Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

Abstract

Extracellular vesicles (EVs) have recently emerged as a highly promising cell-free biotherapeutics. While a range of engineering strategies have been developed to functionalize the EV surface, current approaches fail to address the limitations associated with endogenous surface display, pertaining to the heterogeneous display of commonly used EV-loading moieties among different EV subpopulations. Here we present a novel engineering platform to display multiple protein therapeutics simultaneously on the EV surface. As proof-of-concept, we screened multiple endogenous display strategies for decorating the EV surface with cytokine binding domains derived from tumor necrosis factor receptor 1 (TNFR1) and interleukin 6 signal transducer (IL6ST), which can act as decoys for the pro-inflammatory cytokines TNFα and IL6, respectively. Combining synthetic biology and systematic screening of loading moieties, resulted in a three-component system which increased the display and decoy activity of TNFR1 and IL6ST, respectively. Further, this system allowed for .

Identification of storage conditions stabilizing extracellular vesicles preparations

“…In order to facilitate evaluation of parameters related to general integrity and functionality of stored EVs in addition to basic parameters such as particle concentration and size measured by NTA, we decided to perform stability experiments with two different types of engineered EVs: MSC‐TNFR EVs engineered to suppress TNF‐α signalling (Gupta et al., 2021 ) and HEK293T:CD63mNeonGreen EVs engineered to carry an intravesicular green fluorescent mNeonGreen (mNG) tag. EVs from both cell sources were prepared by TFF/UF including a PBS‐based diafiltration step and purified by BE‐SEC.…”

“…An ideal storage buffer formulation wouldn't only facilitate stable recovery of numbers of EVs and their size and cargo, but also preservation of their surface composition. In another study, we recently described engineered therapeutic EVs displaying cytokine binding domains which can act as decoys for pro‐inflammatory cytokines (Gupta et al., 2020 ). The MSC‐TNFR1 EVs included in this Stage were consequently used to assess the ability of those EVs to decoy TNF‐α over time, after storing EVs in different buffer formulations.…”

“…All cell lines were grown at 37°C, 5% CO 2 in a humidified atmosphere and regularly tested for the presence of mycoplasma. The creation and characterization of the genetically modified stable cell lines HEK293T:CD63eGFP (Corso et al., 2017 ), HEK293T:CD63mNG (Wiklander et al., 2018 ), HEK293FS:CD63mNG (HEK293 FreeStyle:CD63mNeonGreen) (Cavallaro et al., 2021 ), and MSC:TNFR1 (Gupta et al., 2021 ) were described previously.…”

“…After 24 h, 50 μl of complete DMEM were mixed with or without MSC:TNFR1 EVs in combination with hTNF‐α (5 ng/ml, NordicBiosite) and added to the cells. 6 h post‐treatment the cells were lysed using 0.1% Triton X‐100 diluted in PBS (Sigma) and mixed with D‐Luciferin substrate (Promega) prior to luminescence measurement with GloMax‐96 Microplate Luminometer (Promega) as described previously (Gupta et al., 2021 ).…”

Peritoneal M2 macrophage-derived extracellular vesicles as natural multi-target nanotherapeutics to attenuate cytokine storm after severe infections

“…g ., IL-6 or TNF-α blockers) had been developed and shown certain beneficial effects in pre-clinical models[8, 9], but they showed controversial results in clinical trials of COVID-19 treatment[10]. The possible reason is that single cytokine-targeted therapy may well-function when this cytokine is the driving factor in this disease, while the mechanisms of cytokine storm are complicated and many cytokines are involved[11]. Instead, novel therapies that target multiple proinflammatory pathways might be more efficient, since the basis consensus is that cytokine storm is a result of disrupted networks involved multiple immune cells and cytokines.…”