May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
B-Crystallin Promoter as a Novel Delivering Tool for in vivo Targeted Gene Expression to the Trabecular Meshwork Tissue
Author Affiliations & Notes
  • M.-G. Spiga
    Ophthalmology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
  • L. K. Buie
    Ophthalmology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
  • G. Li
    Ophthalmology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
  • C. A. Rasmussen
    Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin
  • P. L. Kaufman
    Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin
  • T. Borras
    Ophthalmology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
  • Footnotes
    Commercial Relationships  M. Spiga, None; L.K. Buie, None; G. Li, None; C.A. Rasmussen, None; P.L. Kaufman, None; T. Borras, None.
  • Footnotes
    Support  NIH Grant EY11906, EY13126, EY15873, EY02698, NIH 1 P30 EY001665, OPREF, RRF (W Helmerich Chair), UW (P Duehr Chair) and RPB challenge grant to the UNC Dept. Ophthalmology
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 1612. doi:https://doi.org/
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      M.-G. Spiga, L. K. Buie, G. Li, C. A. Rasmussen, P. L. Kaufman, T. Borras; B-Crystallin Promoter as a Novel Delivering Tool for in vivo Targeted Gene Expression to the Trabecular Meshwork Tissue. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1612. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Gene targeting to the trabecular meshwork (TM) tissue rather than to the whole anterior segment of the eye could specifically regulate outflow facility without affecting other eye tissues. Our goal was to identify a promoter that could drive expression of selected genes to a relevant outflow region of the TM in vivo. Because αB-crystallin (αBcry) is an abundant TM gene and its expression localizes specifically to the juxtacanalicular region (JCT), we investigated whether its promoter would target a reporter gene to the TM of perfused human anterior segments, living rats and monkeys.

Methods: : The promoter sequence (-700/+44 bp) of αBcry was high-proof amplified from human genomic DNA and fused to the lacZ reporter gene. The expression cassette was inserted into the pShuttle vector in between a transcription blocker and a polyA signal. The adenovirus AdhαBcry.LacZ was generated by recombination. AdhαBcry.LacZ viruses were injected into perfused postmortem human eyes (n=3) (3X1010 VP), the anterior chamber of Wistar rats (n=6) (7X109 VP) and into the anterior chamber of both eyes of one cynomolgous monkey (OD:3.5X1010, OS:3.5X109 VP). Seven to 12 days post infection, β-galactosidase activity was assessed by enzyme histochemistry.

Results: : In all the species studied, LacZ expression (X-gal blue staining) was detected specifically in the TM tissue. In human and monkey eyes, expression was localized to the JCT region beneath Schlemm’s canal. No blue cells were observed in the inner wall of the canal. In the rats, the expression appeared to be extended to the entire TM. First experiments in primary human TM cells showed that the reporter transgene was induced by TGFβ2 (absorbance of standardized enzymatic reaction).

Conclusions: : A targeted and potentially inducible promoter approach represents an attractive alternative to the ubiquitous gene delivery to the anterior segment of the eye. The use of the αBcry promoter for specific expression to the TM could be a novel tool for targeted gene therapy for glaucoma.

Keywords: gene transfer/gene therapy • adenovirus • trabecular meshwork 
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