A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells  Richard H Smith, Justin R Levy, Robert M Kotin  Molecular Therapy  Volume 17, Issue 11, Pages 1888-1896 (November 2009) DOI: 10.1038/mt.2009.128 Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

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 late promoters. Messenger RNA transcripts are represented by wavy lines. All pertinent codons are indicated in a left-to-right orientation for convenience. The modified AAV serotype 2 rep gene is under the transcriptional control of the baculovirus polyhedrin promoter (PPh). The bifunctional rep mRNA transcript utilizes a CUG triplet embedded within a Kozak consensus sequence to direct synthesis of Rep78 polypeptides. A portion of ribosomal subunits fail to initiate translation at the nonstandard start codon and scan to the next AUG start codon to initiate translation of Rep52 polypeptides. The AAV cap gene is under the transcriptional control of the baculovirus p10 promoter (PP10). The three capsid proteins (VP1, VP2, and VP3) are translated from a single mRNA species, as described by Urabe et al.7 Sequences directing 3′-mRNA processing are derived from simian virus 40 (SV40 pA) and the herpes simplex virus thymidine kinase gene (HSV tk pA) as indicated. (b) The DNA sequence encoding the amino-terminus of Rep78 is shown. Translation of Rep78 polypeptides initiates at a CUG (leucine) codon placed within the context of a Kozak consensus sequence (specifically gccgccCUGg). A codon encoding proline at position 2 of Rep78 was altered to encode alanine to accommodate the Kozak consensus sequence. Four in-frame and five out-of-frame AUG triplets were altered to yield either a conservative amino acid change or a silent mutation, respectively. Nucleotide changes and amino acid substitutions (highlighted) are indicated by lower-case font. A BglII recognition site used for cloning purposes is underlined. AAV, adeno-associated virus. Molecular Therapy 2009 17, 1888-1896DOI: (10.1038/mt.2009.128) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

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 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. Molecular Therapy 2009 17, 1888-1896DOI: (10.1038/mt.2009.128) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

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 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. Molecular Therapy 2009 17, 1888-1896DOI: (10.1038/mt.2009.128) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

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, 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. Molecular Therapy 2009 17, 1888-1896DOI: (10.1038/mt.2009.128) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

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 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. Molecular Therapy 2009 17, 1888-1896DOI: (10.1038/mt.2009.128) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

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 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. Molecular Therapy 2009 17, 1888-1896DOI: (10.1038/mt.2009.128) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

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. a and b, c and d, and e and f represent wells receiving 0, 2, and 8 µl, respectively, of the peak elution fraction (fraction 7) of the affinity column. Top row, micrographs taken using a GFP-compatible filter set. Bottom row, micrographs taken using a Hoechst 33342-compatible filter set. rAAV, recombinant adeno-associated virus; GFP, green fluorescent protein. Molecular Therapy 2009 17, 1888-1896DOI: (10.1038/mt.2009.128) Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions