Production of recombinant AAV vectors encoding insulin-like growth factor I is enhanced by interaction among AAV rep regulatory sequences

Shuiliang Shi,Scott A Mercer,Robert Dilley,Stephen B Trippel

doi:10.1186/1743-422x-6-3

Abstract

BackgroundAdeno-associated virus (AAV) vectors are promising tools for gene therapy. Currently, their potential is limited by difficulties in producing high vector yields with which to generate transgene protein product. AAV vector production depends in part upon the replication (Rep) proteins required for viral replication. We tested the hypothesis that mutations in the start codon and upstream regulatory elements of Rep78/68 in AAV helper plasmids can regulate recombinant AAV (rAAV) vector production. We further tested whether the resulting rAAV vector preparation augments the production of the potentially therapeutic transgene, insulin-like growth factor I (IGF-I).ResultsWe constructed a series of AAV helper plasmids containing different Rep78/68 start codon in combination with different gene regulatory sequences. rAAV vectors carrying the human IGF-I gene were prepared with these vectors and the vector preparations used to transduce HT1080 target cells. We found that the substitution of ATG by ACG in the Rep78/68 start codon in an AAV helper plasmid (pAAV-RC) eliminated Rep78/68 translation, rAAV and IGF-I production. Replacement of the heterologous sequence upstream of Rep78/68 in pAAV-RC with the AAV2 endogenous p5 promoter restored translational activity to the ACG mutant, and restored rAAV and IGF-I production. Insertion of the AAV2 p19 promoter sequence into pAAV-RC in front of the heterologous sequence also enabled ACG to function as a start codon for Rep78/68 translation. The data further indicate that the function of the AAV helper construct (pAAV-RC), that is in current widespread use for rAAV production, may be improved by replacement of its AAV2 unrelated heterologous sequence with the native AAV2 p5 promoter.ConclusionTaken together, the data demonstrate an interplay between the start codon and upstream regulatory sequences in the regulation of Rep78/68 and indicate that selective mutations in Rep78/68 regulatory elements may serve to augment the therapeutic value of rAAV vectors.

Highlights

Adeno-associated virus (AAV) vectors are promising tools for gene therapy
When the recombinant AAV (rAAV)-insulin-like growth factor I (IGF-I) was prepared with pAAV-RC/'ATG and used for transduction, Effect of Rep78/68 upstream regulatory sequences in AAV helper plasmid on IGF-I production In pAAV-RC, there is an AAV2 unrelated heterologous sequence in front of the start codon ATG321–323 (Figure 1B)
The AAV helper construct that is in current widespread use for rAAV production was improved by the replacement of its AAV2 unrelated heterologous sequence with the native p5 promoter

Summary

Introduction

Adeno-associated virus (AAV) vectors are promising tools for gene therapy. Currently, their potential is limited by difficulties in producing high vector yields with which to generate transgene protein product. A further potential advantage for gene therapy applications is that, in the absence of a helper virus, wild type (wt) AAV can integrate into the cellular genome, an event that occurs at high frequency into a defined region on the long arm of human chromosome 19 [5,6,7]. This site specificity suggests that AAV may pose a low risk of insertional mutagenesis while providing the potential for long-term gene expression

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Conclusion