Development of Acid-Degradable Lipid Nanoparticles for mRNA Delivery to the Heart

Introduction: Although heart failure remains a leading cause of death globally, advancements in heart therapeutics have been limited. Recent advances in mRNA-based therapies demonstrate promise for treating post-infarct heart failure. However, delivering these therapies to heart tissue is difficult, due to the nonproliferative nature of cardiomyocytes and the diffusional barriers presented by the extracellular matrix of dense cardiac muscle. Recent developments in non-viral drug delivery systems, such as mRNA-lipid nanoparticles (LNPs), offer potential solutions to these challenges; yet, they still face several significant hurdles. The positive charge of cationic lipids used to form mRNA-LNPs can activate pro-apoptotic and pro-inflammatory pathways, leading to toxicity. Endosomal escape of therapeutic cargo represents an additional difficulty in delivering mRNA into the cytosol for effective protein translation. Finally, diffusion of LNPs in heart muscle is poor. In this work, we synthesized novel acid-degradable lipids to create LNPs that take advantage of the acidic environment within endosomes to promote cargo delivery (Fig. 1A, B). To identify acid-degradable lipid compositions that successfully transfect cardiomyocytes, we implemented a high throughput heuristic differential evolutionary algorithm to screen LNPs in 2D monolayers of hiPSC-cardiomyocytes (iCMs) derived from a genetically modified hiPSC line (XR1 hiPSC) containing an eGFP gene downstream of a STOP cassette, which is removed upon CRE recombinase mRNA delivery. Lastly, gene editing efficiency and spatial diffusion of best performing formulations identified in the screening were then evaluated in 3D cardiac micromuscles within a cardiac microphysiological system (MPS) that previously demonstrated the ability to predict LNP transfection in vivo.

Materials and

Methods: Three new acid-degradable lipids containing an azido acetal linkage were synthesized: 1) a cationic lipid, 2) an anionic lipid, and 3) a PEG-lipid (Fig. 1A). Using these acid-degradable lipids and two commercial helper lipids to develop a 5-component LNP chemistry, we aimed to engineer LNPs that match or improve upon the current chemistry previously optimized in our lab, which utilizes 10% 2kDa acid-degradable PEG-lipid (ADP) and four other commercial lipids. To explore the parametric space in an efficient manner and design an optimized LNP delivery system, we implemented a synergistic coupling of a heuristic differential evolutionary algorithm (Fig. 1C). Formulations suggested by the algorithm were first screened in 2D iCM monolayers to assess transfection efficiency via LNP delivery of CRE recombinase mRNA. The %eGFP+ cells were quantified from images of the XR1-iCM monolayers using nuclear segmentation analysis. We selected the best candidates to study their diffusion and gene editing in the 3D cardiac MPS. LNPs were delivered to microtissues composed of 80% XR1-iCMs and 20% XR1 hiPSC-derived cardiac fibroblasts (iCFbs) via the microfluidic media channels within the MPS overnight, where they diffused through the endothelial-like barrier that connects the media channel and tissue chambers. Volumetric nuclear segmentation based on the DAPI signal was used to measure %eGFP+ cells. We determined spatial diffusion of each LNP formulation by determining the number of eGFP+ cells in the center of the cardiac tissue, compared to the number of eGFP+ cells along the edges of the micromuscles.

Results, Conclusions, and Discussions: Using the differential evolutionary algorithm, we screened a total of 180 LNP formulations, equivalent to nearly 8,000 formulations in a traditional parametric analysis. From this high throughput 2D culture screening, we discovered 30 formulations with a transfection efficiency within 10% of the eGFP expression for the 2kDa 10% ADP LNP formulation (Fig. 1D). Our novel lipids either hydrolyze in the endosome to promote fusion of the LNP with the endosomal membrane or generate neutral lipids in the endosomes to decrease toxicity to cells. However, no correlation was observed regarding the molar ratio of acid-degradable components among the best performing formulations. The best 15 formulations were then tested in cardiac micromuscles using the cardiac MPS, where we discovered at least one formulation with improved performance compared to the 2kDa 10% ADP LNP (Fig. 1E). Based upon measurements of tissue contractility following LNP delivery, we showed that acid-degradable LNPs were able to successfully deliver mRNA into and edit the cardiac micromuscles without any significant signs of toxicity or changes in beat metrics. We also observed that the LNP screening in 2D iCM monolayers failed to predict the results observed in the MPS, which suggests that monolayers were unable to provide an evaluation of the spatial diffusion required for robust drug delivery. Collectively, our work shows that the cardiac MPS is a good preclinical model for in vitro screening of LNPs in dense micromuscles, and that we successfully identified a safe LNP formulation with improved efficiency compared previously best performing LNP that previously demonstrated robust in vivo mRNA delivery to the heart with lower off-target levels of liver uptake than other LNPs. Specifically, we identified a densely PEGylated LNP formulation which contains novel acid-degradable lipids with improved diffusivity and functionality upon mRNA delivery into the human heart tissue. Our workflow relied on a non-animal model screening system taking advantage of a phenotypic tissue-level cardiac microfluidic device with genetically encoded human cardiomyocytes. This work will contribute to the progress of new cardiac therapies, and it will stimulate the generation of more advanced non-animal models as preclinical platforms in the mRNA-LNP discovery landscape.

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