Transduction Patterns of Adeno-associated Viral Vectors in a Laser-Induced Choroidal Neovascularization Mouse Model

Abstract

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.

Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice

“…To determine the functional contribution of Dicer1 deficiency to retinal and choroidal neovascularization in JR5558 mice, we developed a gene therapy capable of restoring Dicer1 activity. We selected adenoassociated vector (AAV) because this modality has demonstrated safety and efficacy in treating blinding diseases in human patients (43,44) and in experimental models of CRNV (45)(46)(47)(48)(49)(50). The human and mouse DICER1 genes are encoded by sequences of 5.7 kb, which is too large to be packaged into a traditional AAV with a size limit of ∼5.2 kb (51, 52).…”

Adeno-Associated Viral Vector 2 and 9 Transduction Is Enhanced in Streptozotocin-Induced Diabetic Mouse Retina

“…Immunostaining for various retinal cell markers showed that the targeted retinal cells differed between AAV2- and AAV9-injected diabetic eyes; AAV2 was effectively transduced into RGCs, amacrine cells, bipolar cells, horizontal cells, Müller cells, and microglia, while AAV9 was only transduced into some of the RGCs, Müller cells, and a few horizontal cells. While the different tropisms of AAV serotypes between normal and other pathological retinas were fairly well established in previous studies,14, 22, 23, 24, 25, 26, 27 there has been no report regarding the AAV serotype tropisms in eyes with DR. The results from our study provide further guidance for selecting the most-appropriate AAV serotype when considering targeted ocular gene therapy to treat DR and its related complications.…”

“…Tissue processing for immunohistochemistry was performed according to previously published procedures 14, 22. In brief, at 1 month after AAV administration, mice were deeply anesthetized with a 4:1 mixture of zolazepam and tiletamine and xylazine, and intracardial perfusion was performed with 0.1 M PBS containing 150 U/mL heparin, followed by 4% paraformaldehyde (PFA) in 0.1 M PBS.…”

Translating CRISPR-Cas Therapeutics: Approaches and Challenges

“…Specific cell types in the eyes such as retinal pigment epithelium, ganglion, and photoreceptors can be effectively transduced by AAVs, and surgical processes afford access to the various structures in the eye. 127 Non-invasive methods have been established for evaluating therapeutic effects, whereas the contralateral eye can be used as a convenient control. Lastly, numerous genetic animal models have been generated for diseases in the retina, paving the way for gene therapy development.…”