ABSTRACT
Recombinant adeno-associated viruses (rAAVs) have emerged as promising gene therapy vectors due to their proven efficacy and safety in clinical applications. In non-human primates (NHPs), rAAVs are administered via suprachoroidal injection at a higher dose. However, high doses of rAAVs tend to increase additional safety risks. Here, we present a novel AAV capsid (AAVv128), which exhibits significantly enhanced transduction efficiency for photoreceptors and retinal pigment epithelial (RPE) cells, along with a broader distribution across the layers of retinal tissues in different animal models (mice, rabbits, and NHPs) following intraocular injection. Notably, the suprachoroidal delivery of AAVv128-anti-VEGF vector completely suppresses the Grade IV lesions in a laser-induced choroidal neovascularization (CNV) NHP model for neovascular age-related macular degeneration (nAMD). Furthermore, cryo-EM analysis at 2.1 Å resolution reveals that the critical residues of AAVv128 exhibit a more robust advantage in AAV binding, the nuclear uptake and endosome escaping. Collectively, our findings highlight the potential of AAVv128 as a next generation ocular gene therapy vector, particularly using the suprachoroidal delivery route.
Subject(s)
Choroidal Neovascularization , Dependovirus , Genetic Therapy , Genetic Vectors , Retinal Pigment Epithelium , Animals , Dependovirus/genetics , Genetic Vectors/genetics , Genetic Vectors/administration & dosage , Genetic Therapy/methods , Mice , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/virology , Choroidal Neovascularization/therapy , Choroidal Neovascularization/genetics , Rabbits , Humans , Gene Transfer Techniques , Macular Degeneration/therapy , Macular Degeneration/genetics , Macular Degeneration/pathology , Disease Models, Animal , Capsid Proteins/genetics , Capsid Proteins/metabolism , Transduction, Genetic , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Mice, Inbred C57BL , Retina/metabolism , Retina/virology , Male , HEK293 CellsABSTRACT
The upregulation of vascular endothelial growth factor (VEGF) is strongly associated with the development of choroidal neovascularization (CNV) in patients with neovascular age-related macular degeneration (nAMD). Currently, the standard treatment for nAMD involves frequent intravitreal injections of anti-VEGF agents, which inhibit the growth of new blood vessels and prevent leakage. However, this treatment regimen places a significant burden on patients, their families, and healthcare providers due to the need for repeated visits to the clinic for injections. Gene therapy, which enables the sustained expression of anti-VEGF proteins after a single injection, can dramatically reduce the treatment burden. KH631 is a recombinant adeno-associated virus 8 vector that encodes a human VEGF receptor fusion protein, and it is being developed as a long-term treatment for nAMD. In preclinical studies using non-human primates, subretinal administration of KH631 at a low dose of 3 × 108 vg/eye resulted in remarkable retention of the transgene product in the retina and prevented the formation and progression of grade IV CNV lesions. Furthermore, sustained transgene expression was observed for more than 96 weeks. These findings suggest that a single subretinal injection of KH631 has the potential to offer a one-time, low-dose treatment for nAMD patients.
Subject(s)
Choroidal Neovascularization , Macular Degeneration , Animals , Humans , Vascular Endothelial Growth Factor A/metabolism , Retina/metabolism , Choroidal Neovascularization/genetics , Choroidal Neovascularization/therapy , Primates/genetics , Primates/metabolism , Intravitreal Injections , RNA , Macular Degeneration/pathology , Genetic Therapy/methods , Angiogenesis Inhibitors/pharmacology , Recombinant Fusion ProteinsABSTRACT
Previous research has shown that the pathogenicity and appressorium development of Magnaporthe oryzae can be inhibited by the ATP synthase subunit beta (Atp2) present in the photosynthetic bacterium Rhodopseudomonas palustris. In the present study, transgenic plants overexpressing the ATP2 gene were generated via genetic transformation in the Zhonghua11 (ZH11) genetic background. We compared the blast resistance and immune response of ATP2-overexpressing lines and wild-type plants. The expression of the Atp2 protein and the physiology, biochemistry, and growth traits of the mutant plants were also examined. The results showed that, compared with the wild-type plant ZH11, transgenic rice plants heterologously expressing ATP2 had no significant defects in agronomic traits, but the disease lesions caused by the rice blast fungus were significantly reduced. When infected by the rice blast fungus, the transgenic rice plants exhibited stronger antioxidant enzyme activity and a greater ratio of chlorophyll a to chlorophyll b. Furthermore, the immune response was triggered stronger in transgenic rice, especially the increase in reactive oxygen species (ROS), was more strongly triggered in plants. In summary, the expression of ATP2 as an antifungal protein in rice could improve the ability of rice to resist rice blast.
