Abstract
Although adeno-associated viral (AAV) vector-mediated retinal gene therapies have demonstrated efficacy, the mechanisms underlying dose-dependent retinal inflammation remain poorly understood. Here, we present a quantitative analysis of cellular immune response to subretinal AAV gene therapy in mice using multicolor flow cytometry with a panel of key immune cell markers. A significant increase in CD45+ retinal leukocytes was detected from day 14 post-subretinal injection of an AAV8 vector (1 × 109 genome copies) encoding green fluorescent protein (GFP) driven by a ubiquitous promoter. These predominantly consisted of infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells; no significant change in resident microglia population was detected. This cellular response was persistent at 28 days and suggestive of type 1 cell-mediated effector immunity. High levels (80%) of GFP fluorescence were found in the microglia, implicating their role in viral antigen presentation and peripheral leukocyte recruitment. When compared against AAV.GFP in paired eyes, an equivalent dose of an otherwise identical vector encoding the human therapeutic transgene Rab-escort protein 1 (REP1) elicited a significantly diminished cellular immune response (4.2-fold; p = 0.0221). However, the distribution of immune cell populations remained similar, indicating a common mechanism of AAV-induced immune activation.
Highlights
Adeno-associated viral (AAV) vectors have been widely adopted for gene therapy because of their versatile tissue tropism, ability to stably transduce non-dividing cells without genome integration, and relative low immunogenicity compared to other viral vectors.[1]
CD45+ cell populations were consistently identified: 88.6% of the retinal immune cells were CD45loCD11bhi (P1), 7.0% were CD45hiCD11bhi (P2), and 2.8% were CD45hiCD11bÀ (P3) (Figure 1). Both microglia and macrophages are CD11b+, they can be distinguished by their level of CD45 expression; microglia typically express low levels of CD45, while macrophages express relatively higher levels of CD45.20 The majority of immune cells found in the normal retina were CD45loCD11bhi (P1), suggesting these to be the resident microglial population, while cells defined as CD45hiCD11bhi (P2) were likely to be macrophages
Clinical trials of subretinal AAV gene therapy indicate an association between retinal inflammation and increasing vector dose, with most cases of clinically significant inflammation occurring at doses R 1011 gc.[26]
Summary
Adeno-associated viral (AAV) vectors have been widely adopted for gene therapy because of their versatile tissue tropism, ability to stably transduce non-dividing cells without genome integration, and relative low immunogenicity compared to other viral vectors.[1]. In the phase 1/2 dose-escalation trials for RPE65-associated LCA, choroideremia, and retinitis pigmentosa GTPase regulator (RPGR)-associated X-linked retinitis pigmentosa, patients receiving higher doses of AAV have presented with various signs of retinal inflammation, including vitritis, retinitis, and choroiditis,[2,6,7] implicating the presence of a cell-mediated immune response. Monitoring of such immune responses to AAV in clinical trial patients has primarily relied on clinical examination and retinal imaging (e.g., optical coherence tomography [OCT]); little is known about the mechanisms involved. While patients with signs of intraocular inflammation are generally treated with broad-acting immunosuppression, e.g., systemic or local corticosteroids,[7] a better understanding of the cellular immune responses involved is critical for designing targeted immune intervention and for improving the safety and visual outcomes following retinal gene therapy
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