Lentivirus-Like Particle Delivery of CRISPR RNP for Transient HTT Knockout
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Solution Overview
Problem
Current gene therapies for Huntington's disease, such as AMT-130, face safety concerns due to the long-term integration of exogenous genes into host cells and the stability of these genes is unknown, lacking effective etiological treatments.
Innovation Solution
A lentivirus-like particle (VLP) is developed to deliver CRISPR RNP, specifically targeting the HTT gene using a modified lentiviral envelope, enabling precise knockout of the mutant HTT gene through Cas9 protein and gRNA, either singly or in dual-target configurations to prevent protein aggregation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If AAV vector is used to deliver microRNA for long-term expression, then the therapeutic effect is improved, but the safety risk increases due to unknown gene stability and potential genome integration
Solution Approach 1:
The patent segments the gene editing function into two separate components: the Cas9 protein and the gRNA are delivered independently rather than as a single integrated construct. This segmentation allows for transient expression without permanent genome integration, resolving the contradiction between long-term therapeutic effect and safety.
Solution Approach 2:
The patent uses a lentiviral vector as an intermediary delivery system that carries the Cas9 and gRNA components without integrating them into the host genome. The vector enables transient expression of the gene editing machinery, achieving therapeutic effect while avoiding the safety risks of permanent integration.
2Reliability
If exogenous gene is integrated into host cell genome, then the therapeutic effect is sustained, but the off-target risk increases
Solution Approach 1:
The patent performs preliminary delivery of the Cas9 and gRNA components via lentiviral vector before they exert their gene editing function. This preliminary action allows the components to be present in the cell transiently, perform the desired HTT gene knockout, and then be degraded, avoiding sustained presence that could cause off-target effects.
Solution Approach 2:
The patent employs transient, non-integrated Cas9 and gRNA components that function temporarily and then are degraded by cellular mechanisms. These short-lived components achieve the therapeutic knockout effect without persisting in the cell, thereby minimizing off-target risks while maintaining therapeutic sustainability.
3Reliability
If CRISPR RNP is delivered with VLP for targeted knockout, then the etiological therapy is achieved, but the delivery complexity increases
Solution Approach 1:
The patent uses a lentiviral vector system that has been modified to perform multiple functions: it delivers both Cas9 and gRNA components, enables transient expression, and avoids genome integration. This multi-functional vector simplifies the overall delivery complexity while achieving etiological therapy.
Solution Approach 2:
The patent creates a composite delivery system combining lentiviral vector components with CRISPR-Cas9 machinery. This composite system integrates the advantages of viral delivery (high transduction efficiency) with the precision of CRISPR gene editing, achieving etiological therapy without excessive complexity.
Data Source
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AI summary
The present disclosure provides a lentivirus-like particle for treating Huntington's disease (HD). The lentivirus-like particle VLP-HD is produced by encapsulating an RNP complex including a Cas9 protein and an HTT gene-targeted gRNA with a virus-like particle (VLP) vector. After infecting cells, VLP-HD releases Cas9:gRNA RNP, which can efficiently achieve the targeted knockout of the mutant HTT gene. The present disclosure can prevent the production of the mutant HTT protein with PolyQ at the source, thereby achieving the etiological therapy for HD. VLP-HD can be either single-target VLP that delivers a single CRISPR/Cas9 system to knock out the mutant gene through a frameshift mutation of the mHTT gene, or dual-target VLP that delivers two CRISPR/Cas9 systems to delete disease-causing CAG trinucleotide repeats in the mHTT gene, thereby completely avoiding the production of PolyQ.