Getting lipid nanoparticles into shape for enhanced gene delivery
We discovered that use of plant based cholesterol derivatives in lipid nanoparticles give them a polymorphic shape and improve their capability for intracellular delivery and endosomal escape of mRNA therapeutics.
My lab, in part, specializes in researching the enigma surrounding the intracellular fate of lipid-based nanoparticles (LNPs) used for the delivery of messenger RNA (mRNA). LNPs are potent carriers and have been approved by the FDA for the delivery of small interfering RNA (siRNA) to treat amyloidosis. However, their ability to deliver cargo to target sites within the cytosol remains low. Almost all nanoparticles are sequestered in the endosomal compartments or recycled from the cell. We had previously discovered that a cholesterol transporter is responsible for the efflux of LNPs. Since cholesterol is a key component within the nanoparticles, we hypothesized that through replacement of cholesterol with its derivatives, there is a possibility to restrict exodus of LNPs from the cell and provide them an ample opportunity to timely release their cargo. Nature offers a rich source of cholesterol derivatives, and we rationally selected an array of derivatives found in natural products. We discovered that a cholesterol analogue with just an additional ethyl group in the alkyl tail (at the C24 position) of cholesterol (β-sitosterol) causes a substantial increase in gene delivery. We discovered that a cholesterol analogue with just an additional ethyl group in the alkyl tail (at the C24 position) of cholesterol (β-sitosterol) causes a substantial increase in gene delivery. We found that other C24 alkyl derivatives showed improved intracellular delivery which was independent of the type of ionizable lipid used (active ingredients within LNPs) or cargo (mRNA, siRNA). Thus, began a long journey to uncover the reasons for this increase in gene transfection.
After a lot of failures and months of negative data, we were able to finally piece an important puzzle which came in the form of an image using Cryo-Electron Microscopy, which revealed that these enhanced LNPs (eLNPs) had a multifaceted shape compared to the usual spherical morphology of LNPs, while the internal core as monitored by small-angle x-ray scattering had minor differences. We then posited that the intracellular transport of eLNPs might be different than the traditional counterparts. Conventional microscopy revealed little difference existed in uptake between LNPs and eLNPs. Luckily, our collaborators at Moderna Therapeutics and Duke University have developed powerful tools of imaging that revealed the subtle changes in eLNP delivered mRNA within the vesicular compartments. We found that eLNPs have higher retention and mobility within the cells and readily traversed long distances inside cells while LNPs remain immobilized perhaps inside endosomal compartments. Our study highlights the importance of sterols for efficient intracellular delivery of nanoparticles. Deeper understanding of nanoparticle surface composition, their external and internal structure, and morphology and its interactions with cells can be employed to engineer ever more safe and efficient nanoparticles. We are currently applying these fundamental discoveries for in-vivo gene therapy based applications. We are largely focused our attention for delivery of RNA therapeutics to the lung for the treatment of cystic fibrosis. I would be remiss if I do not mention the incredible persistence of scientists from my lab and from Moderna and Duke University that made this work possible. We thank our funding sources at NIH, Cystic Fibrosis Foundation and Moderna Therapeutics.
Finally, with this work, we learned how to successfully establish collaboration between industry and academia, which I think is ever more essential to overcome the long-standing challenges in drug delivery.
Adapted from Patel, S., Ashwanikumar, N., Robinson, E. et al. Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA. Nat Commun 11, 983 (2020). https://doi.org/10.1038/s41467-020-14527-2. Copyright 2020, Springer Nature.