April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Highly Efficient Subretinal Gene Transfer in the Mouse by AAV Vectors with Capsid Tyrosine Mutations
Author Affiliations & Notes
  • A. Dinculescu
    Ophthalmology,
    University of Florida, Gainesville, Florida
  • H. Petrs-Silva
    Ophthalmology,
    University of Florida, Gainesville, Florida
  • Q. Li
    Ophthalmology,
    University of Florida, Gainesville, Florida
  • J.-J. Pang
    Ophthalmology,
    University of Florida, Gainesville, Florida
  • V. Chiodo
    Ophthalmology,
    University of Florida, Gainesville, Florida
  • L. Zhong
    Pediatrics,
    University of Florida, Gainesville, Florida
  • S. Zolotukhin
    Pediatrics,
    University of Florida, Gainesville, Florida
  • A. Srivastava
    Pediatrics,
    University of Florida, Gainesville, Florida
  • A. S. Lewin
    Molecular Genetics and Microbiology,
    University of Florida, Gainesville, Florida
  • W. W. Hauswirth
    Ophthalmology,
    University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships  A. Dinculescu, None; H. Petrs-Silva, None; Q. Li, None; J.-J. Pang, None; V. Chiodo, None; L. Zhong, UF Office of Technology, P; S. Zolotukhin, UF Office of Technology, P; A. Srivastava, UF Office of Technology, P; A.S. Lewin, None; W.W. Hauswirth, AGTC, P.
  • Footnotes
    Support  EY11123, EY13729, EY07132, EY08571, EY11087, EY0667, EY018335, NS36302, FFB, MVRF, RPB, Inc,CNPq
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 3020. doi:
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    • Get Citation

      A. Dinculescu, H. Petrs-Silva, Q. Li, J.-J. Pang, V. Chiodo, L. Zhong, S. Zolotukhin, A. Srivastava, A. S. Lewin, W. W. Hauswirth; Highly Efficient Subretinal Gene Transfer in the Mouse by AAV Vectors with Capsid Tyrosine Mutations. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3020.

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

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Abstract

Purpose: : To evaluate the transduction efficiency and the pattern of transgene expression after subretinal delivery of AAV vectors with capsid tyrosine-to-phenylalanine (Y-F) mutations in wild-type mice.

Methods: : Several self-complementary (sc) AAV vectors containing mutations in surface-exposed capsid tyrosine residues in serotypes 2, 8 and 9, were used to deliver an enhanced green fluorescent protein (EGFP) transgene under the control of a small CBA promoter to the retina of adult mice by subretinal injection. Fundus photographs were taken at different time-points after injection to monitor EGFP fluorescence in the treated eyes of live animals. Confocal microscopy was used to examine the pattern of EGFP expression in frozen retinal sections, and the identity of specific cells transduced by these vectors was confirmed by immunohistochemistry in co-localization experiments. Western blot analysis was performed to estimate the total amount of EGFP protein expressed in treated eyes.

Results: : Several mutant AAV vectors significantly enhanced transgene expression relative to equivalent doses of each wild-type vector counterpart, as revealed by fundoscopic examination of treated eyes and analysis of retinal sections. Intense EGFP fluorescence was found in photoreceptors and RPE. In addition, a serotype 8 mutant vector strongly transduced Muller cells throughout the retina after subretinal delivery, while a serotype 2 mutant led to widespread EGFP expression in ganglion cells.

Conclusions: : AAV vectors with appropriate tyrosine mutations can be used to enhance vector transduction efficiency over their wild-type counterparts, and could become novel gene delivery tools for a variety of therapeutic proteins to the retina.

Keywords: gene transfer/gene therapy • retina • Muller cells 
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