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Mol Brain. 2016 May 10;9(1):52. doi: 10.1186/s13041-016-0232-4.

rAAV-compatible MiniPromoters for restricted expression in the brain and eye.

Author information

  • 1Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
  • 2Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6H 3N1, Canada.
  • 3Gene Therapy Centre, University of North Carolina, Chapel Hill, NC, 27599, U.S.A.
  • 4Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, V5Z 4S6, Canada.
  • 5Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
  • 6Department of Psychiatry, University of British Columbia, Vancouver, BC, V6T 2A1, Canada.
  • 7Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada. simpson@cmmt.ubc.ca.
  • 8Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6H 3N1, Canada. simpson@cmmt.ubc.ca.
  • 9Department of Psychiatry, University of British Columbia, Vancouver, BC, V6T 2A1, Canada. simpson@cmmt.ubc.ca.

Abstract

BACKGROUND:

Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters-however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo.

METHODS:

For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were "cut down" to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP.

RESULTS:

The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia.

CONCLUSIONS:

Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.

KEYWORDS:

Cornea; Purkinje cells; Raphe nuclei; Retina; rAAV Gene therapy

PMID:
27164903
PMCID:
PMC4862195
DOI:
10.1186/s13041-016-0232-4
[PubMed - in process]
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