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Expanding on previous demonstrations of the therapeutic effects of adeno‐associated virus (AAV) carrying small‐hairpin RNA (shRNA) in downregulating the mechanistic target of rapamycin (mTOR) in in vivo retinal vascular disorders, vascular endothelial growth factor (VEGF)‐stimulated endothelial cells were treated with AAV2‐shmTOR to examine the role of mTOR inhibition in retinal angiogenesis. AAV2‐shmTOR exposure significantly reduced mTOR expression in human umbilical vein endothelial cells (HUVECs) and decreased downstream signaling cascades of mTOR complex 1 (mTORC1) and mTORC2 under VEGF treatment. Moreover, the angiogenic potential of VEGF was significantly inhibited by AAV2‐shmTOR, which preserved endothelial integrity by maintaining tight junctions between HUVECs. These data thus support previous in vivo studies and provide evidence that AAV2‐shmTOR induces therapeutic effects by inhibiting the neovascularization of endothelial cells., Inhibition of mTOR expression by AAV2‐shmTOR suppresses the proliferation and migration of endothelial cells stimulated by VEGF. mTOR is a major activator in endothelial cells under VEGF stimulation, and inhibition by AAV2‐shmTOR blocked the activation of downstream mTOR signaling molecules. Inhibition of the mTOR pathway consequently affected cell proliferation and the remodeling of tight junctions between endothelial cells.

In addition to laser photocoagulation, currently used therapeutic interventions for diabetic retinopathy (DR) include relatively short-lived anti-VEGF drugs targeting vascular endothelial growth factor (VEGF). The latter requires frequent administration via intravitreal injections to effect long-term VEGF suppression. However, due to the patient burden associated with this treatment modality, gene therapy may represent a preferable alternative, providing long-lasting yet patient-friendly effects. Here, we explore the therapeutic efficacy of rAAV2-sVEGFRv-1, a recombinant adeno-associated virus encoding a soluble variant of VEGF receptor-1, upon early DR processes. Bevacizumab, an anti-VEGF agent often prescribed off label to treat DR, was used as an experimental comparator. Administered by intravitreal injection to a streptozotocin-induced diabetic mouse model, rAAV2-sVEGFRv-1 was shown to effectively transduce the mouse retinas and express its transgene therein, leading to significant reductions in pericyte loss and retinal cell layer thinning, two processes that play major roles in DR progression. Acellular capillary formation, vascular permeability, and apoptotic activity, the latter being the cell death mechanism by which retinal neurodegeneration occurs, were also shown to be reduced by the therapeutic virus vector. Immunohistochemistry was used to visualize that rAAV2-sVEGFRv-1 has an effect on cell types important to DR pathophysiology, particularly the ganglion cell layer and glial cells. Combined with our previous work showing that the therapeutic virus vector reduces neovascularization, our current results reveal that rAAV2-sVEGFRv-1 addresses the early aspects of DR as well, thereby demonstrating its potential as a human gene therapeutic versus the condition as a whole.

Adeno-associated viruses (AAVs) are currently the most popular vector platform technology for ocular gene therapy. While transduction efficiency and tropism of intravitreally administered AAV has been fairly well established in various retinal conditions, its transduction pattern in diabetic retinas has not previously been characterized. Here, we describe the transduction efficiencies of four different AAV serotypes, AAV2, 5, 8, and 9, in streptozotocin (STZ)-induced diabetic mouse retinas after intravitreal injections, which differed according to the duration of diabetic induction. STZ was intraperitoneally injected into C57/B6 diabetic mice subjected to unilateral intravitreal injection of AAV2, AAV5, AAV8, and AAV9 packaged with EGFP. Significantly enhanced AAV2 and AAV9 transduction was observed in 2-month-old diabetic mouse retinas compared to the 2-week-old diabetic mouse retinas and nondiabetic, vector uninjected or injected retinas. Intravitreal injection of AAV5 or AAV8 serotype in 2-month- and 2-week-old diabetic mouse retinas did not show any significant vector transduction enhancement compared to the nondiabetic control retinas. The tropism of AAV2 and AAV9 in diabetic mouse retinas differed. AAV2 was transduced into various retinal cells, including Müller cells, microglia, retinal ganglion cells (RGCs), bipolar cells, horizontal cells, and amacrine cells, whereas AAV9 was effectively transduced only into RGC and horizontal cells. The expression levels of receptors and co-receptors for AAV2 and AAV9 were significantly increased in 2-month-old diabetic mouse retinas. The results of our study demonstrated that AAV2 and AAV9 may be the vector of choice in treating diabetic retinopathy (DR) with gene therapy, and DR-related retinal changes may improve AAV vector transduction efficiency. Keywords: adeno-associated virus, diabetes mellitus, diabetic retinopathy, gene therapy

Choroidal neovascularization (CNV) is the defining characteristic feature of the wet subtype of age-related macular degeneration (AMD) and may result in irreversible blindness. Based on anti-vascular endothelial growth factor (anti-VEGF), the current therapeutic approaches to CNV are fraught with difficulties, and mammalian target of rapamycin (mTOR) has recently been proposed as a possible therapeutic target, although few studies have been conducted. Here, we show that a recombinant adeno-associated virus-delivered mTOR-inhibiting short hairpin RNA (rAAV-mTOR shRNA), which blocks the activity of both mTOR complex 1 and 2, represents a promising therapeutic approach for the treatment of CNV. Eight-week-old male C57/B6 mice were treated with the short hairpin RNA (shRNA) after generating CNV lesions in the eyes via laser photocoagulation. The recombinant adeno-associated virus (rAAV) delivery vehicle was able to effectively transduce cells in the inner retina, and significantly fewer inflammatory cells and less extensive CNV were observed in the animals treated with rAAV-mTOR shRNA when compared with control- and rAAV-scrambled shRNA-treated groups. Presumably related to the reduction of CNV, increased autophagy was detected in CNV lesions treated with rAAV-mTOR shRNA, whereas significantly fewer apoptotic cells detected in the outer nuclear layer around the CNV indicate that mTOR inhibition may also have neuroprotective effects. Taken together, these results demonstrate the therapeutic potential of mTOR inhibition, resulting from rAAV-mTOR shRNA activity, in the treatment of AMD-related CNV. Keywords: retinal neovascularization, choroidal neovascularization, adeno-associated virus, mTOR, RNA interference, mTOR shRNA, autophagy

Choroidal neovascularization (CNV) is the defining characteristic of the wet subtype of age-related macular degeneration (AMD), which is a rapidly growing global health problem. Previously, we had demonstrated the therapeutic potential of gene therapy against CNV using short hairpin RNA (shRNA) delivered via recombinant adeno-associated virus (rAAV), which abrogates mammalian-to-mechanistic (mTOR) activity in a novel manner by simultaneously inhibiting both mTOR complexes. Both the target and use of gene therapy represent a novel treatment modality against AMD. Here, the xenogeneic GFP gene used as a reporter in previous studies was removed from the virus vector to further develop the therapeutic for clinical trials. Instead, a stuffer DNA derived from the 3′ UTR of the human UBE3A gene was used to ensure optimal viral genome size for efficient rAAV assembly. The virus vector containing the stuffer DNA, rAAV2-shmTOR-SD, positively compares to one encoding the shRNA and a GFP expression cassette in terms of reducing CNV in a laser-induced mouse model, as determined by fundus fluorescein angiography. These results were confirmed via immunohistochemistry using anti-CD31, while a TUNEL assay showed that rAAV2-shmTOR-SD possesses anti-apoptotic properties as well. The qualities exhibited by rAAV2-shmTOR-SD demonstrate its potential as a human gene therapeutic for the treatment of wet AMD.

Purpose Recent studies have shown that inhibitors of the mechanistic target of rapamycin (mTOR) play important roles in proliferating endothelial cells within the retinal vasculature. Here we explore the effects of inhibiting mTOR as a potential gene therapeutic against pathological retinal angiogenesis in a rat model of oxygen-induced retinopathy (OIR). Methods Sprague-Dawley pups were used to generate the OIR model, with a recombinant adeno-associated virus expressing an shRNA (rAAV2-shmTOR-GFP) being administered via intravitreal injection on returning the rats to normoxia, with appropriate controls. Immunohistochemistry and TUNEL assays, as well as fluorescein angiography, were performed on transverse retinal sections and flat mounts, respectively, to determine the in vivo effects of mTOR inhibition. Results Compared with normal control rats, as well as OIR model animals that were either untreated (20.95 ± 6.85), mock-treated (14.50 ± 2.47), or injected with a control short hairpin RNA (shRNA)-containing virus vector (16.64 ± 4.92), rAAV2-shmTOR-GFP (4.28 ± 2.86, P = 0.00103) treatment resulted in dramatically reduced neovascularization as a percentage of total retinal area. These results mirrored quantifications of retinal avascular area and vessel tortuosity, with rAAV2-shmTOR-GFP exhibiting significantly greater therapeutic efficacy than the other treatments. The virus vector was additionally shown to reduce inflammatory cell infiltration into retinal tissue and possess antiapoptotic properties, both these processes having been implicated in the pathophysiology of angiogenic retinal disorders. Conclusions Taken together, these results demonstrate the strong promise of rAAV2-shmTOR-GFP as an effective and convenient gene therapy for the treatment of neovascular retinal diseases.

Adeno-associated virus (AAV) vector is a promising platform technology for ocular gene therapy. Recently clinical successes to treat choroidal neovascularization (CNV) in wet type age-related macular degeneration have been reported. However, because pathologic conditions of the retina may alter the tropism of viral vectors, it is necessary to evaluate the transduction efficiency of different serotypes of AAV vectors in the retinas with CNVs. Here, we show the patterns and efficacy of transduction of AAV2, -5, and -8 vectors in a laser-induced CNV mouse model. C57BL/6J mice were subjected to unilateral laser photocoagulation on the right eye to induce CNV 5 days prior to intravitreal injection of AAV2, -5, and -8 capsids expressing EGFP. Transduction was increased around CNV lesions for all AAV capsid types, and AAV2 resulted in the highest transduction efficiency. In the absence of CNV, the AAV2 vector transduced ganglion and inner nuclear layer (INL) cells, and AAV5 and AAV8 transduced only a small proportion of cells in the retinal ganglion cell layer. CNV increased AAV2 vector expression throughout the retina and in and around CNVs; the transduced cells included retinal ganglion cells, Müller cells, cells from the INL and outer nuclear layer (ONL), photoreceptors, and retinal pigment epithelium (RPE) cells. Inflammatory cells and endothelial cells in CNVs were also transduced by AAV2. AAV5 and AAV8 were transduced in retinal ganglion, Müller, INL, ONL, and RPE cells in a localized pattern, and only endothelial cells at the surface of CNV lesions showed EGFP expression. Taken together, CNV formation resulted in enhanced transduction of AAV2, -5, and -8, and AAV2 exhibited the highest transduction efficiency in cells in CNV lesions. Keywords: adeno-associated virus, choroidal neovascularization, age-related macular generation, gene therapy

Purpose We determine the prevalence of neutralizing antibodies (NAbs) to adeno-associated virus (AAV) in the vitreous humor and serum of patients with vitreoretinal diseases and investigate the relationship between NAb titers in the vitreous humor and serum. Methods We analyzed NAbs to AAV serotypes 2, 5, 8, and 9 via in vitro neutralization in the vitreous humor and serum from 32 patients requiring vitrectomy for vitreoretinal diseases. The blood-retinal barrier (BRB) was evaluated for integrity based on preoperative examinations, with vitreous hemorrhage (VH) on funduscopy or dye leakage on fluorescein angiography observed indicating disruption. Results NAb levels were much lower in the vitreous humor than in the serum regardless of serotype. Patients with VH had higher levels of NAbs in the vitreous humor than those without VH. The NAb ratio (ratio between NAb titers in the serum and vitreous humor) was much lower in patients with epiretinal membrane with than in those without leakage. A significantly lower NAb ratio was noticed in patients with than in those without BRB disruptions. Conclusions The NAb ratio between levels in serum and vitreous humor varies according to the condition of the BRB. Therefore, in addition to measuring the serum NAb level, physicians should examine BRB integrity when planning retinal gene therapy. Translational relevance This study provides substantial basis for retinal gene therapy using AAVs and how maintenance of BRB integrity in target diseases should be considered.

Purpose With anti-VEGF-based treatments for wet AMD requiring frequent injections, it is often burdensome to both patients and healthcare providers. To explore its possibility as a desirable alternative, we investigated the therapeutic potential of a recombinant adeno-associated virus 2 expressing a soluble variant of VEGF receptor-1 (rAAV2-sVEGFRv-1) in a laser-induced choroidal neovascularization (CNV) model, as CNV is a defining feature of AMD progression. Methods C57/B6 mice were intravitreally administered with rAAV2-sVEGFRv-1, rAAV2-GFP, or clinically used bevacizumab after CNV lesions were induced via laser photocoagulation. Immunostaining was performed with phalloidin and CD31 to measure CNV extensiveness, F4/80 and CD11b for inflammatory cell infiltration, and pan-cytokeratin to visualize fibrotic progression. Results rAAV2-sVEGFRv-1 (5.0 × 107 viral genomes) possesses antiangiogenic, anti-inflammatory, and antifibrotic properties. rAAV2-sVEGFRv-1 was demonstrated to significantly decrease retinal CNV lesion size (1336 ± 186) when compared to rAAV2-GFP-treated (2949 ± 437, P = 0.0043), mock-treated (3075 ± 265, P = 0.0013), and bevacizumab-treated models (995 ± 234). Infiltration by inflammatory cells significantly decreased with rAAV2-sVEGFRv-1 administration, while groups treated with rAAV2-GFP did not. Additionally, antiapoptotic activity was observed via TUNEL assay in rAAV2-sVEGFRv-1 (16.0 ± 3.6) and rAAV2-GFP (46.0 ± 7.5, P = 0.003). Overall, the rAAV2-sVEGFRv-1 viral vector was positively comparable to bevacizumab, indicating it as effective as approved therapeutics. Conclusions The ability of a low dose of rAAV2-sVEGFRv-1 to exert a therapeutically relevant anti-VEGF effect in a CNV model is demonstrated, and strongly suggests gene therapy as an effective and convenient treatment for sustained VEGF suppression.

Retinal transduction by intravitreally administered adeno-associated viral (AAV) vector is previously known to be extremely limited to the neural retina except AAV2 capsid type. Recently, we showed that prior laser photocoagulation enhances retinal transduction of intravitreally administered AAV vectors, including the outer retina and retinal pigment epithelium (RPE). Here, by performing short-pulse laser pretreatment on the mouse retina, we demonstrate RPE cells transduced by three different capsid types of AAV vectors, AAV2, AAV5, and AAV8, using RPE wholemounts. For all capsid types, laser pretreatment effectively induced the transduction of RPE cells in and around the laser site.