Oded Singer

Production and purification of lentiviral vectors

Lentiviral vectors offer unique versatility and robustness as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the transgene both in vitro and in vivo. This protocol describes how lentiviral vectors can be produced, purified and titrated. High titer suspensions can be routinely prepared with relative ease: a low-titer (106 viral particles/ml) unpurified preparation can be obtained 3 d after transfecting cells with lentiviral vector and packaging plasmids; a high-titer (109 viral particles/ml) purified preparation requires 2 more days.

A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA

We describe the use of lentiviral vectors expressing small interfering RNAs (siRNAs) to knock down the expression of specific genes in vitro and in vivo. A lentiviral vector capable of generating siRNA specific for GFP after transduction of 293T-GFP cell lines showed no GFP fluorescence. Furthermore, no GFP-specific RNA could be detected. When eggs from GFP-positive transgenic mice were transduced with lentivirus-expressing siGFP virus, reduced fluorescence could be seen in blastocysts. More interestingly, pups from F1 progeny, which expressed siGFP, showed considerably diminished fluorescence and decreased GFP. We propose that an approach of combining transgenesis by lentiviral vectors expressing siRNAs can be used successfully to generate a large number of mice in which the expression of a specific gene(s) can be down-regulated substantially. We believe that this approach of generating “knockdown” mice will aid in functional genomics.

Intercellular Coupling Confers Robustness against Mutations in the SCN Circadian Clock Network

Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor Per2 gene expression in tissues and cells from clock mutant mice. We discovered that Per1 and Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN). Per2 is also required for sustained rhythms, whereas Cry2 and Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for Per1, Per2, and Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.

Embryonic stem cell potency fluctuates with endogenous retrovirus activity

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.

Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model

In Alzheimer disease, increased β-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.

Dedifferentiation of Neurons and Astrocytes by Oncogenes Can Induce Gliomas in Mice

Cancer Stem Cells: A Moving Target? Glioblastoma multiforme (GBM) is a highly aggressive human brain tumor. The prevailing “cancer stem cell hypothesis” posits that GBMs arise primarily from neuronal stem cells. Now, Friedmann-Morvinski et al. (p. 1080, published online 18 October; see the Perspective by Krivtsov and Armstrong) suggest that this hypothesis may be too restrictive. In mouse models, brain tumors resembling human GBMs could form from fully differentiated cells such as astrocytes and neurons. Upon acquiring certain genetic alterations, mature brain cells may thus be capable of dedifferentiating into a progenitor-like cell, which could then initiate and maintain tumor growth. Murine brain tumors do not necessarily originate from neural stem cells but can arise from mature neurons and astrocytes. 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.

Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells

Significance Pulmonary disease is the third highest cause for morbidity and mortality worldwide. Studies of human lung disease in vivo or in vitro are currently limited. Using induced pluripotent stem cells, we developed a step-wise differentiation protocol ending in an air–liquid interface to generate a pseudostratified polarized layer of endodermal-derived epithelial cells (forkhead box protein A2+ and NK2 homeobox 1+). This layer includes Clara cells with Clara cell 10 kD-positive vesicles, mucin 5A/C-positive goblet cells, multiciliated cells, and isolated cells that have forskolin-induced chloride currents sensitive to cystic fibrosis transmembrane regulator inhibitor 172. The development of this model will enable the future study of many lung diseases (especially those where defective cilia are involved, such as primary ciliary dyskinesia) that have been difficult to study in human models from a developmental perspective. 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.

Design and cloning of lentiviral vectors expressing small interfering RNAs

RNA interference (RNAi) has emerged as a powerful technique to downregulate gene expression. The use of polIII promoters to express small hairpin RNAs (shRNAs), combined with the versatility and robustness of lentiviral vector–mediated gene delivery to a wide range of cell types offers the possibility of long-term downregulation of specific target genes both in vitro and in vivo. The use of silencing lentivectors allows for a rapid and convenient way of establishing cell lines (or transgenic mice) that stably express shRNAs for analysis of phenotypes produced by knockdown of a gene product. Here we present two possible protocols describing the design and cloning of silencing lentiviral vectors. These protocols can be completed in less than 3 weeks.

Applications of Lentiviral Vectors for shRNA Delivery and Transgenesis

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.

Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies.

Significance Chimeric antigen receptor T (CAR-T) cell therapy has produced promising results in clinical trials but has been challenged by the inability to control engineered cells once infused into the patient. Here we present a generalizable method of controlling CAR-T cells using peptide-engrafted antibody-based molecular switches that act as a bridge between the target cell and CAR-T cell. We show that switches specific for CD19 govern the activity, tissue-homing, cytokine release, and phenotype of switchable CAR-T cells in a dose-titratable manner using xenograft mouse models of B-cell leukemia. We expect that this method of tuning CAR-T cell responses will provide improved safety and versatility of CAR–T-cell therapy in the clinic. 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.