DNA AND CELL BIOLOGYVolume 23, Number 5, 2004© Mary Ann Liebert, Inc.Pp. 271–282

Review

Simian Virus-40 as a Gene Therapy Vector

MARIA VERA and PURI FORTES

ABSTRACT

Simian virus-40 (SV40), an icosahedral papovavirus, has recently been modified to serve as a gene deliveryvector. Recombinant SV40 vectors (rSV40) are good candidates for gene transfer, as they display some uniquefeatures: SV40 is a well-known virus, nonreplicative vectors are easy-to-make, and can be produced in titersof 1012 IU/ml. They also efficiently transduce both resting and dividing cells, deliver persistent transgene ex-pression to a wide range of cell types, and are nonimmunogenic. Present disadvantages of rSV40 vectors forgene therapy are a small cloning capacity and the possible risks related to random integration of the viralgenome into the host genome. Considerable efforts have been devoted to modifing this virus and setting upprotocols for viral production. Preliminary therapeutic results obtained both in tissue culture cells and in an-imal models for heritable and acquired diseases indicate that rSV40 vectors are promising gene transfer ve-hicles. This article reviews the work performed with SV40 viruses as recombinant vectors for gene transfer.A summary of the structure, genomic organization, and life cycle of wild-type SV40 viruses is presented. Fur-thermore, the strategies utilized for the development, production, and titering of rSV40 vectors are discussed.Last, the therapeutic applications developed to date are highlighted.

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SIMIAN VIRUS 40

SV40 BELONGS TO THE Polyomaviridae subfamily of Papo-vaviridae, formed by small nonenveloped viruses with

icosahedral capsids. Twelve members of this subfamily havebeen identified. SV40 is the best known, and has been used asa model to analyze several molecular mechanisms (Butel andLednicky, 1999). SV40 virus is formed by an icosahedral cap-sid that packages a single molecule of covalently closed, su-perhelical, doubled-stranded DNA (Cole, 1996).

SV40 virus was first identified as a contaminant of the poliovaccine by screening for viruses in rhesus monkey kidney cellcultures (Hilleman, 1998). The alarm was raised when SV40 wasshown to be able to induce tumors in newborn hamsters and toinfect and immortalize human cells in vitro (Hilleman, 1998).SV40 infection has been related to ependymomas, osteosarcomas,non-Hodgkin’s lymphomas (Malkin, 2002; Shivapurkar et al.,2002; Vilchez et al., 2002) and mesotheliomas of human patientsexposed to asbestos (Carbone, 1999; Bocchetta and Rizzo, 2000).

However, the population vaccinated with polio, and therefore in-fected with SV40, has been studied for years, and a clear link thatcorrelates human tumors with SV40 infection has not been found(Ferber, 2002). Recently, the Immunization Safety Review Com-mittee concluded that “the evidence is inadequate to accept or re-ject a causal relationship between SV40-containing polio vaccinesand cancer” and that “the biological evidence is moderate thatSV40 exposure could lead to cancer in humans under natural con-ditions” [Immunization Safety Review: SV40 Contamination of Polio Vaccine and Cancer (2002). http://www.nap.edu/books/0309086108].

SV40 virion

The SV40 viral capsid, formed by three virus-encoded pro-teins (VP1, VP2, and VP3), packages the SV40 minichromo-some in an icosahedral symmetric structure. One SV40 capsidhas 360 molecules of VP1 arranged as 72 pentamers that facethe outside of the virion. Each VP1 pentamer is associated with

School of Medicine, Foundation for Applied Medical Research, Division of Gene Therapy, Laboratory of Vectors Development, Universityof Navarra, Pamplona, Spain.

either VP2 or VP3, that bridge VP1 to the SV40 DNA (Lid-dington et al., 1991; Cole, 1996). VP1 monomers associate inpentamers via interlocking tertiary structures. VP1 pentamersare tied together through the insertion of carboxyterminal armsinto the cores of neighboring pentamers. Calcium ions are re-quired to stabilize pentamer/pentamer interactions and play arole in virus cell entry, intracellular trafficking, virus uncoat-ing, and capsid assembly (Li et al., 2003). Deletion mapping(Gordon-Shaag et al., 2002) has shown that a region close tothe C-terminus of VP2 or VP3 is joined to the inner cavity ofa VP1 pentamer. This VP15–VP2/VP3 complex is the basicbuilding block of the viral capsid (Sandalon and Oppenheim,1997; Gordon-Shaag et al., 2002).

SV40 genome

The SV40 minichromosome is a circle of doubled-strandedDNA of 5243 bp associated to histones. SV40 genome regula-tory sequences comprise an origin of replication (ori), an en-capsidation signal (ses), and early and late transcriptional pro-moters and enhancers in a region of approximately 500 bp(Oppenheim et al., 1992) (Fig. 1). The origin of replication (coreorigin and two auxiliary elements) binds to the cellular repli-cation machinery (Cole, 1996). The origin is composed of in-verted repeats (IR), a 27-bp sequence repeated four times (27bp 3 4) and a 17-bp AT rich sequence (A/T) (Bergsma et al.,1982) (Fig. 1). The 200-bp encapsidation signal (ses) includespart of the origin of replication, GC-boxes, and 26 bp of theenhancer element and confers specificity for the encapsidationof viral genomes (Oppenheim et al., 1992; 1994; Dalyot-Her-man et al., 1996) (Fig. 1). The transcriptional enhancer isformed by a sequence of 72 bp that has been duplicated in somelaboratory-adapted strains (Wildeman, 1988; Butel and Led-nicky, 1999). Both early and late promoters and polyadenyla-tion sequences are in opposite strands (Fig. 1); thus, the tran-scription extends bidirectionally (Cole, 1996). Transcriptionstarts at the SV40 early promoter that drives the expression ofalternatively spliced mRNAs that encode for the large T anti-gen (Tag), small t antigen (tag), and 17 kT proteins (Fig. 1)(Zerrahn et al., 1993). The 17 kT protein, detected in some Tagexpressing cells, is identical to Tag for the first 131 amino acidsfollowed by four different residues before the stop codon isreached in a different reading frame (Zerrahn et al., 1993; Ray,1995). Tag is essential for regulation of early promoter, acti-vation of replication and late promoter transcription, and virionassembly (Cole, 1996). SV40 late promoter drives the expres-sion of the three structural proteins, VP1, VP2, and VP3, andthe agnoprotein (LP1) (Fig. 1). Alternative splicing producestwo mRNA species that give rise to the four proteins. Transla-tion of VP3 starts in an internal in-frame AUG codon of VP2mRNA, so VP2 and VP3 share the same carboxyterminal do-mains that contain the DNA binding domain and the nuclearlocalization signal (Clever and Kasamatsu, 1991; Clever et al.,1993). LP1 mediates efficient localization of VP1 into the nu-cleus, and facilitates spread of the virus to neighboring cells(Resnick and Shenk, 1986).

SV40 infection cycle

SV40 infects the cell by the caveolar pathway instead of theregular endocytic clathrin-coated vesicle pathway used by most

viruses. SV40 binds the Class-I Major Histocompatibility Com-plex (MHC-I) and specific gangliosides on the cell surface asprimary receptors (Breau et al., 1992; Tsai et al., 2003). SV40associated with flat regions of raft domains, where it transmitsa signal that promotes viral enclosure within caveolar invagi-nations (Dangoria et al., 1996; Chen and Norkin, 1999). Sub-sequently, the virus enters into caveolin-1–containing vesiclesand leaves the plasma membrane to move into caveosomes,larger peripheral organelles with nonacidic pH, where the virusremains for several hours. Virions are then observed in tubularcaveolin-free structures that move along microtubes to thesmooth endoplasmic reticulum (ER) where SV40 is transported(Pelkmans et al., 2001, 2002). Once inside the ER, virion dis-assembly, probably facilitated by ER chaperons, and transport

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FIG. 1. Genomic structure of SV40. Wild-type SV40 genomeis a 5.2-kb double-stranded circularized DNA. Regulatory se-quences are indicated at the top of the figure. The origin of repli-cation is formed by inverted repeats (IR), a 27-bp region repeatedfour times where Tag protein binds (27 3 4) and an AT-rich re-gion (A/T). The transcriptional enhancer contains one 72-bp se-quence that is duplicated in some laboratory-adapted strains. Theencapsidation sequences (ses) include part of the ori and the tran-scriptional enhancer, and a GC-rich region (G/C) contained in3 3 21-bp repeats. The early promoter (EP) is in the AT-rich re-gion, and the late promoter (LP) is downstream to the enhancer.Both promoters and polyadenylation sequences (pA) are in op-posite strands. The SV40 early promoter drives the expression ofthe T antigen alternatively spliced mRNAs that encode for thelarge T antigen (Tag) and small t antigen (tag) proteins. In somecells another alternatively spliced mRNA is produced that en-codes for the 17 kT protein. The large promoter transcribes thecapsid mRNAs that encode for the three structural proteins, VP1,VP2, and VP3, and the agnoprotein (LP1). VP2 and VP3 aretranslated from the same message. Rectangles indicate coding re-gions. Lines indicate introns and noncoding regions. Arrows in-dicate direction of transcription.

to the nucleus occurs (Norkin et al., 2002). SV40 genome is re-leased into the nucleus as a nucleosome-associated minichro-mosome capable of binding the cellular transcription machin-ery. Transcription starts from the early promoter to express theTag genes. Soon the cell nucleus contains high concentrationsof Tag proteins that bind to several regulatory SV40 sequences(Cole, 1996). First, Tag binds to the early promoter sequencesto repress its own transcription (Myers et al., 1981). Then, Tagproteins bind to the 27-bp regions of the origin of replication(Fig. 1, 27 3 4) forming two hexamers capable of recruitingthe cellular replication apparatus and initiating structural alter-ations that result in the opening of the DNA core region (Hur-witz et al., 1990). Each Tag hexamer acts as a DNA helicasethat facilitates replication bidirectionally (Stalh et al., 1986).After replication, Tag activates transcription from the SV40 latepromoter by binding components of the cellular transcriptionmachinery (Gruda et al., 1993). The activated late promoter al-lows expression of capsid proteins that move to the nucleus af-ter being translated. Once in the nucleus, capsid proteins par-ticipate in SV40 DNA nucleosomal rearrangement (Blasquezet al., 1986; Oppenheim et al., 1994). As capsid proteins rec-ognize DNA nonspecifically, specificity is mediated by the tran-scription factor Sp1 that binds the capsid proteins VP1 andVP2/3. Interaction between Sp1 and (VP1)5–VP2/VP3 inhibitsviral promoter activity and allows recruitment of capsid com-plexes to ses (Gordon-Shaag et al., 1998, 2002). There, encap-sidation starts by gradual addition of (VP1)5–VP2/VP3 com-plexes around the SV40 minichromosome (Blasquez et al.,1987). In addition, VP3-activated PARP, a poly (ADP-ribose)polymerase nuclear enzyme, facilitates the release of maturevirions (Gordon-Shaag et al., 2003). VP2 seems to be dispens-able for the formation of infective particles (Gharakhanian etal., 2003).

SV40 normally infects differentiated cells that are growth ar-rested, and these quiescent cells must be stimulated to enter theS phase for efficient viral DNA replication to occur. WhenSV40 infects a cell that is unable to support viral replication,continuous cell division will be activated so cell immortaliza-tion may occur (Carbone et al., 1995). The amount of p53 pres-ent in the host cell could be a determinant of cell transforma-tion, as p53 inhibits SV40 replication by direct interaction withSV40 origin of replication or by blocking the activity of cellu-lar replication factors (Rizzo et al., 2001). In this sense, p53hyperexpression, as in mesothelial cells, aborts replication, andtherefore, SV40 infection could induce cell immortalization(Rizzo et al., 2001). Early genes are responsible for the SV40transformation capacity, as they have to be expressed to induceand maintain the transformation process. Although Tag is suf-ficient for the immortalization of growing cells, efficient trans-formation of growth-arrested cells also depends on the expres-sion of tag (Simmons, 1995). Tag and tag proteins activate cellgrowth by several means. Tag interacts with the DNA bindingregion of p53, blocking cell cycle arrest and apoptosis induc-tion (Pipas and Levine, 2001). Tag activates cell growth medi-ated by E2F by binding to retinoblastoma protein (Rb) or byinducing Rb phosphorylation indirectly (DeCaprio, 1999). Tagalso induces the expression of the insulin-like growth factor I(IGF-I) and requires a functional IGF-I receptor for its growth-stimulating effects (Carbone et al., 1995). Finally, the small tagalso induces cell growth by activating transcription of cyclin A

and altering the substrate specificity of the cellular phosphatasePP2A. PP2A bound to tag increases the activity of MAPK,ERK1, JNK, and of antiapoptotic factors such as AKT (Sontaget al., 1993)

rSV40 VIRUSES AS VECTORS FOR GENE THERAPY

Although SV40 recombinant viruses (rSV40) have been usedas vectors for expression in mammalian cells since the late1970s (Purchio et al., 1979; Elder et al., 1981), rSV40 have re-cently been described as vectors for gene therapy. Several prop-erties make SV40 a good candidate for this purpose: (1) Tagdeletion renders SV40 nonreplicative (Strayer, 2000; Strayer etal., 2001; Arad et al., 2002; Vera et al., submitted); (2) rSV40can be produced in large quantities (Strayer, 2000; Strayer etal., 2001; Arad et al., 2002; Vera et al., submitted); (3) rSV40infects every cell type that has been tested, both dividing andquiescent (Strayer, 1996; Strayer and Milano, 1996; Kimchi-Sarfaty et al., 2002; Strayer et al., 2002b); (4) rSV40 is not im-munogenic, permitting repeated administration in animal mod-els (Strayer and Milano, 1996; Kondo et al., 1998; Strayer andZern, 1999); (5) rSV40 allows long-term expression of thetransgene (Strayer, 1996; Strayer and Milano, 1996; Kondo etal., 1998; Rund et al., 1998; Kimchi-Sarfaty et al., 2002; Strayeret al., 2002b); and (6) the molecular biology of SV40 has beenwell documented (Cole, 1996).

Production of rSV40 virus

Modification of SV40 genome. Tag gene has been removedto generate rSV40 viruses (Strayer, 2000; Strayer et al., 2001;Arad et al., 2002; Vera et al., submitted) (Figs. 2 and 3A). Tagdeletion results in replication-deficient, nononcogenic, and non-immunogenic rSV40 vectors, because the major antigen is notproduced and capsid proteins expression is not activated. Re-moval of the Tag gene also leaves 2.5 kb of available space inSV40 genome, making room to clone exogenous genomic mate-rial. Generation of rSV40 genomes has been performed by sub-stitution of Tag genes by an ampicillin resistance gene (ampr),that allows the rSV40 plasmid to be selected in bacteria andamplified for further modifications (Fig. 2). Different polylink-ers, that share some restriction enzyme target sites, have beencloned at the 59 and 39 ends of the ampr gene. These polylink-ers facilitate both cloning of different transgenes under the con-trol of the early SV40 promoter and the necessary removal ofthe ampr gene. These modifications generate a rSV40 genomecapable of producing rSV40 virus (Fig. 2). Since circular DNAfrom 3.5 kb (Oppenheim and Peleg, 1989) to 5.7 kb (Changand Wilson, 1986) can be encapsidated, transgenes up to 2.5kb can be packaged in Tag-deleted rSV40 vectors, makingtransgene size limitation one of the major drawbacks of the sys-tem.

Different approaches have been developed to overcome thecloning space restriction problem. David Strayer and cowork-ers produced rSV40 vectors without the capsid genes. These“gutless” rSV40 vectors can be obtained with the same proto-col as Tag-deleted vectors, since some packaging cell lines pro-vide in trans both Tag and capsid proteins (see below). Also,

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recombinant adenovirus or wild-type SV40 as helper virusescan provide in trans the capsid proteins for the production ofpseudovirions (Fang et al., 1997; Daylot-Herman et al., 1999).Gutless SV40 can pack transgenes of up to 5 kb (Strayer et al.,2002a). Finally, the group of Ariella Oppenheim has developedan in vitro system to produce rSV40 vectors (see below). En-capsidation can be obtained in the absence of any viral se-quences, and plasmids of 17 kb can be efficiently packaged(Sandalon et al., 1997; Sandalon and Oppenheim 1997, 2001;Rund et al., 1998; Kimchi-Sarfaty et al., 2002, 2003).

When packaging cell lines are used (see below), Tag deletedSV40 vectors should keep the early promoter sequences sinceremoval of the early promoter may affect the regulatory se-quences and the replication of the vector (Fig. 1). When theearly promoter sequences are present, they have been found todrive the expression of a transgene in every cell type tested(Kramer et al., 2002). Alternatively, a foreign promoter clonedin tandem downstream of the early promoter can drive trans-gene expression. In this case, polyadenylation sequences areusually inserted downstream of the early promoter to insulatethe introduced promoter. In this manner, polymerase II pro-moters such as CMV (Jayan et al., 2001; Strayer et al., 2001),

and also other promoters such as methionine tRNA or U6snRNA promoters have been reported to drive transgene ex-pression in rSV40 (Jayan et al., 2001; Strayer et al., 2001). Inaddition, conditional promoters such as HIV LTR can be usedso transgene expression is only obtained in HIV-infected cells(Jayan et al., 2001; Cordelier et al., 2002, 2003a).

Curiously, Tag deleted rSV40 viruses cannot support theexpression of some reporter genes. Different laboratories havefailed to produce rSV40 viruses that express the green fluo-rescent protein, the red fluorescent protein, or the thymidinekinase protein, (Oppenheim and Strayer, personal communi-cation and our unpublished results). It has been suggested thatCpG methylation could be responsible for the lack of expres-sion of these reporter genes, as they are rich in CpG islands,while the SV40 genome is not (Strayer, personal communi-cation). However, rSV40 viruses expressing GFP can be ob-tained with the in vitro packaging system (Kimchi-Sarfaty etal., 2002).

Methods of producing rSV40 vectors. The methods of pro-ducing rSV40 vectors can be divided in those that use a pack-aging cell line (Fig. 3A) or the in vitro packaging system (Fig.

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FIG. 2. rSV40 viruses genome generation. (1) Generation of rSV40 genomes is performed by substitution of Tag genes by anampicillin resistance gene (ampr) (prSVAmp). (2) Polylinkers have been cloned at both sides of the ampr gene (prSVAmpp). (3)Transgenes (X) are cloned under the control of the early SV40 promoter (prSVXAmpp). (4) Removal of the ampr gene and re-circularization of the DNA generates an rSV40 plasmid (prSVX).

3B). Three different protocols used to produce rSV40 vectorsusing a packaging cell line have been described (Strayer, 2000;Arad et al., 2002; Vera et al., submitted). These protocols aresimilar in that a packaging cell line that provides Tag in transmust be transfected with a Tag deleted SV40 plasmid (Fig. 3A).Viruses released from these cells are collected and used to in-fect fresh cells. Several rounds of infection should be performeduntil high yields and good quality recombinant vectors are ob-tained (Fig. 3A). The main differences among the reportedmethods are: the packaging cell line used, the time allowed forvirus production in the packaging cell line and the number ofinfection rounds for virus amplification.

COS-1 and COS-7 cell lines are commonly used for rSV40production, as they constitutively express Tag. COS cells werederived from the monkey kidney fibroblast cell line CV1. It wasdescribed that COS-1 and COS-7 have integrated in theirgenome 1 copy or five to seven copies, respectively, of SV40genome mutated at the origin of replication (Gluzman, 1981).However, recent analysis indicates that some stocks of COS-7

contain one to two copies of Tag sequences per cell (Strayer,personal communication), while others have three to four copiesthat may increase up to 12 copies upon cell passage (our un-published observations). Homologous recombination betweenintroduced vectors and endogenous SV40 sequences in COScells can occur. The more copies of Tag gene present in thepackaging cell, the higher the risk of homologous recombina-tion that can generate replication competent wild-type SV40 re-vertants (Jasin et al., 1985; Arad et al., 2002; Vera et al., sub-mitted). To overcome the problem of wild-type SV40contamination, the CMT4 cell line was developed (Gerard andGluzman, 1985). CMT4 cells lack SV40 promoter sequencesas they express Tag under the control of the mice metallo-thionein promoter. However, CMT4 cells yield low titers ofrSV40 vectors. Recently, a new packaging cell line, calledCOT18, has been developed. COT18 is similar to CMT4, butSV40 sequences common to the rSV40 DNA have been reducedto the early 39UTR, drastically decreasing the probability of ho-mologous recombination (Arad et al., 2002). However, the

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FIG. 3. Methods of producing rSV40 vectors. (A) Production of rSV40 viruses in a packaging cell line. 1. The packaging cellsare transfected with an rSV40 plasmid. 2. A few days after transfection, viruses are collected from transfected cells and super-natants. This crude lysate is used to infect new packaging cells. 3. rSV40 vectors are amplified by performing several rounds ofinfection. 4. When an optimal amount and quality of the rSV40 vector is achieved, viruses are purified by sucrose gradient ul-tracentrifugation. (B) rSV40 vectors production in vitro. 1. The three capsid proteins or only VP1 are purified from baculovirusinfected Sf9 insect cells and capsids are formed. 2. The plasmid with the transgene sequence is amplified in E. coli. 3. Incuba-tion of purified capsids with naked DNA allows the formation of infective competent rSV40 viruses.

rSV40 titers obtained using this packaging cell line are of only107 IU/ml (Arad et al., 2002). The reason for this low titer maybe attributed to the formation of defective interfering particles(DI particles) that affect genome replication and therefore limitvirus passage. DI particles are small pieces of SV40 genomecapable of being encapsidated into SV40 virions and that showreplication advantages over the full-length SV40 genomes. Highconcentrations of DI particles interfere with full-length genomereplication and therefore decrease full-length recombinant virustiter. The presence of DI particles is also detected after threerounds of amplification in COS-1 cells, when each amplifica-tion is only allowed to proceed for 3 days. Under these condi-tions titers of 1011–1012 infective virus/ml are obtained (Veraet al., submitted). Furthermore, quality controls using quanti-tative real-time PCR indicate that revertant wild-type SV40viruses are not detected, and that the number of rSV40 parti-cles is similar to the number of infective viruses (Vera et al.,submitted). Repeating rounds of amplification increases the riskof wild-type SV40 formation and decreases virus titer, proba-bly because the high concentration of DI particles starts to af-fect replication.

The group of Ariella Oppenheim developed an in vitro sys-tem to package recombinant DNA into SV40 capsids (Fig. 3B).In the in vitro packaging system, capside proteins are expressedin baculovirus infected Sf9 insect cells. Incubation of VP1 aloneor in combination with VP2 and VP3 proteins can form cap-sids. Incubation of capsids with naked DNA, amplified in Escherichia coli, allows the formation of pseudovirions (San-dalon et al., 1997; Sandalon and Oppenheim 1997, 2001; Rundet al., 1998; Kimchi-Sarfaty et al., 2002, 2003). This methodhas several advantages. First, plasmids, as large as 17 kb canbe efficiently packaged in VP1 capsides, which seem to be moreflexible than capsids produced from the three VP proteins (Kimchi-Sarfaty et al., 2003). Second, wild-type SV40 se-quences are not required for in vitro production of pseudoviri-ons, so wild-type revertants cannot arise, thus increasing thesafety of the system. Furthermore, as replication occurs in bac-teria, DNA quality can be easily tested, and DI particles are notselected (Sandalon et al., 1997; Rund et al., 1998; Kimchi-Sar-faty et al., 2002). Finally, any sequence that could affect SV40replication can be included and any gene (including reportergenes) can be expressed from SV40 vectors packaged in vitro(Sandalon et al., 1997; Kimchi-Sarfaty et al., 2002). The con-clusion is that these vectors combine the advantages of SV40as a viral gene delivery vehicle and the safety of nonviral vec-tors. However, low yields are obtained with this method: 106

IU/ml compared to 1012 IU/ml using the in vivo packaging sys-tem (Sandalon et al., 1997; Rund et al., 1998; Arad et al., 2002;Kimchi-Sarfaty et al., 2002).

Regardless of the method used for their production, recom-binant vectors need to be purified for further applications. First,rSV40 vectors packaged in cells are separated from membranesin a cell lysate by treatment with detergents. Vectors are thenpurified by sucrose gradient ultracentrifugation and dialyzedagainst sterile PBS (Rosenberg et al., 1981; Strayer, 2000; Veraet al., submitted). Vectors produced in vitro lose activity uponpurification for reasons that are currently under investigation(Kimchi-Sarfaty and Oppenheim, personal communication).

There is no standard method for determining SV40 vectortiter. The titration of wild-type SV40 was performed for many

years by scoring plaques (McCutchun and Pagano, 1968). Scor-ing of infective centers (Rosenberg et al., 1981) can be appliedin the case of origin of replication-containing rSV40 vectors incells that support their replication, such as COS, CMT4, orCOT18 cells (Arad et al., 2002). There is a perfect correlationwith luciferase-expressing SV40 vectors packaged in vitro be-tween infectious centers and luciferase activity measured bylimiting dilutions. However, infectious centers may also formout of wild-type revertants in rSV40 vectors packaged in celllines (Oppenheim, personal communication). More recently,titration of rSV40 vectors by in situ PCR has been described(Strayer et al., 1997; Strayer, 2000; Strayer et al., 2001). Thistechnique amplifies the 59-end of capsid genes in permissiveinfected cells that do not allow replication. Again, with thismethod, not only infective rSV40 vectors but also DI particlesand emerging wild-type revertant SV40 viruses are beingtitered. We have also obtained a good correlation between insitu PCR of infected cells and quantitative real-time PCR ofpurified vector particles with primers that amplify the structuralgenes or the transgene (Vera et al., submitted). Probably thebest titration method would be to check for transgene expres-sion in infected cells. However, this is not possible for all trans-genes. Results from the three methods described, that is, infec-tive centers, in situ PCR and quantitative real-time PCR shouldbe compared to obtain a reliable estimate of the titer of trans-gene containing infectious virus and to control the quality ofviral stocks in terms of revertant wild-type viruses and DI par-ticles.

rSV40 vectors mediated gene transfer

rSV40 vectors are able to deliver transgenes to many cellsfrom different species, because viral entry is mediated by MHC-I, a widely expressed protein. Also, rSV40 can productively in-fect quiescent as well as dividing cells, as disassembly of thenuclear membrane is not required for SV40 entry into the nu-cleus. In addition, rSV40 vectors are poor immunogens for sev-eral reasons: MHC I does not enter the cell with SV40, and itis transiently shed (Anderson et al., 1998); SV40 entry into thecell avoids the endocytic–phagocytic pathway, which involveslysosomal processing of virus antigens; and the absence of Tag,the major antigen protein, does not allow transcription of thecapsid gene. These advantages have encouraged several groupsto study rSV40 vector efficacy in gene therapy models.

In vitro applications. rSV40 vectors can infect and expressfunctional transgenes in different cell lines. All tested tissueculture cells infected with rSV40 vectors express transgenes todifferent levels (Vera, unpublished results). Also, both intra-cellular and secreted functional proteins can be produced fromcells infected by rSV40 vectors (Vera et al., manuscript inpreparation). It has been recently shown that Hepatitis B virusinfected cells are transduced more efficiently by SV40 vectorsthat the parental uninfected cells, suggesting that SV40 vectorscould be useful in the treatment of HBV infections using genetherapy (Arad et al., in press).

rSV40 viruses successfully infect human T lymphocytes invitro. Using this property, rSV40 vectors were designed to tar-get HIV-1 life cycle in both lymphocyte-derived cell lines andprimary human lymphocyte cultures. rSV40 expressing thera-

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peutic transgenes that have been successful in interfering withHIV-1 replication include: (1) single-chain antibodies (SChAb)against HIV-1 integrase (BouHamdan et al., 1999; Strayer etal., 2002a; Goldstein et al., 2002); (2) SChAb against CXCR4,the principal coreceptor of HIV in T-cells (BouHamdan et al.,2001; Strayer et al., 2002a); (3) SChAb against HIV-1 reversetranscriptase (RT) (Strayer et al., 2002a); (4) a dominant neg-ative mutant of HIV-I Rev (RevM10) (Strayer et al., 2002a;Cordelier et al., 2003b); (5) polymeric TAR decoys that bindTAR RNA structure and inhibit transactivation (Jayan et al.,2001; Strayer et al., 2002a); (6) the human interferon alpha 2(INFa2) (Cordelier et al., 2002, 2003a); and (7) the human al-pha-1 antitrypsine gene (a1-AT) that inhibits gp160 and p55processing by different mechanisms (Cordelier et al., 2003b,2003c). Several combinations of some of these therapeutic vec-tors have shown a synergistic inhibition of HIV-1 replication(Strayer et al., 2002a). Furthermore, some of these vectors weresuccessfully tested in terminally differentiated human-derivedneurons, the principal HIV reservoir (Cordelier et al., 2003a;2003c).

rSV40 vectors also efficiently transduce human haematopoi-etic cells (Oppenheim et al., 1986; Rund et al., 1998; Strayer

et al., 2000; Kimchi-Sarfaty et al., 2002). Selection of infectedcells is not required, since more than 95% of fresh human andsimian hematopoietic progenitor cells are infected in vitro byrSV40 vectors. A COS-1 packaged rSV40 pseudovirus, ex-pressing the human b-globin gene, has been used to infect pri-mary human erythroid progenitors from peripheral blood ofb(0)-thalassemia patients. After infection, almost normal lev-els of beta-globin mRNA gene were detected, suggesting thatrSV40 vectors could be used in the treatment of b-thalassemia(Daylot-Herman et al., 1999). Also, SV40 pseudoviruses ex-pressing multidrug resistance gene 1 (SVMDR1) have been pro-duced, with the aim of reducing toxicity to anticancer drugs inpatients undergoing chemotherapy. SVMDR1 were first pro-duced in COS-1 or CMT4 cells (Rund et al. 1998). The effi-ciency of infection of primary human bone marrow cells wasvery high as MDR-1 was expressed in 95% of the cells. Long-term expression was observed in MEL and 3T3 SVMDR1 in-fected cells in the absence of selection (Rund et al., 1998).SVMDR1 vectors have also been produced in vitro (Kimchi-Sarfaty et al., 2002, 2003). Surprisingly, even if in vitro pro-duced vectors also infected bone marrow cells efficiently,MDR-1 expression did not last more than 3 weeks in the ab-

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TABLE 1a. rSV40 VECTORS THAT HAVE BEEN USED IN VITRO FOR THERAPEUTIC APPLICATIONS

Vector Transgene Cells Result Reference

SVSFv IN Single chain antibody Human T lymphocytes Inhibition of HIV-1 BouHamdan et al.,against HIV integrase and SupT1 cells replication 1999; Strayer et al.,

2002c

SVSFvCXCR4 Single chain antibody Human T lymphocytes Inhibition of HIV-1 BouHamdan et al.,against CXCR4 and SupT1 cells replication 2001; Strayer et al.,receptor 2002c

SVRT3 Single chain antibody Human T lymphocytes Inhibition of HIV-1 Strayer et al., 2002cagainst HIV-1 reverse and SupT1 cells replicationtranscriptase

rSVrevM10 Dominant negative Human T lymphocytes, Inhibition of HIV-1 Strayer et al., 2002cmutant against HIV-1 SupT1 cells and replication Cordelier et al., 2003aRev human NT2 derived

neuronsSVPolyTAR Polymeric TAR Human T lymphocytes Inhibition of HIV-1 Jayan et al., 2001;

decoys. and SupT1 cells replication Strayer et al., 2002a

SVhINFa2 Human interferon Human T lymphocytes, Inhibition of HIV-1 Cordelier et al., 2002alpha 2 (INFa2) SupT1 cells replication

human NT2 cellsderived from neurons

SVha1AT Human alpha-1 Human T lymphocytes, Inhibition of HIV-1 Cordelier et al., 2003a;antitrypsine gene SupT1 cells and replication Cordelier et al., 2003b(a1-AT) human NT2 cells

derived from neuronsSVMDR1 Human multidrug Human hematopoietic MDR-1 expression Rund et al., 1998;

resistance gene 1 stem cells. Human Kimchi-Sarfaty et al.,(MDR1) erythroleykemia cells 2002; Kimchi-Sarfaty

(k562) and human et al., 2003alymphoblastoid cells(.45)

SVhb-globin Human b-globin Primary human Human b(0)-globin Daylot-Herman et al.,gene erythroid progenitors expression 1999

sence of selection. It remains unclear if the method of produc-tion of the recombinant virus may affect duration of expression.However, it would be a great advantage to have SV40 vectorsthat could be used as short- or long-term expression, depend-ing on the needs.

From the experiments reviewed above we conclude thatrSV40 vectors are able to efficiently infect both T lymphocytesand hematopoietic cells. Also, the therapeutic gene expressionderived from these vectors appears sufficient for the treatmentof different disorders (Table 1a).

Ex vivo applications. Bone marrow cells from Balb/C miceand human CD341 cells have been used to evaluate rSV40 vec-tors as candidates for cellular adoptive therapies. Bone marrow-derived cells were transduced with a rSV40 vector driving theexpression of the firefly luciferase gene and were transplantedinto syngenic mice. Luciferase expression was evident inmononuclear and polymorphonuclear leukocytes and plateletseven at day 105 postinfection, indicating a long-term expres-sion of the transgene (Strayer and Milano, 1996). Unselectedhuman CD341 cells were transduced with a rSV40 vector ex-pressing a SChAb against HIV-I integrase. These transducedcells were transplanted into sublethally irradiated SCID mice.Three months after implantation almost 100% of the bone mar-

row cells expressed the transgene. The duration and stability ofexpression indicated random integration, as confirmed by PCR(Strayer et al., 2000). Random integration of different rSV40vectors into the genome of other infected cells lines has beendemonstrated (Strayer et al., 2002b).

In vivo applications. The rSV40 vector that drives the ex-pression of the SChAb against HIV-I integrase was also eval-uated in a model of immunodeficient mice implanted with hu-man foetal thymic and liver tissue (thy/liv-SCID-hu mice).Thymocytes from these mice transduced in vivo by the rSV40vector were refractory to HIV-I replication after an in vivo chal-lenge with HIV-I (Goldstein et al., 2002).

In vivo infection with rSV40 vectors could also be useful forvaccination or for the treatment of heritable and acquired dis-eases. For this, it is essential to know the biodistribution, dura-bility of transgene expression, and immunogenicity of thesevectors. These features have been studied by injecting Balb/Cmice with rSV40 viruses that drive the expression of the fire-fly luciferase gene (rSVLuc) or the hepatitis B surface antigen(rSVHB-SAg), (Strayer, 1996; Strayer and Milano, 1996;Strayer and Zern, 1999; Arad et al., submitted; Vera et al., man-uscript in preparation). Mice injected intravenously withrSVLuc showed luciferase activity in the liver, spleen, brain,

VERA AND FORTES278

TABLE 1b. rSV40 VECTORS USED IN VIVO

Vector Transgene Cells/animal Result Reference

SVSFv IN Single chain antibody Unselected human Almost all human cells Strayer et al., 2000against HIV integrase CD341 cells express the transgene

transplanted into three months afterBalb/C mice transplantation

SVSFv IN Single chain antibody Mice implanted with Decreased number of Goldstein et al., 2002against HIV integrase human foetal thymic HIV-I infected

and liver tissue (thy/ thymocytes after an inliv-SCID-hu mice) vivo challenge with

HIV-1

SVLUC Firefly Luciferase Balb/C mice Biodistribution study Strayer, 1996; Strayerand Milano, 1996;Strayer and Zern,1999; Arad et al.,2003; Vera et al.,2003

SVHBV The hepatitis B virus Balb/C mice High immune response Strayer and Zern,capsid protein against the transgen 1999

SVgp130 The SIV envelope Balb/C mice High immune response McKee and Strayer,glycoprotein (gp130) against the transgen 2002

SV-hBUGT Bilirubin-uridine59- Bilirubin-UGT- Normal serum and liver Sauter et al., 2000diphosphate- deficient jaundired parameters afterglucuronosyl- gunn rats intraportal injectiontransferase

SVrIGFI The rat insuline like Wistar rats treated with Normal serum levels of Vera et al., manuscriptgrowth factor I gene carbon tetrachloride transaminases and in preparation(IGF-I) bilirubin. Decrease of

liver fibrosis

kidney, skin, colon, and peripheral blood nuclear cells, fromday 14 to the end of the study at day 105 (Strayer, 1996; Strayerand Milano, 1996). This long-term expression seems to be me-diated by integration (Strayer and Milano, 1996). Inflammationor immune response against the transgene or SV40 capsid pro-teins was not detected after a single viral injection (Strayer,1996). Even after eight repetitive injections of rSVHB-SAg,only significant levels of antibodies specific to hepatitis B sur-face were found (Strayer and Zern, 1999). Similar results wereobtained with rSV40 expressing the SIV enveloped protein(gp130) (McKee and Strayer, 2002). In a recent study, low lev-els of SV40-neutralizing antibodies were detected in the seraof mice injected intravenously or by hydrodynamic injectionwith rSVLuc vectors; however, there was no indication of avector-specific cellular immune response (Arad et al., submit-ted). Biodistribution and analysis of rSV40 driven luciferaseexpression has been also studied in vivo with a CCD camera.Animals that express luciferase are able to emit light when in-jected with luciferin substrate. A CCD camera is able to detectthe photons emitted by the animal, so luciferase expression canbe evaluated periodically in living animals (Massound andGambhir, 2003). Both luciferase expressing SV40 vectors pro-duced in vitro or packaged in COS-1 cells have been used forCCD camera in vivo evaluation of luciferase expression (Aradet al., submitted; Vera, unpublished results). COS-1 packagedrSVLuc vectors injected intravenously in mice are able to ex-press luciferase for longer than 5 months (Vera, unpublishedresults). Luciferase expression in BALB/C mice injected by the

hydrodynamic method with rSVLuc decreases steeply after thefirst day postinjection, but after day 20 remains relatively sta-ble at a lower level that persisted to the end of the study at day107 (Arad et al., submitted). However, luciferase detection inthese animals is only observed in the liver (Fig. 4) (Arad et al.,submitted; Vera, unpublished results).

The lack of antigenicity, the long-term expression and the ef-ficiency of rSV40 virus to infect the liver (Zern et al., 1999) en-couraged some authors to use rSV40 vectors for the treatment ofliver metabolic diseases. 60 to 90% of liver hepatocytes can beefficiently transduced by rSV40 in rats after three to seven in-traportal injections of 3 3 109 infectious particles. Bilirubin-UGT–deficient Jaundired Gunn rats, a model for the Crigler-Na-jjar syndrome 1 (CN-1), were injected seven times intraportallywith a rSV40 vector expressing the human coding region forbilirubin-uridine59-diphosphate-glucuronosyl-transferase (SV-hBUGT). Three weeks after the treatment, serum and liver pa-rameters were normal, and did not change until the end of theexperiment at day 40 (Sauter et al., 2000). Recently, a rSV40vector driving the expression of the rat insulin like growth fac-tor gene (IGF-I), has been used for the prevention of cirrhosisdevelopment in a carbon tetrachloride inhalation model in rats.Rats treated twice intraportally with rSVIGF-I showed low lev-els of fibrosis in liver and normal levels of transaminases andbilirubin in serum (Vera et al., manuscript in preparation). Theseresults indicate that rSV40 vectors could be valuable tools forthe treatment of metabolic and acquired diseases that requirelong-term expression of the transgene (Table 1b).

SV40 AND GENE THERAPY 279

FIG. 4. In vivo evaluation of luciferase expression with a CCD camera. Mice were mock infected (left) or infected with arSVLuc vector (right). The photons emitted by the animal were captured by a CCD camera and plotted using the gray scale codeindicated to the right of the picture. Luciferase expression is evident in the liver.

CONCLUSIONS

rSV40 vectors are good candidates for several gene therapyapplications. Good-quality nonreplicative vectors can be pro-duced with high titers, and they are able to transduce efficientlyboth resting and dividing cells. Furthermore, they can deliverpersistent transgene expression to a wide range of cell typesand they are nonimmunogenic. Although the results obtainedfrom many laboratories in different areas are encouraging, muchwork remains to be done. First, the molecular mechanism ofrSV40 genome integration into the host genome should be elu-cidated. It is still unknown if integrated rSV40 genome can al-ter the expression of neighboring genes. Also, in vivo packagedrSV40 vectors should be developed with an increased cloningcapacity capable of expressing all transgenes, including GFPand other reporters. Techniques to produce high titers of in vitropackaged rSV40 vectors should also be developed. Direct com-parisons between function of in vitro and in vivo packagedrSV40 vectors in animal models should be carried out. Fur-thermore, comparison of expression and therapeutic efficacyshould be made between rSV40 vectors and other vectors com-monly used in gene therapy approaches. Finally, it is essentialto extend rSV40 system usage to novel therapeutic applications.We hope that the unique properties of rSV40 vectors and theresults obtained from different groups will encourage other lab-oratories to exploit such a promising system.

ACKNOWLEDGMENTS

We thank A. Oppenheim, C. Smerdou, S.A. Calarota, G.González-Aseguinolaza, M. Zaratiegui, I. Narvaiza, and R. Her-nandez for the critical reading of the manuscript. We are alsograteful to A. Oppenheim, D. Strayer, and C. Kimchy-Sarfatyfor sharing unpublished results and J. Prieto for helpful dis-cussions. J.M. Ezcurra was essential for the design of Figure 1.The author’s own research was supported by CICYT(PM1999/0091), FIS (01/1310 and 01/0843), Instituto CarlosIII C03/02, Education Department of the Government ofNavarra and through the agreement between FIMA and the“UTE project CIMA.” M.V. is an FPI fellow.

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Address reprint requests to:Maria Vera or Puri Fortes, Ph.D.

School of MedicineFoundation for Applied Medical Research

Division of Gene TherapyLaboratory of Vectors Development

University of NavarraIrunlarrea 1

Pamplona, Spain.

E-mail: mveruga@alumni.unav.espfortes@unav.es

Received for publication December 14, 2003; received in re-vised form January 16, 2004; accepted January 30, 2004.

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