Cells communicate by transferring molecular cargo using nanosized delivery vehicles called extracellular vesicles (EV). As we age, there is loss of EV-mediated communication, leading to the progression of retinal degenerations such as Age-related Macular Degeneration (AMD). We hypothesise that if we can supplement the retina with cargo lost during degeneration, we can restore cellular communication and slow the progression of AMD. 

We propose to use autologous-sourced EV from red blood cells (RBC-EV) as delivery vehicles of these essential retinal EV cargo and develop a novel gene therapy for AMD.

EV were isolated from RBCs using differential ultracentrifugation and characterised using nanoparticle tracking analysis, electron microscopy and western blotting . RBC-EV were labelled with an RNA dye and transfected in-vitro into 4 retinal cell lines. The safety and uptake efficiency was analysed using Fluorescence Live cell imaging. In-vivo safety and uptake efficiency was investigated using functional and histological analysis.

Labelled RBC-EV were safely and efficiently uptaken by all retinal cell types with upto 90% transfection efficiency within 24 hours. In-vivo, intravitreal injection of RBC-EV showed significant protection in murine model of AMD as recorded in both functional readouts and histological analysis. Further, labelled-RBC EV were uptaken by cells in ganglion cell layer, inner nuclear layer and external limiting membrane of the retina within 6 hours of injection.

This work not only supports use of RBC-EV as therapeutic delivery agents but shows their efficacy in native state as therapeutics.

RBC EV loaded with healthy retinal cargo is a novel and promising gene therapy option for AMD.


Rakshanya Sekar, Yvette Wooff, Adrian Cioanca, Riccardo Natoli
 
 

1.     JCSMR, The Australian National University, Acton, ACT, 2601