Skip to main content
2023 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

    Drug Delivery

    Ionizable Lipid Nanoparticles for mRNA Delivery to Human T Cells for Enforced Homing in Bone Marrow

    (L-466) Ionizable Lipid Nanoparticles for mRNA Delivery to Human T Cells for Enforced Homing in Bone Marrow

    Saturday, October 14, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row L - Poster # 466
    Introduction::

    T cell-based immunotherapies, such as chimeric antigen receptor (CAR) T cell therapy, have emerged as potential cancer treatments given their ability to specifically target tumor cells and avoid systemic toxicities of conventional chemotherapies [1]. However, general applicability of this therapeutic approach is limited by the required amount of T cells to achieve therapeutic response and systemic toxicities that result from dose-related off-target dissemination of these cytotoxic lymphocytes [2]. Thus, it is critical to devise strategies to improve tumor site-specific colonization of these “live drugs,” thereby improving their cancer-killing efficacy while reducing off-target effects. Hematologic malignancies typically benefit from the bone marrow microenvironment, where they colonize and migrate to upon progression. In the bone marrow niche, the endothelial cells lining the blood vessels constitutively express the adhesion molecule E-selectin, previously shown to interact with cancer cells via the tetrasaccharide glycan sialyl Lewis-X (sLeX) complex [3]. However, there is evidence showing that T cells do not express sLeX and do not bind to E-selectin [4]. Thus, it is hypothesized that engineering T cells to transiently express sLeX on their surface would enhance their colonization to the bone marrow. Here, we utilized a lipid nanoparticle (LNP) platform for the delivery of human fucusyltransferase 6 (FUT6) mRNA, a gene known to synthesize sLeX, to enhance the expression of sLeX on the T cell surface for improved colonization to the bone marrow. We believe this therapeutic strategy has the potential to enhance CAR T therapies, particularly those against bone marrow-homing cancers.

    Sub-track: Nucleic Acid Delivery



    Materials and Methods::

    The mRNA LNP platform was synthesized by combining an aqueous phase containing mRNA with an ethanol phase containing all lipid components via microfluidic mixing. The aqueous phase was composed of citrate buffer and mRNA, while the ethanol phase contained the ionizable lipid, DOPE, cholesterol, and lipid-anchored polyethylene glycol at the respective molar ratios of 40:30:25:2.5 [5]. The resultant LNPs were dialyzed against PBS for 2 hrs before sterilization with 0.22 um filters.


     


    To measure size, the LNPs were suspended in PBS and analyzed using dynamic light scattering performed on a Zetasizer Nano. The diameter (z-average) and polydispersity index (PDI) of the LNPs were measured in triplicate. The mRNA concentration of the LNPs was measured via A260 absorbance on an Infinite M Plex plate reader. To determine the pKa values of the LNPs, 6-(ptoluidinyl) naphthalene-2-sulfonic acid (TNS) assays were run, and the value was calculated as the pH at which the fluorescence intensity reached 50% of its maximum value.


     


    For cell studies, primary human CD4+ and CD8+ T cells were activated with CD3 and CD28 Dynabeads for 24 hrs. Cells were then plated in triplicate at 1,000,000 cells per 1000 uL in 6-well plates and immediately treated with a dose of 100 ng of mRNA per 100 uL via LNPs diluted in PBS or a positive control. To measure sLeX expression, T cells were stained using an anti-human HECA452 antibody conjugated to FITC. T cells were then washed, and the surface expression of sLeX was evaluated on a flow cytometer.



    Results, Conclusions, and Discussions::

    After formulation of the LNPs, these were characterized for their size, surface charge, mRNA encapsulation, and pKa. The hydrodynamic diameter obtained was about 140 nm with a surface charge of -7 mV, values that have been observed in previous work [5]. The pKa values of the LNPs, the pH at which LNPs are 50% protonated, reflect their ability to change charge in acidic environments to facilitate endosomal escape. In this work, the pKa value measured was about 5.5, confirming the ionizable properties of the LNPs (pKa < 7.2).


     


    To evaluate the efficacy and safety of the LNPs in delivering mRNA, we activated primary human CD4+/CD8+ T cells using CD3 and CD28 Dynabeads for 24 hrs. Following incubation, T cells were plated and immediately treated with LNPs encapsulating FUT6 mRNA. After 24, 48, and 72 hrs of treatment, T cells were washed and stained with HECA452 antibody and Sytox Red Stain to detect the surface expression of sLeX and evaluate cytotoxic effects, respectively, using flow cytometry. Our results show that the most effective T cell transfection using our FUT6 mRNA LNPs at a concentration of 100 ng of mRNA per uL occurs 48 hrs post-treatment. Further, no cytotoxic effects related to the LNP treatment were observed for any of the different time points. In future work, we will evaluate the abilities of these engineered T cells to adhere to bone marrow endothelial cells in vitro, and assess the efficacy of this therapeutic strategy to augment T and CAR T cell infiltration in bone marrow in vivo. We expect these findings will provide a novel approach that could be translated to CAR T cell therapies to enhance their homing to E-selectin-bearing tumor sites such as bone marrow. We believe this strategy has the potential to boost T cell activity to achieve a faster therapeutic response, ultimately improving T cell-based immunotherapies.

    Acknowledgements (Optional): :

    References (Optional): :

    [1] Mai D, et al. PNAS, 2023


    [2] Thomas S, et al. Cell Reports, 2023


    [3] Figueroa-Espada CG, et al. Cell. Mol. Bioeng., 2023


    [4] Mondal N, et al. J. Biol. Chem., 2019


    [5] Billingsley MM, et al. Nano Letters, 2022