Stable and efficient intraocular gene transfer using pseudotyped EIAV lentiviral vectors

Abstract

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.

Absence of ocular malignant transformation after sub-retinal delivery of rAAV2/2 or integrating lentiviral vectors in p53-deficient mice

“…As cell viability is a requisite for malignant transformation, the reporter gene hrGFP was chosen in preference to more commonly used eGFP, as it appears to be less toxic to retinal cells after prolonged in vivo expression. 42,43 An integrating lentiviral vector was chosen as its very high efficiency of integration would be expected to result in a large number of mutagenic events. In vivo reporter gene expression from the viral vectors delivering hrGFP was assayed using a combination of in vivo fundoscopy and post-mortem fluorescence microscopy of retinal cryosections.…”

“…Animals were anaesthetised and pupillary mydriasis achieved as previously described. 42 For Ni 3 S 2 tumour-induction experiments, age-matched adult p53 À/À , p53 +/À and p53 +/+ mice on a mixed C57Bl10J/CBA background received single intravitreal injections of a suspension containing phosphate buffered saline and 100 mg Ni 3 S 2 . (CAS Number: 12035-72, Sigma Aldrich, Gillingham, UK) via a 10-mm 34-gauge needle mounted on a 5-ml Hamilton syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland).…”

“…This method results in vector delivery to approximately two-thirds of the mouse retina. 42 Detection of transgene expression hrGFP fluorescence was detected by fluorescence microscopy of cryosections, similar to the method described for eGFP detection. 42 Briefly, eyes from each hrGFP vector-injected group were retrieved at various intervals following administration, and fixed overnight in 4% paraformaldehyde at 4 1C.…”

“…42 Detection of transgene expression hrGFP fluorescence was detected by fluorescence microscopy of cryosections, similar to the method described for eGFP detection. 42 Briefly, eyes from each hrGFP vector-injected group were retrieved at various intervals following administration, and fixed overnight in 4% paraformaldehyde at 4 1C. Eyes were cryoprotected using 10% sucrose in phosphate buffered saline for 20 min and embedded in optimum cutting temperature medium (OCT, Sakura Finetek Europe BV, Zoeterwoude, The Netherlands).…”

“…Eyes were serially sectioned at 12 mm thickness, and sections counterstained with Hoechst 33342, mounted in aqueous mounting medium (DakoCytomation, Glostrup, Denmark), and viewed using fluorescence microscopy. In vivo retinal imaging of hrGFP fluorescence was performed as previously described 42 at various intervals following subretinal injection of hrGFP-containing vectors using a human ophthalmic slit lamp (Keeler SL-16, Keeler Ltd, Windsor, UK) modified for obtaining colour and fluorescent images of rodent retinas. Immunostaining for RPE65 was performed on 4% paraformaldehyde-fixed 12 mm thick propidium iodide-counterstained cryosections, and RPE65 immunofluorescence detected using fluorescence microscopy as previously described.…”