Linked Life Data

12631245

Source:http://linkedlifedata.com/resource/pubmed/id/12631245

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2003-3-12
pubmed:abstractText
Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome-mediated RNA trans-splicing (SMaRT), which provides intron-specific gene-correction at the pre-RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A beta-galactosidase (beta-gal) trans-splicing assay system was established using intron 51 of COL17A1 as the target for trans-splicing. In this system, intron 51 is flanked by the 5'exon and the 3'exon of the beta-gal gene, the latter containing two in-frame stop codons. Cotransfection of a pre-trans-splicing molecule consisting of the binding domain of intron 51 and the 3'exon of beta-gal without the stop codons resulted in a 300-fold increase of beta-gal activity compared to controls. A 2-3-fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3'end of the COL17A1 gene was shown using a collagen XVII mini-gene construct. The beta-gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte-specific spliceosome background. Reverse transcription polymerase chain reaction and beta-gal activity assay showed functional correction of the stop-codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT is feasible in a keratinocyte-specific context and therefore may be applied in skin gene therapy.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0906-6705
pubmed:author
pubmed:issnType
Print
pubmed:volume
12
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
37-46
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Development of spliceosome-mediated RNA trans-splicing (SMaRT) for the correction of inherited skin diseases.
pubmed:affiliation
Department of Dermatology, General Hospital, Salzburg, Austria.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't