Beta-Globin HDR Editing for E6V Mutation Correction
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Solution Overview
Problem
Current methods for treating hemoglobinopathies such as sickle cell disease and beta-thalassemias, including gene therapy and hematopoietic stem cell transplantation, pose risks like insertional mutagenesis and graft vs. host disease, and often lack effective donor matching.
Innovation Solution
A system and method using a Cas9 endonuclease, single guide RNA (sgRNA), and a recombinant vector to correct the E6V mutation in the beta-globin gene by inducing a double-strand break (DSB) and homologous directed repair (HDR) to replace the E6V mutation with a codon encoding glutamic acid, utilizing specific nucleotide sequences and spacer sequences targeting intron 1 of the HBB gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If gene therapy is used to deliver corrected HBB gene, then treatment of hemoglobinopathies is achieved, but risk of insertional mutagenesis increases
Solution Approach 1:
The patent replaces traditional gene therapy methods (viral vectors) with CRISPR-Cas9 genome editing technology. Instead of inserting a corrected gene into the genome using viral vectors, the system uses Cas9 endonuclease guided by sgRNA to precisely edit the HBB gene at its native location, correcting the E6V mutation through homology-directed repair without requiring external gene insertion.
Solution Approach 2:
The patent introduces a homology template as an intermediary element that mediates the correction process. This template contains the correct DNA sequence (GAG instead of GTG) and serves as a blueprint for the homology-directed repair mechanism, allowing precise correction of the mutation without requiring viral vector insertion.
2Reliability
If hematopoietic stem cell transplantation is performed, then cure of SCD is achieved, but risk of graft vs. host disease and difficulty in donor matching increases
Solution Approach 1:
The patent enables the patient's own cells to correct their own genetic defect through CRISPR-Cas9 editing. The patient's hematopoietic stem cells are extracted, genetically corrected ex vivo using the CRISPR system, and then reinfused. This eliminates the need for allogeneic donors and avoids graft vs. host disease risk, as the patient's immune system recognizes the corrected cells as self.
Solution Approach 2:
The patent performs genetic correction as a preliminary action before cell transplantation. The HBB gene is corrected in the patient's own stem cells before they are reinfused, ensuring that the cells are already functional and corrected prior to transplantation, thereby eliminating the need for donor matching and reducing transplantation-related risks.
3Manufacturing precision
If CRISPR-Cas9 is used to correct E6V mutation, then precision of gene editing is improved, but complexity of the system increases
Solution Approach 1:
The patent divides the CRISPR-Cas9 system into separable components: Cas9 endonuclease, sgRNA with specific spacer sequences targeting intron 1 of HBB, and homology template. These components can be delivered separately or together, allowing flexible implementation and reducing overall system complexity while maintaining high editing precision.
Solution Approach 2:
The patent optimizes specific parameters of the CRISPR system, including the sgRNA spacer sequence length and composition, the PAM sequence requirements, and the homology template design. By carefully tuning these parameters, the system achieves high precision editing while simplifying the overall approach through standardized, optimized components.
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 effectively corrects the E6V mutation in beta-globin, potentially reducing the risk of complications associated with existing treatments and improving the production of functional hemoglobin, thereby addressing the challenges of current therapies.
Implementation Method 1
a Cas9 endonuclease, an mRNA encoding the Cas9 endonuclease, or a recombinant expression vector comprising a nucleotide sequence encoding the Cas9 endonuclease; (b) a single guide RNA (sgRNA) comprising a spacer sequence corresponding to a target sequence adjacent a PAM, the target sequence comprising a target site within intron 1 of HBB
Implementation Method 2
HDR of the DSB results in exchange of the region of the HBB gene encoding the E6V mutation with the nucleic acid, thereby correcting the E6V mutation in the HBB gene in the cell or population of cells
Data Source
AI summary
The disclosure features systems and methods for correcting a mutation in the human beta-globin (HBB) gene in a cell or population of cells. The disclosure also features methods of increasing repair of a DNA double stranded break (DSB) in an HBB gene by the homology-directed repair (HDR) pathway. The disclosure also features compositions for use in the methods.