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

Poppy Datta; Kun Do Rhee; Rylee J Staudt; Jacob M Thompson; Ying Hsu; Salma Hassan; Arlene V Drack; Seongjin Seo

doi:10.1101/2024.04.10.588864

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

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

Poppy Datta, Kun Do Rhee, Rylee J Staudt, Jacob M Thompson, Ying Hsu, Salma Hassan, Arlene V Drack and Seongjin Seo

bioRxiv : the preprint server for biology

Cold Spring Harbor Laboratory

04/10/2024

DOI: 10.1101/2024.04.10.588864

PMCID: PMC11030439

PMID: 38645107

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Preprint (Author's original)This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

Abstract

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 therapy vectors to deliver , , , and and demonstrate efficient production of full-length proteins in cultured mammalian cells and mouse retinas. Notably, this approach significantly surpasses the trans-splicing and split-intein-based reconstitution methods in efficiency, requiring lower doses, minimizing or eliminating the production of truncated protein products, and offering flexibility in selecting splitting positions. The CRE-lox approach described here provides a simple and effective platform for producing AAV gene therapy vectors beyond AAV's packaging capacity.

Details

Title: Subtitle: Delivering large genes using adeno-associated virus and the CRE-lox DNA recombination system
Creators: Poppy Datta - University of Iowa
Kun Do Rhee - University of Iowa
Rylee J Staudt - University of Iowa
Jacob M Thompson - University of Iowa
Ying Hsu - University of Iowa
Salma Hassan - University of Iowa
Arlene V Drack - University of Iowa
Seongjin Seo - University of Iowa
Resource Type: Preprint
Publication Details: bioRxiv : the preprint server for biology
DOI: 10.1101/2024.04.10.588864
PMID: 38645107
PMCID: PMC11030439
Publisher: Cold Spring Harbor Laboratory; United States
Language: English
Date posted: 04/10/2024
Academic Unit: Stead Family Department of Pediatrics; Ophthalmology and Visual Sciences
Record Identifier: 9984618502702771

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