Multi-gRNA CRISPR Vector Editing BCL11a Enhancers
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Solution Overview
Problem
Current treatments for hemoglobinopathies, such as beta-thalassemias and sickle cell anemia, are limited in effectively increasing fetal hemoglobin levels, which is crucial for alleviating the symptoms of these disorders.
Innovation Solution
The use of a vector comprising at least two guide RNAs that target specific genomic DNA locations on human chromosome 2, combined with a DNA endonuclease enzyme, to alter the expression of genes involved in fetal hemoglobin production, specifically targeting and editing the BCL11A gene to reduce its repressive effect on gamma-globin gene transcription.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If a single guide RNA is used in CRISPR-Cas gene editing, then the system is simpler to design and implement, but the gene-editing efficacy is reduced in quiescent cells
Solution Approach 1:
The patent combines multiple guide RNAs (at least two) into a single CRISPR-Cas system to simultaneously target multiple genomic locations or to enhance editing efficiency at a single location. This merging of multiple gRNA functions resolves the contradiction by maintaining system simplicity while improving gene-editing efficacy in quiescent cells through cooperative binding and enhanced nuclease recruitment.
2Productivity
If multiple guide RNAs are used to increase gene-editing efficacy, then the editing efficiency improves, but the system complexity increases
Solution Approach 1:
The patent employs a universal CRISPR-Cas platform that can accommodate variable numbers of guide RNAs (at least two) to perform multiple functions: simultaneous targeting of multiple enhancer regions within the BCL11A locus, enhanced editing efficiency through cooperative binding, and flexible adaptation to different genomic targets. This multi-functionality allows the system to improve gene-editing efficacy while maintaining design flexibility and avoiding excessive complexity.
3Reliability
If CRISPR-Cas gene editing is applied to increase fetal hemoglobin levels, then therapeutic benefit is achieved, but potential genotoxicity and stem cell function impact must be managed
Solution Approach 1:
The patent applies local quality by using multiple guide RNAs to target specific enhancer regions (+55, +58, +62) within the BCL11A locus with high precision. This localized targeting approach ensures that gene editing occurs only at the intended genomic locations, minimizing off-target effects and genotoxicity while achieving the therapeutic goal of increasing fetal hemoglobin levels in beta-hemoglobinopathy patients.
Solution Approach 2:
The patent employs preliminary action by using CRISPR-Cas gene editing to modify hematopoietic stem cells ex vivo before transplantation. This preliminary genetic modification allows for controlled editing under optimized conditions, enabling assessment of editing efficiency and safety before clinical application, thereby reducing potential harmful effects while ensuring therapeutic effectiveness.
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 significantly increases fetal hemoglobin levels in erythroid cells, potentially offering a therapeutic benefit by reducing the severity of hemoglobinopathies and improving the clinical outcomes for patients suffering from these conditions.
Implementation Method 1
a vector comprising at least two guide RNAs (gRNAs) that targets and hybridizes to a target sequence on a DNA molecule
Implementation Method 2
Described herein are CRISPR enzymes in combination with at least two guide RNAs (gRNAs), where the number of gRNAs increases its gene-editing efficacy
Data Source
AI summary
Provided herein are vectors, and compositions thereof, comprising at least two guide RNAs (gRNAs) that targets and hybridizes to a target sequence on a DNA molecule, wherein each of the at least two gRNAs hybridizes at least two genomic DNA locations selected from human chromosome 2 at location 60725424 to 60725688 according to GRCh37/hg19 human genome reference build (+55 functional region); human chromosome 2 at location 60722238 to 60722466 GRCh37/hg19 human genome reference build (+58 functional region); or human chromosome 2 at location 60718042 to 60718186 according to GRCh37/hg19 human genome reference build (+62 functional region). Methods for the use of such vectors are further provided.