Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina.

Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

In vitro cell subtype-specific transduction of adeno-associated virus in mouse and marmoset retinal explant culture.

Adeno-associated virus (AAV) is a non-pathogenic human parvovirus that can infect both non-proliferating and proliferating cells. Owing to its favorable safety profile, AAV is regarded as suitable for clinical purposes such as gene therapy. The target cell types of AAV depend largely on the serotype. In the retina, AAV has been used to introduce exogenous genes into photoreceptors, and photoreceptor-specific enhancers/promoters are used in most cases. Therefore, serotype specificity of AAV in retinal subtypes is unclear, particularly in vitro. We compared its infection profile in mouse and monkey retinas using EGFP under the control of the CAG promoter, which expressed the gene ubiquitously and strongly regardless of cell type. AAV1, 8, and 9 infected the horizontal cells when an embryonic day-17 retina was used as a host. Amacrine cell was also a major target of AAVs, and a small number of rod photoreceptors were infected. When adult retinas were used as a host, the main target of AAV was Müller glia. A small number of rod photoreceptors were also infected. In the adult common marmoset retina, rod and cone photoreceptors were efficiently infected by AAV1, 8, and 9. A portion of the Müller glia and amacrine cells were also infected. In summary, the infection specificity of different AAV serotypes did not differ, but was dependent on the stage of the host retina. In addition, infection specificities differed between mature marmoset retinas and mature mouse retinas.

HIV-1 Tat-mediated neurotoxicity in retinal cells.

The current study was aimed at investigating the effect of HIV-1 protein Tat on the retinal neurosensory cell line R28. Exposure of Tat resulted in induction of pro-inflammatory mediators such as CXCL10 and TNF-α in addition to the activation marker GFAP in these cells. Conditioned media from Tat-treated R28 cells was able to induce monocyte migration, an effect that was blocked by CXCR3 antagonist. Complementary studies in the HIV-1 Tat-transgenic mice, showed a complete absence of the nuclear layer and the outer photoreceptor segments of the retina with a concomitant increase in glial activation. These findings lend support to the observation in post-HAART era of increased incidence of immune response-mediated retinal degeneration. These findings have direct relevance to diseases such as immune response uveitis and patients recovering from CMV retinitis.

Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2.

PURPOSE: We evaluated adeno-associated virus (AAV)-mediated gene transfer of basic fibroblast growth factor (FGF-2) as a therapy for photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. METHODS: Recombinant adeno-associated virus vector (rAAV) incorporating a constitutive cytomegalovirus (CMV) promoter was used to transfer the bovine FGF-2 gene to photoreceptors. AAV was administered by subretinal injection to transgenic rats (TgN S334ter-4) at postnatal day 15 (P15). Control eyes were uninjected, injected with PBS, or AAV-LacZ. Eyes were examined by histopathology, morphometric analysis, and electroretinography at P60. RESULTS: Expression of recombinant FGF-2 slowed the rate of photoreceptor degeneration. Morphologic studies demonstrated significantly more photoreceptors surviving in eyes injected with AAV-FGF-2 than in controls. Insignificant rescue effects were seen in retinas injected with buffer only. No significant inflammatory response or neovascularization was detected. Electroretinographic (ERG) responses of eyes injected with AAV-FGF-2 were increased compared with uninjected eyes; however, these amplitudes were not significantly larger than eyes receiving an AAV-LacZ control vector. CONCLUSIONS: Transduction of retinal cells with AAV-FGF-2 reduces the rate of photoreceptor degeneration in an S334ter-4 animal model. Despite the lack of significantly increased ERG amplitudes from eyes expressing FGF-2, a greater number of surviving photoreceptors was demonstrated. Delivery of FGF-2 using recombinant AAV has potential as a therapy for retinal degeneration.