Combined effect of cyclosporine and sirolimus on improving the longevity of recombinant adenovirus-mediated transgene expression in the retina.
OBJECTIVE
To reevaluate the longevity and intraocular safety of recombinant adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic immunosuppressants.


METHODS
An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expression was monitored in real time by fundus fluorescent photography. Intraocular safety was examined by observation of changes of retinal pigmentation, cell infiltration in virus-contacted area, immunophenotyping for CD4(+) and CD8(+) cytotoxic T lymphocytes, and CD68(+) macrophages, histologic findings, and dark-adapted electroretinography. Two synergistic immunosuppressants, cyclosporine and sirolimus, were used alone or in combination to prolong transgene expression by temporary immunosuppression.


RESULTS
The GFP expression peaked on day 4, dramatically decreased on day 10, and was not detectable on day 14. The decreased GFP expression was coincident with cell infiltration in virus-contacted area. Immunostaining showed that the infiltrating cells were CD4(+) and CD8(+) cytotoxic T lymphocytes and CD68(+) macrophages. Clumped retinal pigmentation and decreased b wave of dark-adapted electroretinogram were observed at 3 to 4 weeks after injection. Histologic examination confirmed rAd-induced retinal degeneration. Transient immunosuppression by cyclosporine and sirolimus, either alone or in combination, improved transgene expression, with the combination being the most efficient. The combined immunosuppression attenuated but did not retard the rAd-induced retinal damage.


CONCLUSIONS
Transgene expression mediated by rAd after subretinal delivery is short-term and toxic to the retina. Combination of cyclosporine and sirolimus may act as an immunosuppressive adjunct to prolong rAd-mediated gene transfer.


CONCLUSIONS
The intraocular safety of rAd should be carefully considered before clinical trials are performed.
Shen
W Y
WY
Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Australia.
Lai
M C
MC
Beilby
J
J
Barnett
N L
NL
Liu
J
J
Constable
I J
IJ
Rakoczy
P E
PE
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000473:pathology; D018414:CD8-Positive T-Lymphocytes / Q000473:pathology; D016572:Cyclosporine / Q000494:pharmacology*; D004338:Drug Combinations; D004596:Electroretinography; D005451:Fluorescein Angiography; D005654:Fundus Oculi; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D016130:Immunophenotyping; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology / Q000473:pathology; D051381:Rats; D011922:Rats, Mutant Strains; D012160:Retina / Q000187:drug effects* / Q000378:metabolism; D012162:Retinal Degeneration / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D020123:Sirolimus / Q000494:pharmacology*; D013602:T-Lymphocytes, Cytotoxic / Q000473:pathology; D019076:Transgenes; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D016572:Cyclosporine; D004338:Drug Combinations; D049452:Green Fluorescent Proteins; D007166:Immunosuppressive Agents; D008164:Luminescent Proteins; D020123:Sirolimus; C067980:CD68 antigen, human; D000097382:CD68 Molecule