Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2.

Significant progress has been made in enhancing recombinant adeno-associated virus (rAAV) for clinical investigation. Despite its versatility as a gene delivery platform, the inherent packaging constraint of 4.7 kb imposes restrictions on the range of diseases it can address. In this context, we present findings of an exceptionally compact and long-term promoter that facilitates the expression of larger genes compared to conventional promoters. This compact promoter originated from the genome of the alphaherpesvirus pseudorabies virus, latency-associated promoter 2 (LAP2, 404 bp). Promoter driving an mCherry reporter was packaged into single strand (ss) AAV8 and AAV9 vectors and injected into adult C57BL/6 mice at a dose of 5 × 1011 vg/mouse by single intravenous or intramuscular administration. An ssAAV8 and ssAAV9 vector with elongation factor-1α promoter (EF1α, 1264 bp) was injected side-by-side for comparison. After 400 days, we sacrificed the mice and examined mCherry expression in liver, kidney, heart, lung, spleen, pancreas, skeletal muscle, and brain. We found that LAP2 exhibited robust transgene expression across a wide range of cells and tissues comparable to the larger EF1α, which is currently recognized as a rather potent and ubiquitous promoter. The AAV8-LAP2 and AAV9-LAP2 constructs displayed strong transduction and transcription in liver, kidney, and skeletal muscle on both route of administration. However, no expression was detected in the heart, lung, spleen, pancreas, and brain. The outcomes of our investigation propose the viability of LAP2 for gene therapy applications demanding the expression of large or multiple therapeutic genes following a single viralvector administration.

Transneuronal Circuit Analysis with Pseudorabies Viruses.

Our ability to understand the function of the nervous system is dependent upon defining the connections of its constituent neurons. Development of methods to define connections within neural networks has always been a growth industry in the neurosciences. Transneuronal spread of neurotropic viruses currently represents the best means of defining synaptic connections within neural networks. The method exploits the ability of viruses to invade neurons, replicate, and spread through the intimate synaptic connections that enable communication among neurons. Since the method was first introduced in the 1970s, it has benefited from an increased understanding of the virus life cycle, the function of viral genomes, and the ability to manipulate the viral genome in support of directional spread of virus and the expression of transgenes. In this article, we review these advances in viral tracing technology and the ways in which they may be applied for functional dissection of neural networks. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Retrograde infection of CNS circuits by peripheral injection of virus Basic Protocol 2: Transneuronal analysis by intracerebral injection Alternate Protocol 1: Transneuronal analysis with multiple recombinant strains Alternate Protocol 2: Conditional replication and spread of PRV Alternate Protocol 3: Conditional reporters of PRV infection and spread Alternate Protocol 4: Reporters of neural activity in polysynaptic circuits Support Protocol 1: Growing and titering a PRV viral stock Support Protocol 2: Immunohistochemical processing and detection Support Protocol 3: Dual-immunofluorescence localization.

Local and systemic administration of AAV vectors with alphaherpesvirus latency-associated promoter 2 drives potent transgene expression in mouse liver, kidney, and skeletal muscle.

Adeno-associated virus (AAV) has great potential as a source of treatments for conditions that might respond to potent and ubiquitous transgene expression. However, among its drawbacks, the genetic "payload" of AAV vectors is limited to <4.9 kb and some commonly used gene promoters are sizeable and susceptible to transcriptional silencing. We recently described a short (404 bp), potent, and persistent promoter obtained from the genome of pseudorabies virus (PrV) called alphaherpesvirus latency-associated promoter 2 (LAP2). Here, we evaluated the biodistribution and potency of transgene expression in mouse peripheral tissues in response to local and systemic administration of AAV8-LAP2 and AAV9-LAP2. We found that administration of these vectors resulted in levels of transgene expression that were similar to the larger EF1α promoter. LAP2 drives potent transgene expression in mouse liver and kidney when administered systemically and in skeletal muscle in response to intramuscular delivery. Notably, in skeletal muscle, administration of vectors with LAP2 and EF1α promoters resulted in preferential transduction of myofibers type 2. A direct side-by-side comparison between LAP2 and the EF1α promoter revealed that, despite its smaller size, LAP2 was equally potent to the EF1α promoter and resulted in widespread gene expression after IV and IM administration of AAV8 or AAV9 vectors. Collectively, these findings suggest that constructs that include LAP2 may have the capacity to deliver large therapeutically effective payloads in support of future gene therapy protocols.

Novel tool to quantify with single-cell resolution the number of incoming AAV genomes co-expressed in the mouse nervous system.

Adeno-associated viral (AAV) vectors are an established and safe gene delivery tool to target the nervous system. However, the payload capacity of <4.9 kb limits the transfer of large or multiple genes. Oversized payloads could be delivered by fragmenting the transgenes into separate AAV capsids that are then mixed. This strategy could increase the AAV cargo capacity to treat monogenic, polygenic diseases and comorbidities only if controlled co-expression of multiple AAV capsids is achieved on each transduced cell. We developed a tool to quantify the number of incoming AAV genomes that are co-expressed in the nervous system with single-cell resolution. By using an isogenic mix of three AAVs each expressing single fluorescent reporters, we determined that expression of much greater than 31 AAV genomes per neuron in vitro and 20 genomes per neuron in vivo is obtained across different brain regions including anterior cingulate, prefrontal, somatomotor and somatosensory cortex areas, and cerebellar lobule VI. Our results demonstrate that multiple AAV vectors containing different transgenes or transgene fragments, can efficiently co-express in the same neuron. This tool can be used to design and improve AAV-based interrogation of neuronal circuits, map brain connectivity, and treat genetic diseases affecting the nervous system.

Optimized formulation buffer preserves adeno-associated virus-9 infectivity after 4 °C storage and freeze/thawing cycling.

Adeno-associated virus (AAV) have long been one of the most common and versatile vectors for in vitro and in vivo gene transfer. AAV production protocols are complex and time consuming, one key concern is the recovery and infectivity of viral vector after purification. The buffer used in the storage of AAV at 4 °C and - 80 °C is a crucial factor and methods to improve it have been thoroughly investigated. Viral core facilities have developed formulas using either 0.001% Pluronic F68 or 5% sorbitol in their storage buffers based on the results of this research. Interestingly, few use formulations that include both a non-ionic surfactant and cryopreservative. In this study, AAV9 stored at 4 °C and at - 80 °C in the standard buffers is compared to a buffer that contains 5% glycerol and 0.001% Pluronic F68. By viral genome quantitation with qPCR, all three formulations show the same extent of viral titer loss at 4 °C, while after several cycles of freeze/thaws at - 80 °C, the viral recovery and infectivity in the preparation with both glycerol and Pluronic F68 was most stable compared to the other buffers.

Eyeblink tract tracing with two strains of herpes simplex virus 1.

BACKGROUND: Neuroinvasive herpes simplex-1 (HSV-1) isolates including H129 and McIntyre cross at or near synapses labeling higher-order neurons directly connected to infected cells. H129 spreads predominately in the anterograde direction while McIntyre strains spread only in the retrograde direction. However, it is unknown if neurons are functional once infected with derivatives of H129 or McIntyre. NEW METHOD: We describe a previously unpublished HSV-1 recombinant derived from H129 (HSV-373) expressing mCherry fluorescent reporters and one new McIntyre recombinant (HSV-780) expressing the mCherry fluorophore and demonstrate how infections affect neuron viability. RESULTS AND COMPARISON WITH EXISTING METHODS: Each recombinant virus behaved similarly and spread to the target 4 days post-infection. We tested H129 recombinant infected neurons for neurodegeneration using Fluoro-jade C and found them to be necrotic as a result of viral infection. We performed dual inoculations with both HSV-772 and HSV-780 to identify cells comprising both the anterograde pathway and the retrograde pathway, respectively, of our circuit of study. We examined the presence of postsynaptic marker PSD-95, which plays a role in synaptic plasticity, in HSV-772 infected and in dual-infected rats (HSV-772 and HSV-780). PSD-95 reactivity decreased in HSV-772-infected neurons and dual-infected tissue had no PSD-95 reactivity. CONCLUSIONS: Infection by these new recombinant viruses traced the circuit of interest but functional studies of the cells comprising the pathway were not possible because viral-infected neurons died as a result of necrosis or were stripped of PSD-95 by the time the viral labels reached the target.

Single-Cell Quantification of Triple-AAV Vector Genomes Coexpressed in Neurons.

Adeno-associated viruses (AAVs) are one of the most widely used types of viral vectors for research and gene therapy. AAV vectors are safe, have a low immunogenic profile, and provide efficient and long-term transgene expression in a variety of tissues and organs targeted by a specific serotype. Despite these unique features, therapeutic applications, as well as basic research studies, of AAVs have been limited by their packaging capacity of less than 5 kb. Multiple strategies have been explored to deliver large genes. One strategy is to split large transgenes into two or three fragments and package them into separate AAV capsids, generating dual or triple AAV vectors. Combining the fragments potentially allows reconstitution of an mRNA transcript containing the complete sequence of transgene in the same cell. The success of AAVs as vectors for the delivery of large or multiple genes depends directly on the efficiency of co-transduction. Here, we describe a method to measure the efficacy of codelivery, quantifying the number of AAV vectors per cell. We detail how to calculate the average number of incoming AAV genomes in neurons, given the distribution of cell fluorescence across in vitro and in vivo experimental models. To validate the method, we simulated a triple AAV strategy using three fluorescent-protein-encoding genes. We provide a general protocol for constructing plasmids and producing and purifying AAV vectors. We also include a protocol for triple AAV vector co-transduction in primary neuronal cultures and mouse brain. The method can be applied to multiple organs and tissues for the treatment of disorders caused by mutations in multiple or large genes. These protocols will be useful for researchers working to develop and improve new gene delivery technologies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Construction of AAV plasmids and production of AAVs Basic Protocol 2: AAV transduction of primary superior cervical ganglia (SCG) neuronal cultures Basic Protocol 3: Mouse surgery, AAV injection, and tissue collection and processing Basic Protocol 4: Image analysis and AAV genome quantification.

Methods and Applications of Campenot Trichamber Neuronal Cultures for the Study of Neuroinvasive Viruses.

The development of compartmentalized neuron culture systems has been invaluable in the study of neuroinvasive viruses, including the alpha herpesviruses Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PRV). This chapter provides updated protocols for assembling and culturing rodent embryonic superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in Campenot trichamber cultures. In addition, we provide several illustrative examples of the types of experiments that are enabled by Campenot cultures: (1) Using fluorescence microscopy to investigate axonal outgrowth/extension through the chambers, and alpha herpesvirus infection, intracellular trafficking, and cell-cell spread via axons. (2) Using correlative fluorescence microscopy and cryo electron tomography to investigate the ultrastructure of virus particles trafficking in axons.

Transneuronal tracing to map connectivity in injured and transplanted spinal networks.

It has become widely appreciated that the spinal cord has significant neuroplastic potential, is not hard-wired, and that with traumatic injury and anatomical plasticity, the networks that we once understood now comprise a new anatomy. Harnessing advances in neuroanatomical tracing to map the neuronal networks of the intact and injured spinal cord has been crucial to elucidating this new spinal cord anatomy. Many new techniques have been developed to identify these networks using a variety of retrograde and anterograde tracers. One method of tracing that has become more widely used to map anatomical changes is transneuronal tracing. Viral tracers are being increasingly used to map spinal networks, leading to an advanced understanding of spinal circuitry and host-donor-host interactions between the injured spinal cord and neural transplants. This review will highlight advances in neuronal tracing, specifically using pseudorabies virus (PRV), and its use in the intact, injured, and transplanted spinal cord.

Homologous organization of cerebellar pathways to sensory, motor, and associative forebrain.

Cerebellar outputs take polysynaptic routes to reach the rest of the brain, impeding conventional tracing. Here, we quantify pathways between the cerebellum and forebrain by using transsynaptic tracing viruses and a whole-brain analysis pipeline. With retrograde tracing, we find that most descending paths originate from the somatomotor cortex. Anterograde tracing of ascending paths encompasses most thalamic nuclei, especially ventral posteromedial, lateral posterior, mediodorsal, and reticular nuclei. In the neocortex, sensorimotor regions contain the most labeled neurons, but we find higher densities in associative areas, including orbital, anterior cingulate, prelimbic, and infralimbic cortex. Patterns of ascending expression correlate with c-Fos expression after optogenetic inhibition of Purkinje cells. Our results reveal homologous networks linking single areas of the cerebellar cortex to diverse forebrain targets. We conclude that shared areas of the cerebellum are positioned to provide sensory-motor information to regions implicated in both movement and nonmotor function.

Deconstructing the origins of sexual dimorphism in sensory modulation of pancreatic ß cells.

The regulation of glucose-stimulated insulin secretion and glucose excursion has a sensory component that operates in a sex-dependent manner. OBJECTIVE: Here, we aim to dissect the basis of the sexually dimorphic interaction between sensory neurons and pancreatic ß cells and its overall impact on insulin release and glucose homeostasis. METHODS: We used viral retrograde tracing techniques, surgical and chemodenervation models, and primary cell-based co-culture systems to uncover the biology underlying sex differences in sensory modulation of pancreatic ß-cell activity. RESULTS: Retrograde transsynaptic labeling revealed a sex difference in the density of sensory innervation in the pancreas. The number of sensory neurons emanating from the dorsal root and nodose ganglia that project in the pancreas is higher in male than in female mice. Immunostaining and confocal laser scanning microscopy confirmed the higher abundance of peri-islet sensory axonal tracts in the male pancreas. Capsaicin-induced sensory chemodenervation concomitantly enhanced glucose-stimulated insulin secretion and glucose clearance in male mice. These metabolic benefits were blunted when mice were orchidectomized prior to the ablation of sensory nerves. Interestingly, orchidectomy also lowered the density of peri-islet sensory neurons. In female mice, capsaicin treatment did not affect glucose-induced insulin secretion nor glucose excursion and ovariectomy did not modify these outcomes. Interestingly, same- and opposite-sex sensory-islet co-culture paradigms unmasked the existence of potential gonadal hormone-independent mechanisms mediating the male-female difference in sensory modulation of islet ß-cell activity. CONCLUSION: Taken together, these data suggest that the sex-biased nature of the sensory control of islet ß-cell activity is a result of a combination of neurodevelopmental inputs, sex hormone-dependent mechanisms and the potential action of somatic molecules encoded by the sex chromosome complement.

Genome Sequence of the Virulent Model Herpes Simplex Virus 1 Strain McKrae Demonstrates the Presence of at Least Two Widely Used Variant Strains.

Herpes simplex virus 1 (HSV-1) strain McKrae was isolated in 1965 and has been utilized by many laboratories. Three HSV-1 strain McKrae stocks have been sequenced previously, revealing discrepancies in key genes. We sequenced the genome of HSV-1 strain McKrae from the laboratory of James M. Hill to better understand the genetic differences between isolates.

Adult-born granule cell mossy fibers preferentially target parvalbumin-positive interneurons surrounded by perineuronal nets.

Adult-born granule cells (abGCs) integrate into the hippocampus and form connections with dentate gyrus parvalbumin-positive (PV+) interneurons, a circuit important for modulating plasticity. Many of these interneurons are surrounded by perineuronal nets (PNNs), extracellular matrix structures known to participate in plasticity. We compared abGC projections to PV+ interneurons with negative-to-low intensity PNNs to those with high intensity PNNs using retroviral and 3R-Tau labeling in adult mice, and found that abGC mossy fibers and boutons are more frequently located near PV+ interneurons with high intensity PNNs. These results suggest that axons of new neurons preferentially stabilize near target cells with intense PNNs. Next, we asked whether the number of abGCs influences PNN formation around PV+ interneurons, and found that near complete ablation of abGCs produced a decrease in the intensity and number of PV+ neurons with PNNs, suggesting that new neuron innervation may enhance PNN formation. Experience-driven changes in adult neurogenesis did not produce consistent effects, perhaps due to widespread effects on plasticity. Our study identifies abGC projections to PV+ interneurons with PNNs, with more presumed abGC mossy fiber boutons found near the cell body of PV+ interneurons with strong PNNs.

Invasion of the Nervous System.

In vertebrates, the nervous system (NS) is composed of a peripheral collection of neurons (the peripheral nervous system, PNS), a central set found in the brain and spinal cord (the central nervous system, CNS). The NS is protected by rather complicated multi-layer barriers that allow access to nutrients and facilitate contact with the peripheral tissues, but block entry of pathogens and toxins. Virus infections usually begin in peripheral tissues and if these barriers are weakened, they can spread into the PNS and more rarely into the CNS. Most viral infections of the NS are opportunistic or accidental pathogens that gain access via the bloodstream (e.g., HIV and various arboviruses). But a few have evolved to enter the NS efficiently by invading neurons directly and by exploiting neuronal cell biology (e.g., rhabdoviruses and alphaherpesviruses). Most NS infections are devastating and difficult to manage. Remarkably, the alphaherpesviruses establish life-long quiescent infections in the PNS, with rare but often serious CNS pathology. In this review, we will focus on how alphaherpesviruses gain access to and spread in the NS, with particular emphasis on bidirectional transport and spread within and between neurons and neural circuits, which is regulated by complex viral-host protein interactions. Finally, we will describe the wide use of alphaherpesviruses as tools to study nerve connectivity and function in animal models.

CRISPR/Cas9-Constructed Pseudorabies Virus Mutants Reveal the Importance of UL13 in Alphaherpesvirus Escape from Genome Silencing.

Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally delivered genomes is facilitated by pseudorabies virus (PRV) tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP), and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N terminus (PRV mTurq-VP16) or the C terminus (PRV VP16-mTurq). Live-cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. However, immunofluorescence staining of particles in mid-axons indicated that UL13 might be cotransported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as adeno-associated virus (AAV)-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins.IMPORTANCE Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude the indirect role of UL13 in PRV escape from latency in primary neurons, along with its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.

A Comparison of Dopaminergic and Cholinergic Populations Reveals Unique Contributions of VTA Dopamine Neurons to Short-Term Memory.

We systematically compare the contributions of two dopaminergic and two cholinergic ascending populations to a spatial short-term memory task in rats. In ventral tegmental area dopamine (VTA-DA) and nucleus basalis cholinergic (NB-ChAT) populations, trial-by-trial fluctuations in activity during the delay period relate to performance with an inverted-U, despite the fact that both populations have low activity during that time. Transient manipulations reveal that only VTA-DA neurons, and not the other three populations we examine, contribute causally and selectively to short-term memory. This contribution is most significant during the delay period, when both increases and decreases in VTA-DA activity impair short-term memory. Our results reveal a surprising dissociation between when VTA-DA neurons are most active and when they have the biggest causal contribution to short-term memory, and they also provide support for classic ideas about an inverted-U relationship between neuromodulation and cognition.

Small Alphaherpesvirus Latency-Associated Promoters Drive Efficient and Long-Term Transgene Expression in the CNS.

Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy to identify three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp), and LAP1_2 (880 bp). They drive chronic transcription of the virus-encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1α promoter (1,264 bp). Our data suggest that the LAPs are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration.

2019 Colorado Alphaherpesvirus Latency Society symposium.

Meeting Report on the 9th Annual Symposium of the Colorado Alphaherpesvirus Latency Society (CALS) held on May 8-11, 2019, in Vail, CO.

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.