PUBLICATION
Successful large gene augmentation of USH2A with non-viral episomal vectors
- Authors
- Toms, M., Toualbi, L., Almeida, P.V., Harbottle, R., Moosajee, M.
- ID
- ZDB-PUB-230620-42
- Date
- 2023
- Source
- Molecular therapy : the journal of the American Society of Gene Therapy 31(9): 2755-2766 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Humans
- Zebrafish/genetics
- HEK293 Cells
- DNA
- Plasmids/genetics
- Animals
- Mutation
- Extracellular Matrix Proteins/genetics
- Usher Syndromes*/genetics
- Usher Syndromes*/therapy
- PubMed
- 37337429 Full text @ Mol. Ther.
Citation
Toms, M., Toualbi, L., Almeida, P.V., Harbottle, R., Moosajee, M. (2023) Successful large gene augmentation of USH2A with non-viral episomal vectors. Molecular therapy : the journal of the American Society of Gene Therapy. 31(9):2755-2766.
Abstract
USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases, however conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7kb, such as the 15.6kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23kb. We assessed the vectors in transfected HEK-293 cells and USH2A patient-derived dermal fibroblasts, in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localisation in the photoreceptors of ush2au507 zebrafish retina injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector which can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping