Engineering Human ARMMs as Therapeutic Non-Viral Vehicles for in vivo Delivery of Genome Editing Payloads

Abstract

The ability to edit the human genome to treat previously incurable diseases or to modify previously undruggable targets has the potential to transform the lives of patients. However, delivery of genome editing machineries in vivo, and in a transient manner, continues to be a major challenge. We engineered human cell-derived ARRDC1-mediated microvesicles (ARMMs) as non-viral vehicles that mediate intracellular delivery of proteins, including DNA-modifying enzymes such as Cre recombinase and Cas9/guide RNA ribonucleoproteins (RNPs). ARMMs packaged multiple payloads per vesicle in our production system, yielding dose-dependent functional delivery of Cre or Cas9/gRNA RNP to a variety of cell types, including primary mouse and human cells. Oropharyngeal aspiration of ARMMs loaded with Cre led to biodistribution to alveolar macrophages (AMs), whereas intravenous administration predominantly to Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and splenocytes. Suprisingly, one administration of ARMMs loaded with Cas9 RNPs by oropharyngeal aspiration or intravenously resulted in efficient knockout of multiple genes in AMs or KCs, suggesting potential therapeutic utility. Through intravenous administration of ARMMs loaded with Cas9/NLRP3 gRNA we showed amelioration of drug-induced liver injury in a mouse model, supporting the use of ARMMs as human cell-derived therapeutic vehicles for genome editor delivery in vivo.