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The interaction between gliomas and the immune system is poorly understood and thus hindering development of effective immunotherapies for glioma patients. The immune response is highly variable during tumor development, and affected by therapies such as surgery, radiation, and chemotherapy. Currently, analysis of these local changes is difficult due to poor accessibility of the tumor and high-morbidity of sampling. In this study, we developed a model for repeat-biopsy in mice to study these local immunological changes over time. Using fine needle biopsy we were able to safely and repeatedly collect cells from intracranial tumors in mice. Ultra-fast cycling technology (FAST) was used for multi-cycle immunofluorescence of retrieved cells, and provided insights in the changing immune response over time. The combination of these techniques can be utilized to study changes in the immune response in glioma or other intracranial diseases over time, and in response to treatment within the same animal.
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Gene editing technologies have the potential to correct genetic disorders by modifying, inserting, or deleting specific DNA sequences or genes, paving the way for a new class of genetic therapies. While gene editing tools continue to be improved to increase their precision and efficiency, the limited efficacy of in vivo delivery remains a major hurdle for clinical use. An ideal delivery vehicle should be able to target a sufficient number of diseased cells in a transient time window to maximize on-target editing and mitigate off-target events and immunogenicity. Here, we review major advances in novel delivery platforms based on cell-derived vesicles - extracellular vesicles and virus-like particles - for transient delivery of gene editing payloads. We discuss major findings regarding packaging, in vivo biodistribution, therapeutic efficacy, and safety concerns of cell-derived vesicles delivery of gene editing cargos and their potential for clinical translation.
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Vesículas Extracelulares , Edición Génica , Técnicas de Transferencia de Gen , Humanos , Edición Génica/métodos , Animales , Vesículas Extracelulares/metabolismo , Terapia Genética/métodosRESUMEN
[This corrects the article DOI: 10.1016/j.omtm.2022.06.012.].
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Glioblastoma (GB) tumors are one of the most insidious cancers which take over the brain and defy therapy. Over time and in response to treatment the tumor and the brain cells in the tumor microenvironment (TME) undergo many genetic/epigenetic driven changes in their phenotypes and this is reflected in the cellular contents within the extracellular vesicles (EVs) they produce. With the result that some EVs try to subdue the tumor (friends of the brain), while others participate in the glioblastoma takeover (foes of the brain) in a dynamic and ever changing process. Monitoring the contents of these EVs in biofluids can inform decisions based on GB status to guide therapeutic intervention. This review covers primarily recent research describing the different cell types in the brain, as well as the tumor cells, which participate in this EV deluge. This includes EVs produced by the tumor which manipulate the transcriptome of normal cells in their environment in support of tumor growth (foes), as well as responses of normal cells which try to restrict tumor growth and invasion, including traveling to cervical lymph nodes to present tumor neo-antigens to dendritic cells (DCs). In addition EVs released by tumors into biofluids can report on the status of living tumor cells via their cargo and thus serving as biomarkers. However, EVs released by tumor cells and their influence on normal cells in the tumor microenvironment is a major factor in immune suppression and coercion of normal brain cells to join the GB "band wagon". Efforts are being made to deploy EVs as therapeutic vehicles for drugs and small inhibitory RNAs. Increasing knowledge about EVs in the TME is being utilized to track tumor progression and response to therapy and even to weaponize EVs to fight the tumor.
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Background: Glioblastoma (GBM) is a highly aggressive and invasive brain tumor associated with high patient mortality. A large fraction of GBM tumors have been identified as epidermal growth factor receptor (EGFR) amplified and ~50% also are EGFRvIII mutant positive. In a previously reported multicenter phase II study, we have described the response of recurrent GBM (rGBM) patients to dacomitinib, an EGFR tyrosine kinase inhibitor (TKI). As a continuation of that report, we leverage the tumor cargo-encapsulating extracellular vesicles (EVs) and explore their genetic composition as carriers of tumor biomarker. Methods: Serum samples were longitudinally collected from EGFR-amplified rGBM patients who clinically benefitted from dacomitinib therapy (responders) and those who did not (nonresponders), as well as from a healthy cohort of individuals. The serum EV transcriptome was evaluated to map the RNA biotype distribution and distinguish GBM disease. Results: Using long RNA sequencing, we show enriched detection of over 10 000 coding RNAs from serum EVs. The EV transcriptome yielded a unique signature that facilitates differentiation of GBM patients from healthy donors. Further analysis revealed genetic enrichment that enables stratification of responders from nonresponders prior to dacomitinib treatment as well as following administration. Conclusion: This study demonstrates that genetic composition analysis of serum EVs may aid in therapeutic stratification to identify patients with dacomitinib-responsive GBM.
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In glioblastoma, a malignant primary brain tumor, liposomes have shown promise in pre-clinical and early phase clinical trials as delivery vehicles for therapeutics. However, external factors influencing cellular uptake of liposomes in glioma cells are poorly understood. Heparin and heparin analogues are commonly used in glioma patients to decrease the risk of thrombo-embolic events. Our results show that heparin inhibits pegylated liposome uptake by U87 glioma and GL261 cells in a dose dependent manner in vitro, and that heparin-mediated inhibition of uptake required presence of fetal bovine serum in the media. In a subcutaneous model of glioma, Cy5.5 labeled liposomes could be detected with in vivo imaging after direct intra-tumoral injection. Ex-vivo analysis with flow cytometry showed a decreased uptake of liposomes into tumor cells in mice treated systemically with heparin compared to those treated with vehicle only.
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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 over time. 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 bdEV transfer at physiological levels, which will shed light on the role of bdEVs in neural communication within the brain and beyond.
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Vesículas Extracelulares , Integrasas , Ratones , Animales , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Integrasas/genética , Integrasas/metabolismo , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismoRESUMEN
X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease caused by a retrotransposon insertion in intron 32 of the TAF1 gene. This insertion causes mis-splicing of intron 32 (TAF1-32i) and reduced TAF1 levels. TAF1-32i transcript is unique to XDP patient cells and can be detected in their extracellular vesicles (EVs). We engrafted patient and control iPSC-derived neural progenitor cells (hNPCs) into the striatum of mice. To track TAF1-32i transcript spread by EVs, we transduced the brain-implanted hNPCs with a lentiviral construct called ENoMi, which consists of a re-engineered tetraspanin scaffold tagged with bioluminescent and fluorescent reporter proteins under an EF-1α promoter. Alongside this improved detection in ENoMi-hNPCs-derived EVs, their surface allows specific immunocapture purification, thereby facilitating TAF1-32i analysis. Using this ENoMi-labeling method, TAF1-32i was demonstrated in EVs released from XDP hNPCs implanted in mouse brains. Post-implantation of ENoMi-XDP hNPCs, TAF1-32i transcript was retrieved in EVs isolated from mouse brain and blood, and levels increased over time in plasma. We compared and combined our EV isolation technique to analyze XDP-derived TAF1-32i with other techniques, including size exclusion chromatography and Exodisc. Overall, our study demonstrates the successful engraftment of XDP patient-derived hNPCs in mice as a tool for monitoring disease markers with EVs.
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Vesículas Extracelulares , Enfermedades Neurodegenerativas , Humanos , Ratones , Animales , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Biomarcadores , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismoRESUMEN
Diseases of the central nervous system (CNS) are challenging to treat, mainly due to the blood-brain barrier (BBB), which restricts drugs in circulation from entering target regions in the brain. To address this issue extracellular vesicles (EVs) have gained increasing scientific interest as carriers able to cross the BBB with multiplex cargos. EVs are secreted by virtually every cell, and their escorted biomolecules are part of an intercellular information gateway between cells within the brain and with other organs. Scientists have undertaken efforts to safeguard the inherent features of EVs as therapeutic delivery vehicles, such as protecting and transferring functional cargo, as well as loading them with therapeutic small molecules, proteins, and oligonucleotides and targeting them to specific cell types for the treatment of CNS diseases. Here, we review current emerging approaches that engineer the EV surface and cargo to improve targeting and functional responses in the brain. We summarize existing applications of engineered EVs as a therapeutic delivery platform for brain diseases, some of which have been evaluated clinically.
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Encefalopatías , Vesículas Extracelulares , Humanos , Sistema Nervioso Central , Encéfalo , Vesículas Extracelulares/metabolismo , Encefalopatías/metabolismo , Barrera Hematoencefálica , Sistemas de Liberación de MedicamentosRESUMEN
Tools to effectively demonstrate and quantify functional delivery in cellular communication have been lacking. This study reports the use of a fluorescently labeled split Nanoluc reporter system to demonstrate and quantify functional transfer between cells in vitro and in a subcutaneous tumor mouse model. Our construct allows monitoring of direct, indirect, and specifically extracellular vesicle-mediated functional communication.
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Vesículas Extracelulares , Ratones , Animales , Vesículas Extracelulares/genética , Luciferasas/genética , Comunicación Celular , ComunicaciónRESUMEN
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.
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Myeloid cells are abundant, create a highly immunosuppressive environment in glioblastoma (GBM), and thus contribute to poor immunotherapy responses. Based on the hypothesis that small molecules can be used to stimulate myeloid cells to elicit anti-tumor effector functions, a synthetic nanoparticle approach is developed to deliver dual NF-kB pathway-inducing agents into these cells via systemic administration. Synthetic, cyclodextrin-adjuvant nanoconstructs (CANDI) with high affinity for tumor-associated myeloid cells are dually loaded with a TLR7 and 8 (Toll-like receptor, 7 and 8) agonist (R848) and a cIAP (cellular inhibitor of apoptosis protein) inhibitor (LCL-161) to dually activate these myeloid cells. Here CANDI is shown to: i) readily enter the GBM tumor microenvironment (TME) and accumulate at high concentrations, ii) is taken up by tumor-associated myeloid cells, iii) potently synergize payloads compared to monotherapy, iv) activate myeloid cells, v) fosters a "hot" TME with high levels of T effector cells, and vi) controls the growth of murine GBM as mono- and combination therapies with anti-PD1. Multi-pathway targeted myeloid stimulation via the CANDI platform can efficiently drive anti-tumor immunity in GBM.
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Neoplasias Encefálicas , Glioblastoma , Ratones , Animales , Glioblastoma/patología , Inmunoterapia , Células Mieloides/metabolismo , Células Mieloides/patología , Adyuvantes Inmunológicos , Microambiente Tumoral , Neoplasias Encefálicas/patologíaRESUMEN
Loss of function of the neurofibromatosis type 2 (NF2) tumor suppressor gene leads to the formation of schwannomas, meningiomas, and ependymomas, comprising â¼50% of all sporadic cases of primary nervous system tumors. NF2 syndrome is an autosomal dominant condition, with bi-allelic inactivation of germline and somatic alleles resulting in loss of function of the encoded protein merlin and activation of mammalian target of rapamycin (mTOR) pathway signaling in NF2-deficient cells. Here we describe a gene replacement approach through direct intratumoral injection of an adeno-associated virus vector expressing merlin in a novel human schwannoma model in nude mice. In culture, the introduction of an AAV1 vector encoding merlin into CRISPR-modified human NF2-null arachnoidal cells (ACs) or Schwann cells (SCs) was associated with decreased size and mTORC1 pathway activation consistent with restored merlin activity. In vivo, a single injection of AAV1-merlin directly into human NF2-null SC-derived tumors growing in the sciatic nerve of nude mice led to regression of tumors over a 10-week period, associated with a decrease in dividing cells and an increase in apoptosis, in comparison with vehicle. These studies establish that merlin re-expression via gene replacement in NF2-null schwannomas is sufficient to cause tumor regression, thereby potentially providing an effective treatment for NF2.
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Non-coding RNAs, including microRNAs (miRNAs), support the progression of glioma. miR-21 is a small, non-coding transcript involved in regulating gene expression in multiple cellular pathways, including the regulation of proliferation. High expression of miR-21 has been shown to be a major driver of glioma growth. Manipulating the expression of miRNAs is a novel strategy in the development of therapeutics in cancer. In this study we aimed to target miR-21. Using CRISPR genome-editing technology, we disrupted the miR-21 coding sequences in glioma cells. Depletion of this miRNA resulted in the upregulation of many downstream miR-21 target mRNAs involved in proliferation. Phenotypically, CRISPR-edited glioma cells showed reduced migration, invasion, and proliferation in vitro. In immunocompetent mouse models, miR-21 knockout tumors showed reduced growth resulting in an increased overall survival. In summary, we show that by knocking out a key miRNA in glioma, these cells have decreased proliferation capacity both in vitro and in vivo. Overall, we identified miR-21 as a potential target for CRISPR-based therapeutics in glioma.
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Cell membrane-based biovesicles (BVs) are important candidate drug delivery vehicles and comprise extracellular vesicles, virus-like particles, and lentiviral vectors. Here, we introduce a non-enzymatic assembly of purified BVs, supercharged proteins, and plasmid DNA called pDNA-scBVs. This multicomponent vehicle results from the interaction of negative sugar moieties on BVs and supercharged proteins that contain positively charged amino acids on their surface to enhance their affinity for pDNA. pDNA-scBVs were demonstrated to mediate floxed reporter activation in culture by delivering a Cre transgene. We introduced pDNA-scBVs containing both a CRE-encoding plasmid and a BV-packaged floxed reporter into the brains of Ai9 mice. Successful delivery of both payloads by pDNA-scBVs was confirmed with reporter signal in the striatal brain region. Overall, we developed a more efficient method to load isolated BVs with cargo that functionally modified recipient cells. Augmenting the natural properties of BVs opens avenues for adoptive extracellular interventions using therapeutic loaded cargo.
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ADN , Vesículas Extracelulares , Animales , ADN/genética , Sistemas de Liberación de Medicamentos , Vesículas Extracelulares/metabolismo , Ratones , Plásmidos , TransgenesRESUMEN
Extracellular vesicles (EVs) are membrane-encapsulated particles that carry genetically active and protein/lipid cargo that can affect the function of the recipient cell. A number of studies have described the effect of these vesicles on recipient cells and demonstrated their promise as therapeutic delivery vectors. Here we demonstrate functional delivery of EV-encapsulated RNA and protein cargo through use of luminescence and fluorescence reporters by combining organelle-targeted nanoluciferase with fluorescent proteins. We highlight a mechanism by which cells retain internalized cargo in the endosomal compartment for days, usually leading to content degradation. We also identify a mode through which recipient cells re-release internalized EVs intact after uptake. Highlighting these different fates of EVs in recipient cells sheds light on critical factors in steering functional cargo delivery and will ultimately allow more efficient use of EVs for therapeutic purposes.
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Vesículas Extracelulares , Transporte Biológico , Comunicación Celular/genética , Endosomas/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas/metabolismo , ARN/metabolismoRESUMEN
The lack of techniques to trace brain cell behavior in vivo hampers the ability to monitor status of cells in a living brain. Extracellular vesicles (EVs), nanosized membrane-surrounded vesicles, released by virtually all brain cells might be able to report their status in easily accessible biofluids, such as blood. EVs communicate among tissues using lipids, saccharides, proteins, and nucleic acid cargo that reflect the state and composition of their source cells. Currently, identifying the origin of brain-derived EVs has been challenging, as they consist of a rare population diluted in an overwhelming number of blood and peripheral tissue-derived EVs. Here, we developed a sensitive platform to select out pre-labelled brain-derived EVs in blood as a platform to study the molecular fingerprints of brain cells. This proof-of-principle study used a transducible construct tagging tetraspanin (TSN) CD63, a membrane-spanning hallmark of EVs equipped with affinity, bioluminescent, and fluorescent tags to increase detection sensitivity and robustness in capture of EVs secreted from pre-labelled cells into biofluids. Our platform enables unprecedented efficient isolation of neural EVs from the blood. These EVs derived from pre-labelled mouse brain cells or engrafted human neuronal progenitor cells (hNPCs) were submitted to multiplex analyses, including transcript and protein levels, in compliance with the multibiomolecule EV carriers. Overall, our novel strategy to track brain-derived EVs in a complex biofluid opens up new avenues to study EVs released from pre-labelled cells in near and distal compartments into the biofluid source.
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Vesículas Extracelulares , Animales , Fenómenos Biofísicos , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismo , Ratones , Tetraspaninas/metabolismoRESUMEN
The central nervous system (CNS) consists of a heterogeneous population of cells with highly specialized functions. For optimal functioning of the CNS, in disease and in health, intricate communication between these cells is vital. One important mechanism of cellular communication is the release and uptake of extracellular vesicles (EVs). EVs are membrane enclosed particles actively released by cells, containing a wide array of proteins, lipids, RNA, and DNA. These EVs can be taken up by neighboring or distant cells, and influence a wide range of processes. Due to the complexity and relative inaccessibility of the CNS, our current understanding of the role of EVs is mainly derived in vitro work. However, recently new methods and techniques have opened the ability to study the role of EVs in the CNS in vivo. In this review, we discuss the current developments in our understanding of the role of EVs in the CNS in vivo.