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rAAV2/5 gene-targeting to rods:dose-dependent efficiency and complications associated with different promoters

Gene Therapy volume 17, pages 1162–1174 (2010)Cite this article

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Abstract

A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken β actin proximal promoter CBA were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor-specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, whereas the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 1011 vg ml–1 were used.

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Acknowledgements

We are grateful to Dr Lingli Zhang (Veterinary Imaging Core Facility, University of Pennsylvania) for assistance with confocal microscopy, Dr Cheryl Craft (University of Southern California, Los Angeles) for providing the human cone arrestin antibody, Dr W Clay Smith (University of Florida) for the polyclonal GFP antibody, Svetlana Savina for histotechnical assistance, the staff of the RDSF facility for help with animal resources, Lydia Melnyk for research coordination, and Mary Leonard (Biomedical Art and Design facility, University of Pennsylvania) for the illustrations. This work was supported by: NIH Grants EY06855, EY13132, EY17549, RR02512, P30EY-001583, The Foundation Fighting Blindness Individual Investigator and Center grants (TA-NP-0607-0393-UPA; C-CMM-04090476-UPA02, and TA-GT-0507-0384-UFL), The Fight for Sight Nowak family grant, Hope for Vision Foundation, and the ONCE International Price for R&D in Biomedicine and New Technologies for the Blind.

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Authors and Affiliations

  1. Department of Clinical Studies School of Veterinary Medicine, Section of Ophthalmology, University of Pennsylvania, Philadelphia, PA, USA

    W A Beltran & G D Aguirre

  2. Department of Ophthalmology, University of Florida, Gainesville, FL, USA

    S L Boye, S E Boye, V A Chiodo & W W Hauswirth

  3. Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA

    A S Lewin

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  1. W A Beltran
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Correspondence to W A Beltran.

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Competing interests

WWH and the University of Florida have a financial interest in the use of rAAV therapies and own equity in a company (AGTC Inc.) that might, in the future commercialize some aspects of this work. All remaining authors declare that they have no competing financial interests.

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Beltran, W., Boye, S., Boye, S. et al. rAAV2/5 gene-targeting to rods:dose-dependent efficiency and complications associated with different promoters. Gene Ther 17, 1162–1174 (2010). https://doi.org/10.1038/gt.2010.56

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