Specific Evaluation of the Oncogenic Potential of Aav2/2 Vectors After Intraocular Delivery in P53 Knockout Mice

Abstract

Purpose:: Iatrogenic malignant transformation using integrating retroviral vectors has been reproducibly demonstrated in vivo, and has also induced leukaemia in 3 children with X-SCID. Recombinant AAV2/2 vectors can integrate into the host chromosomes of cells in vivo, albeit of substantially lower efficiency than retroviral vectors. However, the potential for malignant transformation after intraocular delivery of these vectors remains, considering the high number of infectious viral particles required. An evaluation of the oncogenic potential of these vectors is particularly important in the context of therapy of non-lethal diseases such as retinopathies. We sought to specifically evaluate this possibility using mice highly susceptible to tumour formation, receiving subretinal injections of AAV2/2 vectors.

Methods:: -/- and +/- p53 knockout mice, which readily develop non-ocular malignancies within their natural lifespan were used in this study. Intraocular injections of carcinogenic Ni₃S₂ were used initially to establish the capacity of these animals to develop intraocular tumours. Further groups of animals received subretinal injections of either rAAV.CMV.hrGFP (n = 220 eyes; 4 x 10⁸ vg/eye) or rAAV.hRPE65P.hRPE65 (n = 46 eyes; 4 x 10⁸ vg/eye). Age-matched uninjected -/- and +/- eyes (n=118) were used to establish the background incidence of intraocular malignancy in these animals. Animals were maintained until their natural lifespan or until there were visible signs of ocular or systemic tumours. Wax and cryosections were examined for microscopic evidence of intraocular malignancy and to confirm transgene expression.

Results:: No spontaneous intraocular tumours were detected in uninjected controls, however the capacity for malignant transformation in these mice was confirmed in Ni₃S₂ injected eyes. No intraocular tumours have been identified so far by fundoscopy or histological examination in any -/- or +/- eyes receiving either vector.

Conclusions:: As retinal cells in patients with retinopathies amenable to gene therapy using AAV2/2 vectors are not expected to be p53-deficient, the preliminary results of this study are particularly encouraging. The absence of malignant transformation in these animals, which are highly susceptible to tumour formation, provides important biosafety data specifically applicable to clinical applications of AAV2/2 vectors for retinopathies, a finding especially relevant for the imminent gene therapy trials for RPE65 deficiency.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • retina