Nuclease-Targeted IDLV for Precise Large-Sequence Integration
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Solution Overview
Problem
Current methods for site-specific and non-toxic delivery of transgenes in cells, particularly in hematopoietic stem cells, are inefficient and toxic, with limitations in size variability and integration efficiency, especially for sequences exceeding 4 kb.
Innovation Solution
An integration-defective lentiviral vector (IDLV) is designed with specific components including a nucleus export signaling sequence, a nucleic acid sequence of interest, a nuclease site, and homology arms, enabling precise and non-toxic integration of sequences up to several kilobases in the genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If plasmid DNA is used for transgene delivery, then delivery method is simple, but toxicity is high in primary cells and integration efficiency is low
Solution Approach 1:
The patent uses viral vectors (lentiviral and AAV vectors) as intermediary carriers to deliver transgenes to primary cells. These viral vectors are engineered to be non-toxic while maintaining efficient delivery capability, resolving the contradiction between simple delivery and low toxicity in primary cells like hematopoietic stem cells.
Solution Approach 2:
The patent modifies the physical and chemical parameters of delivery vehicles by using engineered viral vectors with specific properties (integrase-defective lentiviral vectors for stable integration, AAV vectors for episomal persistence). This allows optimization of both delivery efficiency and cellular compatibility, reducing toxicity while improving integration efficiency.
2Quantity of substance
If AAV vectors are used for transgene delivery, then packaging capacity is limited to ~4.7 kb, but transgene size flexibility is reduced
Solution Approach 1:
The patent employs multiple types of viral vectors (both AAV and lentiviral vectors) to cover different transgene size requirements. AAV vectors handle smaller transgenes within 4.7 kb capacity, while lentiviral vectors accommodate larger transgenes, collectively providing universal delivery capability across various transgene sizes and ensuring versatility.
Solution Approach 2:
The patent segments the transgene delivery system into different vector types based on transgene size requirements. By dividing the delivery approach into AAV for small transgenes and lentiviral for large transgenes, the system maintains optimal packaging capacity for each vector type while achieving overall flexibility in transgene size adaptation.
3Manufacturing precision
If HDR mediated KI is used, then precision of integration is high, but efficiency is low in non-dividing cells
Solution Approach 1:
The patent introduces a nuclease (such as CRISPR-Cas9) as an intermediary to create targeted double-strand breaks in the genome at specific loci. This creates high-affinity binding sites for the transgene, enabling precise integration even in non-dividing cells where HDR is normally inefficient, thus resolving the contradiction between precision and productivity.
Solution Approach 2:
The patent performs preliminary genome editing by creating targeted DNA breaks at the integration site before transgene delivery. This preliminary action prepares the genomic landscape to favor precise transgene integration, significantly enhancing integration efficiency in non-dividing cells while maintaining high precision through targeted nuclease activity.
4Adaptability or versatility
If transgene size is increased to exceed 4 kb, then therapeutic protein coverage is improved, but AAV packaging capacity is exceeded
Solution Approach 1:
The patent uses a universal delivery system comprising both AAV and lentiviral vectors that can collectively accommodate transgenes of various sizes. For therapeutic proteins requiring transgenes larger than 4 kb, the lentiviral vector component of the system is used, which has significantly larger packaging capacity, thereby maintaining versatility in therapeutic protein coverage without being constrained by AAV's 4.7 kb limit.
Data Source
AI summary
The present invention relates to an integration-defective lentiviral vector (IDLV) comprising a nucleic acid, the said nucleic acid comprising, between a 5′ LTR sequence and a 3′ LTR sequence, at least one nucleus export signaling sequence; at least one nucleic acid sequence of interest; and at least one nuclease site. The invention further relates to an isolated cell comprising said IDLV, a pharmaceutical composition comprising said IDLV or said isolated cell, and their pharmaceutical use in the treatment of a disease selected from the group consisting of immune diseases, viral infections, tumors and blood diseases; and/or a disease caused by the lack of a protein or by the presence of an aberrant non-functional one in an individual in need thereof.