Lentiviral mediated gene delivery to the anterior chamber of rodent eyes.
OBJECTIVE
To study the in vivo efficiency of lentiviral vectors in delivering genes to the trabecular meshwork (TM) of rodent eyes.


METHODS
Lentiviral vectors were constructed using the elongation factor 1 alpha (EF-1alpha) promoter driving expression of the green fluorescent protein (GFP) gene. The viral construct was injected intracamerally through the peripheral cornea into the anterior chamber of live rodent eyes. Several variables were evaluated to determine the optimal conditions for TM cell transduction. These parameters included viral concentration, injection volume, needle rotation, and the modulation of anterior chamber current convections. Changes in intraocular pressures (IOPs) were monitored using a Tonopen XL. Signs of inflammation and corneal neovascularization were evaluated by slit lamp observation. Three weeks after injection, the eyes were enucleated and analyzed for GFP expression and distribution.


RESULTS
A single intracameral viral dose between 10(7) and 10(8) pfu produced a high and evenly distributed expression of GFP in the TM and corneal endothelial cells. The cornea remained clear and no signs of inflammation were present during the course of the experiment. Moreover, no significant changes in IOPs were observed.


CONCLUSIONS
A high transduction efficiency of TM and corneal endothelial cells can be effectively obtained after a single dose of recombinant lentivirus. The EF-1alpha promoter induces high expression of the reporter gene and is a reliable alternative to the CMV promoter when stable, long term expression is desired.
Department of Ophthalmology, Duke University, Durham, NC 27710, USA. Chall001@mc.duke.edu
Pratap
P
Challa
Coralia
C
Luna
Paloma B
PB
Liton
Beth
B
Chamblin
John
J
Wakefield
Ram
R
Ramabhadran
David L
DL
Epstein
Pedro
P
Gonzalez
D000818:Animals; D000867:Anterior Chamber / Q000821:virology; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D007429:Intraocular Pressure; D016086:Lentivirus / Q000235:genetics*; D023421:Models, Animal; D051381:Rats; D017207:Rats, Sprague-Dawley; D014065:Tonometry, Ocular; D014129:Trabecular Meshwork / Q000378:metabolism*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins