Absence Of Ocular Malignant Transformation After Subretinal Delivery Of Raav2/2 Or Hiv-1 Vectors In P53 Knockout Mice

Abstract

Purpose: : Chromosomal integration of rAAV vectors is a passive, low frequency event, in contrast to efficient active integration observed with HIV vectors. However, evidence suggesting that rAAV vectors may induce insertional mutagenesis (IM) and increase the propensity for hepatocellular carcinogenesis in several models, has resurrected concerns regarding their safety for human application. Although the capacity for vector-induced malignant transformation in non-dividing ocular cells is likely to be lower than in dividing cells, evaluation of the intraocular oncogenicity of these vectors is important in view of the non-lethal nature of most ocular diseases.

Methods: : We investigated the intraocular oncogenicity of these vectors using p53^-/- and ^+/- mice, which readily develop non-ocular tumours within their natural lifespan. Initially, p53^-/-,^+/- and ^+/+ mice received intraocular injections of carcinogenic Ni₃S_2, and the incidence of induced ocular tumours was determined to establish the relative ocular tumour susceptibilities of the different genotypes. Further p53^-/- , ^+/- or ^+/+ mice received subretinal injections of rAAV.CMV.hrGFP (n = 81 eyes; 4 x 10⁹ vg/eye), rAAV.hRPE65P.hRPE65 (n = 61 eyes; 4 x 10⁸ vg/eye) or HIV-1.sFFV.hrGFP (101 eyes; 2 x 10⁶ TU/eye). Age-matched uninjected eyes (n=240) were used to establish the background incidence of intraocular malignancy. Animals were maintained until there were visible signs of ocular or systemic tumours. H&E or tumour marker immunostained histological sections were examined for intraocular malignancies.

Results: : Ni₃S₂ administration gave rise to significantly more intraocular tumours in p53^-/- mice, compared with ^+/- and ^+/+ animals, with tumours consisting primarily of uveal melanomas. No tumours have been identified so far in any vector injected or uninjected eyes.

Conclusions: : The absence of vector-induced tumourigenesis in animals with a higher propensity for ocular malignancy provides important biosafety data supporting the further development of rAAV and HIV-1 vectors for ophthalmic applications, in particular for the treatment of retinal diseases.

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