The cellular tropism and expression kinetics of optimised EIAV vectors after intraocular administration make them attractive vehicles for delivering therapeutic genes in the management of inherited and acquired retinal and anterior segment disorders.

We evaluated the efficacy of equine infectious anaemia virus (EIAV)-based lentiviral vectors encoding endostatin (EIA-V.endostatin) or angiostatin (EIAV.angiostatin) in inhibiting angiogenesis and vascular hyperpermeability in the laserinduced model of choroidal neovascularisation (CNV). Equine infectious anaemia virus.endostatin, EIAV.angiostatin or control (EIAV.null) vectors were administered into the subretinal space of C57Bl/6J mice. Two weeks after laser injury CNV areas and the degree of vascular hyperpermeability were measured by image analysis of in vivo fluorescein angiograms. Compared with EIAV.null-injected eyes, EIAV.endostatin resulted in a 59.5% ( Po0.001) reduction in CNV area and a reduction in hyperpermeability of 25.6% ( Po0.05). Equine infectious anaemia virus.angiostatin resulted in a 50.0% ( Po0.05) reduction in CNV area and a 23.9% ( Po0.05) reduction in hyperpermeability.Equine infectious anaemia virus.endostatin, but not EIA-V.angiostatin significantly augmented the frequency of apoptosis within the induced CNV as compared with injected controls. TdT-dUTP terminal nick end labeling analysis 5 weeks post-injection, and histological and retinal flatmount analysis 12 months post-injection revealed no evidence of vector-or transgene expression-related deleterious effects on neurosensory retinal cells, or mature retinal vasculature in non-lasered eyes. Highly expressing EIAV-based vectors encoding endostatin or angiostatin effectively control angiogenesis and hyperpermeability in experimental CNV without long-term deleterious effects, supporting the use of such a strategy in the management of patients with exudative age-related macular degeneration.

No abstract

Human cytochrome P450 2B6 ( CYP2B6 ) metabolizes the prodrug cyclophosphamide ( CPA ) to produce phosphoramide mustard that cross -links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 ( designated``MetXia -P450'' ) and used it to transduce the human tumor cell lines HT29 and T47D. MetXia -P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide -treated cells transduced with MetXia -P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3 -D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed -enzyme prodrug therapy ( GDEPT ). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450 -based GDEPT strategy mediated by a direct retroviral gene transfer in vivo. Cancer Gene Therapy ( 2001 ) 8, 473 ± 482

A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells.