RAAV-compatible MiniPromoters for restricted expression in the brain and eye. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: de Leeuw, Charles; Korecki, Andrea; Berry, Garrett; Hickmott, Jack; Lam, Siu; Lengyell, Tess; Bonaguro, Russell; Borretta, Lisa; Chopra, Vikramjit; Chou, Alice; D'Souza, Cletus; Kaspieva, Olga; Laprise, Stéphanie; McInerny, Simone; Portales-Casamar, Elodie; Swanson-Newman, Magdalena; Wong, Kaelan; Yang, George; Zhou, Michelle; Jones, Steven; Holt, Robert; Asokan, Aravind; Goldowitz, Daniel; Wasserman, Wyeth; Simpson, Elizabeth
• Is Part Of: Molecular brain. Volume 9:Issue 1(2016)
• Page Start: 1
• Page End: 13
• Publication Date: 2016-12
• Subjects: rAAV Gene therapy -- Raphe nuclei -- Purkinje cells -- Retina -- Cornea; Brain -- Periodicals; Molecular biology -- Periodicals; 573.86
• DOI: 10.1186/s13041-016-0232-4
• Languages: English
• ISSNs: 1756-6606

Abstract

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.