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1.
Front Virol ; 32023.
Artigo em Inglês | MEDLINE | ID: mdl-37383986

RESUMO

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes an acute febrile illness. ZIKV can be transmitted between sexual partners and from mother to fetus. Infection is strongly associated with neurologic complications in adults, including Guillain-Barré syndrome and myelitis, and congenital ZIKV infection can result in fetal injury and congenital Zika syndrome (CZS). Development of an effective vaccine is imperative to protect against ZIKV vertical transmission and CZS. Recombinant Vesicular Stomatitis virus (rVSV) is a highly effective and safe vector for the delivery of foreign immunogens for vaccine purposes. Here, we evaluate an rVSV vaccine expressing the full length pre-membrane (prM) and ZIKV envelope (E) proteins (VSV-ZprME), shown to be immunogenic in murine models of ZIKV infection, for its capacity to induce immune responses in nonhuman primates. Moreover, we assess the efficacy of the rVSVΔM-ZprME vaccine in the protection of pigtail macaques against ZIKV infection. Administration of the rVSVΔM-ZprME vaccine was safe, but it did not induce robust anti-ZIKV T-cell responses, IgM or IgG antibodies, or neutralizing antibodies in most animals. Post ZIKV challenge, animals that received the rVSVΔM control vaccine lacking ZIKV antigen had higher levels of plasma viremia compared to animals that received the rVSVΔM-ZprME vaccine. Anti-ZIKV neutralizing Ab titers were detected in a single animal that received the rVSVΔM-ZprME vaccine that was associated with reduced plasma viremia. The overall suboptimal ZIKV-specific cellular and humoral responses post-immunization indicates the rVSVΔM-ZprME vaccine did not elicit an immune response in this pilot study. However, recall antibody response to the rVSVΔM-ZprME vaccine indicates it may be immunogenic and further developments to the vaccine construct could enhance its potential as a vaccine candidate in a nonhuman primate pre-clinical model.

2.
Proc Natl Acad Sci U S A ; 115(39): E9182-E9191, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30181272

RESUMO

In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is "foreign," and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTRG2 mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.


Assuntos
DNA , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Força Muscular/genética , Distrofia Muscular de Duchenne/terapia , Plasmídeos , Animais , DNA/genética , DNA/farmacocinética , Modelos Animais de Doenças , Distrofina/genética , Distrofina/imunologia , Distrofina/metabolismo , Vetores Genéticos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Força Muscular/imunologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/metabolismo , Plasmídeos/genética , Plasmídeos/farmacologia
3.
Mol Ther ; 20(8): 1501-7, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22692496

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.


Assuntos
Distrofina/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Animais , Western Blotting , Linhagem Celular , Cães , Distrofina/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Distrofia Muscular de Duchenne/genética
4.
J Virol ; 82(15): 7711-5, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18480442

RESUMO

Recombinant adeno-associated virus vectors based on serotype 6 (rAAV6) efficiently transduce skeletal muscle after intravenous administration and have shown efficacy in the mdx model of muscular dystrophy. As a prelude to future clinical studies, we investigated the biodistribution and safety profile of rAAV6 in mice. Although it was present in all organs tested, rAAV6 was sequestered mainly in the liver and spleen. rAAV6 had a minimal effect on circulating blood cells and caused no apparent hepatotoxicity or coagulation activation. rAAV6 caused some neutrophil infiltration into the liver, with a transient elevation in cytokine and chemokine transcription/secretion. In summary, rAAV6 induces transient toxicity that subsides almost completely within 72 h and causes no significant side effects.


Assuntos
Dependovirus , Vetores Genéticos/efeitos adversos , Vetores Genéticos/farmacocinética , Animais , Coagulação Sanguínea , Citocinas/metabolismo , Vetores Genéticos/administração & dosagem , Injeções Intravenosas , Fígado/patologia , Fígado/virologia , Testes de Função Hepática , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Baço/virologia
5.
Hum Mol Genet ; 16(17): 2105-13, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17588958

RESUMO

Duchenne muscular dystrophy and Becker muscular dystrophy (BMD) are caused by mutations in the dystrophin gene. Although many in-frame deletions in the dystrophin gene lead to mild cases of BMD, truncations within the N-terminal actin-binding domain (ABD1) typically decrease dystrophin expression and lead to more severe cases of BMD. Because of the large reduction in protein expression, the functional capacity of dystrophin proteins deleted for subportions of ABD1 has been difficult to ascertain. ABD1 contains three actin-binding sequences designated ABS1-3. In the present study, we examined the pathophysiological effects of in-frame actin-binding sequence deletions in the context of a highly functional microdystrophin (DeltaR4-R23/DeltaCT). We delivered microdystrophins into the tibialis anterior muscles of 2-day-old dystrophin-deficient mdx mice using recombinant adeno-associated viral vectors. Muscles expressing microdystrophin with an intact ABD1 displayed normal morphology and specific force generation and were partially protected from contraction-induced injury when evaluated at 4 months of age. In contrast, muscles expressing microdystrophins lacking ABS2 and 3 or ABS3 alone developed significantly lower levels of specific force and were highly susceptible to contraction-induced injury. Microdystrophins with deletions within ABD1 were also less able to protect myofibers from degeneration than was a microdystrophin with the complete ABD1. We conclude that an intact ABD1 is required to support normal contractile properties of skeletal muscle and to protect against myofiber necrosis.


Assuntos
Actinas/metabolismo , Distrofina/genética , Distrofina/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Dados de Sequência Molecular , Músculo Esquelético/fisiologia , Estrutura Terciária de Proteína/genética , Deleção de Sequência
6.
Mol Ther ; 15(2): 320-9, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17235310

RESUMO

Systemic delivery of recombinant adeno-associated virus (rAAV) 6 vectors mediates efficient transduction of the entire striated musculature, making this an attractive strategy for muscle gene therapy. However, owing to widespread transduction of non-muscle tissues, optimization of this method would benefit from the use of muscle-specific promoters. Most such promoters either lack high-level expression in certain muscle types or are too large for inclusion in rAAV vectors encoding microdystrophin. Here, we describe novel regulatory cassettes based on enhancer/promoter regions of murine muscle creatine kinase (CK) and alpha-myosin heavy-chain genes. The strongest cassette, MHCK7 (770 bp), directs high-level expression comparable to cytomegalovirus and Rous sarcoma virus promoters in fast and slow skeletal and cardiac muscle, and low expression in the liver, lung, and spleen following systemic rAAV6 delivery in mice. Compared with CK6, our previous best cassette, MHCK7 activity is approximately 400-, approximately 50-, and approximately 10-fold higher in cardiac, diaphragm, and soleus muscles, respectively. MHCK7 also directs strong microdystrophin expression in mdx muscles. While further study of immune responses to MHCK7-regulated microdystrophin expression is needed, this cassette is not active in dendritic cell lines. MHCK7 is thus a highly improved regulatory cassette for experimental studies of rAAV-mediated transduction of striated muscle.


Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Clonagem Molecular/métodos , Creatina Quinase/genética , Creatina Quinase/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Miocárdio/citologia , Regiões Promotoras Genéticas/genética , Transfecção , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
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