Toward a gene therapy for dominant disease: validation of an RNA interference-based mutation-independent approach.
The intragenic heterogeneity encountered in many dominant disease-causing genes represents a significant challenge with respect to development of economically viable therapeutics. For example, 25% of autosomal dominant retinitis pigmentosa is caused by over 100 different mutations within the gene encoding rhodopsin, each of which could require a unique gene therapy. We describe here an RNA interference (RNAi)-based mutation-independent approach, targeting as an example murine rhodopsin. Native transcripts are suppressed by a single RNAi molecular species, whereas transcripts from replacement genes engineered at degenerate third-codon wobble positions are resistant to suppression. We demonstrate suppression of murine rhodopsin transcript by up to 90% with full concomitant expression of replacement transcript and establish the validity of this approach in cell culture, retinal explants, and mouse liver in vivo.
Kiang
Anna-Sophia
AS
Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin 2, Ireland. skiang@tcd.ie
Palfi
Arpad
A
Ader
Marius
M
Kenna
Paul F
PF
Millington-Ward
Sophia
S
Clark
Gerry
G
Kennan
Avril
A
O'reilly
Mary
M
Tam
Lawrence C T
LC
Aherne
Aileen
A
McNally
Niamh
N
Humphries
Pete
P
Farrar
G Jane
GJ
D000818:Animals; D019556:COS Cells; D002469:Cell Separation; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018274:Electroporation; D005434:Flow Cytometry; D020868:Gene Silencing; D005799:Genes, Dominant; D015316:Genetic Therapy / Q000379:methods*; D008099:Liver / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D009154:Mutation; D011312:Pressure; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012313:RNA; D012243:Rhodopsin