
a second polynucleotide, which comprises a second nucleotide sequence encoding an RNA-guided DNA endonuclease, or the RNA-guided DNA endonuclease; and (c) a third polynucleotide, which comprises a third nucleotide sequence encoding a guide RNA (gRNA), wherein the gRNA directs cleavage by the RNA-guided DNA endonuclease at a target site
Implementation Method 2
the gRNA directs cleavage by the RNA-guided DNA endonuclease at a target site, which is position 1220, 2068, 3821, 4262, 5041, 5052, 5278, 5343, 5538, or 6150 of intron 10 in the CFTR gene
Implementation Method 3
The 5′ homologous arm may comprise a nucleic acid sequence that is homologous to a region upstream to the target site. Alternatively or in addition, the 3′ homologous arm may comprise a nucleic acid sequence that is homologous to a region downstream to the target site
Data Source
AI summary
Described herein are highly efficient gene-editing systems comprising a nuclease, a guide RNA, and/or a donor template and uses thereof for editing a cystic fibrosis transmembrane regulator (CFTR) gene either in vitro or in vivo.