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.

Engineered compact pan-neuronal promoter from Alphaherpesvirus LAP2 enhances target gene expression in the mouse brain and reduces tropism in the liver.

Small promoters capable of driving potent neuron-restricted gene expression are required to support successful brain circuitry and clinical gene therapy studies. However, converting large promoters into functional MiniPromoters, which can be used in vectors with limited capacity, remains challenging. In this study, we describe the generation of a novel version of alphaherpesvirus latency-associated promoter 2 (LAP2), which facilitates precise transgene expression exclusively in the neurons of the mouse brain while minimizing undesired targeting in peripheral tissues. Additionally, we aimed to create a compact neural promoter to facilitate packaging of larger transgenes. Our results revealed that MiniLAP2 (278 bp) drives potent transgene expression in all neurons in the mouse brain, with little to no expression in glial cells. In contrast to the native promoter, MiniLAP2 reduced tropism in the spinal cord and liver. No expression was detected in the kidney or skeletal muscle. In summary, we developed a minimal pan-neuronal promoter that drives specific and robust transgene expression in the mouse brain when delivered intravenously via AAV-PHP.eB vector. The use of this novel MiniPromoter may broaden the range of deliverable therapeutics and improve their safety and efficacy by minimizing the potential for off-target effects.

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.

NMDA and P2X7 Receptors Require Pannexin 1 Activation to Initiate and Maintain Nociceptive Signaling in the Spinal Cord of Neuropathic Rats.

Pannexin 1 (Panx1) is involved in the spinal central sensitization process in rats with neuropathic pain, but its interaction with well-known, pain-related, ligand-dependent receptors, such as NMDA receptors (NMDAR) and P2X7 purinoceptors (P2X7R), remains largely unexplored. Here, we studied whether NMDAR- and P2X7R-dependent nociceptive signaling in neuropathic rats require the activation of Panx1 channels to generate spinal central sensitization, as assessed by behavioral (mechanical hyperalgesia) and electrophysiological (C-reflex wind-up potentiation) indexes. Administration of either a selective NMDAR agonist i.t. (NMDA, 2 mM) or a P2X7R agonist (BzATP, 150 µM) significantly increased both the mechanical hyperalgesia and the C-reflex wind-up potentiation, effects that were rapidly reversed (minutes) by i.t. administration of a selective pannexin 1 antagonist (10panx peptide, 300 µM), with the scores even reaching values of rats without neuropathy. Accordingly, 300 µM 10panx completely prevented the effects of NMDA and BzATP administered 1 h later, on mechanical hyperalgesia and C-reflex wind-up potentiation. Confocal immunofluorescence imaging revealed coexpression of Panx1 with NeuN protein in intrinsic dorsal horn neurons of neuropathic rats. The results indicate that both NMDAR- and P2X7R-mediated increases in mechanical hyperalgesia and C-reflex wind-up potentiation require neuronal Panx1 channel activation to initiate and maintain nociceptive signaling in neuropathic rats.

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.

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

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.

Astroglial Ca2+-Dependent Hyperexcitability Requires P2Y1 Purinergic Receptors and Pannexin-1 Channel Activation in a Chronic Model of Epilepsy.

Astrocytes from the hippocampus of chronic epileptic rats exhibit an abnormal pattern of intracellular calcium oscillations, characterized by an augmented frequency of long lasting spontaneous Ca2+ transients, which are sensitive to purinergic receptor antagonists but resistant to tetrodotoxin. The above suggests that alterations in astroglial Ca2+-dependent excitability observed in the epileptic tissue could arise from changes in astrocyte-to-astrocyte signaling, which is mainly mediated by purines in physiological and pathological conditions. In spite of that, how purinergic signaling contributes to astrocyte dysfunction in epilepsy remains unclear. Here, we assessed the possible contribution of P2Y1R as well as pannexin1 and connexin43 hemichannels-both candidates for non-vesicular ATP-release-by performing astroglial Ca2+ imaging and dye uptake experiments in hippocampal slices from control and fully kindled rats. P2Y1R blockade with MRS2179 decreased the mean duration of astroglial Ca2+ oscillations by reducing the frequency of slow Ca2+ transients, and thereby restoring the balance between slow (ST) and fast transients (FT) in the kindled group. The potential contribution of astroglial pannexin1 and connexin43 hemichannels as pathways for purine release (e.g., ATP) was assessed through dye uptake experiments. Astrocytes from kindled hippocampi exhibit three-fold more EtBr uptake than controls, whereby pannexin1 hemichannels (Panx1 HCs) accounts for almost all dye uptake with only a slight contribution from connexin43 hemichannels (Cx43 HCs). Confirming its functional involvement, Panx1 HCs inhibition decreased the mean duration of astroglial Ca2+ transients and the frequency of slow oscillations in kindled slices, but had no noticeable effects on the control group. As expected, Cx43 HCs blockade did not have any effects over the mean duration of astroglial Ca2+ oscillations. These findings suggest that P2Y1R and Panx1 HCs play a pivotal role in astroglial pathophysiology, which would explain the upregulation of glutamatergic neurotransmission in the epileptic brain and thus represents a new potential pharmacological target for the treatment of drug-refractory epilepsy.

Heavy Alcohol Exposure Activates Astroglial Hemichannels and Pannexons in the Hippocampus of Adolescent Rats: Effects on Neuroinflammation and Astrocyte Arborization.

A mounting body of evidence indicates that adolescents are specially more susceptible to alcohol influence than adults. However, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity and recently, the opening of hemichannels and pannexons has been found to participate in both processes. Here, we evaluated whether adolescent rats exposed to ethanol exhibit changes in the activity of astrocyte hemichannels and pannexons in the hippocampus, as well as alterations in astrocyte arborization and cytokine levels. Adolescent rats were subjected to ethanol (3.0 g/kg) for two successive days at 48-h periods over 14 days. The opening of hemichannels and pannexons was examined in hippocampal slices by dye uptake, whereas hippocampal cytokine levels and astroglial arborization were determined by ELISA and Sholl analysis, respectively. We found that adolescent ethanol exposure increased the opening of connexin 43 (Cx43) hemichannels and pannexin-1 (Panx1) channels in astrocytes. Blockade of p38 mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS) and cyclooxygenases (COXs), as well as chelation of intracellular Ca2+, drastically reduced the ethanol-induced channel opening in astrocytes. Importantly, ethanol-induced Cx43 hemichannel and Panx1 channel activity was correlated with increased levels of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 in the hippocampus, as well as with profound alterations in astrocyte arbor complexity. Thus, we propose that uncontrolled opening of astrocyte hemichannels and pannexons may contribute not only to the glial dysfunction and neurotoxicity caused by adolescent alcohol consumption, but also to the pathogenesis of alcohol use disorders in the adulthood.

High glucocorticoid levels during gestation activate the inflammasome in hippocampal oligodendrocytes of the offspring.

Exposure to high levels of glucocorticoids (GCs) during early life induces long-lasting neuroinflammation. GCs induce rapid degranulation of mast cells, which release proinflammatory molecules promoting activation of microglia and astrocytes. The possible involvement of oligodendrocytes, however, remains poorly understood. It was studied whether high GC levels during gestation activates the inflammasome in hippocampal oligodendrocytes of mouse offspring. Oligodendrocytes of control pups showed expression of inflammasome components (NLRP3, ACS, and caspase-1) and their levels were increased by prenatal administration of dexamethasone (DEX), a synthetic GC. These cells also showed high levels of IL-1ß and TNF-α, revealing activation of the inflammasome. Moreover, they showed increased levels of the P2X7 receptor and pannexin1, which are associated to inflammasome activation. However, levels of connexins either were not affected (Cx29) or reduced (Cx32 and Cx47). Nonetheless, the functional states of pannexin1 and connexin hemichannels were elevated and directly associated to functional P2X7 receptors. As observed in DEX-treated brain slices, hemichannel activity first increased in hippocampal mast cells and later in microglia and macroglia. DEX-induced oligodendrocyte hemichannel activity was mimicked by urocortin-II, which is a corticotropin-releasing hormone receptor (CRHR) agonist. Response to DEX and urocortin-II was inhibited by antalarmin (a CRHR blocker) or by mast cells or microglia inhibitors. The increase in hemichannel activity persisted for several weeks after birth and cross-fostering with a control mother did not reverse this condition. It is proposed that activation of the oligodendrocyte inflammasome might be relevant in demyelinating diseases associated with early life exposure to high GC levels. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 625-642, 2017.

Cannabinoids prevent the amyloid ß-induced activation of astroglial hemichannels: A neuroprotective mechanism.

The mechanisms involved in Alzheimer's disease are not completely understood and how astrocytes and their gliotransmission contribute to this neurodegenerative disease remains to be fully elucidated. Previous studies have shown that amyloid-ß peptide (Aß) induces neuronal death by a mechanism that involves the excitotoxic release of ATP and glutamate associated to astroglial hemichannel opening. We have demonstrated that synthetic and endogenous cannabinoids (CBs) reduce the opening of astrocyte Cx43 hemichannels evoked by activated microglia or inflammatory mediators. Nevertheless, whether CBs could prevent the astroglial hemichannel-dependent death of neurons evoked by Aß is unknown. Astrocytes as well as acute hippocampal slices were treated with the active fragment of Aß alone or in combination with the following CBs: WIN, 2-AG, or methanandamide (Meth). Hemichannel activity was monitored by single channel recordings and by time-lapse ethidium uptake while neuronal death was assessed by Fluoro-Jade C staining. We report that CBs fully prevented the hemichannel activity and inflammatory profile evoked by Aß in astrocytes. Moreover, CBs fully abolished the Aß-induced release of excitotoxic glutamate and ATP associated to astrocyte Cx43 hemichannel activity, as well as neuronal damage in hippocampal slices exposed to Aß. Consequently, this work opens novel avenues for alternative treatments that target astrocytes to maintain neuronal function and survival during AD. GLIA 2016 GLIA 2017;65:122-137.

Restraint stress increases hemichannel activity in hippocampal glial cells and neurons.

Stress affects brain areas involved in learning and emotional responses, which may contribute in the development of cognitive deficits associated with major depression. These effects have been linked to glial cell activation, glutamate release and changes in neuronal plasticity and survival including atrophy of hippocampal apical dendrites, loss of synapses and neuronal death. Under neuro-inflammatory conditions, we recently unveiled a sequential activation of glial cells that release ATP and glutamate via hemichannels inducing neuronal death due to activation of neuronal NMDA/P2X7 receptors and pannexin1 hemichannels. In the present work, we studied if stress-induced glia activation is associated to changes in hemichannel activity. To this end, we compared hemichannel activity of brain cells after acute or chronic restraint stress in mice. Dye uptake experiments in hippocampal slices revealed that acute stress induces opening of both Cx43 and Panx1 hemichannels in astrocytes, which were further increased by chronic stress; whereas enhanced Panx1 hemichannel activity was detected in microglia and neurons after acute/chronic and chronic stress, respectively. Moreover, inhibition of NMDA/P2X7 receptors reduced the chronic stress-induced hemichannel opening, whereas blockade of Cx43 and Panx1 hemichannels fully reduced ATP and glutamate release in hippocampal slices from stressed mice. Thus, we propose that gliotransmitter release through hemichannels may participate in the pathogenesis of stress-associated psychiatric disorders and possibly depression.

Possible involvement of TLRs and hemichannels in stress-induced CNS dysfunction via mastocytes, and glia activation.

In the central nervous system (CNS), mastocytes and glial cells (microglia, astrocytes and oligodendrocytes) function as sensors of neuroinflammatory conditions, responding to stress triggers or becoming sensitized to subsequent proinflammatory challenges. The corticotropin-releasing hormone and glucocorticoids are critical players in stress-induced mastocyte degranulation and potentiation of glial inflammatory responses, respectively. Mastocytes and glial cells express different toll-like receptor (TLR) family members, and their activation via proinflammatory molecules can increase the expression of connexin hemichannels and pannexin channels in glial cells. These membrane pores are oligohexamers of the corresponding protein subunits located in the cell surface. They allow ATP release and Ca(2+) influx, which are two important elements of inflammation. Consequently, activated microglia and astrocytes release ATP and glutamate, affecting myelinization, neuronal development, and survival. Binding of ligands to TLRs induces a cascade of intracellular events leading to activation of several transcription factors that regulate the expression of many genes involved in inflammation. During pregnancy, the previous responses promoted by viral infections and other proinflammatory conditions are common and might predispose the offspring to develop psychiatric disorders and neurological diseases. Such disorders could eventually be potentiated by stress and might be part of the etiopathogenesis of CNS dysfunctions including autism spectrum disorders and schizophrenia.