Tracking RPE transplants labeled by retroviral gene transfer with green fluorescent protein.
OBJECTIVE
To determine whether human retinal pigment epithelium (RPE) can be modified by retroviral-mediated gene transfer and to monitor the human RPE cells in the subretinal space of living rabbits with scanning laser ophthalmoscopy (SLO).


METHODS
Cultured human fetal retinal pigment epithelium (HFRPE) was exposed to green fluorescent protein (GFP)-transducing retroviral vectors, Moloney murine leukemia virus, and lentivirus. The cultured cells were followed by fluorescence microscopy. Suspensions of GFP-expressing HFRPE were transplanted into the subretinal space of pigmented rabbits, and the transplant sites were examined by SLO for fluorescence, including fluorescein and indocyanine green angiography. The rabbits were euthanatized at different times after transplantation, and the retinas were studied histologically.


RESULTS
Retroviral gene transfer can introduce a foreign gene such as GFP into cultured HFRPE. Gene expression is maintained in cultured RPE for at least 3 months. The lentiviral vector transduced both nondividing and dividing cells; the Moloney vector only transduced the latter. GFP-expressing cells can be followed in the living retina. Their changes reflect the rejection response followed histologically.


CONCLUSIONS
Cultured HFRPE could be transduced to express GFP for long periods of time by retroviral gene transfer. GFP allowed retinal transplants and gene expression to be monitored in vivo. These results provide a model for potential ex vivo gene therapy in the subretinal space.
Lai
C C
CC
Department of Ophthalmology, Columbia University, Howard Hughes Medical Institute, New York, New York 10032, USA.
Gouras
P
P
Doi
K
K
Lu
F
F
Kjeldbye
H
H
Goff
S P
SP
Pawliuk
R
R
Leboulch
P
P
Tsang
S H
SH
D000818:Animals; D002478:Cells, Cultured; D003673:Defective Viruses; D016332:Fetal Tissue Transplantation; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009887:Ophthalmoscopy; D010857:Pigment Epithelium of Eye / Q000378:metabolism / Q000637:transplantation* / Q000821:virology; D011817:Rabbits; D012160:Retina / Q000166:cytology / Q000378:metabolism / Q000601:surgery*; D012190:Retroviridae / Q000235:genetics* / Q000378:metabolism
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins