Targeted gene transfer to corneal endothelium in vivo by electric pulse

Abstract

A novel method of in vivo targeted gene transfer to intenthelial cells as early as day 1 and lasted until day 21. The tionally selected areas of the corneal endothelium was most intense gene expression was observed on days 1 and developed. Plasmid DNA with the lacZ gene coding for ␤-3 (5.21% on day 1 and 6.45% on day 3). The expression galactosidase was injected into the anterior chamber of of ␤-galactosidase on day 3 was most evident following adult Wistar rats, and eight pulses of electricity at intendelivery of 20 V electric pulses (0.09% at 5 V, 0.03% at sities ranging from 5 to 40 V/cm were delivered for 50 ms 10 V, 6.45% at 20 V). ␤-Galactosidase expression was limto the cornea with a specially designed electric probe in ited to the corneal endothelial cells in highly selected areas order to determine the effect of gene transfer on the corand no ␤-galactosidase expression was detected in any neal endothelial cells. Gene expression was visualized by other intra-or extraocular tissues. In addition, no cell damenzymatic color reaction using X-gal in enucleated eyes on age was apparent in the cornea and no inflammation was days 1, 3, 7, 14 and 21 after gene transfer. The treated detected in any other intraocular tissues. Thus, low-voltage eyes were then photographed and the X-gal-positive areas electric pulses successfully transferred the gene of interest were evaluated by an image analyzer. The ratios of the to highly selective areas of the corneal endothelium without areas (X-gal-positive area/area of entire corneal endoinducing any pathological changes. This targeted gene thelium × 100%) were then calculated to determine gene transfer method appears to have great potential for use in transfection efficiency. The expression of ␤-galactosidase gene therapy for ocular diseases. was clearly detected in the cytoplasm of the corneal endo-

Efficient and sustained transgene expression in human corneal cells mediated by a lentiviral vector

“…1-4 Similar short-term expression has been observed with electropulse-mediated gene delivery to rat endothelial cells 5 and liposomemediated transfection of human keratocytes. 6 Lentiviral vectors are integrating vectors with a broad tropic range, able to transduce mitotically active or inactive cells, making them potentially useful for the transfer of genetic information into all corneal cell types.…”

In vivo gene delivery into ocular tissues by eye drops of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles

“…The presence of ␤-Gal activity was determined by histochemical analysis using X-gal substrate (Gibco BRL, Grand Island, NY, USA) following the method of Oshima et al 22 Enucleated eyes were fixed with 4% paraformaldehyde at 4°C for 90 min. Eyes were next exposed to 10 mm K 4 Fe(CN) 6 , 10 mm K 3 Fe(CN) 6 , 0.01% sodium deoxycholate, 0.02% NP40, and 2 mm MgCl 2 in PBS solution containing 1 mg/ml of the X-gal substrate.…”

In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice

“…3 Marker genes were successfully transferred into normal tissues of animals including skeletal muscle, 4,5 liver, 6 skin 7 and cornea. 8 Tumors implanted in animals were also revealed to be good targets for in vivo electroporation; melanoma, 9 hepatocellular carcinoma, 10 and glioma 11 resulting in remarkable therapeutic outcome. The herpes simplex virus-type I thymidine kinase gene was transferred into tumors derived from the CT26 colon carcinoma cell line.…”