ELANE Mutant Allele Knockout for Allele-Specific Neutropenia Therapy
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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 developing myelodysplastic syndrome or leukemia, and require a matched donor, while existing genetic approaches lack specificity in targeting dominant-mutant alleles.
Innovation Solution
Utilizing CRISPR nuclease and guide RNA molecules to target and induce double-strand breaks in the mutant allele of the ELANE gene at specific heterozygous SNP sites, allowing for the inactivation of the dominant-mutant allele and expression of the functional protein, thereby treating SCN and CyN.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If G-CSF therapy is used to treat SCN and CyN, then neutropenia symptoms are improved, but the risk of developing myelodysplastic syndrome or leukemia increases
Solution Approach 1:
The invention extracts and removes the harmful mutant allele from the patient's genome using CRISPR-Cas9 gene editing technology. By specifically targeting and disabling the mutant ELANE allele while preserving the wild-type allele, the treatment eliminates the source of neutropenia without requiring continuous G-CSF therapy, thereby avoiding the long-term risks of leukemia and myelodysplastic syndrome associated with G-CSF treatment.
Solution Approach 2:
The invention changes the genetic parameter of the ELANE gene by introducing targeted mutations via CRISPR-Cas9 that disable only the mutant allele. This genetic parameter change permanently corrects the underlying cause of neutropenia, transforming the disease state from a condition requiring ongoing pharmacological intervention to a cured state, thereby eliminating the harmful long-term effects of continuous G-CSF therapy.
2Reliability
If hematopoietic stem cell transplant is used to treat SCN and CyN, then neutropenia symptoms are improved, but the requirement for a matched donor and treatment complexity increases
Solution Approach 1:
The invention enables the patient's own cells to serve the therapeutic function by editing the patient's autologous hematopoietic stem cells ex vivo and reinfusing them. This eliminates the need for external donor cells and complex immunomatching procedures. The edited patient cells engraft and produce healthy neutrophils, simplifying the treatment to a self-service approach that avoids the complexities of allogeneic transplantation.
3Reliability
If existing genetic approaches are used to target mutant alleles, then gene therapy is achieved, but specificity in targeting dominant-mutant alleles is insufficient
Solution Approach 1:
The invention applies local quality by designing guide RNAs that recognize and bind only to specific sequences present in the mutant allele but not in the wild-type allele. The CRISPR-Cas9 system is directed to a precise location in the mutant ELANE gene, creating a localized edit that disables only the mutant copy. This site-specific targeting ensures high specificity, preserving the wild-type allele's function while eliminating the mutant allele's harmful effects.
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 risks associated with existing treatments, with potential for high coverage in the population due to allele-specific editing.
Implementation Method 1
Utilizing CRISPR nuclease and guide RNA molecules to target and induce double-strand breaks in the mutant allele of the ELANE gene at specific heterozygous SNP sites
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: rs10424470, rs4807932, rs10414837, rs376107533, rs3761010, rs10413889, rs3761007, rs10409474, rs3761005, rs351107, rs3761001, rs740021, rs781452480, rs371057361, rs570466264, rs1041904080, rs7250194, rs17216649, rs199720952, rs6510983, rs17223066, rs7255385, rs112639467, rs141213775, rs28591229, rs10469327, rs3834645, rs1683564, rs71335276, and rs8107095, 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 17-30 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 genethe 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.