Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice.
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10-15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a "molecular tape measure" approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.
EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003 Barcelona, Spain.
Mireia
M
Garriga-Canut
Carmen
C
Agustín-Pavón
Frank
F
Herrmann
Aurora
A
Sánchez
Mara
M
Dierssen
Cristina
C
Fillat
Mark
M
Isalan
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism* / Q000473:pathology*; D033481:Chromosomes, Mammalian / Q000378:metabolism; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics / Q000628:therapy; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D013238:Stereotaxic Techniques; D019680:Trinucleotide Repeat Expansion / Q000235:genetics; D016335:Zinc Fingers
C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D012097:Repressor Proteins; C097188:polyglutamine