J Control Release. 2024 Sep:373:952-961. doi: 10.1016/j.jconrel.2024.07.046. Epub 2024 Aug 8. A perspective on bleb and empty LNP structures Abstract Although lipid nanoparticles (LNPs) have been FDA-approved for mRNA delivery, there is still much to learn about these fascinating multi-component delivery systems. Here, I discuss the presence of "bleb" structures on LNPs and the co-existence of mRNA-empty LNPs in LNP-mRNA-based formulations. Specifically, I discuss key articles on these structural and compositional heterogeneities, whether these features present negative or positive LNP attributes, and how to deal with them in research and quality control settings. Additionally, I present current approaches and propose novel strategies on how to study and quantify bleb and empty LNP structures. With the conflicting views on these features in the literature and limited systematic studies on their impact on safety and efficacy, I hope this Perspective will support current and bring forward new thinking about these matters. I anticipate that novel studies and insights could emerge from these lines of thinking, which could potentially enhance the development of safe and efficient LNP-based drug products that will either embrace, leverage, or mitigate the presence of blebs and empty LNPs. Keywords: Bleb; Empty; LNP; Lipid nanoparticles; Quantifcation; Structures. For more product information, please contact us at: US Tel: 1-844-782-5734 US Tel: 1-844-QUAL-PEG CHN Tel: 400-918-9898 Email: sales@sinopeg.com

J Control Release. 2021 Jul 10:335:237-246. doi: 10.1016/j.jconrel.2021.05.021. Epub 2021 May 18. Impact of lipid nanoparticle size on mRNA vaccine immunogenicity Abstract Lipid nanoparticles (LNP) are effective delivery vehicles for messenger RNA (mRNA) and have shown promise for vaccine applications. Yet there are no published reports detailing how LNP biophysical properties can impact vaccine performance. In our hands, a retrospective analysis of mRNA LNP vaccine in vivo studies revealed a relationship between LNP particle size and immunogenicity in mice using LNPs of various compositions. To further investigate this, we designed a series of studies to systematically change LNP particle size without altering lipid composition and evaluated biophysical properties and immunogenicity of the resulting LNPs. While small diameter LNPs were substantially less immunogenic in mice, all particle sizes tested yielded a robust immune response in non-human primates (NHP). Keywords: Lipid; Nanoparticle; Size; Vaccine; mRNA. For more product information, please contact us at: US Tel: 1-844-782-5734 US Tel: 1-844-QUAL-PEG CHN Tel: 400-918-9898 Email: sales@sinopeg.com

Review Mol Ther. 2017 Jul 5;25(7):1467-1475.   doi: 10.1016/j.ymthe.2017.03.013.   Epub 2017 Apr 13. Lipid Nanoparticle Systems for Enabling Gene Therapies Abstract Genetic drugs such as small interfering RNA (siRNA), mRNA, or plasmid DNA provide potential gene therapies to treat most diseases by silencing pathological genes, expressing therapeutic proteins, or through gene-editing applications.   In order for genetic drugs to be used clinically, however, sophisticated delivery systems are required.   Lipid nanoparticle (LNP) systems are currently the lead non-viral delivery systems for enabling the clinical potential of genetic drugs.   Application will be made to the Food and Drug Administration (FDA) in 2017 for approval of an LNP siRNA drug to treat transthyretin-induced amyloidosis, presently an untreatable disease.   Here, we first review research leading to the development of LNP siRNA systems capable of silencing target genes in hepatocytes following systemic administration.   Subsequently, progress made to extend LNP technology to mRNA and plasmids for protein replacement, vaccine, and gene-editing applications is summarized.   Finally, we address current limitations of LNP technology as applied to genetic drugs and ways in which such limitations may be overcome.   It is concluded that LNP technology, by virtue of robust and efficient formulation processes, as well as advantages in potency, payload, and design flexibility, will be a dominant non-viral technology to enable the enormous potential of gene therapy. Keywords: gene editing;   gene therapy;   genetic drugs;   lipid nanoparticles;   mRNA;   siRNA. For more product information, please contact us at: US Tel: 1-844-782-5734 US Tel: 1-844-QUAL-PEG CHN Tel: 400-918-9898 Email: sales@sinopeg.com

Review Mol Pharm. 2024 Dec 2;21(12):5944-5959. doi: 10.1021/acs.molpharmaceut.4c00826. Epub 2024 Nov 11. Development of Thermally Stable mRNA-LNP Delivery Systems: Current Progress and Future Prospects Abstract The success of mRNA-LNP-based COVID-19 vaccines opens a new era for mRNA-LNP-based therapy. This breakthrough is expected to catalyze the development of more mRNA-LNP-based medicines, not only for preventive vaccines but also for therapeutic purposes. Despite the promising outlook, there are fundamental challenges impeding the progress and widespread application of mRNA-LNP formulations. One of the significant challenges is their thermal instability, requiring these products to be stored at ultralow temperatures for long-term stability. The specific requirements present significant challenges for the storage, transportation, and distribution of mRNA-LNP formulations. To effectively prepare for future infectious disease outbreaks and broaden the application of mRNA-LNP-based therapies for other illnesses, improving the thermostability of mRNA-LNP formulations is critical. In this review, we discuss the potential factors contributing to the thermal instability of mRNA-LNP formulations and examine the roles of key components such as ionizable lipids, cholesterol, pH, buffers, and stabilizing agents like sugars in maintaining their thermal stability, with the goal of providing insights that can guide the future development of thermally stable mRNA-LNP formulations. Keywords: Formulations; Functional stability; Lipid nanoparticles; Physicochemical stability; Thermal stability; mRNA. For more product information, please contact us at: US Tel: 1-844-782-5734 US Tel: 1-844-QUAL-PEG CHN Tel: 400-918-9898 Email: sales@sinopeg.com

J Virol. 2024 Jun 13;98(6):e0057824. doi: 10.1128/jvi.00578-24. Epub 2024 May 20. An mRNA-LNP-based Lassa virus vaccine induces protective immunity in mice Abstract The mammarenavirus Lassa virus (LASV) causes the life-threatening hemorrhagic fever disease, Lassa fever. The lack of licensed medical countermeasures against LASV underscores the urgent need for the development of novel LASV vaccines, which has been hampered by the requirement for a biosafety level 4 facility to handle live LASV. Here, we investigated the efficacy of mRNA-lipid nanoparticle (mRNA-LNP)-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), in mice. Two doses of LASgpc- or LCMnp-mRNA-LNP administered intravenously (i.v.) protected C57BL/6 mice from a lethal challenge with a recombinant (r) LCMV expressing a modified LASgpc (rLCMV/LASgpc2m) inoculated intracranially. Intramuscular (i.m.) immunization with two doses of LASgpc- or LCMnp-mRNA-LNP significantly reduced the viral load in C57BL/6 mice inoculated i.v. with rLCMV/LASgpc2m. High levels of viremia and lethality were observed in CBA mice inoculated i.v. with rLCMV/LASgpc2m, which were abrogated by i.m. immunization with two doses of LASgpc-mRNA-LNP. The protective efficacy of two i.m. doses of LCMnp-mRNA-LNP was confirmed in a lethal hemorrhagic disease model of FVB mice i.v. inoculated with wild-type rLCMV. In all conditions tested, negligible and high levels of LASgpc- and LCMnp-specific antibodies were detected in mRNA-LNP-immunized mice, respectively, but robust LASgpc- and LCMnp-specific CD8+ T cell responses were induced. Accordingly, plasma from LASgpc-mRNA-LNP-immunized mice did not exhibit neutralizing activity. Our findings and surrogate mouse models of LASV infection, which can be studied at a reduced biocontainment level, provide a critical foundation for the rapid development of mRNA-LNP-based LASV vaccines.IMPORTANCELassa virus (LASV) is a highly pathogenic mammarenavirus responsible for several hundred thousand infections annually in West African countries, causing a high number of lethal Lassa fever (LF) cases. Despite its significant impact on human health, clinically approved, safe, and effective medical countermeasures against LF are not available. The requirement of a biosafety level 4 facility to handle live LASV has been one of the main obstacles to the research and development of LASV countermeasures. Here, we report that two doses of mRNA-lipid nanoparticle-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of lymphocytic choriomeningitis virus (LCMV), a mammarenavirus genetically closely related to LASV, conferred protection to recombinant LCMV-based surrogate mouse models of lethal LASV infection. Notably, robust LASgpc- and LCMnp-specific CD8+ T cell responses were detected in mRNA-LNP-immunized mice, whereas no virus-neutralizing acti...

Biomedicines. 2023 Jun 16;11(6):1735. doi: 10.3390/biomedicines11061735. ASL mRNA-LNP Therapeutic for the Treatment of Argininosuccinic Aciduria Enables Survival Benefit in a Mouse Model Abstract Argininosuccinic aciduria (ASA) is a metabolic disorder caused by a deficiency in argininosuccinate lyase (ASL), which cleaves argininosuccinic acid to arginine and fumarate in the urea cycle. ASL deficiency (ASLD) leads to hepatocyte dysfunction, hyperammonemia, encephalopathy, and respiratory alkalosis. Here we describe a novel therapeutic approach for treating ASA, based on nucleoside-modified messenger RNA (modRNA) formulated in lipid nanoparticles (LNP). To optimize ASL-encoding mRNA, we modified its cap, 5' and 3' untranslated regions, coding sequence, and the poly(A) tail. We tested multiple optimizations of the formulated mRNA in human cells and wild-type C57BL/6 mice. The ASL protein showed robust expression in vitro and in vivo and a favorable safety profile, with low cytokine and chemokine secretion even upon administration of increasing doses of ASL mRNA-LNP. In the ASLNeo/Neo mouse model of ASLD, intravenous administration of the lead therapeutic candidate LNP-ASL CDS2 drastically improved the survival of the mice. When administered twice a week lower doses partially protected and 3 mg/kg LNP-ASL CDS2 fully protected the mice. These results demonstrate the considerable potential of LNP-formulated, modified ASL-encoding mRNA as an effective alternative to AAV-based approaches for the treatment of ASA. Keywords: argininosuccinate lyase deficiency (ASLD); argininosuccinic aciduria (ASA); lipid nanoparticle-mRNA (LNP-mRNA); mRNA optimization; mRNA therapeutic; rare disease. For more product information, please contact us at: US Tel: 1-844-782-5734 US Tel: 1-844-QUAL-PEG CHN Tel: 400-918-9898 Email: sales@sinopeg.com

Review Eur J Pharm Biopharm. 2024 Apr:197:114222. doi: 10.1016/j.ejpb.2024.114222. Epub 2024 Feb 20. Kinetics of RNA-LNP delivery and protein expression Abstract Lipid nanoparticles (LNPs) employing ionizable lipids are the most advanced technology for delivery of RNA, most notably mRNA, to cells. LNPs represent well-defined core-shell particles with efficient nucleic acid encapsulation, low immunogenicity and enhanced efficacy. While much is known about the structure and activity of LNPs, less attention is given to the timing of LNP uptake, cytosolic transfer and protein expression. However, LNP kinetics is a key factor determining delivery efficiency. Hence quantitative insight into the multi-cascaded pathway of LNPs is of interest to elucidate the mechanism of delivery. Here, we review experiments as well as theoretical modeling of the timing of LNP uptake, mRNA-release and protein expression. We describe LNP delivery as a sequence of stochastic transfer processes and review a mathematical model of subsequent protein translation from mRNA. We compile probabilities and numbers obtained from time resolved microscopy. Specifically, live-cell imaging on single cell arrays (LISCA) allows for high-throughput acquisition of thousands of individual GFP reporter expression time courses. The traces yield the distribution of mRNA life-times, expression rates and expression onset. Correlation analysis reveals an inverse dependence of gene expression efficiency and transfection onset-times. Finally, we discuss why timing of mRNA release is critical in the context of codelivery of multiple nucleic acid species as in the case of mRNA co-expression or CRISPR/Cas gene editing. For more product information, please contact us at: US Tel: 1-844-782-5734 US Tel: 1-844-QUAL-PEG CHN Tel: 400-918-9898 Email: sales@sinopeg.com

Review Biomedicines. 2021 May 10;9(5):530. doi: 10.3390/biomedicines9050530. Non-Immunotherapy Application of LNP-mRNA: Maximizing Efficacy and Safety Abstract Lipid nanoparticle (LNP) formulated messenger RNA-based (LNP-mRNA) vaccines came into the spotlight as the first vaccines against SARS-CoV-2 virus to be applied worldwide. Long-known benefits of mRNA-based technologies consisting of relatively simple and fast engineering of mRNA encoding for antigens and proteins of interest, no genomic integration, and fast and efficient manufacturing process compared with other biologics have been verified, thus establishing a basis for a broad range of applications. The intrinsic immunogenicity of LNP formulated in vitro transcribed (IVT) mRNA is beneficial to the LNP-mRNA vaccines. However, avoiding immune activation is critical for therapeutic applications of LNP-mRNA for protein replacement where targeted mRNA expression and repetitive administration of high doses for a lifetime are required. This review summarizes our current understanding of immune activation induced by mRNA, IVT byproducts, and LNP. It gives a comprehensive overview of the present status of preclinical and clinical studies in which LNP-mRNA is used for protein replacement and treatment of rare diseases with an emphasis on safety. Moreover, the review outlines innovations and strategies to advance pharmacology and safety of LNP-mRNA for non-immunotherapy applications. Keywords: LNP-mRNA; RNA protein replacement therapy; cytokines; efficacy; in vitro transcription (IVT); innate immunity; lipid nanoparticle; non-immunotherapy applications; rare disease; safety. For more product information, please contact us at: US Tel: 1-844-782-5734 US Tel: 1-844-QUAL-PEG CHN Tel: 400-918-9898 Email: sales@sinopeg.com