INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Adeno-associated virus (AAV) is a parvovirus and is composed of three structural proteins and a linear, single-stranded DNA (ssDNA) genome of approximately 4.7 kb (17). The particle has icosahedral symmetry and a diameter of 20 to 24 nm. The three capsid proteins of AAV, VP1, VP2, and VP3, have molecular masses of 87, 72, and 62 kDa, respectively, and a ratio of approximately 1:1:10 in the mature particle. All three capsid proteins are encoded by one of the two viral open reading frames, cap, and have overlapping amino acid sequences which differ only in the length of the N terminus. As expected, mutations that affect all three capsid proteins are defective for virus production (8, 28). These mutations are capable of synthesizing double-stranded replication intermediates but are not capable of accumulating ssDNA. Genetic studies also show that in the absence of VP1, VP2 and VP3 can encapsidate progeny ssDNA (8, 28). However, such virions appear to have lower infectivity, suggesting that VP1 is required for stability of the viral particle or for efficient infection. Expression of various combinations of the three capsid proteins in insect cells suggests that VP2 is required for some essential role in the assembly of viruslike empty particles (23). In addition, the major capsid protein, VP3, appears to require the presence of one of the minor capsid proteins for efficient nuclear localization (23). Virus assembly is believed to occur in the nucleus, and immunofluorescence studies indicate that the capsid proteins and the viral nonstructural Rep proteins colocalize in the nucleus (11, 30). Studies of the subcellular distribution of AAV capsid proteins suggest that the three capsid proteins form a variety of complexes with sedimentation coefficients of 10S to 180S, with major peaks at approximately 66S and 110S, the positions of empty and mature virus particles (31). Some of these complexes are associated with the Rep proteins (22, 30, 31).

The Rep proteins are a family of four overlapping proteins encoded by the other viral open reading frame, rep. They are required for AAV DNA replication and for the control of AAV gene expression (17). Genetic and biochemical experiments suggest that the two smaller Rep proteins, Rep52 and Rep40, are not required for viral double-stranded DNA (dsDNA) synthesis but are necessary for the accumulation of ssDNA and virus particles (1a), suggesting that Rep52 and Rep40 are involved in virus assembly.

The linear viral genome contains two inverted terminal repeats (TRs) of 145 bases (13). The TRs contain the viral origin of DNA replication and are required for viral packaging (15). It is not clear which sequences within the TR are required for packaging or what the immediate DNA precursor for packaging is.

In vivo pulse-labeling experiments (18) have suggested that newly synthesized AAV capsid proteins are rapidly assembled into empty capsids, which quickly associate with DNA. These intermediates are then converted to mature virions in a slow process that requires several hours. One of the intermediates identified was a 60S particle whose density was identical to that of mature particles but whose DNA component was sensitive to DNase. Inhibition of DNA synthesis with hydroxyurea inhibited packaging, suggesting that some step in DNA synthesis is necessary for packaging. However, addition of hydroxyurea after pulse-labeling suggested that maturation of AAV particles can occur in the absence of ongoing DNA synthesis.

Viral DNA packaging is an essential step in the virus life cycle, yet our knowledge of the mechanism is limited. To understand the mechanism of virus assembly, it is necessary to develop in vitro systems that are capable of assembling mature virions. Recently, significant progress has been made in the development of an in vitro system for the replication-coupled assembly of poliovirus (16). Here we report on an in vitro system for assembling AAV particles.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Materials. Ribonucleotides, deoxyribonucleotides, creatine phosphate, creatine phosphate kinase, and aphidicolin were purchased from Sigma or Pharmacia. Protein G-Sepharose was from Pharmacia. Ascites preparations of anti-Rep monoclonal antibodies (anti-78/68 and anti-52/40 [11]) and anti-capsid monoclonal antibodies (B1 and A20 [30]) were made by Rockland Inc. and purified on protein G-Sepharose prior to use. The ECL Western immunoblot detection kit was purchased from Amersham and used as suggested by the manufacturer. Guinea pig anti-AAV capsid protein polyclonal antibody was provided by R. J. Samulski (University of North Carolina). Cationic liposomes were prepared and used as previously described (5). Restriction endonucleases were purchased from New England BioLabs.

Viruses, plasmids, and cell culture. 293 cells were maintained in Dulbecco modified Eagle medium containing heat-inactivated calf serum and antibiotics. Only cells that had undergone fewer than 100 passages were used, and they were plated 1 day prior to transfection or infection. Plasmid dl63-87/45 was constructed by digesting pIM29+45, which has been renamed pIM45 (14), with ApaI and religating the resulting larger fragment. pTR_BRLacZ (called pTRLacZ throughout this report) and pAB11 were recombinant AAV (rAAV) vectors which contained the Escherichia coli -galactosidase (-gal) gene (lacZ) under the control of the cytomegalovirus (CMV) immediate-early promoter (see Fig. 1). The two plasmids differ only in that pAB11 is missing an internal PstI site and contains a nuclear localization signal in the coding sequence of its lacZ gene. pAB11 was kindly supplied by R. J. Samulski, and its construction has already been described (6). pTR_BRLacZ contains the 3.7-kb BamHI/HindIII fragment carrying the lacZ coding region from pCH110 (Pharmacia) ligated at the HindIII end to the 0.9-kb BamHI/HindIII CMV promoter fragment form pBS-CMV (Pharmacia) and cloned into the BglII site of pTR_BR (24).

Preparation of cell extracts. Ten 150-mm-diameter plates of 293 cells at approximately 60% confluency were transfected with 20 µg of plasmid DNA per plate by using cationic liposomes and infected with adenovirus type 5 at a multiplicity of infection (MOI) of 5. The cells were harvested at 48 h postinfection and washed with 20 ml of cold phosphate-buffered saline (PBS) and then 10 ml of cold hypotonic buffer (20 mM HEPES [pH 7.4]), 5 mM KCl, 1.5 mM MgCl₂, 1 mM dithiothreitol [DTT]). The cell suspension was centrifuged, and the cell pellet was resuspended in a final volume of 4.8 ml of hypotonic buffer and incubated on ice for 10 min. The cell suspension was Dounce homogenized (20 strokes with a type B pestle), and 0.2 ml of 5 M NaCl was added to raise the NaCl concentration to 0.2 M. The suspension was incubated on ice for 1 h, and the extract was cleared by centrifugation at 15,000 × g for 20 min. After dialysis against a buffer containing 20 mM Tris Cl (pH 7.4), 0.1 mM EDTA, 25 mM NaCl, 10% glycerol, and 1 mM DTT, the extract was stored at 80°C.

Depletion of Rep proteins from cell extracts. Anti-78/68 monoclonal antibody was coupled to protein G-Sepharose as previously described (7). To immunoprecipitate Rep proteins, 3 volumes of cell extract was incubated twice with 1 volume of anti-78/68-protein G-Sepharose beads at 4°C for 1 h with rocking.

Immunoprecipitation of in vitro-synthesized recombinant UF2 virus particles. Monoclonal antibody B1 or A20 (30) was added directly to the products of the in vitro packaging reaction, and the mixture was incubated for 30 min. The immune complexes were then precipitated with a 1:1 mixture of protein A-Sepharose and protein G-Sepharose, and the supernatant was tested for the presence of infectious virus by transduction assay for green fluorescence protein expression as described below.

Western analysis. Ten microliters of each cell extract was electrophoresed on 8 to 14% polyacrylamide gradient gels. Proteins were transferred to nitrocellulose membranes, and the Rep and capsid proteins were detected with anti-52/40 monoclonal antibody or guinea pig anti-capsid polyclonal antibody.

Preparation of pTRLacZ and pTR-UF2 replicative-form (RF) DNAs. 293 cells were cotransfected at 10 µg per 10-cm-diameter plate with pIM45 and pTRLacZ DNAs (3:1) or pTR-UF2 DNA by using cationic liposomes and infected with adenovirus at an MOI of 5. Rescued and replicated AAV(lacZ) or UF2 DNA was extracted 48 h later by using the method of Hirt as previously described (15). Briefly, Hirt supernatant DNA was treated with proteinase K and RNase to inactivate marker proteins and prevent the possibility of the marker protein message being translated in the in vitro packaging reaction. The Hirt supernatant, containing the AAV(lacZ) or UF2 DNA intermediate, was then extracted with phenol and chloroform and precipitated with ethanol to remove any remaining protein and prevent the possibility of pseudotransduction (i.e., marker protein transfer) as described by Alexander et al. (1). The concentration of AAV(lacZ) or UF2 DNA was determined by comparison with known amounts of AAV plasmid DNA following electrophoresis in an agarose gel and staining with ethidium bromide.

In vitro packaging of AAV DNA. The complete reaction mixture contained (in 30 µl) 30 mM HEPES (pH 7.5); 7 mM MgCl₂; 0.5 mM DTT; 0.1 mM each dATP, dGTP, dCTP, and dTTP; 4 mM ATP; 0.2 mM each CTP, GTP, and UTP; 40 mM creatine phosphate; 37.5-µg/ml creatine phosphokinase; 0.17-µg/ml pTRLacZ RF DNA; and 15 µl of cell extract. The reaction mixture was incubated at 37°C for 4 h. The products of the reaction were then incubated at 55°C for 30 min and extracted twice with an equal volume of chloroform, unless otherwise indicated.

Determination of the titer of in vitro-synthesized rAAV(lacZ) or recombinant UF2 virus. The efficiency of the in vitro packaging reaction was assessed by infection of cells with aliquots of the packaged virus. The products of the packaging reaction were added to 293 cells in 96-well plates at 5 × 10⁴ cells per well. The cells were coinfected with adenovirus type 5 to enhance the transient expression of the AAV transgenes (4, 31a). The cells were stained and counted for -gal expression at 48 h postinfection by using 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) as previously described (3) or counted for green fluorescence protein expression by fluorescence microscopy as previously described (32).

Determination of the titer of wild-type AAV by infectious-center assay. The titer of wild-type AAV was determined by the infectious-center assay as previously described (15). Aliquots of virus stocks were used to infect 293 cells in a 96-well plate at a density of 5 × 10⁴ cells per well. The cells were coinfected with adenovirus type 5 at an MOI of 5. After 30 h of incubation at 37°C, the cells were trypsinized and transferred onto nylon membranes with a filtration device. The membranes were wetted for 3 min on 3MM paper saturated with a solution containing 0.5 N NaOH and 1.5 M NaCl. This step was repeated once after the membranes had been blotted dry. The membranes were neutralized with 10 mM Tris Cl (pH 7.5) and 1.5 M NaCl and heated in a microwave oven for 5 min. The membranes then were hybridized with a wild-type AAV probe. Each spot on the membrane hybridizing to the probe represented one cell productively infected by AAV.

Cesium chloride gradient centrifugation. The density of the AAV virions was determined by cesium chloride gradient centrifugation. In vitro- or in vivo-packaged AAV was treated at 55°C for 30 min and then added to 4 ml of a cesium chloride solution with a final refractive index of 1.3720. The solution was centrifuged in an SW50.1 rotor at 40,000 rpm for 20 h at 4°C. Two hundred-microliter fractions were taken from the top of the gradient, and the refractive index of each fraction was determined. Fractions were then dialyzed against PBS, and the titer of wild-type AAV or AAV(lacZ) was determined by the infectious-center assay or by staining for -gal activity, respectively.

Sucrose gradient centrifugation. A linear sucrose gradient of 15 to 30% (wt/wt) was prepared in 10 mM Tris Cl (pH 8.8). Peak fractions from the CsCl gradients were analyzed against PBS, adjusted to 100 ml, and loaded on top of the gradients. The gradients were centrifuged at 110,000 × g for 2.5 h at 20°C. Fractions were collected from the top of the gradients, dialyzed against PBS, and analyzed for wild-type or lacZ mutant virus as described for the CsCl gradients.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Capsid protein-mediated gene transfer. Our approach to the development of an in vitro AAV packaging procedure was to isolate two types of extracts. One of these, the packaging extract, contained all of the AAV-encoded and host cell proteins. The other contained only the AAV RF DNA to be packaged.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Diagrams of pTRLacZ, pAB11, wild-type (wt) AAV, pIM45, and dl63-87. pTRLacZ and pAB11 are recombinant plasmids which contain the lacZ coding sequence under the control of the CMV immediate-early promoter and the simian virus 40 early polyadenylation signal (not shown). pAB11 is different from pTRLacZ in that it is missing a PstI site near the junction of the CMV promoter and the left TR and contains a nuclear localization signal in the lacZ coding sequence. Both plasmids were used to generate substrates for in vitro packaging and contain only the AAV 145-bp TRs. Cut sites of certain restriction endonucleases in pTRLacZ and pAB11 are shown. These restriction fragments were used as substrates for the in vitro packaging experiments described in Table 2. pIM45 and dl63-87/45 were used to generate packaging extracts that contain AAV proteins. dl63-87/45 contains a deletion within the capsid coding region that is indicated by the dotted line. Both pIM45 and dl63-87/45 are missing the 145-bp wild-type AAV TRs and have no homologous overlap with pAB11 and pTRLacZ. The wild-type AAV genome is also shown for comparison. The solid lines indicate vector plasmid sequences.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Western analysis of in vitro packaging cell extracts. Extracts were prepared as described in Materials and Methods from 293 cells infected with adenovirus (Ad) alone or infected with adenovirus plus transfected with either dl63-87 or pIM45 plasmid DNA. Partial depletion of Rep proteins from the pIM45 + Ad extract (Rep-depleted lanes) was accomplished by incubating the extract with mouse anti-78/68 monoclonal antibody coupled to protein G-Sepharose beads. Ten microliters of each extract was electrophoresed on a polyacrylamide gel, transferred to a nitrocellulose membrane, and probed for Rep proteins (left side) by using the mouse anti-52/40 monoclonal antibody, which recognizes all four Rep proteins or for capsid proteins (right side) by using the guinea pig polyclonal anti-capsid antibody, which recognizes all three capsid proteins.

Substrate requirement for in vitro packaging. One criterion for determining whether we had reconstituted a faithful in vitro packaging system is the substrate requirements for encapsidation. The cis-active AAV sequences that are required in vivo for both AAV DNA replication and packaging are contained within the 145-base TRs (2, 15, 26). Thus, if the in vitro packaging reaction faithfully mimicked the authentic in vivo reaction, it should be sensitive to modifications of the TR.

In vitro-synthesized particles had the same density as authentic wild-type AAV. Another criterion for determining whether we had reconstituted a faithful in vitro packaging system was the density of the in vitro-synthesized pTRLacZ particle. The pTRLacZ genome was nearly the same size as the wild-type AAV genome (104% of the size of wild-type AAV). Since the density of the virus particle is a function of the protein and the DNA content of the particle, we predicted that the density of the in vitro-synthesized -gal virus particles should be indistinguishable from that of wild-type AAV. To see if this was the case, we compared the density of in vitro-packaged -gal transducing particles with that of authentic wild-type AAV that had been isolated from a cell culture by fractionating the two kinds of particles in parallel cesium chloride equilibrium density gradients. Both kinds of particles were treated with heat (55°C, 30 min) but not with chloroform. The distribution of wild-type AAV was determined by the infectious-center assay, and the -gal particles were located by staining for -gal activity following infection of 293 cell monolayers. As shown in Fig. 3, both types of particles produced a single peak of activity and the densities of the two types of particles were virtually the same. Both the wild-type and -gal particles had a peak refractive index of 1.3698, equivalent to a density of 1.38 g/ml. Thus, although we could distinguish two kinds of particles among the products of the in vitro reaction, both appeared to have the same complement of protein and DNA, as judged by their density.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Density gradient centrifugation of in vitro-packaged pTRLacZ virus and wild-type AAV packaged in vivo. In vitro-packaged pTRLacZ virus and wild-type AAV produced in vivo were centrifuged in parallel cesium chloride density gradients as described in Materials and Methods. The pTRLacZ virus titer (solid circles) was determined with the -gal staining method; the wild-type AAV titer (open circles) was determined by the infectious-center assay. The virus preparations were heated at 55°C for 30 min prior to centrifugation in CsCl but were not treated with chloroform. ×, refractive index.

Precipitation with structure-specific antibodies. Two monoclonal antibodies are available which can recognize either soluble, unassembled capsid protein (B1) or an epitope that is present only in mature or partially assembled virus particles (A20) (30). When the chloroform-resistant products of the in vitro reaction were treated with the A20 antibody, all of the particles were precipitated (Table 3) or neutralized (data not shown). Treatment with the B1 antibody suggested that a small portion (approximately 30%) of the products were either not completely assembled or had denatured capsid protein associated with them. Control precipitation of purified authentic AAV virions demonstrated that mature particles were completely resistant to B1 antibody. Thus, the bulk of the chloroform-resistant fraction appeared to consist of completely mature virus particles.

Sucrose gradient centrifugation. To compare the sedimentation profile of in vivo-synthesized wild-type particles with the products of the in vitro reaction, we applied the peak fractions of the wild-type and lacZ particles from the cesium chloride gradient shown in Fig. 3 to a 15 to 30% sucrose gradient (Fig. 4). In general, the sedimentation profiles of the two particle preparations were similar. Both contained species that sedimented at the position of mature 110S AAV particles. In addition, both preparations contained material that sedimented with either lower or higher sedimentation coefficients. The higher-molecular-weight species are likely to be aggregates of more than one AAV particle. The lower-molecular-weight species (60S to 110S) were consistent with packaging intermediates found in vivo in previous studies (19, 30).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Sucrose gradient centrifugation of in vitro-packaged pTRLacZ virus and wild-type AAV packaged in vivo. Peak fractions of the in vivo-packaged wild-type virus and the in vitro-packaged pTRLacZ virus obtained from the CsCl gradient shown in Fig. 3 were sedimented in linear 15 to 30% sucrose gradients. Virus titers were determined as described in the legend to Fig. 3. , position of mature 110S AAV particles; ×, refractive index.

In vitro encapsidation requires Rep78 or Rep68 and active DNA synthesis. We and others have shown previously that in vitro AAV DNA replication requires the presence of either Rep78 or Rep68 (10, 12, 20) and that DNA synthesis is inhibited by aphidicolin (15a). In addition, a mutant defective for the synthesis of Rep52 and Rep40 was found to be deficient in the accumulation of ssDNA and virus production, suggesting that the two smaller Rep proteins have a role in packaging (1a). To determine whether Rep78 or Rep 68 is required for in vitro packaging, the Rep proteins were depleted from the packaging extract by immunoprecipitation of the extract with anti-78/68 monoclonal antibody conjugated to protein G-Sepharose beads. This procedure was successful in reducing the Rep78 and Rep68 concentration by approximately 10-fold without significantly affecting the concentration of capsid protein in the extract (Fig. 2). Rep52 and Rep40, which were not recognized by the anti-78/68 antibody, were also partially depleted, presumably due to their interaction with the larger Rep proteins (21). When the depleted extract was tested for packaging activity, it was found to be significantly reduced in activity (approximately fourfold) compared to the complete extract (Table 4). Addition of aphidicolin (10 µg/ml) to reaction mixtures containing the complete extract reduced activity approximately ninefold. Finally, addition of aphidicolin to the depleted extract reduced the packaging activity even further, approximately 20-fold. We also measured the level of DNA synthesis under the conditions of reduced Rep concentration and aphidicolin treatment and found that the level of DNA synthesis was reduced to approximately the same extent as the level of in vitro packaging (data not shown). We concluded from these results that the presence of one or more of the Rep proteins and active DNA synthesis was required for in vitro AAV packaging.

Cofactor requirements for in vitro packaging. The divalent cation Mg²⁺ and ATP were essential for packaging activity (Table 5). Omission of the Mg ion completely eliminated packaging activity, while omission of ATP or the ATP-regenerating system, creatine phosphate and creatine phosphokinase, severely inhibited the packaging reaction (approximately 20-fold). The residual activity seen in the absence of ATP or the regenerating system presumably reflects the fact that the cell-free packaging extract contained substantial amounts of ATP. Since both Mg and ATP are necessary for AAV DNA replication, the requirement for these cofactors was not surprising. This also probably explains the modest decrease in activity seen when the four deoxynucleoside triphosphates were omitted from the reaction mixture (approximately 40%). Again, although the deoxynucleoside triphosphates are essential for DNA replication, the cell-free packaging extract is likely to have contained substantial amounts of these nucleotides. Surprisingly, the reaction was also partially dependent on the presence of the other three ribonucleoside triphosphates, UTP, CTP, and GTP. We previously showed that these are not required for in vitro AAV DNA replication (20). Thus, the reason for this dependence was not clear.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We have demonstrated that it is possible to package in vitro an rAAV genome carrying a marker gene. The particles made in this fashion can be used to transduce mammalian cells in vivo. Taken together, our results suggest that the in vitro reaction produces a mixture of virus particles that consist of both mature virions and partially assembled intermediates. At least two kinds of in vitro products can be formed which facilitate DNA transfer of a marker gene. These can be distinguished by their sensitivity to chloroform and DNase I treatment. The chloroform-resistant product is, by several criteria, an authentic AAV particle. First, the in vitro-synthesized particle is resistant to treatment with chloroform, DNase I and heat. Second, the particle is synthesized only in the presence of AAV capsid proteins. Third, only rAAV that contains an intact terminal repeat is efficiently packaged. This is consistent with in vivo genetic studies that have demonstrated the TR to be a cis-active element required for both DNA replication and viral packaging (2, 15, 26). Fourth, the in vitro packaging reaction requires the presence of one or more of the Rep proteins. This is also consistent with the in vivo genetic data and reflects either a need for Rep78 and Rep68 in DNA replication (20) or a possible direct role of Rep52 and Rep40 in packaging (1a). Fifth, the particles have a density, and therefore a protein-to-DNA ratio, that is identical to that of wild-type AAV. Sixth, at least a portion of the in vitro-synthesized product has a molecular weight that is indistinguishable from that of in vivo-synthesized wild-type virus. Finally, most of the chloroform-resistant particles are bound by an antibody that recognizes mature virus and not bound by an antibody that recognizes monomeric or denatured capsid proteins.

The chloroform-sensitive product can also transfer a marker gene to mammalian cells, but it has several features that suggest that it is either an incompletely assembled virus particle or a DNA molecule that is nonspecifically associated with AAV capsid proteins. As already mentioned, it is sensitive to chloroform and DNase I. In addition, although its formation requires the presence of AAV capsid proteins, it facilitates the transfer of DNA molecules that are much larger than those that can be packaged in authentic AAV particles in vitro. Like many other icosahedral particles, AAV particles can package DNA molecules up to 105% of full-length AAV in size with no significant reduction in viral yield or stability. Yet, the chloroform-sensitive in vitro product facilitated transfer of a molecule nearly two times the size of the normal genome. Finally, the chloroform-sensitive product did not require an intact AAV TR and, thus, appears to be capable of transferring any DNA molecule to mammalian cells.

Although the structure of the chloroform- and DNase I-sensitive -gal transfer products was not clear, the most likely possibility was that these were due to incomplete particle formation or a nonspecific association of capsid proteins with DNA, irrespective of its size or the composition of the TRs. In vivo studies of AAV packaging have identified a number of possible packaging intermediates that are consistent with the size, density, antibody sensitivity, and DNase sensitivity of the in vitro products described here. Wistuba et al. (31) identified a number of different capsid complexes in addition to completely assembled empty particles. Some of these (notably in the 60S-to-110S range) were recognized by monoclonal antibody A20 but not by B1. Myers and Carter (18) found evidence for a partially assembled intermediate of approximately 60S (as opposed to 110S for mature particles) which had the same density as mature particles but contained DNA which was DNase sensitive. It is worth noting that the properties of this intermediate are similar to those of the chloroform- and DNase I-sensitive particles that are made in the in vitro assay described here. We note, however, that although it appears that the chloroform-sensitive particles synthesized in the in vitro reaction have the same density as mature particles, we have not ruled out completely the possibility that such particles are unstable in CsCl and were lost during CsCl centrifugation.

Mechanism of AAV packaging. Relatively little is known about the mechanism of AAV packaging, and it is hoped that the packaging system described here will be useful for studying the mechanism of this process. Only one other in vitro packaging system is available for a eukaryotic virus; Wimmer and his colleagues (16) have demonstrated that poliovirus can be packaged in a coupled in vitro translation-and-replication system.

Potential uses of the in vitro packaging system. We showed previously that AAV can be used to stably transfer foreign DNA to mammalian cells, and we suggested that AAV may be useful for human gene therapy (9). One of the difficulties in applying AAV to gene therapy is the cumbersome method currently used for isolating recombinant AAV stocks (15, 25). In addition to producing rAAV titers that are significantly lower than those of wild-type AAV stocks, the method also produces stocks which are contaminated with adenovirus. Furthermore, all AAV vectors produced in cell culture are potentially prone to contamination with adventitious viruses, as well as wild-type rAAV. The in vitro method described here produces rAAV in a cell-free reaction that contains only one kind of DNA substrate, and therefore, there is no possibility of contamination with adventitious agents, adenovirus, or wild-type rAAV. Although the method produces AAV vectors at a level that is approximately 1/10 of the level currently seen with cell culture techniques, we anticipate that the method can be improved significantly as the individual components involved in the reaction are purified and identified.