RNA-Guided Endonuclease Corrects F8 Gene Inversions
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Solution Overview
Problem
Current methods for treating hemophilia A caused by genetic mutations in the blood coagulation factor VIII (F8) gene, particularly large genomic inversions, are inefficient due to challenges in correcting the F8 gene and generating inversion-corrected induced pluripotent stem cells for therapy.
Innovation Solution
A composition and method using RNA-guided endonucleases and TALE effector domains to specifically target and correct inversions in the F8 gene, enabling the generation of inversion-corrected induced pluripotent stem cells for hemophilia A treatment by reprogramming somatic cells and delivering them to patients.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR is used to analyze genotype, then small inversion regions can be detected, but large inversion regions (e.g., 10-kbp int22h) cannot be amplified or analyzed
Solution Approach 1:
The patent divides the large inversion region into smaller analyzable segments by using multiple primer pairs targeting different regions within the inversion. This allows detection of large inversions that would otherwise be too large for single PCR amplification, enabling genotype analysis of inversions up to 10-kbp and larger by analyzing multiple smaller fragments simultaneously
2Reliability
If TALENs are used to invert chromosomal segments, then hemophilia A model cell lines can be created, but the process is complex and time-consuming
Solution Approach 1:
The patent performs preliminary identification and characterization of inversion breakpoints and structures before generating model cell lines. By pre-mapping the inversion regions and designing targeted TALEN pairs based on known inversion structures, the process eliminates trial-and-error steps, significantly reducing the time required to generate accurate hemophilia A model cell lines while maintaining high reliability
3Reliability
If large chromosomal segments are inverted to create hemophilia A models, then accurate disease modeling is achieved, but recovery and correction become more difficult
Solution Approach 1:
The patent implements a feedback mechanism where the inverted structure itself provides information for its own correction. By designing TALEN pairs that target sequences within the inverted region, the system uses the inversion's own structural characteristics as guides for precise excision and correction, making large inversion recovery as efficient as small inversion correction
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 approach effectively corrects genetic inversions in the F8 gene, restoring normal gene function in induced pluripotent stem cells, which can be differentiated and transplanted to express the corrected gene, providing a potential cure for hemophilia A.
Implementation Method 1
an inversion of the F8 gene could be successfully corrected using the guiding RNA that targets an inverted region and the RNA-guided endonuclease that cleaves a site targeted by the guiding RNA
Implementation Method 2
an inversion of the F8 gene could be successfully corrected using TAL effector domains that target an inverted region and a pair of endonuclease that bind to the domains, respectively, to cleave a gene site targeted by the domains
Data Source
AI summary
The present invention provides a method for inducing an inversion of normal blood coagulation factor VIII (F8) gene, a method for correcting an inversion of blood coagulation factor VIII gene in which the inversion has occurred, and a Hemophilia A patient-derived induced pluripotent stem cell in which the inversion is corrected, constructed using the same. The method of the present invention effectively reproduces the inversion of intron 1 and intron 22 of the F8 gene, which is responsible for the majority of severe hemophilia A, and thereby may be effectively used for studying the development mechanism of hemophilia A and as a research tool for screening therapeutic agents. The inversion-corrected induced pluripotent stem cell constructed according the method of the present invention enables an efficient and fundamental treatment for hemophilia A by restoring a genotype in which mutation has occurred to a wild type-like state, without limitation via normal gene or protein delivery.