Introduction of DNA into the Rat and Primate Trabecular Meshwork by Fusogenic Liposomes Masanori Hangai,1 Hidenobu Tanihara,l Yoshihito Honda,1 and Yasufumi Kaneda' PURPOSE. TO evaluate the feasibility of introducing exogenous genes and phosphorothioate oligonucle- otides into the anterior chamber tissues of rats and monkeys using the authors' fusogenic liposomes. METHODS. Hemagglutinating virus of Japan liposomes containing LacZ DNA-high-mobility group 1 complexes or fluorescein isothiocyanate (FITC)-labeled phosphorothioate oligonucleotides were prepared and injected into the anterior chambers of rats (3 /xl) and rhesus monkeys (30 /u-i). The expression of LacZ DNA was visualized histochemically by j3-Galactosidase assay and was followed for as long as 60 days in rats and 30 days in monkeys. FITC-labeled phosphorothioate oligonucle- otides were observed by fluorescence microscopy for as long as 14 days in rats and 7 days in monkeys. RESULTS. Injection of LacZ DNA-high-mobility group 1 complexes encapsulated in hemagglutinat- ing virus of Japan liposomes resulted in blue staining in the trabecular meshwork and iris-ciliary body of rats and selectively in the trabecular meshwork of monkeys at the concentrations used. This LacZ expression lasted for as long as 14 days after injection in both animals. Phosphorothioate oligonucleotides (3 /xM) also were introduced into the rat trabecular meshwork and iris-ciliary body and into the primate trabecular meshwork when encapsulated in hemagglutinating virus of Japan liposomes, although the injection of naked FITC-labeled phosphorothioate oligonucleotides at the same concentration resulted in little fluorescence in any anterior chamber tissue. CONCLUSIONS. This study shows that the use of hemagglutinating virus of Japan liposomes can transfer LacZ DNA and phosphorothioate oligonucleotides to adult rat and primate trabecular meshwork. This system may enable progress in glaucoma research and in the development of nonviral somatic gene therapy of the trabecular meshwork to treat glaucoma. (Lnvest Ophthalmol Vis Set. 1998;39:509-5l6) laucoma is the second leading cause of vision loss in of the trabecular meshwork of eyes from patients with POAG the world; it affects at least 1 in 100 people world- and with glucocorticoid-induced glaucoma.6 The trabecular wide. ' Glaucoma is generally associated with elevated meshwork cells and extracellular matrix components also are G 2 3 intraocular pressure, ' which has been shown to lead to the thought to play a crucial role in the normal resistance to apoptotic death of retinal ganglion cells in experimental glau- aqueous outflow.6'7 It is hypothesized that a disturbed metab- coma.4'5 Therapeutic efforts have been directed largely at in- olism of the trabecular extracellular matrix may be the major traocular pressure reduction, based on the consensus that cause of increased outflow resistance in glaucoma. To make reduced intraocular pressure decreases the risk for the progres- clear the precise role of the extracellular matrix components in sion of visual loss.3 It is generally accepted that elevated in- outflow resistance, depletion, overexpression, or reconstitu- traocular pressure in most common types of glaucoma, such as tion of individual genes for trabecular extracellular matrix primary open-angle glaucoma (POAG), glucocorticoid-induced molecules, their receptors or extracellular matrix modulators, glaucoma, and pseudoexfoliation glaucoma, results from an such as transforming growth factor-/3 and matrix metallopro- increased resistance to aqueous outflow in the trabecular teinases, are necessary.6 This approach could also provide meshwork. Excessive, abnormal accumulations of extracellular evidence for altering the trabecular meshwork to treat glau- matrix materials, such as glycosaminoglycans and elastin, have coma. been noted in the corneoscleral and juxtacanalicular portions It has been recognized that POAG is hereditary, although not necessarily according to the simple Mendelian patterns. From the genetic linkage analysis of patients with POAG and a From the "Department of Ophthalmology and Visual Science, 8 12 Graduate School of Medicine, Kyoto University, Japan, and the insti- strong family history linked to chromosome lq, " several tute for Molecular and Cellular Biology, Osaka University, Japan. candidate genes have been tested, and mutations were recently Supported in part by a Grant-in-Aid for Scientific Research from detected in the gene encoding a trabecular meshwork-induced the Ministry of Education, Science, Sports, and Culture, by a Grant-in- glucocorticoid response protein.13 This protein is known to be Aid for Cancer Research from the Ministry of Health and Welfare of the Japanese Government, and by the Japanese National Society for the expressed by the trabecular meshwork and iris-ciliary body. Prevention of Blindness. Mutations in this gene were found in patients with a strong Submitted for publication April 21, 1997; revised September 16, family history and in unselected patients with POAG. To study 1997; accepted November 18, 1997. the mechanism by which the genetic abnormalities lead to Proprietary interest category: N. POAG, overexpression or inhibition of the gene products in Reprint requests: Masanori Hangai at present address: Doheny Eye Institute, University of Southern California School of Medicine, Room the trabecular meshwork in vivo would be useful. This could 303, 1450 San Pablo Street, Los Angeles, CA CA9OO33. also lead to somatic gene therapy. Investigative Ophthalmology & Visual Science, March 1998, Vol. 39, No. 3 Copyright © Association for Research in Vision and Ophthalmology 509 Downloaded from iovs.arvojournals.org on 09/23/2021 510 Hangai et al. IOVS, March 1998, Vol. 39, No. 3 For these reasons, the introduction of genes or anti- sonicated for 3 seconds and was agitated by vortexing for 30 sense oligomers into trabecular meshwork cells in vivo may seconds. After the addition of 300 jal balanced salt solution, provide an opportunity to study molecular mechanisms of the sample was shaken at 37°C for 30 minutes. Hemagglu- glaucoma and to alter the trabecular meshwork as a treat- tinating virus of Japan (Z strain) was propagated, collected, ment for glaucoma. This approach would depend on target- and purified as described previously.21 The purified hemag- ing the human trabecular meshwork cells. Previous reports glutinating virus of Japan was inactivated by ultraviolet irra- showed that adenoviral vectors could introduce a reporter diation (110 erg/mm2 per second) for 3 minutes just before gene into the murine and rabbit trabecular meshwork in use. The liposome suspension (0.5 ml, containing 10 mg l4 16 vivo. ~ However, the human drainage system through the lipids) was mixed with the inactivated hemagglutinating trabecular meshwork is markedly different in architecture virus of Japan (30,000 hemagglutinating units) in a total from that of other species, except for other higher pri- volume of 4 ml balanced salt solution. The mixture was 718 mates.' Therefore, it is not necessarily appropriate to incubated for 5 minutes at 4°C and was shaken gently for 30 apply results obtained in lower mammals, such as rodents minutes at 37°C. Free hemagglutinating virus of Japan was and rabbits, to humans. The rhesus monkey is considered a removed from the hemagglutinating virus of Japan lipo- useful animal model for investigating the trabecular mesh- somes by sucrose density gradient centrifugation. The hem- work for glaucoma. The morphology, functions, and extra- agglutinating virus of Japan liposome solution was concen- cellular matrix synthesis of the monkey trabecular mesh- trated 8-fold by centrifugation at 27,000g for 30 minutes. work have been well documented. Therefore, we used not only rats but also rhesus monkeys as models for evaluating All animal experiments adhered to the ARVO Statement the ability of nonviral vectors, hemagglutinating virus of for the Use of Animals in Ophthalmic and Vision Research. Japan liposomes, to transfer LacZ DNA and phosphorothio- Sprague-Dawley rats (6 weeks old, male) and adult rhesus ate oligonucleotides into the trabecular meshwork in vivo. monkeys were anesthetized with an intramuscular injection of We show here that, using this system, LacZ DNA and phos- ketamine-HCl. Sixty-six rats were used for gene transfer exper- phorothioate oligonucleotides can be introduced into the iments and 21 for the introduction of oligomers. Twelve mon- rat and nonhuman primate trabecular meshwork cells. keys were used for gene transfer experiments, and another 12 were used for oligomer introduction. All injections were mon- itored under an ophthalmic operating microscope. Anterior chamber injections of hemagglutinating virus of Japan lipo- MATERIALS AND METHODS somes were made through the peripheral cornea. The amount pAct-lacZ (7.2 kb) carrying the gene for /3-Galactosidase (j3-Gal) of plasmid DNA injected was 0.24 jag (3 M>1) per rat and 2.4 jag of Escherichia colt under the control of a chicken /3-actin (30 /xl) per monkey. The amount of phosphorothioate oligo- promoter was constructed previously.l9 The plasmid DNA was nucleotides injected was 0.18 /xg (3 jal) per rat and 1.8 jag (30 purified by equilibrium centrifugation in cesium chloride con- jal) per monkey. Right eyes were used for each transfection, taining ethidium bromide and then was used for injections in and left ones were uninjected to serve as internal controls. naked forms or for preparations of hemagglutinating virus of Three