Modified self-amplifying RNA mediates robust and prolonged gene expression in the mammalian brain
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Abstract
In self-amplifying RNA (saRNA), substitution of cytidine with 5-hydroxymethylcytidine (hm5C) reduces innate immune responses and prolongs protein expression. When formulated as a vaccine and administered intramuscularly, lipid nanoparticles (LNPs) loaded with modified saRNA (saRNA-LNPs) afford robust and long-term protein expression. Here we report the protein expression and cell type tropism of modified saRNA-LNPs, encoding fluorescent proteins, when injected in the mammalian brain. saRNA encapsulated in an LNP formulation comprising ALC-0315 (present in Comirnaty®) efficiently mediates robust and long-lasting protein expression in brain cells beyond five weeks, with detectable expression in some neurons at three months. hm5C saRNA substantially outperforms N1mΨ mRNA. Intriguingly, in addition to transfecting astrocytes and neurons at the injection site, saRNA-LNPs labels neurons retrogradely. Thus, saRNA-LNPs are an exciting nonviral gene transduction method that effectively transduces brain cells with excellent potency and mediates prolonged gene expression.
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Continued efforts on engineering LNP formulations and saRNA designs will further improve transfection efficiency, cell type specific targeting, and brain specific targeting.
It will be interesting to see how organismal age and disease state impact hm5C saRNA stability/efficacy!
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robust mCherry fluorescence was present in some neurons
How does the number of saRNA-positive cells compare to the time points shown in Figure 2? It would be interesting to have micrographs from earlier time points at the same magnification of those in Supp. Fig. 1. I'm also curious if there's something measurably different about the cells still expressing saRNA after three months or if it's just a random decrease in cell cout over time.
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Surprisingly, mCherry expression was also evident throughout the corpus callosum, suggesting robust labeling of neuronal axons or myelin.
These expression patterns between treatments and across time are so striking! How much does this vary between individuals? For example, the patterns between mRNA and saRNA on day 14 are very distinct from one another and I'm curious if these types of differences are consistent.
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