Glycosylphosphatidylinositol Engineered TSG-6 Exosomes for Treatment of Visual Dysfunction after Mild Traumatic Brain Injury

Abstract

Purpose : Traumatic brain injury (TBI) is anticipated to become a significant cause of death and disability, including vision loss, surpassing infectious diseases. Mild TBI (mTBI), such as concussions, lacks effective treatments. Our previous study demonstrated exosome-associated TSG-6 from cytokine-treated mesenchymal stem cells (MSC) improves vision loss in a mouse model. This research investigates whether genetically engineered exosomes displaying surface TSG-6 can effectively reduce retinal inflammation and visual deficits in a murine mTBI model.

Methods : Human TSG-6 was overexpressed in HEK293 cells, with or without a GPI-anchored Fc domain. Exosomes were purified using size exclusion chromatography and characterized. DiI-labeled exosomes were injected into the eye to track their biodistribution. Mild TBI was induced in mice, followed by intravitreal injection of Control (Ctrl-Exo), human recombinant TSG-6 protein (hTSG-6), or TSG-6-Fc-GPI-Exo. The vision was assessed after one month using functional experiments, pro-inflammatory gene transcripts, and GFAP/IBA1 immunohistochemistry of the retina.

Results : Ctrl-Exo and TSG-6-Fc-GPI exosomes expressed CD9, CD63, and CD81 and lacked non-exosomal Calnexin. TSG-6 quantity in exosomes was quantified by ELISA. DiI labeled TSG-6-Fc-GPI-Exo were mainly integrated into RGC and tracked in the ONL after 24 h. Visual acuity and a- and b-wave amplitudes in blast mice significantly improved in those receiving only TSG-6-Fc-GPI-Exo (p<0.05) compared to blast mice receiving Ctrl-Exo. Conversely, blast mice receiving >1500X hTSG-6 showed an improvement trend without statistical significance (p>0.05). Retinal immunohistological analysis revealed increased GFAP and IBA1 expression in the blast group with Ctrl-Exo (p<0.05) compared to the sham, with a reduction observed with TSG-6-Fc-GPI-Exo (p<0.05) post-TBI. While CD44, CD68, TREM2, and APOE were elevated in the blast group with Ctrl-Exo (p<0.05), only TSG-6-Fc-GPI-Exo (p<0.05) reduced retinal pro-inflammatory gene transcripts.

Conclusions : Exosomes are promising biotherapeutic delivery agents for addressing visual impairments in mTBI. TSG-6 linked to exosomes via a GPI anchor enhanced therapeutic efficacy over soluble hTSG-6. Although further research is needed, engineering non-MSC cells to produce TSG-6-expressing exosomes might pave the way for clinical trials in mTBI treatment.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.