To date adeno-associated viral (AAV) vectors are the only gene therapy vectors that have been shown to efficiently transduce photoreceptor cells and have thus become the most commonly used vector for ocular transduction. Various AAV serotypes have been evaluated in the eye, the first of which was AAV2, which is able to transduce photoreceptors, retinal pigment epithelium (RPE) and retinal ganglion cells. AAV serotypes 1 and 4, as well as AAV2 pseudotyped with these capsids, only transduce the RPE. AAV serotype 5 and AAV2/5 transduce the photoreceptors as well as RPE, but not retinal ganglion cells. Here, we assessed the capacity of the novel serotype AAV2/8 to transduce various ocular tissues of the adult murine retina by administering AAV2/8 green fluorescent protein intravitreally, subretinally and intracamerally. We also determined the kinetics and efficiency of self-complementary AAV (scAAV) vectors of serotypes 2/2, 2/5 and 2/8 and compared them with single-stranded AAV (ssAAV). We found that ssAAV2/8 transduces photoreceptors and RPE more efficiently than ssAAV2/2 and ssAAV2/5, and that scAAV2/8 had faster onset and higher transgene expression than ssAAV2/8. This improved transduction efficiency might facilitate the development of improved gene therapy protocols for inherited retinal degenerations, particularly those caused by defects in photoreceptor-specific genes.

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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.

Uveitis is a sight threatening inflammatory disorder that remains a significant cause of visual loss. We investigated lentiviral gene delivery of IL-1ra or IL-10 to ameliorate murine endotoxin-induced uveitis (EIU). An HIV-1-based vector containing the mIL-1ra or mIL-10 cDNA demonstrated high expression of biologically active cytokine. Following administration of Lenti.GFP into the anterior chamber, transgene expression was observed in corneal endothelial cells, trabecular meshwork and iris cells. To treat EIU, mice were injected with Lenti.IL-1ra, Lenti.IL-10 or a combination of these. EIU was induced 14 days after vector administration and mice were culled 12 h following disease induction. Lenti.IL-1ra or Lenti.IL10 treated eyes showed significantly lower mean inflammatory cell counts in the anterior and posterior chambers compared with controls. The aqueous total protein content was also significantly lower in treated eyes, demonstrating better preservation of the blood-ocular barrier. Furthermore, the treated eyes showed less in vivo fluorescein leakage from inner retinal vessels compared with controls. The combination of both IL-1ra and IL-10 had no additive effect. Thus, lentiviral gene delivery of IL-1ra or IL-10 significantly reduces the severity of experimental uveitis, suggesting that lentiviral mediated expression of immunomodulatory genes in the anterior chamber offers an opportunity to treat uveitis.

Aim: To quantify the rate of recurrence of acute anterior uveitis (AAU), and evaluate the influence of associated risk factors. Methods: We retrospectively reviewed the case notes of 185 patients with acute anterior uveitis, from their time of presentation to August 2001. The time to the first three recurrences of AAU from the onset of the disease was recorded, as well as the site of recurrence. Information regarding risk factors (for example (HLA-B27) status, spondyloarthropathy (SpA), family history of AAU/SpA and history of non-specific joint pain) were also collected. Results: Patients were followed up until their third relapse, or up to the censoring date (August 2001) if less than three relapses had occurred. The median length of follow-up was 35 months. One hundred and twentytwo patients (66%) developed at least one relapse and 67 (36%) had three or more relapses. Kaplan-Meier estimate of median interval between disease onset and the first relapse was 24 months 95% CI (16 to 34) and between the first and second relapse was 14 months 95% CI (9 to 22), and was 15 months 95% CI (10 to 25) months between the second and third relapse. Using Cox regression only the number of previous relapses was significantly associated with the risk of AAU recurrence. There was no significant association between other reported risk factors and the risk of relapse, and neither did any risk factor significantly modify the association between previous relapses and AAU recurrence (p.0.066 for all interactions). There was a borderline significant difference in survival according to the laterality pattern of recurrences (ipsilateral, alternate, or bilateral) with a slightly greater than expected number of events in those with bilateral recurrence (p = 0.048). Conclusion: Patients with previous relapse(s) of AAU have a greater risk of AAU recurrence compared to those at disease onset but the risk of recurrence appears not to increase in a dose-response manner with increasing number of previous relapses. Demographic and extraocular features do not appear to influence the rate, or risk of recurrence of AAU.