A Mistry

Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1.

Retinal angiogenesis is a central feature of the leading causes of blindness. Current treatments for these conditions are of limited efficacy and cause significant adverse effects. In this study, we evaluated the angiostatic effect of gene transfer of the soluble VEGF receptor sFlt-1 in a mouse model of ischaemia-induced retinal neovascularisation using adenovirus and adeno-associated virus (AAV) vectors. We induced proliferative retinopathy in mice by exposure to 75% oxygen from postnatal day 7 (p7) to p12 and injected intravitreally recombinant viral vectors expressing the reporter green fluorescent protein (GFP) or vectors expressing the VEGF inhibitor sFlt-1. Efficient adenovirus-mediated GFP expression was evident in cells of the corneal endothelium and iris pigment epithelium. AAV-mediated GFP expression was evident in ganglion cells and cells of the inner nuclear layer of the retina. Vector-mediated sFlt-1 expression was confirmed by ELISA of pooled homogenised whole eyes. Injection of either vector expressing sFlt-1 resulted in a reduction in the number of neovascular endothelial cells by 56% and 52% for adenovirus and AAV vectors, respectively (P < 0.05). Local gene transfer of sFlt-1 consistently inhibits experimental retinal neovascularisation by approximately 50% and offers a powerful novel approach to the clinical management of retinal neovascular disorders.

Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina.

Recombinant adeno-associated virus (rAAV) vectors are attractive candidates for the treatment of inherited and acquired retinal disease. Although rAAV vectors are well characterized in rodent models, a prerequisite to their clinical application in human patients is the thorough evaluation of their efficacy and safety in intermediate animal models. In this study, we describe rAAV-2-mediated expression of GFP reporter gene in retinal cells following local vector delivery in dogs. Subretinal delivery of rAAV.CMV.GFP was performed unilaterally in eight normal dogs from 6 weeks of age. The area of retinal transduction was maximized by the optimization of surgical techniques for subretinal vector delivery by pars-plana vitrectomy and the use of fine-gauge subretinal cannulae to create multiple retinotomies. rAAV-2 vectors mediated efficient stable reporter gene expression in photoreceptors and retinal pigment epithelial cells. We found efficient transduction of cone photoreceptors in addition to rods in both the canine retina and after subretinal vector delivery in another intermediate animal model, the feline retina. GFP expression in dogs was confined to the area of the retinal bleb and was sustained in cells at this site for at least 18 months. Electroretinography demonstrated a modest reduction in global rod-mediated retinal function following subretinal delivery of rAAV.CMV.GFP. Three of the eight animals developed delayed-onset intraocular inflammation, in two cases associated with a serum antibody response to GFP protein. We conclude that rAAV-2 vectors mediate efficient sustained transgene expression in rod and cone photoreceptors following subretinal delivery in this intermediate animal model. The possibility of adverse effects including intraocular immune responses and reduced retinal function requires further investigation prior to clinical applications in patients.

Gene therapy for ocular angiogenesis

Ocular neovascularization is a central feature of diabetic retinopathy and age-related macular degeneration. These conditions are the major causes of blindness in the developed world. Current treatments are of limited efficacy and associated with significant adverse effects. Characterization of the molecular and cellular events involved in angiogenesis has led to the identification of a number of angiostatic molecules with potential therapeutic value. The systemic administration of small molecule angiostatic proteins risks significant systemic adverse effects and the effect of their intraocular injection is short-lived. Local gene transfer, however, offers the possibility of targeted, sustained and regulatable delivery of angiostatic proteins to the retina after a single procedure to introduce a vector to an intraocular site. The effect of intra-ocular delivery of recombinant viruses carrying genes encoding angiostatic proteins has been demonstrated in rodent models of ocular neovascularization. Recombinant adeno-associated virus-mediated local gene transfer of a vascular endothelial growth factor inhibitor controls both retinal and choroidal neovascularization. The clinical application of this approach may require the means to regulate gene expression in order to minimize the potential for adverse effects. Regulation of transgene expression by means of a hypoxia-responsive promoter offers an attractive strategy for the targeted and regulated delivery of angiostatic proteins to the retina in the management of ischaemia-induced ocular neovascularization. Preclinical studies of gene transfer in a large animal model following subretinal delivery of a recombinant adeno-associated virus vector have demonstrated efficient sustained reporter gene expression in cells of the outer retina. Recent progress has enabled the planning of clinical trials of gene therapy for ocular neovascular disorders.

Hypoxia-regulated transgene expression in experimental retinal and choroidal neovascularization

Recombinant AAV vectors mediate efficient and sustained transgene expression in retinal tissues and offer a powerful approach to the local, sustained delivery of angiostatic proteins for the treatment of ocular neovascular disorders. The application of such strategies may also require regulated gene expression to minimize the potential for unwanted adverse effects. In this study, we have evaluated the effect of a hypoxia-responsive element (HRE) on the kinetics of recombinant adeno-associated (rAAV)-mediated reporter gene expression in murine models of retinal and choroidal neovascularization. In murine ischaemia-induced retinal neovascularization, intravitreal delivery of rAAV.HRE.GFP results in reporter gene expression specifically at sites of vascular closure during the period of active neovascularization and not after vector delivery in normal controls. In murine laser-induced choroidal neovascularization, subretinal delivery of rAAV.HRE.GFP results in reporter gene expression at sites of active neovascularization but not elsewhere or after vector delivery in normal controls. HRE-driven gene expression offers an attractive strategy for the targeted and regulated delivery of angiostatic proteins to the retina in the management of neovascular disorders.

Erratum: "Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice" (Molecular Therapy (2005) vol. 12 (763-771) 10.1016/j.ymthe.2005.07.358)

The authors regret that in Table 2 on page 768, one of the insertion sites of the SMART 2 provirus vector identified using LAM-PCR as present on chromosome 5 positioned 32374 bp upstream of Cyp3a11 was incorrectly assigned to Mouse (tumour) 2 T1. This insertion site should be assigned to an independent mouse not listed in Table 2. This animal had only a single provirus insertion found by Southern and LAM-PCR analyses and should be labeled as mouse 7.

Corrigendum to “Oncogenesis Following Delivery of a Nonprimate Lentiviral Gene Therapy Vector to Fetal and Neonatal Mice”

The authors regret that in Table 2 on page 768, one of the insertion sites of the SMART 2 provirus vector identified using LAM-PCR as present on chromosome 5 positioned 32374 bp upstream of Cyp3a11 was incorrectly assigned to Mouse (tumour) 2 T1. This insertion site should be assigned to an independent mouse not listed in Table 2. This animal had only a single provirus insertion found by Southern and LAM-PCR analyses and should be labeled as mouse 7.

827. Oncogenesis Following Delivery of Lentiviral Vectors to Fetal and Neonatal Mice

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases due to the ability of these vectors to integrate in a stable manner into the patients’ chromosomes. Since three cases of T-cell leukaemia have been identified after retrovirus gene therapy for X-linked severe combined immune deficiency as being associated with the integrating vector used for gene therapy the need for animal models to test for vector safety has become of paramount importance. Our previous work has shown that a high frequency of hepatocellular carcinomas has occurred following in utero and neonatal injection with certain lentivirus vectors. It has been hypothesized that the woodchuck post regulatory element (WPRE) carried by the vectors used in this study could be implicated in the tumour development process. Our recent study using novel vectors with mutations in the WPRE shows that mice treated with these vectors still develop liver tumours. In this report we discuss these findings and preliminary data to support an alternative cause for tumorigenesis. We also discuss the fetal and neonatal system as a novel and sensitive in vivo model to test the effects and safety of integrating vectors under consideration for clinical applications.