Allele-Specific ELANE Knockout Using SNP-Guided CRISPR Editing
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Solution Overview
Problem
Current treatments for severe congenital neutropenia (SCN) and cyclic neutropenia (CyN), such as G-CSF therapy and hematopoietic stem cell transplant, are associated with risks and limitations, including increased risk of myelodysplastic syndrome or leukemia, and require a matched donor, while existing genetic approaches lack specificity in targeting dominant-mutant alleles.
Innovation Solution
A method utilizing CRISPR nuclease and guide RNA molecules to target and introduce double-strand breaks specifically at heterozygous SNPs in the ELANE gene, allowing for the inactivation of the mutant allele and preservation of the functional allele, thereby treating SCN or CyN.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If G-CSF therapy is used to treat severe congenital neutropenia, then neutrophil production is stimulated, but the risk of developing myelodysplastic syndrome or leukemia increases
Solution Approach 1:
The patent extracts and removes the harmful mutant ELANE allele from the patient's genome while preserving the functional allele. By using CRISPR-Cas9 to specifically target and inactivate the dominant-negative mutant allele, the treatment eliminates the source of the harmful effect (mutant protein interfering with neutrophil production) without needing continuous G-CSF stimulation that carries malignancy risks
Solution Approach 2:
Instead of stimulating neutrophil production pharmacologically with G-CSF (which has harmful side effects), the patent inverts the approach by directly correcting the genetic defect causing the production failure. The CRISPR system is designed to specifically recognize and disable the mutant allele sequence, thereby restoring normal neutrophil production through genetic correction rather than pharmacological stimulation
2Reliability
If hematopoietic stem cell transplant is performed to treat severe congenital neutropenia, then curative treatment is achieved, but a matched donor is required and the procedure carries significant risks
Solution Approach 1:
The patent enables the patient's own hematopoietic stem cells to be corrected and used for treatment. By modifying the patient's autologous stem cells ex vivo to inactivate the mutant ELANE allele, the treatment eliminates the need for donor matching and reduces transplant-related risks. The corrected patient cells engraft and produce healthy neutrophils, achieving curative intent with reduced complexity
Solution Approach 2:
The patent performs preliminary genetic correction of the mutant allele in the patient's hematopoietic stem cells before transplantation. By pre-inactivating the dominant-negative mutant allele ex vivo using CRISPR-Cas9, the stem cells are prepared in advance to produce only functional neutrophils upon engraftment, eliminating the need for complex donor matching procedures and reducing transplant risks
3Reliability
If existing genetic approaches are used to target mutant alleles, then gene therapy is attempted, but specificity in targeting dominant-mutant alleles is lacking
Solution Approach 1:
The patent applies local quality by designing the CRISPR guide RNA to specifically recognize and bind only to the mutant ELANE allele sequence at the precise location of the dominant-negative mutation. The guide RNA sequence is customized to match the mutant allele while having minimal homology to the functional allele, ensuring that the Cas9 nuclease cuts only the mutant DNA and leaves the functional allele intact. This localized, sequence-specific targeting achieves high manufacturing precision in allele discrimination
Solution Approach 2:
The patent segments the ELANE gene into two distinct targets: the functional allele and the mutant allele. By identifying sequence variations (such as SNPs or indels) that differ between the alleles, the CRISPR system is designed to specifically target and inactivate only the mutant segment while preserving the functional segment. This segmentation allows for precise genetic editing that distinguishes between the two alleles
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 inactivates the mutant ELANE allele, reducing neutropenia symptoms and minimizing the risk of secondary malignancies, with potential for long-term engraftment and differentiated hematopoietic cell production.
Implementation Method 1
A method utilizing CRISPR nuclease and guide RNA molecules to target and introduce double-strand breaks specifically at heterozygous SNPs in the ELANE gene
Data Source
AI summary
Methods for inactivating in a cell a mutant allele of the elastase, neutrophil expressed gene (ELANE gene) gene having a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN) and which cell is heterozygous at one or more polymorphic sites selected from the group consisting of: rs10414837, rs3761005, rs1683564, rs9749274, rs740021, rs201048029, rs199720952, rs28591229, rs71335276, rs58082177, rs3826946, rs10413889, rs761481944, rs3761008, rs10409474, rs3761007, rs17216649, rs10469327, rs8107095, rs10424470 and rs78302854, the method comprisingintroducing to the cell a composition comprising:a CRISPR nuclease or a sequence encoding the CRISPR nuclease; anda first RNA molecule comprising a guide sequence portion having 17-20 nucleotides,wherein a complex of the CRISPR nuclease and the first RNA molecule affects a double strand break in the mutant allele of the ELANE gene,the method optionally further comprising introduction of a second RNA molecule comprising a guide sequence portion capable of complexing with a CRISPR nuclease, wherein the complex of the second RNA molecule and CRISPR nuclease affects a second double strand break in the ELANE gene.