RESUMEN
The structural genes of adeno-associated virus serotype 2 (AAV2) have been altered by linker insertional mutagenesis in order to define critical components of virion assembly and infectivity. An in-frame restriction site linker was inserted across the capsid coding domain of a recombinant plasmid. After complementation in vivo, recombinant AAV2 viruses were generated and assayed for capsid production, packaging, transduction, heparin agarose binding, and morphology. Three classes of capsid mutants where identified. Class I mutants expressed structural proteins but were defective in virion assembly. Class II mutants generated intact virions that protected the viral genome from DNase, but failed to infect target cells. The majority of these mutants bound the heparin affinity matrix, suggesting that attachment to the AAV primary receptor was not rate limiting. One class II mutant, H2634, assembled virions and bound heparin using only Vp3, indicating that this subunit is responsible for mediating AAV receptor attachment. Finally, class III mutants assembled virions, encapsidated DNA, and infected target cells. Infectivity of these mutants ranged from 5 to 100% of that of the wild-type, demonstrating for the first time the ability to alter capsid proteins without interfering with infectivity. These AAV virions with altered capsid subunits will provide critical templates for manipulating AAV vectors for cell-specific gene delivery in vivo. In summary, the AAV capsid variants described here will facilitate further study of virus assembly, entry, and infection, as well as advance the development of this versatile vector system.
Asunto(s)
Cápside/fisiología , Dependovirus/crecimiento & desarrollo , Cápside/análisis , Cápside/genética , Proteínas de la Cápside , Línea Celular Transformada , Dependovirus/genética , Dependovirus/fisiología , Dependovirus/ultraestructura , Células HeLa , Heparina/metabolismo , Humanos , Mutagénesis Insercional , Recombinación Genética , Virión , Replicación ViralRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN , Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Adenoviridae/genética , Animales , Biotecnología , Encéfalo/virología , Línea Celular , ADN Helicasas/genética , Expresión Génica , Genes Reporteros , Terapia Genética , Virus Helper/genética , Humanos , Músculos/virología , Plásmidos/genética , Transactivadores/genéticaRESUMEN
The neural cell adhesion molecule (NCAM) is a membrane-associated member of the immunoglobulin superfamily capable of both homophilic and heterophilic binding. To investigate the significance of this binding, a gene targeting strategy in embryonic stem (ES) cells was used to replace the membrane-associated forms of NCAM with a soluble, secreted form of its extracellular domain. Although the heterozygous mutant ES cells were able to generate low coat color chimeric mice, only the wild-type allele was transmitted, suggesting the possibility of dominant lethality. Analysis of chimeric embryos with high level of ES cell contribution revealed severe growth retardation and morphological defects by E8.5-E9.5. The second allele was also targeted, and embryos derived almost entirely from the homozygous mutant ES cells exhibited the same lethal phenotype as observed with heterozygous chimeras. Together, these results indicate that dominant lethality associated with the secreted NCAM does not require the presence of membrane-associated NCAM. Furthermore, the data indicate that potent bioactive cues or signals can be generated by NCAM.