MacLaren, R. E. | bioRxiv Science

bioRxiv2025

Single-cell and spatial transcriptomic analyses of gene therapy-associatedretinal inflammation in non-human primates

Adeno-associated viral (AAV) vectors are rapidly advancing as gene therapies for inherited and common retinal disorders, but gene therapy-associated uveitis (GTAU) limits their broader application. To investigate the primate ocular immune response, we administered subretinal AAV gene therapy to two non-human primates (NHPs): NHP1 received AAV2-CAG-hRPE65 (voretigene neparvovec) bilaterally at clinical dose; NHP2 received AAV8-GRK1-hRPGRco alongside an analogous mScarlet reporter vector in separate blebs. Longitudinal assessments over three months included multimodal imaging, electroretinography and cytokine profiling, followed by immunohistological, single-cell and spatial transcriptomic analyses of retinal punches. Both therapies were well-tolerated, with preserved retinal structure and function. Single-cell RNA-sequencing revealed that the AAV8 vector transduced 80% of cones/rods in treated areas, while AAV2 targeted 30% of retinal pigment epithelium (RPE)/rods. Transgene expression did not correlate with apoptotic markers. Persistent immune infiltration (dominated by myeloid and T cells) suggested a type 1 cell-mediated response. Adjunctive intravitreal anti-TNF (adalimumab) did not appear to mitigate this anti-viral response. Spatial analysis highlighted microglia migration to the subretinal space, consistent with upregulated cytokines (MCP-1/CCL2, IP-10/CXCL10, IL-8/CXCL8, IL-6), which implicate monocytic phagocytes in driving local inflammation. These findings elucidate the mechanism of GTAU and identify potential therapeutic targets to prevent immune-mediated complications in retinal gene therapy.

immunology↗

bioRxiv2019

The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Author summaryHaving the correct eye size is important, too large and you will be short-sighted and too small and you will be far-sighted. Nanophthalmos, literally very small eye from the Greek, is a condition where the eye is very small but structurally normal. In addition to being farsighted such eyes are prone to glaucoma which can lead to loss of vision. Here we studied a protein called TMEM98 which is found in the membranes of the cells which form a layer at the back of eye called the retinal pigment epithelium (RPE). Mutations in TMEM98 have been found in nanophthalmos patients. Patients have one normal copy of the gene and one carrying a mutation. We removed Tmem98 from the RPE of mice in order to ascertain its function. We found, surprisingly, that rather than having small eyes this led to the development of very large eyes that were structurally fragile. We went to on to identify protein partners of TMEM98 and found that it interacts with a protein called MYRF, mutations in which also causes nanophthalmos. This work demonstrates the importance of TMEM98 in eye size specification.

genetics↗