Therapy (1998) 5, 415–418  1998 Stockton Press All rights reserved 0969-7128/98 $12.00 BRIEF COMMUNICATION Semliki Forest -based DNA expression vector: transient protein production followed by cell death

A Kohno, N Emi, M Kasai, M Tanimoto and H Saito First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan

We have constructed a novel DNA expression vector The expression level of ␤-galactosidase from pSFV3-CMV- based on Semliki Forest virus (SFV). SFV produces non- lacZ-pA was more than 20-fold higher than that obtained structural proteins (nsPs) which replicate genomic RNA from the plasmid with deleted nsPs , pSFV3A5976- and amplify the mRNA encoding the structural proteins of lacZ, demonstrating that the nsPs genes were essential for SFV. A recombinant cDNA of SFV, in which the the high level of expression. Substantial ␤-galactosidase SFV structural genes were replaced by a polylinker cas- activity was detected in the medium of pSFV3-CMV-lacZ- sette to allow for insertion of heterologous DNA, was pA-transfected cells, suggesting that the overproduction of placed under the control of a cytomegalovirus immediate– ␤-galactosidase caused cell death and release of the pro- early enhancer/promoter with a polyadenylation signal. tein into the medium. We have demonstrated a high-level Transfection of mammalian cells with this SFV-based plas- expression of the exogenous ␤-galactosidase gene mid vector, pSFV3-CMV-lacZ-pA, resulted in transient from pSFV3-CMV-lacZ-pA constructed using an SFV high-level expression of a ␤-galactosidase reporter gene. replication system.

Keywords: self-amplifying vector; Semliki Forest virus; plasmid vector

There are many expression vectors which can be used such constructs also encodes the SFV replicase, high lev- in eukaryotic cells. However, transfection of conventional els of expression of the heterologous gene can be ach- plasmid vectors usually results in only a single or a few ieved by directly transfecting the recombinant RNA into recombinant DNA molecules reaching the nucleus. cells. It was reported that recombinant RNA vectors Consequently, only a limited number of transcripts can based on SFV were transfected with high efficiency into be generated. To increase expression levels, several self- animal tissue culture cells by means of electroporation.4 amplifying systems of gene transfer have been Although this system can be used, the preparation of developed. Some of these self-amplifying systems use capped RNA vectors by in vitro transcription is necessary RNA of , such as Sindbis virus1–3 before transfection and the RNA molecules are unstable and Semliki Forest virus (SFV).4,5 in general. Therefore, it is of great use to construct a DNA SFV, a member of the genus, is a small- vector based on self-amplifying systems of SFV, enveloped virus with a single-stranded RNA genome of especially in consideration of in vivo expression. positive polarity. The 5′ two-thirds of the viral genome In this report, we describe the development of an SFV- encode nonstructural (replication) proteins (nsPs1–4) and derived DNA-based expression vector which can initiate the 3′ one-third encodes structural proteins.6,7 Upon the replication cascade in transfected mammalian cells to infection, the RNA genome functions as mRNA for the produce high-level expression of a reporter gene. Fur- translation of nonstructural proteins. These subsequently thermore, we demonstrate that the transfection of cells replicate the virus by copying the plus-strand RNA gen- with this SFV-based DNA vector results in cell death, ome into minus-strand RNA and vice versa. The minus- along with the release of large amounts of heterologous strand RNA also serves as a template for the synthesis of proteins encoded in the vector. a shorter subgenomic RNA which encodes the structural Plasmid pSFV3 (GIBCO BRL, Grand Island, NY, USA), proteins. Transcription starting at the internal subgen- an SFV-based plasmid vector, contains nsPs genes fol- omic promoter in the minus-strand results in the pro- lowed by a subgenomic RNA promoter and a polylinker duction of large amounts of subgenomic mRNA.6,7 cassette which replaces the structural genes of SFV SFV-derived vectors are based on the insertion of a (Figure 1a).4 The nsPs genes of pSFV are under the tran- genomic SFV cDNA into an SP6 promoter plasmid, and scriptional control of the SP6 promoter; therefore, in vitro subsequent modification by deletion of the SFV structural transcription and capping of the 5′ end are required for genes to allow for the insertion of heterologous DNA as the expression of the gene of interest in mammalian cells. part of the SFV replicon.4 Since the in vitro transcript from To place pSFV3 under the control of a RNA polymerase II promoter, a cytomegalovirus (CMV) immediate–early (IE) enhancer/promoter sequence was inserted upstream Correspondence: A Kohno of the nsP1 gene. In addition, to ensure that mRNA tran- Received 8 May 1997; accepted 27 October 1997 scribed from the plasmid was properly terminated, a SFV-based DNA expression vector A Kohno et al 416

Figure 1 Construction of plasmid vectors. (a) Construction of pSFV3. The promoter sequence for the subgenomic RNA is located at the end of nsP4 gene and the BamHI–SmaI–XmaI polylinker cassette is positioned downstream of the subgenomic promoter. (b) To construct pSFV3-CMV-lacZ-pA, the cytomegalovirus (CMV) immediate–early (IE) enhancer/promoter sequence and the simian virus (SV) 40 late polyadenylation signal sequence were inserted upstream of the nsP1 gene and downstream of the polylinker cassette of pSFV3, respectively (pSFV3-CMV-pA), and the ␤-galactosidase gene was positioned in the polylinker cassette. The CMV-IE enhancer/promoter sequence and the SV40 polyadenylation signal sequence were obtained by polymerase chain reaction using the pCI Mammalian Expression Vector (Promega) as a template. Primers for the amplification of the CMV-IE enhancer/promoter region (CI-CMV1; 5′-ACATGCATGCTCAATATTGGCCATTAGC-3′, CI-CMV2; 5′-ACATGCATGCCTGACTGCGTTAGCAATT- 3′) contain an SphI site, and the PCR product was ligated into the unique SphI site of pSFV3. The primers for the SV40 polyadenylation signal (CI- pA1; 5′-ACTAGTCAGACATGATAAGATACA-3′, CI-pA2; 5′-ACTAGTTACCACATTTGTAGAGGT-3′) contain an SpeI site, and the PCR product was ligated into the unique SpeI site of pSFV3. (c) To delete the nsPs genes from pSFV3-CMV-pA, pSFV3-CMV-pA was digested with AccI, and a 5976 bp fragment was self-ligated to construct pSFV3A5976. The ␤-galactosidase gene was inserted into the polylinker cassette to construct pSFV3A5976-lacZ. SFV-based DNA expression vector A Kohno et al 417 constructed by the deletion of the nsPs genes from pSFV3-CMV-lacZ-pA (Figure 1c). To compare the expression level of lacZ from pSFV3- CMV-lacZ-pA and pSFV3A5976-lacZ, baby hamster kid- ney (BHK) cells were transfected with these plasmid vec- tors as follows. BHK cells were plated into a 24-well plate at a density of 1 × 105 cells per well in 0.5 ml of Iscove’s modified Dulbecco’s medium (IMDM; GIBCO BRL) with 10% (v/v) fetal calf serum (FCS) and incubated at 37°Cin a5%CO2 humidified atmosphere. Eighteen hours later, transfection of pSFV3-CMV-lacZ-pA or pSFV3A5976-lacZ was performed. Starburst polyamidoamine dendrimers (generation 4; Yunitika, Osaka, Japan)8 were used as mediators of DNA transduction. Plasmid DNA was pre- pared by diluting either 3.0 ␮g of pSFV3-CMV-lacZ-pA or 1.8 ␮g of pSFV3A976-lacZ (equivalent number of plas- ␤ mid molecules) in 270 ␮l of plain IMDM per well. Twenty Figure 2 Production of -galactosidase in baby hamster kidney (BHK) ␮ cells transfected with pSFV3-CMV-lacZ-pA or pSFV3A5976-lacZ. micrograms of dendrimers were diluted in 130 lof Quantitative levels of ␤-galactosidase expression were determined in 20 ␮l IMDM and added drop by drop to the DNA solution. of cell lysates (total lysates, 200 ␮l) after the indicated time-periods of The solution was then gently mixed and incubated at culture. ␤-Galactosidase activity was assayed using the ␤-galactosidase room temperature for 15 min. The medium in each well Enzyme Assay System (Promega) according to the manufacturer’s − was removed, cells were rinsed twice with IMDM, and instructions. Each assay included a standard curve of 1 to 5 × 10 3 units ␤ 400 ␮l of the DNA–dendrimer mixture were added to of -galactosidase. Quantitative assay of total protein in cell lysates was ° performed using the Bio-Rad Protein Assay Kit (Bio-Rad, Richmond, CA, each well. After 3 h of incubation at 37 C in a 5% CO2 USA) according to the manufacturer’s instructions. ␤-Galactosidase humidified atmosphere, the DNA–dendrimer mixture activity was normalized to 1.0 ␮g of total protein and plotted on a logar- was replaced by 1 ml of IMDM with 10% FCS. After 24 ithmic scale. The data reflect the results of three similar experiments. and 48 h of culture, a quantitative ␤-galactosidase assay was performed. The expression level of ␤-galactosidase obtained following transduction of pSFV3-CMV-lacZ-pA simian virus (SV) 40 late polyadenylation signal sequence was more than 20-fold higher than that obtained with the was inserted downstream of the polylinker cassette pSFV3A5976-lacZ (Figure 2). (pSFV3-CMV-pA). The CMV-IE enhancer/promoter ␤-Galactosidase expressing cells were visualized by sequence and the SV40 polyadenylation signal sequence staining with X-gal (5-bromo-4-chloro-3-indolyl-␤-d- were obtained by polymerase chain reaction using the galactopyranoside) 24 h after transfection. Briefly, after pCI Mammalian Expression Vector (Promega, Madison, the medium was removed, cells were washed twice with WI, USA) as a template. Plasmid pSFV3-CMV-lacZ-pA phosphate-buffered saline (PBS), fixed with 1.25% glutar- was constructed by the insertion of the E. coli ␤-galacto- aldehyde in PBS, and stained with X-gal solution. sidase gene (lacZ) into the polylinker cassette of pSFV3- Approximately 30 min following the addition of X-gal CMV-pA (Figure 1b). Plasmid pSFV3A5976-lacZ was solution, about 20% of the cells incubated with pSFV3-

Figure 3 Expression of ␤-galactosidase in transfected BHK cells. Cytochemical staining for ␤-galactosidase activity was performed for 30 min at 37°C on BHK cells transfected with pSFV3-CMV-lacZ-pA (a) and with pSFV3A5976-lacZ (b). SFV-based DNA expression vector A Kohno et al 418 of expression with pSFV3-CMV-lacZ-pA was higher than with pSFV3A5976-lacZ (data not shown). This is the first article demonstrating a high level of expression of an exogenous gene by a plasmid vector constructed using a SFV replication system. Recently, Herweijer et al9 and Dubensky et al10 independently reported that they constructed plasmid vectors from , a subgroup III Alphavirus with two envel- ope proteins, as compared with SFV which has three envelope proteins. In their systems as well, they showed 10- to 30-fold higher expression compared with conven- tional methods. Comparison of ␤-galactosidase reporter gene expression between pSFV3-CMV-lacZ-pA and pSFV3A5976-lacZ plas- mids showed that the nsPs genes were essential for high- level expression of ␤-galactosidase. After transfection of pSFV3-CMV-lacZ-pA, an initial plus-strand full-length RNA is transcribed in the nucleus, translocated into the cytoplasm, and then translated into the nsPs of SFV. This ␤ initiates the replication cascade, and consequently, the high- Figure 4 Time-course of -galactosidase activity in cell lysates and cul- ␤ ture medium of pSFV3-CMV-lacZ-pA-transfected cells. The medium in level expression of -galactosidase is achieved. However, each culture was replaced by 1 ml of fresh Iscove’s modified Dulbecco’s protein synthesis in the transfected cell is probably medium (IMDM) with 10% fetal calf serum (FCS) at 12 h for a continued inhibited due to competition for translational machinery 36 h of culture and every 24 h for longer cultures. ␤-Galactosidase activity with self-amplified RNA derived from the plasmid. Protein in cell lysates and culture medium was measured after the indicated time- synthesis in host cells infected by wild-type SFV is inhibited periods of culture. Similar results were obtained in two other experiments. by the competitive production of viral proteins,7 conse- quently leading to cell death. A major advantage of the SFV-derived plasmid vector, pSFV3-CMV-pA, is a high-level expression of an exogen- CMV-lacZ-pA showed a dense blue color, while only a ous gene using the self-amplifying systems of SFV. In limited number of cells incubated with pSFV3A5976-lacZ addition, this vector is transfected into cells as double- demonstrated a very faint color (Figure 3). However, stranded DNA. Therefore, there is no need for in vitro tran- after overnight staining there was no difference in the scription and mRNA capping as required for the transfec- frequency of positively stained cells, and it was estimated tion of previously described SFV-derived RNA vectors. that 25–35% of the cells were positively stained in both pSFV3-CMV-lacZ-pA- and pSFV3A5976-lacZ-transfected cells. Unexpectedly, differences in appearance were observed between the cells transfected with pSFV3-CMV- References lacZ-pA and pSFV3A5976-lacZ. Most of the cells trans- fected with pSFV3A5976-lacZ maintained the same shape 1 Levis R, Huang H, Schlesinger S. Engineered defective inter- fering RNAs of Sindbis virus express bacterial chloramphenicol as untransfected cells, while the cells transfected with acetyltransferase in avian cells. Proc Natl Acad Sci USA 1987; 84: pSFV3-CMV-lacZ-pA showed a condensed round form. ␤ 4811–4815. Figure 4 shows the time-course of -galactosidase 2 Xiong C et al. Sindbis virus: an efficient, broad host range vector activity in cell lysates and culture medium of pSFV3- for gene expression in animal cells. Science 1989; 243: 1188–1191. CMV-lacZ-pA-transfected cells. Transfection was perfor- 3 Johanning FW et al. A sindbis virus mRNA polynucleotide vec- med as described above except that the initial number of tor achieves prolonged and high level heterologous gene × 4 ␤ expression in vivo. Nucleic Acids Res 1995; 23: 1495–1501. BHK cells was 2 10 per well. -Galactosidase activity ¨ in the lysates of the cells transfected with pSFV3-CMV- 4 Liljestrom P, Garoff H. A new generation of animal cell lacZ-pA showed a transient increase with a peak 36 h expression vectors based on the Semliki Forest virus replicon. Bio/Technology 1991; 9: 1356–1361. after transfection. In contrast, ␤-galactosidase activity in 5 Berglund P et al. Semliki Forest virus expression system: pro- the medium began to increase 36 h after transfection and duction of conditionally infectious recombinant particles. showed a delayed peak at 72 h. In the medium of Bio/Technology 1993; 11: 916–920. pSFV3A5976-lacZ-transfected cells, measurable activity 6 Schlesinger S, Schlesinger MJ. Togaviridae: the and their of ␤-galactosidase was not detected (data not shown). ␤- replication. In: Fields BN et al (eds). Fields Virology, 3rd edn. Galactosidase is an intracellular protein and is not Lippincott-Raven: Philadelphia, PA, 1996, pp 825–841. released from cells. The leakage of ␤-galactosidase from 7 Strauss JH, Strauss EG. The alphavirus: gene expression, repli- pSFV3-CMV-lacZ-pA-transfected cells suggests that the cation, and evolution. Microbiol Rev 1994; 58: 491–562. overproduction of ␤-galactosidase resulted in cell death 8 Haensler J, Szoka FC Jr. Polyamidoamine cascade polymers and release of the protein into the medium. Another com- mediate efficient transfection of cells in culture. Bioconjugate Chem 1993; 4: 372–379. parative study of transfection using pSFV3-CMV-lacZ-pA 9 Herweijer H et al. A plasmid-based self-amplifying Sindbis virus and pSFV3A5976-lacZ was done in human 293 cell line. vector. Hum Gene Ther 1995; 6: 1161–1167. Although X-gal staining of transfected cell cultures and 10 Dubensky TW Jr et al. Sindbis virus DNA-based expression vec- ␤-galactosidase assay in cell lysates revealed lower tors: utility for in vitro and in vivo gene transfer. J Virol 1996; 70: expression levels in 293 cells than in BHK cells, the level 508–519.