CRISPR-Cas9 RAG1 Gene Editing for Immunodeficiency

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Solution Overview

Problem

Current gene therapy approaches for RAG1 deficiency, such as those using lentiviral vectors, face challenges with suboptimal gene expression levels and immune dysregulation risks, leading to poor immune reconstitution and inflammation in patients with severe combined immunodeficiency.

Innovation Solution

Development of CRISPR-Cas9 gene editing strategies that target the RAG1 gene at its endogenous locus, using specific nucleotide sequences and guide RNAs to introduce RAG1 polypeptides or fragments, ensuring precise correction and minimizing off-target effects.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If lentiviral vectors are used to deliver RAG1 transgene, then gene delivery is achieved, but gene expression levels are suboptimal and immune dysregulation occurs

Engineering Contradiction:

Improvegene expression regulationVSAvoidimmune dysregulation

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The endogenous RAG1 gene locus serves itself as the target for correction, utilizing the cell's own genomic infrastructure to regulate expression. By correcting mutations at the native locus rather than introducing ectopic transgenes, the system leverages endogenous promoters and regulatory elements to achieve physiologically appropriate expression levels, eliminating the immune dysregulation associated with conventional gene therapy vectors

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The invention extracts the problematic elements (ectopic transgene integration, foreign vector sequences) from the gene therapy approach and replaces them with endogenous locus correction. By removing the lentiviral vector system and instead using CRISPR-Cas9 to directly edit the native RAG1 gene, the harmful effects of foreign DNA integration and ectopic expression are eliminated while preserving the beneficial gene correction

Inventive Principle:

Principle #2Taking out (Extraction)

2Manufacturing precision

If CRISPR-Cas9 is used to edit endogenous RAG1 locus, then precise gene correction is achieved, but off-target effects may occur

Engineering Contradiction:

Improvegene correction precisionVSAvoidoff-target effects

Core Design Contradiction:

Manufacturing precisionVSObject-affected harmful factors

Solution Approach 1:

The invention applies local quality by designing guide RNAs with high specificity to the exact target sequence at the RAG1 locus, ensuring that the CRISPR-Cas9 system acts only where needed. By optimizing the guide RNA sequence to match the unique genomic context of the RAG1 gene and using homology-directed repair with specific donor templates, the system achieves precise local correction while minimizing off-target effects through sequence discrimination

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The invention introduces homology-directed repair donor templates as intermediaries between the CRISPR-Cas9 cleavage event and the final gene correction. These donor templates serve as precise blueprints for repair, guiding the cellular repair machinery to restore the wild-type sequence at the exact target location. This intermediary mechanism ensures high-fidelity correction while the specificity of guide RNA binding prevents off-target cleavage events

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS20240425851A1Polynucleotides useful for correcting mutations in the rag1 gene

Publication Date: 2024.12.26 FONDAZIONE TELETHON ETS (50)

AI summary

The present invention relates to an isolated polynucleotide comprising from 5′ to 3′: a first homology region, a nucleotide sequence encoding a RAG1 polypeptide or a RAG1 polypeptide fragment, and a second homology region for use in treating a RAG-deficient immunodeficiency.