Background
In situ programming of immune cells using in vitro-transcribed therapeutic mRNA (IVT-mRNA) is currently under translation as an emerging approach for cancer and immune-related disorder treatment.1 A critical challenge with this approach is the selective delivery of RNAs to organs where target cells reside.2 For instance, macrophages are prominent cellular targets of anti-tumor immunotherapies due to their broad therapeutic effector functions.3 4 However, selective RNA delivery to lymphoid organs where macrophages reside poses a significant barrier to macrophage-reprogramming gene therapy development. Existing FDA-approved lipid nanoparticle (LNP) formulations primarily target the liver when administered systemically, limiting IVT-mRNA’s broad applicability as a systemic therapy.2 To address these challenges, we conducted a multi-tier library screening of 162 mRNA-nanoparticle formulations based on dendrimer-lipid hybrid materials (dLNPs). From this screening, we aim to identify nanoformulations with the greatest therapeutic potential by assessing macrophage transfection in vitro and nanoparticle biodistribution in vivo.
Methods
In vitro Assays: RAW 264.7 cells were seeded in 96-well plates (40,000 cells/well) in DMEM with 10% FBS, 48 hours before transfection. The medium was replaced with 100μL DMEM, and 0.2μg mRNA/well of dLNPs were added. After a 3-hour incubation, cells were washed, and 200μL complete medium was added. Transfection efficiency was evaluated 24 hours post-treatment following the PerkinElmer IVISbrite protocol and bioluminescence was measured with a luminometer. Toxicity was assessed using the Promega CellTiter Blue protocol. Fluorescence was measured via platereader and converted to percent cell viability. Ex vivo Imaging: All animal studies were IACUC-approved. B6(Cg)-Tyrc-2J/J mice received dLNPs via tail vein (0.5 mg/kg mRNA). After 24 hours, mice received 10μL/g D-luciferin (15 mg/ml). After 15 minutes, mice were euthanized, perfused, and target organs were collected. Luminescence was analyzed using an IVIS imaging system and quantified with LivingImage software.
Results
In vitro screening of a dLNP library identified 12 lead formulations (9 novel) with comparable or superior transfection efficacy and safety to clinically relevant formulations (figure 1). In vivo biodistribution studies revealed 5 formulations with tropism for myeloid cell tissue reservoirs, such as the spleen (figure 2). Spleen-targeting dLNPs contained internal secondary amines, while liver-targeting dLNPs contained solely tertiary amines. Two formulations showed superior targeting and transfection efficiency compared to clinically relevant SM-102 and SORT-lipid modified DLin-MC3-DMA (figure 3).
Conclusions
Our findings establish a structure-function relationship between dendrimer internal amine structures and selective in vivo targeting. These insights inform rational nanoformulation design, enhancing the potential for targeted RNA delivery to spleens for cancer and immune-related disorder treatment.
References
Sloas C, Saar G, Michael K. Engineered CAR-macrophages as adoptive immunotherapies for solid tumors. Frontiers in immunology 12 (2021): 783305.
Cheng, Qiang, et al. “Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing.” Nature nanotechnology 15;4(2020):313–320.
Klichinsky M, Ruella M, Shestova O, Best A, Blouch K, Lu XQM, Kenderian SS, Kim MY, O’Connor R, Wallace S, Kozlowski M, Marchione DM, Shestov M, Garcia BA, June C, Gill S, Human chimeric antigen receptor (CAR) macrophages for cancer immunotherapy. Cancer Immunology Research 2020;8(4):29–30 .
Dong, Simon XM, et al. Transfection of hard-to-transfect primary human macrophages with Bax siRNA to reverse Resveratrol-induced apoptosis. RNA biology 17;6(2020):755–764.
Abstract 1110 Figure 1
In vitro library screening yields 12 dendrimer-based lipid nanoparticle (DLNP) formulations with efficient macrophage transfection. A) Identification of 12 DLNPs (blue), 9 novel, with efficient murine macrophage transfection (n=4) and limited toxicity (n=3). Data compared to all controls (orange) and normalized to positive control SM-102
Abstract 1110 Figure 2
Amine structure and apparent pKa determine organ-selectivity of dLNPs. A)Organ-selectivity of i.v. infused dLNPs obtained via bioluminescent imaging, measured in total flux (p/s) (n=3). B)Representative IVIS images of spleen- and liver-targeting formulations emphasized in panel A. (C)Apparent pKa of dLNPs and%expression
Abstract 1110 Figure 3
Spleen-targeting dLNPs demonstrate superior targeting compared to status quo. (A) Unpaired t-test of SM-102 mRNA expression in the spleen compared to select dLNP formulations (n=3). (B) dLNP formulations (n=3) demonstrate higher spleen-selectivity compared to status quo SORT-DLin-MC3-DMA (n=2) and SM-102 (n=3)