ABSTRACT
CD19-targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors. Here, we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short hairpin RNA cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR T cells in the context of four different checkpoint combinations-PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4, and PD-1/TIGIT-and found that CAR T cells with PD-1/TIGIT downregulation uniquely exerted synergistic antitumor effects. Importantly, functional and phenotypic analyses suggested that downregulation of PD-1 enhances short-term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD-1/TIGIT-downregulated CAR T cells generated from diffuse large B cell lymphoma patient-derived T cells also showed robust antitumor activity and significantly improved persistence in vivo. The efficacy and safety of PD-1/TIGIT-downregulated CD19-targeting CAR T cells are currently being evaluated in adult patients with relapsed or refractory large B cell lymphoma (ClinicalTrials.gov: NCT04836507).
Subject(s)
Lymphoma, Large B-Cell, Diffuse , Programmed Cell Death 1 Receptor , Antigens, CD19 , Down-Regulation , Humans , Immunotherapy, Adoptive , Phenotype , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , T-LymphocytesABSTRACT
Traditional approaches to studying Alzheimer's disease (AD) using mouse models and cell lines have advanced our understanding of AD pathogenesis. However, with the growing divide between model systems and clinical therapeutic outcomes, the limitations of these approaches are increasingly apparent. Thus, to generate more clinically relevant systems that capture pathological cascades within human neurons, we generated human-induced neurons (HiNs) from AD and non-AD individuals to model cell autonomous disease properties. We selected an AD patient population expressing mutations in presenilin 1 (mPS1), which is linked to increased amyloid production, tau pathology, and calcium signaling abnormalities, among other features. While these AD components are detailed in model systems, they have yet to be collectively identified in human neurons. Thus, we conducted molecular, immune-based, electrophysiological, and calcium imaging studies to establish patterns of cellular pathology in this patient population. We found that mPS1 HiNs generate increased Aß42 and hyperphosphorylated tau species relative to non-AD controls, and exaggerated ER calcium responses that are normalized with ryanodine receptor (RyR) negative allosteric modulators. The inflammasome product, interleukin-18 (IL-18), also increased PS1 expression. This work highlights the potential for HiNs to model AD pathology and validates their role in defining cellular pathogenesis and their utility for therapeutic screening.
Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/pathology , Mutation/genetics , Neurons/pathology , Presenilin-1/genetics , Allosteric Regulation/physiology , Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Calcium/metabolism , Calcium Signaling/physiology , Endoplasmic Reticulum/metabolism , Humans , Inflammasomes/genetics , Inflammasomes/metabolism , Interleukin-18/metabolism , Neurons/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , tau Proteins/metabolismABSTRACT
Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR-T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens.
Subject(s)
Antigens, CD19/immunology , Antigens, Neoplasm/immunology , Immunotherapy, Adoptive/methods , Leukemia, B-Cell/therapy , Receptors, Antigen, T-Cell/immunology , Sialic Acid Binding Ig-like Lectin 2/immunology , T-Cell Antigen Receptor Specificity , T-Lymphocyte Subsets/immunology , Animals , Azides , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Basic-Leucine Zipper Transcription Factors/immunology , Cell Line, Tumor , Cytokines/metabolism , Cytotoxicity, Immunologic , Dose-Response Relationship, Immunologic , Female , Genes, Reporter , Genetic Vectors , Humans , Immunotherapy, Adoptive/adverse effects , Lymphocyte Activation , Lymphopenia/etiology , Lymphopenia/prevention & control , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Phenylalanine/analogs & derivatives , Protein Engineering/methods , Receptors, Antigen, T-Cell/genetics , Recombinant Fusion Proteins/immunology , Saccharomyces cerevisiae Proteins/immunology , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Structure-Activity Relationship , T-Lymphocyte Subsets/transplantation , Xenograft Model Antitumor AssaysABSTRACT
The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR-T--cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as "switch" molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a "universal" anti-FITC-directed CAR-T cell. Optimization of this CAR-switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR-T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy.
Subject(s)
Antigens, CD19/immunology , Antigens, Neoplasm/immunology , Immunotherapy, Adoptive/methods , Leukemia, B-Cell/therapy , Protein Engineering/methods , Receptors, Antigen, T-Cell/immunology , Sialic Acid Binding Ig-like Lectin 2/immunology , T-Cell Antigen Receptor Specificity , T-Lymphocytes/immunology , Animals , Azides , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Line, Tumor , Cytotoxicity, Immunologic , Female , Fluorescein-5-isothiocyanate , Genetic Vectors , Humans , Immunotherapy, Adoptive/adverse effects , Lentivirus/genetics , Lymphocyte Activation , Lymphopenia/etiology , Lymphopenia/prevention & control , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Models, Molecular , Phenylalanine/analogs & derivatives , Protein Conformation , Receptors, Antigen, T-Cell/genetics , Recombinant Fusion Proteins/immunology , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , T-Lymphocytes/transplantation , Transduction, Genetic , Xenograft Model Antitumor AssaysABSTRACT
Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.
Subject(s)
Cell Dedifferentiation/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Endogenous Retroviruses/genetics , Pluripotent Stem Cells/cytology , Totipotent Stem Cells/cytology , Totipotent Stem Cells/metabolism , Animals , Cell Dedifferentiation/physiology , Cell Lineage/genetics , Chimera/embryology , Chromatin/genetics , Chromatin/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryo, Mammalian/virology , Embryonic Stem Cells/virology , Epigenesis, Genetic , Female , Gene Expression Regulation, Developmental , Genes, Reporter/genetics , Histones/chemistry , Histones/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Lysine/chemistry , Lysine/metabolism , Methylation , Mice , Phenotype , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/virology , Terminal Repeat Sequences/genetics , Totipotent Stem Cells/virology , Transcriptome/geneticsABSTRACT
Despite therapeutic advancement, pulmonary disease still remains a major cause of morbidity and mortality around the world. Opportunities to study human lung disease either in vivo or in vitro are currently limited. Using induced pluripotent stem cells (iPSCs), we generated mature multiciliated cells in a functional airway epithelium. Robust multiciliogenesis occurred when notch signaling was inhibited and was confirmed by (i) the assembly of multiple pericentrin-stained centrioles at the apical surface, (ii) expression of transcription factor forkhead box protein J1, and (iii) presence of multiple acetylated tubulin-labeled cilia projections in individual cells. Clara, goblet, and basal cells were all present, confirming the generation of a complete polarized epithelial-cell layer. Additionally, cAMP-activated and cystic fibrosis transmembrane regulator inhibitor 172-sensitive cystic fibrosis transmembrane regulator currents were recorded in isolated epithelial cells. Our report demonstrating the generation of mature multiciliated cells in respiratory epithelium from iPSCs is a significant advance toward modeling a number of human respiratory diseases in vitro.
Subject(s)
Cilia/metabolism , Epithelial Cells/cytology , Epithelium/metabolism , Induced Pluripotent Stem Cells/cytology , Lung/cytology , Cell Differentiation , Cell Membrane/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Endoderm/cytology , Epithelial Cells/metabolism , Humans , Receptors, Notch/metabolism , Signal TransductionABSTRACT
The choroid plexus (CP) plays a key role in remotely controlling brain function in health, aging, and disease. Here, we report that CP epithelial cells express the brain-specific cholesterol 24-hydroxylase (CYP46A1) and that its levels are decreased under different mouse and human brain conditions, including amyloidosis, aging, and SARS-CoV-2 infection. Using primary mouse CP cell cultures, we demonstrate that the enzymatic product of CYP46A1, 24(S)-hydroxycholesterol, downregulates inflammatory transcriptomic signatures within the CP, found here to be elevated across multiple neurological conditions. In vitro, the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) downregulates CYP46A1 expression, while overexpression of CYP46A1 or its pharmacological activation in mouse CP organ cultures increases resilience to TNF-α. In vivo, overexpression of CYP46A1 in the CP in transgenic mice with amyloidosis is associated with better cognitive performance and decreased brain inflammation. Our findings suggest that CYP46A1 expression in the CP impacts the role of this niche as a guardian of brain immune homeostasis.
Subject(s)
Amyloidosis , Choroid Plexus , Humans , Mice , Animals , Cholesterol 24-Hydroxylase/metabolism , Choroid Plexus/metabolism , Tumor Necrosis Factor-alpha/metabolism , Brain/pathology , Homeostasis/physiology , Mice, Transgenic , Amyloidosis/metabolism , Amyloidosis/pathologyABSTRACT
Multiple studies have identified metabolic changes within the tumor and its microenvironment during carcinogenesis. Yet, the mechanisms by which tumors affect the host metabolism are unclear. We find that systemic inflammation induced by cancer leads to liver infiltration of myeloid cells at early extrahepatic carcinogenesis. The infiltrating immune cells via IL6-pSTAT3 immune-hepatocyte cross-talk cause the depletion of a master metabolic regulator, HNF4α, consequently leading to systemic metabolic changes that promote breast and pancreatic cancer proliferation and a worse outcome. Preserving HNF4α levels maintains liver metabolism and restricts carcinogenesis. Standard liver biochemical tests can identify early metabolic changes and predict patients' outcomes and weight loss. Thus, the tumor induces early metabolic changes in its macroenvironment with diagnostic and potentially therapeutic implications for the host. SIGNIFICANCE: Cancer growth requires a permanent nutrient supply starting from early disease stages. We find that the tumor extends its effect to the host's liver to obtain nutrients and rewires the systemic and tissue-specific metabolism early during carcinogenesis. Preserving liver metabolism restricts tumor growth and improves cancer outcomes. This article is highlighted in the In This Issue feature, p. 1501.
Subject(s)
Liver , Pancreatic Neoplasms , Humans , Liver/metabolism , Carcinogenesis/pathology , Hepatocytes , Pancreatic Neoplasms/pathology , Immunity, Innate , Tumor MicroenvironmentABSTRACT
Anxiety and fear are normal emotional responses to threatening situations. In human anxiety disorders--such as panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, social phobia, specific phobias and generalized anxiety disorder--these responses are exaggerated. The molecular mechanisms involved in the regulation of normal and pathological anxiety are mostly unknown. However, the availability of different inbred strains of mice offers an excellent model system in which to study the genetics of certain behavioural phenotypes. Here we report, using a combination of behavioural analysis of six inbred mouse strains with quantitative gene expression profiling of several brain regions, the identification of 17 genes with expression patterns that correlate with anxiety-like behavioural phenotypes. To determine if two of the genes, glyoxalase 1 and glutathione reductase 1, have a causal role in the genesis of anxiety, we performed genetic manipulation using lentivirus-mediated gene transfer. Local overexpression of these genes in the mouse brain resulted in increased anxiety-like behaviour, while local inhibition of glyoxalase 1 expression by RNA interference decreased the anxiety-like behaviour. Both of these genes are involved in oxidative stress metabolism, linking this pathway with anxiety-related behaviour.
Subject(s)
Anxiety/enzymology , Anxiety/genetics , Brain/metabolism , Gene Expression Profiling , Glutathione Reductase/metabolism , Lactoylglutathione Lyase/metabolism , Animals , Anxiety/physiopathology , Female , Gene Expression Regulation , Glutathione Reductase/genetics , Lactoylglutathione Lyase/genetics , Lentivirus/genetics , Male , Mice , Mice, Inbred Strains , Oxidative Stress/genetics , Phenotype , Transduction, GeneticABSTRACT
Many cancers and leukemias are associated with strong dominant oncogenic mutations that activate tyrosine kinases and other classes of molecules, including transcription factors and antiapoptotic mechanisms. Some of these events can be targeted with small molecules or antibody-based therapeutics, but many remain intractable. In addition, cancer-related enzyme targets can often mutate, and drug-resistant variants are selected. Therapies directed at the mRNA encoding dominant oncogenes could provide a more global set of technologies for cancer treatment. To test this concept, we have used the model of transformation of hematopoietic cells by the chimeric Bcr-Abl oncogene, a highly activated tyrosine kinase. Our results show that tandem arrays of miRNA mimics, but not single miRNA mimics, directed against the Abl portion of the mRNA and introduced by lentiviral vectors can effectively alter the leukemogenic potency when the degree of suppression of expression of Bcr-Abl is reduced >200-fold from control levels. Only methods capable of such dramatic sustained reduction in the level of expression of highly activated kinase oncogenes are likely to be effective in controlling malignant cell populations.
Subject(s)
Fusion Proteins, bcr-abl/antagonists & inhibitors , Genetic Therapy , Leukemia, Lymphoid/therapy , MicroRNAs/genetics , Animals , Apoptosis , Fusion Proteins, bcr-abl/genetics , Lentivirus/genetics , Mice , Mice, Inbred StrainsABSTRACT
The SARS-CoV2 coronavirus responsible for the current COVID19 pandemic has been reported to have a relatively low mutation rate. Nevertheless, a few prevalent variants have arisen that give the appearance of undergoing positive selection as they are becoming increasingly widespread over time. Most prominent among these is the D614G amino acid substitution in the SARS-CoV2 Spike protein, which mediates viral entry. The D614G substitution, however, is in linkage disequilibrium with the ORF1b P314L mutation where both mutations almost invariably co-occur, making functional inferences problematic. In addition, the possibility of repeated new introductions of the mutant strain does not allow one to distinguish between a founder effect and an intrinsic genetic property of the virus. Here, we synthesized and expressed the WT and D614G variant SARS-Cov2 Spike protein, and report that using a SARS-CoV2 Spike protein pseudotyped lentiviral vector we observe that the D614G variant Spike has >1/2 log10 increased infectivity in human cells expressing the human ACE2 protein as the viral receptor. The increased binding/fusion activity of the D614G Spike protein was corroborated in a cell fusion assay using Spike and ACE2 proteins expressed in different cells. These results are consistent with the possibility that the Spike D614G mutant increases the infectivity of SARS-CoV2.
ABSTRACT
In Alzheimer disease, increased beta-secretase (BACE1) activity has been associated with neurodegeneration and accumulation of amyloid precursor protein (APP) products. Thus, inactivation of BACE1 could be important in the treatment of Alzheimer disease. In this study, we found that lowering BACE1 levels using lentiviral vectors expressing siRNAs targeting BACE1 reduced amyloid production and the neurodegenerative and behavioral deficits in APP transgenic mice, a model of Alzheimer disease. Our results suggest that lentiviral vector delivery of BACE1 siRNA can specifically reduce the cleavage of APP and neurodegeneration in vivo and indicate that this approach could have potential therapeutic value for treatment of Alzheimer disease.
Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Endopeptidases/genetics , Endopeptidases/metabolism , RNA, Small Interfering/therapeutic use , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid Precursor Protein Secretases , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Analysis of Variance , Animals , Aspartic Acid Endopeptidases , Brain/drug effects , Brain/metabolism , Brain/pathology , Brain/virology , Cell Line , Cloning, Molecular , Disease Models, Animal , Endopeptidases/chemistry , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Genetic Vectors/physiology , Glial Fibrillary Acidic Protein/metabolism , Humans , Immunohistochemistry/methods , Lentivirus/physiology , Maze Learning/drug effects , Maze Learning/physiology , Mice , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , Molecular Sequence Data , Spatial Behavior/drug effects , Time FactorsABSTRACT
Lentiviral vectors are potent gene delivery vehicles that enable stable expression of transgenes in both dividing and post-mitotic cells. Development of lentiviral vectors expressing small hairpin RNAs generates a system that can be used to down regulate specific target genes in vivo and in vitro. In this review, we will discuss two examples of in vivo applications for the use of lentiviral vectors expressing shRNAs: Gene therapy of neurological disorders and generation of transgenic knockdown animals.
Subject(s)
Genetic Therapy/methods , Lentivirus/genetics , RNA, Viral/metabolism , Transgenes , Animals , Animals, Genetically Modified , Gene Transfer Techniques , Genetic Vectors , Humans , Mitosis , Models, Genetic , Nervous System Diseases/genetics , RNA, Small Interfering/geneticsABSTRACT
Toca 511, a retroviral replicating vector (RRV), uses an internal ribosomal entry site (IRES) to express an optimized yeast cytosine deaminase (yCD2), which converts 5-fluorocytosine to 5-fluorouracil. This configuration is genetically stable in both preclinical mouse models and human clinical trials. However, the use of IRES (â¼600 bp) restricts choices of therapeutic transgenes due to limits in RRV genome size. This study replaced IRES with 2A peptides derived from picornaviruses with or without a GSG linker. The data show that GSG-linked 2A (g2A) peptide resulted in higher polyprotein separation efficiency than non-GSG linked 2A peptide. The study also shows that RRV can tolerate insertion of two separate 2A peptides to allow expression of two transgenes without compromising the assembly and function of the virus in addition to insertion of a single 2A peptide to confirm genetic stability with yCD2, green fluorescent protein, and HSV-1 thymidine kinase. In a parallel comparison of the RRV-IRES-yCD2 and RRV-g2A-yCD2 configurations, the study shows the yCD2 protein expressed from RRV-g2A-yCD2 has higher activity, resulting in a higher survival benefit in an intracranial tumor mouse model. These data enable a wider range of potential product candidates that could be developed using the RRV platform.
Subject(s)
Brain Neoplasms/therapy , Genetic Therapy , Genetic Vectors , Internal Ribosome Entry Sites/genetics , Animals , Brain Neoplasms/genetics , Cytosine Deaminase/genetics , Disease Models, Animal , Green Fluorescent Proteins/genetics , Humans , Mice , Peptides/genetics , Picornaviridae/genetics , Virus Replication/genetics , Xenograft Model Antitumor AssaysABSTRACT
During ageing, microglia acquire a phenotype that may negatively affect brain function. Here we show that ageing microglial phenotype is largely imposed by interferon type I (IFN-I) chronically present in aged brain milieu. Overexpression of IFN-ß in the CNS of adult wild-type mice, but not of mice lacking IFN-I receptor on their microglia, induces an ageing-like transcriptional microglial signature, and impairs cognitive performance. Furthermore, we demonstrate that age-related IFN-I milieu downregulates microglial myocyte-specific enhancer factor 2C (Mef2C). Immune challenge in mice lacking Mef2C in microglia results in an exaggerated microglial response and has an adverse effect on mice behaviour. Overall, our data indicate that the chronic presence of IFN-I in the brain microenvironment, which negatively affects cognitive function, is mediated via modulation of microglial activity. These findings may shed new light on other neurological conditions characterized by elevated IFN-I signalling in the brain.Microglia cells in the brain regulate immune responses, but in ageing can negatively affect brain function. Here the authors show that the chronic presence of type I interferon in aged mouse brain impedes cognitive ability by altering microglia transcriptome and limiting Mef2C, a microglia 'off' signal.
Subject(s)
Aging/immunology , Brain/immunology , Interferon-beta/immunology , MEF2 Transcription Factors/immunology , Microglia/immunology , Animals , Brain/growth & development , Brain/physiopathology , Humans , Interferon-beta/genetics , MEF2 Transcription Factors/genetics , Male , Mice , Mice, Inbred C57BLABSTRACT
Adipose tissue is an important insulin target organ, and 3T3-L1 cells are a model cell line for adipocytes. In this study, we have used lentivirus-mediated short hairpin RNA (shRNA) for functional gene knockdown in 3T3-L1 adipocytes to assess the molecular mechanisms of insulin signaling. We chose to target GLUT4 to validate this approach. We showed that lentiviruses efficiently delivered transgenes and small interfering RNA (siRNA) into fully differentiated 3T3-L1 adipocytes. We established a strategy for identifying efficient siRNA sequences for gene knockdown by transfecting 293 cells with the target gene fluorescent fusion protein plasmid along with a plasmid that expresses shRNA. Using these methods, we identified highly efficient siGLUT4 sequences. We demonstrated that lentivirus-mediated shRNA against GLUT4 reduced endogenous GLUT4 expression to almost undetectable levels in 3T3-L1 adipocytes. Interestingly, insulin-stimulated glucose uptake was only reduced by 50-60%, suggesting that another glucose transporter mediates part of this effect. When siGLUT1 was introduced into GLUT4-deficient adipocytes, insulin-stimulated glucose uptake was essentially abolished, indicating that both GLUT4 and GLUT1 contribute to insulin-stimulated glucose transport in 3T3-L1 adipocytes. We also found that GLUT4 knockdown led to impaired insulin-responsive aminopeptidase protein expression that was dependent on whether GLUT4 was knocked down in the differentiating or differentiated stage. We further found that GLUT4 expression was not required for adipogenic differentiation but was necessary for full lipogenic capacity of differentiated adipocytes. These studies indicate that lentiviral shRNA constructs provide an excellent approach to deliver functional siRNAs into 3T3-L1 adipocytes for studying insulin signaling and adipocyte biology.
Subject(s)
Adipocytes/metabolism , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/physiology , Lentivirus/genetics , 3T3-L1 Cells , Animals , Azo Compounds/pharmacology , Biological Transport , Cell Differentiation , Cell Line , Cloning, Molecular , Electroporation , Fluorescent Dyes/pharmacology , Gene Silencing , Glucose/metabolism , Glucose/pharmacokinetics , Green Fluorescent Proteins/metabolism , Humans , Immunoblotting , Insulin/metabolism , Luminescent Proteins/metabolism , Mice , Models, Biological , Plasmids/metabolism , RNA, Small Interfering/metabolism , Signal Transduction , Time Factors , Transfection , Red Fluorescent ProteinABSTRACT
X-linked Severe Combined Immunodeficiency (SCID-X1) is a genetic disease that leaves newborns at high risk of serious infection and a predicted life span of less than 1 year in the absence of a matched bone marrow donor. The disease pathogenesis is due to mutations in the gene encoding the Interleukin-2 receptor gamma chain (IL-2Rγ), leading to a lack of functional lymphocytes. With the leukemogenic concerns of viral gene therapy there is a need to explore alternative therapeutic options. We have utilized induced pluripotent stem cell (iPSC) technology and genome editing mediated by TALENs to generate isogenic subject-specific mutant and gene-corrected iPSC lines. While the subject-derived mutant iPSCs have the capacity to generate hematopoietic precursors and myeloid cells, only wild-type and gene-corrected iPSCs can additionally generate mature NK cells and T cell precursors expressing the correctly spliced IL-2Rγ. This study highlights the potential for the development of autologous cell therapy for SCID-X1 subjects.
Subject(s)
Genetic Therapy/methods , Immunotherapy, Adoptive , Induced Pluripotent Stem Cells/physiology , Killer Cells, Natural/physiology , Precursor Cells, T-Lymphoid/physiology , Regeneration , Regenerative Medicine , X-Linked Combined Immunodeficiency Diseases/therapy , Antigens, CD/metabolism , Bacterial Proteins/metabolism , Cell Differentiation/genetics , Cell Line , DNA Repair , DNA Repair Enzymes/metabolism , Humans , Induced Pluripotent Stem Cells/transplantation , Infant , Interleukin Receptor Common gamma Subunit/genetics , Killer Cells, Natural/transplantation , Mutation/genetics , Precursor Cells, T-Lymphoid/transplantation , X-Linked Combined Immunodeficiency Diseases/geneticsABSTRACT
Glioblastoma multiforme (GBM) is one of the most common and most malignant of the brain tumors. Gliomas can be classified into four different grades according to their histologic characteristics; the most aggressive of the gliomas is glioblastoma multiforme (grade IV). Despite optimal treatment, the median survival is only 12 to 15 months. In the past few years, important advances were made in understanding the biology and pathology of malignant gliomas. A mouse model of brain tumors using inducible lentiviral vectors is described here. In this approach, a lenti-vector with loxP sites flanking the gene of interest (oncogene) is injected into mice expressing Cre recombinase under the control of a brain-specific promoter. The steps to perform cell-type/region-specific injection of Cre-loxP-controlled lentiviral vectors in the brain of adult mice are described here in detail. Curr. Protoc. Mouse Biol. 3:121-139 © 2013 by John Wiley & Sons, Inc.
ABSTRACT
Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans. Here we show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Transduction by oncogenic lentiviral vectors of neural stem cells (NSCs), astrocytes, or even mature neurons in the brains of mice can give rise to malignant gliomas. All the tumors, irrespective of the site of lentiviral vector injection (the initiating population), shared common features of high expression of stem or progenitor markers and low expression of differentiation markers. Microarray analysis revealed that tumors of astrocytic and neuronal origin match the mesenchymal GBM subtype. We propose that most differentiated cells in the CNS upon defined genetic alterations undergo dedifferentiation to generate a NSC or progenitor state to initiate and maintain the tumor progression, as well as to give rise to the heterogeneous populations observed in malignant gliomas.
Subject(s)
Astrocytes/pathology , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Glioma/genetics , Glioma/pathology , Neurons/pathology , Oncogenes , Animals , Astrocytes/metabolism , Genes, Neurofibromatosis 1 , Genes, p53 , Glial Fibrillary Acidic Protein , Glioblastoma/genetics , Glioblastoma/pathology , Lentivirus , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Neural Stem Cells/metabolism , Neural Stem Cells/pathology , Neurons/metabolism , Transduction, GeneticABSTRACT
Human carcinomas can metabolically incorporate and present the dietary non-human sialic acid Neu5Gc, which differs from the human sialic acid N-acetylneuraminic acid (Neu5Ac) by 1 oxygen atom. Tumor-associated Neu5Gc can interact with low levels of circulating anti-Neu5Gc antibodies, thereby facilitating tumor progression via chronic inflammation in a human-like Neu5Gc-deficient mouse model. Here we show that human anti-Neu5Gc antibodies can be affinity-purified in substantial amounts from clinically approved intravenous IgG (IVIG) and used at higher concentrations to suppress growth of the same Neu5Gc-expressing tumors. Hypothesizing that this polyclonal spectrum of human anti-Neu5Gc antibodies also includes potential cancer biomarkers, we then characterize them in cancer and noncancer patients' sera, using a novel sialoglycan microarray presenting multiple Neu5Gc-glycans and control Neu5Ac-glycans. Antibodies against Neu5Gcα2-6GalNAcα1-O-Ser/Thr (GcSTn) were found to be more prominent in patients with carcinomas than with other diseases. This unusual epitope arises from dietary Neu5Gc incorporation into the carcinoma marker Sialyl-Tn, and is the first example of such a novel mechanism for biomarker generation. Finally, human serum or purified antibodies rich in anti-GcSTn-reactivity kill GcSTn-expressing human tumors via complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity. Such xeno-autoantibodies and xeno-autoantigens have potential for novel diagnostics, prognostics, and therapeutics in human carcinomas.