A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells
Authors: Richard H Smith, Justin R Levy, Robert M Kotin
Journal: Molecular Therapy (2009)

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. 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. IntroductionAs 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. 1Negrete A Kotin RM Strategies for manufacturing recombinant adeno-associated virus vectors for gene therapy applications exploiting baculovirus technology.Brief Funct Genomic Proteomic. 2008; 7: 303-311Crossref PubMed Scopus (16) Google Scholar and 2Cecchini S Negrete A Kotin RM Toward exascale production of recombinant adeno-associated virus for gene transfer applications.Gene Ther. 2008; 15: 823-830Crossref PubMed Scopus (41) Google Scholar). 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,3Meghrous J Aucoin MG Jacob D Chahal PS Arcand N Kamen AA Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor.Biotechnol Prog. 2005; 21: 154-160Crossref PubMed Scopus (59) Google Scholar,4Negrete A Kotin RM Production of recombinant adeno-associated vectors using two bioreactor configurations at different scales.J Virol Methods. 2007; 145: 155-161Crossref PubMed Scopus (34) Google Scholar,5Negrete A Yang LC Mendez AF Levy JR Kotin RM Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system.J Gene Med. 2007; 9: 938-948Crossref PubMed Scopus (46) Google Scholar and rAAV produced via the baculovirus system has been administered in a Phase II human clinical trial for the treatment of lipoprotein lipase deficiency.6Gaudet D Méthot J Essiembre Brisson D van Deventer S Kleefstra A et al.Biodistribution of AAV1-LPLPS447X vector co-administered with immunosuppression to lipoprotein lipase deficient patients in a phase II study.Mol Ther. 2008; 16 (Abstr. 985): s1-s389Abstract Full Text Full Text PDF PubMed Scopus (29) Google ScholarIn the baculovirus-mediated rAAV production strategy, as originally configured by Urabe et al.,7Urabe M Ding C Kotin RM Insect cells as a factory to produce adeno-associated virus type 2 vectors.Hum Gene Ther. 2002; 13: 1935-1943Crossref PubMed Scopus (351) Google Scholar 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,5Negrete A Yang LC Mendez AF Levy JR Kotin RM Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system.J Gene Med. 2007; 9: 938-948Crossref PubMed Scopus (46) Google Scholar,8Kohlbrenner E Aslanidi G Nash K Shklyaev S Campbell-Thompson M Byrne BJ et al.Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system.Mol Ther. 2005; 12: 1217-1225Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar 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. 9Kozak M The scanning model for translation: an update.J Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2794) Google Scholar,10Kozak M Bifunctional messenger RNAs in eukaryotes.Cell. 1986; 47: 481-483Abstract Full Text PDF PubMed Scopus (243) Google Scholar,11Kozak M Initiation of translation in prokaryotes and eukaryotes.Gene. 1999; 234: 187-208Crossref PubMed Scopus (1121) Google Scholar), 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.7Urabe M Ding C Kotin RM Insect cells as a factory to produce adeno-associated virus type 2 vectors.Hum Gene Ther. 2002; 13: 1935-1943Crossref PubMed Scopus (351) Google Scholar 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.12Hermens WTJMC Haast SJP Biesmans DJ Bakker AC Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV in insect cells. World Intellectual Property Organization, Geneva2007Google Scholar 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, Chen13Chen H Intron splicing-mediated expression of AAV Rep and Cap genes and production of AAV vectors in insect cells.Mol Ther. 2008; 16: 924-930Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar 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.14Xiao W Chirmule N Berta SC McCullough B Gao G Wilson JM Gene therapy vectors based on adeno-associated virus type 1.J Virol. 1999; 73: 3994-4003Crossref PubMed Google Scholar,15Chao H Liu Y Rabinowitz J Li C Samulski RJ Walsh CE Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors.Mol Ther. 2000; 2: 619-623Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar,16Rabinowitz JE Rolling F Li C Conrath H Xiao W Xiao X et al.Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity.J Virol. 2002; 76: 791-801Crossref PubMed Scopus (623) Google ScholarResultsModification of the AAV rep gene and consolidation of AAV rep and cap gene expression to a single recombinant baculovirusTo obtain expression of the AAV Rep78 and Rep52 proteins from a single baculovirus construct while avoiding destabilizing genomic duplication of rep coding sequences,5Negrete A Yang LC Mendez AF Levy JR Kotin RM Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system.J Gene Med. 2007; 9: 938-948Crossref PubMed Scopus (46) Google Scholar,8Kohlbrenner E Aslanidi G Nash K Shklyaev S Campbell-Thompson M Byrne BJ et al.Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system.Mol Ther. 2005; 12: 1217-1225Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar 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.9Kozak M The scanning model for translation: an update.J Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2794) Google Scholar,10Kozak M Bifunctional messenger RNAs in eukaryotes.Cell. 1986; 47: 481-483Abstract Full Text PDF PubMed Scopus (243) Google Scholar,11Kozak M Initiation of translation in prokaryotes and eukaryotes.Gene. 1999; 234: 187-208Crossref PubMed Scopus (1121) Google Scholar 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.17Kozak M Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.Nucleic Acids Res. 1984; 12: 857-872Crossref PubMed Scopus (2378) Google Scholar 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. 7Urabe M Ding C Kotin RM Insect cells as a factory to produce adeno-associated virus type 2 vectors.Hum Gene Ther. 2002; 13: 1935-1943Crossref PubMed Scopus (351) Google Scholar) 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.Analysis of rep and cap gene expression during rAAV production in Sf9 insect cellsTo 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.7Urabe M Ding C Kotin RM Insect cells as a factory to produce adeno-associated virus type 2 vectors.Hum Gene Ther. 2002; 13: 1935-1943Crossref PubMed Scopus (351) Google Scholar 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,18Kelly BJ King LA Possee RD Introduction to baculovirus molecular biology Baculovirus and Insect Cell Expression Protocols. Humana Press, Totowa2007: 25-53Google Scholar 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 2Western 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 were taken at 24-hour intervals and subjected to western blot analysis. (a) Rep protein expression detected with anti-Rep monoclonal antibody 303.9 (b) Cap protein expression detected with an anti-AAV capsid protein rabbit polyclonal antiserum (c) Monoclonal antibody–mediated detection of the baculovirus capsid protein VP39 and the ubiquitous cellular protein β-tubulin. AAV, adeno-associated virus; rAAV, recombinant adeno-associated virus; uninfected, uninfected Sf9 cell control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of replicative-form transgene sequencesTo 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 3Bac-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 on a 0.8% agarose–TBE gel. Replicative-form vector genomic intermediates were visualized by ethidium bromide staining. An image negative is shown. AAV, adeno-associated virus; GFP, green fluorescent protein; hpi, hours postinfection; MOI, multiplicity of infection; RFD, replicative-form AAV-GFP dimer (5.30-kbp); RFM, replicative-form AAV-GFP monomer (2.65-kbp); TBE, Tris–borate–EDTA; uninfected, uninfected Sf9 cell control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression stability of the Bac-RepCap construct upon serial passagePrevious reports have noted genetic instability of the first-generation rep-expressing baculovirus construct (Bac-Rep) upon serial passage.5Negrete A Yang LC Mendez AF Levy JR Kotin RM Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system.J Gene Med. 2007; 9: 938-948Crossref PubMed Scopus (46) Google Scholar,8Kohlbrenner E Aslanidi G Nash K Shklyaev S Campbell-Thompson M Byrne BJ et al.Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system.Mol Ther. 2005; 12: 1217-1225Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar This instability was attributed to duplication of rep coding sequences within a single baculovirus genome.8Kohlbrenner E Aslanidi G Nash K Shklyaev S Campbell-Thompson M Byrne BJ et al.Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system.Mol Ther. 2005; 12: 1217-1225Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar 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 4Western 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, this stock was further passaged by inoculation of fresh Sf9 cells with a 0.01× volume of culture supernatant from the previous passage. Samples were taken at the completion of each passage, and equal amounts of total cellular extract (10 µg) were analyzed by western blot analysis with antibodies to the indicated protein. Passage number is indicated above each lane. Identities of the AAV Rep and Cap proteins are indicated at left. AAV, adeno-associated virus; β-tubulin, endogenous cellular protein; MOI, multiplicity of infection; uninfected, uninfected Sf9 cell extract; VP39, baculovirus-encoded capsid protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of rAAV production and affinity column purificationTo 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 5Affinity 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 material was loaded onto a Tricorn 10/100 column packed with AVB Sepharose High Performance chromatography medium, and the column was washed with 1× phosphate-buffered saline (pH 7.4). Beginning at ~230 ml of total elution volume (x-axis), bound rAAV particles were eluted from the affinity column with the application of 50 mmol/l glycine–HCl buffer (pH 2.7). Glycine buffer-eluted material was fractionated into tubes containing 100 µl of 1 mol/l Tris–HCl (pH 8.0) for elution buffer neutralization. The graph represents the column elution profile as detected by absorbance at 280 nm. (b) Enlarged partial chromatogram showing the rAAV elution peak in relation to the fraction number, which is indicated at the bottom of the graph. rAAV, recombinant adeno-associated virus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 6Characterization 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 were separated by SDS-PAGE on a 4–12% polyacrylamide gradient gel, and the separated proteins were visualized by silver staining. (b) For western blot analysis, a polyacrylamide gel similar to that described above was blotted to nitrocellulose and probed with an anti-AAV capsid protein-specific polyclonal rabbit antiserum. (c) Quantitative, real-time PCR analysis was used to determine the number of nuclease-resistant particles (NRP) in each column fraction. (d) Transmission electron micrograph of rAAV-1 particles eluted within the peak AVB affinity column fraction (fraction 7) and negatively stained with a 1% uranyl acetate solution. Bar = 100 nm. A digital enlargement of a portion of the micrograph (bottom left insert) shows morphological detail. PAGE, polyacrylamide gel electrophoresis; rAAV, recombinant adeno-associated virus; SDS, sodium dodecyl sulfate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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).Table 1Yield and recovery of rAAVPrep. numberSerotypeCells/prep.aAt the time of baculovirus inoculation.YieldbSum of nuclease-resistant particles eluted in peak affinity column fractions (fractions 6-8)., cDetermined by quantitative realtime PCR analysis.NRP/mlcDetermined by quantitative realtime PCR analysis., dPeak fraction from affinity column.Tu/mldPeak fraction from affinity column., eTransducing units of rAAV-GFP per ml determined by flow cytometry of HEK-293A cells transduced with rAAV in the presence of recombinant adenovi