Technologies

For decades, DNA was not used in LNPs, because it was found to be “too toxic.” In this paper, we showed that the cause of DNA-LNPs’ toxicity was activation of the cGAS-STING pathway. We then co-loaded the DNA-LNPs with the endogenous inhibitor of STING, nitro-oleic acid (NOA), completely preventing STING activation. While standard DNA-LNPs produce 100% mortality in mice, NOA-DNA-LNPs produce 0% mortality. Further, DNA-LNPs express proteins for 6 months per dose. This opens up the LNP world to chronic diseases, such as Alzheimer’s, emphysema, diabetes, and many more.

We showed that LNPs’ hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We found that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo. We also showed that a unique class of ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway.

LNP innovation has almost exclusively focused on varying the structure of individual molecules, and ignored control of the spatial arrangement of molecules, which is suboptimal because supramolecular structure determines function in biology. To control LNPs’ supramolecular structure, we introduced multi-stage-mixing (MSM) to successively add different molecules to LNPs. We first utilized MSM to create a core-then-shell (CTS) synthesis. CTS-LNPs vastly lowered the frequency of LNPs containing no detectable mRNA, and improved mRNA-LNP expression. With DNA-loaded LNPs, which for decades lagged behind mRNA- LNPs due to low expression, CTS improved DNA-LNPs’ protein expression by 2-3 orders of magnitude, bringing it within range of mRNA-LNPs. MSM/CTS may finally make DNA-LNPs into a practical platform for long-term gene expression.