Biology subjects

Gradinaru, V.

Publications and source records attributed to Gradinaru, V..

At least 19 recordsLinked to original sources

bioRxiv2026

AAV-DJ Enables Targeted Gene Modulation in Human Ovarian Cells

Study question: Can adeno-associated viruses (AAVs) efficiently and safely transduce human primary granulosa cells and human ovarian stromal fibroblasts to modulate the expression of genes involved in follicle recruitment and ovarian microenvironment regulation? Summary answer: AAVs can efficiently transduce human primary granulosa cells and human ovarian stromal cells with low toxicity, with the serotype DJ (AAV-DJ) demonstrating superior performance. AAVs enable targeted modulation of ovarian genes such as AMHR2 in human primary granulosa cells and TGF{beta}1 in human ovarian stromal cells, supporting the feasibility of gene-based approaches to improve ovarian function. What is known already: Infertility affects approximately 15% of couples, with diminished oocyte quality and quantity being major contributors, particularly with advanced reproductive age. Assisted reproductive technologies rely primarily on hormonal stimulation and do not directly target molecular pathways governing folliculogenesis or the ovarian microenvironment. Animal studies suggest that gene therapy using AAVs offers a promising strategy for tissue-specific and durable gene modulation, yet its application in human ovarian cells remains largely unexplored. Study design, size, duration: This was an experimental laboratory study using primary human granulosa cells from patients undergoing in vitro fertilization (IVF) and ovarian stromal cells obtained from post-pubertal patients who donated surplus ovarian tissue from ovarian tissue cryopreservation procedures for research. Sixteen AAV serotypes were evaluated for transduction efficiency, toxicity, and expression dynamics, followed by functional gene knockdown studies using AAV-DJ-based shRNA vectors. Participants/materials, setting, methods: Primary human granulosa cells and ovarian stromal cells were isolated from follicular aspirates and ovarian tissue, respectively. Cells were transduced with GFP-expressing AAV serotypes at varying multiplicities of infection (MOIs), incubation times, and culture days to assess transduction efficiency and cytotoxicity. Lead serotypes were further evaluated in dose-response studies. Functional gene modulation was assessed using AAV-DJ-mediated shRNA knockdown of AMHR2 in granulosa cells and TGF{beta}1 in stromal cells, quantified by RT-qPCR, immunofluorescence, and western blotting. Main results and the role of chance: Six of sixteen AAV serotypes (AAV2, AAV3, AAV6, AAV9, AAV-DJ, and AAV-X1.1) achieved greater than 10% transduction efficiency in human primary granulosa cells. Among these, AAV-DJ demonstrated the highest efficiency across all MOIs tested, reaching up to 48% transduction at 100,000 MOI, significantly outperforming AAV2 and AAV9. Toxicity increased with viral dose for all serotypes but remained comparable across vectors, with AAV-DJ exhibiting higher efficiency with comparable toxicity. The AAV-DJ-shRNA-AMHR2 construct produced a significant reduction in expression of AMHR2 mRNA by ~81% (p<0.05) in human primary granulosa cells as well as at the protein level in HEK293T-AMHR2-ZsGreen engineered cells (~85% knockdown, p<0.01). In human ovarian stromal cells, AAV-DJ preferentially transduced EMILIN1 and ACTA2-positive fibroblast and myofibroblast-like cells and enabled robust knockdown of TGF{beta}1 at both transcript (~89% knockdown, p<0.05) and protein levels (~67% knockdown, p<0.0001) using AAV-DJ-shRNA-TGF{beta}1 construct. Limitations, reasons for caution: These experiments were performed in vitro using primary human ovarian cells, which may not fully recapitulate the in vivo ovarian environment. Functional reproductive outcomes were not assessed, and future studies will be required to evaluate the effects of gene modulation on folliculogenesis and fertility. Wider implications of the findings: These findings establish AAV-DJ as a promising vector for targeted gene modulation in human ovarian cells. This approach introduces a novel framework for addressing infertility and ovarian function by directly targeting molecular pathways involved in follicle recruitment and ovarian stromal signaling, potentially enhancing current assisted reproductive technologies.

developmental biology↗

bioRxiv2026

ESCRT-0 regulates AMPA receptor currents and Ca2+- dependent signaling

Membrane protein trafficking is essential for synaptic growth, maintenance, function, and plasticity, and involves the regulated exocytosis and endocytosis of proteins to and from the pre-and post-synaptic membranes. Defects in the clearance of membrane proteins can lead to the accumulation of ubiquitinated membrane proteins and contribute to neurodegenerative disease. The ESCRT (endosomal sorting complexes required for transport) machinery binds and sorts ubiquitinated membrane proteins into lysosomes for degradation, yet the presence and function of ESCRTs in sorting ubiquitinated AMPA and other receptors at the post-synapse remain unclear. Here we show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and levels are modulated by neuronal activity, increasing and decreasing with higher and lower neuronal activity, respectively. Phosphoproteomic profiling of Hrs-depleted post-synaptic membranes revealed a role for Hrs in glutamatergic synaptic transmission, including long-term potentiation. In addition, Hrs-depleted neurons showed faster AMPAR current kinetics and reduced amplitude in whole-cell patch-clamp recordings. Genetic deletion of neuronal Hgs in mice led to reductions in phosphorylated CaMKII- and -{beta} (T286/T287) and structural proteins, PSD-95 and gephyrin, suggestive of LTD (long-term depression)-like synaptic depression. In contrast, Hrs overexpression led to increases in Ca2+-dependent signaling, including protein kinase C (PKC) and PKC substrate, AMPAR subunit GluA1-S831, a site which increases conductance. Together, these findings identify a dynamic, bidirectional role for Hrs at the post-synapse as it both senses and is modulated by neuronal activity, ultimately impacting excitatory synaptic strength. Significance StatementSynaptic plasticity relies on dynamic trafficking and turnover of membrane proteins, including AMPA-type glutamate receptors (AMPARs), yet how receptor trafficking intersects with ubiquitin-mediated sorting pathways at synapses remains unclear. We show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and its abundance is bidirectionally regulated by neuronal activity. Genetic depletion of Hrs in mice reduces CaMKII phosphorylation and impacts AMPAR channel surface localization. In contrast, neuronal-specific Hrs overexpression led to enhanced GluA1 and protein kinase C substrate phosphorylation, suggesting altered AMPAR trafficking, subunit composition, and/or function. Thus, Hrs emerges as a modulator of glutamatergic signaling, coupling ubiquitin-mediated receptor sorting to the fine-tuning of synaptic transmission, with direct implications for learning and memory in health and disease.

neuroscience↗

bioRxiv2026

AAV tools enable functional modulation and readout of central and peripheral nervous systems in spiny mice

Among mammals, spiny mice (Acomys spp.) exhibit the unique capacity to regenerate parts of their nervous system. Studying this phenomenon has the potential to reveal new targets that can slow or halt human neurodegenerative disorders. Unfortunately, research tools (e.g., transgenic lines, gene delivery vehicles) are lacking compared to those available for other rodent models. Here, we tested systemic adeno-associated viral vectors (AAVs) in Acomys dimidiatus and identified three promising candidates: X1.1, CAP-Mac, and MaCPNS1. Characterizing their tropism following intravenous delivery, we found that in the brain, MaCPNS1 and X1.1 primarily transduced astrocytes. In the peripheral nervous system, MaCPNS1 efficiently transduced dorsal root ganglia, axon bundles of the ear pinnae, and enteric neurons throughout the gastrointestinal tract. As a proof-of-concept, we used MaCPNS1 to chemogenetically modulate the activity of enteric neurons, successfully decreasing gastric motility in vivo and increasing colonic motility ex vivo. We expect these findings to enable functional studies of the uniquely regenerative nervous system of Acomys, which may in turn help advance neuroregenerative therapeutics for humans. Summary StatementIdentification of an AAV tool to efficiently deliver transgenes to the central and peripheral nervous systems of spiny mice enables functional studies of the nervous system in a mammalian model of regeneration.

neuroscience↗

bioRxiv2026

A Perturb-seq screen guided by species divergence uncovers pathways for collateral artery formation

Collateral arteries are natural bypasses that can reroute blood flow around arterial blockages, limiting tissue injury during stroke and coronary artery disease. Despite their clinical effectiveness, therapeutic strategies to stimulate collateral artery growth remain unavailable due to our limited understanding of their developmental mechanisms. Remarkably, guinea pigs display exceptionally dense collateral artery networks across various organs, resulting in complete resistance to ischemic damage in the brain and heart. In this study, we compared single-cell RNA sequencing (scRNA-seq) from guinea pig and mouse tissues to identify endothelial cell (EC) gene expression patterns associated with extensive collateral artery development. We then developed an in vivo Perturb-seq platform in mice to test whether genes differentially expressed in guinea pigs influence artery EC specification. This pipeline identified artery repressors that were downregulated in guinea pigs and increased pial collateral abundance when inhibited in mice. Downstream analysis suggests that artery repressors, including WNT and hypoxia response genes, function in two capillary EC subsets--Esm1+ pre-artery and Apln+ angiogenic tip cells. Reduced activity of these repressors allows more ECs to acquire arterial identity, potentiating collateral artery formation. Collectively, our study establishes a strategy for discovering the genes underlying species-specific traits, suggests that guinea pigs have collaterals due to decreased activity of artery inhibitor pathways and hypoxia responses, and identifies novel targets for stimulating collateral artery formation (Graphical abstract). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/721711v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@18b36cdorg.highwire.dtl.DTLVardef@bafbaforg.highwire.dtl.DTLVardef@1e1a6bdorg.highwire.dtl.DTLVardef@189f585_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2026

Defining a Midgestational Window for In Utero Genome Editing of the Fetal Murine Cortex

Congenital disorders of cortical development arise from genetic lesions that disrupt neurogenesis and neuronal migration. Unfortunately, tools to model or correct these defects before birth are limited. Here we establish a platform for systemic in utero gene delivery and genome editing in the mouse cortex at midgestation. By microdissecting a uterine window over the vitelline vein at embryonic day 12.5 (E12.5), we achieve fetal circulation access, enabling robust AAV-mediated transduction of the central nervous system (CNS) while reducing off-target expression in peripheral organs. Barcoded capsid screens reveal that AAV9 exhibits developmental stage-dependent tropism, with higher CNS penetrance and lower liver transduction at E12.5 than at E15.5. Leveraging this window, we provide a proof-of-concept of efficient cortical editing, using Cre-lox and CRISPR/Cas9 strategies to recapitulate prenatal reeler-like cortical misordering phenotypes following Reln knockout. We further use homology-directed repair to demonstrate precise genome modification, epitope-tagging the endogenous Reln and Actb loci, and installing a human-derived pathogenic allele of PDHA1. Importantly, we show that edited cells span neural progenitors and differentiated neurons across the cortex and hippocampus. These results define a permissive midgestational window for prenatal genome editing, providing a platform for functional modeling of congenital CNS disorders and exploration of early therapeutic interventions with minimized peripheral exposure.

neuroscience↗

bioRxiv2026

Controlled linking of AAV capsids enables coordinated multi-vector delivery

Adeno-associated virus (AAV)-based delivery of large genetic systems splits cargo across vectors, but functional reconstitution depends on stochastic co-delivery and often requires high viral doses. Here, we present a multi-capsid linking strategy that enables coordinated delivery of AAV vectors carrying distinct or split genetic cargos. Using a substrate-confined linking process, we suppress uncontrolled higher-order aggregation while enabling programmable nanoscale capsid coupling via oligonucleotide linkers. These linkers further enable efficient downstream purification of linked AAVs and recycling of unlinked vectors, yielding [~]70% AAV dimers and trimers in <24 hours. By enforcing coordinated delivery, linked AAVs achieve co-transduction at reduced viral doses and with greater expression uniformity than conventional dual-AAV approaches. This improved coordination enhances reconstitution of split genetic systems and increases targeted prime editing efficiency in vitro and in the mouse brain. This capsid coupling strategy expands the functional scope of AAVs toward dose-efficient and uniform delivery of large genetic systems.

bioengineering↗

bioRxiv2025

Parkinsons disease modeling in regenerative spiny mice (Acomys dimidiatus) captures key disease-relevant behavioral, histological, and molecular signatures

Parkinsons disease (PD) is a multifactorial neurodegenerative disorder that has been modeled extensively in animals, primarily rodents, but also in non-human primates and non-mammalian organisms. However, no single animal model fully recapitulates the hallmarks of PD pathology. Here, we extend this work by modeling PD for the first time in the spiny mouse (Acomys dimidiatus), a mammal notable for its robust regeneration of multiple tissues. We show that the nigrostriatal pathway of A. dimidiatus is vulnerable to both acute 6-hydroxydopamine (6-OHDA) toxicity and chronic -synuclein (Syn) preformed fibril-induced aggregation. Mouse Syn PFFs produced widespread pS129-positive Syn inclusions across multiple brain regions, mirroring a key pathological hallmark of PD. Compared to C57BL/6J mice, A. dimidiatus exhibited more pronounced behavioral impairments, greater nigrostriatal degeneration, and higher pS129-Syn inclusion burden within substantia nigra pars compacta (SNpc) neurons. To probe the molecular underpinnings behind the vulnerability, we performed single-cell spatial proteomics, which revealed extensive proteomic alterations in dopaminergic neurons associated with Syn aggregation. Multiple proteins were dysregulated in A. dimidiatus, including those involved in proteasomal function, mitochondrial pathways, and oxidative stress regulation, which are processes commonly implicated in PD. Notably, proteomic analysis identified heightened astrocytic activation in the SNpc, which we validated histologically, suggesting a distinct glial response compared to mice. Together, these findings expand our understanding of PD-relevant pathophysiology across species and establish A. dimidiatus as a model for studying mechanisms of neurodegeneration.

neuroscience↗

bioRxiv2025

Next-generation hybridization chain reaction tools with enhanced sensitivities to detect challenging targets

Compared to traditional enzyme-based in situ amplification methods, Hybridization Chain Reaction v3.0 (HCR v3.0) offers high specificity for spatial RNA visualization but lacks the sensitivity required to robustly detect short or low-abundance targets, particularly in thick tissue with high autofluorescence. Here, we describe three HCR variants that combine the specificity of HCR v3.0 with additional signal amplification through catalytic reporter deposition (HCR-Cat), immunostaining (HCR-Immuno), or iterative HCR and immunodetection (HCR-Multi). These methods substantially enhance detection sensitivity, enabling robust spatial visualization of low-abundance transcripts, improved performance in challenging tissue environments, and compatibility with fluorescence- and alkaline phosphatase-based chromogenic detection. These methods enable transcript-resolution imaging, even when using a limited number of probes, thereby expanding the range of biological applications accessible to HCR.

neuroscience↗

bioRxiv2025

Structural basis of liver de-targeting and neuronal tropism of CNS-targeted AAV capsids

Crossing the blood-brain barrier while minimizing liver transduction is a key challenge in developing safe adeno-associated virus (AAV) vectors for treating brain disorders. In mice, the engineered capsid PHP.eB shows enhanced brain transduction, while the further engineered CAP-B10 is also de-targeted from astrocytes and liver. Here, we solve cryo-EM structures of CAP-B10 and its complex with AAV receptor (AAVR) domain PKD2, at 2.22 and 2.20 [A] resolutions, respectively. These structures reveal a structural motif that hinders AAVR binding, which we confirm by measuring affinities. We show that this motif is transferable to other capsids by solving cryo-EM structures of AAV9-X1 and AAV9-X1.1, without and with PKD2, at 3.09, 2.51, and 2.18 [A], respectively. Using this structural information, we designed and validated novel AAV variants with reduced liver and altered brain cell tropism in vivo. Overall, our findings demonstrate that rationally modulating AAVR affinity can alter liver targeting and cellular tropism.

molecular biology↗

bioRxiv2025

AAVs Targeting Human Carbonic Anhydrase IV Enhance Gene Delivery to the Brain

Clinically approved gene therapies based on natural adeno-associated virus (AAV) serotypes have restricted applications, particularly in the brain, due to their poor targeting, high dose requirements, and resulting safety concerns. Directed evolution of enhanced AAV capsids in mice or non-human primates (NHPs) has resulted in markedly improved performance in those species, but inter-species differences present a serious challenge for translating these vectors into human therapies. Here, we engineer AAVs to target human carbonic anhydrase IV (CA-IV), a recently identified blood-brain barrier (BBB) transcytosis receptor. Among known transcytosis receptors, CA-IV is notable for its relatively specific expression in brain endothelial cells and the potency of AAVs that target it in mice. CA-IVs AAV binding site, and thus the mouse vectors enhanced brain potency, is not conserved across species, so we employed a two-phase engineering strategy to identify AAVs optimized for human CA-IV-dependent gene delivery to the brain. We first used in vitro receptor-based selection of a vast AAV library to exclude capsids that do not bind human CA-IV, followed by in vivo selection in "humanized" mice expressing human CA-IV in brain endothelial cells. Notably, we find that human CA-IV binding capsid variants that were poorly enriched in the pull-down selection outperform strong binders in vivo. The most promising vector, AAV-hCA4-IV77, engages human CA-IV to achieve 100-fold greater brain transduction than AAV9, with robust neuronal and astrocytic coverage throughout multiple brain regions. These results advance our understanding of receptor-targeted capsid design and support the therapeutic potential of human CA-IV-engaging AAVs.

bioengineering↗

bioRxiv2025

Axially decoupled photo-stimulation and two photon readout (ADePT) for mapping functional connectivity of neural circuits

All-optical in vivo physiology enables three-dimensional interrogation of neural circuits function. Here, we introduce ADePT (Axially-Decoupled Photo-stimulation and Two-photon readout), a strategy that combines flexible optogenetic stimulation with neural activity imaging. ADePT achieves axially contained widefield patterned stimulation by coupling a digital micro-mirror device illuminated by a solid-state laser to a motorized holographic diffuser, while in parallel performing multiphoton imaging across different z-planes. As a proof of principle, we apply ADePT to map excitatory and inhibitory functional connectivity in the mouse early olfactory system. By controlling activity in individual glomeruli on the olfactory bulb surface and recording responses from output mitral and tufted cells in deeper layers, we identify cohorts of sister cells driven by the same parent glomerulus and analyze their distinct connectivity patterns. We further report specificity in the inhibitory circuitry, as we find that GABAergic/dopaminergic (DAT+) interneurons associated with different glomeruli selectively suppress baseline and odor-evoked activity in distinct mitral and tufted cell populations. Together, these results establish ADePT as a platform for high-throughput functional connectivity mapping in optically accessible brain regions.

neuroscience↗

bioRxiv2025

Molecularly-guided spatial proteomics captures single-cell identity and heterogeneity of the nervous system

Single-cell spatial proteomics (scSP) holds substantial potential for profiling healthy and diseased tissues. The emerging method of molecularly-guided unbiased scSP has mostly been applied to peripheral somatic tissues. Here, we optimize and apply scSP to the healthy and diseased mammalian brain, using molecularly-guided laser capture microdissection and unbiased mass spectrometry. We systematically evaluate the effects of tissue fixation, marker staining, and sample input size on proteome coverage and quantitative accuracy. We benchmark this workflow by profiling region-specific neuronal proteomes and describing the response of non-neuronal cells to acute brain injury. Across these applications, we integrate complementary transcriptomic resources to evaluate cross-modality trends and refine neuronal proteomic results by filtering out protein signals likely arising from non-neuronal cells, an essential consideration in heterogeneous tissues such as the brain. Finally, we leverage this approach to resolve proteomic differences between dopaminergic neuron subpopulations with differential vulnerability to Parkinsons disease and to uncover disease-specific disruptions in -synuclein-aggregate-bearing single dopaminergic neurons. Together, these data demonstrate the utility of scSP in neuroscience research for understanding fundamental biology and the molecular drivers of neurological conditions.

neuroscience↗

bioRxiv2024

The gut microbiome promotes mitochondrial respiration in the brain of a Parkinson's disease mouse model

The pathophysiology of Parkinsons disease (PD) involves gene-environment interactions that impair various cellular processes such as autophagy, lysosomal function, or mitochondrial dysfunction. Specifically, mitochondria-associated gene mutations increase PD risk, mitochondrial respiration is altered in the PD brain, and mitochondrial-damaging toxins cause PD-like motor and gastrointestinal symptoms in animal models. The gut microbiome is altered in PD patients and represents an environmental risk, however a relationship between mitochondrial function and the microbiome in PD has not been previously established. Herein, we report that striatal mitochondria are functionally overactive in -synuclein-overexpressing (ASO) mice, a model of PD, and that microbiome depletion restores respiration and mitochondria-associated gene expression patterns to wild-type levels. ASO mice harboring a complex microbiome produce increased reactive oxygen species in the striatum whereas germ-free counterparts express elevated levels of antioxidant proteins that may buffer against oxidative damage. Indeed, antioxidant treatment improves motor performance in ASO mice and, remarkably, blocking oxidant scavenging in germ-free mice induces -synuclein-dependent motor deficits. Thus, the gut microbiome increases mitochondrial respiration and oxidative stress in the brain, which enhances motor symptoms in a mouse model of PD.

physiology↗

bioRxiv2024

Synthetic dosage-compensating miRNA circuits for quantitative gene therapy

A longstanding challenge in gene therapy is expressing a dosage-sensitive gene within a tight therapeutic window. For example, loss of MECP2 function causes Rett syndrome, while its duplication causes MECP2 duplication syndrome. Viral gene delivery methods generate variable numbers of gene copies in individual cells, creating a need for gene dosage-invariant expression systems. Here, we introduce a compact miRNA-based, incoherent feed-forward loop circuit that achieves precise control of Mecp2 expression in cells and brains, and improves outcomes in an AAV-based mouse model of Rett syndrome gene therapy. Single molecule analysis of endogenous and ectopic Mecp2 mRNA revealed precise, sustained expression across a broad range of gene dosages. Delivered systemically in a brain-targeting AAV capsid, the circuit strongly suppressed Rett behavioral symptoms for over 24 weeks, outperforming an unregulated gene therapy. These results demonstrate that synthetic miRNA-based regulatory circuits can enable precise in vivo expression to improve the safety and efficacy of gene therapy. One sentence description: A synthetic miRNA-based incoherent feed-forward loop circuit embedded in a gene delivery vector overcomes the challenge of overexpression toxicity in a mouse model of Rett syndrome gene therapy.

synthetic biology↗

bioRxiv2024

Human cell surface-AAV interactomes identify LRP6 as blood-brain-barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder

Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapted an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identified a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density-lipoprotein-receptor-related-protein 6 (LRP6) interactions specific to engineered capsids that cross the blood-brain barrier in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allowed us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors peripheral organ tropism and side effects. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.

bioengineering↗

bioRxiv2023

Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo

Integrating cell type-specific regulatory elements (e.g. enhancers) with recombinant adeno-associated viruses (AAVs) can provide broad and efficient genetic access to specific cell types. However, the packaging capacity of AAVs restricts the size of both the enhancers and the cargo that can be delivered. Transcriptional crosstalk offers a novel paradigm for cell type-specific expression of large cargo, by separating distally-acting regulatory elements into a second AAV genome. Here, we identify and profile transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. To understand transcriptional crosstalk, we develop spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue. Using these methods, we construct detailed views of the dynamics of AAV transduction and demonstrate that transcriptional crosstalk is dependent upon concatemer formation. Finally, we leverage transcriptional crosstalk to drive expression of a large Cas9 cargo in a cell type-specific manner with systemically-administered engineered AAVs and demonstrate AAV-delivered, minimally-invasive, cell type-specific gene editing in wildtype animals that recapitulates known disease phenotypes. HighlightsO_LITranscriptional crosstalk between enhancers and promoters delivered in trans by AAVs is a generalized phenomenon. C_LIO_LISpatial genomics techniques, AAV-Zombie and SpECTr, reveal that AAV genome concatemerization facilitates transcriptional crosstalk. C_LIO_LITranscriptional crosstalk can be leveraged for minimally-invasive, targeted AAV delivery of large cargo, including machinery for CRISPR-based gene editing and manipulation. C_LIO_LITranscriptional crosstalk enables cell-type specific gene disruption in wildtype animals, recapitulating behavioural phenotypes of genetic knockouts. C_LI

bioengineering↗

bioRxiv2023

Removal of a genomic duplication by double-nicking CRISPR restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. Using the ASD and anxiety mouse model Flailer, that contains a duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700bp genomic duplication in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove more gene regions <100bp successfully and with high efficiency. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene edition. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescues some of the mutant behaviors, while intracerebroventricular delivery, completely recovers Flailer animal phenotype associated to anxiety and ASD. Our results demonstrate the potential of DN-CRISPR to efficiently (>60% editing in vivo) remove large genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

neuroscience↗

bioRxiv2023

Dysregulated mammalian estrus cycle rescued by timed activation of VIP neurons in the circadian pacemaker and late afternoon light exposure

Jet lag and shift work disrupt the menstrual cycle and decrease fertility. The circadian pacemaker, the suprachiasmatic nucleus (SCN), is known to modulate ovulation, but the mechanism is unclear. Here we explore this connection by tracking the dynamics of vasoactive intestinal peptide (VIP)-expressing neurons in the SCN in freely-behaving mice. We show that SCNVIP activity is time-of-day- and sex-dependent, and estrous-state-dependent in late afternoon, gating downstream activation of GnRH neurons. Afternoon light, as well as specific activation of SCNVIP neurons, rescues estrous cycle regularity and egg release in animals in altered light conditions, emphasizing the role of SCNVIP neurons as a time-dependent light-responsive switch. Our results reveal the dynamic mechanism by which SCNVIP neurons mediate light responses to regulate estrous states and demonstrate light-induced fertility rescue. One Sentence SummaryModulating and recording the activity of suprachiasmatic VIP neurons in freely behaving mice reveals their regulation of fertility by mediating the response to late afternoon light.

neuroscience↗