(370b) Award Submission: Accelerated Blood Clearance of Lipid Nanoparticles Occurs Regardless of Formulation

Lipid nanoparticle (LNP) based mRNA delivery has the potential to revolutionize medicine as it can be used in vaccines, protein replacement, and cancer therapy. Most viable mRNA therapies will need repeated administration to enact a continuous therapeutic effect. Unfortunately, some LNP formulations lose mRNA delivery efficacy upon repeat dosing due to Accelerated Blood Clearance (ABC). ABC is a tachyphylaxis response against the PEGylated lipid component of LNPs, and correlates with anti-PEG antibody levels in serum. The goal of this study is to investigate whether formulation optimization of an ABC-inducing LNP can enable successful repeated dosage. To this extent, we investigated the effect of different PEGylated lipids, PEG molar ratios, and administration routes on repeat efficacy of an ABC-inducing LNP and the adaptive immune response observed after delivery. Lipid nanoparticles (LNP A) encapsulating Erythropoietin (EPO) mRNA were formulated with varied PEGylated lipids and molar ratios (0-2.5%). Mice were injected intravenously, intramuscularly, or intraperitoneally at a dose of 0.5 mg/kg, two weeks apart. EPO protein expression and serum antibody titers were measured using ELISA. ABC-inducing LNP A elicited efficacy loss after repeat dose regardless of administration route and prompted anti-PEG IgM production that peaked 21 days post injection. Next, we explored if changing the PEG-lipid type would allow for LNP A to successfully repeat delivery. Of the three PEG-lipids tested, none resulted in successful repeated dosage, and all significantly increased anti-PEG IgM on day 21 compared to baseline. Following these findings, we then changed the concentration of PEG-lipid used. Loss of efficacy was seen for LNPs with 1.0-2.5 mol% PEG-lipid. However, LNPs with less than 1.0 mol% PEG-lipid loss their ability to efficaciously deliver EPO. Formulation that showed significant EPO delivery over PBS did have increased anti-PEG IgM on day 21. Our work indicates that altering the PEG-lipid type, PEG-lipid concentration, or route of administration does not alleviate an ABC-inducing LNP’s efficacy loss. Loss of efficacy correlated with elevated anti-PEG IgM levels, suggesting that ABC of LNPs is associated with anti-PEG IgM. However, the reason why anti-PEG IgM increases and causes ABC occurs with one LNP and not another remains unknown. Future work will evaluate the effect of pre-existing anti-PEG levels on de novo efficacy of an FDA-approved LNP. It is crucial to determine if ABC results in elimination of any LNP to successfully deliver mRNA, as it could have clinical implications for future therapeutics.