CRISPR HBD Promoter Editing to Increase HbA2 Expression
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Solution Overview
Problem
Current therapies for haemoglobinopathies, such as sickle cell disease and beta-thalassemia, have focused primarily on increasing fetal haemoglobin (HbF) levels, while little research has been done on enhancing haemoglobin A2 (HbA2) expression to compensate for low or abnormal HbA, which is crucial for treating these conditions.
Innovation Solution
A CRISPR/Cas composition is used to edit the human HBD gene by inserting a KLF1 binding site, a b-DRF motif, and a TFIIB binding site into its promoter region, along with a LDB1 complex binding site, to enhance HbA2 expression, even in the presence of the HBB gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If CRISPR/Cas9 is used to increase HbF levels by downregulating BCL11A, then fetal haemoglobin expression is improved, but research on enhancing HbA2 expression remains limited and undeveloped
Solution Approach 1:
The invention changes the target parameter from HbF to HbA2 expression by modifying the HBD gene promoter region. By inserting specific transcription factor binding sites (KLF1, b-DRF, TFIIB, LDB1), the patent alters the expression parameters of delta-globin to increase HbA2 levels, providing an alternative treatment approach to the conventional HbF-upregulation strategy.
Solution Approach 2:
Instead of following the conventional approach of downregulating repressors (BCL11A) to increase HbF, the invention inverts the strategy by directly upregulating HbA2 through promoter modification. This reverse approach targets the HBD gene activation pathway rather than the HbF repression pathway, diversifying treatment options for haemoglobinopathies.
2Quantity of substance
If multiple transcription factor binding sites are inserted into the HBD promoter region, then HbA2 expression is enhanced, but the complexity of the CRISPR/Cas composition increases
Solution Approach 1:
The DNA donor template is segmented into multiple functional modules: homology arms for precise integration, and distinct transcription factor binding sites (KLF1, b-DRF, TFIIB, LDB1) that can be independently designed and optimized. This modular segmentation allows systematic construction of the promoter region to achieve cumulative enhancement of HbA2 expression while maintaining design flexibility.
Solution Approach 2:
The invention combines multiple transcription factor binding sites within a single DNA donor template, integrating KLF1, b-DRF, TFIIB, and LDB1 sites together with homology arms. This merging approach enables simultaneous recruitment of multiple transcription factors to协同 enhance HbA2 expression from a single edited locus, maximizing therapeutic effect while managing composition complexity.
3Reliability
If HBD gene expression is enhanced to compensate for low HbA, then treatment effectiveness for haemoglobinopathies is improved, but the competitiveness of HBD expression against HBB must be overcome
Solution Approach 1:
The invention changes the regulatory parameters of the HBD promoter by introducing high-affinity binding sites for multiple transcription factors. This parameter modification increases the transcriptional activation strength of HBD, enabling it to compete effectively with the highly expressed HBB gene and achieve therapeutically relevant HbA2 levels despite the natural dominance of beta-globin expression.
Solution Approach 2:
The edited HBD promoter functions as a composite regulatory element combining multiple transcription factor binding sites (KLF1, b-DRF, TFIIB, LDB1) that work synergistically. This composite promoter structure integrates multiple activation mechanisms to overcome the natural expression suppression of HBD relative to HBB, achieving reliable HbA2 production for therapeutic benefit.
Data Source
AI summary
A CRISPR-based genome editing is disclosed. More specifically, nucleic acids, compositions and kits for editing a human HBD gene and their use in the treatment of haemoglobinopathies are disclosed. Methods of making thereof and methods of editing a human HBD gene are also disclosed. The composition and/or kit contains (a) a first component selected from a guide RNA (gRNA) or a DNA polynucleotide encoding the gRNA, (b) a second component selected from a DNA donor template or a vector containing the DNA donor template, and (c) a third component containing a Cas9 polypeptide or a variant thereof or a nucleic acid encoding a Cas9 polypeptide or a variant thereof.