Adeno-associated Virus of a Single-polarity DNA Genome Is Capable of Transduction In Vivo
Authors: Xiaohuai Zhou, Xinghua Zeng, Zhenghong Fan, Chengwen Li, Thomas McCown, R Jude Samulski, Xiao Xiao
Journal: Molecular Therapy (2008)

The adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. AAV vectors with wild-type inverted terminal repeats package either the plus- or the minus-strand DNA genomes with equal frequency. By creating a series of deletions within the, we have developed a genetic approach that can generate an AAV vector that packages its single-stranded DNA genome predominantly in a single polarity (99.4%). This novel reagent efficiently transduced muscle, brain and liver in whole animals. The transduction efficiencies were similar to those of the control mixed-polarity vectors. Our results showed that reannealing of plus- and minus-strand DNA was not required for AAV-mediated transduction in vivo, supporting the hypothesis that second-strand DNA synthesis is a primary pathway in converting the single-stranded AAV genome into double-stranded forms. The availability of the single-polarity AAV vector would aid further studies on the mechanism of AAV transduction as well as the application of AAV vector for gene replacement therapy. The adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. AAV vectors with wild-type inverted terminal repeats package either the plus- or the minus-strand DNA genomes with equal frequency. By creating a series of deletions within the, we have developed a genetic approach that can generate an AAV vector that packages its single-stranded DNA genome predominantly in a single polarity (99.4%). This novel reagent efficiently transduced muscle, brain and liver in whole animals. The transduction efficiencies were similar to those of the control mixed-polarity vectors. Our results showed that reannealing of plus- and minus-strand DNA was not required for AAV-mediated transduction in vivo, supporting the hypothesis that second-strand DNA synthesis is a primary pathway in converting the single-stranded AAV genome into double-stranded forms. The availability of the single-polarity AAV vector would aid further studies on the mechanism of AAV transduction as well as the application of AAV vector for gene replacement therapy. IntroductionAdeno-associated virus (AAV) is a single-stranded DNA virus encapsidating either polarity of its genome at the same frequency.1Berns KI Molecular biology of the adeno-associated viruses.Curr Top Microbiol Immunol. 1974; 65: 1-20PubMed Google Scholar It has been demonstrated that both polarities of the viral genome were equally infectious.2Samulski RJ Chang LS Shenk T A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.J Virol. 1987; 61: 3096-3101Crossref PubMed Google Scholar However, because the study was conducted using a cultured transformed cell line which supported second-strand synthesis, it remained to be determined whether one polarity of the virus was sufficient or both polarities were required for AAV-mediated transgene expression in vivo, where second-strand synthesis was rate limiting. Recently published observations seemed to suggest that the above two different mechanisms were in action. First, we showed that a novel AAV vector containing a self-complementary genome expressed in mouse tissues at a faster onset than the conventional single-stranded AAV vectors did.3McCarty DM Monahan PE Samulski RJ Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis.Gene Ther. 2001; 8: 1248-1254Crossref PubMed Scopus (525) Google Scholar,4Wang Z Ma HI Li J Sun L Zhang J Xiao X Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors, in vitro and in vivo.Gene Ther. 2003; 10: 2105-2111Crossref PubMed Scopus (322) Google Scholar At later time points, the convectional vector had a gradually increased level of gene expression. The difference in the onset of gene expression indicated that the single-stranded genome of the convectional AAV vector required conversion to the double-stranded form prior to transgene expression. The gradual rise of gene expression resulting from the conventional AAV vectors implied that either second-strand synthesis or single-stranded DNA annealing, or both, were taking place though at a slow rate. Srivastava and colleagues reported that the efficiency of AAV transduction correlated with the phosphorylation level of a cellular protein regulating the conversion of the single-stranded AAV genome to its double-stranded form (refs. 5Qing K Hansen J Weigel-Kelley KA Tan M Zhou S Srivastava A Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression.J Virol. 2001; 75: 8968-8976Crossref PubMed Scopus (107) Google Scholar,6Qing K Li W Zhong L Tan M Hansen J Weigel-Kelley KA et al.Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo.J Virol. 2003; 77: 2741-2746Crossref PubMed Scopus (52) Google Scholar and accompanying paper7Zhong L Zhou X Li Y Qing K Xiao Samulski RJ et al.Single-polarity recombinant adeno-associated Virus 2 vector-mediated transgene expression In vitro and In Vivo: mechanism of transduction. Mol Ther. ePUP ahead of print, 2007Google Scholar). These findings suggest that second-strand synthesis was the mechanism responsible for AAV transgene expression in vivo. However, Kay and colleagues used a mixed population of two AAV vectors that contained minor differences labeled with various recognizable features.8Nakai H Storm TA Kay MA Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.J Virol. 2000; 74: 9451-9463Crossref PubMed Scopus (156) Google Scholar After analyzing the proviral structures using methods including restriction digestion, they concluded that reannealing of both polarities of vector genome was responsible for AAV transduction in the mouse liver. However, they could not rule out the possibility of single-stranded vector DNA annealing during the DNA extraction process in the test tubes. In order to further characterize the molecular fate of the AAV genome in vivo, we took a genetic approach to generate AAV vectors that contained vector DNA genome predominantly of a single polarity. This novel reagent, unable to generate any significant reannealing event when used alone, was employed in this study to analyze the mechanism of AAV transduction in animals.ResultsGeneration of single-polarity AAV vectorsThe AAV packaging signal is contained within the terminal repeat and packaging of AAV DNA is unidirectional from the 3′-end to the 5′-end into pre-form empty capsids.1Berns KI Molecular biology of the adeno-associated viruses.Curr Top Microbiol Immunol. 1974; 65: 1-20PubMed Google Scholar,9King JA Dubielzig R Grimm D Kleinschmidt JA DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids.EMBO J. 2001; 20: 3282-3291Crossref PubMed Scopus (199) Google Scholar Thus, if the packaging signal is missing from one end of a double-stranded AAV genome, only one polarity of the DNA will be packaged, resulting in the production of an AAV preparation containing only a single polarity of the genome instead of the conventional two equally mixed polarities. We hypothesized that the AAV packaging signal is located within the D element, and therefore by creating deletion mutants of the D element we should be able to generate AAV vectors with a single polarity. To test this hypothesis, we employed a series of deletion mutants based on a novel 165–base pair (bp) AAV terminal repeat consisting of a regular 145-bp AAV terminal repeat with an additional copy of the D element on the opposite side of the existing D element (Figure 1). Previously, we demonstrated that plasmids containing the 165-bp terminal repeat were resolved at the Holiday junction to form double-stranded AAV genomes.10Xiao X Xiao W Li J Samulski RJ A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.J Virol. 1997; 71: 941-948Crossref PubMed Google Scholar The series of deletion mutants included 8-, 12-, 15-, 18-, and 29-bp deletions into the terminal repeat from one end of the D element (X.X., Ph.D. thesis).11Xiao X Characterization of adeno-associated virus DNA replication and integration. PhD thesis. Univ. Pittsburgh, 1992Google Scholar This series of plasmids, named pDDΔ8-CMV-GFP, pDDΔ12-CMV-GFP, pDDΔ15-CMV-GFP, pDDΔ18-CMV-GFP(+), and pDDΔ29-CMV-GFP respectively, as well as the parental pDDwt-CMV-GFP plasmid, were generated and separately packaged into AAV viral particles. To determine the polarities of the vector genomes, viral DNA genomes were extracted and hybridized, respectively with one of the complementary oligonucleotides derived from the cytomegalovirus (CMV) promoter. The Probe CMV(+) would only hybridize to the plus-strand of the viral DNA, while the CMV(−) would only hybridize to the minus-strand of the viral DNA. As shown in Figure 2, AAV vectors with progressive deletion of the D element contained an increasing proportion of one polarity of the viral genome and a decreasing proportion of the other polarity. With a near complete deletion of the D element as in the pDDΔ18-CMV-GFP(+), 99.4% of the vector genome was of one polarity. Our result confirmed our hypothesis that the D element contained a major AAV packaging signal and that deletion of the D element from one end of a double-stranded AAV genome allowed the generation of an AAV preparation with a single polarity of the genome.Figure 2Determination of the polarity of adeno-associated virus (AAV) vector genome. AAV vector DNA was extracted and dot-blotted to two identical sets of membrane as described in the Methods. The two sets of membrane were then hybridized each with one of the two complementary oligonucleotides derived from the cytomegalovirus (CMV) promoter, Probe CMV(+) or Probe CMV(−), which detect either the plus-strand or the minus-strand of the AAV DNA genomes. The amount of each polarity of the vector genome was determined by comparison with double-stranded, CMV-promoter plasmid standards loaded on each set of the membrane. GFP, green fluorescent protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transduction of mouse muscle by single-polarity AAV vectorsTo determine whether a single-polarity AAV vector is capable of expressing its transgene in muscle, we injected directly into the hindleg muscle of C57BL/6 mice with AAV-DDΔ18-CMV-GFP(+), AAV-DDΔ18-CMV-GFP(−) (containing the CMV-GFP cassette in a flipped orientation), a mix of both the above vectors in a 1:1 ratio, AAV-DDwt-CMV-GFP, or a control AAV-CMV-LacZ. Two weeks later, expression of the GFP transgene from the AAV vectors was imaged in whole mice. Representative images are shown in Figure 3. Regardless of the genome polarity, all four preparations of the AAV expressed the GFP transgene at comparable levels. The result demonstrated that AAV vectors with a single polarity were equally capable of transduction in mouse muscle compared to mixed-polarity AAV vectors, suggesting that annealing of plus- and minus-strands did not play an appreciable role in AAV-GFP gene expression.Figure 3Transduction of mouse muscle by single-polarity adeno-associated virus (AAV) vectors. The following five preparations of AAV vector at a dose of 1011 physical particles per animal were injected into the hindlegs of C57BL/6 mice: AAV-DDΔ18-CMV-GFP, AAV-DDΔ18-CMV-GFP(−), AAV-DDΔ18-CMV-GFP mixed with AAV-DDΔ18-CMV-GFP(−) in a 1:1 ratio, AAV-DDwt-CMV-GFP, and a control AAV-CMV-LacZ. Two weeks later, expression of the green fluorescent protein (GFP) transgene contained in the AAV vectors was imaged as described in the Methods. CMV, cytomegalovirus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transduction of rat brain by single-polarity AAV vectorsTo evaluate the expression of single-polarity AAV vectors in the brain, we injected directly into the striatum of Sprague–Dawley rats with AAV-DDΔ18-CMV-GFP(+), AAV-DDΔ18-CMV-GFP(−), or a mix of both in a 1:1 ratio. Transgene expression was determined 2 weeks after the injection. Representative images are shown in Figure 4. Judging from the number of GFP-positive neurons per field, the three preparations of AAV vectors resulted in similar levels of transduction. This result suggested that single-polarity AAV vectors were as efficient as the conventional AAV vectors in mediating gene transfer in the brain.Figure 4Transduction of mouse brain by single-polarity adeno-associated virus (AAV) vectors. The following three preparations of AAV vector at a dose of 1011 physical particles per animal were injected into the striatum of Sprague–Dawley rats: AAV-DDΔ18-CMV-GFP, AAV-DDΔ18-CMV-GFP(−), and AAV-DDΔ18-CMV-GFP mixed with AAV-DDΔ18-CMV-GFP(−) in a 1:1 ratio. Two weeks later, expression of the green fluorescent protein (GFP) transgene contained in the AAV vectors was examined as described in the Methods. CMV, cytomegalovirus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transduction of mouse liver by single-polarity AAV vectorsIt has been reported that the CMV promoter in the context of AAV vectors was inefficient in driving transgene expression in the liver.12Xiao W Berta SC Lu MM Moscioni AD Tazelaar J Wilson JM Adeno-associated virus as a vector for liver-directed gene therapy.J Virol. 1998; 72: 10222-10226Crossref PubMed Google Scholar Thus, we replaced the CMV promoter in the single-polarity vectors with the murine U1a promoter before assessing the expression of the vectors in the liver. The U1a promoter is a part of the U1 small nuclear RNA gene and is transcribed by RNA polymerase II. The promoter has been shown to efficiently express functional messenger RNAs in a variety of mammalian cell types.13Bartlett JS Sethna M Ramamurthy L Gowen SA Samulski RJ Marzluff WF Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.Proc Natl Acad Sci USA. 1996; 93: 8852-8857Crossref PubMed Scopus (21) Google Scholar We then injected this into mice through the portal vein with 3 × 1011 particles of AAV-DDΔ18-U1a-GFP(+), AAV-DDΔ18-U1a-GFP(−), and a mix of both in a 1:1 ratio, and a control AAV-CMV-LacZ. This amount of AAV was used because Nakai et al. showed that it was the upper limit of a dose range that yielded a linear response of transgene expression in their study.14Nakai H Thomas CE Storm TA Fuess S Powell S Wright JF et al.A limited number of transducible hepatocytes restricts a wide-range linear vector dose response in recombinant adeno-associated virus-mediated liver transduction.J Virol. 2002; 76: 11343-11349Crossref PubMed Scopus (80) Google Scholar Six weeks after injection, the mouse livers were sectioned for GFP imaging. As shown in Figure 5a, all three preparations of the AAV vectors transduced hepatocytes with comparable efficiencies, at ∼60% of the hepatocytes in the liver. This high level of expression far exceeded the reported limit of ∼5% by single-stranded AAV vectors, and was likely due to the use of the U1a promoter for driving the transgene and the sensitive imaging system for capturing the GFP fluorescence. To evaluate the possibility that the transgene expression was due to reannealing of the single-polarity AAV genomes with the 0.06% contaminating polarity genomes, and to demonstrate that the fluorescence observed was indeed true GFP fluorescence, we decreased the injection dose to 1 × 1011 particles per mouse and sectioned the livers for both GFP direct imaging and immunostaining 6 weeks after injection. As shown in Figure 5b, at a decreased vector dose, both the single- and mixed-polarity vectors again resulted in similar levels of GFP expression. The native GFP green fluorescence coincided with the red fluorescence from immunostaining the GFP protein, confirming that the green fluorescence observed was indeed due to expression of the GFP transgene. This set of experiments suggested that the transgene expression was unlikely to have resulted from reannealing of the single-polarity AAV genomes with the contaminating polarity genomes in the vector preparation. In other words, it indicated that the GFP expression was predominantly due to conversion of the single-polarity AAV genome to its double-stranded form.Figure 5Transduction of mouse liver by single-polarity adeno-associated virus (AAV) green fluorescent protein (GFP) vectors. (a) The following four preparations of AAV vector at a dose of 3 × 1011 vector genome (v.g.) particles per animal were injected into C57BL/6 mice through the portal vein: AAV-DDΔ18-U1a-GFP, AAV-DDΔ18-U1a-GFP(−), AAV-DDΔ18-U1a-GFP mixed with AAV-DDΔ18-U1a-GFP(−) in a 1:1 ratio, and a control AAV-CMV-LacZ. Six weeks later, expression of the GFP transgene from the AAV vectors was directly imaged as described in the Methods. (b) Lower vector doses (1 × 1011 v.g. particles) were identically administered and processed as in a, except that an immunofluorescent staining for GFP was also performed on consecutive sections to confirm the GFP expression.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionThe development of AAV vectors with a single polarity allowed us to directly address the question whether one polarity of the genome is sufficient or both polarities are required for AAV-mediated transgene expression in vivo. Our results presented in this study show that AAV vectors of a single polarity are similarly efficient in transduction of muscle, brain, and liver in vivo, compared to the mixed-polarity AAV vectors. These findings suggest that reannealing of the vector genome is not required for transgene expression, and therefore may not be the primary mechanism of AAV transduction in vivo. Our conclusion is different from that of a previous report.8Nakai H Storm TA Kay MA Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.J Virol. 2000; 74: 9451-9463Crossref PubMed Scopus (156) Google Scholar Those authors proposed that the mechanism of AAV transduction was through recruitment and reannealing of the complementary plus and minus single-stranded viral DNA to form the double-stranded genomes.8Nakai H Storm TA Kay MA Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.J Virol. 2000; 74: 9451-9463Crossref PubMed Scopus (156) Google Scholar As a result, their proposed mechanism predicted that single-polarity AAV vectors are incapable of efficient transduction in vivo, and that a mixture of vectors of both polarities render significantly higher transduction. However, our results clearly disagree with the annealing hypothesis as a major mechanism of single-stranded to double-strande DNA conversion. As yet, we still cannot rule out the possibility of reannealing of AAV genomes in vivo as a minor alternative mechanism.Recently, Hauck et al. used labeling with bromodeoxyuridine of viral genomes to physically separate the two different polarity vectors.15Hauck B Zhao W High K Xiao W Intracellular viral processing, not single-stranded DNA accumulation, is crucial for recombinant adeno-associated virus transduction.J Virol. 2004; 78: 13678-13686Crossref PubMed Scopus (70) Google Scholar Using the vectors, their results seem to be inconsistent with our current conclusion. However, there are two major differences between our two studies. First, their studies were conducted using HeLa cells in vitro. Compared to primary cells in vivo, HeLa cells may have a much higher activity for converting the single-stranded AAV genome into the double-stranded form. Second, the multiplicity of infection in their experiment shown in Figure 7 was much higher than in ours, especially in the liver transduction experiment. Therefore, it was more likely for reannealing to take place under their conditions.In addition to serving as a novel reagent for studying mechanisms of AAV transduction, the single-polarity AAV vectors may prove to be useful for gene replacement therapy. Hendrie et al. demonstrated that an autonomous parvovirus which packages only one polarity of the viral genome had improved efficiencies of homologous recombination.16Hendrie PC Hirata RK Russell DW Chromosomal integration and homologous gene targeting by replication-incompetent vectors based on the autonomous parvovirus minute virus of mice.J Virol. 2003; 77: 13136-13145Crossref PubMed Scopus (32) Google Scholar In this context, single-polarity AAV vectors packaged in various serotype capsids would provide a broad spectrum of gene targeting vectors for therapeutic applications.Materials and MethodsPlasmids. The construction of pDD-2, a 2.9-kb plasmid that contains a novel 169-bp AAV terminal repeat with two copies of the D element, has been described.10Xiao X Xiao W Li J Samulski RJ A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.J Virol. 1997; 71: 941-948Crossref PubMed Google Scholar To generate serial deletions from one end of the novel terminal repeat, pDD-2 was digested with XbaI and then nuclease Bal31. The detailed procedures for constructing the deletion mutants have also been described.11Xiao X Characterization of adeno-associated virus DNA replication and integration. PhD thesis. Univ. Pittsburgh, 1992Google Scholar Mutants thus produced include 8-, 12-, 15-, 18-, and 29-bp deletions into the terminal repeat from one end of the D element. These mutants were named pB-1-7, pB-5-6, pB-5-7, pB-1-14, and pB-5-1, respectively. To prepare marker vectors based on these plasmids, a 2.0-kb EcoRI fragment containing a CMV-GFP expression cassette excised from the pAAV-CMV-GFP was inserted into the EcoRI site next to the intact copy of the D element.17Haberman RP McCown TJ Samulski RJ Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element.J Virol. 2000; 74: 8732-8739Crossref PubMed Scopus (96) Google Scholar Only insertions with the polyA end near the terminal repeat were selected, as determined by XbaI and PvuII double digestion. Plasmids thus derived were 4.9 kb in size and named pDDΔ8-CMV-GFP, pDDΔ12-CMV-GFP, pDDΔ15-CMV-GFP, pDDΔ18-CMV-GFP, and pDDΔ29-CMV-GFP, respectively. Similarly, pDDwt-CMV-GFP was constructed by inserting the CMV-GFP fragment into the pDD-2 plasmid. To generate pDDΔ18-CMV-GFP(−), pB-1-14 was digested with HindIII, and the ends were filled in with Klenow and then ligated with an EcoRI linker. After digestion with EcoRI, the mutant terminal repeat fragment was isolated and used to replace the wild-type terminal repeat, which was franked by two EcoRI sites, in the pDD-2. One of the clones in which the terminal repeat had the opposite orientation as that in the pB-1-14 was selected, as determined by MscI and PvuII double digestion. This intermediate plasmid, pDDΔ18(−), was partially digested with EcoRI and then inserted with the 2.0-kb EcoRI fragment containing a CMV-GFP expression cassette excised from the pAAV-CMV-GFP. Only insertions at the same location and orientation as those in the pDDΔ18-CMV-GFP were selected, as determined by XbaI and PvuII double digestion. The resulting plasmid was 4.9 kb in size and named pDDΔ18-CMV-GFP(−). To generate pDDΔ18-U1a-GFP and pDDΔ18-U1a-GFP(−), a 267-bp Kpn I–XbaI fragment containing the U1a promoter from p-trsU1aGFP was used to replace the 599-bp CMV promoter in the pDDΔ18-CMV-GFP and pDDΔ18-CMV-GFP(−) plasmids. The CMV promoter was excised as a Kpn I–XbaI fragment.AAV vector production and characterization. AAV vectors used in this study were generated, purified and titered as described previously.18Xiao X Li J Samulski RJ Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J Virol. 1998; 72: 2224-2232Crossref PubMed Google Scholar Briefly, a mixture of three plasmids consisting of an AAV vector plasmid, an AAV helper plasmid XX2, and an adenovirus helper plasmid XX6-80 was transfected into 293 cells. AAV vectors thus generated were purified with both an iodixanol gradient centrifugation and a heparin affinity chromatography.19Zolotukhin S Byrne BJ Mason E Zolotukhin I Potter M Chesnut K et al.Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield.Gene Ther. 1999; 6: 973-985Crossref PubMed Scopus (1010) Google Scholar The physical particle titer of each AAV vector preparation was then determined with a dot-blot assay. To determine the polarity of the AAV vector genome, two sets of identical dot-blot membrane were hybridized separately with each one of the following two complementary oligonucleotides derived from the CMV promoter, Probe CMV(+): 5′-TCCCGGTGTCTTCTATGGAG-3′ and Probe CMV(−): 5′-CTCCATAGAAGACACCGGGA-3′. The oligonucleotides were labeled prior to the hybridization using the DIG oligonucleotide 3′-end labeling kit (Roche, Basel, Switzerland; cat. no. 1362372) following the manufacturer's instruction. To enable the quantification of the vector genome, a set of known amount of DNA standard, twofold serial dilution from 400 ng down to 3.125 ng, was included in each of the dot-blotted membrane. After hybridization, the membranes were exposed to Kodak Biomax MR films (Kodak, Rochester, NY; cat. no. 8701302) and the density of each signal was captured and quantified using the Bio-Rad Gel Doc EQ (Bio-Rad, Hercules, CA; cat. no. 170-8060) and the Quantity One (version 4.3) software, respectively. By comparing to the DNA standards, titers were determined for each polarity of the AAV vectors. Therefore, the purity of a given polarity vector could be calculated using the following formula: purity % = (+) or (−) polarity titer × 100/{(+) polarity titer + (−) polarity titer}.Vector transduction in muscle and whole-animal GFP imaging. Ten-week-old female C57BL/6 mice from Jackson Laboratory (Bar Harbor, Maine) were injected with 1011 particles of the following five preparations of AAV vectors in a volume of 100 μl into the hindleg muscle: AAV-DDΔ18-CMV-GFP, AAV-DDΔ18-CMV-GFP(−), AAV-DDΔ18-CMV-GFP mixed with AAV-DDΔ18-CMV-GFP(−) in a 1:1 ratio, AAV-DDwt-CMV-GFP, and a control AAV-CMV-LacZ. Each preparation of AAV vector was administered to a group of five mice. Two weeks later, the mice were anesthetized with 2.5% Avertin, applied topically with Nair for hair removal around the injection sites, and imaged for GFP expression using an Illumatool Bright Light System LT-9900 from the Lighttools Research (Encinitas, CA). The parameters for the imaging were consistently set at fluorescence for the White Balance Preset and 800 ms exposure time. All animal studies have been approved by the authors' Institutional Review Board.Vector transduction in brain and GFP imaging. All of the animals were pathogen-free male Sprague–Dawley rats obtained from Harlan (Indianapolis, IN). The animals were maintained in a 12-hour light–dark cycle and had free access to water and food. Virus vector infusions were performed as previously described.20McCown TJ Xiao X Li J Breese GR Samulski RJ Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector.Brain Res. 1996; 713: 99-107Crossref PubMed Scopus (385) Google Scholar Briefly, animals were first anesthetized with 50 mg/kg pentobarbital and placed into a stereotaxic frame. Using a 32-gauge stainless steel injector and a Sage infusion pump, animals received 1 μl of the following three AAV preparations over 10 minutes into the striatum [1.0 mm anterior to bregma, 3.0 mm lateral, 5.0 mm vertical, according to the atlas of Paxinos and Watson, 1988 (ref. 21Paxinos G Watson G The Rat Brain in Stereotaxic Coordinates. Academic Pross, New York1998Google Scholar)]: AAV-DDΔ18-CMV-GFP (n = 4), AAV-DDΔ18-CMV-GFP(−) (n = 4), AAV-DDΔ18-CMV-GFP mixed with AAV-DDΔ18-CMV-GFP(−) in a1:1 ratio (n = 2). The injector was left in place for 2 minutes post-infusion to allow diffusion from the injector. Fourteen days after the virus vector infusions, the animals were anesthetized with 100 mg/kg pentobarbital, intraperitoneally and perfused transcardially with ice-cold 100 mmol/l sodium phosphate-buffered saline (pH = 7.4), followed by 4% paraformaldehyde in 100 mmol/l phosphate buffer (pH = 7.4). After overnight fixation in the paraformaldehyde-phosphate buffer, vibratome sections (40-μm thick) were taken and rinsed in phosphate-buffered saline. The tissue sections were rinsed three times in phosphate-buffered saline, mounted on slides and coverslipped with a fluorescent mounting media. GFP fluorescence was visualized using an Olympus IX 70 microscope.Vector transduction in liver and GFP imaging. Five- to six-week-old female C57BL/6 mice from Jackson Laboratory were injected through the portal vein with AAV vectors at the doses indicated. Each preparation of the AAV vector was administered to a group of five mice. Six weeks later, the livers were perfused, harvested, embedded in OTC compound, and sectioned at 9-μm thickness for direct GFP imaging. Images of liver sections were captured using a Leica DM IRB microscope. The parameters for the imaging were consistently set at max gain and 8-second exposure time. For GFP immunostaining, the livers were perfused, harvested, fixed, and sectioned with a vibratome. The tissue sections were immunostained and imaged as described.22Spector DL Smith HC Redistribution of U-snRNPs during mitosis.Exp Cell Res. 1986; 163: 87-94Crossref PubMed Scopus (74) Google Scholar IntroductionAdeno-associated virus (AAV) is a single-stranded DNA virus encapsidating either polarity of its genome at the same frequency.1Berns KI Molecular biology of the adeno-associated viruses.Curr Top Microbiol Immunol. 1974; 65: 1-20PubMed Google Scholar It has been demonstrated that both polarities of the viral genome were equally infectious.2Samulski RJ Chang LS Shenk T A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.J Virol. 1987; 61: 3096-3101Crossref PubMed Google Scholar However, because the study was conducted using a cultured transformed cell line which supported second-strand synthesis, it remained to be determined whether one polarity of the virus was sufficient or both polarities were required for AAV-mediated transgene expression in vivo, where second-strand synthesis was rate limiting. Recently published observations seemed to suggest that the above two different mechanisms were in action. First, we showed that a novel AAV vector containing a self-complementary genome expressed in mouse tissues at a faster onset than the conventional single-stranded AAV vectors did.3McCarty DM Monahan PE Samulski RJ Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis.Gene Ther. 2001; 8: 1248-1254Crossref PubMed Scopus (525) Google Scholar,4Wang Z Ma HI Li J Sun L Zhang J Xiao X Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors, in vitro and in vivo.Gene Ther. 2003; 10: 2105-2111Crossref PubMed Scopus (322) Google Scholar At later time points, the convectional vector had a gradually increased level of gene expression. The difference in the onset of gene expression indicated that the single-stranded genome of the convectional AAV vector required conversion to the double-stranded form prior to transgene expression. The gradual rise of gene expression resulting from the conventional AAV vectors implied that either second-strand synthesis or single-stranded DNA annealing, or both, were taking place though at a slow rate. Srivastava and colleagues reported that the efficiency of AAV transduction correlated with the phosphorylation level of a cellular protein regulating the conversion of the single-stranded AAV genome to its double-stranded form (refs. 5Qing K Hansen J Weigel-Kelley KA Tan M Zhou S Srivastava A Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression.J Virol. 2001; 75: 8968-8976Crossref PubMed Scopus (107) Google Scholar,6Qing K Li W Zhong L Tan M Hansen J Weigel-Kelley KA et al.Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo.J Virol. 2003; 77: 2741-2746Crossref PubMed Scopus (52) Google Scholar and accompanying paper7Zhong L Zhou X Li Y Qing K Xiao Samulski RJ et al.Single-polarity recombinant adeno-associated Virus 2 vector-mediated transgene expression In vitro and In Vivo: mechanism of transduction. Mol Ther. ePUP ahead of print, 2007Google Scholar). These findings suggest that second-strand synthesis was the mechanism responsible for AAV transgene expression in vivo. However, Kay and colleagues used a mixed population of two AAV vectors that contained minor differences labeled with various recognizable features.8Nakai H Storm TA Kay MA Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.J Virol. 2000; 74: 9451-9463Crossref PubMed Scopus (156) Google Scholar After analyzing the proviral structures using methods including restriction digestion, they concluded that reannealing of both polarities of vector genome was responsible for AAV transduction in the mouse liver. However, they could not rule out the possibility of single-stranded vector DNA annealing during the DNA extraction process in the test tubes. In order to further characterize the molecular fate of the AAV genome in vivo, we took a genetic approach to generate AAV vectors that contained vector DNA genome predominantly of a single polarity. This novel reagent, unable to generate any significant reannealing event when used alone, was employed in this study to analyze the mechanism of AAV transduction in animals.