NLRP3 inflammasome-driven IL-1ß and IL-18 contribute to lipopolysaccharide-induced septic cardiomyopathy.

Sepsis is a life-threatening syndrome, and its associated mortality is increased when cardiac dysfunction and damage (septic cardiomyopathy [SCM]) occur. Although inflammation is involved in the pathophysiology of SCM, the mechanism of how inflammation induces SCM in vivo has remained obscure. NLRP3 inflammasome is a critical component of the innate immune system that activates caspase-1 (Casp1) and causes the maturation of IL-1ß and IL-18 as well as the processing of gasdermin D (GSDMD). Here, we investigated the role of the NLRP3 inflammasome in a murine model of lipopolysaccharide (LPS)-induced SCM. LPS injection induced cardiac dysfunction, damage, and lethality, which was significantly prevented in NLRP3-/- mice, compared to wild-type (WT) mice. LPS injection upregulated mRNA levels of inflammatory cytokines (Il6, Tnfa, and Ifng) in the heart, liver, and spleen of WT mice, and this upregulation was prevented in NLRP3-/- mice. LPS injection increased plasma levels of inflammatory cytokines (IL-1ß, IL-18, and TNF-α) in WT mice, and this increase was markedly inhibited in NLRP3-/- mice. LPS-induced SCM was also prevented in Casp1/11-/- mice, but not in Casp11mt, IL-1ß-/-, IL-1α-/-, or GSDMD-/- mice. Notably, LPS-induced SCM was apparently prevented in IL-1ß-/- mice transduced with adeno-associated virus vector expressing IL-18 binding protein (IL-18BP). Furthermore, splenectomy, irradiation, or macrophage depletion alleviated LPS-induced SCM. Our findings demonstrate that the cross-regulation of NLRP3 inflammasome-driven IL-1ß and IL-18 contributes to the pathophysiology of SCM and provide new insights into the mechanism underlying the pathogenesis of SCM.

Therapeutic strategy for Fabry disease by intravenous administration of adeno-associated virus 2 or 9 in α-galactosidase A-deficient mice.

BACKGROUND: Fabry disease (FD) is an inherited lysosomal storage disease caused by deficiency of α-galactosidase A (α-Gal A) encoded by the GLA gene. The symptoms of FD occur as a result of the accumulation of globotriaosylceramide (Gb3), comprising a substrate of α-Gal A, in the organs. Adeno-associated virus (AAV)-mediated gene therapy is a promising treatment for FD. METHODS: α-Gal A knockout (GLAko) mice were injected intravenously with AAV2 (1 × 1011 viral genomes [vg]) or AAV9 (1 × 1011 or 2 × 1012 vg) vectors carrying human GLA (AAV-hGLA), and plasma, brain, heart, liver and kidney were tested for α-Gal A activity. The vector genome copy numbers (VGCNs) and Gb3 content in each organ were also examined. RESULTS: The plasma α-Gal A enzymatic activity was three-fold higher in the AAV9 2 × 1012 vg group than wild-type (WT) controls, which was maintained for up to 8 weeks after injection. In the AAV9 2 × 1012 vg group, the level of α-Gal A expression was high in the heart and liver, intermediate in the kidney, and low in the brain. VGCNs in the all organs of the AAV9 2 × 1012 vg group significantly increased compared to the phosphate-buffered-saline (PBS) group. Although Gb3 in the heart, liver and kidney of the AAV9 2 × 1012 vg was reduced compared to PBS group and AAV2 group, and the amount of Gb3 in the brain was not reduced. CONCLUSIONS: Systemic injection of AAV9-hGLA resulted in α-Gal A expression and Gb3 reduction in the organs of GLAko mice. To expect a higher expression of α-Gal A in the brain, the injection dosage, administration route and the timing of injection should be reconsidered.

Oxytocin receptor-expressing neurons in the medial preoptic area are essential for lactation, whereas those in the lateral septum are not critical for maternal behavior.

INTRODUCTION: In nurturing systems, the oxytocin (Oxt)-oxytocin receptor (Oxtr) system is important for parturition, and essential for lactation and parental behavior. Among the nerve nuclei that express Oxtr, the lateral septal nucleus (LS) and medial preoptic area (MPOA) are representative regions that control maternal behavior. METHODS: We investigated the role of Oxtr- and Oxtr-expressing neurons, located in the LS and MPOA, in regulating maternal behavior by regulating Oxtr expression in a region-specific manner using recombinant mice and adeno-associated viruses. We quantified the prolactin (Prl) concentrations in the pituitary gland and plasma when Oxtr expression in the MPOA was reduced. RESULTS: The endogenous Oxtr gene in the neurons of the LS did not seem to play an essential role in maternal behavior. Conversely, decreased Oxtr expression in the MPOA increased the frequency of pups being left outside the nest and reduced their survival rate. Deletion of Oxtr in MPOA neurons prevented elevation of Prl levels in plasma and pituitary at postpartum day 2. DISCUSSION/CONCLUSION: Oxtr-expressing neurons in the MPOA are involved in the postpartum production of Prl. We confirmed the essential functions of Oxtr-expressing neurons and the Oxtr gene itself in the MPOA for the sustainability of maternal behavior, which involved Oxtr-dependent induction of Prl.

Dark Rearing in the Visual Critical Period Causes Structural Changes in Myelinated Axons in the Adult Mouse Visual Pathway.

An appropriate sensory experience during the early developmental period is important for brain maturation. Dark rearing during the visual critical period delays the maturation of neuronal circuits in the visual cortex. Although the formation and structural plasticity of the myelin sheaths on retinal ganglion cell axons modulate the visual function, the effects of dark rearing during the visual critical period on the structure of the retinal ganglion cell axons and their myelin sheaths are still unclear. To address this question, mice were reared in a dark box during the visual critical period and then normally reared to adulthood. We found that myelin sheaths on the retinal ganglion cell axons of dark-reared mice were thicker than those of normally reared mice in both the optic chiasm and optic nerve. Furthermore, whole-mount immunostaining with fluorescent axonal labeling and tissue clearing revealed that the myelin internodal length in dark-reared mice was shorter than that in normally reared mice in both the optic chiasm and optic nerve. These findings demonstrate that dark rearing during the visual critical period affects the morphology of myelin sheaths, shortens and thickens myelin sheaths in the visual pathway, despite the mice being reared in normal light/dark conditions after the dark rearing.

Arm movements induced by noninvasive optogenetic stimulation of the motor cortex in the common marmoset.

Optogenetics is now a fundamental tool for investigating the relationship between neuronal activity and behavior. However, its application to the investigation of motor control systems in nonhuman primates is rather limited, because optogenetic stimulation of cortical neurons in nonhuman primates has failed to induce or modulate any hand/arm movements. Here, we used a tetracycline-inducible gene expression system carrying CaMKII promoter and the gene encoding a Channelrhodopsin-2 variant with fast kinetics in the common marmoset, a small New World monkey. In an awake state, forelimb movements could be induced when Channelrhodopsin-2-expressing neurons in the motor cortex were illuminated by blue laser light with a spot diameter of 1 mm or 2 mm through a cranial window without cortical invasion. Forelimb muscles responded 10 ms to 50 ms after photostimulation onset. Long-duration (500 ms) photostimulation induced discrete forelimb movements that could be markerlessly tracked with charge-coupled device cameras and a deep learning algorithm. Long-duration photostimulation mapping revealed that the primary motor cortex is divided into multiple domains that can induce hand and elbow movements in different directions. During performance of a forelimb movement task, movement trajectories were modulated by weak photostimulation, which did not induce visible forelimb movements at rest, around the onset of task-relevant movement. The modulation was biased toward the movement direction induced by the strong photostimulation. Combined with calcium imaging, all-optical interrogation of motor circuits should be possible in behaving marmosets.

Direct reprogramming with Sendai virus vectors repaired infarct hearts at the chronic stage.

Adult hearts have limited regenerative capacity. Hence, after acute myocardial infarction (MI), dead myocardial tissues are digested by immune cells and replaced by fibrosis, leading to ventricular remodeling and heart failure at the chronic stage. Direct reprogramming of the cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) with cardiac transcription factors, including Gata4, Mef2c, and Tbx5 (GMT), may have significant potential for cardiac repair. Sendai virus (SeV) vectors expressing GMT have been reported to reprogram the mouse cardiac fibroblasts into iCMs without any risk of insertional mutagenesis. In vivo reprogramming improved the cardiac function after acute MI in immunodeficient mice. However, it is unknown whether the newly generated iCMs could exist in infarct hearts for a prolonged period and SeV-GMT can improve cardiac function after MI at the chronic stage in immunocompetent mice. Here, we show that SeV vectors efficiently infect CFs in vivo and reprogram them into iCMs, which existed for at least four weeks after MI, in fibroblast-linage tracing mice. Moreover, SeV-GMT improved cardiac function and reduced fibrosis and collagen I expression at 12 weeks after MI in immunocompetent mice. Thus, direct cardiac reprogramming with SeV vectors could be a promising therapy for MI.

Utility of microminipigs for evaluating liver-mediated gene expression in the presence of neutralizing antibody against vector capsid.

Adeno-associated virus (AAV) vectors can transduce hepatocytes efficiently in vivo in various animal species, including humans. Few reports, however, have examined the utility of pigs in gene therapy. Pigs are potentially useful in preclinical studies because of their anatomical and physiological similarity to humans. Here, we evaluated the utility of microminipigs for liver-targeted gene therapy. These pigs were intravenously inoculated with an AAV8 vector encoding the luciferase gene, and gene expression was assessed by an in vivo imaging system. Robust transgene expression was observed almost exclusively in the liver, even though the pig showed a low-titer of neutralizing antibody (NAb) against the AAV8 capsid. We assessed the action of NAbs against AAV, which interfere with AAV vector-mediated gene transfer by intravascular delivery. When a standard dose of vector was administered intravenously, transgene expression was observed in both NAb-negative and low-titer (14×)-positive subjects, whereas gene expression was not observed in animals with higher titers (56×). These results are compatible with our previous observations using nonhuman primates, indicating that pigs are useful in gene therapy experiments, and that the role of low-titer NAb in intravenous administration of the AAV vector shows similarities across species.

Gene therapy improves motor and mental function of aromatic l-amino acid decarboxylase deficiency.

In patients with aromatic l-amino acid decarboxylase (AADC) deficiency, a decrease in catecholamines and serotonin levels in the brain leads to developmental delay and movement disorders. The beneficial effects of gene therapy in patients from 1 to 8 years of age with homogeneous severity of disease have been reported from Taiwan. We conducted an open-label phase 1/2 study of population including adolescent patients with different degrees of severity. Six patients were enrolled: four males (ages 4, 10, 15 and 19 years) and one female (age 12 years) with a severe phenotype who were not capable of voluntary movement or speech, and one female (age 5 years) with a moderate phenotype who could walk with support. The patients received a total of 2 × 1011 vector genomes of adeno-associated virus vector harbouring DDC via bilateral intraputaminal infusions. At up to 2 years after gene therapy, the motor function was remarkably improved in all patients. Three patients with the severe phenotype were able to stand with support, and one patient could walk with a walker, while the patient with the moderate phenotype could run and ride a bicycle. This moderate-phenotype patient also showed improvement in her mental function, being able to converse fluently and perform simple arithmetic. Dystonia disappeared and oculogyric crisis was markedly decreased in all patients. The patients exhibited transient choreic dyskinesia for a couple of months, but no adverse events caused by vector were observed. PET with 6-[18F]fluoro-l-m-tyrosine, a specific tracer for AADC, showed a persistently increased uptake in the broad areas of the putamen. In our study, older patients (>8 years of age) also showed improvement, although treatment was more effective in younger patients. The genetic background of our patients was heterogeneous, and some patients suspected of having remnant enzyme activity showed better improvement than the Taiwanese patients. In addition to the alleviation of motor symptoms, the cognitive and verbal functions were improved in a patient with the moderate phenotype. The restoration of dopamine synthesis in the putamen via gene transfer provides transformative medical benefit across all patient ages, genotypes, and disease severities included in this study, with the most pronounced improvements noted in moderate patients.10.1093/brain/awy331_video1awy331media15991361892001.

Survivin overexpression via adeno-associated virus vector Rh10 ameliorates ischemic damage after middle cerebral artery occlusion in rats.

Survivin, a member of the inhibitors of apoptosis protein gene family, inhibits the activity of caspase, leading to a halt of the apoptotic process. Our study focused on the neuroprotective effect of survivin after transient middle cerebral artery occlusion (MCAO) with intraparenchymal administration of an adeno-associated virus (AAV) vector. His-tagged survivin was cloned and packaged into the AAV-rh10 vector. Four-week-old Sprague-Dawley rats were injected with 4 × 109  vg of AAV-GFP or AAV-His-survivin into the right striatum and were treated 3 weeks later with transient MCAO for 90 min. Twenty-four hours after MCAO, functional and histological analyses of the rats were performed. The result showed that rats that had been treated with AAV-green fluorescent protein (GFP) and those that had been treated with AAV-His-survivin did not show a significant difference in neurological scores 24 hr after MCAO, however, infarction volume was significantly reduced in the AAV-His-survivin group compared to that in the AAV-GFP group. Although the neutrophil marker myeloperoxidase did not show a significant difference in the ischemic boundary zone, cells positive for active caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling were significantly decreased in the AAV-His-survivin group. In conclusion, survivin overexpression in the ischemic boundary zone induced by using an AAV vector has the potential for amelioration of ischemic damage via an antiapoptotic mechanism.

Corticospinal tract development and spinal cord innervation differ between cervical and lumbar targets.

The corticospinal (CS) tract is essential for voluntary movement, but what we know about the organization and development of the CS tract remains limited. To determine the total cortical area innervating the seventh cervical spinal cord segment (C7), which controls forelimb movement, we injected a retrograde tracer (fluorescent microspheres) into C7 such that it would spread widely within the unilateral gray matter (to >80%), but not to the CS tract. Subsequent detection of the tracer showed that, in both juvenile and adult mice, neurons distributed over an unexpectedly broad portion of the rostral two-thirds of the cerebral cortex converge to C7. This even included cortical areas controlling the hindlimbs (the fourth lumbar segment, L4). With aging, cell densities greatly declined, mainly due to axon branch elimination. Whole-cell recordings from spinal cord cells upon selective optogenetic stimulation of CS axons, and labeling of axons (DsRed) and presynaptic structures (synaptophysin) through cotransfection using exo utero electroporation, showed that overgrowing CS axons make synaptic connections with spinal cells in juveniles. This suggests that neuronal circuits involved in the CS tract to C7 are largely reorganized during development. By contrast, the cortical areas innervating L4 are limited to the conventional hindlimb area, and the cell distribution and density do not change during development. These findings call for an update of the traditional notion of somatotopic CS projection and imply that there are substantial developmental differences in the cortical control of forelimb and hindlimb movements, at least in rodents.

Corticospinal axons make direct synaptic connections with spinal motoneurons innervating forearm muscles early during postnatal development in the rat.

KEY POINTS: Direct connections between corticospinal (CS) axons and motoneurons (MNs) appear to be present only in higher primates, where they are essential for discrete movement of the digits. Their presence in adult rodents was once claimed but is now questioned. We report that MNs innervating forearm muscles in infant rats receive monosynaptic input from CS axons, but MNs innervating proximal muscles do not, which is a pattern similar to that in primates. Our experiments were carefully designed to show monosynaptic connections. This entailed selective electrical and optogenetic stimulation of CS axons and recording from MNs identified by retrograde labelling from innervated muscles. Morphological evidence was also obtained for rigorous identification of CS axons and MNs. These connections would be transient and would regress later during development. These results shed light on the development and evolution of direct CS-MN connections, which serve as the basis for dexterity in humans. Recent evidence suggests there is no direct connection between corticospinal (CS) axons and spinal motoneurons (MNs) in adult rodents. We previously showed that CS synapses are present throughout the spinal cord for a time, but are eliminated from the ventral horn during development in rodents. This raises the possibility that CS axons transiently make direct connections with MNs located in the ventral horn of the spinal cord. This was tested in the present study. Using cervical cord slices prepared from rats on postnatal days (P) 7-9, CS axons were stimulated and whole cell recordings were made from MNs retrogradely labelled with fluorescent cholera toxin B subunit (CTB) injected into selected groups of muscles. To selectively activate CS axons, electrical stimulation was carefully limited to the CS tract. In addition we employed optogenetic stimulation after injecting an adeno-associated virus vector encoding channelrhodopsin-2 (ChR2) into the sensorimotor cortex on P0. We were then able to record monosynaptic excitatory postsynaptic currents from MNs innervating forearm muscles, but not from those innervating proximal muscles. We also showed close contacts between CTB-labelled MNs and CS axons labelled through introduction of fluorescent protein-conjugated synaptophysin or the ChR2 expression system. We confirmed that some of these contacts colocalized with postsynaptic density protein 95 in their partner dendrites. It is intriguing from both phylogenetic and ontogenetic viewpoints that direct and putatively transient CS-MN connections were found only on MNs innervating the forearm muscles in infant rats, as this is analogous to the connection pattern seen in adult primates.

Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner.

The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR.

Vasohibin-1 expression inhibits advancement of ovarian cancer producing various angiogenic factors.

Vasohibin-1 (VASH1) is a negative feedback regulator of angiogenesis, the first to be discovered, and was identified in vascular endothelial growth factor (VEGF)-stimulated vascular endothelial cells. Vasohibin-1 inhibits abnormal vascularization induced by various angiogenic factors including fibroblast growth factor and platelet-derived growth factor (PDGF), in addition to VEGF. By focusing on this characteristic of VASH1, we investigated the antitumor effects of VASH1 expression on ovarian cancer cells that produce different angiogenic factors. By using a high VEGF-producing ovarian cancer cell line, SHIN-3, and a high PDGF-producing ovarian cancer cell line, KOC-2S, the cells were transfected with either a VEGF antagonist, soluble VEGF receptor-1 (sVEGFR-1, or sFlt-1), or VASH1 genes to establish their respective cellular expression. The characteristics of these transfectants were compared with controls. We previously reported that the expression of sFlt-1 inhibited tumor vascularization and growth of high VEGF-producing ovarian cancer cells, reduced peritoneal dissemination and ascites development, and prolonged the survival time of the host. However, in the current study, the expression of sFlt-1 had no such effect on the high PDGF-producing ovarian cancer cells used here, whereas VASH1 expression inhibited tumor vascularization and growth, not only in high VEGF-producing cells, but also in high PDGF-producing cells, reduced their peritoneal dissemination and ascites, and prolonged the survival time of the host. These results suggest that VASH1 is an effective treatment for ovarian cancer cells that produce different angiogenic factors.

Vasopressinergic control of stress-related behavior: studies in Brattleboro rats.

Vasopressin, a nonapeptide, signaling both as hormone in the blood and neuromodulator/neurotransmitter in the brain is considered to be causally involved in the pathological changes underlying anxiety and depression. In the present review we summarize experimental data obtained with Brattleboro rats as a model of congenital vasopressin-deficiency to test the hypothesis that central vasopressin signaling contributes to anxiety- and depression-like behavior. Male, female and lactating rats were studied. We focused on the paraventricular nucleus of the hypothalamus (PVN) and the septum, two brain areas in which vasopressin is proposed to control the endocrine and behavioral stress response, respectively. The presented data support the hypothesis that the behavioral changes seen in these rats are brought about by an altered vasopressin signaling at the brain level. Whereas vasopressin synthesized and released within the hypothalamus is primarily involved in endocrine regulation, vasopressin signaling in other brain areas may contribute to anxiety- and depression-like behavioral parameters. Further studies in this context might focus particularly on the interplay between extra-hypothalamic brain areas such as the septum and the medial amygdala.

Glial dysfunction in the mouse habenula causes depressive-like behaviors and sleep disturbance.

The lateral habenula (LHb) regulates the activity of monoaminergic neurons in the brainstem. This area has recently attracted a surge of interest in psychiatry because studies have reported the pathological activation of the habenula in patients with major depression and in animal models. The LHb plays a significant role in the pathophysiology of depression; however, how habenular neurons are activated to cause various depression symptoms, such as reduced motivation and sleep disturbance, remain unclear. We hypothesized that dysfunctional astrocytes may cause LHb hyperactivity due to the defective uptake activity of extracellular glutamate, which induces depressive-like behaviors. We examined the activity of neurons in habenular pathways and performed behavioral and sleep analyses in mice with pharmacological and genetic inhibition of the activity of the glial glutamate transporter GLT-1 in the LHb. The habenula-specific inhibition of GLT-1 increased the neuronal firing rate and the level of c-Fos expression in the LHb. Mice with reduced GLT-1 activity in the habenula exhibited a depressive-like phenotype in the tail suspension and novelty-suppressed feeding tests. These animals also displayed increased susceptibility to chronic stress, displaying more frequent avoidant behavior without affecting locomotor activity in the open-field test. Intriguingly, the mice showed disinhibition of rapid eye movement sleep, which is a characteristic sleep pattern in patients with depression. These results provide evidence that disrupting glutamate clearance in habenular astrocytes increases neuronal excitability and depressive-like phenotypes in behaviors and sleep.

DNA methylation and methyl-binding proteins control differential gene expression in distinct cortical areas of macaque monkey.

Distinct anatomical regions of the neocortex subserve different sensory modalities and neuronal integration functions, but mechanisms for these regional specializations remain elusive. Involvement of epigenetic mechanisms for such specialization through the spatiotemporal regulation of gene expression is an intriguing possibility. Here we examined whether epigenetic mechanisms might play a role in the selective gene expression in the association areas (AAs) and the primary visual cortex (V1) in macaque neocortex. By analyzing the two types of area-selective gene promoters that we previously identified, we found a striking difference of DNA methylation between these promoters, i.e., hypermethylation in AA-selective gene promoters and hypomethylation in V1-selective ones. Methylation levels of promoters of each area-selective gene showed no areal difference, but a specific methyl-binding protein (MBD4) was enriched in the AAs, in correspondence with expression patterns of AA-selective genes. MBD4 expression was mainly observed in neurons. MBD4 specifically bound to and activated the AA-selective genes both in vitro and in vivo. Our results demonstrate that methylation in the promoters and specific methyl-binding proteins play an important role in the area-selective gene expression profiles in the primate neocortex.

Novel anti-tumor mechanism of galanin receptor type 2 in head and neck squamous cell carcinoma cells.

Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40-60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.

The prevalence of neutralizing antibodies against adeno-associated virus capsids is reduced in young Japanese individuals.

Pre-existing antibodies against adeno-associated virus (AAV), caused by natural AAV infections, interfere with recombinant AAV vector-mediated gene transfer. We studied the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 in healthy subjects (n = 85) and hemophilia patients (n = 59) in a Japanese population. For healthy subjects, the prevalence of neutralizing antibodies against AAV serotypes 1, 2, 5, 8, and 9 was 36.5%, 35.3%, 37.6%, 32.9%, and 36.5%, respectively, while that in hemophilia patients was 39.7%, 28.8%, 35.6%, 32.9%, and 27.4%, respectively. There was no difference in the prevalence of neutralizing antibody against each AAV serotype between the healthy subjects and the hemophilia patients. The prevalence of neutralizing antibodies against all AAV serotypes increased with age in both healthy subjects and hemophilia patients. High titers of neutralizing antibodies against AAV2 (≥1:224) and AAV8 (≥1:224) were more evident in older individuals (≥42 years old). Approximately 50% of all screened individuals were seronegative for neutralizing antibodies against each AAV tested, while approximately 25% of individuals were seropositive for each AAV serotype tested. The prevalence of seronegativity for all AAV serotypes was 67.0% (healthy subjects, 68.6%; hemophilia patients, 65.0%) and 18.6% (healthy subjects, 20.5%; hemophilia patients, 15.7%) in young (<42 years old) and older subjects (≥42 years old), respectively. The findings from this study suggested that young subjects are more likely to be eligible for gene therapy based on AAV vectors delivered via an intravascular route because of the low prevalence of antibodies to AAV capsids.

Minimizing the inhibitory effect of neutralizing antibody for efficient gene expression in the liver with adeno-associated virus 8 vectors.

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 ± 2.10-10.1 ± 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 ± 2.59-12.68 ± 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 ± 1.06-9.0 ± 2.37%) in the presence of NAbs (14-56× dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.