Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice

Proc Natl Acad Sci U S A. 2012 Nov 6;109(45):E3136-45. doi: 10.1073/pnas.1206506109. Epub 2012 Oct 10.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Binding, Competitive
  • Brain / metabolism*
  • Brain / pathology*
  • Chromosomes, Mammalian / metabolism
  • Disease Models, Animal
  • Gene Expression Regulation
  • Gene Transfer Techniques
  • Genes, Reporter
  • HEK293 Cells
  • Humans
  • Huntingtin Protein
  • Huntington Disease / genetics
  • Huntington Disease / therapy
  • Mice
  • Mice, Transgenic
  • Molecular Sequence Data
  • Mutant Proteins / metabolism*
  • Nerve Tissue Proteins / metabolism*
  • Nuclear Proteins / metabolism*
  • Peptides / metabolism
  • Phenotype
  • Plasmids / genetics
  • Protein Binding
  • Repressor Proteins / metabolism*
  • Stereotaxic Techniques
  • Trinucleotide Repeat Expansion / genetics
  • Zinc Fingers*

Substances

  • Htt protein, mouse
  • Huntingtin Protein
  • Mutant Proteins
  • Nerve Tissue Proteins
  • Nuclear Proteins
  • Peptides
  • Repressor Proteins
  • polyglutamine