RNA editing: Expanding the potential of RNA therapeutics.

RNA therapeutics have had a tremendous impact on medicine, recently exemplified by the rapid development and deployment of mRNA vaccines to combat the COVID-19 pandemic. In addition, RNA-targeting drugs have been developed for diseases with significant unmet medical needs through selective mRNA knockdown or modulation of pre-mRNA splicing. Recently, RNA editing, particularly antisense RNA-guided adenosine deaminase acting on RNA (ADAR)-based programmable A-to-I editing, has emerged as a powerful tool to manipulate RNA to enable correction of disease-causing mutations and modulate gene expression and protein function. Beyond correcting pathogenic mutations, the technology is particularly well suited for therapeutic applications that require a transient pharmacodynamic effect, such as the treatment of acute pain, obesity, viral infection, and inflammation, where it would be undesirable to introduce permanent alterations to the genome. Furthermore, transient modulation of protein function, such as altering the active sites of enzymes or the interface of protein-protein interactions, opens the door to therapeutic avenues ranging from regenerative medicine to oncology. These emerging RNA-editing-based toolsets are poised to broadly impact biotechnology and therapeutic applications. Here, we review the emerging field of therapeutic RNA editing, highlight recent laboratory advancements, and discuss the key challenges on the path to clinical development.

Basal filopodia and vascular mechanical stress organize fibronectin into pillars bridging the mesoderm-endoderm gap.

Cells may exchange information with other cells and tissues by exerting forces on the extracellular matrix (ECM). Fibronectin (FN) is an important ECM component that forms fibrils through cell contacts and creates directionally biased geometry. Here, we demonstrate that FN is deposited as pillars between widely separated germ layers, namely the somitic mesoderm and the endoderm, in quail embryos. Alongside the FN pillars, long filopodia protrude from the basal surfaces of somite epithelial cells. Loss-of-function of Ena/VASP, α5ß1-integrins or talin in the somitic cells abolished the FN pillars, indicating that FN pillar formation is dependent on the basal filopodia through these molecules. The basal filopodia and FN pillars are also necessary for proper somite morphogenesis. We identified a new mechanism contributing to FN pillar formation by focusing on cyclic expansion of adjacent dorsal aorta. Maintenance of the directional alignment of the FN pillars depends on pulsatile blood flow through the dorsal aortae. These results suggest that the FN pillars are specifically established through filopodia-mediated and pulsating force-related mechanisms.

Multi-scale quantification of tissue behavior during amniote embryo axis elongation.

Embryonic axis elongation is a complex multi-tissue morphogenetic process responsible for the formation of the posterior part of the amniote body. How movements and growth are coordinated between the different posterior tissues (e.g. neural tube, axial and paraxial mesoderm, lateral plate, ectoderm, endoderm) to drive axis morphogenesis remain largely unknown. Here, we use quail embryos to quantify cell behavior and tissue movements during elongation. We quantify the tissue-specific contribution to axis elongation using 3D volumetric techniques, then quantify tissue-specific parameters such as cell density and proliferation. To study cell behavior at a multi-tissue scale, we used high-resolution 4D imaging of transgenic quail embryos expressing fluorescent proteins. We developed specific tracking and image analysis techniques to analyze cell motion and compute tissue deformations in 4D. This analysis reveals extensive sliding between tissues during axis extension. Further quantification of tissue tectonics showed patterns of rotations, contractions and expansions, which are consistent with the multi-tissue behavior observed previously. Our approach defines a quantitative and multi-scale method to analyze the coordination between tissue behaviors during early vertebrate embryo morphogenetic events.

Mapping a multiplexed zoo of mRNA expression.

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.

A transgenic quail model that enables dynamic imaging of amniote embryogenesis.

Embryogenesis is the coordinated assembly of tissues during morphogenesis through changes in individual cell behaviors and collective cell movements. Dynamic imaging, combined with quantitative analysis, is ideal for investigating fundamental questions in developmental biology involving cellular differentiation, growth control and morphogenesis. However, a reliable amniote model system that is amenable to the rigors of extended, high-resolution imaging and cell tracking has been lacking. To address this shortcoming, we produced a novel transgenic quail that ubiquitously expresses nuclear localized monomer cherry fluorescent protein (chFP). We characterize the expression pattern of chFP and provide concrete examples of how Tg(PGK1:H2B-chFP) quail can be used to dynamically image and analyze key morphogenetic events during embryonic stages X to 11.

In vivo maintenance of human regulatory T cells during CD25 blockade.

Regulatory T cells (Tregs) mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction, and instability are implicated in the pathogenesis of numerous autoimmune diseases. There is considerable interest in therapeutic modulation of the IL-2 pathway to treat autoimmunity, facilitate transplantation tolerance, or potentiate tumor immunotherapy. Daclizumab is a humanized mAb that binds the IL-2 receptor a subunit (IL-2R a or CD25) and prevents IL-2 binding. In this study, we investigated the effect of daclizumab-mediated CD25 blockade on Treg homeostasis in patients with relapsing-remitting multiple sclerosis. We report that daclizumab therapy caused an ~50% decrease in Tregs over a 52-wk period. Remaining FOXP3+ cells retained a demethylated Treg-specific demethylated region in the FOXP3 promoter, maintained active cell cycling, and had minimal production of IL-2, IFN- g, and IL-17. In the presence of daclizumab, IL-2 serum concentrations increased and IL-2R bg signaling induced STAT5 phosphorylation and sustained FOXP3 expression. Treg declines were not associated with daclizumab-related clinical benefit or cutaneous adverse events. These results demonstrate that Treg phenotype and lineage stability can be maintained in the face of CD25 blockade.

Anti-CD25 monoclonal antibody Fc variants differentially impact regulatory T cells and immune homeostasis.

Interleukin-2 (IL-2) is a critical regulator of immune homeostasis through its non-redundant role in regulatory T (Treg) cell biology. There is major interest in therapeutic modulation of the IL-2 pathway to promote immune activation in the context of tumour immunotherapy or to enhance immune suppression in the context of transplantation, autoimmunity and inflammatory diseases. Antibody-mediated targeting of the high-affinity IL-2 receptor α chain (IL-2Rα or CD25) offers a direct mechanism to target IL-2 biology and is being actively explored in the clinic. In mouse models, the rat anti-mouse CD25 clone PC61 has been used extensively to investigate the biology of IL-2 and Treg cells; however, there has been controversy and conflicting data on the exact in vivo mechanistic function of PC61. Engineering antibodies to alter Fc/Fc receptor interactions can significantly alter their in vivo function. In this study, we re-engineered the heavy chain constant region of an anti-CD25 monoclonal antibody to generate variants with highly divergent Fc effector function. Using these anti-CD25 Fc variants in multiple mouse models, we investigated the in vivo impact of CD25 blockade versus depletion of CD25(+) Treg cells on immune homeostasis. We report that immune homeostasis can be maintained during CD25 blockade but aberrant T-cell activation prevails when CD25(+) Treg cells are actively depleted. These results clarify the impact of PC61 on Treg cell biology and reveal an important distinction between CD25 blockade and depletion of CD25(+) Treg cells. These findings should inform therapeutic manipulation of the IL-2 pathway by targeting the high-affinity IL-2R.

Dimethyl fumarate inhibits dendritic cell maturation via nuclear factor κB (NF-κB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen stress-activated kinase 1 (MSK1) signaling.

Dimethyl fumarate (DMF) is an effective novel treatment for multiple sclerosis in clinical trials. A reduction of IFN-γ-producing CD4(+) T cells is observed in DMF-treated patients and may contribute to its clinical efficacy. However, the cellular and molecular mechanisms behind this clinical observation are unclear. In this study, we investigated the effects of DMF on dendritic cell (DC) maturation and subsequent DC-mediated T cell responses. We show that DMF inhibits DC maturation by reducing inflammatory cytokine production (IL-12 and IL-6) and the expression of MHC class II, CD80, and CD86. Importantly, this immature DC phenotype generated fewer activated T cells that were characterized by decreased IFN-γ and IL-17 production. Further molecular studies demonstrated that DMF impaired nuclear factor κB (NF-κB) signaling via reduced p65 nuclear translocalization and phosphorylation. NF-κB signaling was further decreased by DMF-mediated suppression of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and its downstream kinase mitogen stress-activated kinase 1 (MSK1). MSK1 suppression resulted in decreased p65 phosphorylation at serine 276 and reduced histone phosphorylation at serine 10. As a consequence, DMF appears to reduce p65 transcriptional activity both directly and indirectly by promoting a silent chromatin environment. Finally, treatment of DCs with the MSK1 inhibitor H89 partially mimicked the effects of DMF on the DC signaling pathway and impaired DC maturation. Taken together, these studies indicate that by suppression of both NF-κB and ERK1/2-MSK1 signaling, DMF inhibits maturation of DCs and subsequently Th1 and Th17 cell differentiation.

TGF-ß signaling via Smad4 drives IL-10 production in effector Th1 cells and reduces T-cell trafficking in EAE.

Effector Th1 cells perpetuate inflammatory damage in a number of autoimmune diseases, including MS and its animal model EAE. Recently, a self-regulatory mechanism was described in which effector Th1 cells produce the immunomodulatory cytokine IL-10 to dampen the inflammatory response in both normal and autoimmune inflammation. While the presence of TGF-ß has been suggested to enhance and stabilize an IFN-γ(+) IL-10(+) phenotype, the molecular mechanism is poorly understood. Additionally, in the context of adoptive transfer EAE, it is unclear whether IL-10 acts on the transferred Th1 cells or on endogenous host cells. In the present study, using myelin-specific TCR-Tg mice, we show that repetitive Ag stimulation of effector Th1 cells in the presence of TGF-ß increases the population of IFN-γ(+) IL-10(+) cells, which correlates with a decrease in EAE severity. Additionally, TGF-ß signaling causes binding of Smad4 to the IL-10 promoter, providing molecular evidence for TGF-ß-mediated IL-10 production from Th1 effector cells. Finally, this study demonstrates that IL-10 not only reduces encephalitogenic markers such as IFN-γ and T-bet on Th1 effector cells expressing the IL-10R but also prevents recruitment of both transferred and host-derived inflammatory T cells. These data establish a regulatory mechanism by which highly activated Th1 effector cells modulate their pathogenicity through the induction of IL-10.

TGF-beta enhances effector Th1 cell activation but promotes self-regulation via IL-10.

Myelin-specific effector Th1 cells are able to perpetuate CNS inflammation in experimental autoimmune encephalomyelitis, an animal model representative of multiple sclerosis. Although the effects of cytokines in the CNS microenvironment on naive CD4(+) T cells have been well described, much less is known about their ability to influence Ag-experienced effector cells. TGF-beta is a multifunctioning cytokine present in the healthy and inflamed CNS with well-characterized suppressive effects on naive T cell functions. However, the effects of TGF-beta on effector Th1 cells are not well defined. Using myelin-specific TCR transgenic mice, we demonstrate that TGF-beta elicits differential effects on naive versus effector Th1 cells. TGF-beta enhances cellular activation, proliferation, and cytokine production of effector Th1 cells; however, adoptive transfer of these cells into naive mice showed a reduction in encephalitogenicity. We subsequently demonstrate that the reduced encephalitogenic capacity is due to the ability of TGF-beta to promote the self-regulation of Th1 effector cells via IL-10 production. These data demonstrate a mechanism by which TGF-beta is able to suppress the encephalitogenicity of myelin-specific Th1 effector cells that is unique from its suppression of naive T cells.

Avian Primordial Germ Cells Contribute to and Interact With the Extracellular Matrix During Early Migration.

During early avian development, primordial germ cells (PGC) are highly migratory, moving from the central area pellucida of the blastoderm to the anterior extra-embryonic germinal crescent. The PGCs soon move into the forming blood vessels by intravasation and travel in the circulatory system to the genital ridges where they participate in the organogenesis of the gonads. This complex cellular migration takes place in close association with a nascent extracellular matrix that matures in a precise spatio-temporal pattern. We first compiled a list of quail matrisome genes by bioinformatic screening of human matrisome orthologs. Next, we used single cell RNA-seq analysis (scRNAseq) to determine that PGCs express numerous ECM and ECM-associated genes in early embryos. The expression of select ECM transcripts and proteins in PGCs were verified by fluorescent in situ hybridization (FISH) and immunofluorescence (IF). Live imaging of transgenic quail embryos injected with fluorescent antibodies against fibronectin and laminin, showed that germinal crescent PGCs display rapid shape changes and morphological properties such as blebbing and filopodia while surrounded by, or in close contact with, an ECM fibril meshwork that is itself in constant motion. Injection of anti-ß1 integrin CSAT antibodies resulted in a reduction of mature fibronectin and laminin fibril meshwork in the germinal crescent at HH4-5 but did not alter the active motility of the PGCs or their ability to populate the germinal crescent. These results suggest that integrin ß1 receptors are important, but not required, for PGCs to successfully migrate during embryonic development, but instead play a vital role in ECM fibrillogenesis and assembly.

Imaging CAR T cell therapy with PSMA-targeted positron emission tomography.

Chimeric antigen receptor (CAR) T cell therapy for hematologic malignancies is fraught with several unknowns, including number of functional T cells that engage target tumor, durability and subsequent expansion and contraction of that engagement, and whether toxicity can be managed. Non-invasive, serial imaging of CAR T cell therapy using a reporter transgene can address those issues quantitatively. We have transduced anti-CD19 CAR T cells with the prostate-specific membrane antigen (PSMA) because it is a human protein with restricted normal tissue expression and has an expanding array of positron emission tomography (PET) and therapeutic radioligands. We demonstrate that CD19-tPSMA(N9del) CAR T cells can be tracked with [18F]DCFPyL PET in a Nalm6 model of acute lymphoblastic leukemia. Divergence between the number of CD19-tPSMA(N9del) CAR T cells in peripheral blood and bone marrow and those in tumor was evident. These findings underscore the need for non-invasive repeatable monitoring of CAR T cell disposition clinically.

Phenotypic characterization of individuals with SYNGAP1 pathogenic variants reveals a potential correlation between posterior dominant rhythm and developmental progression.

BACKGROUND: The SYNGAP1 gene encodes for a small GTPase-regulating protein critical to dendritic spine maturation and synaptic plasticity. Mutations have recently been identified to cause a breadth of neurodevelopmental disorders including autism, intellectual disability, and epilepsy. The purpose of this work is to define the phenotypic spectrum of SYNGAP1 gene mutations and identify potential biomarkers of clinical severity and developmental progression. METHODS: A retrospective clinical data analysis of individuals with SYNGAP1 mutations was conducted. Data included genetic diagnosis, clinical history and examinations, neurophysiologic data, neuroimaging, and serial neurodevelopmental/behavioral assessments. All patients were seen longitudinally within a 6-year period; data analysis was completed on June 30, 2018. Records for all individuals diagnosed with deleterious SYNGAP1 variants (by clinical sequencing or exome sequencing panels) were reviewed. RESULTS: Fifteen individuals (53% male) with seventeen unique SYNGAP1 mutations are reported. Mean age at genetic diagnosis was 65.9 months (28-174 months). All individuals had epilepsy, with atypical absence seizures being the most common semiology (60%). EEG abnormalities included intermittent rhythmic delta activity (60%), slow or absent posterior dominant rhythm (87%), and epileptiform activity (93%), with generalized discharges being more common than focal. Neuroimaging revealed nonspecific abnormalities (53%). Neurodevelopmental evaluation revealed impairment in all individuals, with gross motor function being the least affected. Autism spectrum disorder was diagnosed in 73% and aggression in 60% of cases. Analysis of biomarkers revealed a trend toward a moderate positive correlation between visual-perceptual/fine motor/adaptive skills and language development, with posterior dominant rhythm on electroencephalogram (EEG), independent of age. No other neurophysiology-development associations or correlations were identified. CONCLUSIONS: A broad spectrum of neurologic and neurodevelopmental features are found with pathogenic variants of SYNGAP1. An abnormal posterior dominant rhythm on EEG correlated with abnormal developmental progression, providing a possible prognostic biomarker.

The acquisition of robust and flexible cognitive skills.

The authors introduce a model of skill acquisition that incorporates elements of both traditional models and models based on embedded cognition by striking a balance between top-down and bottom-up control. A knowledge representation is used in which pre- and postconditions are attached to actions. This model captures improved performance due to learning not only in terms of shorter solution times and lower error rates during the task but also in an increased flexibility to solve similar problems and robustness against unexpected events. In 3 experiments using a complex aviation task, the authors contrasted instructions that explicitly stated pre- and postconditions with conventional instructions that did not. The instructions with pre- and postconditions led to better and more robust performance than other instructions, especially on problems that required transfer. The parameters of the model were estimated to obtain a quantitative fit of the results of Experiment 1, which was then successfully used to predict the results of Experiments 2 and 3.

Japanese quail (Coturnix japonica) as a laboratory animal model.

For the past 50 years, the Japanese quail (Coturnix japonica) has been a popular animal model in numerous fields of research. The quail's 16-d developmental period and its easily accessible embryo make C. japonica a convenient model for studies of developmental biology. Because its lifespan is relatively short and its physiology is comparable to that of humans, the adult quail is useful for studies of aging and disease. The authors describe the Japanese quail as an animal model and, drawing on their experience raising a quail colony at the California Institute of Technology, present detailed guidelines for the husbandry of the species.

Asymmetric localization of Vangl2 and Fz3 indicate novel mechanisms for planar cell polarity in mammals.

Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell-cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates.

Fluorescent Quail: A Transgenic Model System for the Dynamic Study of Avian Development.

Real-time four-dimensional (4D, xyzt) imaging of cultured avian embryos is an ideal method for investigating the complex movements of cells and tissues during early morphogenesis. While methods that transiently label cells, such as electroporation, are highly useful for dynamic imaging, they can also be limiting due to the number and type of cells that can be effectively targeted. In contrast, the heritable, stable, and long-term expression of a fluorescent protein driven by the exogenous promoter of a transgene overcomes these challenges. We have used lentiviral vectors to produce several novel transgenic quail lines that express fluorescent proteins either ubiquitously or in a cell-specific manner. These lines have proven to be useful models for dynamic imaging and analysis. Here, we provide detailed protocols for generating transgenic quail with the emphasis on producing high titer lentivirus , effectively introducing it into the early embryo and efficiently screening for G1 founder birds .

Circulating innate lymphoid cells are unchanged in response to DAC HYP therapy.

Innate lymphoid cells (ILCs) play an important role in immunity, inflammation, and tissue remodeling and their dysregulation is implicated in autoimmune and inflammatory disorders. We analyzed the impact of daclizumab, a humanized monoclonal anti-CD25 antibody, on circulating natural killer (NK) cells and ILCs in a cohort of multiple sclerosis patients. An increase in CD56(bright) NK cells and CD56(hi)CD16(intermediate) transitional NK cells was observed. No significant change in total ILCs or major ILC subpopulations was observed. These results refine our understanding of the impact of daclizumab on innate lymphoid cell populations.

Caspase inhibitors promote vestibular hair cell survival and function after aminoglycoside treatment in vivo.

The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.