Development of lentiviral vectors for gene therapy for Usher syndrome type 1B

Autor: R Groisberg, X.–J. Yang, Concepción Lillo, Sm Azarian, T. Hashimoto, Ds Williams, Daniel Gibbs, Xm Zhang
Rok vydání: 2007
Předmět:
Zdroj: Acta Ophthalmologica Scandinavica. 85
ISSN: 1395-3907
DOI: 10.1111/j.1600-0420.2007.01062_3343.x
Popis: Purpose: Usher 1B, one of the major subtypes of a combined blindness and deafness disease, is caused by mutations in the MYO7A gene, which encodes a large unconventional myosin expressed in the retinal pigment epithelium (RPE) and photoreceptor (PR) cells. This study aims at developing viral vectors expressing the wild type human MYO7A at an adequate level in order to rescue cellular phenotypes of MYO7A mutation. Methods: The full-length (7 kb) human MYO7A cDNA was cloned into the third generation, self-inactivating lentiviral vector under different promoters and enhancers. Human genomic 4-kb DNA fragment including exon 1 through 2 was cloned by PCR. Activities of different promoters and enhancers were tested by reporter assays using ARPE-19 cells. Previously identified Myo7a-null phenotypes in shaker-1 mouse were used to test the efficacy of various lentiviruses. Results: Lentiviral vectors could successfully transduce large genes (up to 7.6 kb) in vitro and in vivo for the purpose of gene therapy. Reporter assay indicated that regions with a suppressor activity and an enhancer activity existed within intron 1. The CMV promoter drove excessive MYO7A expression in the RPE, and thus caused cell death. A chimeric promoter that consists of partial CMV promoter with 160-bp MYO7A enhancer could direct moderate levels of gene expression in RPE and PR in vivo, and rescued a number of phenotypes in the mutant mice. Conclusions: These results illustrate the importance of regulating transgene expression levels in achieving therapeutic outcomes. They demonstrate the efficacy of lentivirus-mediated expression of the large MYO7A cDNA as a gene therapy strategy for correcting the MYO7A deficiency underlying Usher 1B.
Databáze: OpenAIRE
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