Subretinal delivery of adeno‐associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice

Abstract

BackgroundAdeno-associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long-term therapy is the development of immune responses against the vector or transgene product.MethodsWe evaluated cellular and humoural responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression.ResultsFollowing subretinal administration of vector, splenocytes and T-cells from draining lymph nodes showed minimal activation following stimulation by co-culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low-dose AAV.hRPE65.hRPE65 to both eyes of RPE65−/− mice resulted in transgene expression and functional rescue, but re-administration of high-dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye.ConclusionsThese data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose-dependent. Low-dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration. Copyright © 2009 John Wiley & Sons, Ltd.

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function

“…Consistent with this, we have previously observed transgene expression in both eyes of Rd12 mice when a lower concentration (1Â10 11 vg ml À1 ) of vector construct was used, whereas a higher concentration (5Â10 11 vg ml À1 ) resulted in higher NAb titers and variable transgene expression in the second eye. 27 In the larger canine eye, we have achieved successful photoreceptor rescue at both high and low concentrations (1Â10 11 and 1Â10 12 vg ml À1 ) in both first and second treated eyes without evidence of any significant variation in NAb response. Similarly, administration of a larger total dose of vector did not have a major effect on immune responses or degree of rescue in the first or second treated eyes.…”

“…26 Similarly in the rd12 mouse, administration of rAAV-RPE65 to the subretinal space of the second eye also achieved rescue similar to that obtained in the first eye. 27 In addition, prior intravitreal injection of the first eye did not interfere with the degree of reporter gene expression achieved by subretinal injection in the second eye of C57BL/6J mice. 28 Short duration of rAAV-mediated transgene expression in a number of clinical trials has been linked to the very high level of pre-exposure to wild type AAV in the human population.…”

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

“…49 VSV-G pseudotyped HIV-1.SFFV.hrGFP vectors (5Â10 8 TU ml À1 ) were generated and titrated as previously described. 24 …”

Advanced Combinatorial Radioimmunogenetic Therapy for Cancer

“…Refinement of various clinical complications regarding radio/immune/gene therapy approach towards cancer is a major concern in future. Achieving the best radio sensitization, maximal cytotoxicity and exploring the combination of radiation with different genetic and immunogenic approach using stylist technology like monoclonal antibodies, CRISPR, TALENS, SmaRT will be highly appreciated [38][39][40][41].…”