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Elizabeth M Simpson, Charles N de Leeuw, Andrea J. Korecki, Jack W Hickmott, Siu Ling Lam, Tess C Lengyell, Kaelan Wong, Michelle Zhou, Daniel Goldowitz, Wyeth W. Wasserman; rAAV-Compatible MiniPromoters for Restricted Expression in the Brain and Eye. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1176. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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 – but it is anticipated that 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 research and clinical need for such promoters by developing a high-throughput pipeline that goes from bioinformatic design of promoters to their rapid testing in vivo.
For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs, ~4 kb human DNA regulatory elements), which had previously been 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 in non- or slowly-dividing cells typical 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 and characterized by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP.
We initially established a two-step screening system, assuming we would need the sensitivity of a historical-indirect reporter system to detect expression from restricted promoters. However, we found disadvantages in such a sensitive system, and that detection of the direct emerald GFP reporter was not limiting. We also showed a positive impact of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) mut6, which was independent of MiniP, or open reading frame. 16 of the 19 (84%) MiniPs, tested in rAAV, recapitulated the expression pattern of their design source. This included expression for: 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.
The MiniPs presented here are powerful tools for basic and preclinical research, and may enable a paradigm shift in clinical gene therapy. This work establishes a pipeline for MiniP development, and presents new MiniPs positioned to positively impact therapies for such disorders as Parkinson Disease, congenital stationary night blindness, and macular degeneration.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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