INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Adeno-associated virus type 2 (AAV-2) is a nonpathogenic (10, 11), 4.7-kb single-stranded DNA virus that requires coinfection with helper adenovirus or herpes simplex virus for efficient replication (5, 12, 30). The viral genome contains two open reading frames that express four Rep and three Cap proteins and is flanked by the 145-bp terminal repeats, which are the only cis-acting elements that are essential for replication, packaging, or integration (reviewed in reference 47). Thus, the entire AAV genome, except for these repeats, can be replaced by a transgene to form a recombinant AAV (rAAV) (43, 57). The natural tissue tropism of wild-type AAV is for lung epithelial cells; however, the recombinant vector has been used to also efficiently target skeletal muscle, liver, retina, brain, and gut epithelium (15, 20, 23, 24, 32, 34, 35, 60, 71, 77). This nearly ubiquitous tropism can be partly explained by the fact that AAV-2 apparently uses widely expressed molecules as coreceptors, including heparan sulfate proteoglycan (primary attachment receptor), fibroblast growth factor receptor 1, and _V5-integrin (52, 62, 63; J. Qui, H. Mizukami, and K. E. Brown, Letter, Nat. Med. 5:467-468, 1999).

Either strand of the virus can be packaged in virions as a single-stranded DNA (47). In an infected cell, this single-stranded virion DNA is converted to a double-stranded form by poorly defined mechanisms. In the absence of helper virus coinfection, wild-type AAV integrates as concatamer and preferentially but not exclusively at the AAVS1 locus on human chromosome 19 by a process that requires viral Rep protein(s) (33, 36, 40, 58). Upon subsequent helper virus infection, the wild-type AAV genome is rescued and the virus enters the lytic cycle, forming progeny virions and thus completing the viral infection cycle. On the other hand, the genome maturation kinetics is not well understood for the rAAV vector genome. rAAV, delivered in the absence of helper virus and Rep protein, persists as a single-stranded genome for a certain period in transduced tissues. Using host cell enzymes, the single-stranded rAAV genome is converted to double-stranded forms that may persist as linear or circular episomes and may also appear in the high-molecular-weight (HMW) DNA. Integration of rAAV occurs at a low frequency, as monomers or low-copy-numbers concatamers, and with a loss of chromosome 19 specificity (13, 33, 43, 48, 51, 53, 55). Poor understanding of rAAV genome maturation has added to the confusion regarding what form(s) of rAAV, integrated or episomal, is truly responsible for long-term transgene expression and persistence, both of which are the hallmarks of rAAV-mediated gene transfer in muscle, liver, and other target organs.

Skeletal muscle has turned out to be a promising tissue for rAAV-mediated systemic delivery of transgene products. Efficient systemic delivery of transgene products in skeletal muscle has been clearly demonstrated (6, 16, 18, 73). High rAAV transducibility, easy accessibility, low turnover of cells, and high vascularity are salient features that make skeletal muscle a preferred rAAV delivery tissue. The ability of skeletal muscle to exhibit persistent expression of the rAAV transgene has been well described (15, 23, 34, 71). The lifelong transgene expression obtained in mice after rAAV-mediated gene transfer is in part due to the inability to elicit a cell-mediated immune response (31). These features of rAAV in muscle have led to preclinical testing with hemophilia B mouse and dog models and now to initial translational clinical testing (for a review, see reference 54). Hemophilia B is caused by deficiency of blood coagulation factor IX (FIX), a key protein that occupies a pivotal position in the coagulation cascade (17, 37, 41).

A single intramuscular injection of rAAV containing the human, mouse, or canine FIX (hFIX, mFIX, or cFIX) expression unit into immunocompetent normal or hemophiliac mice or dogs can result in the production of biologically active FIX (27, 28, 76). In mice intramuscularly injected with rAAV vector containing the hFIX expression unit (rAAV/hFIX), the level of hFIX increases gradually over 5 to 10 weeks after a 1- to 2-week initial lag phase. Using rAAV/-galactosidase vector, such gradual increase in expression of transgene has also been correlated with a concomitant increase in the integrated form of the rAAV vector (15). Similarly, the integrated form of rAAV correlates with transgene expression in liver-directed rAAV in mice (44). Other groups have suggested a role for episomal rAAV forms in the long-term persistence and expression in muscle (19). To clarify these issues, we analyzed the kinetics of rAAV transduction using a human skeletal muscle cell assay system. Primary human skeletal muscle cells can be maintained for almost a month in culture, much longer than mouse primary muscle cells, allowing us to analyze the detailed initial kinetics of rAAV transduction.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

rAAV vectors. Plasmid pTR/Me4AhFIXm1 containing the 2.8-kb human FIX minigene (hFIXm1) (38) under the transcriptional control of four tandem copies of muscle creatine kinase enhancer (Me4) and -actin promoter (A) (69) was constructed by inserting the Me4-A-hFIXm1 cassette between the AAV terminal repeats in pTR-UF2 (described in reference 77). The Me4-A-hFIXm1 fragment (4.4-kb) was obtained from pBS/Me4AhFIXm1 by KpnI-BamHI digestion. This fragment was inserted at the KpnI-BamHI sites of pTR-UF2 containing AAV terminal repeats and bovine growth hormone (bGH) poly(A) signal, thus generating pTR/Me4AhFIXm1 (Fig. 1). pTR/CMVhFIXm1 was constructed by inserting hFIXm1 (2.8-kb) into pTR-UF2 at the BamHI site between the human cytomegalovirus (CMV) immediate-early gene enhancer/promoter and bGH poly(A) signal (Fig. 1). The rAAV genomes produced from these vectors, rAAV/CMVhFIXm1 and rAAV/Me4AhFIXm1, were 4.0 and 4.9 kb in size, respectively.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Structure of rAAV/Me4AhFIXm1 and rAAV/CMVhFIXm1 constructs. rAAV/Me4AhFIXm1 contains the hFIX minigene under the transcriptional control of four tandem copies of the minimal MCK enhancer (Me4; total size 1.2 kb) and 280-bp -actin promoter. rAAV/CMVhFIXm1 contains the 620-bp CMV immediate-early gene enhancer-promoter, hFIX minigene, and bGH poly(A) signal, flanked by AAV inverted terminal repeats. The CMV promoter probe (0.62-kb EcoRI-BamHI fragment) used for Southern analysis is shown by a dark line. The internal EcoRI fragment detected by the CMV promoter probe is 1.9 kb (arrow a in Fig. 4 and 5). The line at the bottom represents a 5-kb scale, with each mark representing 1 kb.

Isolation and maintenance of human myoblasts. Primary human myoblasts were isolated from biopsy samples of healthy abdominal muscle tissue (rectus abdominus) which were obtained from patients undergoing surgery (at sites unrelated to abdominal muscle) at the Department of Surgery. Written consent was obtained in accordance with institutional guidelines. Muscle tissue biopsy specimens freshly obtained from 44- or 2-year-old individuals were immediately placed in ice-cold human myoblast growth medium (HMB-GM) (see below) and left overnight at 4°C. Myoblasts were isolated by using anti-CD56 antibody linked to magnetic beads (B.-G. Chen and K. Kurachi, unpublished data) or by a cloning method as previously described (74). The primary myoblasts obtained were more than 99% free of fibroblasts, as judged by desmin immunofluorescence staining (data not shown), and were capable of complete differentiation into myotubes. The primary myoblasts can be maintained in culture for at least 25 to 30 doublings (about 8 to 10 passages) (data not shown). The cells used in this report were between passages 3 and 5. Myoblasts were routinely maintained on 0.25% gelatin (Sigma, St. Louis, Mo.)-coated tissue culture dishes. The tissue culture dishes were coated with gelatin by pipetting 0.25% (wt/vol) solution of gelatin made in water, swirling the dishes to wet the surface, and removing the solution before plating the cells. The growth-factor rich media, HMB-GM, used for myoblasts in this paper were (i) Ham's F10 medium (Sigma) supplemented with 20% heat-inactivated fetal bovine serum (FBS; Gibco-BRL, Gaithersburg, Md.), 5 ng of bovine fibroblast growth factor basic (R & D Systems, Minneapolis, Minn.) per ml, and penicillin-streptomycin (pen-strep; Gibco-BRL); and (ii) skeletal muscle growth medium (SKGM; Clonetics/Whittakar, San Diego, Calif.) supplemented with 20% Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL), 5% FBS, and pen-strep. The differentiation medium used was DMEM supplemented with 2.5% FBS and pen-strep.

Recombinant AAV transduction. For rAAV transduction, 3 × 10⁵ human myoblasts were routinely plated on 60-mm-diameter gelatin-coated culture dishes and grown for 3 days to 90% confluency (~10⁶ cells). The myoblasts were then differentiated by being grown for 3 days in DMEM containing 2.5% FBS. The medium was replaced every day. Fully differentiated myotubes or 90% confluency myoblasts were infected with rAAV at a multiplicity of infection (MOI) of 1 × 10⁴, 1 × 10⁵, or 5 × 10⁵ particles/cell (ca. 1 × 10¹⁰, 1 × 10¹¹, or 5 × 10¹¹ particles/dish) for 2 h at 37°C with continuous rocking in 0.5 ml made up with Opti-MEM-I medium (Gibco-BRL). The volume was then adjusted to 2 ml with medium containing BaSO₄-treated FBS (75) and supplemented with 10 µg of vitamin K₁ per ml (AquaMEPHYTON; Merck, West Point, Pa.). The medium was collected daily, and fresh medium was added. In some experiments, the infections were done in six-well plates. The medium collected was centrifuged for 30 s in a microcentrifuge to remove cell debris and stored at 70°C until use. Levels of hFIX produced in the medium were estimated by enzyme-linked immunosorbent assay (ELISA), using pooled normal human plasma (George King Bio-Medical Inc., St. Overland Park, Kans.) as the standard, as previously described (38). All samples were assayed in duplicate, and the sensitivity of this ELISA was of the subnanogram order.

Isolation and analysis of HMW and Hirt DNAs. HMW (cellular genomic and possibly some very large concatameric forms of rAAV) DNA was isolated from infected cells at various times postinfection (p.i.). Cells were gently rinsed with ice-cold phosphate-buffered saline and were harvested by scraping into 2 ml of ice-cold phosphate-buffered saline. Cells from three 60-mm-diameter dishes were pooled for each time point, and the genomic DNA fraction was obtained by proteinase digestion of cells as described previously (45, 56). Stringy DNA (HMW DNA), precipitated upon addition of 70% ethanol (final volume) to cell lysates, was spooled out using a plastic pipette tip, transferred to a tube containing 70% ethanol, and pelleted by brief centrifugation at room temperature. The DNA pellet was air dried and allowed to resuspend overnight in 20 to 100 µl of TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) at 4°C. After removal of HMW DNA, the remaining original 70% ethanol solution was centrifuged at room temperature for 15 min in a microcentrifuge to obtain Hirt DNA (unintegrated low-molecular-weight DNA). The Hirt DNA pellet was washed with 70% ethanol and resuspended in 25 µl of TE containing RNase A (at 0.1 mg/ml [final concentration]). This spooling method, which is routinely used for obtaining high-quality genomic DNA from culture cells and various tissues, was used rather than conventional Hirt precipitation (29) because this enabled us to prepare both high-quality HMW and Hirt DNA preparations from the same cells. Southern blot analysis of restricted genomic DNA (4 to 10 µg) was carried out on a 0.75% agarose gel using ³²P-radiolabelled probes (56). Specifically hybridized bands were quantitated on a PhosphorImager (STORM system; Molecular Dynamics, Sunnyvale, Calif.).

Transfection of human myoblasts. Primary myoblasts were transfected using FuGENE 6 (Roche, Indianapolis, Ind.) as recommended by the manufacturer, with modifications. Briefly, 2 × 10⁵ myoblasts were plated per well of a six-well plate coated with 0.25% gelatin and incubated overnight at 37°C. After 1 day, cells at 70 to 80% confluency were transfected by centrifugation (30 min at 1,180  g and 32°C) after addition of a mixture of 3 µg of plasmid DNA (precondensed by incubating with 3 µg of histone H1 protein [Roche] for 5 min at room temperature) and 9 µl of FuGENE 6 on cells in a final volume of 1 ml of medium. Centrifugation was performed using microtiter plate carrier. After 4 h, the transfection medium was replaced with 1 ml of growth medium (SKGM) supplemented with BaSO₄-treated FBS and 10 µg of vitamin K₁ per ml. The medium was collected every 24 h, and the hFIX produced was assayed by ELISA. Expression obtained in the 24- to 48-h posttransfection period in repeated experiments was reported.

Western blot analysis of rAAV/CMVhFIXm1 virions. rAAV preparations were diluted to 10¹¹ particles/ml in 50 mM Tris (pH 6.7). Sodium dodecyl sulfate (SDS)-polyacrylamide gel-loading buffer (5×) was added to the samples and boiled for 5 min, and 2 or 4 µl was resolved on an SDS-10% polyacrylamide gel and electroblotted onto polyvinylidene difluoride membranes as previously described (42). The hFIX proteins on blots were visualized by sequential treatment with goat anti-hFIX polyclonal antibody (obtained from Enzyme Research Laboratories Inc., South Bend, Ind.) (used at 1:1,000 dilution in Tris-buffered saline [TBS] [pH 7.5]), horseradish peroxidase-conjugated swine antigoat immunoglobulin G (Roche) (1:5,000 dilution in TBS) and chemiluminescent reagents (ECL-PLUS [Amersham]). The titers of rAAV/CMV--gal and wild-type AAV-2 used as controls in these experiments were 5.5 × 10¹¹ and 1.3 × 10¹¹ particles/ml, respectively. These vectors were produced from rAAV plasmids, pAB-11 and pSub201, respectively, using the helper-free system as described above.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Transduction of human myotubes with rAAV/CMVhFIXm1 results in much higher expression of transgene than does that of rAAV/Me4AhFIXm1. Previously we showed that Me4A muscle-specific promoter can drive high hFIX expression in primary mouse myoblasts (69). To evaluate the expression of hFIX transgene from the Me4A promoter in rAAV-mediated gene transfer, we constructed rAAV/Me4AhFIXm1 vector and compared it with the constitutive vector rAAV/CMVhFIXm1 (Fig. 1). Figure 2 shows the kinetics of hFIX production from human myotubes after transduction with rAAV/Me4AhFIXm1 or rAAV/CMVhFIXm1. It is important to note that longitudinal and detailed analysis of kinetics of rAAV transduction and transgene expression was possible only by using human muscle cells, which could be maintained viable in culture for as long as 1 month. Mouse myotubes, on the other hand, could not be maintained for more than 7 to 9 days in culture (data not shown).

View larger version (2727K): [in this window] [in a new window]   FIG. 2.   Production of hFIX from human muscle cells transduced with rAAV/Me4AhFIXm1 or rAAV/CMVhFIXm1 vector. (A, C, E, G, I, K, and M) hFIX production after direct transduction of myotubes (solid triangles); (B, D, F, H, J, L, and N) hFIX production from myotubes generated from transduced myoblasts (open squares). Cells were transduced with rAAV/Me4AhFIXm1 at a MOI of 1 × 104 (A and B) or rAAV/CMVhFIXm1 at a MOI of 1 × 104 (C to F), 1 × 105 (G to L), and 5 × 105 (M and N). Cells were maintained in differentiation medium (A to D, G and H), F10-based medium (E, F, I, J, M, and N), or SKGM-based medium (K and L). hFIX secreted per day was assayed by ELISA and is shown as mean and standard error of the mean (n = 2 for panels A, B, E, and F; n = 3 for panels C, D, K, L, M, and N). For panels G, H, I, and J, the experiment was started with nine 60-mm dishes, and three dishes were used at each time point as described in the text. For panels F, J, L, and N, myoblasts were differentiated by being transferred to differentiation medium for 3 days p.i., and then maintaining the cells in F10- or SKGM-based rich medium as described in the text. No high or transient expression of hFIX was seen in panels K and L, presumably because a different rAAV batch was used for them, which is different from those used for other experiments.

rAAV transduction of human myotubes and myoblasts results in similar and high hFIX production. Human skeletal myotubes were transduced with rAAV/CMVhFIXm1 at a MOI of 1 × 10⁴, 1 × 10⁵, or 5 × 10⁵ and maintained in differentiation medium (supplemented with 2.5% FBS) or in rich medium (F10- or SKGM-based medium) (Fig. 2C to N). Both F10- and SKGM-based media are routinely used as muscle cell growth media. We tested both to see if these different media have an effect on transgene expression. In addition, we chose to maintain cells throughout in differentiation medium because cells survive longer under these culture conditions, allowing us to extend the duration of transgene expression. As shown in Table 1 and Fig. 2C to N, a viral dose-response relationship was obtained from transduced myotubes maintained in the F10-based medium. These cells showed maximal hFIX production of ~600, ~800, or ~1,700 ng of hFIX/10⁶ cells/day when transduced at a MOI of 1 × 10⁴, 1 × 10⁵, or 5 × 10⁵, respectively (Fig. 2E, I, and M; Table 1). Myotubes in differentiation medium produced lower levels of hFIX (~200 ng of hFIX/10⁶ cells/day) (Fig. 2C and G). Similar to the results with myotubes maintained in F10-based medium, the myotubes in SKGM-based medium also produced high levels of hFIX (Fig. 2K; Table 1). A similar dose-response relationship was also obtained from myotubes derived from myoblasts, which were transduced at 90% confluency prior to differentiation (Fig. 2D, F, H, J, L, and N; and Table 1). However, unlike direct transduction of myotubes, the expression of hFIX did not necessarily increase linearly. This may be in part due to the rAAV transduction process and subtle unknown effects of induction of differentiation, although further studies are required to find the reason. The overall maximal hFIX expression in these experiments was 1,500 to 1,900 ng of hFIX/10⁶ cells/day (Table 1). This supports the high capability of skeletal muscle to express hFIX transgene, agreeing with previous observations in mouse muscle cells (68, 73).

Expression of hFIX from rAAV-transduced human skeletal muscle cells occurs with no lag phase. As shown in Fig. 2, the rAAV/CMVhFIXm1-transduced human myotubes showed no appreciable lag period but showed a linear increase in hFIX production (Fig. 2C, E, G, I, K, and M). In both mice and dogs intramuscularly injected with rAAV/FIX, a lag phase of about 2 weeks followed by a slow rise over 5 to 8 weeks before reaching stable constant levels is observed (27, 28). A similar lag phase was also reported in -galactosidase production in mice intramuscularly injected with rAAV/-gal (15). However, in some experiments, wherein rAAV/erythropoitin was intramuscularly delivered to mice, no such lag phase was observed and transgene expression was observed from as early as 4 days p.i. (61, 67). One possible explanation for the observed lag in detectable hFIX production in mice is a need to saturate hFIX binding sites on endothelial cells and vascular tissue prior to the appearance of detectable hFIX in the circulation (14, 27) whereas the in vitro culture assay system is not affected by such hFIX binding. Our observations with human muscle cells strongly support the notion that there is no appreciable initial lag phase in hFIX production and are consistent with the conceivable mechanism of slow but steady conversion of single- to double-stranded rAAV genome.

rAAV virions can copackage hFIX protein. In some experiments, as shown in Fig. 2, rAAV/CMVhFIXm1-transduced muscle cells exhibited a high and transient hFIX production during the first 2 days of transduction. Some previous reports suggested that the CMV promoter has a differential activity in different tissues and at various stages of organogenesis and differentiation (7, 8, 59). The initial spike in hFIX levels is probably not due to myoblast differentiation-specific effects, because this transient expression was also observed in transduction of fully differentiated myotubes (Fig. 2C, E, I, and M). Since rAAV vectors can package either plus or minus strands (9), the single-stranded DNA of both polarities must be present in infected cells. Therefore, it is possible that some of these strands anneal and form transcriptionally active double-stranded forms. This scenario, however, provides only a partial explanation because of the transient nature of hFIX expression. The notion of carryover of the rAAV plasmid vector used for transfection of the 293 packaging cells to target cell transduction medium is inconceivable, because of the several specific steps involved in rAAV purification.

View larger version (50K): [in this window] [in a new window]   FIG. 3.   Western blot analysis of rAAV virion-packaged proteins. rAAV preparations diluted to 1011 particles/ml were resolved on an SDS-10% polyacrylamide gel and electroblotted onto polyvinylidene difluoride membranes. The hFIX proteins on blots were visualized by the chemiluminescent detection system. Lanes 2 to 4 and 7 to 9 were loaded with 4 or 2 µl of rAAV vectors, respectively. Lanes 1 and 6 were loaded with 8 and 4 ng of purified plasma-derived hFIX protein, respectively. Lanes 2 and 7 were loaded with rAAV/CMVhFIXm1. Lanes 3 and 8 and lanes 4 and 9 were loaded with wild-type AAV or rAAV/CMV--gal, respectively. Lanes 5 and 10 contain the dilution buffer alone. The arrow indicates the position of the hFIX protein (56 kDa).

Integration of the rAAV transgene in cellular genomes does not correlate with transgene expression. One of the major unresolved issues regarding the rAAV transduction mechanism is whether rAAV genome integration into the host cell genome is needed for transgene expression. The human muscle cell model described above provides a powerful system to study the mechanisms governing rAAV transduction, because the cell number (nucleus number) is effectively maintained unchanged upon differentiation and thus any alterations observed in the rAAV genome in these cells can be directly correlated to the changes in transgene expression. Furthermore, it permits us to analyze actively proliferating cells and terminally differentiated cells derived from them.

View larger version (72K): [in this window] [in a new window]   FIG. 4.   Southern blot analysis of DNA isolated from rAAV/CMVhFIXm1-transduced cells. HMW and Hirt DNA were isolated from muscle cells transduced at a MOI of 105 and maintained in F10-based medium (Fig. 2I and J). HMW or Hirt DNA was isolated 4 days p.i. (lanes 2, 6, 9, 12, 15, and 18), 10 days p.i. (lanes 3, 7, 10, 13, 16, and 19), or 19 days p.i. (lanes 4, 8, 11, 14, 17, and 20). Lanes: 2 to 4 and 9 to 11, HMW DNA from cells transduced at the myoblast stage; 6 to 8 and 12 to 14, HMW DNA from cells transduced at the myotube stage; 1 and 5 respective mock-infected control DNA isolated on day 4. HMW DNA (4 µg) was digested with EcoRI and subjected to Southern blot analysis with the CMV promoter probe. Lanes 1 to 8 contain EcoRI-digested HMW DNAs, and lanes 9 to 14 contain undigested HMW DNAs. Arrow a indicates the position of the 1.9-kb internal EcoRI fragment. The possible position for the >12-kb genomic DNA signal, more prominant in Fig. 5, is shown by an asterisk. Lanes 15 to 17 contain Hirt DNA from cells transduced at the myoblast stage; lanes 18 to 20 contain Hirt DNA from cells transduced at the myotube stage. Hirt DNA isolated from cells cultured in the rich medium tends to give less discernible bands, and undigested or digested genomic DNA show bands of >4-kb (lanes 9 to 14) and <1.9 kb (lanes 2 to 4 and 6 to 8), respectively. These bands, however, are not seen when genomic DNA prepared from rAAV-transduced cells was grown in differentiation medium (see Fig. 5). At present, little is known about why genomic DNA from cells maintained in rich medium with much active metabolism tends to have these bands.

View larger version (73K): [in this window] [in a new window]   FIG. 5.   Southern blot analysis of DNA isolated from rAAV/CMVhFIXm1-transduced cells. HMW and Hirt DNA was isolated from muscle cells transduced at a MOI of 105 and maintained in differentiation medium (Fig. 2G and H). HMW or Hirt DNA was isolated 4 days p.i. (lanes 1, 4, 7, 10, 13, and 16), 17 days p.i. (lanes 2, 5, 8, 11, 14, and 17), or 28 days p.i. (lanes 3, 6, 9, 12, 15, and 18). Lanes: 1 to 6, HMW DNA from cells transduced at the myoblast stage; 7 to 12, HMW DNA from cells transduced at the myotube stage. HMW DNA samples (10 µg) were digested with EcoRI and subjected to Southern blot analysis with the CMV promoter probe. The undigested samples are shown in lanes 4 to 6 and 10 to 12. The Hirt DNA profile of rAAV/CMVhFIXm1-transduced cells is shown in lanes 13 to 18. Lanes: 13 to 15, Hirt DNA from cells transduced at the myoblast stage; 16 to 18, Hirt DNA from cells transduced at the myotube stage. Arrow a indicates the position of the 1.9-kb internal EcoRI fragment. Arrow b indicates the single-stranded rAAV genomes, and arrows c, d, and e indicate possible double-stranded forms of rAAV. Determination of which band refers to monomer, dimer, or circular forms has yet to be done. The source of faint bands migrating at <1.9 kb in some digested genomic DNA samples (lanes 1 to 3 and 7 to 9) is not known. Since these <1.9-kb and ~4-kb bands appear resistant, at least partially, to restriction digestion, they may be some trapped single-stranded DNAs.

The HMW DNA fraction contains head-to-tail junctions indicative of integrated and/or concatameric forms of rAAV. The HMW DNA fraction may contain integrated as well as some large concatameric forms of rAAV and/or small amounts of trapped episomal forms. To show if a junction fragment could be released from the HMW DNA, we digested HMW DNA with an enzyme with a single recognition site, BstBI, and analyzed it by Southern blotting using the CMV promoter probe as described above. The head-to-tail, head-to-head, or tail-to-tail junctions should release a 4-, 6.3-, and 1.8-kb fragment, respectively, and the end fragment should be detected as a 3.1-kb band upon BstBI digestion (Fig. 1). As shown in Fig. 6, we detected only the 4-kb fragment, which is indicative of a head-to-tail junction (lanes 2 to 4). Such junctions are most probably released from the integrated and/or cellular genome-associated large concatameric forms of rAAV. Although it may not be significant, the possible presence of some circular episomes trapped in the HMW DNA fraction cannot be ruled out at this stage of study.

View larger version (119K): [in this window] [in a new window]   FIG. 6.   Detection of head-to-tail junctions in HMW DNA. HMW DNA (4 µg) isolated from transduced myotubes (Fig. 2I) were digested with BstBI (lanes 1 to 4) or NotI (lanes 5 to 8) and analyzed by Southern blot analysis with the CMV promoter-specific probe. HMW DNA was isolated 4 days p.i. (lanes 2 and 6), 10 days p.i. (lanes 3 and 7), or 19 days p.i. (lanes 4 and 8). Lanes 1 and 5 contain DNA from mock-infected controls. BstBI recognizes a single site in the vector. The arrow indicates the expected head-to-tail junction. No NotI site exists in the vector. NotI digestion did not release any distinct band and showed a profile similar to that of undigested samples (Fig. 4, lanes 9 to 14).

The conversion of single-stranded vector genomes to double-stranded DNA forms in Hirt DNA, but not the stably integrated form, correlates with the expression of transgene. The slow but linear rise in hFIX level (in transduced myotubes in this study) suggests that the pathway for conversion of the single-stranded input viral genome to the transcriptionally active form may be limited by several steps. The conversion of single-stranded input vector to a double-stranded form is suggested to be one of the primary rate-limiting steps (21, 22). To determine if conversion of the single-stranded input viral genome to episomal forms is responsible for the slow linear rise in transgene expression in myotubes, we analyzed the Hirt DNA fraction. The HMW DNA was isolated by spooling, and the remaining DNA was considered the Hirt fraction (see Materials and Methods). The Hirt DNA was fractionated on an agarose gel and subjected to Southern blot analysis using a CMV promoter-specific probe.

Possible significance of nonintegrated rAAV in transgene expression in muscle. Several reports have concluded that the integrated from of rAAV is responsible for the transgene expression and persistence in muscle (15, 23, 27, 71). In these studies, the head-to-tail concatamers of rAAV often detected by PCR or Southern blotting were taken as indicators of integrated rAAV (15, 23, 27, 71). Miao et al. (44) reached a similar conclusion in studies of transduced mouse liver. Up to 70-copy concatamers of rAAV were found by pulsed-field gel electrophoresis and fluorescent in situ hybridization of metaphase or interphase chromosome in transduced mouse liver, suggesting the presence of an integrated form of rAAV (44). Although no data exist so far, others have raised the possibility that rAAV concatamers are not integrated but are in a tight association with cellular DNA and/or nuclear matrix (2, 26, 67). An expected feature of rAAV genome maturation, which is generally observed, is the gradual loss of single-stranded DNA in transduced mouse muscle (15, 67). Clark et al. (15) observed increasing transgene expression correlating with increasing levels of transgenes in genomic DNA fraction (presumably integrated), whereas Vincent-Lacaze et al. (67) found a somewhat inverse relationship. Such conflicting observations demonstrate the difficulty in performing a reliable analysis of DNA isolated from tissues of animals. The site of rAAV injection and isolation of vector after the animal has aged for a period will introduce numerous variables, including organ growth and the possibility that cells processed for DNA do not truly represent the site of injection.