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Targeted CNS Delivery Using Human MiniPromoters and Demonstrated Compatibility with Adeno-Associated Viral Vectors.

Author information

1
Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada V5Z 4H4 ; Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.
2
Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, FL, USA 32610.
3
Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.
4
Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 4S6.
5
Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4 ; Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 4S6 ; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada V5A 1S6.
6
Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4 ; Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 4S6 ; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada V5A 1S6 ; Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada V6T 2A1.
7
Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada V5Z 4H4 ; Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4 ; Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada V6T 2A1.

Abstract

Critical for human gene therapy is the availability of small promoter tools to drive gene expression in a highly specific and reproducible manner. We tackled this challenge by developing human DNA MiniPromoters using computational biology and phylogenetic conservation. MiniPromoters were tested in mouse as single-copy knock-ins at the Hprt locus on the X Chromosome, and evaluated for lacZ reporter expression in CNS and non-CNS tissue. Eighteen novel MiniPromoters driving expression in mouse brain were identified, two MiniPromoters for driving pan-neuronal expression, and 17 MiniPromoters for the mouse eye. Key areas of therapeutic interest were represented in this set: the cerebral cortex, embryonic hypothalamus, spinal cord, bipolar and ganglion cells of the retina, and skeletal muscle. We also demonstrated that three retinal ganglion cell MiniPromoters exhibit similar cell-type specificity when delivered via adeno-associated virus (AAV) vectors intravitreally. We conclude that our methodology and characterization has resulted in desirable expression characteristics that are intrinsic to the MiniPromoter, not dictated by copy number effects or genomic location, and results in constructs predisposed to success in AAV. These MiniPromoters are immediately applicable for pre-clinical studies towards gene therapy in humans, and are publicly available to facilitate basic and clinical research, and human gene therapy.

KEYWORDS:

AAV; gene therapy; promoters

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