Preservation of aqueous outflow facility after second-generation FIV vector-mediated expression of marker genes in anterior segments of human eyes. Invest Ophthalmol Vis Sci.

PURPOSE. Feline immunodeficiency virus (FIV)-based lentiviral vectors produce effective genetic modification of the trabecular meshwork (TM) of human eyes in organ-perfusion culture, resulting in high-level expression of a ␤-galactosidase marker gene (lacZ) without loss of TM cellularity or architecture. However, effects on aqueous outflow physiology have not been determined, and the ability to monitor FIV vector transgene expression in living TM in situ has not been established. In the current study, transgene expression and outflow facility were evaluated in perfused human anterior segments after FIV vector transduction of lacZ or of a marker gene that can be monitored noninvasively, enhanced green fluorescent protein (eGFP). METHODS. Second-generation FIV vectors were made with a protocol for scaled-up production that requires 10 times less input DNA and allows simplified concentration. One vector encodes ␤-galactosidase (vector CT26), and the other (bicistronic) encodes eGFP and neomycin phosphotransferase (vector GiNWF). Three pairs of eyes were injected with 1 ϫ 10 8 transducing units (TU) of CT26 in the right eye and with a control (mock lacZ) vector in the left eye. Three others were injected with 1 ϫ 10 8 TU GiNWF in the right eye only, with the left eye serving as an uninjected control. Intraocular pressure was recorded and transduction efficiency was determined. RESULTS. The modified protocol produced high-titer FIV vectors, and coordinate expression of marker genes was observed with the bicistronic vector. In human eyes, the eGFP and lacZ vectors transduced 79% Ϯ 15% and 82% Ϯ 4% of TM cells, respectively, without cell loss compared with control eyes. Transduction and marker gene expression caused a transient decrease of outflow facility (30% Ϯ 22%, P ϭ 0.02), which resolved after 48 to 72 hours. CONCLUSIONS. FIV vectors produce high-level expression of eGFP in the TM of the cultured human eye, with transduction efficiency similar to that obtained with ␤-galactosidase vectors. Transduction and expression of these marker genes results in small and transient changes in outflow facility, suggesting suitability of this class of vectors for glaucoma gene therapy. (Invest Ophthalmol Vis Sci. 2002;43:3686 -3690) H igh intraocular pressure is the most common causal risk factor for glaucoma and is the key variable for clinical management. Intraocular pressure is primarily determined by aqueous humor production and outflow. The trabecular meshwork (TM) generates most of the resistance to outflow, which normally maintains intraocular pressure within the narrow range (10 -21 mm Hg) that is essential for viability of the neuroretina. Impairment of TM-regulated outflow is believed to be the primary physiological derangement in the majority of primary open-angle glaucoma patients

Unintegrated Lentivirus DNA Persistence and Accessibility to Expression in Nondividing Cells: Analysis with Class I Integrase Mutants

The circumstances under which unintegrated lentivirus DNA can persist and be a functional template for transcription and protein expression are not clear. We constructed and validated the first class I (nonpleiotropic) integrase (IN) mutants for a non-human lentivirus (feline immunodeficiency virus [FIV]) and analyzed both these and known class I human immunodeficiency virus type 1 IN mutants. The FIV IN mutants (D66V and D66V/D118A) had class I properties: Gag/Pol precursor expression, proteolytic processing, particle formation, and reverse transcriptase (RT) production were normal, while the transduction of dividing fibroblasts was prevented and integration was blocked. When injected into rat retinas, the wild-type (WT) vector produced extensive, persistent transgene expression, compared with only rare positive neuronal cells for the IN mutant vector. In contrast, both WT and mutant vectors produced entirely equivalent, effective transduction levels of primary rat neurons (retinal ganglion cells). By testing the hypothesis that the unexpected retinal neuron transduction was related to cell cycle status, we found that when fibroblasts were growth arrested, transduction and internally promoted transgene expression were not inhibited at all by the class I FIV or HIV-1 IN mutations. Cells were then transduced under aphidicolin arrest and were released from the block 48 h later. Vector expression was stable and durable during repeated passaging in WT vector-transduced cells, while the release of cells transduced with equivalent RT units of class I IN mutant FIV or HIV vector resulted in a steady decline of expression, from 97 to 0% of cells by day 10. Southern blot and PCR analyses showed a lack of integration, irrespective of cell cycle, for the class I mutants and an increase in one- and two-long terminal repeat circular and linear unintegrated DNAs in growth-arrested cells. We conclude that if cell division is prevented, unintegrated FIV and HIV-1 vector DNAs can produce high-level internally promoted transgene expression equivalent to WT vectors. The expression correlates with the unintegrated DNA levels. These observations may facilitate the study of the roles of IN and other preintegration complex components in preintegration phases of infection by (i) providing an alternative way to monitor unintegrated nuclear cDNA forms, (ii) restricting ascertainment to the transcriptionally functional subset of unintegrated DNA, (iii) enabling analysis in individual, nondividing cells, and (iv) uncoupling other potential functions of IN from integration