Adeno-associated virus vectors can be efficiently produced without helper virus

Abstract

The purpose of this work was to develop an efficient Therefore the minimum set of genes required to produce method for the production of adeno-associated virus (AAV) AAV helper activity equivalent to that provided by adenvectors in the absence of helper virus. The adenovirus ovirus infection consists of, or is a subset of, the following regions that mediate AAV vector replication were identified genes: the E4orf6 gene, the 72-M r , E2A protein gene, the and assembled into a helper plasmid. These included the VA RNA genes and the E1 region. AAV vector preparations VA, E2A and E4 regions. When this helper plasmid was made with adenovirus and by the helper virus-free method cotransfected into 293 cells, along with plasmids encoding were essentially indistinguishable with respect to particle the AAV vector, and rep and cap genes, AAV vector was density, particle to infectivity ratio, capsimer ratio and produced as efficiently as when using adenovirus infection efficiency of muscle transduction in vivo. Only AAV vector as a source of help. CMV-driven constructs expressing the preparations made by the helper virus-free method were E4orf6 and the 72-M r , E2A proteins were able to funcnot reactive with anti-adenovirus sera. tionally replace the E4 and E2A regions, respectively.

Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease

“…AAV vectors (AAV-GDNFflag and AAV-LacZ) were purified using two sequential continuous CsCl gradients, as described previously. 40 The final particle titer of the Gene Therapy AAV-GDNFflag was 1.6 × 10 13 vector genome copies/ml and AAV-LacZ was 2.1 × 10 13 vector genome copies/ml, as estimated by quantitative DNA dot-blot hybridization analysis.…”

Novel adenovirus-based helper system to support production of recombinant parvovirus

“…4,22,24 The mechanism(s) underlying the effect of the adenovirus genes on recPV production and which elements, among the ones expressed by the pXX6 plasmid, are required for the amplification, remain uncharacterized. Similar studies conducted with adeno-associated viruses showed that E2a is the major contributor to the adenoviral helper effect on AAV-2, 23 whereas VA RNA sustains the AAV-5 replication by blocking double-stranded RNA-activated protein kinase activation and suppressing the antiviral cellular response. 20 This study shows that pXX6 significantly increases and accelerates viral DNA replication during the production of recPVs.…”

“…Transfection of HEK 293T cells with either pAd5DE1DE3 (containing all adenovirus-5 genes with the exception of E1 and E3), pXX6 or pVAE2AE4-5 (both containing the adenovirus-5 genes E2a, E4(orf6) and VA RNA) led to a comparable increase in recPV production. These results indicate that, similarly to AAV and B19, 22,23 the adenoviral genes contained in pXX6 are sufficient to increase recPV production. It is likely that the protocols used here can be further improved for instance by determining the most appropriate timing for the delivery of the adenovirus genes to producer cells or the optimal molar ratio of the three plasmids used for the production (parvovirus vector: Adenovirus genes enhance parvovirus production N El-Andaloussi et al capsid vector: adenoviral helper).…”

“…In parallel, we also tested the ability of pVAE2AE4-5, a vector carrying the same adenoviral genes as pXX6 but in a different plasmid backbone, to increase parvovirus production. 23 Furthermore, we determined whether the adenoviral helper genes could be applied by infection by using Ad5DE1DE3-CMV/GFP (Ad-GFP). Cells were treated with phH-1-Luc plus pCMV/VP and either co-transfected with one of the adenovirus helper plasmids (pXX6 or pVAE2AE4-5), or infected with Ad-GFP virus at multiplicity of infections ranging from 0.1 to 5 GFU per cell.…”

“…In HEK 293 cells, which provide the E1 gene in trans, the minimal set of genes for efficient recombinant AAV-2 production is E2a, E4(orf6) and the VA RNA gene. [21][22][23] A helper plasmid named pXX6, containing this set of genes, is currently used for the production of adenovirus-free recombinant AAV-2. 22 This plasmid allowed for AAV-2 production in a similar way as infectious adenovirus.…”