Southern blot analysis to detect BamHI- and EcoRI-nondigestible plasmids in mouse livers transfected with plasmids. Twenty-five micrograms of either pAAV.ΔBamHI (mice 1 and 2) or pAAV.ΔEcoRI (mice 11 and 12), or a combination of both (mice 6, 7, and 8), was infused through the tail vein as described in Materials and Methods, and liver samples were analyzed at 6 weeks postinjection in the same manner as that for the experiment shown in Fig. 3G. There were no BamHI- and EcoRI-nondigestible genomes detected in these mice. 1.0-copy/cell standards, marker 1, and the probe used were the same as for the experiment shown in Fig. 3G.

Schematic representation of rAAV transduction in mouse hepatocytes. rAAV virions containing either a plus or minus ss genome enter the nucleus, creating a pool of ss rAAV genomes. Plus- and minus-strand rAAV genomes combine to form ds linear rAAV genomes. This step is presumed to be crucial for rAAV transduction in vivo, and most of the ss rAAV genomes that fail to partner are gradually degraded over a period of 1 to 2 months. Successfully annealed ds rAAV genomes are converted into a more stable form (i.e., ds circular monomer). Some of these ds circular monomers combine with each other, forming ds circular dimers or larger concatemers by intermolecular recombination through the ITRs (42). Some of the ds linear monomer may also form episomal linear concatemers. It is not yet known how integrated concatemers are formed, but possible pathways are indicated by dotted arrows with question marks. All of the ds forms—i.e., circular monomers, dimers, and larger concatemers; ds linear monomers and concatemers; and integrated concatemers—may be capable of expressing transgene (26, 42). However, which form(s) contributes to persistent transgene expression from hepatocytes is not known.

Structures of the rAAV vectors, restriction enzyme sites, and DNA probes. (A) AAV-EF1α-LacZ and AAV-EF1α-LacZ.PMT. (B) AAV-EF1α-F.IX, AAV-EF1α-F.IX.PMT, and AAV-EF1α-F.IX.dam. The GATC sequences present between the two BglII sites are indicated by asterisks, and the location of a hemimethylated GATC sequence in pV4.le-hF.IX.hemi is indicated by a perpendicular arrow. (C and D) AAV.ΔBamHI (C) and AAV.ΔEcoRI (D). The 0.3-kb deletion within EF1α-P is shown. A unique BamHI or EcoRI site has been destroyed. Abbreviations: EF1α-P, EF1α gene enhancer-promoter; δEF1α-P, a truncated EF1α-P with a 1.3-kb SpeI-MunI deletion (28); sδEF1α-P, a truncated EF1α-P with a 0.3-kb SpeI-ClaI deletion; lacZ, the E. coli β-galactosidase gene; pA(SV40), the simian virus 40 early polyadenylation signal [poly(A)]; hFIXcDNA; the human coagulation factor IX cDNA; pA(hGH), the human growth hormone gene poly(A); A, AflII; B, BamHI; Bg, BglII; C, ClaI; D, DrdI; E, EcoRI; H, HindIII; Nd, NdeI; No, NotI; S, SpeI; Xo, XhoI.

Southern blot analysis of the AAV.PMT vectors and transduced mouse livers. (A) Determination of methylation status at the XhoI sites on AAV-EF1α-LacZ (no PMT or not methylated) and AAV-EF1α-LacZ.PMT vectors (left panel) and on AAV-EF1α-F.IX (no PMT) and AAV-EF1α-F.IX.PMT vectors (right panel). ss viral DNA corresponding to 2.0 × 10⁹ particles was extracted from purified stocks, annealed, and digested with the enzyme(s) indicated above the lanes prior to Southern analysis using XhoI-NdeI lacZ and XhoI F.IX probes (left and right panels, respectively). Note that no XhoI-digestible genomes were observed in the PMT lanes. (B) Methylation status of rAAV genomes from the livers of mice 6 weeks after AAV-EF1α-LacZ or AAV-EF1α-LacZ.PMT injection. The liver DNA was digested with NdeI and XhoI prior to Southern analysis using the XhoI-NdeI lacZ fragment as a probe. Faint XhoI-digestible genomes were observed, but most of the genomes remained uncut in the AAV-EF1α-LacZ.PMT-injected mice (mice 1, 2, and 3), while all the genomes were cut with XhoI in AAV-EF1α-LacZ-injected mice (mice 4 and 5). One-unit-length 4.4-kb bands represent head-to-tail molecules because NdeI results in a single cut in the absence of XhoI digestion. (C) The number of vector genomes per cell (diploid genomic equivalent) before and after two-thirds partial hepatectomy (PHx) in the mice injected with AAV-EF1α-F.IX or AAV-EF1α-F.IX.PMT. Mice 1 and 2 were injected with 2.4 × 10¹¹ particles of AAV-EF1α-F.IX.PMT, mice 3 and 4 were injected with 7.2 × 10¹¹ particles of AAV-EF1α-F.IX.PMT, and mice 5 and 6 were injected with 2.4 × 10¹¹ particles of AAV-EF1α-F.IX. The liver DNA was digested with BglII and probed with a BglII F.IX fragment. (D) Methylation status of rAAV genomes from livers of mice injected with AAV-EF1α-F.IX or AAV-EF1α-F.IX.PMT before and after two-thirds hepatectomy. The liver DNA was digested with BglII and XhoI and probed with a BglII F.IX fragment. Virtually all of the genomes remained uncut by XhoI before hepatectomy, but XhoI-digestible genomes were present after the procedure. The mouse DNAs used were the same as those employed for the experiment shown in panel C.

PCR-based detection of mismatch repair of heteroduplex rAAV genomes in mice injected with a combination of AAV.ΔBamHI and AAV.ΔEcoRI vectors. (A) Primer locations and lengths of the products. EF1αP21 and hFIXP21 were used for PCR, and hFIXP22 was used for primer extension of the PCR products. The 422-bp product was cut into 257- and 165-bp fragments at the EcoRI-ΔEcoRI site and into 272- and 150-bp fragments at the BamHI-ΔBamHI site. pA, polyadenylation signal; B and ΔB, Bam HI and ΔBamHI sites, respectively; E and ΔE, EcoRI and ΔEcoRI sites, respectively. (B) Total liver DNA from mouse 9 (group 7), which was injected with both AAV.ΔBamHI and AAV.ΔEcoRI vectors (sample 1), and a 1:1 mixture of two liver DNAs from mouse 1 (group 4) and mouse 10 (group 7), which were injected with only one vector (sample 2), were digested with a combination of BamHI and EcoRI and amplified by PCR with primer set EF1αP21-hFIXP21. Radioactive homoduplexes were synthesized by primer extension with a ³²P-labeled hFIXP22 primer, digested with BamHI and EcoRI, and electrophoresed on a 1.2% agarose gel. The products were stained with ethidium bromide (left panel), and radioactive products were detected by a Molecular Imager System (middle and right panels). Lanes with odd numbers contained undigested products, while lanes with even numbers contained completely digested products. Complete BamHI-EcoRI double digestion was confirmed by cleavage of linearized plasmid DNA of a 1:1 mixture of pAAV.ΔBamHI and pAAV.ΔEcoRI added in the reaction mixtures (lanes 2 and 4). In sample 2 (which serves as a negative control with no mismatch-repaired genomes), all of the homoduplex products were cleavable with either BamHI or EcoRI, generating 165- and 150-bp band (lanes 8 and 12), while some of the homoduplexes in sample 1 remained uncut (lanes 6 and 10). In the rightmost panel, the signals from smear homoduplexes were emphasized to demonstrate that radioactive homoduplex rAAV genomes amplified independently of the PCR primers in sample 1 (lane 10), but not in sample 2 (lane 12), also contained mismatch-repaired genomes. (C) An analysis identical to that of panel B was carried out with gel-purified PCR products as a template for the primer extension reaction. A considerable number of radioactive homoduplex genomes remained uncut (lane 6). Sample 2 served as a negative control with no BamHI- and EcoRI-nondigestible radioactive homoduplexes (lane 8).

(A) DpnI cutting activity on hemimethylated DNA. Plasmid pV4.le-hF.IX.hemi, which carried one adenine-hemimethylated DpnI recognition sequence (see Fig. 1B for its location), was synthesized with TaqI methylase. Nineteen copies of this plasmid (lanes 1 and 2) or a control fully methylated plasmid, pV4.le-hFIX.dam+ (pV4.le-hF.IX propagated in a dam-positive strain of E. coli), per cell were added to 20 μg of naïve-mouse genomic DNA and digested with a combination of DpnI and BglII (lanes 1 and 3) or BglII only (lanes 2 and 4) under the same conditions used for the analyses shown in Fig. 4C, and the digest was subjected to Southern blot analysis with the BglII F.IX probe. Nineteen copies of pV4.le-hF.IX.hemi per cell represents the same molar ratio of hemimethylated DpnI sites as that in 1.0 copy of ds vector genomes per cell. DpnI completely digested the fully methylated plasmid, while about 50% activity was observed on hemimethylated plasmid. (B and C) Southern blot analysis of the AAV-EF1α-F.IX.dam vector. (B) Analysis of methylation status at the GATC sites on the AAV-EF1α-F.IX and AAV-EF1α-F.IX.dam vectors by Southern blotting. ss viral DNAs corresponding to 2.0 × 10⁹ particles were annealed, digested with the enzyme(s) indicated above the lanes, and electrophoresed on a 1.2% agarose gel along with the markers (BglII-digested [lanes 1 and 12], BglII- and DpnI-digested [lane 4], BglII- and MboI-digested [lane 11], or Sau3AI-digested [lane 13] pV4.le-hF.IX.dam+ or BglII-digested [lane 7] or BglII- and MboI-digested [lane 8] pV4.le-hF.IX.dam− [pV4.le-hF.IX propagated in a dam-negative strain of E. coli]). The DNA blot was probed with the BglII F.IX probe. Lanes 2, 5, 9, and 14, AAV-EF1α-F.IX.dam; lanes 3, 6, 10, and 15, AAV-EF1α-F.IX. DpnI-digestible genomes are clearly observed in AAV-EF1α-F.IX.dam (lane 5). Under the conditions used here for DpnI digestion (10 U of DpnI for 2.0 × 10⁹ ss rAAV genomes or 5.4 ng of viral DNA), DpnI showed some cutting activity on fully unmethylated ds DNA; this resulted in a faint 2.3-kb band in lane 6 due to partial digestion of the DpnI sites, but no discrete bands (except for the 2.3-kb band). However, this activity was completely repressed in the presence of mouse genomic DNA at a concentration of 0.25 μg per unit of enzyme (see panel C). (C) Methylation status of ds rAAV genomes in mouse liver at 6 weeks after injection of AAV-EF1α-F.IX.dam. The liver DNA was extracted and electrophoresed on a 1.2% agarose gel after digestion with the enzyme(s) indicated above the lanes, and the blot was analyzed with the BglII F.IX probe. Mice 1 to 3 received AAV-EF1α-F.IX.dam vector, and mice 4 and 5 received AAV-EF1α-F.IX vector. Most of the ds rAAV genomes in mice 1 to 3 remain fully methylated. The 1.0-copy-number standards were prepared using pV4.le-hF.IX.dam+ except for the right-most lane, which contained pV4.le-hF.IX.dam−.

Southern blot analyses of liver DNA from mice transduced with AAV.ΔBamHI and/or AAV.ΔEcoRI. (A) Schematic representation of HMW rAAV concatemer structural analysis using AAV.ΔBamHI and AAV.ΔEcoRI. Possible concatemer structures are shown on the left, and the corresponding Southern blot results after BamHI or EcoRI digestion are shown on the right. The solid line and dotted line with arrowheads represent AAV.ΔBamHI (ΔB) and AAV.ΔEcoRI (ΔE) genomes, respectively. If the HMW concatemers consist of multiple copies from a single rAAV genome amplified by a rolling-circle mechanism (schemes a and c), Southern blot results for liver DNA from the mice injected with both vectors at a 1:1 ratio should show a mixture of scheme a and c products, 1-unit-length fragments, and a genomic-DNA-sized signal for either the BamHI or EcoRI digest. If the recruitment of input rAAV genomes is the mechanism (scheme b), a ladder consisting of n-unit fragments would be observed for either enzyme digest. In the actual DNA samples from both vector-injected mice, the presence of BamHI- and EcoRI-nondigestible rAAV genomes (see Results) would complicate the interpretation of each n-unit-sized fragment in the ladder. However, a ladder should never be observed in a rolling-circle amplification mechanism of concatemerization. (B and C) BamHI and EcoRI digestion, respectively. BamHI in group 4 (G4) and EcoRI in group 7 (G7) served as noncutter controls. Discrete genomic-DNA-sized signals in DNA indicate the presence of large concatemers. The ladder formation consisting of n-unit fragments in groups 5 (G5) and 6 (G6) indicates the presence of heteroconcatemers. Supercoiled ds circular monomer (SdsCM) increases and relaxed ds circular monomer (RdsCM) decreases in size with restriction enzyme digestion to the 1.0-unit-size position when cut with BamHI or EcoRI, while ds linear monomer (dsLM) moves down to the 0.5-unit-size position when cut with either enzyme. The position of the ss monomeric rAAV genome is indicated as ssM, although it was not observed. Open arrowheads indicate positions of n-unit-length framents. Marker 1 was a 7.3-kb marker; marker 2 was a 4.7-kb marker representing 1 unit length. The ClaI-HindIII F.IX probe was used for these blots. (D) Southern blot analysis demonstrating the presence of covalently linked heteroconcatemers. BamHI-digested total mouse liver DNAs were prepared and electrophoresed on a 0.8% gel in the same manner as that used for the experiment shown in panel B, except for the inclusion of heat denaturation of digested mouse liver DNA in 50% formamide before the samples were loaded on the gel. Ladders were observed in mice injected with both vectors (groups 5 and 6), as indicated by open arrowheads, but not in a mouse injected with a single AAV.ΔEcoRI vector (group 7), demonstrating the presence of covalently linked heteroconcatemers. Black arrows indicate head-to-head or tail-to-tail molecules that could be cut with BamHI, resulting in 1.0-unit-sized ss molecules that folded back to 0.5-unit-sized ds molecules with a hairpin structure by intramolecular annealing during electrophoresis under neutral conditions. This was confirmed by alkaline gel electrophoresis, since no >1.0-unit-sized fragment was observed in group 7 mice after BamHI digestion (data not shown). Although we demonstrated the formation of ladders, we could not define some of the bands because of the folding-back phenomenon observed with head-to-head or tail-to-tail molecules. Marker 1, BamHI-digested naïve-mouse liver DNA (20 μg) with 1.0 copy of pV4.le-hF.IX per cell, serving as a monitor for complete digestion and as an approximate 0.5-unit-length size marker. Marker 2, PvuII-digested pAAV.ΔBamHI added to BamHI-digested naïve-mouse liver DNA (20 μg) at 1.0 copy per cell, serving as an approximate 1.0-unit-length size marker. The size markers on the left represent the positions of ds DNA fragments. (E) Southern blot analysis of fractionated HMW DNA (≥17 kb) following KpnI digestion. The HMW DNAs of one or two mice from groups G5, G6, and G7 were digested with BamHI and subjected to Southern blot analysis with a ClaI-HindIII F.IX probe. The intensities of 1.0- and 2.0-unit-length bands were almost the same in G6, and the intensity of the 2.0-unit-length band was higher than that of the 1.0-unit-length band in G5, which is suggestive of random recruitment of input rAAV genomes in a concatemer. (F) Southern blot analysis of total DNA (left panel) or KpnI-digested gel-fractionated HMW DNA (right panels) to establish linking orientations in HMW concatemers. Liver samples from two mice in group 4 (mice 2 and 3) were chosen for this analysis. The purified HMW DNA was digested with AflII or HindIII (see Fig. 1C for their cleavage site locations) and subjected to Southern blot analysis with the ClaI-HindIII F.IX probe. Only the results of AflII digestion are shown here. Open and closed arrowheads indicate positions of head-to-tail (H-T) and tail-to-tail (T-T) molecules, respectively. Although the bands were fuzzy and covered with the enriched smear hybridization indicative of integrated genomes, the head-to-tail form was not the predominant form of HMW concatemers, unlike the situation for total DNA. (G) Southern blotting of DNA from mice in groups 5 and 6 to look for BamHI- and EcoRI-nondigestible ds rAAV genomes. BamHI-EcoRI-XhoI triple digestion generated a 1.7-kb band when rAAV genomes had either a BamHI or an EcoRI site, while a 1.9-kb band was seen when they lacked both sites. All four mice analyzed had a 1.9-kb band in addition to the major 1.7-kb band. The 2.0-unit-length band, RdsCM, and SdsCM, which remained uncut when treated with BamHI-EcoRI, indicate the presence of BamHI- and EcoRI-nondigestible ds rAAV genomes in concatemers and circular monomers. Incomplete enzyme digestion can be ruled out from the complete digestion of the 1.0-copy-number standards (20 μg of naïve-mouse genomic DNA containing a 1:1 mixture of pAAV.ΔBamHI and pAAV.ΔEcoRI equivalent to 1.0 copy per cell). Marker 1 is the 1.9-kb XhoI-XhoI fragment. The XhoI F.IX probe was used.