![]() No human disease has been found to be associated with AAV infection and the virus has a low immunogenicity in humans. AAV is capable of infecting dividing and non-dividing cells in vitro and in vivo and of infecting cells originating from multiple species and tissue types. The human adeno-associated virus (AAV) has attracted attention as a vector for gene therapy because it possesses several favorable characteristics. Genetic modification of cells is a promising approach to generating gene products that have therapeutic potential. 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. 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. ![]() 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. 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. ![]() 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. rAAV vectors carrying the human IGF-I gene were prepared with these vectors and the vector preparations used to transduce HT1080 target cells. We constructed a series of AAV helper plasmids containing different Rep78/68 start codon in combination with different gene regulatory sequences. We further tested whether the resulting rAAV vector preparation augments the production of the potentially therapeutic transgene, insulin-like growth factor I (IGF-I). 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. AAV vector production depends in part upon the replication (Rep) proteins required for viral replication. Currently, their potential is limited by difficulties in producing high vector yields with which to generate transgene protein product. ![]() Adeno-associated virus (AAV) vectors are promising tools for gene therapy. ![]()
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