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BACKGROUND: Pathogenic variants in PKP2 (plakophilin-2) cause arrhythmogenic right ventricular cardiomyopathy, a disease characterized by life-threatening arrhythmias and progressive cardiomyopathy leading to heart failure. No effective medical therapy is available to prevent or arrest the disease. We tested the hypothesis that adeno-associated virus vector-mediated delivery of the human PKP2 gene to an adult mammalian heart deficient in PKP2 can arrest disease progression and significantly prolong survival. METHODS: Experiments were performed using a PKP2-cKO (cardiac-specific, tamoxifen-activated PKP2 knockout murine model). The potential therapeutic, adeno-associated virus vector of serotype rh.74 (AAVrh.74)-PKP2a (PKP2 variant A; RP-A601) is a recombinant AAVrh.74 gene therapy viral vector encoding the human PKP2 variant A. AAVrh.74-PKP2a was delivered to adult mice by a single tail vein injection either before or after tamoxifen-activated PKP2-cKO. PKP2 expression was confirmed by molecular and histopathologic analyses. Cardiac function and disease progression were monitored by survival analyses, echocardiography, and electrocardiography. RESULTS: Consistent with prior findings, loss of PKP2 expression caused 100% mortality within 50 days after tamoxifen injection. In contrast, AAVrh.74-PKP2a-mediated PKP2a expression resulted in 100% survival for >5 months (at study termination). Echocardiographic analysis revealed that AAVrh.74-PKP2a prevented right ventricle dilation, arrested left ventricle functional decline, and mitigated arrhythmia burden. Molecular and histological analyses showed AAVrh.74-PKP2a-mediated transgene mRNA and protein expression and appropriate PKP2 localization at the cardiomyocyte intercalated disc. Importantly, the therapeutic benefit was shown in mice receiving AAVrh.74-PKP2a after disease onset. CONCLUSIONS: These preclinical data demonstrate the potential for AAVrh.74-PKP2a (RP-A601) as a therapeutic for PKP2-related arrhythmogenic right ventricular cardiomyopathy in both early and more advanced stages of the disease.
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Displasia Arritmogênica Ventricular Direita , Adulto , Humanos , Camundongos , Animais , Displasia Arritmogênica Ventricular Direita/genética , Displasia Arritmogênica Ventricular Direita/terapia , Displasia Arritmogênica Ventricular Direita/metabolismo , Placofilinas/genética , Miócitos Cardíacos/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/terapia , Arritmias Cardíacas/metabolismo , Tamoxifeno/metabolismo , Progressão da Doença , Mamíferos/metabolismoRESUMO
Aldehyde dehydrogenase 2 (ALDH2) deficiency affects 35% to 45% of East Asians and 8% of the world population. ALDH2 is the second enzyme in the ethanol metabolism pathway. The common genetic variant ALDH2*2 allele has a glutamic acid-to-lysine substitution at position 487 (E487K) that reduces the enzyme activity, resulting in an accumulation of acetaldehyde after ethanol consumption. The ALDH2*2 allele is associated with increased risk of osteoporosis and hip fracture. Our prior study showed that administration of an adeno-associated virus (AAV) serotype rh.10 gene transfer vector expressing the human ALDH2 cDNA (AAVrh.10hALDH2) before initiation of ethanol consumption prevented bone loss in ALDH2-deficient homozygous knockin mice carrying the E487K mutation (Aldh2 E487K+/+). We hypothesized that AAVrh.10hALDH2 administration after establishment of osteopenia would be able to reverse bone loss due to ALDH2 deficiency and chronic ethanol consumption. To test this hypothesis, male and female Aldh2 E487K+/+ mice (n = 6) were given ethanol in the drinking water for 6 weeks to establish osteopenia and then administered AAVrh.10hALDH2 (1011 genome copies). Mice were evaluated for an additional 12 weeks. AAVrh.10hALDH2 administration after osteopenia was established corrected weight loss and locomotion phenotypes and, importantly, increased midshaft femur cortical bone thickness, the most important component of bone in the resistance to fractures, and showed a trend toward increased trabecular bone volume. AAVrh.10hALDH2 is a promising therapeutic for osteoporosis in ALDH2-deficient individuals. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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Intravenous (IV) administration of naturally occurring adeno-associated virus (AAV) vectors are liver tropic, with a significant proportion of the total vector dose mediating gene expression in liver hepatocytes. AAV capsids that are directed toward other organs such as lung may be useful for therapy of nonliver-based diseases. Based on the knowledge that the lung capillary endothelium is the first capillary bed encountered by an intravenously administered AAV vector, and that the lung endothelium glycocalyx is enriched in negatively charged sialic acid, we hypothesized that adding positively changed lysine residues to the AAV capsid would enhance AAV biodistribution to the lung after IV administration. Using site-directed mutagenesis, two lysine residues were inserted into variable loop VIII of the AAV serotype 5 capsid (AAV5-PK2). Organ distribution of AAV5-PK2 was compared with that of AAV5, AAV2, and AAV2-7m8 4 weeks after IV administration (1011 gc) to C57Bl/6 male mice. As predicted, after IV administration, AAV5-PK2 had the highest biodistribution in the lung (p < 0.02 compared with AAV5, AAV2, and AAV2-7m8). Furthermore, biodistribution to liver of AAV5-PK2 was 2 logs decreased compared with AAV5 (p < 10-4) with a ratio of AAV5-PK2 lung to liver of 62-fold compared with AAV5 of 0.2-fold (p < 0.0003). The AAV5-PK2 capsid represents a lung-tropic AAV vector that is also significantly detargeted from the liver, a property that may be useful in lung-directed gene therapies.
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Capsídeo , Parvovirinae , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Vetores Genéticos/genética , Fígado/metabolismo , Pulmão/metabolismo , Lisina/análise , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos , Parvovirinae/genética , Distribuição Tecidual , Transdução GenéticaRESUMO
Approximately 8% of the world population and 35-45% of East Asians are carriers of the hereditary disorder aldehyde dehydrogenase 2 (ALDH2) deficiency. ALDH2 plays a central role in the liver to metabolize ethanol. With the common E487K variant, there is a deficiency of ALDH2 function; when ethanol is consumed, there is a systemic accumulation of acetaldehyde, an intermediate product in ethanol metabolism. In ALDH2-deficient individuals, ethanol consumption acutely causes the "Alcohol Flushing Syndrome" with facial flushing, tachycardia, nausea, and headaches. With chronic alcohol consumption, ALDH2 deficiency is associated with a variety of disorders, including a remarkably high risk for aerodigestive tract cancers. Acetaldehyde is a known carcinogen. The epidemiologic data relating to the association of ALDH2 deficiency and cancer risk are striking: ALDH2 homozygotes who are moderate-to-heavy consumers of ethanol have a 7-12-fold increased risk for esophageal cancer, making ALDH2 deficiency the most common hereditary disorder associated with an increased cancer risk. In this review, we summarize the genetics and biochemistry of ALDH2, the epidemiology of cancer risk associated with ALDH2 deficiency, the metabolic consequences of ethanol consumption associated with ALDH2 deficiency, and gene therapy strategies to correct ALDH2 deficiency and its associated cancer risk. With the goal of reducing the risk of aerodigestive tract cancers, in the context that ALDH2 is a hereditary disorder and ALDH2 functions primarily in the liver, ALDH2 deficiency is an ideal target for the application of adeno-associated virus-mediated liver-directed gene therapy to prevent cancer.
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Aldeído Desidrogenase , Neoplasias , Acetaldeído/metabolismo , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Etanol/efeitos adversos , Etanol/metabolismo , Terapia Genética , Humanos , Neoplasias/genética , Neoplasias/prevenção & controleRESUMO
Chronic eosinophilic leukemia-not otherwise specified (CEL-NOS) is a rare, aggressive, fatal disease characterized by blood eosinophilia and dysfunction of organs infiltrated with eosinophils. Clinically, the disease manifests with weight loss, cough, weakness, diarrhea, and multi-organ dysfunction that is unresponsive to therapy. We developed a one-time gene therapy for CEL-NOS using an adeno-associated virus (AAV) expressing an anti-eosinophil monoclonal antibody (AAVrh.10mAnti-Eos) to provide sustained suppression of eosinophil numbers in blood, thus reducing eosinophil tissue invasion and organ dysfunction. A novel CEL-NOS model was developed in NOD-scid IL2rγnull (NSG) mice by administration of AAV expressing the cytokine IL5 (AAVrh.10mIL5), resulting in marked peripheral and tissue eosinophilia of the heart, lung, liver, and spleen, and eventually death. Mice were administered AAVrh.10mAnti-Eos (1011 genome copies) 4 wk after administration of AAVrh.10mIL5 and evaluated for anti-eosinophil antibody expression, blood eosinophil counts, organ eosinophil invasion, and survival. AAVrh.10mAnti-Eos expressed persistent levels of the anti-eosinophil antibody for >24 wk. Strikingly, CEL-NOS treated mice had markedly lower blood eosinophil levels and reduced mortality when compared with control treated mice. These results suggest that a single treatment with AAVrh.10mAnti-Eos has the potential to provide substantial therapeutic benefit to patients with CEL-NOS, a fatal malignant disorder.
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Anticorpos Monoclonais/farmacologia , Dependovirus/genética , Modelos Animais de Doenças , Eosinófilos/imunologia , Terapia Genética , Síndrome Hipereosinofílica/terapia , Interleucina-5/genética , Leucemia/terapia , Animais , Eosinófilos/efeitos dos fármacos , Feminino , Síndrome Hipereosinofílica/genética , Síndrome Hipereosinofílica/imunologia , Leucemia/genética , Leucemia/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
BACKGROUND: The rapid movement of mental health services on the internet following the onset of the COVID-19 pandemic has demonstrated the potential advantages of digital delivery and has highlighted the need to learn from prepandemic digital services. OBJECTIVE: The aim of this study is to explore the different elements of interconnected digital mental health and neurodevelopmental services of a well-established provider to the UK National Health Service and how web-based delivery enables young people and their families to access high-quality assessments and interventions in a more timely, flexible, and person-centered manner than in-person delivery. METHODS: A realist evaluation multiple case-study design was used, with 9 pediatric cases (aged 8-15 years) identified as representative of the services provided by Healios. Presenting concerns included autism and ADHD, anxiety and panic attacks, low self-esteem, anger and self-harm. The research literature was used to define the program theory and six context-mechanism-outcome (CMO) statements. The CMOs formed the basis for the initial data extraction, with novel elements added via an iterative process. RESULTS: We identified 10 key elements of web-based services: flexible delivery and timely response, personalized care to the individual, comprehensive care enabled by multiple interconnected services, effective client engagement and productive therapeutic alliances, use of multiple communication tools, client satisfaction with the service, good clinical outcomes, ease of family involvement throughout sessions or from different locations, facilitation of multi-agency working and integration with National Health Services, and management of risk and safeguarding. These elements supported the six CMOs; there was clear evidence that young people and their families valued the responsiveness and flexibility of the web-based mental health service and, in particular, how quickly they were seen. There was also clear evidence of individual needs being met, good therapeutic alliances, and client satisfaction. Multiple communication tools appeared to maximize engagement and working digitally facilitated multi-agency communication and delivery of safe care. The abovementioned factors may be related to the finding of good clinical outcomes, but the methodology of this study does not allow any conclusions to be drawn regarding causality. CONCLUSIONS: This study demonstrates the effectiveness of interconnected digital mental health and neurodevelopmental services as well as how web-based delivery enables young people and their families to access assessments and interventions in a more timely, flexible, and person-centered manner than in-person delivery. The 10 key elements of web-based service delivery identified through the 9 case studies suggest the potential advantages of web-based work. These elements can inform future research and aid in the delivery of high-quality digital services.
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BACKGROUND: Eosinophils are specialized granulocytic effector cells that store and release highly active mediators used in immune defense. Eosinophils are also implicated in the pathogenesis of allergic disorders, including eosinophilic esophagitis (EoE), a chronic disorder characterized by infiltration of eosinophils into the esophagus and release of mediators that damage tissue, resulting in gastrointestinal morbidity, food impaction, and dysphagia. Treatment with elimination diets and/or topical corticosteroid therapy slow disease progression, but are complicated by adverse effects, limited compliance, and loss of response to therapy. We hypothesized that a single administration of an adeno-associated virus (AAV) coding for an anti-eosinophil monoclonal antibody that induces eosinophil clearance (anti-Siglec-F) would treat on a persistent basis a murine model of EoE. METHODS: A mouse model of peanut-induced EoE that mimics the human disease was established by sensitization and challenge with peanut extract. After challenge, these mice exhibited an EoE phenotype demonstrated by elevated levels of blood eosinophils, infiltration of eosinophils in the esophagus with associated esophageal remodeling and food impaction. RESULTS: The mice were treated with a single intravenous administration (1011 genome copies) of AAVrh.10mAnti-Eos, a serotype rh.10 AAV vector coding for an anti-Siglec-F monoclonal antibody. Vector administration resulted in persistent, high levels of anti-Siglec-F antibody expression. Administration of AAVrh.10mAnti-Eos to the mouse model of EoE reduced blood (P < 0.02) and esophageal eosinophil numbers (P < 0.002) protected from esophageal tissue remodeling and minimized food impaction. CONCLUSION: These results suggest that a single treatment with AAVrh.10mAnti-Eos has the potential to provide persistent therapeutic benefit to patients with EoE.
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Esofagite Eosinofílica , Animais , Anticorpos Monoclonais , Modelos Animais de Doenças , Esofagite Eosinofílica/genética , Esofagite Eosinofílica/terapia , Eosinófilos , Terapia Genética , Humanos , CamundongosRESUMO
Alpha 1-antitrypsin (AAT) deficiency, a hereditary disorder characterized by low serum levels of functional AAT, is associated with early development of panacinar emphysema. AAT inhibits serine proteases, including neutrophil elastase, protecting the lung from proteolytic destruction. Cigarette smoke, pollution, and inflammatory cell-mediated oxidation of methionine (M) 351 and 358 inactivates AAT, limiting lung protection. In vitro studies using amino acid substitutions demonstrated that replacing M351 with valine (V) and M358 with leucine (L) on a normal M1 alanine (A) 213 background provided maximum antiprotease protection despite oxidant stress. We hypothesized that a onetime administration of a serotype 8 adeno-associated virus (AAV8) gene transfer vector coding for the oxidation-resistant variant AAT (A213/V351/L358; 8/AVL) would maintain antiprotease activity under oxidant stress compared with normal AAT (A213/M351/M358; 8/AMM). 8/AVL was administered via intravenous (IV) and intrapleural (IPL) routes to C57BL/6 mice. High, dose-dependent AAT levels were found in the serum and lung epithelial lining fluid (ELF) of mice administered 8/AVL or 8/AMM by IV or IPL. 8/AVL serum and ELF retained serine protease-inhibitory activity despite oxidant stress while 8/AMM function was abolished. 8/AVL represents a second-generation gene therapy for AAT deficiency providing effective antiprotease protection even with oxidant stress.
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Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Oxidantes , Transgenes , Deficiência de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/administração & dosagem , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologiaRESUMO
Friedreich's ataxia (FA), an autosomal recessive disorder caused by a deficiency in the expression of frataxin (FXN), is characterized by progressive ataxia and hypertrophic cardiomyopathy. Although cardiac dysfunction is the most common cause of mortality in FA, the cardiac disease remains subclinical for most of the clinical course because the neurologic disease limits muscle oxygen demands. Previous FXN knockout mouse models exhibit fatal cardiomyopathy similar to human FA, but in contrast to the human condition, untreated mice become moribund by 2 months of age, unlike humans where the cardiac disease often does not manifest until the third decade. The study was designed to create a mouse model for early FA disease relevant to the time for which a gene therapy would likely be most effective. To generate a cardiac-specific mouse model of FA cardiomyopathy similar to the human disease, we used a cardiac promoter (αMyhc) driving CRE recombinase cardiac-specific excision of FXN exon 4 to generate a mild, cardiac-specific FA model that is normal at rest, but exhibits the cardiac phenotype with stress. The hearts of αMyhc mice had decreased levels of FXN and activity of the mitochondrial complex II/complex IV respiratory chain. At rest, αMyhc mice exhibited normal cardiac function as assessed by echocardiographic assessment of ejection fraction and fractional shortening, but when the heart was stressed chemically with dobutamine, αMyhc mice compared with littermate control mice had a 62% reduction in the stress ejection fraction (p < 2 × 10-4) and 71% reduction in stress-related fractional shortening (p < 10-5). When assessing functional cardiac performance using running on an inclined treadmill, αMyhc mice stayed above the midline threefold less than littermate controls (p < 0.02). A one-time intravenous administration of 1011 genome copies of AAVrh.10hFXN, an adeno-associated virus (AAV) serotype rh10 gene transfer vector expressing human FXN, corrected the stress-induced ejection fraction and fractional shortening phenotypes. Treated αMyhc mice exhibited exercise performance on a treadmill indistinguishable from littermate controls (p > 0.07). These αMyhc mice provide an ideal model to study long-term cardiac complications due to FA and AAV-mediated gene therapy correction of stress-induced cardiac phenotypes typical of human FA.
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Cardiomiopatias/terapia , Dependovirus/genética , Ataxia de Friedreich/complicações , Terapia Genética , Vetores Genéticos/administração & dosagem , Proteínas de Ligação ao Ferro/genética , Estresse Fisiológico , Animais , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fenótipo , FrataxinaRESUMO
Aldehyde dehydrogenase type 2 (ALDH2), a key enzyme in ethanol metabolism, processes toxic acetaldehyde to nontoxic acetate. ALDH2 deficiency affects 8% of the world population and 35-45% of East Asians. The ALDH2*2 allele common genetic variant has a glutamic acid-to-lysine substitution at position 487 (E487K) that reduces the oxidizing ability of the enzyme resulting in systemic accumulation of acetaldehyde with ethanol ingestion. With chronic ethanol ingestion, mutations in ALDH2 are associated with a variety of hematological, neurological, and dermatological abnormalities, and an increased risk for esophageal cancer and osteoporosis. Based on our prior studies demonstrating that a one-time administration of an adeno-associated virus (AAV) serotype rh.10 gene transfer vector expressing the human ALDH2 cDNA (AAVrh.10hALDH2) prevents the acute effects of ethanol administration (the "Asian flush syndrome"), we hypothesized that AAVrh.10hALDH2 would also prevent the chronic disorders associated with ALDH2 deficiency and chronic ethanol ingestion. To assess this hypothesis, AAVrh.10hALDH2 (1011 genome copies) was administered intravenously to two models of ALDH2 deficiency, Aldh2 knockout homozygous (Aldh2-/-) and knockin homozygous (Aldh2E487K+/+) mice (n = 10 per group). Four weeks after vector administration, mice were given drinking water with 10-15% ethanol for 12 weeks. Strikingly, compared with nonethanol drinking littermates, AAVrh.10hALDH2 administration prevented chronic ethanol-induced serum acetaldehyde accumulation and elevated liver malondialdehyde levels, loss of body weight, reduced hemoglobin levels, reduced performance in locomotor activity tests, accumulation of esophageal DNA damage and DNA adducts, and development of osteopenia. AAVrh.10hALDH2 should be considered as a preventative therapy for the increased risk of chronic disorders associated with ALDH2 deficiency and chronic alcohol exposure.
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Alcoolismo/genética , Alcoolismo/terapia , Aldeído-Desidrogenase Mitocondrial/deficiência , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Alcoolismo/metabolismo , Animais , Comportamento Animal , Biomarcadores , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Linhagem Celular , Adutos de DNA , Dano ao DNA , Modelos Animais de Doenças , Esôfago/metabolismo , Esôfago/patologia , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Transdução Genética , Transgenes , Resultado do TratamentoRESUMO
Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder caused by repetitive trauma to the central nervous system (CNS) suffered by soldiers, contact sport athletes, and civilians following accident-related trauma. CTE is a CNS tauopathy, with trauma-induced inflammation leading to accumulation of hyperphosphorylated forms of the microtubule-binding protein Tau (pTau), resulting in neurofibrillary tangles and progressive loss of neurons. At present, there are no therapies to treat CTE. We hypothesized that direct CNS administration of an adeno-associated virus (AAV) vector coding for an anti-pTau antibody would generate sufficient levels of anti-pTau in the CNS to suppress pTau accumulation thus interrupting the pathogenic process. Using a serotype AAVrh.10 gene transfer vector coding for a monoclonal antibody directed against pTau, we demonstrate the feasibility of this strategy in a murine CTE model in which pTau accumulation was elicited by repeated traumatic brain injury (TBI) using a closed cortical impact procedure over 5 days. Direct delivery of AAVrh.10 expression vectors coding for either of the two different anti-pTau antibodies to the hippocampus of these TBI mice significantly reduced pTau levels across the CNS. Using doses that can be safely scaled to humans, the data demonstrate that CNS administration of AAVrh.10anti-pTau is effective, providing a new strategy to interrupt the CTE consequences of TBI.
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Encefalopatia Traumática Crônica/genética , Encefalopatia Traumática Crônica/terapia , Terapia Genética , Proteínas tau/genética , Animais , Anticorpos Monoclonais/farmacologia , Encéfalo/metabolismo , Encéfalo/patologia , Dependovirus/genética , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Ligação Proteica , Proteínas tau/antagonistas & inibidores , Proteínas tau/metabolismoRESUMO
Aldehyde dehydrogenase 2 (ALDH2) deficiency causes "Asian flush syndrome," presenting as alcohol-induced facial flushing, tachycardia, nausea, and headaches. One of the most common hereditary enzyme deficiencies, it affects 35%-40% of East Asians and 8% of the world population. ALDH2 is the key enzyme in ethanol metabolism; with ethanol challenge, the common ALDH2*2 (E487K) mutation results in accumulation of toxic acetaldehyde. ALDH2*2 heterozygotes have increased risk for upper digestive tract cancers, compounded by smoking and drinking alcohol. We hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the human ALDH2 coding sequence (AAVrh.10hALDH2) would correct the deficiency state. AAVrh.10hALDH2 was administered intravenously to Aldh2 knockout (Aldh2 -/-) and Aldh2 E487K knockin homozygous (Aldh2 E487K+/+) mice. Following acute ethanol ingestion, untreated ALDH2-deficient mice had elevated acetaldehyde levels and performed poorly in behavioral tests. In contrast, treated Aldh2 -/- and Aldh2 E487K+/+ mice had lower serum acetaldehyde levels and improved behavior. Thus, in vivo AAV-mediated ALDH2 therapy may reverse the deficiency state in ALDH2*2 individuals, eliminating the Asian flush syndrome and reducing the risk for associated disorders.
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Alpha-1 antitrypsin deficiency (AATD) manifests primarily as early-onset emphysema caused by the destruction of the lung by neutrophil elastase due to low amounts of the serine protease inhibitor alpha-1 antitrypsin (AAT). The current therapy involves weekly intravenous infusions of AAT-derived from pooled human plasma that is efficacious, yet costly. Gene therapy applications designed to provide constant levels of the AAT protein are currently under development. The challenge is for gene therapy to provide sufficient amounts of AAT to normalize the inhibitor level and anti-neutrophil elastase capacity in the lung. One strategy involves administration of an adeno-associated virus (AAV) gene therapy vector to the pleural space providing both local and systemic production of AAT to reach consistent therapeutic levels. This review focuses on the strategy, advantages, challenges, and updates for intrapleural administration of gene therapy vectors for the treatment of AATD.
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Genetic modification therapy is a promising therapeutic strategy for many diseases of the lung intractable to other treatments. Lung gene therapy has been the subject of numerous preclinical animal experiments and human clinical trials, for targets including genetic diseases such as cystic fibrosis and α1-antitrypsin deficiency, complex disorders such as asthma, allergy, and lung cancer, infections such as respiratory syncytial virus (RSV) and Pseudomonas, as well as pulmonary arterial hypertension, transplant rejection, and lung injury. A variety of viral and non-viral vectors have been employed to overcome the many physical barriers to gene transfer imposed by lung anatomy and natural defenses. Beyond the treatment of lung diseases, the lung has the potential to be used as a metabolic factory for generating proteins for delivery to the circulation for treatment of systemic diseases. Although much has been learned through a myriad of experiments about the development of genetic modification of the lung, more work is still needed to improve the delivery vehicles and to overcome challenges such as entry barriers, persistent expression, specific cell targeting, and circumventing host anti-vector responses.
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Terapia Genética , Vetores Genéticos/administração & dosagem , Pneumopatias/terapia , Vírus/genética , Animais , Humanos , Pneumopatias/genéticaRESUMO
UNLABELLED: Alphaviruses are small enveloped RNA viruses that infect cells via clathrin-mediated endocytosis and low-pH-triggered fusion in the early endosome. Using a small interfering RNA (siRNA) screen in human cells, we previously identified TSPAN9 as a host factor that promotes infection by the alphaviruses Sindbis virus (SINV), Semliki Forest virus (SFV), and chikungunya virus (CHIKV). Depletion of TSPAN9 specifically decreases SFV membrane fusion in endosomes. TSPAN9 is a member of the tetraspanin family of multipass membrane proteins, but its cellular function is currently unknown. Here we used U-2 OS cells stably overexpressing TSPAN9 to show that TSPAN9 is localized at the plasma membrane and in early and late endosomes. Internalized SFV particles colocalized with TSPAN9 in vesicles early during infection. Depletion of TSPAN9 led to reductions in the amounts of the late endosomal proteins LAMP1 and CD63 and an increase in the amount of LAMP2. However, TSPAN9 depletion did not alter the delivery of SFV to early endosomes or change their pH or protease activity. Comparative studies showed that TSPAN9 depletion strongly inhibited infection by several viruses that fuse in early endosomes (SFV, SINV, CHIKV, and vesicular stomatitis virus [VSV]), while viruses that fuse in the late endosome (recombinant VSV-Lassa and VSV-Junin), including an SFV point mutant with a lower pH threshold for fusion (SFV E2 T12I), were relatively resistant. Our data suggest that TSPAN9 modulates the early endosome compartment to make it more permissive for membrane fusion of early-penetrating viruses. IMPORTANCE: Alphaviruses are spread by mosquitoes and can cause serious human diseases such as arthritis and encephalitis. Recent outbreaks of CHIKV infection are responsible for millions of cases of acute illness and long-term complications. There are no vaccines or antiviral treatments for these important human pathogens. Alphaviruses infect host cells by utilizing the endocytic machinery of the cell and fusing their membrane with that of the endosome. Although the mechanism of virus-membrane fusion is well studied, we still know relatively little about the host cell proteins that are involved in alphavirus entry. Here we characterized the role of the host membrane protein TSPAN9 in alphavirus infection. TSPAN9 was localized to early endosomes containing internalized alphavirus, and depletion of TSPAN9 inhibited virus fusion with the early endosome membrane. In contrast, infection of viruses that enter through the late endosome was relatively resistant to TSPAN9 depletion, suggesting an important role for TSPAN9 in the early endosome.
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Infecções por Alphavirus/genética , Infecções por Alphavirus/virologia , Alphavirus/fisiologia , Endossomos/virologia , Interações Hospedeiro-Patógeno , Tetraspaninas/genética , Internalização do Vírus , Infecções por Alphavirus/metabolismo , Linhagem Celular , Colesterol/metabolismo , Endocitose , Endossomos/metabolismo , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Espaço Intracelular , Peptídeo Hidrolases/metabolismo , Transporte Proteico , Vírus da Floresta de Semliki/fisiologia , Tetraspaninas/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
UNLABELLED: Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in the Old World and a highly fatal hantavirus cardiopulmonary syndrome (HCPS) in the New World. No vaccines or antiviral therapies are currently available to prevent or treat hantavirus disease, and gaps in our understanding of how hantaviruses enter cells challenge the search for therapeutics. We performed a haploid genetic screen in human cells to identify host factors required for entry by Andes virus, a highly virulent New World hantavirus. We found that multiple genes involved in cholesterol sensing, regulation, and biosynthesis, including key components of the sterol response element-binding protein (SREBP) pathway, are critical for Andes virus entry. Genetic or pharmacological disruption of the membrane-bound transcription factor peptidase/site-1 protease (MBTPS1/S1P), an SREBP control element, dramatically reduced infection by virulent hantaviruses of both the Old World and New World clades but not by rhabdoviruses or alphaviruses, indicating that this pathway is broadly, but selectively, required by hantaviruses. These results could be fully explained as arising from the modest depletion of cellular membrane cholesterol that accompanied S1P disruption. Mechanistic studies of cells and with protein-free liposomes suggested that high levels of cholesterol are specifically needed for hantavirus membrane fusion. Taken together, our results indicate that the profound dependence on target membrane cholesterol is a fundamental, and unusual, biophysical property of hantavirus glycoprotein-membrane interactions during entry. IMPORTANCE: Although hantaviruses cause important human diseases worldwide, no specific antiviral treatments are available. One of the major obstacles to the development of new therapies is a lack of understanding of how hantaviruses hijack our own host factors to enter cells. Here, we identified multiple cellular genes that control the levels of cholesterol in cellular membranes to be important for hantavirus entry. Our findings suggest that high concentrations of cholesterol in cellular membranes are required at a specific step in the entry process-fusion between viral and cellular membranes-that allows escape of the hantavirus genome into the host cell cytoplasm to initiate infection. Our findings uncover a fundamental feature of the hantavirus infection mechanism and point to cholesterol-lowering drugs as a potential new treatment of hantaviral infections.
Assuntos
Colesterol/metabolismo , Orthohantavírus/fisiologia , Internalização do Vírus , Animais , Chlorocebus aethiops , Testes Genéticos , Células HEK293 , Haploidia , Humanos , Células VeroRESUMO
The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.
Assuntos
Infecções por Alphavirus/genética , Alphavirus/fisiologia , Endocitose/genética , Interações Hospedeiro-Patógeno/genética , Interferência de RNA , Internalização do Vírus , Infecções por Alphavirus/virologia , Animais , Células Cultivadas , Vírus Chikungunya/fisiologia , Cricetinae , Endocitose/efeitos dos fármacos , Genoma Humano , Células HeLa , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , RNA Interferente Pequeno/genética , Vírus da Floresta de Semliki/fisiologia , Sindbis virus/fisiologia , TranscriptomaRESUMO
The identity of the receptors that mediate alphavirus entry into host cells has been elusive. In this issue of Cell Host & Microbe, Rose et al. (2011) use a Drosophila RNAi screen to identify NRAMP, an iron transporter with 12 transmembrane domains, as a receptor for Sindbis virus in both insect and mammalian cells.
RESUMO
To initiate membrane fusion and virus entry, herpes simplex virus (HSV) gD binds to a cellular receptor such as herpesvirus entry mediator (HVEM). HVEM is a tumor necrosis factor (TNF) receptor family member with four natural ligands that either stimulate (LIGHT and LTα) or inhibit (BTLA and CD160) T cell function. We hypothesized that the interaction of gD with HVEM affects the binding of natural ligands, thereby modulating the immune response during infection. Here, we investigated the effect that gD has on the interaction of HVEM with its natural ligands. First, HSV gD on virions or cells downregulates HVEM from the cell surface. Similarly, trans-interaction with BTLA or LIGHT also downregulates HVEM from the cell surface, suggesting that HSV may subvert a natural mechanism for regulating HVEM activity. Second, we showed that wild-type gD had the lowest affinity for HVEM compared with the four natural ligands. Moreover, gD directly competed for binding to HVEM with BTLA but not LTα or LIGHT, indicating the possibility that gD selectively controls HVEM signals. On the other hand, natural ligands influence the use of HVEM by HSV. For instance, soluble BTLA, LTα, and LIGHT inhibited the binding of wild-type gD to HVEM, and soluble BTLA and LTα blocked HSV infection of HVEM-expressing cells. Thus, gD is at the center of the interplay between HVEM and its ligands. It can interfere with HVEM function in two ways, by competing with the natural ligands and by downregulating HVEM from the cell surface.
Assuntos
Regulação para Baixo , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Animais , Ligação Competitiva , Linhagem Celular , Expressão Gênica , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Humanos , Camundongos , Ligação Proteica , Membro 14 de Receptores do Fator de Necrose Tumoral/química , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Entry of herpes simplex virus (HSV) occurs either by fusion at the plasma membrane or by endocytosis and fusion with an endosome. Binding of glycoprotein D (gD) to a receptor such as nectin-1 is essential in both cases. We show that virion gD triggered the rapid down-regulation of nectin-1 with kinetics similar to those of virus entry. In contrast, nectin-1 was not constitutively recycled from the surface of uninfected cells. Both the nectin-1alpha and beta isoforms were internalized in response to gD despite having different cytoplasmic tails. However, deletion of the nectin-1 cytoplasmic tail slowed down-regulation of nectin-1 and internalization of virions. These data suggest that nectin-1 interaction with a cytoplasmic protein is not required for its down-regulation. Overall, this study shows that gD binding actively induces the rapid internalization of various forms of nectin-1. We suggest that HSV activates a nectin-1 internalization pathway to use for endocytic entry.