Multipartite AAV Vectors for Large-Gene Recombination Delivery
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Solution Overview
Problem
Adeno-associated viral (AAV) vectors face limitations in delivering genes larger than 4.7 kb, such as the ABCA4 gene for Stargardt disease, which is crucial for treating inherited retinal diseases like juvenile macular degeneration.
Innovation Solution
AAV vector genomes are designed with a 5' to 3' orientation comprising a 5' AAV inverted terminal repeat, promoter, splice donor site, recombination site, polynucleotide encoding a recombinase, poly-A site, and 3' AAV inverted terminal repeat, allowing for multipartite gene delivery through recombinase-mediated recombination of transgenes, including the ABCA4 gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional AAV vectors are used for gene delivery, then packaging capacity is limited to about 4.7 kb, but this limitation prevents delivery of larger genes such as the ABCA4 gene (6.8 kb) required for treating Stargardt disease
Solution Approach 1:
The patent divides the large gene (ABCA4, 6.8 kb) into two separate transgene portions (5' portion and 3' portion) that can each fit within the 4.7 kb packaging capacity of conventional AAV vectors. These segmented portions are delivered by separate AAV vectors and then recombined in the target cell to reconstruct the full functional gene, thereby overcoming the packaging size limitation while maintaining compatibility with standard AAV delivery systems
2Quantity of substance
If the transgene is split into multiple portions for multipartite delivery, then packaging capacity limitation is overcome, but recombination efficiency and correct assembly of the full transgene become challenging
Solution Approach 1:
The patent introduces a recombinase enzyme (such as Cre recombinase) as an intermediary to catalyze the recombination between the 5' and 3' transgene portions. The recombinase recognizes specific recombination sites (e.g., LoxP sites) flanking each transgene portion and facilitates their precise joining to reconstruct the full transgene. This intermediary enzyme significantly enhances recombination efficiency and reliability compared to relying on spontaneous homologous recombination alone
Solution Approach 2:
The patent incorporates recombination sites (e.g., LoxP sites) and recombinase expression cassettes into the AAV vector constructs before delivery. The recombinase is co-delivered with the transgene portions and is already positioned and ready to act on the recombination sites upon entry into the target cell. This preliminary preparation ensures that recombination can occur efficiently and correctly without requiring additional steps or external factors
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
Enables effective expression of large genes like ABCA4, overcoming the packaging limitations of conventional AAV vectors and facilitating therapeutic interventions for genetic disorders.
Implementation Method 1
administration of the first AAV viral particle and the second AAV viral particle leads to recombination of the first AAV vector genome and the second AAV vector genome via the recombination site
Data Source
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AI summary
Provided herein are recombinant AAV vectors, AAV viral vectors, and capsid proteins for improved gene therapy, and methods for their manufacture and use in a multipartite (e.g., bipartite) delivery system. The vectors provide portions of transgene and direct their recombination in a cell to provide complete transgenes.