Using genetically modified extracellular vesicles as a non-invasive strategy to evaluate brain-specific cargo

Rufino-Ramos, David; Lüle, Sevda; Mahjoum, Shadi; Ughetto, Stefano; Bragg, D. Cristopher; Almeida, Luís Pereira de; Breakefield, Xandra O.; Breyne, Koen

doi:10.1016/j.biomaterials.2022.121366

Cited by 24 publications

(30 citation statements)

References 90 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous groups have utilized genetic strategies to label EVs endogenously (e.g., tetraspanins fused with fluorescent or bioluminescent proteins); however, the overexpression of these proteins, driven by exogenous promoters, combined with the large size of the tags raises questions about how these transgenes can impact endogenous EV production 37 – 45 . Overexpression of tetrapanins, and specifically CD63, can generate off-target effects at the level of EV production and even changes in physiology 37 – 39 , 42 .…”

Section: Resultsmentioning

confidence: 99%

“…Endogenous labeling of EVs with EV-enriched tetraspanin fusion proteins avoids many of these pitfalls, though common approaches come with their own drawbacks 35 , 36 . Tetraspanins (e.g., CD9, CD81, CD63) have been labeled with a fluorescent protein or luciferase of approximately the same size as the proteins targeted for labeling, and the expression of the tagged protein is generally driven by an exogenous promoter resulting in elevated levels and off-target expression 37 – 45 . Unfortunately, these manipulations have largely impacted the functionality of the tagged tetraspanins that play a fundamental role in EV biology and facilitate EV budding by inducing membrane curvature and regulating EV cargo by recruiting other functional proteins into vesicles 46 , 47 .…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, as a first step toward examining epididymal EVs in the somatic-to-germline transmission of paternal stress experience in vivo, we developed a transgenic construct that would allow for cell-type specific labeling of EVs via Cre-induced hemagglutinin (HA) tagging of the endogenous CD63 protein. While genetic strategies have been used to label EVs in cell lines and rodent models, our construct has advantages over previously reported transgenes 37 – 45 . First, CD63 is a tetraspanin protein enriched in predominantly small MVB-derived EVs (i.e., exosomes) widely expressed in tissues throughout the body, and we have designed our construct to be introduced via homologous recombination 40 , 46 , 48 – 52 .…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

HA-tag CD63 is a novel conditional transgenic approach to track extracellular vesicle interactions with sperm and their transfer at conception

Morgan

Meadows

Marx-Rattner

et al. 2023

Sci Rep

View full text Add to dashboard Cite

Extracellular vesicles (EVs) are a unique mode of intercellular communication capable of specificity in transmitting signals and cargo to coordinate local and distant cellular functions. A key example of this is the essential role that EVs secreted by epithelial cells lining the lumen of the male reproductive tract play in post-spermatogenic sperm maturation. We recently showed in a preclinical mouse model that this fundamental process had a causal role in somatic-to-germline transmission of biological information regarding prior stress experience capable of altering the rate of fetal development. However, critical mechanistic questions remain unanswered as to the processes by which signaling occurs between EVs and sperm, and whether EVs or their cargo are delivered at conception and are detectable in the early embryo. Unfortunately, notable methodological limitations shared across EV biology, particularly in the isolation and labeling of EVs, complicate efforts to answer these important questions as well as questions on EV targeting specificity and mechanisms. In our current studies, we developed a novel approach to track EVs using a conditional transgenic construct designed to label EVs via conditional Cre-induced hemagglutinin (HA) tagging of the EV endogenous tetraspanin, CD63. In our exhaustive validation steps, this internal small molecular weight tag did not affect EV secretion or functionality, a common problem found in the previous design of EV tags using larger molecular weight proteins, including fluorescent proteins. Utilizing a stably transfected immortalized epididymal epithelial cell line, we first validated key parameters of the conditional HA-tagged protein packaged into secreted EVs. Importantly, we systematically confirmed that expression of the CD63-HA had no impact on the production, size distribution, or surface charge of secreted EVs, nor did it alter the tetraspanin or miRNA composition of these EVs. We also utilized the CD63-HA EVs to verify physical interactions with sperm. Finally, using in vitro fertilization we produced some of the first images confirming sperm delivered EV cargo at conception and still detectable in the early-stage embryo. As such, this construct serves as a methodological advance and as a valuable tool, with applications in the study of EV function across biomedical research areas.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

HA-tag CD63 is a novel conditional transgenic approach to track extracellular vesicle interactions with sperm and their transfer at conception

Morgan

Meadows

Marx-Rattner

et al. 2023

Sci Rep

View full text Add to dashboard Cite

show abstract

“…The analysis of tdTomato expression in peripheral organs would also be informative to evaluate to what extent bdEVs can deliver functional cargo beyond the CNS. Additionally, to confirm peripheral diffusion of bdEVs secreted from neural cells into the bloodstream, future analyses should focus on methods avoiding direct cell transduction to prevent transduction of other cell types present in the brain ( Rufino-Ramos et al 2022 ). Definitive surface markers for bdEVs would be helpful to distinguish different subpopulations of neural EVs, as neuronal markers such as L1CAM or NCAM were previously shown to be present in EVs from other tissues ( Norman et al 2021 ; Ter-Ovanesyan et al 2021 ) and are thus not exclusive of neural cells.…”

Section: Discussionmentioning

confidence: 99%

“…BdEVs detected in biofluids, such as serum and cerebrospinal fluid (CSF) have been studied as potential diagnostic and prognostic biomarkers for brain diseases (Badhwar and Haqqani 2020; Hill 2019; Rufino-Ramos et al 2022; Street et al 2012). However, the physiological role of bdEVs in brain communication to near and long distances remains largely unknown.…”

Section: Discussionmentioning

confidence: 99%

Extracellular communication between brain cells through functional transfer of Cre mRNA

Rufino-Ramos

Leandro

Perdigão

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo overtime. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEVs transfer at physiological levels which will shed light on the role of bdEVs in neural communication within the brain and beyond.

show abstract

The Landscape of Biomimetic Nanovesicles in Brain Diseases

You,

Liang,

et al. 2023

Advanced Materials

View full text Add to dashboard Cite

Brain diseases, such as brain tumors, neurodegenerative diseases, cerebrovascular diseases and brain injuries, are caused by various pathophysiological changes such as excessive or impaired angiogenesis, neuroinflammation, immune activation or suppression, neurodegenerative disorders, and neurovirulent protein deposition, which poses a serious health threat. Brain disorders are often difficult to treat due to the presence of the blood‐brain barrier (BBB), which hinders the delivery of drugs to the brain. Biomimetic nanovesicles (BNVs), including endogenous extracellular vesicles (EVs) derived from various cells and artificial nanovesicles (ANVs), possess the ability to penetrate the BBB and thus can be utilized for drug delivery to the brain. BNVs, especially endogenous EVs, are widely distributed in body fluids and usually carry various disease‐related signal molecules such as proteins, RNA and DNA, and may therefore also be analyzed to understand the etiology and pathogenesis of brain diseases. This review will cover the exhaustive classification and characterization of BNVs and pathophysiological roles involved in various brain diseases, and emphatically focus on nanotechnology‐integrated BNVs for brain disease theranostics, including various diagnosis strategies and precise therapeutic regulations (e.g., immunity regulation, disordered protein clearance, anti‐neuroinflammation, neuro‐regeneration, angiogenesis and the gut‐brain axis regulation). We also discuss and outline the remaining challenges and future perspectives regarding the nanotechnology‐integrated BNVs for the diagnosis and treatment of brain diseases.This article is protected by copyright. All rights reserved

show abstract

Using genetically modified extracellular vesicles as a non-invasive strategy to evaluate brain-specific cargo

Cited by 24 publications

References 90 publications

HA-tag CD63 is a novel conditional transgenic approach to track extracellular vesicle interactions with sperm and their transfer at conception

HA-tag CD63 is a novel conditional transgenic approach to track extracellular vesicle interactions with sperm and their transfer at conception

Extracellular communication between brain cells through functional transfer of Cre mRNA

The Landscape of Biomimetic Nanovesicles in Brain Diseases

Contact Info

Product

Resources

About