Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment ...strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
Memories are integrated into interconnected networks; nevertheless, each memory has its own identity. How the brain defines specific memory identity out of intermingled memories stored in a shared ...cell ensemble has remained elusive. We found that after complete retrograde amnesia of auditory fear conditioning in mice, optogenetic stimulation of the auditory inputs to the lateral amygdala failed to induce memory recall, implying that the memory engram no longer existed in that circuit. Complete amnesia of a given fear memory did not affect another linked fear memory encoded in the shared ensemble. Optogenetic potentiation or depotentiation of the plasticity at synapses specific to one memory affected the recall of only that memory. Thus, the sharing of engram cells underlies the linkage between memories, whereas synapse-specific plasticity guarantees the identity and storage of individual memories.
The global number of patients with depression increases in correlation to exposure to social stress. Chronic stress does not trigger depression in all individuals, as some remain resilient. The ...underlying molecular mechanisms that contribute to stress sensitivity have been poorly understood, although revealing the regulation of stress sensitivity could help develop treatments for depression. We previously found that striatal Shati/Nat8l, an N-acetyltransferase, was increased in a depression mouse model. We investigated the roles of Shati/Nat8l in stress sensitivity in mice and found that Shati/Nat8l and brain-derived neurotrophic factor (BDNF) levels in the dorsal striatum were increased in stress-susceptible mice but not in resilient mice exposed to repeated social defeat stress (RSDS). Knockdown of Shati/Nat8l in the dorsal striatum induced resilience to RSDS. In addition, blockade of BDNF signaling in the dorsal striatum by ANA-12, a BDNF-specific receptor tropomyosin-receptor-kinase B (TrkB) inhibitor, also induced resilience to stress. Shati/Nat8l is correlated with BDNF expression after RSDS, and BDNF is downstream of Shati/Nat8l pathways in the dorsal striatum; Shati/Nat8l is epigenetically regulated by BDNF via histone acetylation. Our results demonstrate that striatal Shati/Nat8l-BDNF pathways determine stress sensitivity through epigenetic regulation. The striatal Shati/Nat8l-BDNF pathway could be a novel target for treatments of depression and could establish a novel therapeutic strategy for depression patients.
The Keap1-Nrf2 system plays a central role in the oxidative stress response; however, the identity of the reactive oxygen species sensor within Keap1 remains poorly understood. Here, we show that a ...Keap1 mutant lacking 11 cysteine residues retains the ability to target Nrf2 for degradation, but it is unable to respond to cysteine-reactive Nrf2 inducers. Of the 11 mutated cysteine residues, we find that 4 (Cys226/613/622/624) are important for sensing hydrogen peroxide. Our analyses of multiple mutant mice lines, complemented by MEFs expressing a series of Keap1 mutants, reveal that Keap1 uses the cysteine residues redundantly to set up an elaborate fail-safe mechanism in which specific combinations of these four cysteine residues can form a disulfide bond to sense hydrogen peroxide. This sensing mechanism is distinct from that used for electrophilic Nrf2 inducers, demonstrating that Keap1 is equipped with multiple cysteine-based sensors to detect various endogenous and exogenous stresses.
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•Keap1 H2O2 sensor is distinct from that used for sensing electrophilic inducers•Keap1 uses Cys226, Cys613, and Cys622/624 residues to sense H2O2•Keap1 uses these cysteine residues to set up an elaborate fail-safe mechanism
The Keap1-Nrf2 system plays a central role in the oxidative stress response. Suzuki et al. show that the H2O2 sensor of Keap1 is distinct from those used for electrophilic inducers. Keap1 exploits Cys226, Cys613, and Cys622/624 residues for sensing H2O2, and these residues set up an elaborate fail-safe mechanism.
Shati/Nat8l is a novel N‐acetyltransferase identified in the brain of mice treated with methamphetamine (METH). Shati/Nat8l mRNA is expressed in various brain areas, including the prefrontal cortex ...(PFC), where the expression level is higher than that in other brain regions. Shati/Nat8l overexpression in the nucleus accumbens (NAc) attenuates the pharmacological response to METH via mGluR3. Meanwhile, dopamine (DA) and glutamate dysregulations have been reported in the medial prefrontal cortex (mPFC) and NAc after METH self‐administration and during reinstatement. However, the mechanism, the reward system, and function of Shati/Nat8l in the mPFC is unclear. Here, we injected an adeno‐associated virus (AAV) vector containing Shati/Nat8l into the mPFC of mice, to overexpress Shati/Nat8l in the mPFC (mPFC‐Shati/Nat8l). Interestingly, the METH‐induced conditioned place preference (CPP) was attenuated in the mPFC‐Shati/Nat8l mice, but locomotor activity was not. Additionally, immunohistochemical results from mice that were injected with AAV‐GFP showed fluorescence in the mPFC and other brain regions, mainly the NAc, indicating an mPFC‐NAc top‐down connection. Finally, in vivo microdialysis experiments revealed that Shati/Nat8l overexpression in the mPFC reduced extracellular DA levels and suppressed the METH‐induced DA increase in the NAc. Moreover, decreased extracellular glutamate levels were observed in the NAc. These results indicate that Shati/Nat8l overexpression in the mPFC attenuates METH‐induced CPP by decreasing extracellular DA in the NAc. In contrast, Shati/Nat8l‐mPFC overexpression did not alter METH‐induced hyperlocomotion. This study demonstrates that Shati/Nat8l in the mPFC attenuates METH reward‐seeking behaviour but not the psychomotor activity of METH.
Shati/Nat8l overexpression in the medial prefrontal cortex attenuates methamphetamine‐induced reward behaviour by decreasing extracellular glutamate and dopamine levels in the nucleus accumbens. We consider that extracellular dopamine was reduced by the decrease of glutamate in the nucleus accumbens. Indeed, glutamate acts on medium spiny neurons that project to GABA interneurons in the ventral tegmental area. These interneurons inhibit the dopaminergic neurons projecting to the nucleus accumbens.
Gene therapy using adeno-associated virus (AAV) is a rapidly developing technology with widespread treatment potential. AAV2 vectors injected directly into the brain by stereotaxic brain surgery have ...shown good results in treating aromatic l-amino acid decarboxylase deficiency. Moreover, gene therapy using the AAV9 vector, which crosses the blood–brain barrier, has been performed in more than 2000 patients worldwide as a disease-modifying therapy for spinal muscular atrophy. AAV vectors have been applied to the development of gene therapies for various pediatric diseases. Gene therapy trials for hemophilia and ornithine transcarbamylase deficiency are underway. Clinical trials are planned for glucose transporter I deficiency, Niemann-Pick disease type C, and spinocerebellar ataxia type 1. The genome of AAV vectors is located in the episome and is rarely integrated into chromosomes, making the vectors safe. However, serious adverse events such as hepatic failure and thrombotic microangiopathy have been reported, and ongoing studies are focusing on developing more efficient vectors to reduce required dosages.
The timing and characteristics of neuronal death in Alzheimer's disease (AD) remain largely unknown. Here we examine AD mouse models with an original marker, myristoylated alanine-rich C-kinase ...substrate phosphorylated at serine 46 (pSer46-MARCKS), and reveal an increase of neuronal necrosis during pre-symptomatic phase and a subsequent decrease during symptomatic phase. Postmortem brains of mild cognitive impairment (MCI) rather than symptomatic AD patients reveal a remarkable increase of necrosis. In vivo imaging reveals instability of endoplasmic reticulum (ER) in mouse AD models and genome-edited human AD iPS cell-derived neurons. The level of nuclear Yes-associated protein (YAP) is remarkably decreased in such neurons under AD pathology due to the sequestration into cytoplasmic amyloid beta (Aβ) aggregates, supporting the feature of YAP-dependent necrosis. Suppression of early-stage neuronal death by AAV-YAPdeltaC reduces the later-stage extracellular Aβ burden and cognitive impairment, suggesting that preclinical/prodromal YAP-dependent neuronal necrosis represents a target for AD therapeutics.
The natural serotypes of adeno-associated virus (AAV) or their variants, such as AAV8 and AAV5, are commonly used as vectors in the clinical programs for liver-targeted gene therapy. While AAV8 ...vectors are not highly efficient at targeting primary human hepatocytes, AAV3 vectors have recently demonstrated remarkable efficiency at targeting both human and non-human primate hepatocytes. However, the presence of high levels of neutralizing antibodies (NAbs) impedes transduction into hepatocytes, representing a major obstacle to the clinical application of AAV3 vectors. Herein, we engineered the viral capsid to reduce its reactivity with pre-existing NAbs, thereby enhancing the transduction efficiency. By introducing three substitutions (S472A, S587A, and N706A) on the surface loop of AAV3B capsid protein, we generated a triple mutant AAV3 (AAV.GT5) vector with less reactivity to anti-AAV capsid NAbs. While the transduction efficiency of AAV.GT5 into human hepatocellular cell lines was similar to those of parental AAV3B, it was 50-fold higher for hepatocytes derived from humanized mice compared to AAV8 vectors. Moreover, the AAV.GT5 vector yield was similar to those of the AAV2 and AAV3B vectors. Thus, high resistance to pre-existing NAbs makes AAV.GT5 a promising candidate for future liver-targeted gene therapy clinical trials.
AADC deficiency causes severe motor and intellectual disability as a result of reduced catecholamine levels. Kojima et al. report beneficial effects of gene therapy in six patients with heterogeneous ...genetic backgrounds. Gene delivery into putamen improved motor function in all patients, plus verbal and cognitive skills in one moderate-phenotype patient.
Abstract
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 6-18Ffluoro-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_video1
awy331media1
5991361892001
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