[Triple helix: a new promise for gene therapy].
OBJECTIVE
This publication focuses on the inhibition of gene expression by triplex-forming oligonucleotides, which is a promising approach to the treatment of dominant genetic diseases. The most common cause for autosomal dominant retinitis pigmentosa is a mutation in the rhodopsin gene.


METHODS
Two psoralen-linked triplex-forming oligonucleotides were used to inhibit the expression from a plasmid carrying the rhodopsin and green fluorescent protein fusion gene. Following in vitro triplex formation, UVA irradiation was used to activate the psoralen moiety to form covalent photoadducts between the three strands. The samples where then transfected into human fibrosarcoma cells and analysed for green fluorescence.


RESULTS
Photoadducted triple helix formation resulted in reduction of gene expression by as much as 90% and this effect persisted for at least 72 hours. However, expression levels from a cotransfected control plasmid were unaffected. Mutations at one of the triplex binding sites within the rhodopsin gene also abolished the effect of the corresponding triplex forming oligonucleotide, without diminishing the inhibition by the other oligo. Northern blots indicated that photoadducted triplex formation blocked the progression of the RNA polymerase, resulting in truncated transcripts.


CONCLUSIONS
The authors conclude, that psoralen linked triplex forming oligonucleotides are efficient and specific tools for blocking gene expression from the human rhodopsin gene.
Intödy
Zsófia
Z
Semmelweis Egyetem, Altalános Orvostudományi Kar, I. Sz. Szemészeti Klinika, Budapest. imtody@matavnet.hu
Perkins
Brian D
BD
Wilson
John H
JH
D018736:DNA Adducts; D005354:Fibrosarcoma; D005363:Ficusin / Q000627:therapeutic use*; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics*; D017319:Photosensitizing Agents / Q000627:therapeutic use*; D010957:Plasmids / Q000235:genetics; D012174:Retinitis Pigmentosa / Q000235:genetics* / Q000628:therapy*; D012243:Rhodopsin / Q000235:genetics*; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014466:Ultraviolet Rays
D018736:DNA Adducts; D008164:Luminescent Proteins; D009841:Oligonucleotides; D017319:Photosensitizing Agents; D049452:Green Fluorescent Proteins; D012243:Rhodopsin; D005363:Ficusin