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16. Single-Polarity Recombinant Adeno-Associated Virus 2 Vector-Mediated Transgene Expression In Vitro and In Vivo: Mechanism of Transduction
AAV VECTORS: VECTOR BIOLOGY 16. Single-Polarity Recombinant AdenoAssociated Virus 2 Vector-Mediated Transgene Expression In Vitro and In Vivo: Mechanism of Transduction Li Zhong,1 Xiaohuai Zhou,2 Yanjun Li,3 Keyun Qing,4 Richard J. Samulski,2 Arun Srivastava.1 1 Pediatrics, University of Florida College of Medicine, Alachua, FL; 2Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC; 3Pharmacutics, University of Florida College of Medicine, Gainesville, FL; 4Eli Lilly & Company, Indianapolis, IN. Recombinant AAV vectors encapsidate single-stranded genomes of both polarities in separate mature virions with equal frequency. Since single-stranded viral genomes of either polarity are transcriptionally inactive, it has been suggested that failure to undergo DNA strand-annealing is responsible for low efficiency of transgene expression. Although a number of recent reports (Gene Ther., 10: 2105, 2003; Gene Ther., 10: 2112, 2003; Gene Ther., 11, 1165; Mol. Ther., 10: 950, 2004) support the conclusion that viral secondstrand DNA synthesis is the major rate-limiting step, we wished to remove any and all ambiguities by using viral stocks that contain genomes of single-polarity, and therefore, incapable of strandannealing. We reasoned that if DNA strand-annealing is the predominant mechanism, then no transgene expression would be expected with single-polarity AAV vectors, since DNA strands of same polarity cannot undergo annealing. However, if viral secondstrand DNA synthesis is a prerequisite for transgene expression, then we would expect to see efficient transduction of HeLa cells stably transfected with a TC-PTP expression plasmid in vitro, and primary hepatocytes in TC-PTP-transgenic (TC-PTP-TG) and FKBP52-knockout (FKBP52-KO) mice in vivo since tyrosinedephosphorylation of FKBP52 in TC-PTP-TG, or the absence of FKBP52 in FKBP52-KO mice, will allow viral second-strand DNA synthesis. We compared transduction efficiencies of conventional AAV vectors, containing both [-] and [+] polarity genomes, with those containing either [-], or [+] polarity genomes in HeLa cells in vitro and in murine hepatocytes in vivo. Our data document the following: (i) both single-polarity AAV vectors fail to transduce HeLa cells efficiently, but co-infection with adenovirus enhances the transduction efficiency up to 40-fold, similar to that obtained with conventional AAV vectors; (ii) siRNA-mediated downmodulation of a cellular protein, FKBP52, tyrosine-phosphorylated forms of which have previously been shown to inhibit AAV secondstrand DNA synthesis, also allows a significant increase in the transduction efficiency of single-polarity AAV vectors; (iii) in HeLa cells, over-expression of a cellular protein tyrosine phosphatase, TC-PTP, known to catalyze tyrosine-dephosphorylation of FKBP52, also leads to a significant increase in the transduction efficiency of single-polarity AAV vectors; and (iv) single-polarity AAV vectors are equally efficient as conventional AAV vectors in transducing primary hepatocytes in transgenic mice over-expressing the TC-PTP gene, or in mice deficient in FKBP52. These data are consistent with the interpretation that viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction by AAV vectors, which has implications in the use of these vectors in human gene therapy. LZ and XZ contributed equally to this work.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
17. Visualization of the Intranuclear rAAV Replication Centers and Their Co-Localization with Double-Stranded DNA Break Repair Proteins Cervelli Tiziana, Lorena Zentilin, Alessandro Marcello, Mauro Giacca. 1 LTGM, Scuola normale Superiore, Pisa, Italy; 2Lab of Molecular Medicine, ICGEB, Trieste, Italy; 3Lab of Molecular Medicine, ICGEB, Trieste, Italy; 4Lab of Molecular Medicine, ICGEB, Trieste, Italy. We have developed a method for the visualization of the sites of conversion of the ssDNA AAV genome to dsDNA inside the cell nucleus. This method is based on the utilization of a recombinant AAVlacO.14 vector, containing 112 LacR binding sites (lacO) cloned between the viral ITRs, which is used for the infection of cells that constitutively express a LacR-GFP fusion protein. The rationale of this approach is that the fluorescent sequence-specific DNA binding protein only binds its target site when this is present in a dsDNA form; this permits visualization and monitoring of ss to ds AAV genome conversion over time. A first set of experiments was carried out in HeLa cells and MRC5 cells expressing a LacR-GFP protein containing a nuclear localization signal. These cells were infected with AAVlacO.14, either without, or after overnight incubation with, 1 mM hydroxyurea (HU), and analyzed by real-time confocal microscopy in live cells at different times after transduction. Specific regions of AAV ss to dsDNA conversion started to be detected as early as 3 hours post infection as tiny bright foci. The dynamics of foci formation was followed by tracking the trajectories of individual foci during the first 24 hours after transduction, through a z-series of images captured at 0.45 um intervals. Most of the tracked AAV foci showed random movement in a restricted area of the nucleus. Occasionally, individual spots traveled short distances to join another immobile spot. Most of the AAV foci were found to progressively grow larger in size over time. It has been observed that cells treated with DNA damaging agents are more permissive to AAV infection, and that HU increase transduction efficiency by a post-entry mechanism, possibly involving ss to ds AAV genome conversion. To establish whether a relationship exists between AAV genome maturation and DSB repair proteins, we visualized the AAVlacO.14 foci and the localization of some of these proteins, including Rad51, Rad50, Mre11 and histone H2AX. HeLa and MRC5 cells, treated with HU, were infected with AAVlacO.14 and the co-localization experiments were performed at several times post infection. The vast majority of Mre11 and Rad50 foci were found to co-localize with AAVlacO.14 at 24 hours post infection. In contrast, we observed colocalization of the Rad51 foci and AAVlacO only at early times post infection. After 24 hours, the Rad51 foci appeared juxtaposed to, but clearly separated from, the AAVlacO dots. Finally, the AAVlacO foci were found to be distinct from those formed by the accumulation of H2AX. Collectively, these results suggest that the Rad51 and Mre11 complex proteins, may facilitate conversion of the rAAV genomes from single ss to ds and that they interact with these genomes with different dynamics. In contrast, g-H2AX, apparently does not participate in the maturation of the AAV genomes.
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Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
17. Visualization of the Intranuclear rAAV Replication Centers and Their Co-Localization with Double-Stranded DNA Break Repair Proteins Cervelli Tiziana, Lorena Zentilin, Alessandro Marcello, Mauro Giacca. 1 LTGM, Scuola normale Superiore, Pisa, Italy; 2Lab of Molecular Medicine, ICGEB, Trieste, Italy; 3Lab of Molecular Medicine, ICGEB, Trieste, Italy; 4Lab of Molecular Medicine, ICGEB, Trieste, Italy. We have developed a method for the visualization of the sites of conversion of the ssDNA AAV genome to dsDNA inside the cell nucleus. This method is based on the utilization of a recombinant AAVlacO.14 vector, containing 112 LacR binding sites (lacO) cloned between the viral ITRs, which is used for the infection of cells that constitutively express a LacR-GFP fusion protein. The rationale of this approach is that the fluorescent sequence-specific DNA binding protein only binds its target site when this is present in a dsDNA form; this permits visualization and monitoring of ss to ds AAV genome conversion over time. A first set of experiments was carried out in HeLa cells and MRC5 cells expressing a LacR-GFP protein containing a nuclear localization signal. These cells were infected with AAVlacO.14, either without, or after overnight incubation with, 1 mM hydroxyurea (HU), and analyzed by real-time confocal microscopy in live cells at different times after transduction. Specific regions of AAV ss to dsDNA conversion started to be detected as early as 3 hours post infection as tiny bright foci. The dynamics of foci formation was followed by tracking the trajectories of individual foci during the first 24 hours after transduction, through a z-series of images captured at 0.45 um intervals. Most of the tracked AAV foci showed random movement in a restricted area of the nucleus. Occasionally, individual spots traveled short distances to join another immobile spot. Most of the AAV foci were found to progressively grow larger in size over time. It has been observed that cells treated with DNA damaging agents are more permissive to AAV infection, and that HU increase transduction efficiency by a post-entry mechanism, possibly involving ss to ds AAV genome conversion. To establish whether a relationship exists between AAV genome maturation and DSB repair proteins, we visualized the AAVlacO.14 foci and the localization of some of these proteins, including Rad51, Rad50, Mre11 and histone H2AX. HeLa and MRC5 cells, treated with HU, were infected with AAVlacO.14 and the co-localization experiments were performed at several times post infection. The vast majority of Mre11 and Rad50 foci were found to co-localize with AAVlacO.14 at 24 hours post infection. In contrast, we observed colocalization of the Rad51 foci and AAVlacO only at early times post infection. After 24 hours, the Rad51 foci appeared juxtaposed to, but clearly separated from, the AAVlacO dots. Finally, the AAVlacO foci were found to be distinct from those formed by the accumulation of H2AX. Collectively, these results suggest that the Rad51 and Mre11 complex proteins, may facilitate conversion of the rAAV genomes from single ss to ds and that they interact with these genomes with different dynamics. In contrast, g-H2AX, apparently does not participate in the maturation of the AAV genomes.
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