CTLA4 Gene Editing via Intron 1 Targeting
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Solution Overview
Problem
Current gene therapy approaches for CTLA4 haploinsufficiency, such as viral vector-mediated gene addition, risk supraphysiological expression and insertional mutagenesis, and are ineffective due to the need for close regulation of the CTLA4 gene, particularly in disorders with heterogeneous mutational landscapes like CTLA4 deficiency.
Innovation Solution
A method involving site-directed nucleases, like CRISPR-associated protein Cas9, to target and edit the CTLA4 gene at the 3′-end of intron 1, allowing for the insertion of replacement CTLA4 sequences using homology-directed repair, preserving endogenous promoter regulation and intronic machinery, thereby modulating and restoring CTLA4 expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If viral vector-mediated gene addition is used to correct CTLA4 deficiency, then the immune defect can be corrected, but supraphysiological expression and insertional mutagenesis occur
Solution Approach 1:
The invention extracts and removes the harmful elements (viral vector and its associated risks of supraphysiological expression and insertional mutagenesis) while retaining the beneficial function of correcting CTLA4 deficiency through targeted gene editing using site-directed nucleases and homology-directed repair
Solution Approach 2:
The invention changes the approach from gene addition (viral vector) to gene editing (site-directed nuclease) with precise control of expression levels through endogenous promoter regulation, transforming the parameter of gene delivery mechanism to eliminate harmful effects while maintaining therapeutic benefit
2Reliability
If viral vector-mediated gene addition is used to correct CTLA4 deficiency, then the immune defect can be corrected, but insertional mutagenesis risk increases
Solution Approach 1:
The invention extracts and eliminates the viral vector component that causes insertional mutagenesis, replacing it with a non-viral gene editing approach using site-directed nucleases that perform precise edits without integrating foreign genetic material into the host genome
Solution Approach 2:
The invention substitutes the viral vector-mediated gene addition mechanism with a biochemical gene editing mechanism using site-directed nucleases and homology-directed repair, replacing the harmful mechanical process of viral integration with a precise molecular editing process that avoids insertional mutagenesis
3Reliability
If germline heterozygous mutations in CTLA4 are present, then CTLA4 haploinsufficiency occurs, but the mutational landscape is heterogeneous with over 45 different mutations
Solution Approach 1:
The invention creates a universal gene editing approach using site-directed nucleases with customizable guide RNAs that can target and correct any of the over 45 different germline heterozygous mutations in CTLA4, making the therapy adaptable to the heterogeneous mutational landscape through a single platform
Solution Approach 2:
The invention changes the therapeutic approach from mutation-specific treatments to a general gene editing platform where the site-directed nuclease and guide RNA can be designed to match any specific mutation, transforming the parameter of therapy specificity to accommodate the heterogeneous mutational landscape
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
This approach ensures regulated CTLA4 expression, maintains functional protein levels, and addresses the heterogeneous mutational landscape of CTLA4 deficiency, offering a potential cure for CTLA4-related immune disorders with reduced risks of immunological complications and insertional mutagenesis.
Implementation Method 1
Gene insertion is typically facilitated by the repair of nuclease-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR)
Data Source
AI summary
The present invention provides a method of engineering a cell, comprising the steps of introducing into the cell: i) a site-directed nuclease which is capable of cleaving a target nucleotide sequence at the 3′-end of Intron 1 of a Cytotoxic T-Lymphocyte Associated Protein 4 gene (CTLA4); and ii) a nucleic acid construct which comprises a nucleic acid sequence comprising one or more of exon 2, exon 3 and exon 4 of CTLA4, or a sequence with at least 70% identity to exon 2, exon 3 and/or exon 4 of the CTLA4 gene, and 5′- and 3′-homology arms, wherein each of the 5′ and '3 homology arms is essentially complementary to a sequence flanking the target nucleotide sequence at the 3′-end of Intron 1 of CTLA4.