The effects of PEGylation on LNP based mRNA delivery to the eye

Abstract

Gene therapy is now an effective approach to treat many forms of retinal degeneration. Delivery agents that are cell-specific, allow for multiple dosing regimens, and have low immunogenicity are needed to expand the utility of gene therapy for the retina. We generated eight novel lipid nanoparticles (LNPs) ranging in size from 50 nm to 150 nm by changing the PEG content from 5% to 0.5%, respectively. Subretinal injections of LNP-mRNA encoding luciferase revealed that 0.5% PEG content within nanoparticles elicits the highest expression. Similar injections of LNP delivered cre mRNA into Ai9 mice revealed cell-specific protein expression in the retinal pigment epithelium (RPE), confirmed by fundus photography and immunohistochemistry of whole globe cross-sections. To investigate mechanisms of LNP delivery to the eye, we injected mCherry mRNA using the subretinal approach in apoE -/- and Mertk -/- mice. RPE transfection was observed in both mouse models suggesting that LNP intracellular delivery is not solely dependent on apolipoprotein adsorption or phagocytosis. To investigate LNP penetration, particles were delivered to the vitreous chamber via an intravitreal injection. The 0.5% PEG particles mediated the highest luciferase activity and expression was observed in the Müller glia, the optic nerve head and the trabecular meshwork, but failed to reach the RPE. Overall, particles containing less PEG (~150 nm in size) mediated the highest expression in the eye. Thus far, these particles successfully transfect RPE, Müller cells, the optic nerve head and the trabecular meshwork based on route of administration which can expand the utility of LNP-mediated gene therapies for the eye.

Lipid nanoparticles for mRNA delivery

“…Further optimization of the amine head group of DLin-KC2-DMA led to (6 Z ,9 Z ,28 Z ,31 Z )-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA; MC3), which is a key delivery component of Onpattro, the first United States Food and Drug Administration (FDA)-approved siRNA drug 18 , 66 . MC3-based lipid nanoparticles have also been tested for mRNA therapeutics, such as protein replacement therapies 56 , 67 – 72 and antiviral therapies 73 – 75 . Incorporation of biodegradable lipids improves the tolerability of lipid nanoparticles, by allowing fast metabolism while retaining mRNA delivery efficacy.…”

“…PEG-lipids can have multiple effects on the properties of lipid nanoparticles 14 , 17 , 72 , 127 – 129 . The amount of PEG-lipids can affect particle size and zeta potential 17 , 72 .…”

“…PEG-lipids can have multiple effects on the properties of lipid nanoparticles 14 , 17 , 72 , 127 – 129 . The amount of PEG-lipids can affect particle size and zeta potential 17 , 72 . PEG-lipids can further contribute to particle stability by decreasing particle aggregation 14 , 17 , 127 , and certain PEG modifications prolong the blood circulation time of nanoparticles by reducing clearance mediated by the kidneys and the mononuclear phagocyte system (MPS) 14 , 17 , 127 – 129 .…”

mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability

“…Aggregation of LNPs during storage and fusion of LNPs has been reported ( Ayat et al, 2019 , Ball et al, 2016 ). To increase stability on the shelf, LNPs are often formulated with PEG-lipids ( Burke et al, 2013 , Ryals et al, 2020 ). The PEG-molecules at the surface prevent the individual LNPs from aggregating.…”

Are the Allergic Reactions of COVID-19 Vaccines Caused by mRNA Constructs or Nanocarriers? Immunological Insights

“…Another stabilizer PEG is non-toxic approved by the FDA in 1990. PEG is commonly used for surface components of lipid nanoparticles reported to have higher stability, circulation half-life, and reduced immunogenicity [ 13 ]. In general, mRNAs are short and easily degraded by the natural enzymes in the human body.…”