Novel Transcriptional Regulatory Signals in the Adeno-Associated Virus Terminal Repeat A/D Junction Element

Abstract

Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604-1611, 1998) demonstrated the use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the "off" state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other cis-acting elements (AAV terminal repeats [TR]) were contributing to this activity. In the present study, we identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using deletion analysis and specific PCR-derived TR reporter gene templates, we mapped this activity to a 37-nucleotide stretch in the A/D elements of the TR. Although the mRNA derived from the TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green fluorescent protein expression both in vitro and in vivo in brain by using recombinant virus carrying only the TR element. Since the AAV terminal repeat is a necessary component of all recombinant AAV vectors, this TR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel TR split gene vectors currently being considered for genes too large to be packaged.

Herpes Simplex Virus Type 1 ICP0 Protein Mediates Activation of Adeno-Associated Virus Type 2 rep Gene Expression from a Latent Integrated Form

“…Under this latter condition, as in cells infected with Ad5, a larger protected Rep band was also detected. This signal likely corresponded to transcripts initiated from the 5Ј ITR, as previously documented by other investigators (29,34). Taken together, these data indicated that the activation of rep gene expression by ICP0 occured at the transcriptional level.…”

Separate Basic Region Motifs within the Adeno-Associated Virus Capsid Proteins Are Essential for Infectivity and Assembly

“…Confluent 293 cells were split 1:3 24 h prior to transfection. Triple transfections with pACG2 (31), pTrufEGFP (16), and pXX6-80 (adenovirus helper plasmid) were carried out by calcium phosphate precipitation as previously described (16a). At 48 h posttransfection, cells were scraped, pelleted, and resuspended in phosphate-buffered saline (PBS).…”

Efficient gene expression by self-complementary adeno-associated virus serotype 2 and 5 in various human cancer cells

“…Previous studies have suggested that long-term gene expression by ssAAV is due to either the integration of rAAV genome to chromosome (27,28) or genomic persistence in its episomal conformation (29)(30)(31). Even though ssAAV vectors preferentially integrate into actively transcribed regions (32), the terminal repeats in each end of the rAAV vector have minimal promoter activity, unlike that of retroviruses characterized by strong promoter activity (33). More importantly, there is no evidence indicating that the rAAV vector can cause cancers in vivo (34,35).…”