A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties

Dooley, Kevin; McConnell, Russell E.; Xu, Ke; Lewis, Nuruddeen D.; Haupt, Sonya; Youniss, Madeleine R.; Martin, Shelly; Sia, Chang Ling; McCoy, Christine; Moniz, Raymond J.; Burenkova, Olga; Sanchez-Salazar, Jorge; Jang, Su Chul; Choi, Bryan D.; Harrison, Rane A.; Houde, Damian J.; Burzyn, Dalia; Leng, Charan; Kirwin, Katherine; Ross, Nikki L.; Finn, Jonathan D.; Gaidukov, Leonid; Economides, Kyriakos D.; Estes, Scott; Thornton, James E.; Kulman, John D.; Sathyanarayanan, Sriram; Williams, Douglas E.

doi:10.1016/j.ymthe.2021.01.020

Cited by 187 publications

(209 citation statements)

References 48 publications

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“…To further explore the biology of the MAAP-dependent AAV secretory process, we adopted a gradient centrifugation method to purify EVs from large volumes of cell culture supernatant 28,29 . Cell culture medium from suspension adapted HEK293 rAAV8/MAAPΔ producing cells (complemented in trans with MAAP8-HA) was processed by successive filtration and centrifugation steps to generate a crude EV pellet that was then separated on an iodixanol gradient (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The most notable attribute of MAAP is a cationic amphipathic C-terminal domain, which is critical for membrane anchoring and extracellular secretion. Recent studies have revealed that PTGFRN and BASP1 can be utilized as scaffolds for loading various molecules onto the surface or into the lumen of EVs 29 . BASP1 is an inner membrane leaflet localized protein that utilizes a polybasic effector domain (PED) to bind to the EV membrane.…”

Section: Discussionmentioning

confidence: 99%

“…For Figures 5C-D and Supplementary Figure 7D-H, a previously established EV isolation protocol was utilized 29 . Suspension adapted HEK293 (human embryonic kidney cells obtained from the University of North Carolina Vector Core) cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin, 100ug/ml streptomycin and inoculated at a density of 5E+5 viable cells per milliliter.…”

Section: Methodsmentioning

confidence: 99%

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The membrane associated accessory protein is an adeno-associated viral egress factor

Elmore

Havlik

et al. 2021

Preprint

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Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some recombinant AAV serotypes are secreted in a pre-lytic manner as extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a (+1) frameshifted open reading frame (ORF) in the AAV capsid (cap) gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with recombinant MAAP restored extracellular secretion of multiple AAV/MAAPΔ serotypes. MAAP is sorted into recycling Rab11+ vesicles and strongly associates with EV markers upon fractionation. In addition to characterizing a novel viral egress factor, these studies highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The membrane associated accessory protein is an adeno-associated viral egress factor

Elmore

Havlik

et al. 2021

Preprint

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“…S7A-B, noise subtracted and normalised). SV also displayed reduced levels of ADAM10 and of contaminants, including Histone 3, LGALS3BP [41], and RNAse 4 (Fig. S7C).…”

Section: Rbpsmentioning

confidence: 98%

Synthetic antigen-presenting cells reveal the diversity and functional specialisation of extracellular vesicles composing the fourth signal of T cell immunological synapses

Jainarayanan

Fernández-Messina

et al. 2021

Preprint

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The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of synaptic vesicles (SVs) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with SVs and steadily shed EVs (sEVs). Altogether, our data provide evidence for a higher specialisation of SVs which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the SV output, which is also accompanied by SMAPs, we propose their classification as signal four.

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“…It is a single-pass transmembrane glycoprotein that enables the high-density surface display of fused proteins of interest, including cytokines, antibody fragments, and other immunomodulatory proteins or specific antigens up to 170 kDa. Due to its high abundance in EVs, PTGFRN showed better packaging and antigen display efficiencies than conventional scaffold proteins, such as CD81, LAMP2B, and the vector system called "pDisplay" based on platelet-derived growth factor receptor (PDGFR) [63]. However, exoVACC™ is still in the research phase, as multiple combinations of SARS-CoV-2 antigens and adjuvants can be produced, and their effectiveness and specificity in vitro and in animal models should be assessed (Figure 3c).…”

Section: Ev-based Vaccines: Focus On Covid-19mentioning

confidence: 99%

Promising Extracellular Vesicle-Based Vaccines against Viruses, Including SARS-CoV-2

Sabanovic¹,

Piva²,

Cecati³

et al. 2021

Biology

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Extracellular vesicles (EVs) are secreted from almost all human cells and mediate intercellular communication by transferring heterogeneous molecules (i.e., DNA, RNAs, proteins, and lipids). In this way, EVs participate in various biological processes, including immune responses. Viruses can hijack EV biogenesis systems for their dissemination, while EVs from infected cells can transfer viral proteins to uninfected cells and to immune cells in order to mask the infection or to trigger a response. Several studies have highlighted the role of native or engineered EVs in the induction of B cell and CD8(+) T cell reactions against viral proteins, strongly suggesting these antigen-presenting EVs as a novel strategy for vaccine design, including the emerging COVID-19. EV-based vaccines overcome some limitations of conventional vaccines and introduce novel unique characteristics useful in vaccine design, including higher bio-safety and efficiency as antigen-presenting systems and as adjuvants. Here, we review the state-of-the-art for antiviral EV-based vaccines, including the ongoing projects of some biotech companies in the development of EV-based vaccines for SARS-CoV-2. Finally, we discuss the limits for further development of this promising class of therapeutic agents.

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A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties

Cited by 187 publications

References 48 publications

The membrane associated accessory protein is an adeno-associated viral egress factor

The membrane associated accessory protein is an adeno-associated viral egress factor

Synthetic antigen-presenting cells reveal the diversity and functional specialisation of extracellular vesicles composing the fourth signal of T cell immunological synapses

Promising Extracellular Vesicle-Based Vaccines against Viruses, Including SARS-CoV-2

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