Reprogramming extracellular vesicles with engineered proteins

Journal Pre-proofs Reprogramming Extracellular Vesicles with Engineered Proteins Xiaojing Shi, Qinqin Cheng, Yong Zhang PII: DOI: Reference:

S1046-2023(19)30217-8 https://doi.org/10.1016/j.ymeth.2019.09.017 YMETH 4804

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Please cite this article as: X. Shi, Q. Cheng, Y. Zhang, Reprogramming Extracellular Vesicles with Engineered Proteins, Methods (2019), doi: https://doi.org/10.1016/j.ymeth.2019.09.017

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Reprogramming Extracellular Vesicles with Engineered Proteins Xiaojing Shi1, Qinqin Cheng1, and Yong Zhang*,1,2,3,4 1Department

of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089 2Norris

Comprehensive Cancer Center, University of Southern California, Los Angeles, CA

90089 3Department

of Chemistry, Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA 90089 4Research

Center for Liver Diseases, University of Southern California, Los Angeles, CA 90089

*Corresponding

author. Email: [email protected]

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Abstract: Extracellular vesicles (EVs) have been emerging as a new class of cell-free therapy for the treatment of a variety of diseases, including cancer, tissue injuries, and inflammatory diseases. Reprograming native EVs by genetic engineering and other approaches offers an attractive prospect of extending therapeutic capabilities of EVs beyond their natural functions and properties. In this review article, we survey the state-of-the-art methods of EVs engineering and summarize major therapeutic applications of the reprogrammed EVs. Keywords: extracellular vesicles; protein engineering; fusion protein; drug delivery; cell-free therapy 1. Introduction Extracellular vesicles (EVs) are nanoscale particles released from host cells that are delimited by lipid bilayer membranes and incapable of replication1. Cells may secrete different types of EVs that all consist of cell-derived membranes with associated proteins and encapsulated cytosolic materials2. On the basis of their sizes and biogenic mechanisms, EVs can be categorized into three types, including (1) apoptotic bodies formed during apoptosis; (2) microvesicles generated through direct budding from plasma membranes; and (3) exosomes produced inside of endosomes with subsequent secretion2-7. Exosomes are derived from endosomes where endosomal membranes invaginate to form multiple smaller intraluminal vesicles inside of endosomes, resulting in subcellular structures called multivesicular bodies (MVBs). Upon fusing with plasma membranes, MVBs release the intraluminal vesicles to extracellular space. These released nanovesicles are thus named as exosomes5-7. Due to their distinct biogenesis routes, the three EVs differ in size, composition, and biological function. Apoptotic bodies are the largest EVs with a diameter of up to several micrometers and pack a considerable amount of cell debris resulted from apoptosis3,8. The sizes of microvesicles are between 50 nm to 1 μm and their compositions are much similar to the donor cells because of the direct membrane budding7,9,10. Diameters of exosomes are within the range of 30-150 nm. Since exosomes are generated from a highly regulated endosomal pathway, certain types of biomolecules, especially proteins associated with lysosomes, endosomes, and endosomal sorting complex required for transport (ESCRT) complexes, are enriched in exosomes from various types of cells and often are recognized as exosomal markers, such as CD9, CD63, CD81, Lamp2b, TSG101, and ALIX11,12. Given the fact that exosomes undergo two membrane invaginations, they are topologically equivalent to their donor cells, meaning that the orientations of membrane proteins on exosomes and exosome-producing cells are the same. This is very important for modifying exosomes through protein engineering, especially for displaying functional proteins and peptides on exosome surfaces. Since there exists significant overlaps among EV populations concerning size, density, and composition, making it difficult to isolate and characterize a pure population1, both exosomes and microvesicles are referred to as EVs in this review unless for referring studies that specifically analyzed particular populations. Ever since their discovery, extensive research studies have been performed to decipher EVs’ functions in physiological and pathological processes13-19. Research interests are mainly focused 2

on two types of EVs: exosomes and microvesicles. EVs are found to mediate cell-to-cell communications through distinct mechanisms13,20. By directly interacting with target cells via their surface ligands and receptors, EVs can trigger downstream intracellular signaling pathways of target cells21-23. They can also transfer membrane receptors to target cells through membrane fusion and/or deliver soluble cargoes, such as functional proteins24 and nucleic acids25,26, to target cells via direct fusion, endocytosis, or phagocytosis27-29. Different types of cells can produce EVs with inherent but distinct properties and therapeutic potentials. Tumor cells-derived EVs are shown to play important roles in cancer progression, including metastasis30-32, angiogenesis33,34, and immune suppression35,36. Mesenchymal stem cells (MSCs)-derived EVs are known to have therapeutic potentials for cardiovascular diseases and tissue injuries16,37. Dendritic cells (DC)-derived EVs enable antigen presentations and are under development for cancer vaccines38-40. To maximize EVs’ therapeutic potentials, extensive protein engineering studies have been conducted to reprogram EVs for new and/or enhanced functions and properties. This review is focused on recent progresses in the studies of protein engineering in EVs. 2. Strategies for Engineering Proteins in EVs 2.1 Genetic Manipulation of EV-Producing Cells Synthetic and engineered biomolecules have been introduced to cells to augment therapeutic functions for several decades41. Considering the biogenesis processes of EVs, it is possible that the incorporated biomolecules will be unwittingly packaged into EVs produced by engineered cells. Moreover, since membranes and membrane proteins of EVs are derived from plasma membranes and formation of intraluminal vesicles in endosomes allows to engulf cytosol to the lumens of exosomes, various approaches have been generated to reprogram EVs through engineering proteins in host cells. Genetic manipulation is a well-established strategy for protein engineering in cells. Exogenous proteins could be expressed in target cells through transduction or transfection and then loaded into EVs through passive packaging. EVs released from cells expressing the Cre recombinase carry Cre and can induce fluorescent color switch in reporter-expressing cells upon uptake42. Cells transduced with a gene encoding for GFP-tagged cystic fibrosis transmembrane conductance regulator (CFTR) were shown to secret EVs containing both mRNA and GFPCFTR glycoprotein. The resulting EVs facilitate the delivery of the CFTR fusion protein to recipient cells in a dose-dependent manner43. In addition to cytosolic proteins, EVs could also be engineered to carry membrane proteins via expression of target proteins in parental cells44-46. Notably, simple protein overexpression in EV-producing cells is usually inefficient and nonspecific for those without tailoring to EVs. To express protein in EVs, fusion proteins consisting of an EV anchor protein domain and one or more function domains have been constructed. Various EV anchor proteins were tested and compared (Figure 1). The most straightforward candidates are exosomal marker membrane proteins, including Lamp2b47-54, CD955, CD63, and CD81.

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Figure 1. Methods for genetic engineering of EV proteins. Exogenous cargo proteins (yellow stars) can be fused to the N-terminus of Lamp2b; N-terminus of C1C2 domain of lactadherin; Nterminus of PDGFR TMD; GPI anchor at C-terminus; palmitoylation peptide sequence at Nterminus; and tetraspanins. 2.1.1 Lamp2b Lysosome-associated membrane glycoprotein 2 (Lamp2) is a highly-glycosylated singletransmembrane protein involved in lysosomal protein degradation and is found abundantly in exosomal membrane47,56. Alternative splicing of Lamp2 gene can result in multiple transcript variants encoding different isoforms, including Lamp2a, Lamp2b, and Lamp2c. One pioneer study performed by Alvarez-Erviti et al. isolated EVs bearing neuron-targeting rabies viral glycoprotein (RVG) peptide from mouse DCs transduced with a gene encoding for mouse Lamp2b fused with N-terminal RVG. Such engineered EVs were shown to mediate cargo delivery to neurons47. Following studies adapted this approach in the human system by using human Lamp2b as the fusion partner53. Another study also demonstrated the feasibility of using human Lamp2a for the same purpose57. Furthermore, it was found that engineering peptideLamp2b fusion protein with a glycosylation motif helps protect the peptide from degradation and also increase overall Lamp2b fusion expression54. Currently, Lamp2b-based fusion proteins are only used for displaying target peptides. No larger proteins are displayed on EVs through Lamp2b.

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2.1.2 Tetraspanins All tetraspanins share similar ‘M’-shape topology on exosomal surface, including two short intra-vesicle termini, two extra-vesicle loops, a small intra-vesicle loop, and four transmembrane domains58-60. Tetraspanins are relatively small (200-350 aa), making them particularly suitable for EV display via molecular engineering in mammalian cells. Of their two extra-vesicular loops, the second larger one potentially allows for engineering for protein display on EVs surface61. By utilizing these structural features, Stickney et al. identified sites on CD63 that allow stable expression of a fluorescent protein on either the inner or outer membrane of EVs61. A GFP molecule was inserted between Ala 133 and Ser 134 of CD63 for outer surface display and fused to the N-terminus of CD63 for inner surface display. Similarly, exogenous proteins were incorporated into other tetraspanins55,62-65. Since CD9 and CD63 exist in both large and small EVs11, they could serve as suitable candidates for engineering all types of EVs. 2.1.3 PDGFR While tetraspanins are smaller than Lamp2b, they lack free termini on the outer leaflet of the membrane, making them suboptimal candidates to display peptides or proteins on EV surfaces. In comparison to Lamp2b and tetraspanins, the transmembrane domain (TMD) of plateletderived growth factor receptors (PDGFR) that has been widely used to display peptides or proteins on cellular surface may improve the display of functional proteins on EV surfaces given its small size (~50 aa)66,67. Although PDGFR TMD has no specific enrichments in EVs, it has been successfully used to display functional peptides and proteins that were fused to its Nterminus on EV outer surfaces68-71. 2.1.4 C1C2 Domain In addition to those transmembrane proteins, several peripheral proteins/protein domains were engineered as EV anchors to display proteins on the outer/inner EV surfaces. One example is C1C2 domain of lactadherin, which is a secreted protein with an epidermal growth factor (EGF)like domain at the N-terminus and a C1C2 domain at the C-terminus72. C1C2 domain binds with a high affinity to lipid membranes, especially in the presence of phosphatidylserine, which is found to be more abundant in EVs than in plasma membrane72,73. Because of the C1C2 domain, lactadherin is secreted in association with membrane vesicles and was recently identified on EVs secreted by mammary epithelial cells and mouse DCs73-75. Upon fusion to the N-terminus of C1C2 domain of lactadherin, intracellular soluble proteins are no longer found intracellularly but are released in extracellular compartments associated with EVs76-78. Additionally, the extracellular domains (ECDs) of membrane proteins could be functionally displayed on EV surfaces through the fusion with C1C2 domain79. Due to the lack of transmembrane domains, the C1C2 fusion proteins may exhibit reduced association with EV membranes in comparison to those with transmembrane fusion partners54,80. 2.1.5 GPI Anchor EVs are described to be selectively enriched in cholesterol, sphingolipids, glycerophospholipids, and lipid raft-associated proteins such as glycosylphosphatidylinositol (GPI)-anchored 5

proteins18,81,82. Proteins are singled out for GPI anchoring due to the presence of a GPI signaling sequence (GSS)83. GPI-anchored protein decay-accelerating factor (DAF, also known as CD55) was found to be specifically secreted in EVs during reticulocyte maturation84. Using the GGS (37 amino acids) of DAF, a variety of proteins could be tethered to the outer leaflet of EV membranes85,86. 2.1.6 Palmitoylation Peptide As a reversible post-translational modification, S-palmitoylation results in the addition of a 16carbon saturated fatty acid chain to cytoplasmic cysteine residues87. Palmitoylation of soluble proteins allows association with membranes87,88. Fusion of a consensus peptide sequence for palmitoylation to protein N-termini could tether them to cell membranes, enabling whole-cell labeling89,90, as well as EV labeling91-93. Palmitoylated proteins were shown to predominantly associate with the inner membranes of EVs 91. Therefore, this strategy is more suitable for packing proteins into EVs instead of surface display92. Genetic engineering represents an effective approach for incorporation of target functional proteins into EVs at varied locations. It also need be noted that limitations exist for genetic modification of EV-producing cells. This strategy is typically applicable only to genetically encodable molecules. The design and generation of fusion proteins with retained activity and specificity could be challenging. Additionally, transfections of EV-producing cells may affect the contents or protein composition of the engineered EVs. Fitzgerald et al. reported that stimulations to EV-producing cells could alter the patterns of cytokines encapsulated in EVs94, suggesting that protein composition of EVs could be changed upon systemic activation at cellular levels. The genetically-introduced proteins may alter protein repertoire of EVs. Wang et al. found that expression of arrestin domain-containing protein 1 (ARRDC1) in cells can induce incorporation of NOTCH2, ITCH and ADAM10 to ARMMs (ARRDC1-mediated microvesicles) and mediate non-canonical intercellular NOTCH signaling95. 2.2 Non-Genetic Manipulation of EV-Producing Cells Biochemical stimulation and metabolic labeling of EV-producing cells were also exploited to generate engineered EVs. It was found that EVs produced in response to cytokine exposure or inflammation stimuli contain altered RNAs and protein compositions and exert novel functions in recipient cells upon uptake 96,97. Riazifar et al. reported that exosomes from mesenchymal stem cells stimulated by IFNγ (IFNγ-Exo) carry anti-inflammatory and neuroprotective proteins and RNAs98. Intravenous administration of IFNγ-Exo reduces demyelination and neuroinflammation in the experimental autoimmune encephalomyelitis (EAE) mouse models, demonstrating that IFNγ-Exo can potentially serve as cell-free therapies in inducing tolerogenic immune responses to treat autoimmune and central nervous system disorders. Metabolic labeling utilizes selective cellular biosynthesis pathways to introduce modified biomolecules99. Wang et al. combined metabolic labeling of glycan or protein synthesis of EVproducing cells with click chemistry-mediated conjugation to engineer EVs100. Using azidobearing saccharides and L-azidohomoalanine (AHA), an azide-containing amino acid analogue of methionine, clickable groups were incorporated into EVs for bioconjugation, which reveal 6

little effects on EV’s integrity and protein expression100. Compared with genetic modification of EV-producing cells, non-genetic manipulation provides a rapid and efficient approach to generate engineered EVs. But the composition changes or modification sites in the resulting EVs are less defined and need further characterization studies. 2.3 Surface Functionalization of EVs Physical and chemical methods were developed for engineering EVs postproduction. Peptides or protein domains with high affinities to membrane lipids were used to attach proteins to EV surfaces. By conjugating to ApoA-I mimetic peptide (L-4F)101 that binds membrane lipids, targeting and therapeutic peptides could be associated with EVs via simple incubation101,102. C1C2 domain that possesses a high affinity to lipid membranes was also used to anchor proteins to EV surfaces72. Wang et al. demonstrated that native EVs incubated with C1C2-based fusion proteins could carry more fusion proteins than EVs produced by cells transfected with the genes of the fusion proteins103. Anchor peptides that specifically bind to EV-specific membrane proteins were also synthesized for EV surface functionalization. Using a peptide CP05 identified by phage display that showed specific and tight binding to CD63, Gao et al. successfully functionalized EV surfaces with therapeutic biomolecules104. Additionally, nanoparticles coated with antibodies that target EV surface proteins were used to decorate EVs105. In addition to anchor peptides, membrane anchor lipids were exploited to mediate non-covalent attachments to EV surfaces. Glycerol-phospholipid dimyristoyl phosphatidylethanolamine (DMPE), which was used as a cell membrane anchor106, was conjugated with polyethylene glycol (PEG) and nanobody. DMPE-PEG enables to anchor nanobodies on EV surfaces and also protects EVs from clearance by the reticulo-endothelial system (RES)107. Further exploitation of the DMPE-PEG system demonstrated a modular EV membrane engineering platform where streptavidin (STVDN) was conjugated to the DMPE-PEG59. The DMPE-PEG-STVDN conjugate could directly embed in EV surfaces and serve as an anchor for coupling biotinylated molecules, including fluorescent dyes, tissue-homing peptides, and antibodies59. Aptamers are short nucleic acids oligomers with high affinity and specificity for target molecules. Functioning as attractive alternatives to antibodies, aptamers have been used as versatile anchors for the development of biosensors and molecular imaging tools in physiological environments. A DNA aptamer LZH8 that selectively targets EVs from liver cancer cells (HepG2) was combined with DNA-based nanotechnologies for surface functionalization of target EVs.108 Additionally, several studies explored covalent methods to decorate EVs with peptides or proteins, primarily via click chemistry, a robust and bioorthogonal reaction involving alkyne and azide groups to form a triazole linkage109,110. Smyth et al. cross-linked EVs with alkyne groups using carbodiimide chemistry111. The resulting EVs with conjugated fluorescent dyes demonstrated the feasibility of chemically engineering EVs112. The authors reported that the conjugation process caused no impact on EVs’ size and association with recipient cells112. Furthermore, targeting peptides were conjugated to EV surfaces via copper-free click chemistry. The reactive dibenzylcyclootyne (DBCO) groups were attached to amine-containing molecules on EVs. The generated DBCO-conjugated EVs were then covalently linked to azide-containing molecules via copper-free click chemistry113. 7

Direct functionalization of isolated EVs allows to covalently and noncovalently attach diverse types of molecules to EV surfaces, resulting in EVs with new and/or enhanced functions. However, the physical and chemical modifications may inadvertently affect stability of EVs and/or decrease biological activity of proteins at EV surface. The modification processes along with additional purification procedures may cause reduced yields. 3. Applications of EVs with Engineered Proteins Protein engineering adds exogenous peptides/proteins to EVs, resulting in novel and/or enhanced functions and properties and consequent transformation of EVs into unique and important research and therapeutic tools61. Here we review the applications of reprogrammed EVs by protein engineering. 3.1 Engineering EVs for In Vivo Tracking Engineering fluorescent molecules59/proteins or luciferase into EVs could enable visualization of cellular internalization of EVs and to track of EVs in animal models. GFP-labeling of EVs via CD63 fusion was utilized to visualize cellular uptake of EVs64, to characterize encapsulation of fluorophore-labeled cargoes62, and to image the fate of cancer-cell-derived EVs in orthotopic breast cancer mouse models63. Gaussia luciferase (Gluc) fused with C1C2 domain-labeled EVs revealed tissue distribution and pharmacokinetics of intravenously injected EVs in the mouse models114. Lai et al. developed a multimodal imaging reporter, termed as GlucB, consisting of Gluc fused to a biotin acceptor domain, which is metabolically biotinylated upon expression in mammalian cells in the presence of biotin ligase70,91. GlucB-labeled EVs exhibit strong bioluminescent signals when incubated with the Gluc substrate. In addition, the surface biotin allows EVs to be conjugated to any labeled streptavidin, which could then be imaged noninvasively in vivo using different techniques including fluorescence-mediated tomography (FMT)70,91. 3.2 Engineering EVs with Targeting Moieties EVs possess intrinsic tissue specificity and tropism conferred by the repertoire of their membrane proteins. Tetraspanin-integrin receptors on EV membranes play an important role in target cell selection, as EVs expressing Tspan8-alpha4 complexes could be readily taken up by CD54-positive endothelial and pancreatic cells115. Hoshino et al. reported that tumor cell-derived EVs carry different types of integrins depending on tumor origins and the integrins mediate tumor metastasis toward different organs116. EVs with integrin (ITG) αVβ5 specifically bind to Kupffer cells of liver, whereas EVs expressing ITGα6β4 and ITGα6β1 show lung tropism with binding specificity for lung-resident fibroblasts and epithelial cells116. The tropisms of EVs could be modulated by introducing tissue-specific peptides or proteins onto EV surfaces. Multiple targeting peptides were used for this purpose, including RVG peptide fused with Lamp2b recognizing acetylcholine receptors on neurons47,48,50,51,54, iRGD peptide specific for ITGαVpositive breast cancer cells49, ischemic myocardium-targeting peptide CSTSMLKAC52, fragments of interleukin 3 (IL-3) toward the IL-3 receptor53, GE11 peptide fused to PDGFR TMD for epidermal growth factor receptor (EGFR)68, PC94 peptide fused to Lamp2a against 8

hepatocellular carcinoma57, and low-density lipoprotein (LDL) for LDL receptor overexpressed on blood brain barrier and glioblastoma101. Moreover, larger targeting moieties such as singlechain variable region (scFv) antibodies and nanobodies were displayed on EV surfaces, exemplified by anti-EGFR nanobodies85,107 and anti-human epidermal growth factor receptor 2 (HER2) scFv78,103 (Table 1). Cheng et al. reported that by fusing two scFv antibodies to PDGFR TMD, EVs were constructed for simultaneously targeting both T-cell CD3 and cancer cellassociated EGFR71. The resulting synthetic multivalent antibodies retargeted exosomes (SMART-Exos) were shown to not only induce cross-linking of T cells and EGFR-expressing breast cancer cells but also elicit potent antitumor immunity both in vitro and in vivo. This proofof-concept study provides a novel and versatile platform for EV engineering. Table 1. Targeting moieties displayed on EV surfaces. EV Anchor PDGFR TMD

Lamp2b/a

C1C2 domain GPI anchor CP05 peptide

Targeting Moiety

Target(s)

GE11 (peptide) SIRPα variant Dual scFv antibodies iRGD (peptide) RVG (peptide) IMTP (peptide) PC94 (peptide) IL3 (protein domain) scFv Nanobody M12 (peptide)

EGFR CD47 CD3 and EGFR ITGαV-positive breast cancer cells Neuron Ischemic Myocardium Hepatocellular carcinoma IL3 receptor HER2 EGFR Muscle

Ref(s). 68 69 71 49 47,50,51 52 57 53 78,103 85 104

3.3 Engineering EVs with Therapeutic Peptides/Proteins EVs can induce intracellular signaling through receptor-ligand interactions, especially in the context of immune modulation in tumors. Tumor-derived EVs could induce apoptosis of T cells and natural killer (NK) cells through receptor-mediated signaling pathways involving Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), and programmed death-ligand 1 (PDL1)117-123. Such mechanism was borrowed to arm EVs to induce apoptosis of cancer cells. EVs isolated from TRAIL-overexpressing cells could induce apoptosis in cancer cells and control tumor progression in vivo when administered intratumorly45. CD47 frequently over-expressed on tumor surface binds to the signal regulatory protein a (SIRPα) on macrophages, activating “do not eat me” signals and leading to tumor escape from phagocytosis124. EVs with genetically displayed SIRPα could occupy CD47-binding sites on tumor cells and disrupt CD47-SIRPα interactions to increase tumor cell engulfment by macrophages, inducing remarkably augmented tumor phagocytosis and leading to effective anti-tumor T-cell responses69. Genetic display of hyaluronidase PH20 on EVs facilitates the degradation of hyaluronan of extracellular matrix and hence augments EVs penetration into solid tumors86. Compared with other forms of 9

nanoparticles, EVs are characterized by biocompatible membrane scaffolds for displaying native or engineered peptides and proteins. Functional expression of therapeutic peptides and proteins on EV surfaces may help preserve their three-dimensional structures and post-translational modifications, therefore enabling maximal therapeutic efficacies.

Figure 2. Applications of EVs with engineered proteins. 3.4 Engineering EVs for Enhanced Cargo Loading Despite extensive studies on use of EVs as potential drug delivery vehicles125,126, conventional cargo-loading methods, including incubation127, sonication128, freeze-thaw,128 and electroporation129, seem to have inefficient loading efficiency and are limited to cargoes with low molecular weights. Reprograming EVs with engineered proteins can facilitate loading of therapeutic molecules, including proteins and nucleic acids. Loading protein cargoes into purified EVs is challenging. Several genetic engineering approaches were developed to actively load soluble proteins into EVs during production. Nedd4 family-interacting protein 1 (Ndfip1) was identified to mediate protein packaging into exosomes130. Fusion of Cre recombinase with the WW domains of Nedd4, which interacts Ldomain of Ndfip1, resulted in efficient loading of Cre into exosomes131. The loading capacities correlates with the expression levels of Ndfip1. It was also found that WW-Cre fusion was ubiquitinated in the presence of Ndfip1, with monoubiquitination as the predominant form. The idea of using ubiquitin as an exosome-loading signal was also explored. Both soluble proteins and membrane proteins were loaded into exosomes through genetic fusion with ubiquitin at Ctermini132. Yim et al. developed a reversible protein packaging system, named as “exosomes for protein loading via optically reversible protein–protein interactions” (EXPLORs), which consists of two parts, the truncated cryptochrome (CRY)-interacting protein (CIBN) tethered to exosome membranes through CD9-based fusions and the photoreceptor cryptochrome 2 (CRY2) fused 10

with cargo proteins. CRY2 could bind to CIBN in the presence of blue light illumination, therefore promoting the docking of cargoes to exosome membranes. Upon removal of blue light, cargoes could be disassociated from CIBN-CD9 fusion and released into intraluminal space of exosomes65. In addition to protein cargoes, nucleic acids were actively loaded into EVs through exosome membrane-tethered RNA-binding domains. Several RNA-binding protein domains were fused to EV membrane proteins, including MS2 bacteriophage coat protein recruiting RNAs that contain cognate MS2 stem loop92,133, TAT peptide recognizing TAR RNA loop57, archaeal ribosomal protein L7Ae binding to the C/Dbox RNA structure134, and human antigen R (HuR) interacting with AU-rich elements of RNA cargoes55. It was shown that MS2 domain fused to CD63 increases RNA loading by six-fold and the active loading becomes more efficient for smaller (~0.5 kb) RNA molecules133. Using the MS2 bacteriophage coat protein coupled with the CIBNCRY2-based light-inducible approach, endogenous RNAs were actively sorted into exosomes for functional delivery to leukemia cells135. Lamp2a fused with TAT peptide could enhance the loading of miRNA into EVs by 65-fold. However, the resulting EVs were ineffective at delivering active miRNA to recipient cells57. Co-transfection of the constitutively active Cx43 S368A mutant with L7Ae-based RNA packaging device was demonstrated to facilitate the delivery of mRNA into the cytosol of target cells134. EVs expressing CD9-HuR fusion proteins were shown to not only actively encapsulate miRNAs but also functionally deliver miRNAs into recipient cells55.

Figure 3. Engineering methods for enhanced cargo loading. CIBN is expressed on EV membrane through fusion with EV-anchoring domain. CRY2 is fused with protein cargoes or RNA binding domain to recruit RNA cargoes. Upon blue light induction, CRY2 and tethered cargoes are recruited to EV. Protein cargoes fused to the WW domain and ubiquitin are packaged into EVs through binding to Ndfip1 and ESCRT complex, respectively. 11

4. Conclusions EVs function as natural nanocarriers of biomolecules to mediate intercellular communications. Owing to their advantageous properties, EV emerges as a potentially new form of delivery vehicle and therapeutic modality. By exploiting various protein engineering strategies, native EVs have been successfully reprogrammed for a range of research and therapeutic applications. Despite significant progresses on EV engineering, challenges remain on production, characterization, and cargo encapsulation. Since EVs carry functional biomolecules from EVproducing cells, cell sources for EV production are critical for EV engineering. It becomes more difficult and less efficient for genetic modification in primary cells. MSCs produce high levels of EVs compared to other types of cells, even when they are immortalized136. Dendritic cells (DC)derived EVs have demonstrated safety in human clinical trials137,138. Comprehensive characterization and safety evaluations of EVs produced from various types of cells are needed for clinical applications. EVs contain heterogeneous populations, which are characterized by different molecular compositions because of distinct biogenesis pathways. The healthy state of EV-producing cells could also contribute to their molecular heterogeneity. However, it remains difficult to isolate EVs based on their molecular composition or to characterize individual EVs. Since engineered EVs may contain subpopulations that show low therapeutic efficacy or cause unwanted side effects, more sensitive and precise analytical methods are needed to detect and characterize single vesicles for full exploration of subset heterogeneity139. Moreover, better understanding the structure, molecular composition, and biogenesis of EV subpopulations with high levels of cargoes will provide insights into optimization of EV engineering methods and generation of therapeutic EVs in a more controlled manner. Drug delivery is one of the major applications of EVs. By engineering EVs with targeting moieties on EV surface, targeted delivery to diseased cells or tissues could be achieved. Whether and how the targeting moieties change the uptake mechanism(s) of EVs remain unknown. Knowledge on the cellular uptake mechanism(s) of EVs will facilitate the design and engineering of EVs with improved potency and specificity. To load and deliver protein and RNA cargoes, current methods utilize EV anchoring domains to tether protein or RNAs into EVs, which limit the size and forms of cargo molecules. Further studies of cargo sorting during EV biogenesis may offer new directions for efficient loading of distinct types of cargos into EVs, leading to the development of engineered EVs with improved pharmacological activities or expanded therapeutic applications. Acknowledgements This work was supported by University of Southern California School of Pharmacy Start-Up Fund for New Faculty, University of Southern California Ming Hsieh Institute for Engineering Medicine for Cancer, STOP CANCER Research Career Development Award (to Y. Z.), and PhRMA Foundation Research Starter Grant in Translational Medicine and Therapeutics (to Y. Z).

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