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de Leeuw, Charles N.; Korecki, Andrea J.; Berry, Garrett E.; Hickmott, Jack W.; Lam, Siu L.; Lengyell, Tess C.; Bonaguro, Russell J.; Borretta, Lisa J.; Chopra, Vikramjit; Chou, Alice Y.; D’Souza, Cletus A.; Kaspieva, Olga; Laprise, Stéphanie; McInerny, Simone C.; Portales-Casamar, Elodie; Swanson-Newman, Magdalena I.; Wong, Kaelan; Yang, George S.; Zhou, Michelle; Jones, Steven J. M.; Holt, Robert A.; Asokan, Aravind; Goldowitz, D.; Wasserman, Wyeth W.; Simpson, Elizabeth M.

BioMed Central

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.

Article

eng

Vancouver : University of British Columbia Library

10.14288/1.0361796

Medicine, Faculty of; Other UBC; Non UBC; Medical Genetics, Department of; Psychiatry, Department of

10.1186/s13041-016-0232-4

Faculty

http://creativecommons.org/licenses/by/4.0/