To optimize mRNA delivery to monocytes in vivo, Mukalel et al. generated oxidized (o) and unoxidized (u) LNPs. Compared to uLNPs, oLNPs differed in charge and morphology, improved mRNA delivery to macrophages in vitro, and had better splenic biodistribution in vivo. An LNP library carrying barcoded mCherry mRNA was screened in vivo, identifying specific LNP formulations that enriched mCherry expression in monocytes. One of these oLNPs was used in vivo to deliver anti-CD19 CAR mRNA, resulting in CAR expression in monocytes (~2%) moreso than in lymphocytes (~1%), and CD19+ B cell depletion.
Contributed by Alex Najibi
ABSTRACT: Chimeric antigen receptor (CAR) monocyte and macrophage therapies are promising solid tumor immunotherapies that can overcome the challenges facing conventional CAR T cell therapy. mRNA lipid nanoparticles (mRNA-LNPs) offer a viable platform for in situ engineering of CAR monocytes with transient and tunable CAR expression to reduce off-tumor toxicity and streamline cell manufacturing. However, identifying LNPs with monocyte tropism and intracellular delivery potency is difficult using traditional screening techniques. Here, ionizable lipid design and high-throughput in vivo screening are utilized to identify a new class of oxidized LNPs with innate tropism and mRNA delivery to monocytes. A library of oxidized (oLNPs) and unoxidized LNPs (uLNPs) is synthesized to evaluate mRNA delivery to immune cells. oLNPs demonstrate notable differences in morphology, ionization energy, and pKa, therefore enhancing delivery to human macrophages, but not T cells. Subsequently, in vivo library screening with DNA barcodes identifies an oLNP formulation, C14-O2, with innate tropism to monocytes. In a proof-of-concept study, the C14-O2 LNP is used to engineer functional CD19-CAR monocytes in situ for robust B cell aplasia (45%) in healthy mice. This work highlights the utility of oxidized LNPs as a promising platform for engineering CAR macrophages/monocytes for solid tumor CAR monocyte therapy.
Author Info: (1) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. (2) Department of Bioengineering, University of Pennsylvania, Philade
Author Info: (1) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. (2) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. (3) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. (4) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. (5) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. (6) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. (7) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. (8) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. (9) Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. (10) Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. (11) Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. (12) Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
