Production of recombinant AAV vectors encoding insulin-like growth factor-I is enhanced by interaction among AAV rep regulatory sequences
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.
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).
Results: We constructed a series of AAV helper plasmids containing different Rep78/68 start codon in combination with different gene regulatory regulatory sequences.
rAAV vectors carrying the human IGF-I gene were prepared with these vectors and the vector preparations used to transfect 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 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.
Conclusion: Taken 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.
Author: Shuiliang Shi, Scott A. Mercer, Robert Dilley and Stephen B. Trippel
Credits/Source: Virology Journal 2009, 6:3
Published on: 2009-01-07
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
make sure to read our disclaimer prior to contacting 7thSpace Interactive. To contact our editors, visit our online helpdesk. If you wish submit your own press release, click here.
Social Bookmarking
RETWEET This! | Digg this! | Post to del.icio.us | Post to Furl | Add to Netscape | Add to Yahoo! | Rojo
|
|
