Unlocking the Therapeutic Applicability of LNP-mRNA: Chemistry, Formulation, and Clinical Strategies

Abstract

Messenger RNA (mRNA) has emerged as an innovative therapeutic modality, offering promising avenues for the prevention and treatment of a variety of diseases. The tremendous success of mRNA vaccines in effectively combatting coronavirus disease 2019 (COVID-19) evidences the unlimited medical and therapeutic potential of mRNA technology. Overcoming challenges related to mRNA stability, immunogenicity, and precision targeting has been made possible by recent advancements in lipid nanoparticles (LNPs). This review summarizes state-of-the-art LNP-mRNA-based therapeutics, including their structure, material compositions, design guidelines, and screening principles. Additionally, we highlight current preclinical and clinical trends in LNP-mRNA therapeutics in a broad range of treatments in ophthalmological conditions, cancer immunotherapy, gene editing, and rare-disease medicine. Particular attention is given to the translation and evolution of LNP-mRNA vaccines into a broader spectrum of therapeutics. We explore concerns in the aspects of inadequate extrahepatic targeting efficacy, elevated doses, safety concerns, and challenges of large-scale production procedures. This discussion may offer insights and perspectives on near- and long-term clinical development prospects for LNP-mRNA therapeutics.

Fig.  1.

Establishing chemistry, formulation, targeted delivery, and CMC approaches to tackle the hurdles hindering LNP-mRNA clinical translation. Created with BioRender.com.

Fig. 2.

Examples of ionizable lipids, sterols, phospholipids, and PEG lipids used in LNP formulations. Created with BioRender.com.

Fig. 3.

High-throughput screening of the three-component ionizable lipidoids facilitates the potent mRNA delivery in vitro and in vivo. (A to E) Schematic illustration of reaction mechanism and component structures of the screened lipidoids, along with representative LNP morphology via cryogenic electron microscopy images. (F to H) In vitro luciferase mRNA delivery efficacy among a range of 48 top performing lipids in HeLa cell line and mouse bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs). (I to K) In vivo luciferase mRNA delivery for selecting optimal ketone, isocyanide, and amine structures, resulting in three top-performing lipids. Reproduced with permission [39]. Copyright 2019, Springer Nature.

Fig. 4.

The effect of phosphate lipids (A to D) and cholesterol (E to J) on LNP-mRNA targeting and delivery efficacy. (A to D) Targeted mRNA delivery into specific organs (lung, spleen, and liver) via charged phospholipid-incorporated LNPs. Reproduced with permission [56]. Copyright 2022, Elsevier. LNPs were prepared using one of three helper lipids [chemical structure shown in (A)]: DOPE (with a net neutral charge), phosphatidylserine (PS) (with a net negative charge), or DOTAP (with a positive charge). (B) Ex vivo images demonstrated that luciferase expression occurred predominantly in the liver, regardless of helper lipid charge, for a standard LNP formulation (16 mol % helper lipid) incorporating the ionizable lipidoid 306O₁₀ (0.75 mg kg⁻¹ Luc mRNA, intravenous administration, 3 h). (C) Ex vivo images demonstrated that luciferase expression shifted from the liver to the spleen or lungs when LNPs were formulated with 40 mol % PS or DOTAP instead of DOPE (0.75 mg kg⁻¹ Luc mRNA, intravenous administration, 3 h). (D) Quantitative luciferase signal from In Vivo Imaging System that presented the charge of the helper lipid influenced the organ site of protein expression regardless of the identity of the ionizable lipidoid (200O_i10, 205O_6,10, and 306O₁₀) in LNPs. (E to H) Enhanced mRNA delivery in hepatic cells with altered stereochemistry of cholesterol. Reproduced with permission [59]. Copyright 2023, Springer Nature. (E and F) Size, polydispersity index (PDI), pKa, and morphology characterization of LNPs formulated with 20α or 20mix. (G) 20-Hydroxycholesterol chemical structure, with stereocenter highlighted in dark blue. (H) The 20α LNPs facilitated significantly higher liver-targeted delivery among all types of cells compared to 20mix LNPs, as shown by the percentage of tdTom⁺ cells, measured by flow cytometry, 3 days after Ai14 mice were injected with 0.3 mg/kg (body weight) of LNPs formulated with 20α or 20mix. (I and J) LNP formulated with hydroxycholesterol substitutes mediated higher mRNA delivery to human T cells. Reproduced with permission [60]. Copyright 2022, Elsevier. (I) Chemical structures of the hydroxycholesterol substitutes. (J) LNPs formulated with substitution of 7α -hydroxycholesterol by 25% (A1–25) and 50% (A1–50) assisted higher primary human T cells compared to standard LNP formulation (S2) at various doses, characterized by the luciferase mRNA expression.

Fig. 5.

Selective organ targeting (SORT) enables the systematic and reliable engineering of lipid nanoparticles (LNPs) for precise mRNA delivery into specific organ. Reproduced with permission [62]. Copyright 2020, Springer Nature. (A) When a supplementary component, referred to as a SORT molecule, is introduced to conventional LNPs, it systematically modifies the in vivo delivery profile, facilitating tissue-specific delivery based on the percentage and biophysical properties of the SORT molecule. This strategy effectively redirected various categories of nanoparticles. (B) Ex vivo images demonstrate luminescence in major organs using DLin-MC3-DMA SORT LNPs and C12-200 SORT LNPs, incorporating cationic lipid (DOTAP) and anionic lipid (18PA) in the formulations (0.1 mg kg⁻¹ Luc mRNA, intravenous administration, 6 h). (C to E) SORT molecules could be divided into specific groups with defined biophysical properties despite distinct chemical structures. (C) Permanently cationic SORT lipids (dimethyldioctadecylammonium (bromide salt) （DDAB）, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC), and DOTAP) all resulted in the same mRNA delivery profile to the lung. (D) Anionic SORT lipids (14PA, 18BMP, and 18PA) all resulted in the same mRNA delivery profile to the spleen. (E) Ionizable cationic SORT lipids with tertiary amino groups (DODAP, C12-200) enhanced liver delivery without luciferase expression in the lungs (0.1 mg kg⁻¹ Luc mRNA, intravenous administration, 6 h).

Fig. 6.

Schematic illustration of in vivo fate of LNP-mRNA modality after administration into the body, including encompassing systemic circulation, cellular uptake, mRNA release into the cytosol, and subsequent translation into proteins. Created by Biorender.com.

Fig. 7.

Surface modification strategies for tissue targeting of LNP-mRNA. Created with BioRender.com.

Fig. 8.

The characterization and in vivo T cell targeting delivery efficacy of anti-CD3 (aCD3)-conjugated LNP [75]. (A) Cryo-TEM images of the nontargeted LNPs and aCD3-conjugated LNPs with magnified regions in which white rectangles presented the conjugated 16% aCD3 on the surface of LNPs. (B to D) Delivery efficiency in vivo analyzed by mCherry-mRNA expression in the splenic CD3e⁺ (B), CD4⁺ (C), and CD8a⁺ T cells (D) at 24 and 48 h after control [no treatment, isotype-conjugated LNPs, or aCD3 F(ab′)2] or 1, 2, or 16% aCD3-LNP treatment, respectively.

Fig. 9.

(A) Schematic illustration of CD5-targeted LNPs achieved in vivo production of CAR T cells. (B) Delivery efficiency demonstrated by Cre-mRNA expression among the splenocytes. Ai6 mice (Rosa26CAG-LSL-ZsGreen) were injected with either saline, IgG/LNP-Cre (30 mg), or CD5/LNP-Cre (30 mg). After 24 h, they observed ZsGreen expression in 81.1% of CD4⁺ splenocytes and in 75.6% of CD8⁺ splenocytes, whereas only 15.0% of CD3 splenocytes exhibited such expression. The bar graphs depict data from two independent biological replicates. (C and D) Images (C) and quantitative analysis (D) of fibrosis of ventricles in coronal cardiac sections of mock uninjured animals (3 weeks after saline pump implant + saline injection at week 1), injured control animals (AngII/PE + saline), and treated animals (AngII/PE + CD5/LNP-FAPCAR). Picrosirius red staining highlights collagen (pink). Reproduced with permission [80]. Copyright 2022, The American Association for the Advancement of Science.

Fig.  10.

Schematic illustration of innovative strategies for accelerating LNP design. (A) Schematic illustration of high-throughput screening of ionizable lipids. (B) Barcode-based screening of LNP formulations for the advanced targeting efficiency. (C) Corona effect and the formation mechanisms for LNPs. Created with BioRender.com.

Fig.  11.

Instrumentation of multi-color CICS platform and methodology for characterization of LNP formulations. Reproduced with permission [111,133]. Copyright 2022, Springer Nature. (A) Three fluorescent tags were used for single-particle fluorescence detection and categorization: Cy5 tag for mRNA, TMR tag for help lipids, and YOYO-1 for unloaded mRNA. (B) Instrumental setup of three-color CICS. (C) For all the detecting species of interest, the fluorescence was classified as mRNA-loaded LNPs (circles, TMR-Cy5 coincident), empty LNPs (crosses, TMR only), and free mRNAs (asterisks, Cy5-YOYO-1 coincident). (D and E) The Cy5 intensity profile of single free mRNA molecules, and the profile shifted right when multiplexed mRNAs were expected in LNPs. (F) TMR intensity profiles of LNP formulations correlate with their relative helper lipid content. (G to I) Cryo-TEM images of mRNA LNPs of the benchmark formulation at pH 7.4. (G) LNPs formulated with nonlabeled mRNA and nonlabeled DSPC. (H) LNP formulated with Cy5-mRNA and 0.5% (mol % to total lipid content) TMR-PC. (I) Empty LNPs formulated in the absence of mRNA. All scale bars, 200 nm.