(M0930-03-13) Tuning the Organ Selectivity of mRNA Expression through Dendrimer-Based Lipid Nanoparticles

Purpose: Lipid nanoparticles (LNPs) are effective nanocarriers for nucleic acids with clinically proven safety and efficacy, as evidenced by the FDA approval of ONPATTRO^® and mRNA COVID-19 vaccines. However, achieving organ-selective delivery remains challenging, as systemically-administered LNPs predominantly accumulate in the liver and preferentially target liver hepatocytes¹. For example, ONPATTRO^® treats hereditary transthyretin amyloidosis by systemically directing siRNA-LNPs to the liver to silence transthyretin protein production^2,3. Most current clinical and preclinical research on systemically administered LNP therapeutics targets liver hepatocytes or liver-related diseases⁴. To broaden their use in systemic therapies, such as for immunomodulation by targeting the spleen, it is crucial to efficiently deliver nucleic acid cargos beyond the liver⁵. Previously explored strategies for this purpose include adjusting the mRNA-to-cationic lipids ratio, adding additional excipient lipid molecules, and attaching targeting ligands to the LNP surface^6-8. However, these strategies have inherent limitations, including the need for extra lipid excipients, reduced transfection efficiency, decreased encapsulation and stability, and difficulties in clinical translation. Therefore, there is an unmet need to develop novel LNP formulations that leverage the existing formulation scheme to mediate extrahepatic mRNA expression.

Methods: To address this need, we constructed a library of 162 dendrimer-based ionizable lipids and formulated them with mRNA to form dendrimer-based lipid nanoparticles (DLNPs) for screening. Dendrimers have broad theoretical space for flexible structural tuning, similar to polydisperse polymers, yet they exhibit high molecular uniformity comparable to small molecules⁹. These features have allowed us to analyze the impact of dendrimer structural properties on the performance of mRNA-DLNPs. Therefore, we modified dendrimer initiator cores, generations, lipid types, and number of lipid tails and internal amines. We then performed in vitro screening on murine-derived RAW 264.7 macrophages for transfection efficiency and cell viability and in vivo evaluation of organ-selective mRNA expression using B6 albino mice via tail vein injection. We also analyzed the physicochemical properties of DLNPs with a series of techniques, including dynamic light scattering for particle size, zeta potential, and polydispersity index; RiboGreen binding assay for encapsulation efficiency; TNS fluorescent for apparent pKa; and negative stain electron microscopy for morphology. We then analyzed the structure-function relationship among internal amines, physicochemical properties, and organ-selectivity of mRNA expression.

Results: Our established library of dendrimer-based lipids, comprising 162 compounds, has been constructed as shown in Figure 1. Starting with 6 initiator cores, which we referred to as generation 0 (G0) dendrimers, we expanded them through a two-step reaction to two additional generations (G1 and G2), obtaining a total of 18 dendrimers¹⁰. These 18 dendrimers contain primary (1º) and tertiary (3º) amine functional groups and initially have no lipid modification (0% lipid conjugation). We then selectively modified the 1º amines of these dendrimers with four different types of lipid-terminated epoxides to yield dendrimer-based lipids¹¹. Consequently, we obtained 72 dendrimer-based lipids that contain both secondary (2º) amines and 3º amines (representing 50% lipid conjugation), and an additional 72 dendrimer-based lipids featuring exclusively 3º amines (representing 100% lipid conjugation). We identified 12 DLNPs with superior in vitrotransfection efficiency comparable to FDA-approved LNPs and these formulations are exclusively DLNPs derived from 50% and 100% lipid conjugated dendrimer-lipids. Further in vivo study identified five DLNP formulations with liver-selective luciferase mRNA expression and four DLNP formulations with spleen-selective mRNA expression (Figure 2). Subsequent analyses of physicochemical properties revealed a correlation between the type of amine functional group, the apparent pKa of DLNPs, and the percentage of mRNA expression in different organs (Figure 3A). Our results suggest that DLNPs derived from dendrimer-lipids containing both 2º amines and 3º amines (50% lipid conjugation) tend to exhibit a higher apparent pKa and predominantly accumulate in the spleen following systemic administration. Conversely, DLNPs from dendrimer-lipids containing only 3º amines (100% lipid conjugation) display slightly lower apparent pKa values and preferentially accumulate in the liver (Figure 3B).

Conclusion: This study established a structure-function relationship between different internal amine types of dendrimer-based ionizable lipids and their organ selectivity for RNA delivery in vivo, while simultaneously aiming to unravel the underlying mechanisms by analyzing plasma proteins adsorbed to DLNP surfaces using mass spectrometry. This study will provide guiding principles for the rational design of LNPs focused on extrahepatic targeting upon systemic administration. When implemented in clinical settings, this knowledge will facilitate the organ-selective delivery of therapeutic RNAs, presenting a new strategy for the development of LNP therapeutics.

References: 1. Dilliard SA, Cheng Q, Siegwart DJ. On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proceedings of the National Academy of Sciences 2021; 118(52): e2109256118.
2. Adams D, Gonzalez-Duarte A, O’Riordan WD, et al. Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. New England Journal of Medicine 2018; 379(1): 11-21.
3. Kristen AV, Ajroud-Driss S, Conceição I, Gorevic P, Kyriakides T, Obici L. Patisiran, an RNAi therapeutic for the treatment of hereditary transthyretin-mediated amyloidosis. Neurodegenerative Disease Management 2019; 9(1): 5-23.
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8. Smith TT, Stephan SB, Moffett HF, et al. In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers. Nature Nanotechnology 2017; 12(8): 813-20.
9. Zhou K, Nguyen LH, Miller JB, et al. Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proceedings of the National Academy of Sciences 2016; 113(3): 520-5.
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Acknowledgements: This project is supported by American Cancer Society Institutional Research Grant (IRG202203 Zhang), Ocala Royal Dames for Cancer Research (AGR DTD 06152022) and National Cancer Institute (1R21CA277263-01). The authors declare no conflict of interests.


Figure 1: Schematic illustration of constructing the dendrimer-based ionizable lipids library.


Figure 2: Representative pie charts for the organ-specificity of FLuc mRNA expression mediated by two distinct dendrimer-based lipids (0.5 mg/kg, 24 h post-injection, n = 3)


Figure 3: (A) The performance metrics (in vitro transfection efficiency, cell viability, and encapsulation efficiency) of DLNPs grouped by internal amine types. (B) Pearson Correlation between DLNP apparent pKa (measured by TNS fluorescent assay, n = 5) and luciferase mRNA expression in spleen and liver (0.5 mg/kg, measured by IVIS 24 h post-injection, n = 3).