Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

Regulation of Energy Expenditure by Brainstem GABA Neurons.

Homeostatic control of core body temperature is essential for survival. Temperature is sensed by specific neurons, in turn eliciting both behavioral (i.e., locomotion) and physiologic (i.e., thermogenesis, vasodilatation) responses. Here, we report that a population of GABAergic (Vgat-expressing) neurons in the dorsolateral portion of the dorsal raphe nucleus (DRN), hereafter DRNVgat neurons, are activated by ambient heat and bidirectionally regulate energy expenditure through changes in both thermogenesis and locomotion. We find that DRNVgat neurons innervate brown fat via a descending projection to the raphe pallidus (RPa). These neurons also densely innervate ascending targets implicated in the central regulation of energy expenditure, including the hypothalamus and extended amygdala. Optogenetic stimulation of different projection targets reveals that DRNVgat neurons are capable of regulating thermogenesis through both a "direct" descending pathway through the RPa and multiple "indirect" ascending pathways. This work establishes a key regulatory role for DRNVgat neurons in controlling energy expenditure.

Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells.

Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events-RF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics-reveals detailed operational parameters of gene-regulatory mechanisms hitherto-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.

VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells.

Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10-3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system's name: viral evolution of genetically actuating sequences, or "VEGAS." Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks' time.

Gene Therapy for Retinal Degeneration.

Biallelic mutations in the RPE65 gene are associated with inherited retinal degenerations/dystrophies (IRD) and disrupt the visual cycle, leading to loss of vision. A new adenoviral vector-based gene therapy surgically delivered to retinal cells provides normal human RPE65 protein that can restore the visual cycle and some vision. To view this Bench to Bedside, open or download the PDF.

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.

The Neuropeptide Tac2 Controls a Distributed Brain State Induced by Chronic Social Isolation Stress.

Chronic social isolation causes severe psychological effects in humans, but their neural bases remain poorly understood. 2 weeks (but not 24 hr) of social isolation stress (SIS) caused multiple behavioral changes in mice and induced brain-wide upregulation of the neuropeptide tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB expression and release phenocopied SIS in group-housed mice, promoting aggression and converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2 coordinates a pleiotropic brain state caused by SIS via a distributed mode of action. These data reveal the profound effects of prolonged social isolation on brain chemistry and function and suggest potential new therapeutic applications for Nk3R antagonists.

Multiplexed activation of endogenous genes by CRISPRa elicits potent antitumor immunity.

Immunotherapy has transformed cancer treatment. However, current immunotherapy modalities face various limitations. In the present study, we developed multiplexed activation of endogenous genes as an immunotherapy (MAEGI), a new form of immunotherapy that elicits antitumor immunity through multiplexed activation of endogenous genes in tumors. We leveraged CRISPR activation (CRISPRa) to directly augment the in situ expression of endogenous genes, and thereby the presentation of tumor antigens, leading to dramatic antitumor immune responses. Deploying this as a cell-based vaccination strategy showed efficacy in both prophylactic and therapeutic settings. Intratumoral adeno-associated virus delivery of CRISPRa libraries elicited strong antitumor immunity across multiple cancer types. Precision targeting of mutated gene sets eradicated a large fraction of established tumors at both local and distant sites. This treatment modality led to alterations in the tumor microenvironment, marked by enhanced T cell infiltration and antitumor immune signatures. Multiplexed endogenous gene activation is a versatile and highly scalable strategy to elicit potent immune responses against cancer, distinct from all existing cancer therapies.

Dopamine Neuron-Specific Optogenetic Stimulation in Rhesus Macaques.

Optogenetic studies in mice have revealed new relationships between well-defined neurons and brain functions. However, there are currently no means to achieve the same cell-type specificity in monkeys, which possess an expanded behavioral repertoire and closer anatomical homology to humans. Here, we present a resource for cell-type-specific channelrhodopsin expression in Rhesus monkeys and apply this technique to modulate dopamine activity and monkey choice behavior. These data show that two viral vectors label dopamine neurons with greater than 95% specificity. Infected neurons were activated by light pulses, indicating functional expression. The addition of optical stimulation to reward outcomes promoted the learning of reward-predicting stimuli at the neuronal and behavioral level. Together, these results demonstrate the feasibility of effective and selective stimulation of dopamine neurons in non-human primates and a resource that could be applied to other cell types in the monkey brain.

In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma.

CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.

A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.

Split intein-mediated protein trans-splicing to express large dystrophins.

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases1,2. However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases)3, limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO4, we demonstrate that low total doses (2 × 1013 viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that exceed the AAV capacity.

Pathophysiology and Mechanisms of Zika Virus Infection in the Nervous System.

In 2015, public awareness of Zika virus (ZIKV) rose in response to alarming statistics of infants with microcephaly being born to women who were infected with the virus during pregnancy, triggering global concern over these potentially devastating consequences. Although we have discovered a great deal about the genome and pathogenesis of this reemergent flavivirus since this recent outbreak, we still have much more to learn, including the nature of the virus-host interactions and mechanisms that determine its tropism and pathogenicity in the nervous system, which are in turn shaped by the continual evolution of the virus. Inevitably, we will find out more about the potential long-term effects of ZIKV exposure on the nervous system from ongoing longitudinal studies. Integrating clinical and epidemiological data with a wider range of animal and human cell culture models will be critical to understanding the pathogenetic mechanisms and developing more specific antiviral compounds and vaccines.

Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system.

The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.

When size matters: A novel compact Cas12a variant for in vivo genome editing.

A new study characterizes and improves a novel small Cas12a variant before adapting it for in vivo genome editing by delivery via adeno-associated virus (AAV) vectors, showcasing the potential of small CRISPR systems and their compatibility with viral vectors.

A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.

Correcting inborn errors of immunity: From viral mediated gene addition to gene editing.

Allogeneic hematopoietic stem cell transplantation is an effective treatment to cure inborn errors of immunity. Remarkable progress has been achieved thanks to the development and optimization of effective combination of advanced conditioning regimens and use of immunoablative/suppressive agents preventing rejection as well as graft versus host disease. Despite these tremendous advances, autologous hematopoietic stem/progenitor cell therapy based on ex vivo gene addition exploiting integrating γ-retro- or lenti-viral vectors, has demonstrated to be an innovative and safe therapeutic strategy providing proof of correction without the complications of the allogeneic approach. The recent advent of targeted gene editing able to precisely correct genomic variants in an intended locus of the genome, by introducing deletions, insertions, nucleotide substitutions or introducing a corrective cassette, is emerging in the clinical setting, further extending the therapeutic armamentarium and offering a cure to inherited immune defects not approachable by conventional gene addition. In this review, we will analyze the current state-of-the art of conventional gene therapy and innovative protocols of genome editing in various primary immunodeficiencies, describing preclinical models and clinical data obtained from different trials, highlighting potential advantages and limits of gene correction.

Developmental hearing loss-induced perceptual deficits are rescued by genetic restoration of cortical inhibition.

Even a transient period of hearing loss during the developmental critical period can induce long-lasting deficits in temporal and spectral perception. These perceptual deficits correlate with speech perception in humans. In gerbils, these hearing loss-induced perceptual deficits are correlated with a reduction of both ionotropic GABAA and metabotropic GABAB receptor-mediated synaptic inhibition in auditory cortex, but most research on critical period plasticity has focused on GABAA receptors. Therefore, we developed viral vectors to express proteins that would upregulate gerbil postsynaptic inhibitory receptor subunits (GABAA, Gabra1; GABAB, Gabbr1b) in pyramidal neurons, and an enzyme that mediates GABA synthesis (GAD65) presynaptically in parvalbumin-expressing interneurons. A transient period of developmental hearing loss during the auditory critical period significantly impaired perceptual performance on two auditory tasks: amplitude modulation depth detection and spectral modulation depth detection. We then tested the capacity of each vector to restore perceptual performance on these auditory tasks. While both GABA receptor vectors increased the amplitude of cortical inhibitory postsynaptic potentials, only viral expression of postsynaptic GABAB receptors improved perceptual thresholds to control levels. Similarly, presynaptic GAD65 expression improved perceptual performance on spectral modulation detection. These findings suggest that recovering performance on auditory perceptual tasks depends on GABAB receptor-dependent transmission at the auditory cortex parvalbumin to pyramidal synapse and point to potential therapeutic targets for developmental sensory disorders.

Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression.

The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

Vector integration and fate in the hemophilia dog liver multiple years after AAV-FVIII gene transfer.

ABSTRACT: Gene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity. Here, we describe the mechanisms of AAV vector persistence in the liver of a severe hemophilia A dog model (male = 4, hemizygous; and female = 4, homozygous), more than a decade after portal vein delivery. The predominant vector form was nonintegrated episomal structures with levels correlating with long-term transgene expression. Random integration was seen in all samples (median frequency, 9.3e-4 sites per cell), with small numbers of nonrandom common integration sites associated with open chromatin. No full-length integrated vectors were found, supporting predominant episomal vector-mediated long-term transgene expression. Despite integration, this was not associated with oncogene upregulation or histopathological evidence of tumorigenesis. These findings support the long-term safety of this therapeutic modality.