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John Neidhardt, Fabian Schmid, Esther Glaus, Romain Da Costa, Wolfgang Berger; Gene Therapy To Rescue Splice Defects In Eye Diseases Using U1 snRNA. Invest. Ophthalmol. Vis. Sci. 2011;52(14):485.
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Retinitis pigmentosa (RP) initially affects the mid-peripheral retina and ultimately causes severe visual impairment. The disease is associated with mutations in over 40 different genes. Frequently, screening of affected families reveals splice site mutations as the cause of the disease. Here, we evaluate a gene therapy to correct splice defects in two patient-derived cell lines from independent families.
We performed linkage in a large consanguine RP family and candidate sequencing in a five generation family showing X-chromosomal RP. Sequencing and RT-PCR was used to identify the causative mutations and to analyze transcripts. The complementarity of the U1 snRNA (U1) to the mutated splice donor site (SDS) was increased by site directed mutagenesis. Primary skin fibroblasts were cultured from affected and unaffected family members and transduced with lentiviral particles containing U1.
The two novel splice donor site mutations c.1245+3A>T and c.479G>A were identified in the ciliary genes of RPGR and BBS1, respectively, and found to cosegregate with the disease of RP. Both mutations cause splice defects. Affected family members show late-onset forms of RP without additional clinical features. In agreement with these mild phenotypes, we did not detect obvious ciliary defects in patient-derived cell lines.To correct the splice defect, we developed a gene therapeutic approach using mutation-adapted U1. U1 is required for SDS recognition of pre-mRNAs and initiates the splice process. The two RP mutations interfere with the recognition of the SDS by U1. To overcome the deleterious effects of the mutations, we generated four U1 isoforms with increasing complementarity to the SDS. Lentiviral particles were used to transduce patient-derived fibroblasts with these U1 variants. Full complementarity of U1 partially corrects the splice defects and selectively increases recognition of the mutated SDS. The therapeutic effect is dose dependent.
U1-based gene therapy constitutes a promising technology to treat SDS mutations in inherited diseases including autosomal recessive and X-linked RP. The technique might be applicable to many inherited gene defects.
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