Functional delivery of synthetic naked siRNA to the human trabecular meshwork in perfused organ cultures.
OBJECTIVE
To investigate whether naked short-interfering RNA (siRNA) molecules could be directly delivered to perfused intact human trabecular meshwork (TM) tissue, whether this siRNA could silence a trabecular meshwork preferred gene, and whether it could counteract the downstream effect of a deleterious agent (dexamethasone, DEX) by silencing its receptor.


METHODS
Anterior segments from post-mortem normal human donors were perfused at 3.4+/-0.3 microl/min-constant flow or 15 mmHg-constant pressure to stable baseline (outflow facility, C=0.22+/-0.19 microl/min/mmHg; n=14). Commercial siRNAs were diluted in DMEM (Dulbecco's Modified Eagle's Medium) perfusion medium and used without coupling to transfection reagents ("naked"). Perfusion of Cy3-labeled siRNA was performed at 100 nM for 48 h followed by 24 h with DMEM medium (two pairs). Perfusions of Matrix GLA protein (MGP) siRNA (100 nM; right eye [Oculus Dexter]; OD) and scramble-siRNA (control; left eye [Oculus Sinster]; OS) were performed for 48 h (two pairs). Perfusions of glucocorticoid receptor (GR)-siRNA (OD) and scramble-control (OS) were performed for 48 h and continued by adding 100 nM DEX to the perfusion media for an additional 24 h (two pairs). Frozen sections of labeled anterior segments were analyzed by confocal fluorescence microscopy. Differential expression of GR, MGP, myocilin (MYOC), cornea-derived transcript 6 (CDT6), and 18S genes was determined by reverse-transcriptase TaqMan polymerase chain reacion (RT-TaqMan PCR) on RNA extracted from dissected trabecular meshwork. Primary human trabecular meshwork cells were generated from single individuals and transfected using the nucleofector electroporator with program T-23. Levels of secreted MYOC in the effluents were analyzed by western blot.


RESULTS
Histological evaluation of anterior segments perfused with Cy3 labeled siRNA followed by unlabeled medium showed intense fluorescence in the trabecular meshwork region. MGP gene expression was silenced in the trabecular meshwork perfused with naked MGP siRNA. MGP transcripts were reduced 94.7% +/- 0.62 (individual 3) and 93.6% +/- 0.13 (individual 4) from those present in the contralateral eye perfused with the scramble control. Pretreatment of GR siRNA followed by DEX treatment caused a reduction of the MYOC and CDT6 gene expressions when compared with eyes pretreated with scramble-control (percent silencing: 99.3% +/- 0.005 and 97.3% +/- 0.25, respectively, for individual 5 and 98.2% +/- 0.06 and 85.6% +/- 0.88, respectively, for individual 6). Western blots revealed the decrease of MYOC secreted by GR siRNA-treated cell and organ cultures.


CONCLUSIONS
Readily available siRNA can be delivered to the intact human trabecular meshwork by intracameral perfusion. The delivered naked siRNA is functional, inhibiting not only the targeted gene but also their downstream effectors. This functional intracameral delivery might be of use to protect the trabecular meshwork from unwanted insults and could have important therapeutic applications.
Comes
Nuria
N
Department of Ophthalmology, University of North Carolina, Chapel Hill, NC 27599-7041, USA.
Borrás
Teresa
T
D000328:Adult; D000368:Aged; D002135:Calcium-Binding Proteins / Q000235:genetics; D002461:Cell Line, Transformed; D003598:Cytoskeletal Proteins / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005136:Eye Proteins / Q000235:genetics; D020868:Gene Silencing / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D008875:Middle Aged; D009924:Organ Culture Techniques; D010477:Perfusion; D034741:RNA, Small Interfering / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D000097568:Matrix Gla Protein; D000099179:Myocilin
D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D003907:Dexamethasone; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D034741:RNA, Small Interfering; D011965:Receptors, Glucocorticoid; D000097568:Matrix Gla Protein; D000099179:Myocilin