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1.
Nat Commun ; 12(1): 697, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514733

RESUMO

Mutations in voltage-gated potassium channel KCNE1 cause Jervell and Lange-Nielsen syndrome type 2 (JLNS2), resulting in congenital deafness and vestibular dysfunction. We conducted gene therapy by injecting viral vectors using the canalostomy approach in Kcne1-/- mice to treat both the hearing and vestibular symptoms. Results showed early treatment prevented collapse of the Reissner's membrane and vestibular wall, retained the normal size of the semicircular canals, and prevented the degeneration of inner ear cells. In a dose-dependent manner, the treatment preserved auditory (16 out of 20 mice) and vestibular (20/20) functions in mice treated with the high-dosage for at least five months. In the low-dosage group, a subgroup of mice (13/20) showed improvements only in the vestibular functions. Results supported that highly efficient transduction is one of the key factors for achieving the efficacy and maintaining the long-term therapeutic effect. Secondary outcomes of treatment included improved birth and litter survival rates. Our results demonstrated that gene therapy via the canalostomy approach, which has been considered to be one of the more feasible delivery methods for human inner ear gene therapy, preserved auditory and vestibular functions in a dose-dependent manner in a mouse model of JLNS2.


Assuntos
Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Síndrome de Jervell-Lange Nielsen/terapia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais Semicirculares/cirurgia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Vetores Genéticos/genética , Audição/genética , Humanos , Injeções/métodos , Síndrome de Jervell-Lange Nielsen/genética , Masculino , Camundongos , Camundongos Knockout , Parvovirinae/genética , Propriocepção/genética
2.
Theranostics ; 11(2): 649-664, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33391497

RESUMO

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.


Assuntos
Antivirais/administração & dosagem , RNA Guia/genética , Glicoproteína da Espícula de Coronavírus/genética , /virologia , Sistemas CRISPR-Cas/genética , Biologia Computacional , Avaliação Pré-Clínica de Medicamentos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células Hep G2 , Humanos , Simulação de Acoplamento Molecular , Homologia de Sequência de Aminoácidos
3.
Nat Commun ; 12(1): 391, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452250

RESUMO

Spinal cord injury (SCI) often causes severe and permanent disabilities due to the regenerative failure of severed axons. Here we report significant locomotor recovery of both hindlimbs after a complete spinal cord crush. This is achieved by the unilateral transduction of cortical motoneurons with an AAV expressing hyper-IL-6 (hIL-6), a potent designer cytokine stimulating JAK/STAT3 signaling and axon regeneration. We find collaterals of these AAV-transduced motoneurons projecting to serotonergic neurons in both sides of the raphe nuclei. Hence, the transduction of cortical neurons facilitates the axonal transport and release of hIL-6 at innervated neurons in the brain stem. Therefore, this transneuronal delivery of hIL-6 promotes the regeneration of corticospinal and raphespinal fibers after injury, with the latter being essential for hIL-6-induced functional recovery. Thus, transneuronal delivery enables regenerative stimulation of neurons in the deep brain stem that are otherwise challenging to access, yet highly relevant for functional recovery after SCI.


Assuntos
Terapia Genética/métodos , Interleucina-6/genética , Locomoção/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/terapia , Animais , Axônios/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Janus Quinases/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microinjeções , Neurônios Motores/fisiologia , PTEN Fosfo-Hidrolase/genética , Núcleos da Rafe/citologia , Núcleos da Rafe/fisiologia , Recuperação de Função Fisiológica , Fator de Transcrição STAT3/metabolismo , Neurônios Serotoninérgicos/fisiologia , Índice de Gravidade de Doença , Transdução de Sinais , Medula Espinal/citologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/diagnóstico , Traumatismos da Medula Espinal/fisiopatologia , Transdução Genética
4.
Anticancer Res ; 41(1): 81-89, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33419801

RESUMO

BACKGROUND/AIM: Breast cancer is the most common cancer in women worldwide, and triple-negative breast cancer (TNBC) is highly refractory to current standard therapies. Oncolytic virotherapy has recently gathered attention as a new treatment candidate for refractory cancers. MATERIALS AND METHODS: We previously developed a new Coxsackievirus B3 (CVB3) virotherapy targeting lung cancers, and demonstrated that miRNA target sequence insertion into CVB3 reduced its pathogenicity, retaining its original oncolytic activity. In this study, we examined the oncolytic effects of CVB3 against breast cancer cells including TNBC cells. RESULTS: CVB3 infection killed breast cancer cells in a time- and titer-dependent manner, and induced apoptosis. Nude mice transplanted with human TNBC cells were successfully treated with both CVB3-WT and CVB3-HP. Importantly, mice treated with CVB3-HP showed very few adverse events. CONCLUSION: CVB3-HP is a strong oncolytic virus candidate for breast cancer, including TNBC, due to its remarkable oncolytic efficacy and improved safety profile.


Assuntos
Neoplasias da Mama/genética , Enterovirus Humano B/genética , Terapia Genética , Vetores Genéticos/genética , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Animais , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Células Cultivadas , Efeito Citopatogênico Viral , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Humanos , Imuno-Histoquímica , Camundongos , Terapia Viral Oncolítica/métodos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nat Commun ; 12(1): 447, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469018

RESUMO

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation, such as hydrocephalus, can negatively affect perinatal neurodevelopment. The mechanisms regulating CSF clearance during the postnatal critical period are unclear. Here, we show that CSF K+, accompanied by water, is cleared through the choroid plexus (ChP) during mouse early postnatal development. We report that, at this developmental stage, the ChP showed increased ATP production and increased expression of ATP-dependent K+ transporters, particularly the Na+, K+, Cl-, and water cotransporter NKCC1. Overexpression of NKCC1 in the ChP resulted in increased CSF K+ clearance, increased cerebral compliance, and reduced circulating CSF in the brain without changes in intracranial pressure in mice. Moreover, ChP-specific NKCC1 overexpression in an obstructive hydrocephalus mouse model resulted in reduced ventriculomegaly. Collectively, our results implicate NKCC1 in regulating CSF K+ clearance through the ChP in the critical period during postnatal neurodevelopment in mice.


Assuntos
Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/patologia , Hidrocefalia/patologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Animais , Animais Recém-Nascidos , Plexo Corióideo/diagnóstico por imagem , Plexo Corióideo/crescimento & desenvolvimento , Plexo Corióideo/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Hidrocefalia/congênito , Hidrocefalia/diagnóstico , Hidrocefalia/fisiopatologia , Injeções Intraventriculares , Pressão Intracraniana/fisiologia , Imagem por Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Membro 2 da Família 12 de Carreador de Soluto/genética
7.
Viruses ; 12(10)2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33092124

RESUMO

RNA interference (RNAi) represents a novel approach for alternative antiviral therapy. However, issues related to RNA delivery and stability have presented serious obstacles for obtaining good therapeutic efficacy. Viral vectors are capable of efficient delivery of RNAi as short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA). Efficacy in gene silencing for therapeutic applications against viral diseases has been demonstrated in various animal models. Rotavirus (RV) miR-7 can inhibit rotavirus replication by targeting the RV nonstructural protein 5. Viral gene silencing by targeting the RNAi pathway showed efficient suppression of hepatitis B virus replication by adeno-associated virus (AAV)-based delivery of RNAi hepatitis B virus (HBV) cassettes. Hepatitis C virus replication has been targeted by short hairpin RNA molecules expressed from lentivirus vectors. Potentially, RNAi-based approaches could be suitable for antiviral drugs against COVID-19.


Assuntos
Infecções por Coronavirus/terapia , Vetores Genéticos/administração & dosagem , Pneumonia Viral/terapia , Interferência de RNA , Terapêutica com RNAi/métodos , Animais , Antivirais/farmacologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Vetores Genéticos/genética , Humanos , MicroRNAs/genética , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética
8.
Nat Commun ; 11(1): 4524, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913280

RESUMO

Traumatic brain injury (TBI) is a leading global cause of death and disability. Here we demonstrate in an experimental mouse model of TBI that mild forms of brain trauma cause severe deficits in meningeal lymphatic drainage that begin within hours and last out to at least one month post-injury. To investigate a mechanism underlying impaired lymphatic function in TBI, we examined how increased intracranial pressure (ICP) influences the meningeal lymphatics. We demonstrate that increased ICP can contribute to meningeal lymphatic dysfunction. Moreover, we show that pre-existing lymphatic dysfunction before TBI leads to increased neuroinflammation and negative cognitive outcomes. Finally, we report that rejuvenation of meningeal lymphatic drainage function in aged mice can ameliorate TBI-induced gliosis. These findings provide insights into both the causes and consequences of meningeal lymphatic dysfunction in TBI and suggest that therapeutics targeting the meningeal lymphatic system may offer strategies to treat TBI.


Assuntos
Lesões Encefálicas/fisiopatologia , Gliose/fisiopatologia , Sistema Glinfático/fisiologia , Meninges/fisiopatologia , Animais , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Lesões Encefálicas/terapia , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Gliose/etiologia , Gliose/patologia , Gliose/prevenção & controle , Sistema Glinfático/patologia , Humanos , Masculino , Meninges/patologia , Camundongos , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/uso terapêutico
9.
Pediatrics ; 146(3)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32843442

RESUMO

BACKGROUND AND OBJECTIVES: Historically, autosomal recessive 5q-linked spinal muscular atrophy (SMA) has been the leading inherited cause of infant death. SMA is caused by the absence of the SMN1 gene, and SMN1 gene replacement therapy, onasemnogene abeparvovec-xioi, was Food and Drug Administration approved in May 2019. Approval included all children with SMA age <2 years without end-stage weakness. However, gene transfer with onasemnogene abeparvovec-xioi has been only studied in children age ≤8 months. METHODS: In this article, we report key safety and early outcome data from the first 21 children (age 1-23 months) treated in the state of Ohio. RESULTS: In children ≤6 months, gene transfer was well tolerated. In this young group, serum transaminase (aspartate aminotransferase and alanine aminotransferase) elevations were modest and not associated with γ glutamyl transpeptidase elevations. Initial prednisolone administration matched that given in the clinical trials. In older children, elevations in aspartate aminotransferase, alanine aminotransferase and γ glutamyl transpeptidase were more common and required a higher dose of prednisolone, but all were without clinical symptoms. Nineteen of 21 (90%) children experienced an asymptomatic drop in platelets in the first week after treatment that recovered without intervention. Of the 19 children with repeated outcome assessments, 11% (n = 2) experienced stabilization and 89% (n = 17) experienced improvement in motor function. CONCLUSIONS: In this population, with thorough screening and careful post-gene transfer management, replacement therapy with onasemnogene abeparvovec-xioi is safe and shows promise for early efficacy.


Assuntos
Terapia Genética/métodos , Proteínas Recombinantes de Fusão/genética , Atrofias Musculares Espinais da Infância/genética , Atrofias Musculares Espinais da Infância/terapia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Adenovírus Humanos , Fatores Etários , Alanina Transaminase/metabolismo , Aspartato Aminotransferases/metabolismo , Produtos Biológicos , Terapia Genética/efeitos adversos , Vetores Genéticos/administração & dosagem , Glucocorticoides/administração & dosagem , Humanos , Lactente , Ohio , Avaliação de Resultados em Cuidados de Saúde , Prednisolona/administração & dosagem , gama-Glutamiltransferase/metabolismo
10.
PLoS Pathog ; 16(8): e1008326, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804988

RESUMO

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.


Assuntos
Botrytis/fisiologia , Sistemas CRISPR-Cas , Edição de Genes , Oryza/microbiologia , Doenças das Plantas/microbiologia , Ribonucleoproteínas/antagonistas & inibidores , Telômero/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Oryza/genética , Doenças das Plantas/genética , Ribonucleoproteínas/genética
11.
PLoS Pathog ; 16(8): e1008646, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776974

RESUMO

Inositol hexakisphosphate (IP6) potently stimulates HIV-1 particle assembly in vitro and infectious particle production in vivo. However, knockout cells lacking inositol-pentakisphosphate 2-kinase (IPPK-KO), the enzyme that produces IP6 by phosphorylation of inositol pentakisphosphate (IP5), were still able to produce infectious HIV-1 particles at a greatly reduced rate. HIV-1 in vitro assembly can also be stimulated to a lesser extent with IP5, but until recently, it was not known if IP5 could also function in promoting assembly in vivo. Here we addressed whether there is an absolute requirement for IP6 or IP5 in the production of infectious HIV-1 particles. IPPK-KO cells expressed no detectable IP6 but elevated IP5 levels and displayed a 20-100-fold reduction in infectious particle production, correlating with lost virus release. Transient transfection of an IPPK expression vector stimulated infectious particle production and release in IPPK-KO but not wildtype cells. Several attempts to make IP6/IP5 deficient stable cells were not successful, but transient expression of the enzyme multiple inositol polyphosphate phosphatase-1 (MINPP1) into IPPK-KOs resulted in near ablation of IP6 and IP5. Under these conditions, we found that HIV-1 infectious particle production and virus release were essentially abolished (1000-fold reduction) demonstrating an IP6/IP5 requirement. However, other retroviruses including a Gammaretrovirus, a Betaretrovirus, and two non-primate Lentiviruses displayed only a modest (3-fold) reduction in infectious particle production from IPPK-KOs and were not significantly altered by expression of IPPK or MINPP1. The only other retrovirus found to show a clear IP6/IP5 dependence was the primate (macaque) Lentivirus Simian Immunodeficiency Virus, which displayed similar sensitivity as HIV-1. We were not able to determine if producer cell IP6/IP5 is required at additional steps beyond assembly because viral particles devoid of both molecules could not be generated. Finally, we found that loss of IP6/IP5 in viral target cells had no effect on permissivity to HIV-1 infection.


Assuntos
Vetores Genéticos/administração & dosagem , Infecções por HIV/virologia , Fosfatos de Inositol/metabolismo , Lentivirus de Primatas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ácido Fítico/metabolismo , Vírion/fisiologia , Animais , Vetores Genéticos/genética , HIV/fisiologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Fosforilação , Primatas
12.
Anticancer Res ; 40(8): 4425-4444, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727773

RESUMO

BACKGROUND/AIM: Patients with metastasized melanoma have limited treatment options and poor diagnosis. Therefore, the development of treatments requires a new therapeutic approach, of which gene therapy using rAAV vectors can be proposed. The aim of the study was to examine the efficiency of the rAAV vector to transduce mouse melanoma cells both in vitro and in vivo. MATERIALS AND METHODS: Different rAAV serotypes encoding GFP under the control of both chicken beta-actin and cytomegalovirus promoters were used in the experiments. Intranasal, intraperitoneal, intravenous and intratumoral pathways of administration of rAAV vectors were tested using quantitative-PCR and immunohistochemical staining. RESULTS: The highest transduction efficiency in metastatic cells in vivo was observed 7 days after intranasal administration of a 1010 gc/0.03 ml dose of rAAV/DJ-CAG. CONCLUSION: Melanoma gene therapy based on rAAV vectors is a possible treatment option.


Assuntos
Dependovirus/genética , Proteínas de Fluorescência Verde/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Melanoma Experimental/genética , Administração Intranasal , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Vetores Genéticos/administração & dosagem , Neoplasias Pulmonares/terapia , Masculino , Melanoma Experimental/terapia , Camundongos , Células NIH 3T3 , Regiões Promotoras Genéticas , Transdução Genética , Resultado do Tratamento
13.
PLoS One ; 15(7): e0236175, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32697798

RESUMO

Adenoviruses cause upper respiratory infections, conjunctivitis, keratitis, and gastrointestinal illness. These can be fatal in immunocompromised individuals. Adenoviruses have also been engineered into viral vectors to deliver therapeutic genes or induce immunity as vaccine carriers. The success of ocular gene therapy is driven partly by the immunologic and biochemical influences of the intraocular environment. We have shown that versican and hyaluronan modulate adenoviral vector transgene expression through CD44 signaling. Herein we explored the role of these pathways on virus replication and viral protein expression of wild type adenovirus. We report that the addition of vitreous humor (which contains both versican and hyaluronan) increases viral hexon protein levels. Vitreous humor also increased wild type adenovirus DNA replication in vitro. Metalloproteinase and γ-secretase inhibitors, which inhibit CD44 proteolytic activation, blocked adenoviral replication in vitro. Similarly, protein kinase C and RhoA kinase inhibitors, both proteins associated with CD44 mediated pathways, also inhibited wild type adenoviral replication in vitro. Application of metalloproteinase and γ-secretase inhibitors to human conjunctival explants sharply decreased adenoviral vector gene expression. Our results demonstrate that pharmacologic delivery of these inhibitors is easily achievable. The inhibition of these enzymes should be explored as potential therapies of wild type adenoviral infections.


Assuntos
Infecções por Adenoviridae/tratamento farmacológico , Adenoviridae/efeitos dos fármacos , Antivirais/farmacologia , Vetores Genéticos/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Adenoviridae/fisiologia , Infecções por Adenoviridae/virologia , Administração Oftálmica , Amidas/farmacologia , Amidas/uso terapêutico , Antivirais/uso terapêutico , Túnica Conjuntiva/metabolismo , DNA Viral/genética , DNA Viral/isolamento & purificação , Diaminas/farmacologia , Diaminas/uso terapêutico , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/fisiologia , Células HeLa , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Maleimidas/farmacologia , Maleimidas/uso terapêutico , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Permeabilidade , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteólise/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Versicanas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Corpo Vítreo/metabolismo , Quinases Associadas a rho/metabolismo
14.
Lancet ; 396(10249): 467-478, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32702298

RESUMO

BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. METHODS: We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18-55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 × 1010 viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT50]; a microneutralisation assay [MNA50, MNA80, and MNA90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. FINDINGS: Between April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p<0·05). There were no serious adverse events related to ChAdOx1 nCoV-19. In the ChAdOx1 nCoV-19 group, spike-specific T-cell responses peaked on day 14 (median 856 spot-forming cells per million peripheral blood mononuclear cells, IQR 493-1802; n=43). Anti-spike IgG responses rose by day 28 (median 157 ELISA units [EU], 96-317; n=127), and were boosted following a second dose (639 EU, 360-792; n=10). Neutralising antibody responses against SARS-CoV-2 were detected in 32 (91%) of 35 participants after a single dose when measured in MNA80 and in 35 (100%) participants when measured in PRNT50. After a booster dose, all participants had neutralising activity (nine of nine in MNA80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R2=0·67 by Marburg VN; p<0·001). INTERPRETATION: ChAdOx1 nCoV-19 showed an acceptable safety profile, and homologous boosting increased antibody responses. These results, together with the induction of both humoral and cellular immune responses, support large-scale evaluation of this candidate vaccine in an ongoing phase 3 programme. FUNDING: UK Research and Innovation, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research (NIHR), NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and the German Center for Infection Research (DZIF), Partner site Gießen-Marburg-Langen.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas Virais/efeitos adversos , Vacinas Virais/imunologia , Acetaminofen/uso terapêutico , Adenovirus dos Símios/genética , Adulto , Analgésicos não Entorpecentes/uso terapêutico , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Humanos , Imunização Secundária , Imunoglobulina G/sangue , Masculino , Pneumonia Viral/tratamento farmacológico , Método Simples-Cego , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/imunologia , Reino Unido , Vacinas Virais/administração & dosagem
15.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510495

RESUMO

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV's easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3' arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.


Assuntos
Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde/metabolismo , Rim/metabolismo , Infecções por Poxviridae/metabolismo , Poxviridae/genética , eIF-2 Quinase/metabolismo , Animais , Células Cultivadas , Especificidade de Hospedeiro , Humanos , Rim/citologia , Rim/virologia , Infecções por Poxviridae/virologia , Coelhos , Vírus Vaccinia/genética
16.
Protein Cell ; 11(10): 707-722, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32519302

RESUMO

The 2019 novel coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has occurred in China and around the world. SARS-CoV-2-infected patients with severe pneumonia rapidly develop acute respiratory distress syndrome (ARDS) and die of multiple organ failure. Despite advances in supportive care approaches, ARDS is still associated with high mortality and morbidity. Mesenchymal stem cell (MSC)-based therapy may be an potential alternative strategy for treating ARDS by targeting the various pathophysiological events of ARDS. By releasing a variety of paracrine factors and extracellular vesicles, MSC can exert anti-inflammatory, anti-apoptotic, anti-microbial, and pro-angiogenic effects, promote bacterial and alveolar fluid clearance, disrupt the pulmonary endothelial and epithelial cell damage, eventually avoiding the lung and distal organ injuries to rescue patients with ARDS. An increasing number of experimental animal studies and early clinical studies verify the safety and efficacy of MSC therapy in ARDS. Since low cell engraftment and survival in lung limit MSC therapeutic potentials, several strategies have been developed to enhance their engraftment in the lung and their intrinsic, therapeutic properties. Here, we provide a comprehensive review of the mechanisms and optimization of MSC therapy in ARDS and highlighted the potentials and possible barriers of MSC therapy for COVID-19 patients with ARDS.


Assuntos
Betacoronavirus , Infecções por Coronavirus/complicações , Transplante de Células-Tronco Mesenquimais , Pandemias , Pneumonia Viral/complicações , /terapia , Transferência Adotiva , Células Epiteliais Alveolares/patologia , Animais , Apoptose , Líquidos Corporais/metabolismo , Linfócitos T CD4-Positivos/imunologia , Ensaios Clínicos como Assunto , Coinfecção/prevenção & controle , Coinfecção/terapia , Infecções por Coronavirus/imunologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Oxigenação por Membrana Extracorpórea , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/uso terapêutico , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/prevenção & controle , Pneumonia Viral/imunologia , /patologia , Pesquisa Médica Translacional
17.
PLoS Pathog ; 16(6): e1008522, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32589686

RESUMO

DNA vectors have been widely used as a priming of poxvirus vaccine in prime/boost regimens. Whether the number of DNA impacts qualitatively or quantitatively the immune response is not fully explored. With the aim to reinforce T-cell responses by optimizing the prime-boost regimen, the multicentric EV03/ANRS VAC20 phase I/II trial, randomized 147 HIV-negative volunteers to either 3xDNA plus 1xNYVAC (weeks 0, 4, 8 plus 24; n = 74) or to 2xDNA plus 2xNYVAC (weeks 0, 4 plus 20, 24; n = 73) groups. T-cell responses (IFN-γ ELISPOT) to at least one peptide pool were higher in the 3xDNA than the 2xDNA groups (91% and 80% of vaccinees) (P = 0.049). In the 3xDNA arm, 26 (37%) recipients developed a broader T-cell response (Env plus at least to one of the Gag, Pol, Nef pools) than in the 2xDNA (15; 22%) arms (primary endpoint; P = 0.047) with a higher magnitude against Env (at week 26) (P<0.001). In both groups, vaccine regimens induced HIV-specific polyfunctional CD4 and CD8 T cells and the production of Th1, Th2 and Th17/IL-21 cytokines. Antibody responses were also elicited in up to 81% of vaccines. A higher percentage of IgG responders was noted in the 2xDNA arm compared to the 3xDNA arm, while the 3xDNA group tended to elicit a higher magnitude of IgG3 response against specific Env antigens. We show here that the modulation of the prime strategy, without modifying the route or the dose of administration, or the combination of vectors, may influence the quality of the responses.


Assuntos
Vacinas contra a AIDS/imunologia , Vetores Genéticos/imunologia , Antígenos HIV/imunologia , Poxviridae/imunologia , Vacinas de DNA/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Adolescente , Adulto , Linfócitos T CD8-Positivos/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Antígenos HIV/administração & dosagem , Antígenos HIV/genética , Humanos , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Poxviridae/genética , Linfócitos T Auxiliares-Indutores/metabolismo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
18.
PLoS One ; 15(5): e0233373, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453743

RESUMO

Targeted gene integration via precise homologous recombination (HR)-based gene editing has the potential to correct genetic diseases. AAV (adeno-associated virus) can mediate nuclease-free gene integration at a disease-causing locus. Therapeutic application of AAV gene integration requires quantitative molecular characterization of the edited sequence that overcome technical obstacles such as excess episomal vector genomes and lengthy homology arms. Here we describe a novel molecular methodology that utilizes quantitative next-generation sequencing to characterize AAV-mediated targeted insertion and detects the presence of unintended mutations. The methods described here quantify targeted insertion and query the entirety of the target locus for the presence of insertions, deletions, single nucleotide variants (SNVs) and integration of viral components such as inverted terminal repeats (ITR). Using a humanized liver murine model, we demonstrate that hematopoietic stem-cell derived AAVHSC15 mediates in vivo targeted gene integration into human chromosome 12 at the PAH (phenylalanine hydroxylase) locus at 6% frequency, with no sign of co-incident random mutations at or above a lower limit of detection of 0.5% and no ITR sequences at the integration sites. Furthermore, analysis of heterozygous variants across the targeted locus using the methods described shows a pattern of strand cross-over, supportive of an HR mechanism of gene integration with similar efficiencies across two different haplotypes. Rapid advances in the application of AAV-mediated nuclease-free target integration, or gene editing, as a new therapeutic modality requires precise understanding of the efficiency and the nature of the changes being introduced to the target genome at the molecular level. This work provides a framework to be applied to homologous recombination gene editing platforms for assessment of introduced and natural sequence variation across a target site.


Assuntos
Dependovirus/fisiologia , Edição de Genes/métodos , Fígado/química , Fenilalanina Hidroxilase/genética , Animais , Vetores Genéticos/administração & dosagem , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Modelos Animais , Mutação , Análise de Sequência de DNA , Integração Viral
19.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 622-631, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347057

RESUMO

Small interfering RNA (siRNA) has been used to treat various skin diseases. However, siRNA is limited in application due to its electronegativity, strong polarity, easy degradation by nuclease and difficulty in breaking through the skin barrier. Therefore, safe and efficient siRNA delivery vector is the premise of effective treatment of skin diseases by siRNA. In recent years, with the deepening of research on siRNA, great progress has been made in the development of delivery systems based on lipids, polymers, peptides and nanoparticles, some new transdermal delivery vectors of siRNA have emerged, such as liposomes, dendrimers, cell penetrating peptides, and spherical nucleic acid nanoparticles. This review will focus on the recent advance in siRNA transdermal delivery vectors.


Assuntos
Administração Cutânea , RNA Interferente Pequeno , Dermatopatias , Vetores Genéticos/administração & dosagem , Humanos , RNA Interferente Pequeno/administração & dosagem , Dermatopatias/terapia
20.
PLoS One ; 15(4): e0230711, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240193

RESUMO

Vaccinia virus (VACV) has been used extensively as the vaccine against smallpox and as a viral vector for the development of recombinant vaccines and cancer therapies. Replication-competent, non-attenuated VACVs induce strong, long-lived humoral and cell-mediated immune responses and can be effective oncolytic vectors. However, complications from uncontrolled VACV replication in vaccinees and their close contacts can be severe, particularly in individuals with predisposing conditions. In an effort to develop replication-competent VACV vectors with improved safety, we placed VACV late genes encoding core or virion morphogenesis proteins under the control of tet operon elements to regulate their expression with tetracycline antibiotics. These replication-inducible VACVs would only express the selected genes in the presence of tetracyclines. VACVs inducibly expressing the A3L or A6L genes replicated indistinguishably from wild-type VACV in the presence of tetracyclines, whereas there was no evidence of replication in the absence of antibiotics. These outcomes were reflected in mice, where the VACV inducibly expressing the A6L gene caused weight loss and mortality equivalent to wild-type VACV in the presence of tetracyclines. In the absence of tetracyclines, mice were protected from weight loss and mortality, and viral replication was not detected. These findings indicate that replication-inducible VACVs based on the conditional expression of the A3L or A6L genes can be used for the development of safer, next-generation live VACV vectors and vaccines. The design allows for administration of replication-inducible VACV in the absence of tetracyclines (as a replication-defective vector) or in the presence of tetracyclines (as a replication-competent vector) with enhanced safety.


Assuntos
Vetores Genéticos/administração & dosagem , Tetraciclinas/farmacologia , Vírus Vaccinia/crescimento & desenvolvimento , Vaccinia/prevenção & controle , Vírion/crescimento & desenvolvimento , Replicação Viral , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinas Sintéticas/administração & dosagem , Vaccinia/genética , Vaccinia/virologia , Vírus Vaccinia/efeitos dos fármacos , Vírus Vaccinia/genética , Proteínas Virais/genética , Vírion/efeitos dos fármacos
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