Extracellular vesicles (EVs), also known as exosomes, are nanosized membrane-bound vesicles that are secreted by various types of cells. They can carry various biomolecules, such as proteins, lipids, and nucleic acids, and mediate intercellular communication and material exchange. EVs have been recognized as promising vehicles for mRNA delivery due to their low immunogenicity, high biocompatibility, ability to cross biological barriers, and natural targeting specificity. In addition, EVs can encapsulate mRNA molecules and protect them from degradation while facilitating their delivery and release into the recipient cells.
EVs can carry mRNA molecules from the donor cells to the recipient cells, where they can be translated into proteins and modulate cellular functions. However, the mechanisms of mRNA origin, encapsulation, and release in EVs are not fully understood. Several hypotheses have been proposed to explain how mRNA molecules are sorted and packaged into EVs, such as passive diffusion, active sorting, and membrane invagination.
Fig.1 mRNA-encapsulated exosomes. 1
One hypothesis is that mRNA molecules are passively diffused into EVs by random chance, depending on their abundance and localization in the cytoplasm. This hypothesis implies that the mRNA profile in EVs reflects the mRNA profile in the donor cells and that there is no specific selection or regulation of mRNA sorting into EVs. However, this hypothesis is challenged by the evidence that the mRNA profile in EVs is different from the mRNA profile in the donor cells, and that some mRNA molecules are preferentially enriched or excluded in EVs.
Another hypothesis is that mRNA molecules are actively sorted into EVs by specific RBPs or miRNAs, which recognize and bind to the mRNA molecules and direct them to the EV biogenesis sites. This hypothesis implies that the mRNA profile in EVs is determined by the expression and activity of RBPs and miRNAs in the donor cells, and that there is a specific selection and regulation of mRNA sorting into EVs. Several RBPs and miRNAs have been identified as potential regulators of mRNA sorting into EVs, such as HNRNPA2B1, ELAVL1, AGO2, and miR-128.
A third hypothesis is that mRNA molecules are encapsulated into EVs by membrane invagination, which involves the formation of intraluminal vesicles (ILVs) inside the multivesicular bodies (MVBs) or the plasma membrane. This hypothesis implies that the mRNA profile in EVs is influenced by the membrane composition and curvature, and that there is a physical constraint and regulation of mRNA encapsulation into EVs. Several membrane-associated proteins and lipids have been implicated in the membrane invagination process, such as ALIX, TSG101, CD63, and ceramide.
The release of mRNA-containing EVs from the donor cells can occur by two main pathways—the exosomal pathway and the microvesicular pathway. The exosomal pathway involves the fusion of MVBs with the plasma membrane, which releases the ILVs as exosomes into the extracellular space. The microvesicular pathway involves the budding of vesicles directly from the plasma membrane, which releases the vesicles as microvesicles into the extracellular space. The release of EVs can be regulated by various factors, such as cellular stress, hypoxia, inflammation, and signaling molecules.
mRNA molecules carried by EVs can exert various biological functions in the recipient cells, depending on the type, amount, and sequence of the mRNA, as well as the cell type, state, and environment of the recipient cells. mRNA in EVs can be translated into proteins in the recipient cells, which can modulate cellular functions and phenotypes. Alternatively, mRNA in EVs can interact with other RNAs or proteins in the recipient cells, which can affect gene expression and regulation. mRNA in EVs can also induce immune responses in the recipient cells, which can influence inflammation and immunity.
The biological functions of mRNA in EVs have been demonstrated in both normal physiological and pathological conditions, especially in cancer. EVs carrying mRNA can mediate various aspects of cancer progression and microenvironment remodeling, such as cell proliferation, migration, invasion, angiogenesis, hypoxia, immunosuppression, and metastasis. For example, EVs derived from breast cancer cells can transfer mRNA encoding HER2, a receptor tyrosine kinase that promotes cell growth and survival, to other breast cancer cells or normal cells, which can increase HER2 expression and confer resistance to HER2-targeted therapy. EVs derived from hypoxic prostate cancer cells can transfer mRNA encoding HIF-1α, a transcription factor that regulates the cellular response to hypoxia, to normoxic prostate cancer cells, which can enhance HIF-1α expression and activity and induce the expression of angiogenic factors. EVs derived from melanoma cells can transfer mRNA encoding PD-L1, a ligand that binds to the PD-1 receptor on T cells and inhibits their activation, to dendritic cells, which can suppress dendritic cell maturation and function and impair the anti-tumor immune response.
The biological functions of mRNA in EVs have also been explored for therapeutic applications, such as gene therapy, vaccine development, and tissue regeneration. EVs carrying mRNA can be used as natural and biocompatible vehicles to deliver the therapeutic gene to the target cells and tissues without causing adverse effects such as immunogenicity, toxicity, or integration into the host genome. For example, EVs derived from mesenchymal stem cells can transfer mRNA encoding CXCR4, a chemokine receptor that mediates the homing and migration of stem cells, to cardiac stem cells, which can improve cardiac stem cell engraftment and survival and enhance cardiac function after myocardial infarction. EVs derived from dendritic cells can transfer mRNA encoding tumor antigens, such as MAGE-3, to antigen-presenting cells, which can stimulate the antigen-specific T cell response and induce anti-tumor immunity. EVs derived from induced pluripotent stem cells can transfer mRNA encoding Oct4, Sox2, Klf4, and c-Myc, the transcription factors that induce the reprogramming of somatic cells into pluripotent stem cells, to fibroblasts, which can induce the generation of induced pluripotent stem cells and facilitate tissue regeneration.
mRNA in EVs has great potential for therapeutic applications, as it can deliver the desired gene sequence to the target cells and tissues and induce the expression of the therapeutic protein. mRNA in EVs can be used for gene therapy, vaccine development, and tissue regeneration, among other purposes. However, there are some challenges and limitations that need to be overcome, such as the low loading efficiency, the insufficient targeting specificity, and the possible immune reactions.
Fig.2 Exosome-mediated mRNA delivery for personalized medicine. 1
One of the challenges is to increase the loading efficiency of mRNA into EVs, as the natural encapsulation of mRNA into EVs is often low and variable. Several methods have been developed to enhance the loading efficiency of mRNA into EVs, such as electroporation, sonication, saponin, and transfection. Electroporation is a physical method that uses electric pulses to create pores in the EV membrane, allowing the mRNA to enter the EVs. Sonication is another physical method that uses ultrasound waves to disrupt the EV membrane, facilitating mRNA incorporation. Saponin is a chemical method that uses a natural detergent to permeabilize the EV membrane, enabling mRNA delivery. Transfection is a biological method that uses viral or non-viral vectors to introduce the mRNA into the EV-producing cells, which then secrete the mRNA-loaded EVs.
Another challenge is to improve the targeting specificity of mRNA-loaded EVs, as the natural tropism of EVs may not be sufficient or desirable for certain applications. Several strategies have been proposed to modify the surface of EVs, such as genetic engineering, chemical conjugation, and membrane fusion. Genetic engineering is a strategy that uses genetic manipulation to overexpress or insert specific ligands or receptors to the EV membrane, which can bind to the corresponding receptors or ligands on the target cells. Chemical conjugation is a strategy that uses chemical reactions to attach synthetic molecules or antibodies on the EV surface, which can recognize and interact with the target cells. Membrane fusion is a strategy that uses the fusion of EVs with other vesicles or cells that have the desired targeting molecules on their surface, which can transfer the targeting molecules to the EVs.
A third challenge is to reduce the immune reactions of mRNA-loaded EVs, as the mRNA may be recognized as foreign or viral by the immune system and trigger inflammatory or antiviral responses. Several approaches have been suggested to minimize the immune reactions of mRNA-loaded EVs, such as modifying the mRNA structure, coating the EV surface, and selecting the EV source. Modifying mRNA structure is an approach that uses chemical modifications or synthetic analogs to alter mRNA nucleotides, which can reduce the recognition and degradation by immune cells or enzymes. Coating the EV surface is an approach that uses biocompatible or stealth materials to cover the EV membrane, which can prevent the opsonization and clearance by the immune system. Selecting the EV source is an approach that uses autologous or allogeneic EVs from the same or similar species or tissues as the recipient, which can avoid immune rejection or incompatibility.
In summary, mRNA in EVs is a promising strategy for various therapeutic applications, but it also faces some challenges and limitations that need to be addressed. Future research should focus on optimizing the loading efficiency, targeting specificity, and immune compatibility of mRNA-loaded EVs, as well as evaluating their safety and efficacy in preclinical and clinical studies.
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