(T1430-04-20) In Situ Engineering of Tumor-Infiltrating Myeloid Cells Through Dendrimer-Based Lipid Nanoparticles

Tuesday, October 22, 2024

2:30 PM – 3:30 PM MT

Presenting Author(s)

KJ

Kevon Jolly, BS

PhD Student/Graduate Assistant
University of Florida
Gainesville, Florida, United States

Main Author(s)

KJ

Kevon Jolly, BS

PhD Student/Graduate Assistant
University of Florida
Gainesville, Florida, United States

Co-Author(s)

Zijing Xu, M.S.

PhD Candidate/Graduate Assistant
University of Florida College of Pharmacy
Gainesville, Florida, United States
OG

Owen Gillen, BS

Research Volunteer
University of Florida
Gainesville, Florida, United States
Fan Zhang, PhD

Assistant Professor
University of Florida, United States
CS

Chenikkayala Siva Sankara, Ph.D.

Postdoctoral Researcher
University of Florida
Gainesville, Florida, United States

Purpose: In situ programming of immune cells using in vitro-transcribed therapeutic mRNA (IVT-mRNA) is currently under translation as an emerging approach to treat cancer and immune-related disorders¹. A critical challenge with this approach is how to selectively deliver RNAs to organs where target cells reside². For instance, macrophages have emerged as a prominent cellular target 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 therapy². 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 library 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 Transfection Efficiency Assay: RAW 264.7 cells were seeded into 96-well plates at a density of 40,000 cells/well in complete media two days prior to transfection. Before transfection, complete medium (DMEM supplied with 10% FBS) was replaced with 100μL basal medium (DMEM without FBS). The DLNPs prepared were pipetted into DMEM medium at 0.2μg mRNA/well. A 3-hour incubation was followed to allow sufficient DLNP uptake. Cells were then washed with DMEM, and 200μl complete medium was added to each well. A 24-hour incubation was followed to allow sufficient mRNA expression. When characterizing luciferase as the reporter, cells were incubated for 10 minutes in 100μl luciferin assay solution (150μg/mL concentration, PerkinElmer) and the bioluminescence measured by luminometer. In vitro Cell Viability Assay: The DLNP transfection procedures follow the same protocol described in the In Vitro Transfection Efficiency Assay. To evaluate the toxicity of DLNPs, DLNP-containing media was aspirated, and cells were incubated for 3 hours in CellTiter Blue reagent (Promega). The fluorescence signal was measured via a plate reader and was then converted to percent cell viability. Cells being cultured in basal media for 3 hours but did not receive DLNP-treatment were used as the negative control (100% viability). Cells that were exposed to 0.1μL of TritonX-100 treatment for 3 hours were used as the positive control (0% viability). In vivo Bioluminescence Imaging: All animal studies were approved by the University of Florida Institutional Animal Care and Use Committee and were consistent with local, state, and federal regulations as applicable. B6(Cg)-Tyrc-2J/J mice (Jackson Laboratories, 20-25g) were intravenously injected with DLNPs via tail vein at the dose of 0.5 mg/kg mRNA). After 24 hours, mice were administered an intraperitoneal injection of 10μL/g D-luciferin (15 mg/ml in DPBS). After 15 min, the mice were euthanized and perfused to remove DLNPs in the blood, and six organs were collected (lung, liver, spleen, heart, femur, and kidneys). The luminescence signal of each organ was analyzed using an IVIS imaging system (PerkinElmer IVIS Spectrum) and quantified using LivingImage software (PerkinElmer). The in vivo transfection efficiency of each DLNP in these organs was quantified using total flux (p/s).

Results: In vitro DLNP library screening on a murine macrophage cell line has identified 12 lead formulations, 9 novel, with comparable or superior transfection efficacy and safety profile to the clinically relevant formulations (Fig. 1). An in vivo biodistribution study of these identified DLNPs has revealed 5 lead formulations suggest tropism for myeloid cell tissue reservoirs, such as the spleen. Importantly, our results suggest a structure-function relation between dendrimer internal amines and their selective in vivo targeting, with spleen-targeting DLNPs containing internal secondary amine structures while other liver-targeting DLNPs contain solely tertiary amines (Fig. 2). We have also identified two DLNP formulations with superior targeting and transfection efficiency compared to clinically-relevant SM-102 and selective organ targeting (SORT)-lipid modified DLin-MC3-DMA (Fig. 3).

Conclusion: Our results lay the foundation for a structure-function relation between dendrimer internal amine structure and selective in vivo targeting, with spleen-targeting DLNPs containing internal secondary amine structures while other liver-targeting DLNPs contain solely tertiary amines. These findings inform rational nanoformulation design, offering insights into fine-tuning the organ retention of DLNPs. If clinically translated, our delivery platform may be used for targeted therapeutic RNA delivery to spleens for immune-related disorder treatment. Future Directions: Our ongoing experiments employ flow cytometry analysis to identify in vivo engineered-cell subpopulations in a GL261 glioma-bearing Ai14 mouse model. Interestingly, these early results suggest that within the spleen, our secondary-amine formulations target terminally differentiated myeloid subsets, such as macrophages and dendritic cells. However, our tertiary-amine formulations target more immature myeloid subsets, such as monocytes and potential MDSCs. These differences highlight potential applications, where our secondary-amine formulations would be ideal for cancer vaccine application, and our tertiary-amine formulations ideal for targeting tumor-infiltrating myeloid cells.

References: 1. Sloas, Christopher, Saar Gill, and Michael Klichinsky. "Engineered CAR-macrophages as adoptive immunotherapies for solid tumors." Frontiers in immunology 12 (2021): 783305.
2. 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.
3. Klichinsky, M.; Ruella, M.; Shestova, O.; Best, A.; Blouch, K.; Lu, X. Q. M.; Kenderian, S. S.; Kim, M. Y.; O'Connor, R.; Wallace, S.; Kozlowski, M.; Marchione, D. M.; Shestov, M.; Garcia, B. A.; June, C.; Gill, S., Human chimeric antigen receptor (CAR) macrophages for cancer immunotherapy. Cancer Immunology Research 2020, 8 (4), 29-30.
4. Dong, Simon Xin Min, 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.

Acknowledgements: This work was supported by funding from the 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 interest.

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

Figure 2. Amine structure and apparent pKa determine organ-selectivity of DLNPs. (A) Organ-selectivity of intravenously infused DLNP formulations 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 spleen-targeting and liver-targeting DLNP formulations and their correlation with %organ-expression (n=3).

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).