Abstract
Purpose :
Stereotaxic brain injections are commonly used to target neurons projecting to defined locations. Adeno-associated viruses (AAVs) are the standard for retro labeling RGCs with genes or Cre-dependent constructs but are limited by their inability to transport large transgenes, immune activation, genomic integration, and comparatively long expression times. We propose that lipid-nanoparticles (LNPs) encapsulating mRNA are a viable method of retrograde gene delivery due to their rapid expression time and lack of immune activation in experimental animals.
Methods :
Ai140egfp mice (n=17), which express eGFP when Cre is present, were injected bilaterally in the lateral geniculate nucleus (LGN) with either conventional (LNPN ; [417.2 ng/µL]) or carboxylated (LNPX; [372.9 ng/µL]) LNPs encapsulating Cre-mRNA. Seven bilateral injections of 20 nL were made at decreasing depths in the LGN. Mice were euthanized via perfusion with 4% paraformaldehyde (PFA) two days (n=6), four days (n=6), and seven days (n=5) post-injection. The brains were coronally sectioned in 100 µm and retinas were stained for GFP. Brains were imaged to recover the injection location. Retinas were imaged to identify and count the retinal ganglion cells (RGCs) retro-labeled with LNPs.
Results :
LNP injections to the LGN labeled RGCs and their axons at all time points post-injection. Successful injections labeled 32 ± SEM 6 (n=6) RGCs and missed injections labeled 7 ± SEM 4 (n=6) RGCs, likely labeled via hitting axons of passage. Bright eGFP fluorescence was present in RGCs at all time points post injection, including two days, illustrating rapid retrograde gene expression. Multiple subtypes of RGCs with unique morphologies were labeled, illustrating LNPs do not appear to show tropism for a particular cell type. RGC morphologies were consistent with subtypes projecting to the LGN.
Conclusions :
We demonstrate a non-viral gene delivery method to retro-infect RGCs via their axon terminals. While the total number of RGCs labeled using this approach is qualitatively lower than other methods, our future goal is to modify the LNPs and injection volumes to increase gene-expression selectively in RGCs. The rapid gene expression using this approach serves as an advantage for the experimental manipulation of genes in mouse models. Additionally, LNPs serve as a more flexible approach for gene editing in mouse RGCs allowing for custom constructs that are not available using AAVs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.