Viral delivery of multiple miRNAs promotes retinal ganglion cell survival and functional preservation after optic nerve crush injury

Abstract

Bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (sEV) but not fibroblast sEV provide retinal ganglion cell (RGC) neuroprotection both in vitro and in vivo, with miRNAs playing an essential role. More than 40 miRNAs were more abundant in BMSC-sEV than in fibroblast-sEV. The purpose of this study was to test the in vitro and in vivo neuroprotective and axogenic properties of six candidate miRNAs (miR-26a, miR-17, miR-30c-2, miR-92a, miR-292, and miR-182) that were more abundant in BMSC-sEV than in fibroblast-sEV. Adeno-associated virus (AAV)-2 expressing a combination of three of the above candidate miRNAs were added to heterogenous adult rat retinal cultures or intravitreally injected into rat eyes one week before optic nerve crush (ONC) injury. Survival and neuritogenesis of βIIItubulin + RGCs was assessed in vitro, as well as the survival of RBPMS + RGCs and regeneration of their axons in vivo. Retinal nerve fiber layer thickness (RNFL) was measured to assess axonal density whereas positive scotopic threshold response electroretinography amplitudes provided a readout of RGC function. Qualitative retinal expression of PTEN, a target of several of the above miRNAs, was used to confirm successful miRNA activity. AAV2 reliably transduced RGCs in vitro and in vivo. Viral delivery of miRNAs in vitro showed a trend towards neuroprotection but remained insignificant. Delivery of selected combinations of miRNAs (miR-17-5p, miR-30c-2 and miR-92a; miR-92a, miR-292 and miR-182) before ONC provided significant therapeutic benefits according to the above measurable endpoints. However, no single miRNA appeared to be responsible for the effects observed, whilst positive effects observed appeared to coincide with successful qualitative reduction in PTEN immunofluorescence in the retina. Viral delivery of miRNAs provides a possible neuroprotective strategy for injured RGCs that is conducive to therapeutic manipulation.

miRNA Changes in Retinal Ganglion Cells after Optic Nerve Crush and Glaucomatous Damage

“…In this study, we used lipofectamine to deliver miRNA mimics/AntagomiRs into RGC, an approach feasible in vitro but less so in vivo. Alternative approaches would need to be considered, and include the use of viral vectors [ 43 ] or exosome/small extracellular vesicle carriers [ 44 ].…”

Mesenchymal stem cell-derived extracellular vesicles protect retina in a mouse model of retinitis pigmentosa by anti-inflammation through miR-146a-Nr4a3 axis

“…In an optic nerve crush rat model, Mead et al found that exosomes protect retinal neurons by exosome delivery, and that the protective effects were dependent on miRNAs for the diminished therapeutic effects of MSC-Exos after knockdown of Argonaute-2 [ 35 ]. Thereafter, they identified six candidate miRNAs (miR-26a, miR-30c-2 and miR-92a; miR-292, miR-17 and miR-182) and expressed a combination of three of these candidate miRNAs to treat optic nerve crush injury, finding that these candidate miRNAs promoted RGC survival [ 40 ]. Actually, miR-146a has mostly been documented in the context of central nervous system (CNS) diseases, especially in Alzheimer's disease (AD).…”

Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

“…Electroretinography (ERG) was performed as described by us previously (Mead et al ., 2020). Briefly, rats were dark adapted for 12 hours and after anaesthesia eyes were dilated with tropicamide.…”