Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products

Abstract

Adeno-associated virus (AAV) is a human parvovirus currently being developed as a vector for gene therapy applications. Because the gene transfer vector commonly retains only the AAV terminal repeats, propagation of recombinant AAV (rAAV) requires that the viral replication (Rep) and capsid (Cap) proteins be supplied in trans. In an effort to optimize the production of these vectors, a panel of helper plasmids was constructed to determine if expression of the rep and/or cap genes is a limiting factor for rAAV packaging. Expression of the Rep and Cap proteins was increased by replacing the endogenous AAV promoters, p5 and p40, with the Rous sarcoma virus (RSV) long terminal repeat (LTR) and the cytomegalovirus immediate-early promoter, respectively. Increased synthesis of the Cap proteins resulted in an approximately 10-fold increase in the yield of rAAV, indicating that production of capsid proteins is one limiting factor for rAAV packaging. Expression of the rep gene from the RSV LTR not only failed to increase the yield of rAAV but also prevented activation of p40 transcription with adenovirus infection, resulting in a reduced level of capsid protein synthesis.

The Use of Heterologous Promoters for Adeno‐Associated Virus (AAV) Protein Expression in AAV Vector Production

“…These results suggest that sufficient expression of capsid protein is important for AAV vector production and that a large amount of Rep78/68 and/or reduced amount of Rep52/40 have deleterious effects on AAV vector production. These findings are compatible with recent reports (20,37). Recent studies also showed that overexpression of Rep78/68 inhibits Cap expression, but the effect on Rep52/40 expression was unclear (20,37).…”

“…Moreover, we investigated the relation between Rep expression patterns and the efficiency of AAV vector production by adjusting differences in the amount of Cap from various helper plasmids. The production of capsid proteins is one limiting factor for rAAV packaging and overexpression of Rep78/68 inhibits rAAV DNA replication and cap gene expression, resulting in the reduction of rAAV titer (20,37). These previous findings were confirmed by this study, and we found that overexpression of Rep78/68 inhibited not only Cap expression but also Rep52/40 expression in the presence of adenoviruses.…”

Efficient Production of Adeno‐associated Virus Vectors Using Split‐type Helper Plasmids

“…A previous report showed that the production of capsid proteins is a limiting factor for packaging the AAV vector genome and that overexpression of Rep78/68 leads to a much reduced AAV vector titer. 31) On the other hand, down regulation of Rep78/68 expression by using an inefficient translation initiation start signal caused increased capsid protein expression, resulting in much higher AAV vector yields. 32) Thus, our findings are compatible with these earlier reports.…”

Viral Vectors

“…This is usually done by removal of all viral genes between the 5 Ј and 3 Ј terminal repeat (TR) sequences and replacing them with the desired transgene(s) ( Dong et al, 1996 ). Since the only viral DNA sequences the rAAV vectors retain are the TRs, the vectors must be propagated in a special packaging cell lines that express the AAV Rep and Cap proteins, in addition to the helper virus co-infection ( Vincent et al, 1997 ). This has both advantages and disadvantages.…”