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Molecular Brain
Published in: Molecular Brain 1/2016

Open Access 01-12-2016 | Research

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

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

Published in: Molecular Brain | Issue 1/2016

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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.
Appendix
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Metadata
Title
rAAV-compatible MiniPromoters for restricted expression in the brain and eye
Authors
Charles N. de Leeuw
Andrea J. Korecki
Garrett E. Berry
Jack W. Hickmott
Siu Ling Lam
Tess C. Lengyell
Russell J. Bonaguro
Lisa J. Borretta
Vikramjit Chopra
Alice Y. Chou
Cletus A. D’Souza
Olga Kaspieva
Stéphanie Laprise
Simone C. McInerny
Elodie Portales-Casamar
Magdalena I. Swanson-Newman
Kaelan Wong
George S. Yang
Michelle Zhou
Steven J. M. Jones
Robert A. Holt
Aravind Asokan
Daniel Goldowitz
Wyeth W. Wasserman
Elizabeth M. Simpson
Publication date
01-12-2016
Publisher
BioMed Central
Published in
Molecular Brain / Issue 1/2016
Electronic ISSN: 1756-6606
DOI
https://doi.org/10.1186/s13041-016-0232-4

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