Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus

Abstract

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high tit-

Differential Contribution of Adeno-Associated Virus Type 2 Rep Protein Expression and Nucleic Acid Elements to Inhibition of Adenoviral Replication in cis and in trans

“…To eliminate the minor inhibition of adenoviral vector propagation mediated by the Rep proteins, future approaches for rAD-mediated expression of the Rep proteins based on inactivation of the RIS-Ad may rather use inducible promoters silenced during the propagation step to achieve higher titers. Minimization of Rep protein expression may also limit the selective loss of Rep sequences, as encountered in the sole study so far reporting the successful incorporation of the complete AAV-2 genome into a first-generation adenoviral vector (15). Such a loss of Rep sequences, maybe preferentially over the RIS-Ad element, probably also accounts for the small size or complete lack of Rep-reactive bands we observed after infections with the rAd/p5-Rep wild-type preparations originating from the adenoviral plasmids carrying the RIS-Ad.…”

“…With regard to the long-pursued goal of using rAD/AAV vectors to provide all the helper functions needed in trans for the simplified high-titer production of recombinant AAVs (rAAVs) by an infection step (15), both efficient Rep and Cap protein expression is required. Since inactivation of the RIS-Ad by introduction of the recoded sRep sequence seems to inhibit p40 activity and also affects the splice donor required for generation of the spliced transcripts encoding the Cap proteins, no major Cap expression levels can be expected from the corresponding adenoviral constructs.…”

“…The inhibitory effects of the rep gene on adenoviral replication also hampered the generation of rAds expressing both the AAV Rep and structural (Cap) proteins, which would represent easily scalable systems for high-titer production of conventional rAAV vectors. Such rAd/AAVrep-cap vectors either could not be propagated at all or were genetically unstable (15). The observed inhibition has been attributed mainly to the expression of the large Rep proteins.…”

Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies

“…46 Briefly, 293HEK cells were cotransfected with 25 mg of the cis-acting plasmid (pAAV-MCK-muSeAP, pSubhSgca or pAAV-U7-SD23-BP22), 12.5 mg of the transcomplementing rep-cap1 (pLT-RCO 2 ), and adenovirus (pXX6) plasmids. 31,47 Vector particles were harvested 3 days post-transfection and purified through two sequential CsCl gradients and dialyzed against PBS.…”

Interactions Between AAV-2 and HSV-1: Implications for Hybrid Vector Design

“…Infectious rAAV particles are separated from helper virus particles by density gradient centrifugation. There are many variations to this general packaging strategy with respect to cellular delivery of the three components, the rAAV plasmid vector, rep/cap and helper virus functions [39][40][41][42][43][44]. Other recent developments in rAAV vector production include the use of: helper virus-free packaging systems [45], affinity chromatography for vector purification [19,46] and the combination of AAV-2 genomes with capsids of other AAV serotypes [47][48][49].…”