CRISPR Nuclease Editing Mutant ADA2 Allele
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Solution Overview
Problem
Current methods are inadequate for effectively treating Adenosine Deaminase 2 (ADA2) deficiency, a genetic disorder characterized by abnormal inflammation, as they fail to efficiently correct the mutant ADA2 gene leading to functional protein expression.
Innovation Solution
The use of an RNA-guided DNA nuclease, such as CRISPR, to edit the ADA2 gene by introducing a guide sequence and a CRISPR nuclease to cleave and correct the mutant allele through homology-directed repair (HDR), resulting in the expression of a functional ADA2 protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current treatment methods are used for ADA2 deficiency, then the disorder symptoms persist, but the mutant ADA2 gene cannot be effectively corrected
Solution Approach 1:
The patent uses CRISPR-Cas9 system as an intermediary tool to mediate the correction of mutant ADA2 gene. The guide RNA acts as a mediator that directs the Cas9 nuclease to the specific target sequence, enabling precise gene editing without requiring complex manual manipulation of the gene structure.
Solution Approach 2:
The patent replaces traditional mechanical gene manipulation methods with a biochemical system. The CRISPR-Cas9 mechanism uses RNA-guided molecular recognition and enzymatic cleavage to achieve gene correction, substituting physical/mechanical approaches with molecular-level biochemical processes that are more precise and efficient.
2Manufacturing precision
If CRISPR nuclease is used to cleave the mutant allele, then gene editing precision is improved, but the complexity of the treatment protocol increases
Solution Approach 1:
The patent segments the gene editing function into distinct modular components: the Cas9 nuclease module, the guide RNA module with specific 20-nucleotide spacer sequence, and the donor template module. This segmentation allows each component to be optimized independently and facilitates precise targeting through the modular guide RNA design.
Solution Approach 2:
The CRISPR-Cas9 system provides universal applicability for correcting various mutant alleles of the ADA2 gene. The same Cas9 nuclease can target different mutant sequences by simply changing the guide RNA sequence, making the system universally applicable across different mutation types without requiring development of entirely different editing tools.
3Reliability
If homology-directed repair is used to correct the allele, then functional ADA2 protein expression is achieved, but the time required for effective treatment increases
Solution Approach 1:
The patent employs preliminary action by providing the donor template sequence in advance, containing the correct wild-type ADA2 gene sequence flanked by homology regions. This pre-prepared template is ready to guide the repair process immediately after cleavage, reducing the time required for the cell to search for or construct the repair template de novo.
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 allows for the targeted correction of the ADA2 gene, potentially ameliorating or preventing ADA2 deficiency by ensuring the expression of a functional ADA2 protein, thereby reducing inflammation and improving symptoms.
Implementation Method 1
The allele cleavage is selected from the group consisting of: a double strand break (DSB) and a single strand break. In some embodiments, the CRISPR nuclease affects a double strand break (DSB).
Implementation Method 2
In some embodiments, the method further comprises the step of correction of the allele such that the corrected allele result in an expression of a functional ADA2 protein. In some embodiments, the correction is performed by homology directed repair (HDR).
Data Source
AI summary
RNA molecules comprising a guide sequence portion having 17-25 nucleotides in the sequence of 20-22 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-12655 and compositions, methods, and uses thereof.