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

Abstract

Although adeno-associated virus (AAV) vectors are potentially useful gene transfer vehicles for gene therapy, the vector production system is currently at the developmental stage. We constructed AAV helper plasmids (Rep and Cap expression plasmids) by replacing a native AAV promoter, p5, with various heterologous promoters to examine whether the efficiency of AAV vector production was influenced by modulating the AAV protein expression pattern. The helper plasmids containing heterologous promoters (EF, CMV, SV40, B19p6, and CAG promoters, respectively) expressed Rep78/68 more efficiently than a conventional helper plasmid (pIM45), but the expression of Rep52/40 and Cap decreased, resulting in a significant reduction in AAV vector production. Furthermore, the efficiency of vector production never fully recovered even if the Cap proteins were supplied by an additional expression plasmid. A large amount of Rep78/68 and/or a reduced level of Rep52/40 may have deleterious effects on AAV vector production. The present findings will aid in the development of a more efficient AAV vector production system.

Adeno-Associated Viral Vectors for Gene Transfer and Gene Therapy

“…In one case, a ten-fold improvement in the packaging efficiency of rAAV was reported when the p5 promoter was replaced by the HIV-1 LTR (Flotte et al, 1995). However, several other groups observed that increasing Rep78/68 synthesis decreased both the production of capsid proteins and the yield of rAAV, while artificially decreasing the synthesis of Rep78/68 had the opposite effect, suggesting that capsid protein synthesis is the limiting factor in the generation of rAAV (Li et al, 1997;Vincent et al, 1997;Grimm et al, 1998;Ogasawara et al, 1998).…”

Promoter effects of adeno-associated viral vector for transgene expression in the cochlea in vivo

“…SpeI-AgeI fragment containing the Myo promoter was subcloned into pAAV2-CAG to obtain pAAV2-Myo-EGFP-WPRE (pAAV2-Myo). PAAV2-CMV-EGFP-WPRE (pAAV2-CMV), pAAV2-EF1 -EGFP-WPRE (pAAV2-EF1 ), pAAV2-NSE-EGFP-WPRE (pAAV2-NSE), and pAAV2-RSV-EGFP-WPRE (pAAV2-RSV) were constructed as previously described (Ogasawara et al, 1998;Nomoto et al, 2003;Mochizuki et al, 2004). A schematic illustration of the AAV1 vectors is shown in Figure 1.…”

Recombinant adeno-associated virus vector-transduced vascular endothelial cells express the thrombomodulin transgene under the regulation of enhanced plasminogen activator inhibitor-1 promoter

“…The helper plasmid pIM45, containing rep and cap genes for replication and capsid formation, and pladeno-1 containing E2A, E4, and VA genes of the adnovirus genome were used to produce recombinant AAV vectors as described previously. 36 AAV vector production For viral packaging, 293 cells (a human embryonic kidney cell line) were cultured in DMEM/HAM-F12 (Sigma, St Louis, MO, USA) media in the presence of 10% FBS for transfection. Co-transfection of 293 cells grown in flasks with pIM45, pladeno-1, and one of pWCEPT, pWCAGT, pWPTIG, or pWCEPTIG, was done by a calcium phosphate co-precipitation method as described previously.…”

“…Co-transfection of 293 cells grown in flasks with pIM45, pladeno-1, and one of pWCEPT, pWCAGT, pWPTIG, or pWCEPTIG, was done by a calcium phosphate co-precipitation method as described previously. 36 After 72-h incubation, cells were harvested and lysed by repeated freezing and thawing in Tris-buffered saline (10 mm Tris, 150 mm NaCl) pH 8.0. After DNase I (200 U/ml) treatment at 37°C for 30 min to degrade plasmid DNA, the virus particle containing sample was subjected to two rounds of CsCl-density gradient ultracentrifugation in HEPES-buffered saline pH 7.4 in the presence of 25 mm EDTA at 21°C for 24 h as described.…”

“…Expression of β-galactosidase was detected by incubation of cells in buffer containing X-gal as described. 36 Transduction of vascular endothelial cells by rAAV2 vectors in vivo rAAV2/CEPT was injected in the left common carotid artery of Mongolian gerbils under anesthesia by Nembutal. The contralateral carotid artery was temporarily clamped for 5 min after which the clamp was removed and cerebral vascular flow was re-initiated.…”