An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells

Abstract

Production of clinical-grade gene therapy vectors for human trials remains a major hurdle in advancing cures for a number of otherwise incurable diseases. We describe a system based on a stably transformed insect cell lines harboring helper genes required for vector production. Integrated genes remain silent until the cell is infected with a single baculovirus expression vector (BEV). The induction of expression results from a combination of the amplification of integrated resident genes (up to 1,200 copies per cell) and the enhancement of the expression mediated by the immediate-early trans-regulator 1 (IE-1) encoded by BEV. The integration cassette incorporates an IE-1 binding target sequence from wild-type Autographa californica multiple nuclear polyhedrosis virus, a homologous region 2 (hr2). A feed-forward loop is initiated by one of the induced proteins, Rep78, boosting the amplification of the integrated genes. The system was tested for the coordinated expression of 7 proteins required to package recombinant adeno-associated virus (rAAV)2 and rAAV1. The described arrangement provided high levels of Rep and Cap proteins, thus improving rAAV yield by 10-fold as compared with the previously described baculovirus/rAAV production system.

Fig. 1.

Expression of rep52/78-encoding cassettes. (A) Schematic representation of lane 1, Rep-expression cassettes in recombinant BEV Bac-Rep from Urabe et al. (2); lane 2, transfer plasmid used to derive Bac-Rep; lanes 3–5, plasmid constructs derived for the current project; polh, late polyhedrin promoter; ΔIE-1, attenuated OpMNPV immediate early promoter (33); P19, WT AAV2 P19 promoter; RBE, Rep-binding element (WT AAV2 nucleotides 87–126); leftmost numbers correspond to lanes in B. (B) Western blotting analysis of Rep52/78 proteins in Sf9 cells after transient transfection with various plasmid DNAs. Cells in the lane marked (-) are mock-transfected; lane 1, infected with Bac-Rep (multiplicity of infection, moi, of 5); cells in lanes 2–5 are transfected with the respective plasmids shown in A and infected with Bac-VP (2) (moi of 5) to supply trans-regulator IE-1. (C) Western blotting analysis of Rep proteins extracted from individual stable BS^R cell lines after infection with Bac-VP (moi of 5) and harvested 72 hr postinfection.

Fig. 2.

Analysis of AAV2 P19 promoter activation in Sf9 cells. (A) AAV2 P19 promoter nucleotides are indicated above the sequence. Shaded boxes outline sequences with homology to SP1, GGT, and TATA transcription factor binding elements (9, 25). The P19 transcription initiation sites in mammalian (HeLa) and insect cells (Sf9) are indicated by bent arrows. (B) EMSA assay. 5′-End-labeled 128-bp PCR fragment of P19 promoter (AAV2 nucleotides 704–831, lanes 1–8) and 5′-end-labeled IE-1 consensus-binding element (34) (lanes 9–13) are incubated with crude nuclear extract derived from Sf9 infected with BEV. Samples in the control lanes 1 and 9 contain no nuclear extracts, whereas in lane 2 the probe is incubated with cell extract from uninfected Sf9. In lanes 4 and 5 [³²P]P19 DNA probe is challenged with unlabeled P19 promoter probe (5- and 15-fold excess, respectively); in lanes 6, 7, and 9, with unlabeled IE-1 probe (5-, 25-, and 100-fold excess); in lanes 11, 12, and 13, with unlabeled IE-1 probe (5-, 25-, and 100-fold excess). Different DNA–protein complexes are marked by arrows and labeled with letters. Nonspecific DNA band, a by-product of PCR in lane 1, is marked with *. The presumed IE-1 monomer and dimer/DNA–protein complexes are marked at the right edge of the gel.

Fig. 3.

Expression of AAV2 cap-encoding cassettes. (A) Schematic representation of lane 1, cap expression cassette in BacVP as described by Urabe et al. (2); lane 2, same cassette in a shuttle plasmid backbone; lanes 3–5, plasmid constructs derived for the current project. Genetic element designations are the same as in the legend of Fig. 2. The leftmost numbers correspond to lanes in B. (B) Western blotting analysis of VP proteins in Sf9 cells after transient transfection with various plasmid DNAs. Cells in lane 1 are infected with BacVP (moi of 5), cells in lanes 2–5 are transfected with the respective plasmids (Fig. 3A) and either infected (+) or not infected (−) with Bac-Rep (2) (moi of 5) to supply trans-regulator IE-1 and Rep78. (C) Western blotting analysis of VP proteins extracted from individual stable BS^R cell lines after infection with Bac-Rep (moi of 5) and harvested 72 hr postinfection.

Fig. 4.

Characterization of rep/cap-packaging cell lines F3 and G18. (A) Average yields of purified rAAV-GFP tabulated in vector genomes per cell. Typical run is conducted in a 100-mL suspension culture; cells are infected with BEVs at an moi of 3: for Sf9 cells, with 3 BEV helpers (2); for cap stable cell line, with Bac-Rep and Bac-rAAV-GFP; for rep stable line, with Bac-VP and Bac-rAAV-GFP; for AAV2 (F3) and AAV1 (G18) rep/cap-packaging line, with Bac-rAAV-GFP. rAAV-GFP was purified as described earlier (35). Four runs per cell line had been conducted. (B) Western blotting analysis of AAV2 and AAV1 VP capsid proteins in crude lysates and purified rAAV-GFP. VP protein content in uninfected packaging line F3 (lane F3), F3 infected with Bac-rAAV-GFP (next lane) are compared to Sf9 cells infected with 3 BEV helpers (Sf9/3x). Crude lysates and purified rAAV-GFP are analyzed. (C) Western blotting analysis of Rep proteins in crude lysates in F3, F3 infected with Bac-rAAV-GFP, and Sf9 cells infected with 3 BEVs.

Fig. 5.

Analysis of the rescue and amplification of the integrated rep and cap genes. (A) Southern blotting analysis of the integrated rep and cap genes in F3 (rep/cap) and E5 (cap) stable lines. The parental plasmids are digested with a single cutter (XbaI), per lane amounts loaded are equivalent to 500, 50, or 5 copies of the plasmid DNA in 5 μg of chromosomal DNA. Chromosomal DNA samples from uninfected (5 μg, lanes F3 and E5) or BEV-infected (0.5 μg, lanes F3/BacGFP, E5/BacGFP, and E5/BacRep) are digested with XbaI (a single-cutter, the positions of the XbaI sites are schematically shown below the respective panels), separated in an 1.2% agarose gel, transferred to a nylon filter, and hybridized to ³²P-labeled rep or cap ORF DNA probes (left and right, respectively). White double arrowhead shows the form comigrating with a linearized parent plasmid vector; black double arrowhead indicates a position of a DNA fragment hypothetically derived from the Rep-mediated nicking at the RBE. Rep- and VP-encoding transcripts and their respective ORFs are diagrammed below the integrating cassettes. (B) Diagram depicting a postulated feed-forward loop. The transcription of both integrated rep and cap genes is induced by BEV-encoded IE-1 trans-regulator. One of the products, most likely Rep68/78 protein, returns to interact with RBE, inducing rescue/amplification and mediating the transcription.