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Lentiviral Vectors Pseudotyped with Baculovirus Gp64 Efficiently

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Lentiviral Vectors Pseudotyped with Baculovirus Gp64 Efficiently Gene Therapy (2004) 11, 266–275 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00 www.nature.com/gt RESEARCH ARTICLE Lentiviral vectors pseudotyped with baculovirus gp64 efficiently transduce mouse cells in vivo and show tropism restriction against hematopoietic cell types in vitro CA Schauber, MJ Tuerk, CD Pacheco1, PA Escarpe and G Veres Cell Genesys Inc., South San Francisco, CA, USA The envelope glycoprotein from vesicular stomatitis virus found that gp64-pseudotyped lentiviral vectors could effi- (VSV-G) has been used extensively to pseudotype lentiviral ciently transduce a variety of cell lines in vitro, although gp64 vectors, but has several drawbacks including cytotoxicity, showed a more restricted tropism than VSV-G, with potential for priming of immune responses against transgene especially poor ability to transduce hematopoietic cell types products through efficient transduction of antigen-presenting including dendritic cells (DCs). Although we found that gp64- cells (APCs) and sensitivity to inactivation by human pseudotyped vectors are also sensitive to inactivation by complement. As an alternative to VSV-G, we extensively human complement, gp64 nevertheless has advantages characterized lentiviral vectors pseudotyped with the gp64 over VSV-G, because of its lack of cytotoxicity and narrower envelope glycoprotein from baculovirus both in vitro and in tropism. Consequently, gp64 is an attractive alternative to vivo. We demonstrated for the first time that gp64-pseudo- VSV-G because it can efficiently transduce cells in vivo and typed vectors could be delivered efficiently in vivo in mice via may reduce immune responses against the transgene portal vein injection. Following delivery, the efficiency of product or viral vector by avoiding transduction of APCs mouse cell transduction and the transgene expression is such as DCs. comparable to VSV-G-pseudotyped vectors. In addition, we Gene Therapy (2004) 11, 266–275. doi:10.1038/sj.gt.3302170 Keywords: lentivirus; pseudotype; baculovirus; gp64; complement Introduction expression of the gene to specific cell types. Following systemic administration, the VSV-G pseudotyped vectors The G glycoprotein from vesicular stomatitis virus (VSV- transduce target cells of interest, but can also transduce G) has been used extensively for pseudotyping retroviral other cell types that would have undesirable effects in and lentiviral vectors.1,2 VSV-G is advantageous for gene- gene-therapy protocols. For example, antigen-presenting transfer protocols because of its broad species and tissue cells (APCs) could cause deleterious immune responses tropism, and its ability to confer physical stability and if they are transduced inadvertently. Unfortunately, VSV- high infectivity to vector particles.3 However, the high G-pseudotyped lentivectors can efficiently transduce fusogenicity of VSV-G causes rapid syncytia formation APCs from mice and humans, and can elicit immune and cell death, making it difficult to generate stable cell responses against the transgene product, thus potentially lines expressing the protein.4 A number of groups have negating the therapeutic effects of the protein expres- generated stable packaging and producer cell lines using sion.7–10 VSV-G, but in all cases a repressible or inducible Systemic administration of viral vectors also exposes promoter regulates the expression of VSV-G.4–6 While the particles to possible inactivation by serum comple- regulated expression avoids the problem of VSV-G ment.11 Complement inactivation of vectors is dependent cytotoxicity, it makes stable production of viral vectors on the species derivation of the cell line used to produce more complicated and cell line generation more time- the vectors, as well as the identity of the envelope consuming. glycoprotein used for pseudotyping.12,13 VSV-G pseudo- The broad tissue tropism of VSV-G has its disadvan- types, regardless of producer cell type, will be inacti- tages as well. For certain in vivo gene-therapy applica- vated by human serum complement, while vectors tions, it may be important to restrict the transfer and pseudotyped with other envelope glycoproteins such as the MLV amphotropic envelope (Ampho) or RD114 are resistant to inactivation.14,15 Correspondence: PA Escarpe, Cell Genesys Inc., 500 Forbes Blvd. South Owing to the many disadvantages of VSV-G, there is San Francisco, CA 94080, USA 1Department of Neuroscience, University of Michigan, Ann Arbor, MI, considerable interest in examining alternative envelope USA glycoproteins that might have more suitable properties Received 14 May 2003; accepted 03 September 2003 for use with retroviral or lentiviral vectors in gene- gp64-pseudotyped lentiviral vectors in vivo CA Schauber et al 267 transfer applications. Of particular advantage would be transduce three human cell lines. Several independent identification of an envelope that could confer high titer batches of third-generation HIV-1 vector particles carry- in vivo, low cytotoxicity to enable producer cell line ing the gene-encoding green fluorescent protein (GFP) development, resistance to complement and reduced were prepared by transient transfection, and pseudo- ability to transduce APCs. typed with the VSV-G envelope glycoprotein or baculo- We became interested in testing the major envelope virus gp64. The unconcentrated viral supernatants from protein of baculovirus, gp64, for its ability to pseudotype the transfected cells were tested for infectious titer using lentiviral vectors based on the infectivity characteristics 293T, HuH7 human hepatoma and HeLa cells as targets of baculoviral vectors. Baculovirus (Autographa californica (Figure 1a). We found that the gp64-pseudotyped vector nuclear polyhedrosis virus, AcNPV) is an enveloped was infectious in all the three cell lines, with average insect virus that is being developed as a vector for gene titers ranging from 8.1 Â 104 to 1.2 Â 106 transducing therapy.16 Early studies showed that baculovirus vectors units (TU)/ml. The titers of the VSV-G-pseudotyped could efficiently transduce human hepatocyte cell lines vectors ranged from 7.8 Â 105 to 4 Â 106 TU/ml on the and explanted human liver tissue.17,18 Subsequently, it same cell types. The gp64-pseudotyped vector titers were was demonstrated that baculovirus vectors could enter a reduced 25-fold on 293T cells compared to VSV-G, but broader variety of human cell types.19–21 We set out to were only three-fold lower than VSV-G on HuH7 cells. determine whether gp64 could be used to pseudotype The relative titer was the highest on HeLa cells, with other viral vectors and confer high infectivity in cells of gp64 titer (9.3 Â 105 TU/ml) slightly higher than the VSV- hepatic origin, which are primary targets for many gene- G titer (7.8 Â 105 TU/ml). In addition, we found that the therapy applications. Very recently, a report was published that described the use of baculovirus gp64 for pseudotyping HIV-1 a 22 VSVG gp64 vectors. These studies demonstrated that gp64-pseu- 7 dotyped vectors had high titer on several cell lines, and 10 could be concentrated 100-fold by ultracentrifugation. Importantly, it was also shown that gp64 could be 6 constitutively expressed in a stable cell line with no 10 cytotoxic effects.22 The authors showed that this stable cell line could be used to package lentiviral vectors and Titer might enable large-scale production of vectors without 105 regulated expression of the envelope.22 To further explore the utility of gp64 pseudotyping, we carried units/ml) (Transducing out extensive analysis of gp64 for cell and species 4 tropism in vitro, using 24 different target cell lines, and 10 determined the sensitivity of gp64-pseudotyped vectors 293T HeLa 293T HeLa to human complement inactivation. We also scaled up HuH7 HuH7 production of gp64-pseudotyped vectors, and deter- Target cell line mined the binding and elution properties of these vectors b VSVG gp64 during particle purification by anion-exchange chroma- 105 tography. Finally, we carried out two distinct experi- ments to analyze the in vivo transduction efficiency of gp64-pseudotyped vectors. Here we show that gp64 104 confers efficient gene transfer in vivo, and that gp64- pseudotyped vectors are human complement sensitive and have tropism restricted against hematopoietic cells 22 3 including human dendritic cells (DCs). Infectivity 10 Results units/ng p24) (Transducing 102 Baculovirus gp64 envelope protein confers high 293T HuH7 HeLa 293T HuH7 HeLa infectivity to third-generation HIV-1 lentiviral vectors Target cell line Kumar et al22 recently reported that the baculovirus gp64 envelope protein could pseudotype viral vectors based Figure 1 Comparison of VSV-G and gp64 pseudotype titers and on HIV-1. While our vector of choice (also derived from infectivities on three target cell lines. (a) Unconcentrated preparations of third-generation lentiviral vectors carrying a GFP transgene were HIV-1) is slightly different from that used in their produced by transient transfection, and were pseudotyped with either the published studies, we were also interested in using VSV-G (left) or gp64 (right) envelope glycoprotein. These preparations gp64 for pseudotyping, based on the ability of baculo- were assayed for end-point titers on 293T (black bars; n¼3), HuH7 human virus vectors to infect human cell types. Our third- hepatoma (gray bars; n¼5) and HeLa cells (hatched bars; n¼2). Titer generation vector differs in that there are three additional values (TU/ml) were calculated based on % GFP-positive cells, as HIV genes deleted (tat, nef and vpu) and the vector is determined by FACS analysis. Standard deviations of titers determined for 23 independent vector preparations are shown as error bars. (b) To normalize produced through expression of Rev protein in trans. To the TU to total particles in the lentiviral vector preparations, p24Gag confirm that gp64 could also pseudotype this more concentrations in ng/ml were determined by ELISA. Infectivity values advanced HIV vector, pseudotyped lentiviral vector (TU/ng p24Gag) of the preparations were calculated by dividing the titer particles were prepared and tested for their ability to values in (a) in TU/ml by the p24Gag concentrations in ng/ml.
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