Journal
MOLECULAR BRAIN
Volume 9, Issue -, Pages -Publisher
BMC
DOI: 10.1186/s13041-016-0232-4
Keywords
rAAV Gene therapy; Raphe nuclei; Purkinje cells; Retina; Cornea
Categories
Funding
- Genome British Columbia [AGCP-CanEuCre-01]
- Genome Canada
- GlaxoSmithKline RD Ltd.
- BC Mental Health and Addiction Services
- Child and Family Research Institute
- University of British Columbia (UBC) Institute of Mental Health
- UBC Office of the Vice President Research
- NIH National Heart, Lung and Blood Institute
- Brain Canada
- Quebec Consortium for Drug Discovery
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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; similar to 4 kb human DNA regulatory elements), previously tested in knock-in mice, were cut down to similar 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 Muller 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.
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