(M1330-02-06) Establishing Cellular Mechanisms Underlying Species-Related Differences in the Effective Delivery of LNP-mRNA to Overcome Limitations of Human Dose Translation

Purpose: Pharmacological evaluation of lipid nanoparticle-mRNA (LNP-mRNA) therapeutics relies on time- and resource-intensive in vivo studies in rodents and non-human primates, and little is known about how well the mechanisms of LNP mediated mRNA delivery are conserved across species. Importantly, it remains unclear if outcomes in preclinical models provide a reliable basis for the projection of potency and dose-response of LNP-mRNA therapies in humans. We hypothesized that species-related differences in functional delivery of LNP-mRNA could be recapitulated and mechanistically explained using physiologically relevant in vitro cellular systems derived from organs of biotherapeutic interest, such as the liver.

Methods: To compare LNP-mediated transfection efficiency in pre-clinical species and humans, we employed primary hepatocytes isolated from rat, cynomolgus monkey (cyno) and human livers. We adopted a “sandwich-culture” technique to maintain hepatocyte viability and ameliorate cell metabolic remodeling with time in culture (up to 3 days). To investigate the potential species-dependence of cellular processes governing LNP-mRNA transfection, LNPs were formulated, in addition to phospholipids, cholesterol and a helper lipid, with D-Lin-MC3-DMA (MC3) ionizable lipid, due to a documented role of this ionizable lipid in hepatic LNP delivery. Luciferase-encoding mRNA was encapsulated in the LNPs (MC3-Luciferase LNPs) since luciferase is commonly used in preclinical studies for characterizing transgene delivery. For quantification of transgene expression level in primary hepatocytes treated with MC3-Luciferase LNPs, we developed a calibration approach for correlating relative units of luminescence generated by luciferase enzymatic activity to luciferase protein concentration in cell lysates. The luciferase measurements in hepatocytes were then normalized to the cell metabolic activity, determined using an AlamarBlue™ assay. To assess LNP binding and internalization in species hepatocytes using flow- and imaging flow- cytometry, fluorescently labeled MC3-Luciferase LNPs were generated by incorporating rhodamine-labeled cholesterol (structural lipid) into particles’ formulation.

Results: Doses for MC3-Luciferase LNP for in vitro transfection were selected to approximate typical in vivo exposure range, estimated as LNP-mRNA quantity per hepatocyte. Measurements of transgene level after LNP treatment of rat, cyno and human hepatocytes showed a linear dose-response in luciferase expression across all three species, however, the magnitude and time kinetics of expression varied as follows: In human and rat hepatocytes, luciferase protein was expressed at a comparably high level with biphasic kinetics, whereas only minimal luciferase signal was detected in cyno hepatocytes (Fig. 1). Additionally, in human and, to a lesser extent, cyno hepatocytes, donor-to-donor variability in the efficiency of LNP transfection was observed. Phenotypic differences in primary cells from different individuals are anticipated and, if better understood, might inform on the biological pathways impacting LNP interactions with target cells. Investigation of the precursory steps for effective LNP delivery uncovered species-dependence in cellular processes governing LNP-mRNA binding and internalization in primary hepatocytes. Specifically, following addition of LNPs, both the frequency of cells exhibiting membrane-bound LNPs and the relative amount of bound LNPs per cell, varied substantially between species hepatocytes (Fig. 2). Surprisingly, there was no apparent correlation between the propensity of LNP binding to cells and efficiency of transgene expression in hepatocytes from the examined species (Figs.1 and 2). Cyno hepatocytes exhibited the highest magnitude of MC3-Luciferase LNP membrane binding, yet the luciferase transgene level was lowest, as compared to human and rat cells treated with the same LNP dose. These data suggest that LNP binding capacity was not translatable to the efficacy of transgene delivery in species hepatocytes, pointing to the need of characterizing downstream cellular pathways governing LNP transfection. Preliminary experiments comparing LNP-mRNA uptake in hepatocytes from different species indicated that rat and human cells exhibited a greater potential, compared to cyno cells, for the internalization of MC3-Luciferase LNPs from the membrane into the cytosol (Fig. 3).

Conclusion: Our data, obtained by comparing primary hepatocytes from rodents (rat), non-human primates (cyno) and humans, show that the efficiency of LNP-mRNA transfection (dose- and time- dependency) substantially varied across species, and provide new evidence that cell-intrinsic mechanisms, such as differential binding and intracellular uptake, play a critical role in LNP delivery in pre-clinical species and humans. We believe these studies in physiologically relevant cellular systems, can generate key information in support of in vitro-to-in vivo extrapolation modeling aiming to fill a critical gap in the clinical translation of preclinical efficacy and dose potency of LNP therapies.

Acknowledgements: All authors are employees of Pfizer, Inc. and might hold equity in the company.

Fig. 1: Specie-specific difference in transgene expression in MC3-Luciferase LNP-transfected human, cyno and rat hepatocytes. Cells were treated with different doses of MC3-Luciferase mRNA LNPs for the indicated time periods, as specified in the figure. Data shown as mean and SD of technical duplicates. *, below lower limit of detection and #, below lower limit of quantification for this assay. Each row represents a different donor (lot of cells).

Fig. 2: Comparison of dose-dependent LNP binding in hepatocytes from different species. Cells were incubated with increasing doses of fluorescently-tagged MC3-Luciferase LNPs, as specified in the figure, for 60 min on wet ice to allow binding and prevent internalization. Frequency of LNP bound cells (A) and relative amount of bound LNP per cell (B) of the labeled LNP signal was analyzed by flow cytometry.

Fig. 3: LNP intracellular uptake in species hepatocytes. Representative images acquired with an imaging flow-cytometer, depicting membrane and intracellular distribution of fluorescently-tagged MC3-Luciferase LNPs (red) after incubation with cells at 2 pg/cell dose for 120 min at 37°C to allow particle internalization. Note a more prominent LNP signal on the cell membrane (arrow) in cyno hepatocytes.