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Currently, the most effective outflow drugs approved for clinical use are prostaglandin F2α analogues, but these require daily topical self-dosing and have various intraocular, ocular surface and extraocular side effects. Lentiviral vector-mediated delivery of the prostaglandin F synthase (PGFS) gene, resulting in long-term reduction of intraocular pressure (IOP), may eliminate off-target tissue effects and the need for daily topical PGF2α self-administration. Lentiviral vector-mediated delivery of the PGFS gene to the anterior segment has been achieved in cats and non-human primates. Although these results are encouraging, our studies have identified a number of challenges that need to be overcome for prostaglandin gene therapy to be translated into the clinic. Using examples from our work in non-human primates, where we were able to achieve a significant reduction in IOP (2 mm Hg) for 5 months after delivery of the cDNA for bovine PGF synthase, we identify and discuss these issues and consider several possible solutions.

Purpose: To determine if an attenuated herpes simplex virus (HSV) lacking the large subunit of ribonucleotide reductase has antitumor effects in a transgenic mouse model of retinoblastoma (LHbetaTAg)., Methods: LHbetaTAg mice were injected ocularly with 1 x 10(6) pfu of the hrR3 virus and tumor sizes were measured 3 weeks later. Replication of the virus in the eye and cultured murine retinoblastoma cells was tested by titration. Distribution of the virus in tumor was measured by X-gal staining., Results: Intraocular injection of mice with hrR3 (n = 24) did not result in a significant reduction in tumor size compared to uninjected (n = 24) or PBS injected controls (n = 16). Neither the hrR3, nor the HSV RE6 mutant, which was previously shown to have antitumor effects in vivo, replicated in cultured murine tumor cells in vitro, compared to wild-type HSV. The hrR3 virus also did not replicate significantly in tumor cells in vivo, compared to normal eye tissue., Conclusions: These results suggest that mutant HSV lacking ribonucleotide reductase do not display oncolytic activity in the LHbetaTAg mouse and that this model may not be suitable for studying viral oncolysis as a therapy for retinoblastoma.

Purpose: To compare promoter usage in primary differentiated and SV40 TAg transformed human trabecular meshwork cells (HTM and TM1 cells)., Methods: Cultured HTM and TM1 cells were transfected with vectors expressing MYOC/TIGR from the CMV-IE, IE4/5 (HSV immediate early 4/5), ICP6 (early gene ICP6 of HSV), EF-1 alpha (human elongation factor 1 alpha-subunit), or the UB6 (human ubiquitin) promoters, respectively. Immunoblotting was used to measure MYOC/TIGR protein expression. MYOC/TIGR expression at the RNA level was detected by Northern blotting., Results: In primary HTM cells, CMV was the only promoter displaying substantial activity. In TM1 cells, several promoters were functional with the order in decreasing activity being EF-1 alpha > or = CMV > or = UB6 >> IE4/5., Conclusions: The difference between the normal and transformed HTM cells suggests that the latter cell type has alterations that influence cellular promoter function. The type of cell used is likely to be a crucial factor in evaluating the functions of promoter elements for genes expressed in the trabecular meshwork and in screening promoters for use in gene delivery studies, especially for evaluations of the MYOC/TIGR gene in relation to glaucoma mechanisms.

It has been previously shown that the strain of virus, immune competence of the host, and innate resistance of the host have an effect on the severity of ocular disease induced by topical infection with herpes simplex virus type 1 (HSV-1). This study has expanded on earlier work by examining the effect of virus inoculum and host age on mortality, incidence of ocular disease, and severity of ocular disease. BALB/c mice were infected with inocula ranging from 2 x 10(3) to 1 x 10(6) pfu of HSV-1 strain CJ394. The most significant effect of variation in the inoculum was on the percent of mice developing disease. Increasing the inoculum resulted in significantly increased disease incidence, but at 5 x 10(3) pfu/mouse or higher, there was little difference in disease severity in those animals exhibiting symptoms. Decreasing host age also resulted in a significant increase in the incidence of ocular disease, but the dependence of disease severity on host age varied with the symptom being scored. In animals exhibiting disease, the peak severity of stromal keratitis and vascularization of the cornea were unaffected by host age. However, the severity of blepharitis was significantly reduced in older mice. Increasing host age also resulted in increased resistance to encephalitis. Three to four-week old mice were very susceptible to encephalitis (100% mortality), while only 20% of 4-5 week old mice died by day 15 post-infection. Mice older than 5 weeks were completely resistant to lethal encephalitis after corneal infection.

Components of the Renin Angiotensin System (RAS) have been detected in ocular tissues and fluids. The source of the ocular RAS proteins is unknown but possibilities include diffusion or leakage from the systemic circulation, specific uptake from the blood, or local synthesis. We have used RT-PCR and in situ hybridization (ISH) to show that renin mRNA is present in ocular tissues from 3 strains of rats. By RT-PCR, we found 10 of 15 ciliary body samples, 13 of 16 iris samples, and 1 of 3 retina samples were positive for renin mRNA. Also, 6 of 6 brain and 7 of 8 kidney samples were positive. Using ISH, we found renin mRNA in the ciliary muscle adjacent to the sclera extending into the choroid. Tissue near the outflow channels of the anterior chamber angle also labeled. Retinal labeling was weak but present in the nerve fiber layer. Clusters of grains, possibly representing blood vessels, were also seen in the ciliary body, iris, and retina using ISH. These results suggest the presence of a local ocular RAS.