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Delivering large genes using adeno-associated virus and the CRE-lox DNA recombination system

Human Molecular Genetics. 2024-04; 
Poppy Datta, Kun Do Rhee, Rylee J Staudt, Jacob M Thompson, Ying Hsu, Salma Hassan, Arlene V Drack, Seongjin Seo
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摘要

Adeno-associated virus (AAV) is a safe and efficient gene delivery vehicle for gene therapies. However, its relatively small packaging capacity limits its use as a gene transfer vector. Here, we describe a strategy to deliver large genes that exceed the AAV's packaging capacity using up to four AAV vectors and the CRE-lox DNA recombination system. We devised novel lox sites by combining non-compatible and reaction equilibrium-modifying lox site variants. These lox sites facilitate sequence-specific and near-unidirectional recombination of AAV vector genomes, enabling efficient reconstitution of up to 16 kb of therapeutic genes in a pre-determined configuration. Using this strategy, we have developed AAV gene th... More

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