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A Reporter-Based Screen to Identify Potent 3’ Trans-Splicing Molecules for Endogenous RNA Repair
- Source :
- Human Gene Therapy Methods. 24:19-27
- Publication Year :
- 2013
- Publisher :
- Mary Ann Liebert Inc, 2013.
-
Abstract
- In the treatment of genetic disorders, repairing defective pre-mRNAs by RNA trans-splicing has become an emerging alternative to conventional gene therapy. Previous studies have made clear that the design of the binding domains of the corrective RNA trans-splicing molecules (RTMs) is crucial for their optimal functionality. We established a reporter-based screening method that allows for selection of highly functional RTMs from a large pool of variants. The efficiency and functionality of the screen were validated in the COL7A1 gene, in which mutations are the cause of the skin disease dystrophic epidermolysis bullosa. Comparison of RTMs containing different binding domains hybridizing to COL7A1 intron 64/exon 65 revealed highly different trans-splicing efficiencies. Isolated RTMs were then adapted for endogenous trans-splicing in a recessive dystrophic epidermolysis bullosa (RDEB) keratinocyte cell line expressing reduced levels of COL7A1 mRNA. Our results confirm the applicability and relevance of prescreening reporter RTMs, as significant levels of endogenous COL7A1 mRNA repair were seen with RTMs identified as being highly efficient in our screening system.
- Subjects :
- Keratinocytes
Collagen Type VII
Blotting, Western
Genetic Vectors
Green Fluorescent Proteins
Molecular Sequence Data
Trans-splicing
Genes, Recessive
Biology
Transfection
medicine.disease_cause
Applied Microbiology and Biotechnology
Trans-Splicing
Exon
Genes, Reporter
Genetics
medicine
Humans
RNA, Messenger
Cloning, Molecular
Genetics (clinical)
Pharmacology
Messenger RNA
Mutation
Base Sequence
HEK 293 cells
Intron
Epidermolysis bullosa dystrophica
RNA
Exons
Flow Cytometry
medicine.disease
Molecular biology
Epidermolysis Bullosa Dystrophica
Cell biology
HEK293 Cells
Retroviridae
Molecular Medicine
Subjects
Details
- ISSN :
- 19466544 and 19466536
- Volume :
- 24
- Database :
- OpenAIRE
- Journal :
- Human Gene Therapy Methods
- Accession number :
- edsair.doi.dedup.....c231ea4dc0e375ca688f35ff619aceab
- Full Text :
- https://doi.org/10.1089/hgtb.2012.180