Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases

Abstract

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.

Keywords: Heart, lung, blood and sleep (HLBS) diseases; extracellular vesicles (EVs); therapeutics and diagnostics.

FIGURE 1

Heterogenous populations of EVs and non‐vesicular extracellular particles. Based on the biogenesis pathways, EVs can be classified into two basic categories, including exosomes and ectosomes. Exosomes are vesicles ranging ∼40–160 nm in diameter generated by the endocytic pathway and released upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. Ectosomes are released by the outward budding of the plasma membrane and include migrasomes, microvesicles, exophers, apoptotic bodies, large oncosomes and others, in the size range of ∼50 nm–∼5 μm. Non‐vesicular extracellular particles are non‐membranous complexes of proteins and nucleic acids with a diameter of less than 80 nm, that include exomeres, supermeres, chromatimeres, lipoproteins and several others. The mechanisms of non‐vesicular extracellular particle biogenesis are unknown, and such particles were not the focus of this workshop. All the above groups may overlap in size

FIGURE 2

Strategies for engineering therapeutic EVs. Both EV membrane and cargo can be engineered either endogenously or exogenously for therapeutical applications. Endogenous EV engineering refers to modulating EV‐secreting (parent) cells by exposing them to stress‐induced conditions or transfecting these parent cells with exogenous compounds, such as nucleic acids, small molecules, lipids and proteins. Exogenous EV engineering is based on the modifications of isolated EVs that include exploiting the hydrophobicity of EV membranes to carry a cargo of interest on the EV surface or permeabilizing the EV membranes using approaches, such as electroporation, freeze‐thaw procedures, sonication, surfactant treatment, and chemical transfection to carry the cargo of interest as the luminal cargo in EVs. EV membranes can also be used to encapsulate cargo‐carrying nanoparticles (NPs) or EVs can be fused to cargo‐carrying lipid nanoparticles (LNPs)