Host Endoplasmic Reticulum Stress and Interferon Responses Contribute to AAV-Induced Ocular Toxicity
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eLife Assessment
This fundamental study provides convincing evidence that distinct molecular mechanisms underlie AAV-associated retinal toxicity in retinal pigment epithelial cells and photoreceptors, advancing our understanding of gene therapy-related retinal injury. The authors employ a rigorous and comprehensive experimental approach, including multiple knockout mouse models, transcriptomic analyses, and genetic loss-of-function studies, which substantially strengthen the mechanistic conclusions. Some concerns remain regarding vector characterization, the absence of procedural injection controls, and the limited interpretation of adult versus neonatal studies; nevertheless, the study makes a substantial contribution to the field and provides a strong foundation for future translational investigations.
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Abstract
Adeno-associated viruses (AAVs) are popular gene therapy vectors, but AAVs can cause toxicity. This is particularly evident following expression of some transgenes, e.g. GFP, in the retinal pigment epithelium (RPE), which leads to loss of RPE cells and photoreceptors. Here, we sought to unravel the toxicity mechanism(s). Several transgenes, self and non-self, were tested for toxicity, with no clear correlation for this variable. RPE RNA-sequencing revealed upregulation of translational processes, cell stress, cytokine release, antiviral responses, and leukocyte infiltration pathways. Toxicity-inducing pathways were explored for causality by injecting toxic AAVs into mice deficient for intrinsic, innate, or adaptive immune pathways. The CHOP KO partially alleviated toxicity for RPE but not photoreceptors, whereas the type I interferon receptor KO partially alleviated toxicity for photoreceptors but not RPE. In situ hybridization of interferon pathway transcripts (IFNB1, IFNAR1) revealed that the RPE and retina can produce and potentially respond to interferon. These data suggest that transgene-induced cell stress responses in the RPE lead to RPE cell death, while interferon signaling contributes to the death of photoreceptors.
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eLife Assessment
This fundamental study provides convincing evidence that distinct molecular mechanisms underlie AAV-associated retinal toxicity in retinal pigment epithelial cells and photoreceptors, advancing our understanding of gene therapy-related retinal injury. The authors employ a rigorous and comprehensive experimental approach, including multiple knockout mouse models, transcriptomic analyses, and genetic loss-of-function studies, which substantially strengthen the mechanistic conclusions. Some concerns remain regarding vector characterization, the absence of procedural injection controls, and the limited interpretation of adult versus neonatal studies; nevertheless, the study makes a substantial contribution to the field and provides a strong foundation for future translational investigations.
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Reviewer #1 (Public review):
This study examines the mechanisms underlying retinal toxicity associated with certain AAV gene therapy vectors, particularly in the retinal pigment epithelium (RPE) and photoreceptors following expression of transgenes such as GFP. The findings suggest that AAV-related retinal toxicity is driven less by transgene identity itself and more by distinct pathogenic mechanisms, including stress-induced injury in RPE cells and interferon-mediated damage in photoreceptors. The comments are as follows:
(1) The AAV vectors were manufactured in-house, and the production method is described in sufficient detail. However, were any characterization assays performed beyond qPCR-based titer determination, such as vector genome titer, capsid titer, empty/full capsid ratio, sterility, bioburden, endotoxin, mycoplasma, …
Reviewer #1 (Public review):
This study examines the mechanisms underlying retinal toxicity associated with certain AAV gene therapy vectors, particularly in the retinal pigment epithelium (RPE) and photoreceptors following expression of transgenes such as GFP. The findings suggest that AAV-related retinal toxicity is driven less by transgene identity itself and more by distinct pathogenic mechanisms, including stress-induced injury in RPE cells and interferon-mediated damage in photoreceptors. The comments are as follows:
(1) The AAV vectors were manufactured in-house, and the production method is described in sufficient detail. However, were any characterization assays performed beyond qPCR-based titer determination, such as vector genome titer, capsid titer, empty/full capsid ratio, sterility, bioburden, endotoxin, mycoplasma, residual host cell DNA, residual plasmid DNA, or residual host cell protein testing? These analyses, particularly those assessing residual impurities and microbial contamination, are critical, as such contaminants may provoke inflammatory responses following subretinal injection. This, in turn, could confound the interpretation of the results, including the identification of the molecular pathways contributing to toxicity as well as the specific role of GFP-associated toxicity. Please provide any characterization information for the AAV vectors.
(2) The study uses contralateral or uninjected eyes as controls, but this choice may not adequately account for changes induced by the subretinal injection procedure itself. Because the earliest assessment of RPE toxicity was performed at 2 weeks post-injection, any injury, inflammation, retinal detachment-related stress, or wound-healing responses triggered by the surgical procedure could have contributed to the observed phenotype. As a result, comparisons to uninjected eyes alone make it difficult to distinguish vector or transgene-specific toxicity from procedure related effects. Inclusion of a more appropriate procedural control, such as sham-injected eyes or eyes injected with vehicle/buffer alone, would have strengthened the study by enabling clearer discrimination between injection-related retinal responses and toxicity attributable to the AAV construct or transgene expression.
(3) The authors used phalloidin staining on RPE-choroid flatmounts to evaluate RPE toxicity, which provides useful information on RPE morphology and structural disruption. However, it would be highly informative to also assess the presence and distribution of subretinal microglia/macrophages, for example, by Iba1 immunostaining, in the same preparations. Specifically, determining whether Iba1-positive cells accumulate in or around areas of RPE dystrophy would help clarify the contribution of local inflammatory responses to the observed pathology. Such analysis could strengthen the interpretation of the toxicity phenotype by revealing whether RPE degeneration is accompanied by focal immune cell recruitment and whether these cells spatially associate with regions of tissue damage. This would also provide additional insight into whether inflammation is likely to be a downstream consequence of RPE injury or a more direct contributor to disease progression, especially in light of publications by Danial Saban's group regarding the characterization of microglia phenotypes using RNA-seq analysis.
(4) The Discussion should also address the anatomical and procedural differences between neonatal and adult mouse eyes, particularly with respect to retinal thickness and the potential impact of subretinal injection-related injury. Because the RPE toxic effects appeared less severe in adult mice, it would be valuable for the authors to consider whether this difference reflects true age-dependent biological susceptibility or, at least in part, differences in the mechanical consequences of the injection procedure. Neonatal retinas are thinner and structurally less mature than adult retinas, which may render them more vulnerable to injection-associated stress, retinal detachment, or secondary tissue injury following subretinal delivery. In contrast, the greater retinal thickness and maturity of the adult eye may provide some degree of resilience to procedural trauma, thereby reducing the apparent severity of RPE damage. Expanding the Discussion to consider these factors would strengthen the interpretation of the age-related differences observed in toxicity and help distinguish vector- or transgene-driven effects from potential confounding effects introduced by the delivery method itself.
Overall, this manuscript presents a detailed and comprehensive analysis of transgene-induced retinal toxicity and makes effective use of multiple mouse models to dissect the contribution of relevant molecular pathways. The study is particularly strengthened by its systematic approach, combining histologic, transcriptomic, and genetic loss-of-function strategies to distinguish the mechanisms underlying toxicity in the RPE versus photoreceptors. By evaluating several knockout mouse lines, the authors can move beyond descriptive observations and begin to assign causality to specific stress and immune signaling pathways, thereby providing important mechanistic insight into AAV-associated retinal injury. These findings are timely and relevant to the broader field of ocular gene therapy, as they highlight the complexity of vector- and transgene-related toxicity and underscore the need for careful pathway-level evaluation during preclinical development.
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Reviewer #2 (Public review):
Summary:
Adeno-associated viruses (AAVs) are popular gene therapy vectors, but AAVs can cause toxicity. This is particularly evident following expression of some transgenes, e.g., GFP, in the retinal pigment epithelium (RPE), which leads to loss of RPE cells and photoreceptors. Here, we sought to unravel the toxicity mechanism(s). Several transgenes, self and non-self, were tested for toxicity, with no clear correlation for this variable. RPE RNA-sequencing revealed upregulation of translational processes, cell stress, cytokine release, antiviral responses, and leukocyte infiltration pathways. Toxicity-inducing pathways were explored for causality by injecting toxic AAVs into mice deficient for intrinsic, innate, or adaptive immune pathways. The CHOP KO partially alleviated toxicity for RPE but not …
Reviewer #2 (Public review):
Summary:
Adeno-associated viruses (AAVs) are popular gene therapy vectors, but AAVs can cause toxicity. This is particularly evident following expression of some transgenes, e.g., GFP, in the retinal pigment epithelium (RPE), which leads to loss of RPE cells and photoreceptors. Here, we sought to unravel the toxicity mechanism(s). Several transgenes, self and non-self, were tested for toxicity, with no clear correlation for this variable. RPE RNA-sequencing revealed upregulation of translational processes, cell stress, cytokine release, antiviral responses, and leukocyte infiltration pathways. Toxicity-inducing pathways were explored for causality by injecting toxic AAVs into mice deficient for intrinsic, innate, or adaptive immune pathways. The CHOP KO partially alleviated toxicity for RPE but not photoreceptors, whereas the type I interferon receptor KO partially alleviated toxicity for photoreceptors but not RPE. In situ hybridization of interferon pathway transcripts (IFNB1, IFNAR1) revealed that the RPE and retina can produce and potentially respond to interferon. These data suggest that transgene-induced cell stress responses in the RPE lead to RPE cell death, while interferon signaling contributes to the death of photoreceptors.
Strengths:
This manuscript used numerous KO mouse models to evaluate the interferon pathway, inflammatory cytokine pathways, the complement pathway, toll-like receptor signaling, cytosolic DNA sensing, double-stranded RNA sensing strain, intrinsic cellular stress pathways, as well as strains deficient for B cells and T cells or B cells, T cells, and natural killer cells. This is a robust piece of work with rigorous controls, groups, and timepoints tested. The RNA-sequencing data provided helpful guidance on the pathways that should be assessed when analyzing AAV toxicity to the retina.
Weaknesses:
The main weakness of the study is that it focuses on subretinal administration to neonatal mice, and the canonical TLR9-MyD88 was not found to have an impact on the AAV toxicity measured. More information could have been provided to understand the discrepancy.
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