RESUMO
Patients with neutralizing antibodies (Nabs) against adeno-associated virus (AAV) are usually excluded from the treatment with AAV vectors. To develop a standard assay for detecting Nab inhibition activity, we systematically studied current AAV Nab assays in vitro and in vivo. Several factors were found that influence the Nab titers based on the in vitro assay, including sera volume, AAV dose per cell, cell number and choice of transgenes. When the Nab titer assay was performed in vivo via intramuscular (IM) or systemic administration, a fourfold increase in sensitivity for measurement of Nab titers was observed compared with an identical in vitro test. To better mimic the clinical setting, after passively transferring human Nabs into mice, blood was collected before systemic injection of AAV vector and used for Nab titer analysis in vitro or via IM injection. The results showed that AAV delivered via IM injection had a similar inhibition pattern to systemic administration. These studies indicate critical parameters necessary for optimizing Nab sensitivity and that an in vivo Nab assay is more sensitive than an in vitro assay for inclusion/exclusion criteria. The variables identified by this study may explain some of the compounding clinical data seen to date with respect to efficiency of AAV transduction in various phase I clinical trials.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Dependovirus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução Genética , TransgenesRESUMO
Developing continuous systemic delivery of endostatin has been a goal of many laboratories. We have employed a method of gene therapy utilizing different viral constructs. Here, we report that a new serotype of adeno-associated viruses, which incorporates canine endostatin, provides dose-dependent transgene expression in the circulation after intramuscular injection in mice. Elevated levels of endostatin remained stable in the circulation for at least 4 months. In vitro assays determined that the protein expressed was biologically active. Antitumor activities of the above construct demonstrated a U-shape curve, where the maximum activity was observed within a certain critical concentration range. These data suggest that an optimum dose range may be required to achieve therapeutic efficacy in large animal models.
Assuntos
Antineoplásicos/uso terapêutico , Dependovirus/genética , Endostatinas/uso terapêutico , Terapia Genética/métodos , Neoplasias/tratamento farmacológico , Sequência de Aminoácidos , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Endostatinas/administração & dosagem , Endostatinas/genética , Vetores Genéticos , Humanos , Injeções Intramusculares , Masculino , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Alinhamento de SequênciaRESUMO
In contrast to other gene delivery systems, adeno-associated virus vectors show long term gene expression without immune response or toxicity. New production methods have increased vector titers and eliminated adenovirus contamination, thereby facilitating effective in vivo use. These advancements will expedite additional animal model studies providing validation for use of this vector in human clinical trials.