Lipid nanoparticle (LNP) packaged mRNA vaccines have been deployed against infectious diseases such as COVID-19, yet their structural features remain unclear. Cholesterol, a major constituent within LNPs, contributes to their morphology that influences gene delivery. Herein, we examine the structure of LNPs containing cholesterol derivatives using electron microscopy, differential scanning calorimetry, and membrane fluidity assays. LNPs formulated with C24 alkyl derivatives of cholesterol show a polymorphic shape and various degrees of multilamellarity and lipid partitioning, likely due to phase separation. The addition of methyl and ethyl groups to the C24 alkyl tail of the cholesterol backbone induces multilamellarity (>50% increase compared to cholesterol), while the addition of a double bond induces lipid partitioning (>90% increase compared to cholesterol). LNPs with multilamellar and faceted structures, as well as a lamellar lipid phase, showed higher gene transfection. Unraveling the structure of mRNA-LNPs can enable their rational design toward enhanced gene delivery.

Despite lipid nanoparticles' (LNPs) success in the effective and safe delivery of mRNA vaccines, an inhalationbased mRNA therapy for lung diseases remains challenging. LNPs tend to disintegrate due to shear stress during aerosolization, leading to ineffective delivery. Therefore, LNPs need to remain stable through the process of nebulization and mucus penetration, yet labile enough for endosomal escape. To meet these opposing needs, we utilized PEG lipid to enhance the surficial stability of LNPs with the inclusion of a cholesterol analog, β-sitosterol, to improve endosomal escape. Increased PEG concentrations in LNPs enhanced the shear resistance and mucus penetration, while β-sitosterol provided LNPs with a polyhedral shape, facilitating endosomal escape. The optimized LNPs exhibited a uniform particle distribution, a polyhedral morphology, and a rapid mucosal diffusion with enhanced gene transfection. Inhaled LNPs led to localized protein production in the mouse lung without pulmonary or systemic toxicity. Repeated administration of these LNPs led to sustained protein production in the lungs. Lastly, mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) was delivered after nebulization to a CFTR-deficient animal model, resulting in the pulmonary expression of this therapeutic protein. This study demonstrated the rational design approach for clinical translation of inhalable LNP-based mRNA therapies.

Introduction-Lipid based nanoparticles (LNPs) are clinically successful vectors for hepatic delivery of nucleic acids. These systems are being developed for non-hepatic delivery of mRNA for the treatment of diseases like cystic fibrosis or retinal degeneration as well as infectious diseases. Localized delivery to the lungs requires aerosolization. We hypothesized that structural lipids within LNPs would provide features of integrity which can be tuned for attributes required for efficient hepatic and non-hepatic gene delivery. Herein, we explored whether naturally occurring lipids that originate from the cell membrane of plants and microorganisms enhance mRNA-based gene transfection in vitro and in vivo and whether they assist in maintaining mRNA activity after nebulization. Methods-We substituted DSPC, a structural lipid used in a conventional LNP formulation, to a series of naturally occurring membrane lipids. We measured the effect of these membrane lipids on size, encapsulation efficiency and their impact on transfection efficiency. We further characterized LNPs after nebulization and measured whether they retained their transfection efficiency. Results-One plant-derived structural lipid, DGTS, led to a significant improvement in liver transfection of mRNA. DGTS LNPs had similar transfection ability when administered in the nasal cavity to conventional LNPs. In contrast, we found that DGTS LNPs had reduced transfection efficiency in cells pre-and post-nebulization while maintaining size and encapsulation similar to DSPC LNPs. Conclusions-We found that structural lipids provide differential mRNA-based activities in vitro and in vivo which also depend on the mode of administration. Understanding influence of structural lipids on nanoparticle morphology and structure can lead to engineering potent materials for mRNA-based gene therapy applications.

Lipid-based nanoparticles (LNPs) for the delivery of mRNA have jumped to the forefront of non-viral gene delivery. Despite this exciting development, poor endosomal escape after LNP cell entry remains an...

Lipid nanoparticle (LNP)–based mRNA delivery holds promise for the treatment of inherited retinal degenerations. Currently, LNP-mediated mRNA delivery is restricted to the retinal pigment epithelium (RPE) and Müller glia. LNPs must overcome ocular barriers to transfect neuronal cells critical for visual phototransduction, the photoreceptors (PRs). We used a combinatorial M13 bacteriophage–based heptameric peptide phage display library for the mining of peptide ligands that target PRs. We identified the most promising peptide candidates resulting from in vivo biopanning. Dye-conjugated peptides showed rapid localization to the PRs. LNPs decorated with the top-performing peptide ligands delivered mRNA to the PRs, RPE, and Müller glia in mice. This distribution translated to the nonhuman primate eye, wherein robust protein expression was observed in the PRs, Müller glia, and RPE. Overall, we have developed peptide-conjugated LNPs that can enable mRNA delivery to the neural retina, expanding the utility of LNP-mRNA therapies for inherited blindness.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters through the airways and infects the lungs, causing lethal pulmonary damage in vulnerable patients. This virus contains spike proteins on its envelope that binds to human angiotensin-converting enzyme 2 (hACE2) expressed on the surface of airway cells, enabling entry of the virus for causing infection1,2. In severe cases, the virus enters the circulatory system, contributing to multiorgan failure. Soluble form of hACE2 binds to SARS-CoV-2 spike protein and prevents viral entry into target cells3. Moreover, soluble recombinant ACE2 ameliorates lung injury4 but its short half-life limits its therapeutic utility5. Here, we engineered synthetic mRNA to encode a soluble form of hACE2 (hsACE2) to prevent viral infection. Novel lipid nanoparticles (LNPs) were used to package mRNA and transfect mammalian cells for enhanced production of secreted proteins. Intravenously administered LNP led to hepatic delivery of the mRNA. This elicited secretion of hsACE2 into the blood circulation within 2 h, and levels of circulating hsACE2 peaked at 6 h and gradually decreased over several days. Since the primary site of entry and pathogenesis for SARS-CoV-2 is the lungs, we instilled LNPs into the lungs and were able to detect hsACE2 in the bronchoalveolar lavage fluid within 24 h and lasted for 48 h. Through co-immunoprecipitation, we found that mRNA-generated hsACE2 was able to bind with the receptor binding domain of the SARS-CoV-2 spike protein. Furthermore, hsACE2 was able to strongly inhibit (over 90%) SARS-CoV-2 pseudovirus infection. Our proof of principle study shows that mRNA-based nanotherapeutics can be potentially deployed for pulmonary and extrapulmonary neutralization of SARS-CoV-2 and open new treatment opportunities for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) can cause lethal pulmonary damage in humans. It contains spike proteins on its envelope that bind to human angiotensin‐converting enzyme 2 (hACE2) expressed on airway cells, enabling entry of the virus, and causing infection. The soluble form of hACE2 binds SARS‐CoV‐2 spike protein, prevents viral entry into target cells, and ameliorates lung injury; however, its short half‐life limits therapeutic utilities. Here, synthetic mRNA is engineered to encode a soluble form of hACE2 (hsACE2) to prevent viral infection. A novel lipid nanoparticle (LNP) is used for packaging and delivering mRNA to cells to produce hsACE2 proteins. Intravenously administered LNP delivers mRNA to hepatocytes, leading to the production of circulatory hsACE2 initiated within 2 h and sustained over several days. Inhaled LNP results in lung transfection and secretion of mucosal hsACE2 to lung epithelia, the primary site of entry and pathogenesis for SARS‐CoV‐2. Furthermore, mRNA‐generated hsACE2 binds to the receptor‐binding domain of the viral spike protein. Finally, hsACE2 effectively inhibits SARS‐CoV‐2 and its pseudoviruses from infecting host cells. The proof of principle study shows that mRNA‐based nanotherapeutics can be potentially deployed to neutralize SARS‐CoV‐2 and open new treatment opportunities for coronavirus disease 2019 (COVID‐19).

Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19 pandemic. Here, we investigate the impact of commonly used biological buffers on the performance and durability of mRNA-LNPs. We tested the compatibility of three common buffers�HEPES, Tris, and phosphate-buffered saline�with a DLin-MC3-DMA mRNA-LNP formulation before and after a single controlled freeze−thaw cycle. We hypothesized that buffer composition would affect lipid-aqueous phase separation. Indeed, the buffers imposed structural changes in LNP morphology as indicated by electron microscopy, differential scanning calorimetry, and membrane fluidity assays. We employed in vitro and in vivo models to measure mRNA transfection and found that Tris or HEPES-buffered LNPs yielded better cryoprotection and transfection efficiency compared to PBS. Understanding the effects of various buffers on LNP morphology and efficacy provides valuable insights into maintaining the stability of LNPs after long-term storage.