AAV-mediated knockdown of peripherin-2 in vivo using miRNA-based hairpins.
Gene therapy for inherited retinal degeneration in which expression of a mutant allele has a gain-of-function effect on photoreceptor cells is likely to depend on efficient silencing of the mutated allele. Peripherin-2 (Prph2, also known as peripherin/RDS) is an abundantly expressed photoreceptor-specific gene. In humans, gain-of-function mutations in PRPH2 result in both autosomal dominant retinitis pigmentosa and dominant maculopathies. Gene-silencing strategies for these conditions include RNA interference by short hairpin RNAs (shRNAs). Recent evidence suggests that microRNA (miRNA)-based hairpins may offer a safer and more effective alternative. In this study, we used for the first time a virally transferred miRNA-based hairpin to silence Prph2 in the murine retina. The results show that an miRNA-based shRNA can efficiently and specifically silence Prph2 in vivo as early as 3 weeks after AAV2/8-mediated subretinal delivery, leading to a nearly 50% reduction of photoreceptor cells after 5 weeks. We conclude that miRNA-based hairpins can achieve rapid and robust gene silencing after efficient vector-mediated delivery to the retina. The rationale of using an miRNA-based template to improve the silencing efficiency of a hairpin may prove valuable for allele-specific silencing in which the choice for an RNAi target is limited and offers an alternative therapeutic strategy for the treatment of dominant retinopathies.
Georgiadis
A
A
Department of Molecular Therapy, Institute of Ophthalmology, University College London, London, UK.
Tschernutter
M
M
Bainbridge
J W B
JW
Robbie
S J
SJ
McIntosh
J
J
Nathwani
A C
AC
Smith
A J
AJ
Ali
R R
RR
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D000229:Dependovirus; D015316:Genetic Therapy / Q000379:methods*; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D064531:Peripherins; D034622:RNA Interference; D012162:Retinal Degeneration / Q000235:genetics / Q000628:therapy*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D035683:MicroRNAs; D009419:Nerve Tissue Proteins; C578994:PRPH protein, human; C578997:PRPH2 protein, human; D064531:Peripherins; C578996:Prph2 protein, mouse