title: rAAV-compatible MiniPromoters for restricted expression in the brain and eye.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.|Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6H 3N1, Canada.
authors.forename: Charles N
authors.initials: CN
authors.lastname: de Leeuw
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Korecki
authors.affiliation: Gene Therapy Centre, University of North Carolina, Chapel Hill, NC, 27599, U.S.A.
authors.forename: Garrett E
authors.initials: GE
authors.lastname: Berry
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Jack W
authors.initials: JW
authors.lastname: Hickmott
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Siu Ling
authors.initials: SL
authors.lastname: Lam
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Tess C
authors.initials: TC
authors.lastname: Lengyell
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Bonaguro
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Borretta
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, V5Z 4S6, Canada.
authors.forename: Vikramjit
authors.initials: V
authors.lastname: Chopra
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Chou
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, V5Z 4S6, Canada.
authors.forename: Cletus A
authors.initials: CA
authors.lastname: D'Souza
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Olga
authors.initials: O
authors.lastname: Kaspieva
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Laprise
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Simone C
authors.initials: SC
authors.lastname: McInerny
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Elodie
authors.initials: E
authors.lastname: Portales-Casamar
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Magdalena I
authors.initials: MI
authors.lastname: Swanson-Newman
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Kaelan
authors.initials: K
authors.lastname: Wong
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, V5Z 4S6, Canada.
authors.forename: George S
authors.initials: GS
authors.lastname: Yang
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.
authors.forename: Michelle
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6H 3N1, Canada.|Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, V5Z 4S6, Canada.|Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
authors.forename: Steven J M
authors.initials: SJ
authors.lastname: Jones
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6H 3N1, Canada.|Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, V5Z 4S6, Canada.|Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.|Department of Psychiatry, University of British Columbia, Vancouver, BC, V6T 2A1, Canada.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Holt
authors.affiliation: Gene Therapy Centre, University of North Carolina, Chapel Hill, NC, 27599, U.S.A.
authors.forename: Aravind
authors.initials: A
authors.lastname: Asokan
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.|Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6H 3N1, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Goldowitz
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.|Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6H 3N1, Canada.
authors.forename: Wyeth W
authors.initials: WW
authors.lastname: Wasserman
authors.affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada. simpson@cmmt.ubc.ca.|Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6H 3N1, Canada. simpson@cmmt.ubc.ca.|Department of Psychiatry, University of British Columbia, Vancouver, BC, V6T 2A1, Canada. simpson@cmmt.ubc.ca.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Simpson
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D000229:Dependovirus / Q000378:metabolism*; D065847:Dorsal Raphe Nucleus / Q000378:metabolism; D005123:Eye / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism; D019426:Integrases / Q000378:metabolism; D008810:Mice, Inbred C57BL; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics; D051245:Retinal Bipolar Cells / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: C045073:Cre recombinase; D019426:Integrases
keywords: Cornea; Purkinje cells; Raphe nuclei; Retina; rAAV Gene therapy