RESUMO
BACKGROUND: Glioblastomas are the most common and lethal primary brain tumors. Microglia, the resident immune cells of the brain, survey their environment and respond to pathogens, toxins, and tumors. Glioblastoma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Despite the presence of large numbers of microglia in glioblastoma, the tumors continue to grow, and these neuroimmune cells appear incapable of keeping the tumor in check. To understand this process, we analyzed gene expression in microglia interacting with glioblastoma cells. METHODS: We used RNASeq of isolated microglia to analyze the expression patterns of genes involved in key microglial functions in mice with glioblastoma. We focused on microglia that had taken up tumor-derived EVs and therefore were within and immediately adjacent to the tumor. RESULTS: We show that these microglia have downregulated expression of genes involved in sensing tumor cells and tumor-derived danger signals, as well as genes used for tumor killing. In contrast, expression of genes involved in facilitating tumor spread was upregulated. These changes appear to be in part EV-mediated, since intracranial injection of EVs in normal mice led to similar transcriptional changes in microglia. We observed a similar microglial transcriptomic signature when we analyzed datasets from human patients with glioblastoma. CONCLUSION: Our data define a microgliaGlioblastoma specific phenotype, whereby glioblastomas have hijacked gene expression in the neuroimmune system to favor avoiding tumor sensing, suppressing the immune response, clearing a path for invasion, and enhancing tumor propagation. For further exploration, we developed an interactive online tool at http://www.glioma-microglia.com with all expression data and additional functional and pathway information for each gene.
Assuntos
Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Microglia/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Feminino , Técnicas de Introdução de Genes/métodos , Glioblastoma/genética , Glioblastoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Carga Tumoral/fisiologiaRESUMO
BACKGROUND: Anti-psychotics are often prescribed off-licence for personality and affective disorders. The aim of this audit was to identify patients on specific wards within St Andrews Hospital and to determine how many of the Glasgow Antipsychotic Side-Effect Scale (GASS) forms had been completed as per policy. METHODS: Thirteen patients of Dr Boris Iankov, Consultant Psychiatrist within the adolescent service at St Andrews, were identified. Their records were subsequently reviewed in order to determine how many had completed GASS forms. RESULTS: It was found only 31% of patients had completed the GASS forms. The remaining patients were supported to do so. As a result 92% of patients are compliant with the policy. RECOMMENDATIONS: The suitability of the form for adolescents was brought into question due to the sensitive nature of certain side effects. Furthermore the form should be completed routinely on admission and at regular intervals.
Assuntos
Antipsicóticos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Adolescente , Antipsicóticos/efeitos adversos , Hospitalização , Humanos , Transtornos do Humor/tratamento farmacológico , Cooperação do PacienteRESUMO
Extracellular vesicles present an attractive delivery vehicle for therapeutic proteins. They intrinsically contain many proteins which can provide information to other cells. Advantages include reduced immune reactivity, especially if derived from the same host, stability in biologic fluids, and ability to target uptake. Those from mesenchymal stem cells appear to be intrinsically therapeutic, while those from cancer cells promote tumor progression. Therapeutic proteins can be loaded into vesicles by overexpression in the donor cell, with oligomerization and membrane sequences increasing their loading. Examples of protein delivery for therapeutic benefit in pre-clinical models include delivery of: catalase for Parkinson's disease to reduce oxidative stress and thus help neurons to survive; prodrug activating enzymes which can convert a prodrug which crosses the blood-brain barrier into a toxic chemotherapeutic drug for schwannomas and gliomas; and the apoptosis-inducing enzyme, caspase-1 under a Schwann cell specific promoter for schwannoma. This therapeutic delivery strategy is novel and being explored for a number of diseases.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Vesículas Extracelulares/metabolismo , Glioma/tratamento farmacológico , Neurilemoma/tratamento farmacológico , Doença de Parkinson/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Humanos , Neurilemoma/metabolismo , Neurilemoma/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologiaRESUMO
In recent years, mGlu4 has received great research attention because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinson's disease (PD). A specific mGlu4 PET radioligand could be an important tool in understanding the role of mGlu4 in both healthy and disease conditions, and also for the development of new drugs. In this study, we synthesized four new N-(methylthiophenyl)picolinamide derivatives 11-14. Of these ligands, 11 and 14 showed high in vitro binding affinity for mGlu4 with IC50 values of 3.4nM and 3.1nM, respectively, and suitable physicochemical parameters. Compound 11 also showed enhanced metabolic stability and good selectivity to other mGluRs. [(11)C]11 and [(11)C]14 were radiolabeled using the [(11)C]methylation of the thiophenol precursors 20a and 20c with [(11)C]CH3I in 19.0% and 34.8% radiochemical yields (RCY), and their specific activities at the end of synthesis (EOS) were 496±138GBq/µmol (n=6) and 463±263GBq/µmol (n=4), respectively. The PET studies showed that [(11)C]11 accumulated fast into the brain and had higher uptake, slower washout and 25% better contrast than [(11)C]2, indicating improved imaging characteristics as PET radiotracer for mGlu4 compared to [(11)C]2. Therefore, [(11)C]11 will be a useful radioligand to investigate mGlu4 in different biological applications.
Assuntos
Ácidos Picolínicos/síntese química , Ácidos Picolínicos/metabolismo , Tomografia por Emissão de Pósitrons , Ensaio Radioligante , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Tiofenos/síntese química , Tiofenos/metabolismo , Animais , Relação Dose-Resposta a Droga , Ligantes , Masculino , Estrutura Molecular , Ácidos Picolínicos/química , Ligação Proteica , Compostos Radiofarmacêuticos/análise , Compostos Radiofarmacêuticos/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Especificidade por Substrato , Tiofenos/químicaRESUMO
X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was achieved in primary mixed brain glial cells from Abcd1-/- mice as well as X-ALD patient fibroblasts. Importantly, human ABCD1 localized to the peroxisome, and AAV-ABCD1 transduction showed a dose-dependent effect in reducing VLCFA. In vivo, AAV9-ABCD1 was delivered to Abcd1-/- mouse CNS by either stereotactic intracerebroventricular (ICV) or intravenous (IV) injections. Astrocytes, microglia and neurons were the major target cell types following ICV injection, while IV injection also delivered to microvascular endothelial cells and oligodendrocytes. IV injection also yielded high transduction of the adrenal gland. Importantly, IV injection of AAV9-ABCD1 reduced VLCFA in mouse brain and spinal cord. We conclude that AAV9-mediated ABCD1 gene transfer is able to reach target cells in the nervous system and adrenal gland as well as reduce VLCFA in culture and a mouse model of X-ALD.
Assuntos
Adrenoleucodistrofia/genética , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Transdução Genética , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adrenoleucodistrofia/terapia , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Dependovirus/classificação , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/administração & dosagem , Glutationa Peroxidase/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Neuroglia/metabolismo , Transporte Proteico , Sorogrupo , Glutationa Peroxidase GPX1RESUMO
We examined the potential benefit of gene therapy in a mouse model of tuberous sclerosis complex (TSC) in which there is embryonic loss of Tsc1 (hamartin) in brain neurons. An adeno-associated virus (AAV) vector (serotype rh8) expressing a tagged form of hamartin was injected into the cerebral ventricles of newborn pups with the genotype Tsc1(cc) (homozygous for a conditional floxed Tsc1 allele) SynI-cre(+), in which Tsc1 is lost selectively in neurons starting at embryonic day 12. Vector-treated Tsc1(cc)SynIcre(+) mice showed a marked improvement in survival from a mean of 22 days in non-injected mice to 52 days in AAV hamartin vector-injected mice, with improved weight gain and motor behavior in the latter. Pathologic studies showed normalization of neuron size and a decrease in markers of mTOR activation in treated as compared to untreated mutant littermates. Hence, we show that gene replacement in the brain is an effective therapeutic approach in this mouse model of TSC1. Our strategy for gene therapy has the advantages that therapy can be achieved from a single application, as compared to repeated treatment with drugs, and that AAV vectors have been found to have minimal to no toxicity in clinical trials for other neurologic conditions. Although there are many additional issues to be addressed, our studies support gene therapy as a useful approach in TSC patients.
Assuntos
Encéfalo/patologia , Terapia Genética/métodos , Esclerose Tuberosa/terapia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/uso terapêutico , Animais , Modelos Animais de Doenças , Camundongos , Mutação , Neurônios/patologia , Fenótipo , Resultado do Tratamento , Esclerose Tuberosa/genética , Esclerose Tuberosa/patologia , Proteína 1 do Complexo Esclerose TuberosaRESUMO
[This corrects the article DOI: 10.1016/j.omtm.2022.06.012.].
RESUMO
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.
RESUMO
Adeno-associated virus (AAV)-based gene therapy is gaining popularity owing to its excellent safety profile and effective therapeutic outcomes in a number of diseases. Intravenous (IV) injection of AAV into the tail vein, facial vein and retro-orbital (RO) venous sinus have all been useful strategies to infuse the viral vector systemically. However, tail vein injection is technically challenging in juvenile mice, and injection at young ages (≤ postnatal day-(P)21) is essentially impossible. The temporal or facial vein is localized anterior to the ear bud and is markedly visible in the first couple of days postnatally. However, this method is age-dependent and requires a dissecting microscope. Retro-orbital injection (ROI), on the other hand, is suitable for all murine ages, including newborn and older mice, and is relatively less stressful to animals compared to tail vein injection. Although many reports have shown ROI as an effective route of AAV delivery, herein we aim to highlight and summarize the methods and benefits of ROI. To capture the full spectrum of transduction efficiency mediated by ROI, we transduced the editing-dependent reporter mice (Ai9 Cre reporter mice) with the AAV9 vector, which targets a wide range of peripheral tissues with exceptional brain tropism. We also provide a comprehensive description of the ROI technique to facilitate viral vector administration without complications.
Assuntos
Animais Recém-Nascidos , Dependovirus/fisiologia , Transdução Genética , Animais , Vetores Genéticos , Injeções/classificação , CamundongosRESUMO
Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and proliferation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a "condensed" form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demonstrates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.
RESUMO
Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome caused by mutations in TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins act as a complex that inhibits mammalian target of rapamycin (mTOR)-mediated cell growth and proliferation. Loss of either protein leads to overgrowth in many organs, including subependymal nodules, subependymal giant cell astrocytomas, and cortical tubers in the human brain. Neurological manifestations in TSC include intellectual disability, autism, hydrocephalus, and epilepsy. In a stochastic mouse model of TSC1 brain lesions, complete loss of Tsc1 is achieved in homozygous Tsc1-floxed mice in a subpopulation of neural cells in the brain by intracerebroventricular (i.c.v.) injection at birth of an adeno-associated virus (AAV) vector encoding Cre recombinase. This results in median survival of 38 days and brain pathology, including subependymal lesions and enlargement of neuronal cells. Remarkably, when these mice were injected intravenously on day 21 with an AAV9 vector encoding hamartin, most survived at least up to 429 days in apparently healthy condition with marked reduction in brain pathology. Thus, a single intravenous administration of an AAV vector encoding hamartin restored protein function in enough cells in the brain to extend lifespan in this TSC1 mouse model.
RESUMO
Gene therapy for schwannomas was evaluated in two mouse models of neurofibromatosis type 2 (NF2): (1) a transgenic model in which mice express a dominant mutant form of merlin and spontaneously develop schwannomas, and (2) a xenograft model in which human schwannoma tissue is implanted subcutaneously into immune- compromised mice. In both models, schwannoma volumes were monitored by magnetic resonance imaging (MRI) and showed strong gadolinium enhancement typical of these tumors in humans. Both types of tumor were positive for the Schwann cell marker S100, and highly infectable with herpes simplex virus (HSV) vectors. Schwannomas were injected with an oncolytic HSV-1 recombinant virus vector, G47Delta, which has deletions in genes for ribonucleotide reductase (ICP6), gamma34.5, and ICP47. In the NF2 transgenic model, schwannomas were reduced by more than half their original size by 10 days after infection. In the case of subcutaneous schwannoma xenografts, reduction in size after infection occurred more slowly, with a mean reduction of onethird by 42 days after treatment. Schwannomas injected with control vehicles continued to grow slowly over time in both schwannoma models. These studies demonstrate the ability of an oncolytic recombinant HSV vector to reduce the volume of schwannoma tumors in NF2 tumor models in mice and extend the possible therapeutic applications of oncolytic vectors for benign tumors to reduce mass while minimizing nerve damage.
Assuntos
Neoplasias do Sistema Nervoso Central/terapia , Terapia Genética/métodos , Neurilemoma/terapia , Neurofibromatose 2/terapia , Simplexvirus/fisiologia , Animais , Neoplasias do Sistema Nervoso Central/patologia , Neoplasias do Sistema Nervoso Central/virologia , DNA Recombinante/genética , Modelos Animais de Doenças , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Vetores Genéticos/toxicidade , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Transplante de Neoplasias , Neurilemoma/classificação , Neurilemoma/patologia , Neurilemoma/virologia , Neurofibromatose 2/patologia , Neurofibromatose 2/virologia , Simplexvirus/genéticaRESUMO
BACKGROUND: To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo. METHODS: Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels. RESULTS: Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA. CONCLUSIONS: Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs.
Assuntos
Neoplasias Encefálicas/metabolismo , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Microglia/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Tuberous sclerosis (TSC) is an autosomal dominant genetic disorder characterized by abnormalities in cellular migration, proliferation, and differentiation in many tissues. Benign hamartomas develop in multiple organs, believed to be caused by somatic mutation in addition to germ line mutation to cause loss of both alleles of either the TSC1 or TSC2 tumor suppressor gene, with resultant dysregulated growth due to loss of hamartin or tuberin function, respectively. This study focuses on detecting spontaneous lesions in a knockout mouse model of TSC2 by magnetic resonance imaging (MRI) and exploring the efficiency of introducing gene products into lesions, using transduced endothelial cells as gene vehicles. MRI was shown to be effective in detecting spontaneous lesions in multiple tissues as a means of assessing the prevalence of tumors. Tsc(2+/) heterozygous mice were screened at 12-24 months of age. MRI detected 100% of the renal lesions (cystadenomas, renal cell carcinomas) and 75% of the hepatic lesions (hemangiosarcomas), later identified by histology. Cell-mediated gene delivery was evaluated by immunohistochemical analysis of renal, hepatic, and lung lesions after intravenous delivery of MS1 mouse endothelial cells, transduced to express an enhanced form of green fluorescent protein (EGFP). Preliminary immunohistochemical analysis, using a polyclonal antibody to EGFP and a horseradish peroxidase-diaminobenzidine detection system, revealed these cells throughout liver, kidney, and lung sections from injected animals, organs that are frequently affected in TSC2 patients, as well as within the lesions themselves.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Esclerose Tuberosa/patologia , Esclerose Tuberosa/terapia , Proteínas Supressoras de Tumor/genética , Animais , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Células Endoteliais , Genes Supressores de Tumor , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Neoplasias Renais/patologia , Pulmão/patologia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Transdução Genética , Transgenes , Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismoRESUMO
Here we describe a novel method for imaging apoptosis in cells using a near-infrared fluorescent (NIRF) probe selective for caspase-1 (interleukin 1beta-converting enzyme, ICE). This biocompatible, optically quenched ICE-NIRF probe incorporates a peptide substrate, which can be selectively cleaved by caspase-1, resulting in the release of fluorescence signal. The specificity of this probe for caspase-1 is supported by various lines of evidence: 1) activation by purified caspase-1, but not another caspase in vitro; 2) activation of the probe by infection of cells with a herpes simplex virus amplicon vector (HGC-ICE-lacZ) expressing a catalytically active caspase-1-lacZ fusion protein; 3) inhibition of HGC-ICE-lacZ vector-induced activation of the probe by coincubation with the caspase-1 inhibitor YVAD-cmk, but not with a caspase-3 inhibitor; and 4) activation of the probe following standard methods of inducing apoptosis with staurosporine, ganciclovir, or ionizing radiation in culture. These results indicate that this novel ICE-NIRF probe can be used in monitoring endogenous and vector-expressed caspase-1 activity in cells. Furthermore, tumor implant experiments indicate that this ICE-NIRF probe can be used to detect caspase-1 activity in living animals. This novel ICE-NIRF probe should prove useful in monitoring endogenous and vector-expressed caspase-1 activity, and potentially apoptosis in cell culture and in vivo.
Assuntos
Apoptose/fisiologia , Caspase 1/metabolismo , Corantes Fluorescentes , Glioma/patologia , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Ganciclovir/farmacologia , Glioma/metabolismo , Humanos , Radiação Ionizante , Ratos , Simplexvirus/genética , Estaurosporina/farmacologiaRESUMO
Extracellular vesicles (EVs) are nanosized vesicles released by normal and diseased cells as a novel form of intercellular communication and can serve as an effective therapeutic vehicle for genes and drugs. Yet, much remains unknown about the in vivo properties of EVs such as tissue distribution, blood levels, and urine clearance, important parameters that will define their therapeutic effectiveness and potential toxicity. Here we combined Gaussia luciferase and metabolic biotinylation to create a sensitive EV reporter (EV-GlucB) for multimodal imaging in vivo, as well as monitoring of EV levels in the organs and biofluids ex vivo after administration of EVs. Bioluminescence and fluorescence-mediated tomography imaging on mice displayed a predominant localization of intravenously administered EVs in the spleen followed by the liver. Monitoring EV signal in the organs, blood, and urine further revealed that the EVs first undergo a rapid distribution phase followed by a longer elimination phase via hepatic and renal routes within six hours, which are both faster than previously reported using dye-labeled EVs. Moreover, we demonstrate systemically injected EVs can be delivered to tumor sites within an hour following injection. Altogether, we show the EVs are dynamically processed in vivo with accurate spatiotemporal resolution and target a number of normal organs as well as tumors with implications for disease pathology and therapeutic design.
Assuntos
Genes Reporter , Vetores Genéticos , Imagem Multimodal , Animais , Células HEK293 , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Distribuição TecidualRESUMO
Schwannomas are tumors formed by proliferation of dedifferentiated Schwann cells. Patients with neurofibromatosis 2 (NF2) and schwannomatosis develop multiple schwannomas in peripheral and cranial nerves. Although benign, these tumors can cause extreme pain and compromise sensory/motor functions, including hearing and vision. At present, surgical resection is the main treatment modality, but it can be problematic because of tumor inaccessibility and risk of nerve damage. We have explored gene therapy for schwannomas, using a model in which immortalized human NF2 schwannoma cells expressing a fluorescent protein and luciferase are implanted in the sciatic nerve of nude mice. Direct injection of an adeno-associated virus (AAV) serotype 1 vector encoding caspase-1 (ICE) under the Schwann-cell specific promoter, P0, leads to regression of these tumors with essentially no vector-mediated neuropathology, and no changes in sensory or motor function. In a related NF2 xenograft model designed to cause measurable pain behavior, the same gene therapy leads to tumor regression and concordant resolution of tumor-associated pain. This AAV1-P0-ICE vector holds promise for clinical treatment of schwannomas by direct intratumoral injection to achieve reduction in tumor size and normalization of neuronal function.
Assuntos
Caspase 1/administração & dosagem , Dependovirus/metabolismo , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Neurilemoma/terapia , Células de Schwann/patologia , Animais , Caspase 1/genética , Caspase 1/metabolismo , Dependovirus/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neurilemoma/metabolismo , Neurilemoma/prevenção & controle , Neurofibromatose 2/patologia , Neurofibromatose 2/terapia , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Desempenho Psicomotor , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Transgenes , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to mutations in either TSC1 or TSC2 that affects many organs with hamartomas and tumors. TSC-associated brain lesions include subependymal nodules, subependymal giant cell astrocytomas and tubers. Neurologic manifestations in TSC comprise a high frequency of mental retardation and developmental disorders including autism, as well as epilepsy. Here, we describe a new mouse model of TSC brain lesions in which complete loss of Tsc1 is achieved in multiple brain cell types in a stochastic pattern. Injection of an adeno-associated virus vector encoding Cre recombinase into the cerebral ventricles of mice homozygous for a Tsc1 conditional allele on the day of birth led to reduced survival, and pathologic findings of enlarged neurons, cortical heterotopias, subependymal nodules, and hydrocephalus. The severity of clinical and pathologic findings as well as survival was shown to be dependent upon the dose and serotype of Cre virus injected. Although several other models of TSC brain disease exist, this model is unique in that the pathology reflects a variety of TSC-associated lesions involving different numbers and types of cells. This model provides a valuable and unique addition for therapeutic assessment.
Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Modelos Animais de Doenças , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Mutantes , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
Benign schwannomas are common tumors of the cranial and peripheral nerves, causing pain and loss of function. The development of effective therapy for these tumors has been hampered by the lack of relevant experimental in vivo models for convenient testing. Here, we describe a novel schwannoma model in which an immortalized human schwannoma cell line, HEI-193, established from an neurofibromatosis type 2 patient, has been stably transduced with fluorescent protein and luciferase reporters and implanted within the sciatic nerve of nude mice. These cells reliably formed a tumor within several weeks which had pathologic characteristics of schwannoma tumors. This model system will be useful for investigation of schwannoma biology and for preclinical assessment of therapeutic agents.