August 1998
Volume 39, Issue 9
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Articles  |   August 1998
Retroviral cDNA transfer to the RPE: stable expression and modification of metabolism.
Author Affiliations
  • J Ray
    James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.
  • J H Wolfe
    James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.
  • G D Aguirre
    James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.
  • M E Haskins
    James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.
Investigative Ophthalmology & Visual Science August 1998, Vol.39, 1658-1666. doi:
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    • Get Citation

      J Ray, J H Wolfe, G D Aguirre, M E Haskins; Retroviral cDNA transfer to the RPE: stable expression and modification of metabolism.. Invest. Ophthalmol. Vis. Sci. 1998;39(9):1658-1666.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

PURPOSE: To determine the versatility of retroviral vector-mediated rat beta-glucuronidase cDNA expression in the normal retinal pigment epithelium (RPE) of eyes of various species and in RPE of eyes with three types of mucopolysaccharidosis (MPS types I, VI, and VII) and to evaluate the effect of multiple transductions and long-term stable expression in the RPE. METHODS: A retroviral construct containing a rat beta-glucuronidase cDNA (NTK-BGEO) was used to infect RPE cells at subconfluence. The transduced cells were selected in G418, an antibiotic toxic to normal mammalian cells. Beta-glucuronidase activity was measured in transduced cells and media, using a fluorogenic substrate. Glycosaminoglycan profiles were examined by metabolically labeling RPE with Na2(35)SO4. RESULTS: Transduced RPE cells, regardless of species or disease status, expressed rat beta-glucuronidase. The expressed enzyme restored normal levels of glycosaminoglycans in the RPE cells of homozygous MPS VII-affected dogs by metabolizing stored glycosaminoglycans. The expressed enzyme failed to metabolize stored glycosaminoglycans of MPS I and MPS VI, indicating that overexpression could not bypass the exoglycosidase restriction. Multiple transductions increased beta-glucuronidase activity several times in the cell layer and in the media. The expression was stable in vitro for at least 12 weeks. CONCLUSIONS: A retroviral vector can mediate transfer of beta-glucuronidase in various species of normal and MPS-affected RPE. The expression is stable in vitro. The metabolism of stored glycosaminoglycans in MPS needs replacement of only the deficient enzyme to reverse the storage.

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