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Marius Ader, Lynn Ebner, Sylvia Gasparini, Tiago Santos-Ferreira, Oliver Borsch; Photoreceptors harbor the ability for vector-free uptake of molecules following subretinal injection in mice. Invest. Ophthalmol. Vis. Sci. 2020;61(7):734.
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Viral vectors or transfection reagents are commonly used for the delivery of molecules in gene therapy and gene editing approaches for retinal degenerative diseases. While viral vectors provide the desired long and stable expression in gene supplementation, this raises concerns when expressing gene editing tools whose prolonged activity increases the risk of detrimental off-target effects. Here we assessed whether different molecule classes including mRNA, proteins, or polysaccharides can be uptaken and functionally processed in mouse photoreceptors after direct subretinal injection.
To evaluate the uptake potential of retinal cells after subretinal or vitreal delivery, (I) GFP-protein or chemically stabilized GFP-mRNA (5’-capped, polyadenylated, and modified nucleotides), or (II) different sizes (10, 40, 70, 150, and 250 kDa) and concentrations (1, 0.5, 0.25, 0.1, 0.05nmol) of fluorescently-labelled dextran were injected into wild-type mice, as well as (III) Cre-recombinase protein or stabilized mRNA was injected into Cre-dependent conditional fluorescent reporter mice (Ai9, nTnG, mTmG, MuX). Host retinas were subsequently analyzed for the presence and number of fluorescent cells 48h after mRNA, 72h after dextran or 3 weeks after Cre injections.
Subretinal injection of reporter mRNA, dextran, and Cre-protein and Cre-mRNA, lead to the presence of abundant numbers of fluorescent photoreceptors, while vitreal injections resulted in labeling of few Müller glia cells. Effective expression of GFP or Cre-dependent reporter protein from injected mRNAs was dependent on their stabilization by chemical modifications resulting in labeling of »3800 photoreceptors. Dextran-injections yielded fluorescent host photoreceptors in a size- and concentration-dependent manner with 10kDa at 1nmol resulting in »8800 fluorescent photoreceptors with stepwise decrease to »160 photoreceptors at 0.05nmol. While 10, 40, 70 and 150kDa dextran (0.1nmol) labeled 1000-2000 photoreceptors, a significant decrease in fluorescent photoreceptors was observed when using 250kDa.
Our results indicate that photoreceptors are capable of uptaking and physiologically processing mRNA, proteins and polysaccharides directly injected into the subretinal space in vivo, which may be leveraged for non-viral delivery and temporary action of therapy-relevant molecules in retinal degenerative diseases.
This is a 2020 ARVO Annual Meeting abstract.
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