Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.

Therapeutic in vivo delivery of gene editing agents.

In vivo gene editing therapies offer the potential to treat the root causes of many genetic diseases. Realizing the promise of therapeutic in vivo gene editing requires the ability to safely and efficiently deliver gene editing agents to relevant organs and tissues in vivo. Here, we review current delivery technologies that have been used to enable therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles, and virus-like particles. Since no single delivery modality is likely to be appropriate for every possible application, we compare the benefits and drawbacks of each method and highlight opportunities for future improvements.

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.

Designer AAV muscle up.

Directed evolution of AAV capsids has been a successful strategy for generating bespoke serotypes to target gene therapies more specifically to the intended tissue. This has now been achieved for the largest organ, skeletal muscle, by selecting for an RGD containing integrin binding heptamer in a hypervariable region of the capsid of AAV9.

Novel approaches for vaccine development.

Vaccines are critical tools for maintaining global health. Traditional vaccine technologies have been used across a wide range of bacterial and viral pathogens, yet there are a number of examples where they have not been successful, such as for persistent infections, rapidly evolving pathogens with high sequence variability, complex viral antigens, and emerging pathogens. Novel technologies such as nucleic acid and viral vector vaccines offer the potential to revolutionize vaccine development as they are well-suited to address existing technology limitations. In this review, we discuss the current state of RNA vaccines, recombinant adenovirus vector-based vaccines, and advances from biomaterials and engineering that address these important public health challenges.

Molecular Mechanisms of Natural Rubber Biosynthesis.

Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.

CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors.

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.

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.

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.

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.

Adenovirus vector vaccination reprograms pulmonary fibroblastic niches to support protective inflating memory CD8+ T cells.

Pathogens and vaccines that produce persisting antigens can generate expanded pools of effector memory CD8+ T cells, described as memory inflation. While properties of inflating memory CD8+ T cells have been characterized, the specific cell types and tissue factors responsible for their maintenance remain elusive. Here, we show that clinically applied adenovirus vectors preferentially target fibroblastic stromal cells in cultured human tissues. Moreover, we used cell-type-specific antigen targeting to define critical cells and molecules that sustain long-term antigen presentation and T cell activity after adenovirus vector immunization in mice. While antigen targeting to myeloid cells was insufficient to activate antigen-specific CD8+ T cells, genetic activation of antigen expression in Ccl19-cre-expressing fibroblastic stromal cells induced inflating CD8+ T cells. Local ablation of vector-targeted cells revealed that lung fibroblasts support the protective function and metabolic fitness of inflating memory CD8+ T cells in an interleukin (IL)-33-dependent manner. Collectively, these data define a critical fibroblastic niche that underpins robust protective immunity operating in a clinically important vaccine platform.

The transcription factor CREB1 is a mechanistic driver of immunogenicity and reduced HIV-1 acquisition following ALVAC vaccination.

Development of effective human immunodeficiency virus 1 (HIV-1) vaccines requires synergy between innate and adaptive immune cells. Here we show that induction of the transcription factor CREB1 and its target genes by the recombinant canarypox vector ALVAC + Alum augments immunogenicity in non-human primates (NHPs) and predicts reduced HIV-1 acquisition in the RV144 trial. These target genes include those encoding cytokines/chemokines associated with heightened protection from simian immunodeficiency virus challenge in NHPs. Expression of CREB1 target genes probably results from direct cGAMP (STING agonist)-modulated p-CREB1 activity that drives the recruitment of CD4+ T cells and B cells to the site of antigen presentation. Importantly, unlike NHPs immunized with ALVAC + Alum, those immunized with ALVAC + MF59, the regimen in the HVTN702 trial that showed no protection from HIV infection, exhibited significantly reduced CREB1 target gene expression. Our integrated systems biology approach has validated CREB1 as a critical driver of vaccine efficacy and highlights that adjuvants that trigger CREB1 signaling may be critical for efficacious HIV-1 vaccines.

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.

Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing.

A major challenge in genetics is to identify genetic variants driving natural phenotypic variation. However, current methods of genetic mapping have limited resolution. To address this challenge, we developed a CRISPR-Cas9-based high-throughput genome editing approach that can introduce thousands of specific genetic variants in a single experiment. This enabled us to study the fitness consequences of 16,006 natural genetic variants in yeast. We identified 572 variants with significant fitness differences in glucose media; these are highly enriched in promoters, particularly in transcription factor binding sites, while only 19.2% affect amino acid sequences. Strikingly, nearby variants nearly always favor the same parent's alleles, suggesting that lineage-specific selection is often driven by multiple clustered variants. In sum, our genome editing approach reveals the genetic architecture of fitness variation at single-base resolution and could be adapted to measure the effects of genome-wide genetic variation in any screen for cell survival or cell-sortable markers.

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.

FIX It in One Go: Enhanced Factor IX Gene Therapy for Hemophilia B.

A phase 1/2 clinical trial of AAV-mediated gene therapy in patients with hemophilia B using an enhanced specific activity factor IX (FIX) transgene reports sustained levels of FIX levels, leading to the near elimination of bleeding for more than a year and without serious adverse side effects. These results are the best outcome to date for hemophilia gene therapy.

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.

Adeno-Associated Virus Toolkit to Target Diverse Brain Cells.

Recombinant adeno-associated viruses (AAVs) are commonly used gene delivery vehicles for neuroscience research. They have two engineerable features: the capsid (outer protein shell) and cargo (encapsulated genome). These features can be modified to enhance cell type or tissue tropism and control transgene expression, respectively. Several engineered AAV capsids with unique tropisms have been identified, including variants with enhanced central nervous system transduction, cell type specificity, and retrograde transport in neurons. Pairing these AAVs with modern gene regulatory elements and state-of-the-art reporter, sensor, and effector cargo enables highly specific transgene expression for anatomical and functional analyses of brain cells and circuits. Here, we discuss recent advances that provide a comprehensive (capsid and cargo) AAV toolkit for genetic access to molecularly defined brain cell types.