Journal article
Delivering large genes using adeno-associated virus and the CRE-lox DNA recombination system
Human molecular genetics, Vol.33(24), pp.2094-2110
12/06/2024
DOI: 10.1093/hmg/ddae144
PMCID: PMC11630788
PMID: 39393808
Appears in UI Libraries Support 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 IFT140, PCDH15, CEP290, and CDH23 and demonstrate efficient production of full-length proteins in cultured mammalian cells and mouse retinas. Notably, AAV-IFT140 gene therapy vectors ameliorated retinal degeneration and preserved visual functions in an IFT140-associated retinitis pigmentosa mouse model. The CRE-lox approach described here provides a simple, flexible, and effective platform for generating 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 IowaKun-Do Rhee - University of Iowa, Ophthalmology and Visual SciencesRylee J Staudt - University of IowaJacob M Thompson - University of IowaYing Hsu - University of IowaSalma Hassan - University of IowaArlene V Drack - University of IowaSeongjin Seo - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Human molecular genetics, Vol.33(24), pp.2094-2110
- DOI
- 10.1093/hmg/ddae144
- PMID
- 39393808
- PMCID
- PMC11630788
- NLM abbreviation
- Hum Mol Genet
- ISSN
- 1460-2083
- eISSN
- 1460-2083
- Publisher
- Oxford University Press
- Grant note
- University of Iowa Flagella Vision Foundation Retina Research Foundation Pilot R01-EY034176 / NIH HHS Ronald Keech professorship
- Language
- English
- Electronic publication date
- 10/11/2024
- Date published
- 12/06/2024
- Academic Unit
- Stead Family Department of Pediatrics; Ophthalmology and Visual Sciences
- Record Identifier
- 9984722939302771
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