Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues.
OBJECTIVE
Heme oxygenase-1 (HO-1) is a stress protein induced up to 100-fold within a few hours after exposure to oxidative stress, and it has been shown to counteract oxidative injury induced by ultraviolet light or free radicals. The current study was undertaken to determine whether the HO-1 gene can be introduced into adult rabbit ocular tissues by microinjection of a recombinant replication-deficient adenovirus human HO-1 cDNA (Adv-HHO).


METHODS
Human HO-1 gene was used for transfection studies to differentiate endogenous from transfected HO. The purified Adv-HHO construct (10(8) pfu/ml) was mixed with lipofectamine and microinjected into the anterior chamber, vitreous cavity, and subretinal space of New Zealand rabbit eyes. After 2 weeks, total RNA was extracted from different ocular tissues, reverse transcription-polymerase chain reaction was performed using specific human HO-1 primers, and amplification products were subjected to Southern hybridization.


RESULTS
Transfection with the Adv-HHO construct into rabbit corneal epithelial cells in culture resulted in a functional expression of the human HO-1 gene; the human HO-1 mRNA was detected, and enzyme activity increased threefold. Human HO-1 mRNA was detected in the retina after microinjection of the Adv-HHO construct into the subretinal space. Microinjection into the vitreous resulted in HO-1 mRNA expression in the corneal endothelium, iris, lens, and retina; after intracameral injection of the Adv-HHO construct, human HO-1 mRNA was detected in corneal epithelium and endothelium, ciliary body, lens, and iris. Regardless of the injection site, transfected human HO-1 mRNA was undetectable in tissues outside the eye, that is, brain, liver, and kidney.


CONCLUSIONS
These results demonstrated a tissue-selective functional transfer of the human HO-1 gene into rabbit ocular tissues in vivo. This technique may be a promising means for delivering HO-1 gene in vivo as a protective mechanism against oxidative stress that contributes to the pathogenesis of ocular diseases such as cataract, light-induced injury, age-related macular degeneration, and diabetic retinopathy.
Abraham
N G
NG
Laboratory of Clinical Pharmacology, Rockefeller University, New York, NY 10021, USA.
da Silva
J L
JL
Lavrovsky
Y
Y
Stoltz
R A
RA
Kappas
A
A
Dunn
M W
MW
Schwartzman
M L
ML
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000201:enzymology; D017931:DNA Primers / Q000737:chemistry; D004848:Epithelium / Q000201:enzymology; D005123:Eye / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008845:Microinjections; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D012333:RNA, Messenger; D012313:RNA; D006419:Heme Oxygenase (Decyclizing)