Dual Hybrid AAV Vector System for Large Gene Delivery
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Solution Overview
Problem
Adeno-associated virus (AAV) vectors have limited cargo capacity, making it difficult to effectively transfer genes larger than 5 kb for gene therapy, particularly for retinal diseases, as existing strategies like AAV Oversize (OZ) and dual AAV systems face inefficiencies in transduction levels and gene expression.
Innovation Solution
A dual hybrid AAV vector system with a recombinogenic region AP, comprising a pair of nucleic acid sequences with splicing donor and acceptor signals, enhances full-length transcript expression in retinal cells by facilitating recombination and splicing, overcoming previous limitations with the dual AAV hybrid AP approach.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If AAV vectors are used for gene therapy, then transduction effectiveness is improved, but cargo capacity is limited to genes smaller than 5 kb
Solution Approach 1:
The patent divides the large gene (greater than 5 kb) into two separate nucleic acid sequences, each fitting within AAV cargo capacity. The first sequence contains the 5' end portion with splicing donor signal and recombinogenic region AP, while the second sequence contains the 3' end portion with splicing acceptor signal and recombinogenic region AP. This segmentation allows both sequences to be packaged into separate AAV vectors that can subsequently recombine in target cells to restore the full-length functional gene.
2Quantity of substance
If dual AAV hybrid AP strategy is used to overcome cargo capacity limitation, then gene transfer is enabled, but transgene expression levels are reduced
Solution Approach 1:
The patent introduces splicing donor and acceptor signals as intermediary elements between the 5' and 3' portions of the large gene. These splicing signals act as mediators that enable the reconstituted full-length transcript to be properly processed and expressed. The recombinogenic region AP serves as another intermediary that facilitates precise recombination between the two AAV sequences, ensuring correct gene reconstitution and optimal expression levels in retinal cells.
3Quantity of substance
If AAV Oversize (OZ) vectors are used to transfer large genes, then cargo capacity is exceeded, but transduction efficiency decreases
Solution Approach 1:
Instead of using oversized AAV vectors that exceed cargo capacity and result in poor transduction, the patent segments the large gene into two appropriately-sized nucleic acid sequences that each fit within standard AAV packaging limits. This segmentation strategy maintains high transduction efficiency while enabling delivery of genes greater than 5 kb through subsequent recombination of the two sequences in target cells.
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
Significantly improves full-length ABCA4 mRNA production in retinal cells, demonstrating enhanced gene therapy efficacy for large gene reconstitution in photoreceptors and retinal pigment epithelium, as shown in mouse models of STGD and USH1B, offering improved treatment options for blinding conditions.
Implementation Method 1
a first nucleic acid sequence comprising the 5' end portion of a nucleic acid sequence of a synthetic intron comprising a nucleic acid sequence of a splicing donor (SD) signal and a nucleic acid sequence of a recombinogenic region AP, and a second nucleic acid sequence comprising a nucleic acid sequence of a recombinogenic region AP, and the 3' end portion of a nucleic acid sequence of a synthetic intron comprising a branch site and a polypyrimidine tract and a nucleic acid sequence of a splicing acceptor (SA) signal
Implementation Method 2
the 5' end portion of a nucleic acid sequence of a synthetic intron comprising a nucleic acid sequence of a splicing donor (SD) signal and the 3' end portion of a nucleic acid sequence of a synthetic intron comprising a branch site and a polypyrimidine tract and a nucleic acid sequence of a splicing acceptor (SA) signal
Data Source
AI summary
The invention relates to constructs, vectors, relative host cells and pharmaceutical compositions which allow an effective gene therapy, in particular of genes larger than 5 Kb by using an improved hybrid dual recombinant AAV vector system.