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1.
Hum Gene Ther ; 26(2): 69-81, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25419787

RESUMEN

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100-500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).


Asunto(s)
Dependovirus/genética , Factor IX/genética , Terapia Genética/métodos , Vectores Genéticos/farmacocinética , Hemofilia B/terapia , Hemorragia/prevención & control , Animales , Anticuerpos Neutralizantes/análisis , Cápside/química , Cápside/inmunología , Ensayos Clínicos como Asunto , Dependovirus/inmunología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Factor IX/metabolismo , Factor IX/farmacocinética , Expresión Génica , Ingeniería Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/química , Hemofilia B/sangre , Hemofilia B/genética , Hemofilia B/fisiopatología , Hemorragia/sangre , Hemorragia/genética , Hemorragia/fisiopatología , Humanos , Hígado/inmunología , Hígado/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Cola (estructura animal) , Distribución Tisular , Virión/genética
2.
Mol Ther ; 20(2): 443-55, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22068425

RESUMEN

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.


Asunto(s)
Dependovirus/genética , Terapia Genética , Vectores Genéticos/genética , Distrofia Muscular de Duchenne/terapia , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Línea Celular , Niño , Preescolar , Dependovirus/fisiología , Distrofina/genética , Distrofina/metabolismo , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/inmunología , Conformación Proteica , Alineación de Secuencia , Linfocitos T/inmunología , Transducción Genética , Tropismo Viral
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