ABSTRACT The success of inhaled mRNA therapeutics remains limited by the absence of aerosol optimized ionizable lipids and incomplete understanding of formulation device interactions. Here we present an integrated platform for pulmonary mRNA delivery combining novel lipid chemistry, systematic formulation engineering, and device optimization. From a novel phenolic acid derived ionizable lipid library, we identified SY‐3 as a potent lead candidate, based on a biodegradable syringic acid‐based core. Design‐of‐experiments optimization yielded LNP 5, a formulation exhibiting potential for improved cytosolic delivery along with nebulization stability. Repeated nebulization of LNP 5 encapsulating human CFTR mRNA in Cftr ‐knockout mice produced robust protein expression and restored correct apical CFTR localization in airway epithelia. Device characterization revealed that vibrating‐mesh nebulizer selection critically influences LNP integrity, aerosol output, and mass median aerodynamic diameter (MMAD). Collectively, these results demonstrate an integrated development strategy encompassing ionizable lipid design, formulation optimization, and device engineering to enable efficient mRNA delivery to the lungs. While the work successfully shows robust mRNA expression, functional CFTR activity in vitro, and appropriate in vivo tissue localization, further validation is needed—specifically physiological assessments such as nasal potential difference and mucus clearance measurements—before advancing to clinical trials.

mRNA delivery using lipid nanoparticles (LNPs) has become a cornerstone of modern biological therapeutics. During the formulation of LNPs, uniform mixing of LNP components is critical to ensuring desirable functional properties. This study employs simple bi-directional T-mixing of lipids in ethanol with mRNA in buffer to evaluate the effects of mixing chamber turbulence on mRNA-LNP biophysical attributes and develops a mechanistic model relating the mixing processes to biological performance. LNPs encapsulating hEPO mRNA formulated under turbulent mixing conditions with ionizable lipid OF-02 demonstrate higher hEPO expression than those formulated under laminar mixing conditions via two routes of administration in an in vivo mouse model. By measuring LNP zeta potential, fusogenicity, and lipid fluidity as functions of pH, we propose a hypothetical model for increased pH-sensitivity of the turbulently formulated LNPs, presumably resulting in improved intracellular release of mRNA. Unique profiles measured by small-angle X-ray scattering (SAXS) and greater homogeneity observed by cryo-TEM for LNPs formulated under turbulent mixing conditions further support this model. Increased serum protein binding for these turbulently mixed mRNA-LNPs suggests an additional mode of action involving receptor-mediated uptake following systemic delivery. A follow-up study with LNPs made with reduced lipid : mRNA mass ratios indicates that turbulent mixing may preserve LNP function with lower lipid load, compared to LNPs made with higher lipid load under laminar flow conditions. Altogether, these findings underscore important connections between LNP performance and formulation process, offering valuable insights for optimization of mRNA-LNP formulations.

Lipid nanoparticles (LNPs) have emerged as effective carriers for mRNA delivery in vaccine and therapeutic applications, attracting substantial attention since the COVID-19 pandemic. Continued efforts are crucial to optimize LNP composition for improved delivery efficacy and to elucidate the underlying mechanisms driving differences in protein expression. This study systematically screened PEGylated lipids for intramuscular mRNA delivery, followed by optimization of the formulation composition, physicochemical characterization, and investigation of the structure-activity relationship (SAR). Using a model ionizable lipid, we initially evaluated twenty-nine PEGylated lipids from four lipid families (glyceride, phosphoethanolamine (PE), cholesterol, and ceramide), each varying in linker chemistries, tail structures, or PEG molecular weights. 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol - 5000 (DMG-PEG5k) was identified as a promising candidate from this screening. Using a design of experiments (DoE) approach, we further optimized the formulation to increase in vivo transfection efficacy, achieving an increase in protein expression over the DMG-PEG2k benchmark. To explore the SAR of the DoE formulations, advanced physicochemical characterization was conducted including Laurdan assay, SAXS, Cryo-TEM, and QCM-D, alongside standard LNP analysis. Among the key factors examined, high mRNA encapsulation efficiency, LNP membrane integrity (especially under acidic conditions), and ordered internal structures were identified as the critical parameters for transfection efficiency. mRNA encapsulation efficiency increased with a lower PEG-lipid fraction. LNP membrane integrity, assessed by the generalized polarization (GP) ratio at pH 7.5 and 4.5 from the Laurdan assay, was strongly affected by the ionizable lipid ratio and, to a lesser extent, the cholesterol ratio. A lower GP7.5/GP4.5 ratio correlated with enhanced protein expression, primarily driven by a higher GP4.5 observed with lower ionizable lipid and higher cholesterol fractions. Overall, balancing the ratios of all LNP components is critical for maximizing LNP functionality. This study presents a systematic evaluation and characterization of LNPs with different PEG-lipid moieties, deepens SAR understanding, and provides valuable guidelines for rationally designing more effective next-generation LNPs.

The development of nucleic acid therapeutics using non-viral delivery systems requires efficient payload delivery to target organs for higher potency and tolerability. While lipid nanoparticle (LNP) formulations influence biodistribution, cellular uptake, and therapeutic efficacy, underlying mechanisms remain incompletely understood. This study develops potent mRNA-LNP formulations and investigates determinants of liver tropism using ornithine transcarbamylase (OTC) deficiency as a protein replacement therapy model. Systematic screening of ionizable and helper lipids, optimization of composition and process, and biophysical characterization identify a liver-tropic helper lipid-1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) that modulates LNP structure and apolipoprotein E (ApoE) binding, enhancing liver-specific delivery. Analysis of ionizable lipid chemistry reveals its role in cellular uptake mechanisms, leading to the identification of a novel ionizable lipid designed with N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS) core that enables efficient delivery independent of the low-density lipoprotein receptor (LDLR) pathway. The optimized formulation achieves robust dose responsiveness, sustained therapeutic expression, and favorable tolerability in preclinical models. Therapeutic levels of OTC protein expression are observed with minimal toxicity, as indicated by stable liver function markers and cytokine levels. These findings provide mechanistic insights and establish a platform for mRNA-based protein replacement therapies, supporting broader applications in rare genetic diseases requiring hepatic gene expression.

Lipid nanoparticle (LNP) components can impact the safety and immunogenicity of mRNA vaccines. Here we examine the mechanisms contributing to the performance of mRNA-LNP vaccines by exploring the impact of nucleoside modifications and LNP components on translational efficiency, innate immune activation, and immunogenicity. Our data reveals several molecular and immunological parameters affected by nucleoside modification including a synergistic effect of the mRNA and ionizable lipid composition on the immune activation triggered by the mRNA-LNP formulation. Our results indicate changes in the LNP composition, independent from whether the mRNA is modified or unmodified, caused differential expression of genes associated with innate and antiviral immunity. We believe these findings offer valuable insights into mRNA vaccine function and offer strategies for enhancing vaccine efficacy and reducing the reactogenicity of next generation mRNA vaccines.

INTRODUCTION:One of the biggest challenges in the mRNA-LNP vaccine field is product stabilization to overcome the logistical hurdles linked to the ultra-cold distribution chain associated with first-generation mRNA SARS-CoV-2 vaccines. Despite recent progress in the field, many R&D efforts remain focused on the development of mRNA-LNP vaccines that would be as stable as liquid formulations for storage at refrigerated or room temperatures. AREAS COVERED:After an overview of the underlying mechanisms of mRNA-LNP instability, this review provides an update on the different approaches that are currently explored to improve mRNA-LNP thermostability, encompassing mRNA sequence optimization, nucleotide modification and mRNA-LNP design strategies as well as formulation process optimization. Alternative approaches for mRNA-LNP stabilization such as lyophilization, dual-vial formulations and the replacement of water with deep eutectic solvents in the mRNA-LNP process and products are also discussed. EXPERT OPINION:Achieving robust thermostability of mRNA vaccines will require a multifactorial optimization strategy, integrating advances in sequence engineering, novel formulation designs, buffer composition, excipient selection and manufacturing processes.

Rational design and robust formulation processes are critical for optimal delivery of mRNA by lipid nanoparticles (LNPs). Varying degrees of heterogeneity in mRNA-LNPs can affect their biophysical and functional properties. Given the profound complexity of mRNA-LNPs, it is critical to develop comprehensive and orthogonal analytical techniques for a better understanding of these formulations. To this end, we developed a robust ultracentrifugation method for density-based separation of subpopulations of mRNA-LNPs. Four LNP formulations encapsulating human erythropoietin (hEPO) with varying functionalities were synthesized using two ionizable lipids, A and B, and two helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), along with cholesterol and DMG-PEG-2K. Upon ultracentrifugation on a sucrose gradient, a distinct pattern of "fractions" was observed across the gradient, from the less dense topmost fraction to the increasingly denser bottom fractions, which were harvested for comprehensive analyses. Parent LNPs, A-DOPE and B-DOPE, were resolved into three density-based fractions, each differing significantly in the hEPO expression following intravenous and intramuscular routes of administration. Parent B-DEPE LNPs resolved into two density-based fractions, with most of the payload and lipid content being attributed to the topmost fraction compared to the lower one, indicating some degree of heterogeneity, while parent A-DEPE LNPs showed remarkable homogeneity, as indicated by comparable in vivo potency, lipid numbers, and particle count among the three density-based fractions. This study is the first to demonstrate the application of density gradient-based ultracentrifugation (DGC) for a head-to-head comparison of heterogeneity as a function of biological performance and biophysical characteristics of parent mRNA-LNPs and their subpopulations.

Evaluate treatment outcomes among patients with mantle cell lymphoma (MCL) treated with the Bruton's tyrosine kinase inhibitors (BTKis) ibrutinib or acalabrutinib in second- or later-line therapy (2L+).

Lipid nanoparticles (LNPs) have shown great promise as delivery vehicles to transport messenger ribonucleic acid (mRNA) into cells and act as vaccines for infectious diseases including COVID-19 and influenza. The ionizable lipid incorporated within the LNP is known to be one of the main driving factors for potency and tolerability. Herein, we describe a novel family of ionizable lipids synthesized with a piperazine core derived from the HEPES Good buffer. These ionizable lipids have unique asymmetric tails and two dissimilar degradable moieties incorporated within the structure. Lipids tails of varying lengths, degrees of unsaturation, branching, and the inclusion of additional ester moieties were evaluated for protein expression. We observed several key lipid structure activity relationships that correlated with improved protein production in vivo, including lipid tails of 12 carbons on the ester side and the effect of carbon spacing on the disulfide arm of the lipids. Differences in LNP physical characteristics were observed for lipids containing an extra ester moiety. The LNP structure and lipid bilayer packing, visualized through Cryo-TEM, affected the amount of protein produced in vivo. In non-human primates, the Good HEPES LNPs formulated with an mRNA encoding an influenza hemagglutinin (HA) antigen successfully generated functional HA inhibition (HAI) antibody titers comparable to the industry standards MC3 and SM-102 LNPs, demonstrating their promise as a potential vaccine.

Pulmonary delivery of mRNA via inhalation is a very attractive approach for RNA-based therapy for treatment of lung diseases. In this work, we have demonstrated successful development of an mRNA-lipid nanoparticle (LNP) dry powder product (DPP), wherein the LNPs were spray dried using hydroalcoholic solvent along with mannitol and leucine as excipients. The desired critical attributes for the DPP were accomplished by varying the excipients, lipid composition, concentration of LNPs, and weight percentage of mRNA. Leucine alone or in combination with mannitol improved the formulation by increasing the mRNA yield as well as decreasing the particle size. Intratracheal administration of the DPP in mice resulted in luciferase expression in the trachea and lungs indicating successful delivery of functional mRNA. Our results show formulation optimization of mRNA LNPs administered in the form of DPP results in an efficacious functional delivery with great promise for future development of mRNA therapeutics for lung diseases.

Several tyrosine kinase inhibitors (TKIs) targeting EGFR are approved for first-line (1L) treatment of EGFR MU NSCLC. Most pts progress on 1L EGFR TKIs and, unless they stay on current treatment or switch to other targeted therapy, will advance to receive systemic therapies. This study evaluated real-world treatment patterns and outcomes of pts with EGFR MU NSQ a/mNSCLC. This retrospective observational study used pooled, de-identified EHR-derived data from US nationwide databases Flatiron Health, ConcertAI Patient360, and ConcertAI RWD360nlp. To exclude duplicate EHR, overlap assessment was performed via tokenization. EHR from pts diagnosed with EGFR MU NSQ a/mNSCLC between 2017–2022 who received earlier-line osimertinib (Osi), followed by 2L platinum-based chemotherapy, or 3L docetaxel (Doc) were included. Times from treatment line initiation until death (OS), within-line progression or death (PFS), treatment discontinuation (TTD), and initiation of next line or death (TTNTD) were assessed. Data from 256 pts were included: 227 (89%) in 2L and 29 (11%) in 3L. Median age at 2L/3L start was 67 (33–87) yr, 176 (69%) pts were female, 155 (61%) had ECOG PS of 0–1, and 225 (88%) had stage 3B or 4 NSCLC at diagnosis. Most common treatments were carboplatin + pemetrexed + pembrolizumab (39%) or carboplatin + pemetrexed (28%) in 2L, and Doc + ramucirumab (69%) or Doc mono (31%) in 3L. All 2L and 86% of 3L pts had received 1L Osi alone (2L: 68%; 3L: 52%) or Osi after older-generation EGFR TKI or a few rounds of chemotherapy ± IO (2L: 32%; 3L: 34%). Time-to-event outcomes are shown in the table.Table: 573PReal-world clinical outcomesOutcomeNEventsMedian time to event, months (95% CI)Median follow-up, months2L ptsOS22712315.7 (12.9, 20.2)23.3PFS2091564.9 (4.2, 6.4)24.8TTNTD2271766.1 (5.2, 7.9)26.7TTD2271804.4 (3.5, 5.9)24.03L ptsOS291910.1 (5.2, NE)14.7PFS27214.4 (3.0, 9.8)13.0TTNTD29225.2 (3.5, 10.5)12.7TTD29233.7 (1.6, 9.2)12.72L, second-line; 3L, third-line; CI, confidence interval; NE, not estimable; OS, overall survival; PFS, progression-free survival; pts, patients; TTD, time to treatment discontinuation; TTNTD, time to next treatment or death. Open table in a new tab 2L, second-line; 3L, third-line; CI, confidence interval; NE, not estimable; OS, overall survival; PFS, progression-free survival; pts, patients; TTD, time to treatment discontinuation; TTNTD, time to next treatment or death. RWE shows that 2L/3L treatment options for pts with EGFR MU NSQ a/mNSCLC are mostly limited to chemotherapy and are associated with poor outcomes, demonstrating a high unmet need for novel treatments.

For patients with EGFR WT NSQ aNSCLC who are not eligible for tyrosine kinase inhibitors (TKIs), platinum-based chemotherapy with or without immunotherapy is 1L standard of care followed by 2L docetaxel at disease progression. Newer biomarker driven therapies are being developed in this patient population; thus, the goal of this study was to assess outcomes with the current standard of care using real world data. This retrospective cohort study assesses real world overall survival (OS), progression-free survival (PFS), time to next treatment or death (TTNTD), and time to treatment discontinuation (TTD) for patients with EGFR WT NSQ aNSCLC receiving 2L docetaxel post 1L platinum-based chemotherapy plus pembrolizumab. This study used the nationwide Flatiron Health electronic health record (HER)-derived de-identified database to select adult community-based patients who had a confirmed diagnosis between Jan-01, 2017 and Dec-31, 2021 and with an ECOG PS of 0-1 at 2L initiation. Time to event outcomes were analyzed by Kaplan–Meier method. A total of 225 patients (58.2% male, mean [SD=8.6], age of 66 years) were included in the analysis; 138 (61.3%) patients were 65 years or older, 199 (88.4%) had an initial diagnosis at stage III or above, and 41 (18.2%) had brain metastasis. Among the 225 patients eligible for the analysis, the number of events observed for OS, PFS, TTNTD, and TTD were 151, 167, 180, and 185, respectively. The overall median OS (mOS) was 7.4 months (95% CI: 6.6-9.3), and mPFS was 4.3 months (95% CI: 3.8-5.3). The mTTNTD was 5.5 months and mTTD was 3.5 months. This study provides valuable information on real world treatment characteristics associated with standard of care and demonstrates that high unmet need still exists for novel treatment options that further improve OS in patients with EGFR WT NSQ aNSCLC.

The emergence of SARS-CoV-2 variants, especially Beta and Delta, has raised concerns about the reduced protection from previous infection or vaccination based on the original Wuhan-Hu-1 (D614) virus. To identify promising regimens for inducing neutralizing titers towards new variants, we evaluated monovalent and bivalent mRNA vaccines either as primary vaccination or as a booster in nonhuman primates (NHPs). Two mRNA vaccines, D614-based MRT5500 and Beta-based MRT5500β, tested in sequential regimens or as a bivalent combination in naïve NHPs produced modest neutralizing titers to heterologous variants. However, when mRNA vaccines were administered as a booster to pre-immune NHPs, we observed a robust increase in neutralizing titers with expanded breadth towards all tested variants, and notably SARS-CoV-1. The breadth of the neutralizing response was independent of vaccine sequence or modality, as we further showed either MRT5500 or recombinant subunit Spike protein (with adjuvant) can serve as boosters to induce broadly neutralizing antibodies in the NHPs primed with MRT5500. The data support the notion that a third vaccination is key to boosting existing titers and improving the breadth of antibodies to address variants of concern, including those with an E484K mutation in the Receptor Binding Domain (RBD) (Beta, Gamma).

Emergency use authorization of COVID vaccines has brought hope to mitigate pandemic of coronavirus disease 2019 (COVID-19). However, there remains a need for additional effective vaccines to meet the global demand and address the potential new viral variants. mRNA technologies offer an expeditious path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on the spike (S) glycoprotein of SARS-CoV-2. Several mRNA constructs of S-protein, including wild type, a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), as well as others, were tested in animal models for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques, and for efficacy in a Syrian golden hamster model. The selected 2P/GSAS vaccine formulation, designated MRT5500, elicited potent nAbs as measured in neutralization assays in all three preclinical models and more importantly, protected against SARS-CoV-2-induced weight loss and lung pathology in hamsters. In addition, MRT5500 elicited TH1-biased responses in both mouse and non-human primate (NHP), thus alleviating a hypothetical concern of potential vaccine-associated enhanced respiratory diseases known associated with TH2-biased responses. These data position MRT5500 as a viable vaccine candidate for entering clinical development.