Adeno-associated virus (AAV) undergoes site-specific integration into human chromosome 19 through a deletion-substitution mechanism at the well characterized AAVS1 site. We have shown previously that a cis element within the left end of the AAV genome enhances the efficiency of Rep-mediated site-specific integration into chromosome 19 when present in inverted terminal repeat-containing recombinant AAV (rAAV) plasmids. We now demonstrate that a 138-bp cis element, the p5 integration efficiency element (p5IEE), mediates efficient integration. The p5IEE is not only required for efficient site-specific integration, it is also sufficient. Integration mediated by the p5IEE occurs in the absence of the AAV inverted terminal-repeat elements. The data presented in this study demonstrate that the p5IEE is a multifunctional element, serving as the highly regulatable Rep promoter and the primary substrate for targeted integration.A deno-associated virus (AAV) is a single-stranded parvovirus with a 4.7-kb genome (1). The DNA contains two ORFs, rep and cap, that are flanked by two inverted terminal repeats (ITRs; refs. 2-4). AAV is the only known virus that sitespecifically integrates into the human genome, and this targeted integration of the AAV genome occurs at the AAVS1 site on chromosome 19q13.3-qter (5-8).The Rep protein (specifically Rep 68͞78) is essential in mediating recombination between the AAV genome and the AAVS1 chromosomal target (9-13). Rep 68͞78 functions by binding to the Rep-binding elements (RBEs) situated in both the AAV genome and at the AAVS1 site (7,14,15). Through poorly understood interactions, the Rep protein͞AAV-DNA complex localizes to the AAVS1 site, and a nonhomologous deletioninsertion recombination event occurs, resulting in integration of the AAV genome (16)(17)(18)(19)(20)(21)(22). It has been shown that head-to-tail concatemers of the wild-type (wt)AAV genome are able to site-specifically integrate in this manner (23).Rep-mediated integration of recombinant A AV (rAAV)(ITR ϩ ) plasmids [such as pTRUF2 (24) or p2ITRLacZ (10)] is extremely inefficient, with between 0.1 and 1% of transduced cells demonstrating rAAV genome persistence after 6 weeks (25). In contrast, plasmids that carry the entire AAV genome integrate at efficiencies of greater than 10%. This difference between rAAV plasmids and wtAAV plasmids led to the discovery of a previously unknown cis sequence domain present in the left end of the AAV genome that enhances integration efficiency of rAAV(ITR ϩ ) constructs by 10-100-fold (25). The current study extends the characterization of the left-end cis element in mediating site-specific integration. We have discovered a 138-bp AAV integration efficiency element (p5IEE) that is not only necessary for efficient site-specific integration but is also sufficient. Data presented in this study clearly demonstrate efficient Rep-mediated integration of p5IEE-containing plasmids that do not contain AAV ITR elements. Furthermore, in nearly 100% of cases the integration event was targeted to t...

The structurally related somatic and germinal isoforms of angiotensin-converting enzyme (ACE) contain the same catalytic active center and are encoded by the same gene, whose disruption causes renal atrophy, hypotension, and male sterility. The reason for the evolutionary conservation of both isozymes is an enigma, because, in vitro, they have very similar enzymatic properties. Despite the common enzymatic properties, discrete expression of both isoforms is maintained in alternate cell types. We have previously shown that sperm-specific expression of transgenic germinal ACE in Ace ؊/؊ male mice restores fertility without curing their other abnormalities (Ramaraj, P., Kessler, S. P., Colmenares, C. & Sen, G. C. (1998) J. Clin. Invest. 102, 371-378). In this report we tested the biological equivalence of somatic ACE and germinal ACE utilizing an in vivo isozymic substitution approach. Here we report that restoration of male fertility was not achieved by the transgenic expression of enzymatically active, somatic ACE in the sperm of Ace ؊/؊ mice. Therefore, the requisite physiological functions of the two tissue-specific isozymes of ACE are not interchangeable.

The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10-to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.Adeno-associated virus (AAV) is a human parvovirus with a single-stranded DNA genome of 4.7 kb (4). It contains two open reading frames (ORFs), rep and cap, which are flanked by two inverted terminal repeats (ITRs) (14, 32). The ITRs are 160 nucleotides including the D element and are considered to be the only cis elements required for replication and sitespecific integration of the AAV genome. In addition to the ITR elements, the rep gene is necessary in trans to target the integration event to the AAVS1 site (2,5,16,25,30,34). The rep ORF encodes four nonstructural proteins, Rep 40, Rep 52, Rep 68, and Rep 78, which are involved in the replication of the AAV genome (33,35). Rep 68 and Rep 78 are gene products of alternatively spliced mRNA transcribed from the AAV p5 promoter. These larger Rep proteins have DNA binding, site-specific, and strand-specific endonuclease activities as well as ATPase and DNA-DNA and DNA-RNA helicase functions (12,13,41,45). Transcription from the p19 promoter generates Rep 52 and Rep 40; Rep 52 is known to have helicase and ATPase activities but no DNA binding or endonuclease activity (31). Rep 68 and Rep 78 down-regulate the p5 and p19 promoters (3), and this stringent control of Rep expression helps to minimize cell death caused by the cytotoxicity of the Rep prote...