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Mol Ther. Nov 2009; 17(11): 1888–1896.
Published online Jun 16, 2009. doi:  10.1038/mt.2009.128
PMCID: PMC2835045

A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells

Abstract

Scalable methods of recombinant adeno-associated virus (rAAV) production have gained much recent interest as the field of rAAV-mediated gene therapy approaches the clinic. In particular, the production of rAAV vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of rAAV production. Here, we describe a method for the production of rAAV serotypes 1 and 2 in insect cells using a simplified baculovirus-AAV expression vector system coupled with particle purification via affinity chromatography. The number of separate baculovirus constructs required for rAAV production was reduced by genetically modifying the AAV rep gene to allow expression of the AAV-encoded replication enzymes, Rep78 and Rep52, from a single mRNA species and combining the modified rep gene with an AAV cap gene expression cassette in a single baculovirus construct. Additionally, we describe lysis, binding, and elution conditions compatible with a commercially available affinity medium (AVB Sepharose High Performance) used to purify rAAV particles to near homogeneity in a single chromatography step. Using the described method, we obtained an average yield of 7 × 104 purified rAAV particles per cell (range: 3.7 × 104 to 9.6 × 104) from suspension cultures of recombinant baculovirus–infected insect cells.

Introduction

As the field of recombinant adeno-associated virus (rAAV)–mediated gene therapy progresses, the need for scalable methods of rAAV production becomes of growing importance to the translation of successful preclinical investigations to human clinical trials. Baculovirus-mediated production of rAAV vectors in insect cells is especially well suited for the production of large quantities of rAAV (reviewed in refs. 1 and 2). The baculovirus-insect cell rAAV production strategy takes advantage of the efficiency of viral infection coupled with the high cell density and scalability achievable with Spodoptera frugiperda Sf9 insect cells grown in serum-free suspension culture. Successful baculovirus-mediated production of recombinant AAV vectors in stirred-tank bioreactors and disposable, multi-liter “wave” devices has been described,3,4,5 and rAAV produced via the baculovirus system has been administered in a Phase II human clinical trial for the treatment of lipoprotein lipase deficiency.6

In the baculovirus-mediated rAAV production strategy, as originally configured by Urabe et al.,7 Sf9 insect cells are infected with three different recombinant baculovirus constructs: one recombinant baculovirus, designated Bac-Rep, expresses the major AAV replication enzyme, Rep78, and its amino-truncated form, Rep52, from two separate transcription units via partial duplication of rep coding sequences, a second recombinant baculovirus, designated Bac-VP, expresses the AAV virion coat proteins from a modified AAV cap gene, and a third recombinant baculovirus bears the gene of interest flanked by the AAV inverted terminal repeat (ITR) elements, which provide cis-acting elements required for rescue, replication, and packaging of transgene sequences. Although effective, the Bac-Rep construct demonstrates genetic instability upon serial passage due to tandem duplication of homologous regions of the rep78 and rep52 genes,5,8 thus hindering amplification of Bac-Rep stocks for large-scale rAAV production.

We have sought to simplify the production of rAAV vectors using the baculovirus-mediated production strategy in such a way that would increase stability of rep-expressing baculovirus constructs, reduce the number of separate, recombinant baculoviruses required for AAV production, and increase the overall robustness of the system by maintaining genetic linkage between the AAV rep and cap open reading frames. To achieve this goal, the AAV type 2 rep gene was genetically modified to encode a bifunctional mRNA transcript that directs the synthesis of the AAV Rep78 and Rep52 polypeptides from a single mRNA species via a “leaky scanning” mechanism of translational initiation (reviewed in refs. 9,10,11), thus allowing expression of the AAV Rep and Cap proteins from the same recombinant baculovirus genome without destabilizing intramolecular duplication of rep coding sequences. In the leaky scanning mechanism of translational initiation, 40S ribosomal subunits load onto the capped 5′-end of an mRNA transcript and scan the message in a 5′-to-3′ direction in search of a suitable initiation codon. If a suboptimal translational initiation signal is encountered (e.g., an AUG codon occurring within a weak translational initiation sequence context or a non-AUG codon occurring within a strong translational initiation sequence context), a portion of ribosomal subunits initiate translation at this site, while the remainder of 40S subunits continues scanning to the next translational initiation signal, thus resulting in full-length and specific amino-truncated forms of a given protein. The leaky scanning mechanism was utilized by Urabe et al.7 to achieve stoichiometric expression levels of the three AAV capsid proteins (VP1, VP2, and VP3) from a single species of recombinant baculovirus–encoded cap mRNA, and, more recently, by Hermens et al.12 who have described the use of suboptimal rep78 start codons to mediate leaky scanning of recombinant baculovirus–encoded rep mRNA transcripts. In an alternative approach to obtain expression of overlapping AAV polypeptide sequences in insect cells, Chen13 utilized strategic placement of a synthetic, insect promoter–containing intron to facilitate Rep78/52 and VP1/2/3 expression from either a single recombinant baculovirus containing both intron-modified genes or from separate recombinant baculoviruses.

Consistent with our interest in the development of rAAV-based therapeutics for the treatment of human muscular disorders, particularly Duchenne muscular dystrophy, the AAV type 1 cap gene was chosen for the proof-of-principle characterization of the consolidated rep- and cap-expressing baculovirus construct, as this AAV serotype demonstrates highly efficient transduction of muscle tissue.14,15,16

Results

Modification of the AAV rep gene and consolidation of AAV rep and cap gene expression to a single recombinant baculovirus

To obtain expression of the AAV Rep78 and Rep52 proteins from a single baculovirus construct while avoiding destabilizing genomic duplication of rep coding sequences,5,8 the AAV rep gene was modified to allow expression of the Rep78 and Rep52 polypeptides from a single mRNA species via an mRNA leaky scanning mechanism.9,10,11 The AUG initiation codon of the rep78 open reading frame, the adjacent proline codon, and nine downstream AUG triplets occurring before the start codon of the rep52 open reading frame were altered via synthetic gene synthesis (Figure 1). The rep78 initiation codon and proximal flanking nucleotides were mutated to an inefficient translation initiation signal composed of a CUG triplet presented in the context of a Kozak consensus sequence.17 AUG triplets occurring between the initiation codon of the rep78 open reading frame and the AUG initiation codon of the rep52 open reading frame were altered to bear either a silent mutation (in the case of out-of-frame AUG codons), or to encode a conservative amino acid substitution (in the case of in-frame AUG codons). The modified rep gene along with a serotype-specific AAV cap gene bearing a non-AUG-initiated VP1 open reading frame (see ref. 7) were cloned in opposite transcriptional orientations into a prokaryotic transfer plasmid for bacmid-mediated generation of an Autographa californica multiple nuclear polyhedrosis virus–based chimeric baculovirus-AAV expression vector.

Figure 1
Schematic representation of the rep and cap transcription units within the chimeric baculovirus construct, Bac-RepCap, and modifications to the AAV type 2 rep78 open reading frame. (a) The Rep and Cap proteins of AAV are expressed from divergent baculovirus ...

Analysis of rep and cap gene expression during rAAV production in Sf9 insect cells

To characterize the temporal occurrence and relative abundance of the AAV Rep and Cap proteins during recombinant baculovirus–mediated rAAV production in insect cells using a consolidated rep- and cap-expressing baculovirus construct, a time-course analysis was performed in which Sf9 cells grown in suspension culture were co-infected with Bac-RepCap1, a baculovirus-AAV chimera bearing modified AAV type 2 rep and AAV type 1 cap genes, and Bac-GFP, a recombinant baculovirus bearing an ITR-flanked enhanced green fluorescent protein reporter gene.7 Sf9 cells were sampled at various times postinfection and analyzed for AAV protein expression by western blot analysis (Figure 2). Consistent with transcriptional regulation by the baculovirus polyhedrin promoter, a member of the “very late” temporal class of baculovirus promoters,18 expression of the AAV Rep78 and Rep52 proteins demonstrated a delayed onset (Figure 2a). Minimal levels of Rep78 and Rep52 polypeptides were detected at 24 hours postinfection, but were relatively abundant by the 48-hour time-point, and persisted throughout the remaining 96-hour time-course. The AAV Cap proteins, under the transcriptional control of the baculovirus p10 promoter, demonstrated an earlier onset of expression relative to the AAV Rep proteins. AAV Cap proteins were detectable at 24 hours postinfection, peaked at the 48-hour time-point, and slowly decreased during the remaining 48 hours of the time-course (Figure 2b). The overall progression of baculovirus infection was followed by western blot analysis of the A. californica multiple nuclear polyhedrosis virus capsid protein, VP39, and the ubiquitous cellular protein, β-tubulin (Figure 2c).

Figure 2
Western blot analysis of Rep and Cap protein expression in Sf9 insect cells during Bac-RepCap1-mediated rAAV production. Sf9 insect cells (3.6 × 107) grown in suspension culture were infected with Bac-RepCap1 and Bac-GFP at an MOI of 1 each. Samples ...

Analysis of replicative-form transgene sequences

To determine the ability of the consolidated Bac-RepCap baculovirus construct to mediate rescue and replication of AAV ITR-flanked transgene sequences, Sf9 cells were co-infected with Bac-RepCap1 and Bac-GFP, sampled at 24-hour intervals postinfection, and analyzed for the presence of rAAV replicative-form DNA intermediates by agarose gel electrophoresis and ethidium bromide staining (Figure 3). Concomitant with the appearance of abundant levels of the AAV Rep proteins (see Figure 2), DNA bands consistent with monomeric and dimeric replicative-form rAAV-GFP genomes were first detected at 48 hours postinfection (Figure 3, lane 4). In a control experiment, rescue and replication of rAAV transgene sequences was not observed in the absence of Bac-RapCap1 co-infection (Figure 3, lane 2).

Figure 3
Bac-RepCap-mediated rescue of vector genome sequences. Sf9 insect cell were infected with Bac-RepCap1 and Bac-GFP at an MOI of 1 each. Episomal DNA sequences were isolated from culture samples taken at 24-hour intervals and subjected to electrophoresis ...

Expression stability of the Bac-RepCap construct upon serial passage

Previous reports have noted genetic instability of the first-generation rep-expressing baculovirus construct (Bac-Rep) upon serial passage.5,8 This instability was attributed to duplication of rep coding sequences within a single baculovirus genome.8 To examine the stability of Rep and Cap protein expression mediated by the consolidated Bac-RepCap baculovirus construct, which expresses Rep78 and Rep52 from a single open reading frame without intramolecular duplication of rep coding sequences, Sf9 cells were inoculated with a plaque-titered passage 3 Bac-RepCap1 stock at a multiplicity of infection (MOI) of 0.1 plaque-forming units per cell to generate a P4 stock, which was harvested at 3 days postinfection. The P4 stock was further serially propagated at 3-day intervals by volumetric inoculation (1/100th culture volume) of fresh Sf9 suspension cultures to obtain a total of eight serial passages. Passage samples were analyzed for Rep and Cap protein expression by western blot analysis (Figure 4). Stable expression of the AAV Rep and Cap proteins was observed to passage 7. This level of stability will support sufficient baculovirus stock amplification for an MOI ≥1 inoculation of large-scale, stirred-tank bioreactor preparations of rAAV in insect cells.

Figure 4
Western blot analysis of Rep and Cap protein expression during serial passage. Sf9 insect cells in suspension culture (30 ml volume, 1.2 × 106 cells/ml) were inoculated with a passage 3 (P3) stock of Bac-RepCap1 (MOI = 0.1). At 3-day intervals, ...

Characterization of rAAV production and affinity column purification

To evaluate Bac-RepCap-mediated rAAV production in insect cells and purification of vector particles by AVB Sepharose affinity chromatography, suspension cultures of Sf9 cells were co-infected with Bac-RepCap1 and a recombinant baculovirus bearing an ITR-flanked GFP reporter gene at an MOI of 1 plaque-forming unit/cell for each baculovirus construct (total MOI = 2). Recombinant baculovirus–infected cells were collected at ~ 72 hours postinfection and subjected to detergent extraction. The infected-cell extracts were combined with the corresponding culture medium and treated with Benzonase nuclease to digest non-encapsidated DNA. Nuclease-treated material was loaded onto a 10 mm × 100 mm chromatography column packed with AVB Sepharose High Performance affinity medium. Following a phosphate-buffered saline (PBS, pH 7.4) column wash, bound material was eluted with low-pH glycine–HCl buffer (pH 2.7) and collected as 1-ml fractions into sample tubes containing one-tenth volume neutralization buffer. An example chromatogram of a recombinant AAV-1 preparation is shown in Figure 5. Elution of the affinity column with low-pH glycine–HCl buffer yielded a sharp peak (Figure 5b) that, by peak integration, represented ~0.08% of total UV280-absorbing material. A leading-edge sub-peak was reproducibly observed in fraction 6. Upon examination of this fraction by a variety of methods (data not shown), we were unable to definitively identify a salient feature that distinguishes vector in this fraction from that of the major elution peak.

Figure 5
Affinity chromatography. (a) A 200 ml culture of Bac-RepCap1- and Bac-GFP-infected Sf9 insect cells (2.4 × 108 total cells at the time of inoculation) was processed for affinity chromatography as described in Materials and Methods. Nuclease-treated ...

To characterize further the material eluting from the AVB Sepharose affinity medium, samples of each fraction were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and silver staining (Figure 6a). Three protein bands, corresponding to the theoretical molecular weights of the AAV-1 VP1, -2, and -3 capsid proteins (81.4, 66.2, and 59.6 kd, respectively), represented the majority of the eluting material (Figure 6a, fraction 7), and coincided with the major UV absorption peak of the column chromatogram. Densitometry of the silver-stained gel image indicated a purity of >90%. The peak and shoulder elution fractions (fractions 6–8) represented an ~67-fold volumetric concentration of rAAV-1 particles in a single step. Western blot analysis of a duplicate polyacrylamide gel using an anti-AAV capsid antiserum confirmed the identity of the AAV capsid proteins (Figure 6b). Taken in combination with the peak integration analysis of the chromatogram, these data indicate a one-step bulk purification factor of >1000-fold. In agreement with the protein staining and immunoblot results, quantitative, real-time PCR analysis of the AVB column elution fractions using vector-specific primers mapped the peak of rAAV vector genomes to fraction 7 (Figure 6c), and indicated a nuclease-resistant, genome-containing particle titer of 1.7 × 1013 particles/ml for the peak fraction. To examine the morphology of the rAAV particles, samples of the peak column fraction were negatively stained with uranyl acetate and viewed by transmission electron microscopy. Electron micrographs revealed dense clusters of non-enveloped, icosahedral particles with a diameter of ~20–25 nm that are characteristic of the Parvoviridae family (Figure 6d).

Figure 6
Characterization of baculovirus-produced rAAV-1 purified from insect cell extracts by affinity chromatography. (a) Samples of the crude lysate (0.2 µg), column flow-through material (0.2 µg), and 15 µl of each column elution fraction ...

Repeated preparations of rAAV-GFP produced using the consolidated Bac-RepCap baculovirus system were characterized in terms of yield, concentration, particle-to-transducing unit ratio, and purified particle recovery using a combination of quantitative, real-time PCR analysis of vector genome content and flow cytometric analysis of rAAV-mediated GFP expression in vector-transduced human embryonic kidney cells (HEK-293A cells) (Table 1). An example of rAAV-mediated GFP expression in HEK-293A cells is shown in Figure 7. To examine the utility of the consolidated baculovirus production strategy for AAV serotypes other than type 1, a Bac-RepCap2 baculovirus, which contains the codon-modified AAV type 2 rep gene in combination with an ACG-initiated AAV type 2 cap open reading frame, was constructed and characterized in terms of end-point rAAV production parameters following AVB Sepharose affinity purification (Table 1, preparations 5 and 6). Examining all preparations, vector yield ranged from 7.6 × 1012 to 2.3 × 1013 purified, nuclease-resistant particles with an average production of 7.2 (±2.5) × 104 particles per cell (n = 6).

Figure 7
Transduction of HEK-293A cells. HEK-293A cells in 24-well cluster plates were transduced with various amounts of affinity-purified rAAV-1 and examined by fluorescence microscopy at three days post-transduction. Cell nuclei were stained with Hoechst 33342. ...
Table 1
Yield and recovery of rAAV

Discussion

In this paper, we have described the construction and characterization of second-generation rep- and cap-expressing chimeric baculovirus constructs for the production of rAAV particles in insect cells. Additionally, we have evaluated the utility of a commercially available affinity medium, AVB Sepharose High Performance, for the purification of rAAV virions from insect cell extracts. We have used a “leaky scanning” strategy of translational initiation to facilitate consolidation of individual rep- and cap-expressing baculovirus constructs into a single recombinant baculovirus.

Rep modification

The Rep78 initiation codon and 10 additional downstream triplets within the unique portion of the rep78 open reading frame were mutated to facilitate expression of the Rep78 and Rep52 polypeptides from a single species of bifunctional mRNA transcript. Peabody19 demonstrated that, in reticulocyte lysates, wheat germ extracts, and recombinant simian virus 40–transduced CV1 monkey kidney cells, most triplets differing from the canonical AUG initiation codon by a single nucleotide can function, with varying degrees of efficiency, as start codons when presented in the context of a Kozak consensus sequence (CCG/ACCAUGG; ref. 17). ACG and CUG were found to be among the most efficient non-AUG initiation codons. As demonstrated in this report, a CUG triplet presented in a Kozak sequence context functioned efficiently as a translational initiation codon for the rep78 open reading frame within Sf9 insect cells. In addition to alteration of the rep78 initiation codon, the CCG codon encoding proline at amino acid position 2 of Rep78 was mutated to a GCG (alanine) codon in order to support the Kozak consensus sequence context of the adjacent nonstandard rep78 initiation codon. An alanine residue occurs naturally at the equivalent position of the homologous Rep78 protein of AAV serotype 5 (ref. 20), suggesting that this amino acid substitution is compatible with Rep function. To facilitate efficient ribosomal scanning to the AUG initiation codon of the Rep52-encoding portion of the rep open reading frame, four in-frame and five out-of-frame AUG triplets downstream of the Rep78 initiation codon were altered to contain either a silent mutation in the case of out-of-frame AUG triplets, or to encode a conservative amino acid substitution of leucine for methionine in the case of in-frame AUG triplets. An alignment of the amino acid sequences of the Rep78 equivalents of AAV serotypes 1–8 (data not shown) indicates that, in three of the four instances of methionine replacement within the AAV serotype 2 Rep78 protein (at amino acid positions 43, 91, and 172), a leucine residue occurs naturally at an analogous position within the Rep78 protein of at least one other AAV serotype. In contrast, the methionine residue at amino acid position 103 is invariant among the Rep78 proteins of the AAV serotypes examined; however, as demonstrated in this report, a modified Rep78 protein bearing a leucine substitution at this amino acid position (as well as positions 43, 91, and 172) was able to efficiently support rescue, replication, and packaging of ITR-flanked transgene sequences in insect cells.

Affinity chromatography

Scalable methods of rAAV production require equally scalable methods of rAAV purification. Commonly used ultracentrifugation-based procedures for rAAV particle isolation, such as the use of iodixanol or cesium chloride gradients, are limited by capacity and difficulty of scale-up. Accordingly, liquid chromatography–based protocols for rAAV purification have gained much recent interest. Several reports have described chromatography-based methods for the purification of rAAV particles from mammalian cell extracts,21,22,23,24,25,26,27 and a chromatographic procedure for the purification of rAAV particles from insect cell extracts has recently been described.28 Many of these methods, however, require multiple chromatography steps or are limited in applicability to a subset of AAV serotypes (e.g., those with high affinity for immobilized heparin).

We have evaluated AVB Sepharose High Performance, a commercially available, AAV-specific affinity medium, for the purification of recombinant AAV serotypes 1 and 2 from Sf9 insect cell extracts. Advantages of the AVB Sepharose medium include recognition of a variety of AAV serotypes (regardless of heparin-binding efficiency), a relatively high linear flow-rate (up to 150 cm/hour), and a large vector binding capacity (≥1012 particles/ml of medium).29 One disadvantage, however, is cost of the material which may be significant for quantities of medium sufficient for recovery of rAAV vectors from multi-liter, bioreactor-based preparations which, with use of the baculovirus-AAV expression vector system, can approach 1014 genome-containing particles per liter of culture volume.5

When used in conjunction with recombinant baculovirus–infected insect cell extracts, AVB Sepharose affinity medium demonstrated acceptable levels of rAAV purification, concentration, and recovery. Using binding and elution conditions described in this report, the affinity medium provided a >1000-fold bulk purification of rAAV-1 from insect cell extracts in a single chromatography step, with an average recovery of 49.9%. Use of low-pH glycine–HCl elution buffer provided sharp elution peaks that resulted in purified vector concentrations in the range of 5 × 1012 to 2 × 1013 particles/ml (recovered from 100 to 200 ml suspension cultures of recombinant baculovirus–infected Sf9 cells) without the need for additional concentration steps.

In conclusion, the use of the described recombinant baculovirus-AAV expression vectors in combination with a commercially available AAV-specific affinity medium provides a robust, streamlined, and scalable platform for the production and purification of high-titer rAAV vectors.

Materials and Methods

Cell culture. Suspension cultures of S. frugiperda Sf9 insect cells were maintained with constant orbital agitation at 28 °C in polycarbonate Erlenmeyer flasks (Corning, Corning, NY) containing serum-free HyClone SFX-INSECT medium (HyClone Laboratories, Logan, UT). HEK-293A cells were cultured at 37 °C in a humidified, 5% CO2 atmosphere in tissue culture flasks containing Dulbecco's modified Eagle's medium supplemented with 4.5 g/liter glucose, 100 µg/ml streptomycin, 100 U/ml penicillin G, and 10% (vol/vol) heat-inactivated fetal calf serum.

Plasmid and recombinant baculovirus construction. Recombinant baculoviruses were constructed using the Bac-to-Bac Baculovirus Expression System (Invitrogen, Carlsbad, CA), which uses site-specific transposition to insert transfer vector sequences into an A. californica multiple nuclear polyhedrosis virus–derived bacmid DNA maintained in Escherichia coli strain DH10Bac. Bacmid DNA isolated from amplified bacterial colonies is used to transfect Sf9 cells to generate recombinant baculovirus. Recombinant baculovirus Bac-GFP, which bears a cytomegalovirus immediate early promoter-driven GFP reporter gene flanked by AAV-2 ITRs, has been described previously.7 Recombinant baculovirus Bac-GFP/neo (SR652) was constructed by ligating the 4.4-kbp BglII fragment of pSR460A, which contains a cytomegalovirus promoter–driven GFP reporter gene and simian virus 40 promoter–driven aminoglycoside phosphotransferase (neor) gene flanked by AAV-2 ITRs, between the BbsI–BamHI sites of the transfer vector, pFastBac-1 (Invitrogen). To create a modified AAV-2 rep gene expressing a bifunctional Rep78- and Rep52-encoding mRNA, a synthetic gene fragment (produced by Integrated DNA Technologies, Coralville, IA) containing a codon-modified partial rep78 open reading frame (see Figure 1) was excised from its plasmid backbone as a 0.75-kbp XmaI–BamHI fragment and cloned between the XmaI–BamHI sites of pRep(1-621) (ref. 30) to create pSR645. Plasmid pSR645 thus contains the full-length, codon-modified rep open reading frame in a pGEM-3Z (Promega, Madison, WI) backbone. To construct a consolidated recombinant baculovirus expressing the AAV-2 Rep and AAV-1 Cap proteins from the same baculovirus genome, the 2.3-kbp BamHI–XbaI fragment of pFBAAV1VPm11, which contains an ACG-initiated AAV-1 cap open reading frame, was cloned between the BbsI–NheI sites of pFastBac-Dual (Invitrogen) to create the intermediate plasmid pSR648. The 1.9-kbp BglII–XbaI fragment of pSR645 (above) was cloned between the BamHI–XbaI sites of pSR648 to create the baculovirus transfer vector pSR651. Plasmid pSR651 was used to generate recombinant baculovirus Bac-RepCap1. To create a consolidated baculovirus construct expressing the codon-modified AAV-2 rep gene in combination with an AAV-2 cap expression cassette, an intermediate plasmid, pSR653, was constructed by cloning the 1.9-kbp BglII–XbaI fragment of pSR645 (above) between the BamHI–XbaI sites of pFastBac-Dual. The ACG-initiated AAV-2 cap gene of pFBDVPm11 (ref. 7) was PCR amplified (using primers: 5′-GCCCCCGGGGGATCCTGTTAAGACGGC-3′ and 5′-GCCGCTAGCTTACAGATTACGAGTCAGGTATCTG-3′), digested with NheI–XmaI, and cloned between the NheI–XmaI sites of pSR653, to generate the transfer vector pSR657. Plasmid pSR657 was used to generate recombinant baculovirus Bac-RepCap2. Baculovirus stocks were titered by plaque assay on Sf9 monolayers.

Expression time-course, genome rescue, and stability. To examine Bac-RepCap-mediated expression of the AAV Rep and Cap proteins during rAAV production and to evaluate Rep-mediated rescue of vector genomes, 3.6 × 107 Sf9 suspension cells were collected by low-speed centrifugation and resuspended in a 1.8 ml combined inoculum volume of Bac-RepCap1 and Bac-GFP virus stocks (MOI = 1 for each recombinant baculovirus). The inoculated cells were incubated at room temperature on a rocking platform for 1 hour. The infected cells were then diluted in serum-free medium to a final density of 1.2 × 106 cells/ml and further incubated at 28 °C. Samples (1 ml) were taken at 24-hour intervals. Infected cells were pelleted by brief centrifugation, the medium was discarded, and the cell pellets were stored at −80 °C. For western blot analysis, thawed cell pellets were lysed by the addition of 0.4 ml of 1× NuPAGE sample buffer (Invitrogen), and then processed with a QIAshredder Mini Spin Column (Qiagen, Valencia, CA) to reduce viscosity. Equal volumes were loaded onto polyacrylamide gels for western blot analysis as described below. To analyze rescue of vector genomes, episomal DNA was isolated from thawed, infected-cell pellets using a QIAprep Spin Miniprep Kit (Qiagen) following the manufacturer's suggested protocol for plasmid recovery from bacterial cells. Equal sample volumes were separated on a 0.8% agarose gel in 1× Tris–borate–EDTA buffer. DNA was visualized by ethidium bromide staining. To examine Bac-RepCap stability during serial passage, Sf9 suspension cells (30 ml culture; cell density = 1.2 × 106 cells/ml) were inoculated with a passage 3 stock of Bac-RepCap1 at an MOI of 0.1, and then incubated at 28 °C for an additional 3 days to generate a P4 stock. The P4 stock was serially propagated at 3-day intervals (for a total of eight passages) by volumetric (1:100) inoculation of Sf9 suspension cultures with a previous passage supernatant. Samples (1 ml) were taken at each passage, and the cells were pelleted by brief centrifugation. The infected-cell pellets were lysed by the addition of 1 ml of 1× NuPAGE sample buffer and then processed with a QIAshredder device. Portions of each lysate were passed over a MicroSpin G-25 column (GE Healthcare, Piscataway, NJ), and the total protein content was determined using BCA Protein Assay Reagent (Thermo Scientific, Waltham, MA). Equal amounts of total protein were analyzed by polyacrylamide gel electrophoresis and western blot analysis as described below.

rAAV production in Sf9 cells and purification by affinity chromatography. For affinity column purification of Bac-RepCap1-produced rAAV using AVB Sepharose High Performance medium, 2.4 × 108 Sf9 suspension cells were collected by centrifugation at 300 g for 10 minutes and resuspended in a sufficient combined inoculum volume of Bac-RepCap1 and Bac-GFP virus stocks to yield an MOI of 1 for each recombinant baculovirus construct (total MOI = 2). After 1 hour of incubation at room temperature on a rocking platform, the infected cells were diluted into an Erlenmeyer flask containing 200 ml of serum-free medium (final cell density of 1.2 × 106 cells/ml) and further incubated at 28 °C with constant orbital agitation. Bac-RepCap2-mediated rAAV preparations were performed in a similar fashion, but with 1.2 × 108 total Sf9 cells in a final culture volume of 100 ml.

At ~72 hours postinfection, recombinant baculovirus–infected cells were collected by centrifugation at 300 g for 10 minutes, and the culture medium was retained for further processing. The infected-cell pellet was resuspended in 10 ml of TNT extraction buffer [20 mmol/l Tris–HCL (pH 7.5), 150 mmol/l NaCl, 1% Triton X-100, 10 mmol/l MgCl2], and incubated at room temperature for 10 minutes to lyse the outer cell membrane. Insoluble material was pelleted at 2,100 g for 10 minutes, and the supernatant was added back to the original culture medium. Benzonase nuclease (Sigma, St Louis, MO) was added to a final concentration of 20 U/ml, and the crude material was incubated at 37 °C for 1 hour. Just before column loading, the crude nuclease-treated material was filtered through a 0.2 µm polyethersulfone membrane. Affinity chromatography was performed using an AKTA-FPLC chromatography system equipped with a Tricorn 10/100 column packed with an ~8 ml bed volume of AVB Sepharose High Performance affinity medium (all from GE Healthcare, Piscataway, NJ). The packed column was equilibrated with 1× PBS, pH 7.4 before use. Crude material was loaded in sequential applications using a 50 ml or, in some instances, a 150 ml “Superloop” (GE Healthcare) at a flow rate of 2 ml/min. This flow rate was maintained throughout the chromatographic procedure. The column was washed with PBS (pH 7.4) until the OD280 approached baseline. Bound material was eluted from the column by application of 50 mmol/l glycine–HCl (pH 2.7) elution buffer. Elution fractions (1 ml) were collected into tubes containing 100 µl (one-tenth fraction volume) of 1 mol/l Tris–HCl (pH 8.0) to neutralize the low-pH elution buffer. Fractions were stored at 4 °C for further analysis.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis, western blot analysis, and silver staining. Protein separations were performed using NuPAGE Gel System reagents (Invitrogen). Proteins were separated by electrophoresis on 4–12% NuPAGE bis–Tris polyacrylamide gradient gels using 1× 3-[N-morpholino] propane sulfonic acid–sodium dodecyl sulfate running buffer. For sodium dodecyl sulfate–polyacrylamide gel electrophoresis analyses, affinity column fraction samples were passed over MicroSpin G-25 columns to remove excess glycine. For silver staining, fixed gels were stained using the SilverXpress silver stain kit (Invitrogen) according to the manufacturer's protocol. For western blot analysis, polyacrylamide gels were soaked briefly in 1× NuPAGE Transfer Buffer before electrophoretic transfer to nitrocellulose filters using the iBlot Dry Blotting System (Invitrogen). After transfer, the nitrocellulose filters were blocked for a minimum of 1 hour in PBST (PBS, pH 7.4, with 0.05% Tween-20) containing 5% nonfat dry milk. Blots were incubated with the appropriate primary detection antibody (diluted in PBST-5% nonfat dry milk) for 1.5 hours with gentle agitation, washed three times with PBST, and then incubated an additional 1.5 hours with a horseradish peroxidase–conjugated secondary antibody diluted in PBST-5% nonfat dry milk. After three washes in PBST, the filters were incubated with chemiluminescent detection reagent (SuperSignal West Dura Extended Duration Substrate, Pierce, Rockford, IL) for 3 minutes, covered in plastic and exposed to X-ray film. Filters were stripped for reprobing using Restore Western Blot Stripping Buffer (Pierce) according to the manufacturer's instructions. Rep proteins were detected using an anti-Rep monoclonal antibody (clone 303.9; American Research Products, Belmont, MA) at a 1:200 dilution. An anti-AAV-5 capsid antiserum27 that recognizes multiple AAV serotypes was used at a 1:2,000 to 1:2,500 dilution. A monoclonal antibody recognizing the baculovirus VP39 capsid protein (kind gift of Loy Volkman) and a monoclonal antibody recognizing β-tubulin (Boehringer Mannheim, Mannheim, Germany) were both used at a 1:500 dilution. Secondary detection antibodies were used at a 1:2,000 dilution.

Quantification of rAAV. Nuclease-resistant rAAV genome titers were determined by quantitative, real-time PCR analysis of affinity column fractions using the cytomegalovirus promoter-specific oligonucleotide pair 5′-TCCGCGTTACATAACTTACGG-3′ and 5′-GGGCGTACTTGG CATATGAT-3′. Quantitative, real-time PCR analysis was performed with a primary denaturation step of 3 minutes at 95 °C, followed by 40 cycles of 95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds on an iCycler iQ RT-PCR thermocycler using iQ SYBR Green Supermix reagent (both from BioRad Laboratories, Hercules, CA). Biological titers of rAAV expressing a GFP reporter gene were determined by flow cytometric analysis of serially diluted rAAV on HEK-293A cells in the presence of adenovirus co-infection. Briefly, HEK-293A cells were seeded into 24-well cluster plates at a density of 1 × 105 cells per well and allowed to attach overnight. The next day, the medium was removed and replaced with 0.5 ml per well of Dulbecco's modified Eagle's medium-10% fetal calf serum containing recombinant adenovirus, AdCMVLacZ (Quantum Biotechnologies, Montreal, Canada), diluted to an MOI of 1 plaque-forming unit per cell. The HEK-293A cells were then inoculated with a tenfold serial dilution of rAAV and incubated at 37 °C for 24 hours. The cells were harvested for flow cytometric analysis by first transferring the tissue culture supernatant of each well to individual microfuge tubes, followed by the addition of 0.2 ml of a 0.05% trypsin–0.53 mmol/l EDTA solution to each well and incubation for 5 minutes at 37 °C. The detached cells were removed and added to the corresponding tissue culture supernatant. Each sample was filtered using a 5 ml polystyrene Falcon tube with cell-strainer cap (Becton Dickinson, Franklin Lakes, NJ) before flow cytometry. The number of GFP-positive cells was determined using a Guava EasyCyte flow cytometer (Guava Technologies, Hayward, CA) at an excitation wavelength of 488 nm. For fluorescence microscopy, HEK-293A cells were seeded into 24-well cluster plates at 1.2 × 105 cells per well, allowed to attach overnight, and then inoculated with various amounts of rAAV in the absence of adenovirus. At 3 days post-transduction, cell nuclei were stained by the addition of the cell-permeable dye Hoechst 33342 to the culture medium (5 µmol/l final concentration) followed by incubation at 37 °C for 3 hours. Transduced cells were photographed using a Zeiss Axiovert fluorescence microscope (Carl Zeiss Microimaging, Thornwood, NY) equipped with a digital CCD camera.

Transmission electron microscopy. Before electron microscopy, 50 µl aliquots of the peak AVB column fractions were dialyzed against two 0.5-l volumes of 20 mmol/l bis–Tris (pH 6.0), 10 mmol/l NaCl buffer using a mini-dialysis unit (Slide-A-Lyzer MINI Dialysis unit, 20K MWCO, regenerated cellulose membrane; Thermo Scientific). Two to five microliters of each sample were spotted onto formvar-coated, carbon-stabilized copper grids (200-mesh) that had been plasma-discharged just before use. After 1-minute incubation, the samples were negatively stained by drop-wise application of a 1% uranyl acetate solution. Excess staining solution was removed by adsorption to filter paper, and the samples were allowed to air-dry. Grids were examined using a JEM1200EX transmission electron microscope (JEOL, Tokyo, Japan) at a magnification setting of 50,000× and an accelerating voltage of 80 kV.

Acknowledgments

We thank Loy Volkman for providing the anti-VP39 monoclonal antibody. We also thank Mathew P. Daniels and the National Heart, Lung, and Blood Institute (NHLBI) Electron Microscopy Core Facility for assistance with the transmission electron microscopy procedure. This work was supported by the Intramural Research Program of the NHLBI, National Institutes of Health. The authors declare no conflict of interest.

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