375. A Glaucoma Gene Therapy Model: Eighteen Cat Study Revealing Long-Term Targeted Genetic Modification of the Anterior Chamber by Lentiviral Vectors
Authors: Pranay D. Khare, Nils Loewen, Roman Barraza, Michael Fautsch, Douglas Johnson, Eric Poeschla
Journal: Molecular Therapy (2006)

PURPOSE: Glaucoma is a promising candidate for lentiviral vector-based gene therapy, since its main cause, intraocular pressure elevation, results from pathophysiology that is i) chronic; ii) anatomically restricted to a discrete region in the anterior chamber, the trabecular meshwork (TM); iii) readily accessible via a transcorneal approach; and iv) frequently refractory to conventional therapies, such that glaucoma remains a leading cause of irreversible blindness. We present long-term results of transcorneal lentiviral vector injections in 18 domestic cats, which were studied as the most accessible large animal having anatomic similarity of the anterior chamber to the human eye. Animals were followed for 1.2 to 2.2 years. Both marker genes (eGFP) and wild-type and mutant versions of the myocilin gene, which has been implicated in uncommon forms of familial glaucoma, were studied. METHODS: 18 domestic cats received single bolus anterior chamber injections of FIV vector in doses of 106 (16 eyes) or 107 (19 eyes) transducing units (TU). Vectors encoded either eGFP, or a combination of feline myocilin (fMYOC or fMYOC with a Y423H mutation, equivalent to human MYOC Y437H) plus eGFP. A control vector lacked a gene in position 1. Follow-up included IOP measurements, slit lamp biomicroscopy, and gonioscopy. Eyes were then enucleated, examined with fluorescence microscopy, immunostaining for fMYOC and eGFP, and quantitative real-time vector PCR for vector DNA. RESULTS: Single gene eGFP vectors resulted in long-term, high-grade eGFP expression that was remarkably well targeted to the trabecular meshwork and was readily detected non-invasively for over two years. IRES-translated eGFP resulted in less eGFP expression in vitro and in vivo, and was detected in 29 of 32 eyes by gonioscope but was less than seen with translation initiated at the 5' cap site. However, considerably more extensive GFP expression was discovered when eyes were examined as dissected flat mounts after sacrifice. Expression of myocilins and eGFP was further confirmed by immunohistochemistry. Vector DNA copy number correlated with transgene expression. Mild AC inflammation was observed in some animals but resolved after 1|[ndash]|2 weeks. In all animals, IOP at end of study did not differ significantly from initial IOP (19|[ndash]|25 mmHg). CONCLUSIONS: Sustained, high-grade eGFP expression lasting over two years was achieved in TM. Vector-mediated eGFP fluorescence can be detected non-invasively via gonioscope, allowing real-time serial monitoring of transgene expression over time. This nevertheless underestimates long-term in vivo transgene expression, since considerably more eGFP expression was discovered after enucleation. Transduction of fMYOC or Y423H fMYOC did not cause elevated IOP. In summary, high level, long-term, and tissue- targeted gene expression suitable for glaucoma gene therapy is feasible with transcorneally-injected lentiviral vectors. Similar vector tests in macaques have been initiated.