April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Regulation And Quantification Of Intraocular Epo Levels Towards Identification Of The Therapeutic Dose Range
Author Affiliations & Notes
  • Siddharth N. Desai
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee
  • Jessica Hines
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee
  • Rachel Haag
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee
  • Tonia S. Rex
    Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee
  • Footnotes
    Commercial Relationships  Siddharth N. Desai, None; Jessica Hines, None; Rachel Haag, None; Tonia S. Rex, None
  • Footnotes
    Support  Unrestricted funds from Research to Prevent Blindness; NIH 5P30EY13080; NIH 1T35DK07405; Roche Foundation for Anemia Research; Glaucoma Research Foundation; DoD W81XWH-10-1-0528
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1396. doi:
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    • Get Citation

      Siddharth N. Desai, Jessica Hines, Rachel Haag, Tonia S. Rex; Regulation And Quantification Of Intraocular Epo Levels Towards Identification Of The Therapeutic Dose Range. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1396.

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

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Abstract

Purpose: : Successful retinal gene therapy can depend on delivery of the correct amount of protein. An example is erythropoietin (EPO), a neuroprotective agent that has a bell-shaped dose curve, and the therapeutic range in the retina is unknown. The goal of this project was to determine the therapeutic dose range of EPO in the retina using a gene delivery approach in the retinal degeneration slow (rds) mouse.

Methods: : Adult mice that express the tetracycline transactivators (rtTA) from the retinal pigment epithelium (RPE) specific vitelliform macular dystrophy 2 promoter (VMD2.rtTA mice) were injected with a serotype 2/1 recombinant adeno-associated viral vector containing enhanced green fluorescent protein and enhanced yellow fluorescent protein controlled by a tetracycline inducible promoter (rAAV2/1.tet.egfp.eyfp). Three weeks post-injection, mice received a single intraperitoneal (IP) injection of escalating doses of doxycycline (dox), and EGFP fluorescence was quantified using a Kodak 4000MM imaging station. Additional VMD2.rtTA mice were crossed with homozygous rds mice (VMD2.rtTA:rds) and were subretinally injected with rAAV2/1.tet.egfp.Epo at postnatal day 7. From postnatal day 21-60, the mice received escalating doses of dox in the drinking water. EGFP fluorescence was measured on postnatal day 60, then mice were euthanized and eyes were collected for quantification of ocular EPO levels or processed for histological analysis to determine cell rescue in terms outer nuclear layer (ONL) thickness.

Results: : Peak EGFP fluorescence intensity was reached at 7 hours post IP dox injection. EGFP fluorescence intensity increased with increasing concentration of dox. Mice treated with rAAV2/1.tet.egfp.Epo and 2mg/ml dox show a similar level of protection as mice treated with a single intramuscular injection of rAAV.CMV.Epo. This protection correlated to at most, 19mU/ml EPO in the eye.

Conclusions: : Transgene product levels in the eye can be controlled using the tetracycline inducible promoter system in a dose dependent manner in vivo. This methodology can be used to ascertain the therapeutic range for EPO in the eye.

Keywords: neuroprotection • gene/expression • gene transfer/gene therapy 
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