Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model.

Introduction: Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo. Methods: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. Results: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice. Conclusion: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.

A simple and effective genotyping workflow for rapid detection of CRISPR genome editing.

Genetically engineered mouse models play a pivotal role in the modeling of diseases, exploration of gene functions, and the development of novel therapies. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing technology has revolutionized the process of developing such models by enabling precise genome modifications of the multiple interested genes simultaneously. Following genome editing, an efficient genotyping methodology is crucial for subsequent characterization. However, current genotyping methods are laborious, time-consuming, and costly. Here, using targeting the mouse trypsinogen genes as an example, we introduced common applications of CRISPR-Cas9 editing and a streamlined cost-effective genotyping workflow for CRISPR-edited mouse models, in which Sanger sequencing is required only at the initial steps. In the F0 mice, we focused on identifying the presence of positive editing by PCR followed by Sanger sequencing without the need to know the exact sequences, simplifying the initial screening. In the F1 mice, Sanger sequencing and algorithms decoding were used to identify the precise editing. Once the edited sequence was established, a simple and effective genotyping strategy was established to distinguish homozygous and heterozygous status by PCR from tail DNA. The genotyping workflow applies to deletions as small as one nucleotide, multiple-gene knockout, and knockin studies. This simplified, efficient, and cost-effective genotyping shall be instructive to new investigators who are unfamiliar with characterizing CRISPR-Cas9-edited mouse strains.NEW & NOTEWORTHY This study presents a streamlined, cost-effective genotyping workflow for clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) edited mouse models, focusing on trypsinogen genes. It simplifies initial F0 mouse screening using PCR and Sanger sequencing without needing exact sequences. For F1 mice, precise editing is identified through Sanger sequencing and algorithm decoding. The workflow includes a novel PCR strategy for distinguishing homozygous and heterozygous statuses in subsequent generations, effective for small deletions, multiple-gene knockouts, and knockins.

GetPrimers: A generalized PCR-based genetic targeting primer designer enabling easy and standardized targeted gene modification across multiple systems.

Genetic targeting (e.g., gene knockout and tagging) based on polymerase chain reaction (PCR) is a simple yet powerful approach for studying gene functions. Although originally developed in classic budding and fission yeast models, the same principle applies to other eukaryotic systems with efficient homologous recombination. One-step PCR-based genetic targeting is conventionally used but the sizes of the homologous arms that it generates for recombination-mediated genetic targeting are usually limited. Alternatively, gene targeting can also be performed via fusion PCR, which can create homologous arms that are orders of magnitude larger, therefore substantially increasing the efficiency of recombination-mediated genetic targeting. Here, we present GetPrimers (https://www.evomicslab.org/app/getprimers/), a generalized computational framework and web tool to assist automatic targeting and verification primer design for both one-step PCR-based and fusion PCR-based genetic targeting experiments. Moreover, GetPrimers by design runs for any given genetic background of any species with full genome scalability. Therefore, GetPrimers is capable of empowering high-throughput functional genomic assays at multipopulation and multispecies levels. Comprehensive experimental validations have been performed for targeting and verification primers designed by GetPrimers across multiple organism systems and experimental setups. We anticipate GetPrimers to become a highly useful and popular tool to facilitate easy and standardized gene modification across multiple systems.

Reconsidering the role of blood-brain barrier in Alzheimer's disease: From delivery to target.

The existence of a selective blood-brain barrier (BBB) and neurovascular coupling are two unique central nervous system vasculature features that result in an intimate relationship between neurons, glia, and blood vessels. This leads to a significant pathophysiological overlap between neurodegenerative and cerebrovascular diseases. Alzheimer's disease (AD) is the most prevalent neurodegenerative disease whose pathogenesis is still to be unveiled but has mostly been explored under the light of the amyloid-cascade hypothesis. Either as a trigger, bystander, or consequence of neurodegeneration, vascular dysfunction is an early component of the pathological conundrum of AD. The anatomical and functional substrate of this neurovascular degeneration is the BBB, a dynamic and semi-permeable interface between blood and the central nervous system that has consistently been shown to be defective. Several molecular and genetic changes have been demonstrated to mediate vascular dysfunction and BBB disruption in AD. The isoform ε4 of Apolipoprotein E is at the same time the strongest genetic risk factor for AD and a known promoter of BBB dysfunction. Low-density lipoprotein receptor-related protein 1 (LRP-1), P-glycoprotein, and receptor for advanced glycation end products (RAGE) are examples of BBB transporters implicated in its pathogenesis due to their role in the trafficking of amyloid-ß. This disease is currently devoid of strategies that change the natural course of this burdening illness. This unsuccess may partly be explained by our misunderstanding of the disease pathogenesis and our inability to develop drugs that are effectively delivered to the brain. BBB may represent a therapeutic opportunity as a target itself or as a therapeutic vehicle. In this review, we aim to explore the role of BBB in the pathogenesis of AD including the genetic background and detail how it can be targeted in future therapeutic research.

Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo.

Adeno-associated viral (AAV) vectors, used as vehicles for gene transfer into the brain, are a versatile and powerful tool of modern neuroscience that allow identifying specific neuronal populations, monitoring and modulating their activity. For consistent and reproducible results, the AAV vectors must be engineered so that they reliably and accurately target cell populations. Furthermore, transgene expression must be adjusted to sufficient and safe levels compatible with the physiology of studied cells. We undertook the effort to identify and validate an AAV vector that could be utilized for researching the inferior olivary (IO) nucleus, a structure gating critical timing-related signals to the cerebellum. By means of systematic construct generation and quantitative expression profiling, we succeeded in creating a viral tool for specific and strong transfection of the IO neurons without adverse effects on their physiology. The potential of these tools is demonstrated by expressing the calcium sensor GCaMP6s in adult mouse IO neurons. We could monitor subtle calcium fluctuations underlying two signatures of intrinsic IO activity: the subthreshold oscillations (STOs) and the variable-duration action potential waveforms both in-vitro and in-vivo. Further, we show that the expression levels of GCaMP6s allowing such recordings are compatible with the delicate calcium-based dynamics of IO neurons, inviting future work into the network dynamics of the olivo-cerebellar system in behaving animals.

A perspective on astrocyte regulation of neural circuit function and animal behavior.

Studies over the past two decades have demonstrated that astrocytes are tightly associated with neurons and play pivotal roles in neural circuit development, operation, and adaptation in health and disease. Nevertheless, precisely how astrocytes integrate diverse neuronal signals, modulate neural circuit structure and function at multiple temporal and spatial scales, and influence animal behavior or disease through aberrant excitation and molecular output remains unclear. This Perspective discusses how new and state-of-the-art approaches, including fluorescence indicators, opto- and chemogenetic actuators, genetic targeting tools, quantitative behavioral assays, and computational methods, might help resolve these longstanding questions. It also addresses complicating factors in interpreting astrocytes' role in neural circuit regulation and animal behavior, such as their heterogeneity, metabolism, and inter-glial communication. Research on these questions should provide a deeper mechanistic understanding of astrocyte-neuron assemblies' role in neural circuit function, complex behaviors, and disease.

Targeting Subsets of Mammalian Neurons.

Functional dissection of mammalian neuronal circuits depends on accurate targeting of constituent cell classes. Transgenic mice offer precise and predictable access to genetically defined cell populations, but there is the pressing need to target neuronal assemblies in species less amenable to genomic manipulations, such as the primate, which is an important animal model for human perception, cognition, and action. We have developed several virus-based methods for accessing all forebrain inhibitory interneurons as well as the major excitatory and inhibitory neuron subclasses. These methods rely on the wealth of emerging single-cell transcriptome data and harness gene expression variations to refine neuron targeting. Our approach enables nuanced functional studies, including in vivo imaging and manipulation, of the diverse cell populations of the mammalian neocortex, and it represents a timely blueprint for transgenics-independent interrogation of functionally significant cell classes.

Genetic and pharmacological targeting of A2a receptor improves function of anti-mesothelin CAR T cells.

BACKGROUND: CAR T cell-based therapies have shown promising results in hematological malignancies. Results of CAR T cell projects in solid tumors have been less impressive, and factors including lack of targetable antigens and immunosuppressive tumor microenvironment (TME) have been suggested as culprits. Adenosine, a metabolite which is highly produced in TME, is known to mediate the suppression of anti-tumor T cell responses via binding and signaling through adenosine 2a receptor (A2aR). METHODS: In this study, the expression of A2aR and the effects of its activation on the function of fully human anti-mesothelin CAR T cells (MSLN-CAR T), were analyzed. Afterwards, the molecular and pharmacological means to overcome the inhibitory effects of A2aR signaling on CAR T cell function were used. This was performed by targeting A2aR expression in MSLN-CAR T cells using various anti-A2aR shRNA sequences embedded in the CAR vector and an A2aR pharmacological antagonist, SCH-58261. Statistical analyses were performed Prism 7 software. RESULTS: Our experiments showed significant A2aR upregulation on T cells during the CAR T cell production procedure (cell activation and transduction). Activation of adenosine signaling using adenosine analog led to the suppression of all major anti-tumor functions in MSLN-CAR T cells. Interestingly, CAR T cells that carried the anti-A2aR shRNA sequences were resistant to the inhibitory effects of adenosine signaling. Pharmacological inhibition of A2aR reversed the reduction in CAR T cell proliferation and cytokine response caused by the adenosine analog; however, it failed to rescue the cytotoxic function of the cells. CONCLUSION: Altogether, our results indicate that mitigating A2aR signaling by genetic targeting of the receptor might be a promising approach in improving CAR T cell function in an unreceptive microenvironment and could potentially improve the outcome of treatment in clinical settings.

A Chemogenetic Approach for the Optical Monitoring of Voltage in Neurons.

Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for the interrogation of the cellular and molecular logic of the nervous system. Herein, a semisynthetic tethered voltage indicator (STeVI1) based upon nile red is described that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash-free imaging and faithfully detects supra- and sub-threshold activity in neurons.

Mechanosensory neurons control the timing of spinal microcircuit selection during locomotion.

Despite numerous physiological studies about reflexes in the spinal cord, the contribution of mechanosensory feedback to active locomotion and the nature of underlying spinal circuits remains elusive. Here we investigate how mechanosensory feedback shapes active locomotion in a genetic model organism exhibiting simple locomotion-the zebrafish larva. We show that mechanosensory feedback enhances the recruitment of motor pools during active locomotion. Furthermore, we demonstrate that inputs from mechanosensory neurons increase locomotor speed by prolonging fast swimming at the expense of slow swimming during stereotyped acoustic escape responses. This effect could be mediated by distinct mechanosensory neurons. In the spinal cord, we show that connections compatible with monosynaptic inputs from mechanosensory Rohon-Beard neurons onto ipsilateral V2a interneurons selectively recruited at high speed can contribute to the observed enhancement of speed. Altogether, our study reveals the basic principles and a circuit diagram enabling speed modulation by mechanosensory feedback in the vertebrate spinal cord.

Noncanonical connections between the subiculum and hippocampal CA1.

The hippocampal formation is traditionally viewed as having a feedforward, unidirectional circuit organization that promotes propagation of excitatory processes. While the substantial forward projection from hippocampal CA1 to the subiculum has been very well established, accumulating evidence supports the existence of a significant backprojection pathway comprised of both excitatory and inhibitory elements from the subiculum to CA1. Based on these recently updated anatomical connections, such a backprojection could serve to modulate information processing in hippocampal CA1. Here we review the published anatomical and physiological studies on the subiculum to CA1 backprojection, and present recent conclusive anatomical evidence for the presence of noncanonical subicular projections to CA1. New insights into this understudied pathway will improve our understanding of reciprocal CA1-subicular connections and guide future studies on how the subiculum interacts with CA1 to regulate hippocampal circuit activity and learning and memory behaviors. J. Comp. Neurol. 524:3666-3673, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Genetic control of astrocyte function in neural circuits.

During the last two decades numerous genetic approaches affecting cell function in vivo have been developed. Current state-of-the-art technology permits the selective switching of gene function in distinct cell populations within the complex organization of a given tissue parenchyma. The tamoxifen-inducible Cre/loxP gene recombination and the doxycycline-dependent modulation of gene expression are probably the most popular genetic paradigms. Here, we will review applications of these two strategies while focusing on the interactions of astrocytes and neurons in the central nervous system (CNS) and their impact for the whole organism. Abolishing glial sensing of neuronal activity by selective deletion of glial transmitter receptors demonstrated the impact of astrocytes for higher cognitive functions such as learning and memory, or the more basic body control of muscle coordination. Interestingly, also interfering with glial output, i.e., the release of gliotransmitters can drastically change animal's physiology like sleeping behavior. Furthermore, such genetic approaches have also been used to restore astrocyte function. In these studies two alternatives were employed to achieve proper genetic targeting of astrocytes: transgenes using the promoter of the human glial fibrillary acidic protein (GFAP) or homologous recombination into the glutamate-aspartate transporter (GLAST) locus. We will highlight their specific properties that could be relevant for their use.

Multiplexed modular genetic targeting of quantum dots.

While DNA-directed nanotechnology is now a well-established platform for bioinspired nanoscale assembly in vitro, the direct targeting of various nanomaterials in living biological systems remains a significant challenge. Hybrid biological systems with integrated and targeted nanomaterials may have interesting and exploitable properties, so methods for targeting various nanomaterials to precise biological locations are required. Fluorescence imaging has benefited from the use of nanoparticles with superior optical properties compared to fluorescent organic dyes or fluorescent proteins. While single-particle tracking (SPT) in living cells with genetically encoded proteins is limited to very short trajectories, the high photon output of genetically targeted and multiplexed quantum dots (QDs) would enable long-trajectory analysis of multiple proteins. However, challenges with genetic targeting of QDs limit their application in these experiments. In this report, we establish a modular method for targeting QD nanoparticles selectively to multiple genetically encoded tags by precomplexing QD-streptavidin conjugates with cognate biotinylated hapten molecules. This approach enables labeling and SPT of multiple genetically encoded proteins on living cells at high speed and can label expressed proteins in the cytosol upon microinjection into living cells. While we demonstrate labeling with three distinct QD conjugates, the approach can be extended to other specific hapten-affinity molecule interactions and alternative nanoparticles, enabling precise directed targeting of nanoparticles in living biological systems.

Finding degrees of separation: experimental approaches for astroglial and oligodendroglial cell isolation and genetic targeting.

The study of CNS glial cell function requires experimental methods to detect, purify, and manipulate each cell population with fidelity and specificity. With the identification and cloning of cell- and stage-specific markers, glial cell analysis techniques have grown beyond physical methods of tissue dissociation and cell culture, and become highly specific with immunoselection of cell cultures in vitro and genetic targeting in vivo. The unique plasticity of glial cells offers the potential for cell replacement therapies in neurological disease that utilize neural cells derived from transplanted neural stem and progenitor cells. In this mini-review, we outline general physical and genetic approaches for macroglial cell generation. We summarize cell culture methods to obtain astrocytes and oligodendrocytes and their precursors, from developing and adult tissue, as well as approaches to obtain human neural progenitor cells through the establishment of stem cells. We discuss popular targeting rodent strains designed for cell-specific detection, selection and manipulation of neuroglial cell progenitors and their committed progeny. Based on shared markers between astrocytes and stem cells, we discuss genetically modified mouse strains with overlapping expression, and highlight SOX-expressing strains available for targeting of stem and progenitor cell populations. We also include recently established mouse strains for detection, and tag-assisted RNA and miRNA analysis. This discussion aims to provide a brief overview of the rapidly expanding collection of experimental approaches and genetic resources for the isolation and targeting of macroglial cells, their sources, progeny and gene products to facilitate our understanding of their properties and potential application in pathology.