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Faisst S, Rommelaere J (eds): Parvoviruses. From Molecular Biology to Pathology and
Therapeutic Uses. Contrib Microbiol. Basel, Karger, 2000, vol 4, pp 178±202
..............................
Autonomous Parvoviruses as Gene Transfer
Vehicles
Gene A. Palmer a, Peter Tattersall a,b
a
Departments of Laboratory Medicine and b Genetics,
Yale University School of Medicine, New Haven, Conn., USA
The parvoviruses are a family of small, non-enveloped, icosahedral
animal viruses which are unique in the known biosphere in that they contain a single-stranded DNA genome which is linear. Members of the
Adeno-associated viruses (AAV), or Dependovirus subgroup of parvoviruses, integrate at a specific location on human chromosome 19 [1, 2],
in the absence of their helper virus. In contrast, while members of the
autonomous, or helper-virus independent subgroup of parvoviruses, have
yet to be shown to integrate in their natural host cell [3], they may persist for long periods of time in the infected host animal by an unknown
mechanism [4]. In general, the parvoviruses require their host cells to be
of a particular differentiated phenotype and actively traversing the Sphase of the cell cycle, in order to establish a productive infectious cycle.
These viruses display several characteristics which have recommended
them as viral vectors for the delivery of therapeutic protein molecules
into target cells.
A particularly important potential niche for parvoviral vectors is in the
treatment of neoplastic disease. Scenarios in which transient high level
transgene expression could be effective include tumor-specific immunotherapy, wherein immune costimulatory molecules and/or cytokine expression is
targeted to tumor cells. These viruses would also make ideal vectors for tumor-targeted expression of toxin(s) or suicide genes. The bystander effect
from toxin or suicide gene expression could help in reducing tumor burden
and possibly even stimulate natural tumor-specific immunity by providing a
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Introduction
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critical mass of dying tumor cells for efficient antigen presentation on
scavenging phagocytic cells.
Autonomous parvovirus vectors could also be armed to express an antigen not efficiently presented under the conditions of natural infection or exposure to a given pathogen. Classical immunizations with massive doses of
purified protein might be replaced by a single inoculation with a parvovirus
vector that expresses antigen over an extended period, effectively priming
and boosting a sustained immune response.
Parvoviruses are also attractive as delivery vehicles for therapeutic gene
delivery in humans because they comprise the only family, among DNA
viruses, in which there are no tumorigenic members ± indeed, they are
markedly oncosuppressive under many circumstances [5]. This lack of potential for causing cellular transformation should give the parvoviruses a distinct safety advantage over other viral vector systems such as retroviruses
and adenoviruses. Since autonomous parvoviruses are not known to establish a permanent relationship with the host cell genome [3, 6], they therefore
likely pose very little risk for insertional mutagenesis. Furthermore, the first
viral gene expressed is the anti-proliferative non-structural protein NS1,
which inhibits further cell cycle progression, presumably to arrest the host
cell in S-phase, where it is competent for viral DNA replication [7]. These
features confer inherent safety on the parvoviruses in general, and the
autonomous subgroup in particular.
Their potential utility in a clinical setting ± especially in developing
countries ± is considerably greater than that of many other viral vectors because they package their genomes in such extremely rugged virions. Unlike
most viral vectors, parvoviral vectors should require little or no sophisticated equipment to maintain them as viable stocks. For example, the halflife for MVM (Minute Virus of Mice) infectivity is more than nine months
when stored in a simple diluent solution in an ordinary domestic refrigerator
at about 8 °C [8].
About 80% of the human population is seropositive for human AAV
strains by adulthood [9, 10], suggesting that vectors based on these viruses
will find little use in direct injection strategies. However, the frequency of
positive antibody titer to rodent parvoviruses in human populations has
been reported to be extremely low [11]. Despite the virus' apparent lack of
natural infectivity for humans, it is well established that rodent parvoviruses,
including MVM, can grow in many human cell lines in vitro, and, while
there is little evidence for rodent parvoviruses ever infecting humans by any
natural route, direct parenteral inoculation of H-1 virus, a close relative of
MVM, has been shown to result in transient viremia [12].
Table 1 lists some of the advantages and disadvantages of parvoviral
Table 1. Criteria for effective gene transfer agent
Property
Advantages of parvoviruses
Potential disadvantages
Safety
No disease association
Inherent oncotropism
No relation with tumor viruses
Low risk of insertional mutagenesis
Replication competent
recombinants may arise
Stability
Exceptionally durable capsid
Easy to administer
Stable shelf life
Purified particles tend to
adhere to many surfaces
Host range
Broad cell and species tropism
Potential for rational targeting
Loss of `effective titer' by
uptake in non-targeted cells
Transgene
expression
High level, transient expression
possibly sustained from cycling cells
Expression in non-targeted cells
no integration ± not lifelong
Effective titer
Easy to concentrate
Relatively low titers produced
vectors, compared against a list of some of the criteria by which an effective
gene transfer agent must be judged. The focus of this chapter, therefore, will
be on the development of autonomous parvoviruses as vehicles for gene
transfer in this context. We will discuss the pros and cons of individual types
of parvoviral vector, with particular emphasis on how vector design and potential application have been directed toward exploiting the unique features
of these viruses.
Autonomous Parvoviruses
The following is a brief overview of the genome organization and mode
of replication of the autonomous parvoviruses. Interested readers are referred to the many comprehensive reviews on the subject for more detail [7,
13]. Autonomous parvoviruses are small (18±26 nm in diameter), non-enveloped, icosahedral viruses containing an approximately 5 kb single-stranded
DNA genome terminating in short palindromic sequences capable of folding
into hairpin duplexes. By convention, the left end of the genome is that of
the minus sense strand with respect to transcription. Of the twenty members
of the genus currently recognized, only the rodent parvoviruses H-1, LuIII,
and MVM have thus far been exploited for use as vehicles for gene transfer,
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Mode of Replication
therefore, this section will discuss replication of these viruses, which are remarkably conserved in their genome organization [14]. The only striking difference among these viruses lies in their encapsidation patterns. Whereas
MVM and H1 selectively encapsidate predominantly (100:1) strands that
are minus sense with respect to transcription, LuIII efficiently encapsidates,
in separate virions, equal numbers of strands of both sense [15±17].
The genome organization and transcription strategies for these rodent
viruses is shown in figure 1. Upon entering the permissive cell, infectious
particles appear to undergo proteolytic cleavage events and structural rearrangements of the virion that allow its translocation to the nucleus. Synthesis of the complementary strand in S-phase creates a linear duplex intermediate with covalently closed `turn around' ends, which is likely to be the
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Fig. 1. Parvoviral Genome Organization. The coding strategy of the MVM genome,
showing the positions of the two promoters, P4 and P38, on the viral genome, displayed
above the mRNA splicing strategy. The three size classes of transcript are denoted R1, R2
and R3, with their poly A tails shown as AAAAA. The stippled boxes show a 20-fold
magnification [relative to the rest of the figure] of the terminal DNA hairpin structures.
The slashed blocks indicate reading frames 1
,2
, and 3
, encoding individual segments of each viral polypeptide, which are named on the left of the figure.
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DNA duplex used as the initial substrate for transcription. Gene expression
and genome replication now ensue in a coordinated fashion. Transcription
begins from the moderately strong, constitutive P4 promoter [18, 19], at
map unit 4 from the left end of the genome. This promoter drives expression
of a transcript whose spliced derivatives, R1 and R2, encode the non-structural proteins NS1 and NS2, respectively [20±22]. NS1 is an 83 kD, cytotoxic
[23], multifunctional nuclear phosphoprotein [24±27] with nickase, helicase
and ATPase activities [28±31], which participates in driving viral replication,
upregulates the late promoter at map position 38 (P38) [32±34], upregulates
the P4 promoter [35, 36], and likely acts in concert with NS2 to direct the
packaging of progeny single-strand DNA [37, 38]. In the presence of ATP,
NS1 is a site-specific DNA binding protein, which recognizes a (ACCA)2±3
motif [39, 40] distributed widely throughout the genome, in addition to the
viral origins of DNA replication at each terminus and the transactivation region (TAR) upstream of the P38 promoter. As a consequence of its nickase
activity, NS1 is found attached covalently to the 5'-ends of all viral DNA
strands, including those which are packaged [31, 41].
NS2 is a family of minor, predominantly cytoplasmic, short-lived nonstructural phosphoproteins generated in three isoforms by alternative splicing of the small intron [22]. Site-specific mutagenesis of the NS2 coding region has revealed that these proteins are not essential for productive infection of some cell types, notably SV40-transformed human fibroblasts such as
NB324K [37], although in these cells NS2 is required for a late virion release
step [38]. The minor form NS2Y, is dispensable for growth in all cell types
currently tested [42]. However, NS2, presumably in its major form, NS2P, is
required for an essential step(s) in virus replication in mouse cells. One of
these steps is involved in capsid assembly, and in its absence viral replicative
form (RF) DNA replication is severely diminished and progeny singlestrand synthesis is undetectable [38]. The transcript encoding NS2P terminates the coding sequence with a single amber codon which is then followed
by an open reading frame which could encode a C-terminal extension of
some 90 amino acids, with a significantly hydrophobic predicted sequence.
This putative read-through protein, designated NS3, is highly conserved
throughout the rodent parvoviruses, and, although its function is not known
at present, mutational analysis shows it to be dispensable for virus growth in
culture [42].
NS1 transactivates the P38 promoter to express the coat protein gene,
which produces two primary translation products, VP1 and VP2, with molecular weights of 83 and 64 kD, respectively [43]. The latter of these is the
more abundant, and comprises the C-terminal three-quarters of VP1 [44].
VP2 can also be processed to VP3, in full virions only, by proteolytic clea-
vage of approximately 25 amino acids from its N-terminus [44]. The crystal
structure of the parvoviral virion, currently solved for canine, feline and
murine members of this group, reveals that the icosahedral shell of the virion comprises sixty copies of the polypeptide chain common to VP1, VP2
and VP3 [45±48]. No direct structural information has been obtained for the
VP1-specific region, which is internally located and probably interacts with
the viral DNA. This region is highly basic, containing a number of putative
nuclear localization signals, and has recently been shown to be capable of
extrusion from the virion without disassembly, suggesting that it may act to
traffic the entering virion into the nucleus [49±51].
Efficiency of genome usage is characteristic of all parvoviruses and involves encoding proteins in overlapping reading frames within sequences
that also contain regulatory elements for transcription and RNA splicing.
The economy of genome size necessitates intimate interactions between the
host cell replication machinery and the viral non-structural protein NS1.
Synthesis of the complementary strand is followed by amplification of
monomer-length duplex RF DNA molecules through dimer, tetramer and
perhaps larger intermediates, by a rolling-hairpin mechanism unique to the
parvoviruses and similar to the rolling-circle processes of prokaryotic replicons [7, 52]. The nickase activity of NS1 is required for the resolution of
monomeric duplex forms of the genome from these concatemeric RF intermediates, and becomes covalently attached to the 5'-end of each strand. The
capsid proteins are required for the displacement of progeny single-strand
viral genomes from these monomer duplex DNA intermediates [53, 54],
which is encapsidated concomitantly with viral DNA synthesis.
Recent work in our laboratory has shown that cis-acting elements in palindromic sequences at opposite ends of the MVM genome act as origins for
viral replication, but associate with very different host factors. The left-end
origin requires a two protein subunit activity called PIF, for parvovirus initiation factor, to activate the site-specific nicking function of NS1 [55, 56].
The right-end, on the other hand, requires proteins of the high mobility
group 1/2 (HMG 1 or 2) family to activate the same NS1 function [57],
further underscoring the complex interaction of this virus with its host.
For a virus to be an effective vehicle for the transfer of genetic material,
the tropism of that virus should be well established. Autonomous parvoviruses as a group show a broad host range infecting many vertebrate and
avian species. Most significantly, the rodent parvoviruses also infect many
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Host Range
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human cell lines including NB324K, 293, HeLa, and a number of T cell
lines. Indeed, many of these parvoviruses were originally isolated as contaminants of human transplantable neoplasms [4]. H-1 was originally isolated as a contaminant of a human tumor cell line (Hep-1) that had been
passaged in laboratory rats [58], and LuIII as a contaminant of a human
lung carcinoma [59]. The first MVM was isolated from a stock of mouse
adenovirus [60] and more recently designated MVMp, for prototype [61], to
distinguish it from an allotropic variant subsequently isolated from a transplantable mouse lymphoma [62]. This distinct MVM strain inhibited several
lymphocyte functions in vitro and was therefore designated MVMi, for immunosuppressive [63, 64].
The two isolates of MVM exhibit a reciprocal tropism in cultured cell
types. Whereas MVMp productively infects murine fibroblasts and MVMi
productively infects T-lymphocytes, the two strains are restricted for growth
in their reciprocal host cells [61]. This restriction occurs at an early step in
the infectious cycle, after penetration, but prior to the establishment of viral
transcription [65, 66]. Virions from both strains compete for binding on
either cell type, arguing against a receptor-mediated restriction at the cell
surface [65]. Evidence suggesting that this tropism was particle-mediated
came from transfection studies in murine fibroblasts demonstrating efficient
single-cycle virus production irrespective of whether MVMp or MVMi infectious plasmid clones were used [67]. The determinants of this tropism
were mapped to a small region of the capsid coding sequence shared by
both VP1 and VP2 where substituting two amino acids in MVMi with those
from MVMp was sufficient to confer a fibrotropic phenotype to an otherwise lymphotropic MVMi genotype [66±69]. A number of mutants which
have switched between the two phenotypes have also been mapped and analyzed. While these mutations may be distant in the primary sequence of
VP2, the crystal structure reveals that they are clustered at the surface of
the virion [48], in what has been dubbed the allotropic determinant. This determinant can be interpreted as the footprint of an intracellular, developmentally-regulated host receptor, interaction with which is essential in the
establishment of productive infection.
Maxwell and coworkers further determined that this tropism difference
is mediated by VP2, by packaging a luciferase-transducing LuIII vector genome into particles containing combinations of VP1 and/or VP2 from either
MVMp or MVMi [70]. Luciferase transduction was solely dependent on the
particle type contributing the VP2 and was observed in the predicted cell
types. Since 85% of the surface of the virion is contributed by its component
VP2 molecules, and the amino acid residues involved in tropism appear to
encompass an interface between at least two symmetry-related capsid pro-
teins, this probably means that few, if any, VP1 molecules interact this way
at the virion surface to form the allotropic determinant.
Evidence that a mutable host range is mediated by capsid suggests that
with more structural data it may be possible to design vectors with unique
cell-specific tropisms. There have been a number of rodent parvoviruses isolated relatively recently [71, 72]. It is likely that the fibrotropic and lymphotropic variants of MVM currently known represent merely a subset of possible host range variants. A careful analysis of tropism for this group of
viruses could form the foundation for a rational design of parvovirus vectors
specifically targeted to many individual cell types.
Toolan [73] first described a protective effect against spontaneous tumor formation in hamsters after severe infection by H-1 over 30 years ago.
The oncosuppressive effect was greatest in surviving hamsters showing the
characteristic osteolytic effects of H-1 virus on developing teeth, palate, and
skulls, producing `mongoloid' pups. These experiments were extended and
showed that such pups resisted tumor induction by carcinogenic chemicals
as well as by oncogenic viruses [74]. These results encouraged the first use
of a parvovirus to treat human cancer. H-1 was used to treat two patients
with advanced disseminated osteosarcomas that were resistant to conventional therapies. Although this treatment was not able to ablate tumor development, H-1 viremia was established in both cases and serum alkaline
phosphatase levels were stabilized in one case [12].
Protection against tumor growth has also been demonstrated in a murine model with MVM. Infection with MVMp dramatically inhibited growth
of the transplantable Ehrlich ascites tumor in 90% of mice challenged with
tumor inoculations distant to the site of virus injection [75]. MVMp infected
mice were also found to be resistant to subsequent rechallenge with this tumor, while mice infected with MVMi, the lymphotropic strain to which this
tumor is resistant, showed no such protection to the initial tumor inoculation. These studies support a possible direct cytopathic interaction between
virus and tumor cells in vivo, but do not address the issue of immune or
other pathophysiological mechanisms that likely contribute to the inhibition
of tumor growth. Therefore, Dupressoir and coworkers [76] undertook a
study to determine whether H-1 could inhibit the tumorigenic potential of
SV40-transformed human mammary epithelial cells transplanted into nude
mice. They carried out in vitro studies in parallel with tumor-derived or virally transformed cells and showed that H-1 virus infection prevented trans-
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Oncotropic Properties
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formed human mammary cells from forming tumors in vivo and colonies in
vitro. This inhibition correlated with and supported parallel in vitro findings
[77] of a stimulation of parvovirus replication and transcription in transformed cells, as compared to normal, human fibroblasts and epithelial cells.
To separate possible host immune responses from oncosuppressive effects of
parvovirus infection, Faisst and colleagues [78] showed a dose-dependent regression of HeLa cell-derived tumors when injected into SCID (severe combined immunodeficient) mice and subsequently inoculated with H-1 parvovirus.
Oncotropism and oncosuppression of autonomous parvoviruses has
been functionally defined (in vitro and in vivo) as the preferential killing of
transformed cells compared with untransformed parental cells which have
been of human and murine origin. Thus, sensitization of host cells to parvovirus-induced killing correlates both with an increased capacity of transformed cells to support intracellular steps in the parvovirus life cycle, and
an enhanced susceptibility to the cytotoxic action of the non-structural protein NS1 [for review see 5].
Autonomous parvoviruses are known to inhibit cellular transformation
by SV40 in vitro [79], arguing for an interaction between parvoviral genes
and host cell factors controlling the cell cycle. This idea was first supported
by experiments which demonstrated re-expression of a functional wildtype
p53 product in K562 cells selected for resistance to the cytotoxic effect of
H-1 infection [80].
In further studies on H-1 resistant cells, Lopez-Guerrero and colleagues
[81] were able to use virus to select from U937 promonocytic cells, which
overexpress c-myc, clones which were coordinately resistant to lytic infections by H-1 and had reduced levels of c-myc expression and tumorigenicity
in nude mice. Interestingly, these resistant clones also resisted the induction
of cell death in the face of TPA treatment and all exhibited a pattern of nitric oxide (NO) and superoxide anion (O2±) production consistent with a
constitutively activated state. Inhibitors of NO and O2± produced a sensitized phenotype in these cells for H-1-mediated killing. The authors proposed that multiple pathways of cell differentiation and proliferation may
exist for sensitizing cells to parvovirus lytic infections [82].
The mechanism(s) of parvovirus-mediated oncosuppression remain elusive, but it will be interesting to see what influence tumor suppressor gene
products and other members of the cyclin and cyclin-dependent kinase family play in making the switch between a resistant or permissive phenotype
for parvovirus cytotoxicity. As a first step toward unraveling this complex interaction, Van Pachterbeke et al. [83, 84] observed a predictive value for expression of estrogen receptor (ER) on human mammary carcinomas and
their sensitivity to the cytopathic effects of H-1 virus. These studies demonstrated a sensitized phenotype for ER+ tumor-derived cells when compared
with ER± tumor-derived cells and was increased by estradiol treatment, indicating that more differentiated and highly proliferative tumor cells were
more susceptible to the oncolytic effects of parvovirus infection. By focusing
on intracellular events downstream of the engagement of certain hormone
receptors, a better understanding of how parvoviruses induce cell death
should become clearer. In any event, the intrinsic oncosuppressive potential
of autonomous parvoviruses suggest that their modified derivatives will be
exceptionally good candidates as gene transfer vehicles for cancer therapy.
Vector Design
In the absence of efficient packaging cell lines for parvovirus vectors,
two predominant packaging strategies, diagrammed in figure 2, have
evolved, both involving co-transfection of permissive cells with two plasmids
[85]. The first strategy, which we call here complete coding replacement, is
to have one plasmid contain the transgene construct sandwiched between
the terminal sequences of the virus, and the other contain a combination of
the viral non-structural and structural genes [86, 87]. This effectively
packages the transgene in the parvovirus coat in the absence of other viral
genes. The advantages of this strategy are that it cuts down on homology between vector and helper plasmid, and thus, as discussed below, the generation of replication competent virus (RCV). Also, the transgene can be expressed under the control of a promoter of choice [88]. However, such
vectors do not replicate in transduced cells, and transduction with them does
not take advantage of several unique aspects of parvoviral infection, such as
their inherent oncotropism [89, 90]. Probably most important for the strategies explored here is that these vectors do not mimic viral infection, and its
complex interaction with host defenses, as does the second strategy. For this
second type of vector, which we call here capsid replacement, the vector itself contains the viral non-structural genes, with the transgene substituted
for the coat proteins, usually VP2. Packaging is promoted by co-transfection
with another plasmid containing the coat protein genes alone, either under a
strong heterologous promoter, or under the viral P38 promoter.
In each case, the basic strategy for producing recombinant transducing
autonomous parvovirus vectors involves co-transfecting a helper and a
transducing plasmid into a permissive cell line. The helper plasmid supplies,
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General Considerations
Fig. 2. Vector and Helper Design. Diagrams of the arrangement of viral
and
non-viral
sequences in the three types of vector/packaging system described in the
text. The position of the transgene is indicated by the thin-lined clear box, and the transactivation region upstream of the P38 promoter by a thick-lined box. The terminal palindrome sequences are indicated by filled boxes , at the junctions between vector and
plasmid
DNA. 3'UTR denotes the sequences downstream of the viral capsid genes
which contain the polyadenylation signal and the IRS. The region substituted in the promoter replacement construct is indicated by the unfilled box .
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in the first strategy, both the viral proteins required for virus replication and
those for encapsidation of the transducing genome, and, in the second case,
the capsid proteins alone. The helper plasmid may also contain a heterologous origin of replication to amplify the capsid genes. The transducing plasmid carries the transgene of interest typically downstream of an authentic
188
parvovirus, or in some cases an inducible, promoter and stuffer DNA, usually in a genome-length configuration, all flanked by the autonomous parvovirus terminal palindromes for efficient excision from the plasmid, replication, and encapsidation. A variety of promoters and genome configurations
have been developed and are discussed individually in the following sections. Co-transfected producer cells are typically incubated for 2±5 days,
then frozen and thawed three times in an extraction buffer to release cell-associated transducing virus. This lysate is clarified by centrifugation and can
be further purified by isopycnic gradient centrifugation and concentrated, or
used as crude lysate.
Maxwell and colleagues [86, 87] have developed several successful vector systems based on the parvovirus LuIII, in which the entire coding sequence of the original virus is replaced with transgene in the vector. The
theoretical maximum coding capacity of such vectors would be about 4,350
nucleotides, sufficient for a polypeptide 1,450 amino acids in length. The
prototype vectors transduced either the bacterial LacZ gene or luciferase
under the control of the P4 promoter into a variety of human cell types.
These contructs required the co-transfection of helper plasmids that supplied the non-structural protein NS1 to drive expression of structural genes
from the P38 promoter. NS1 expression was constitutive from either the viral P4 promoter or the simian virus SV40 early promoter on plasmids that
lacked the terminal hairpins of the parental virus. In both cases RCV were
generated, limiting the utility of such vectors to in vitro use.
In an effort to eliminate the DNA±DNA recombination between plasmid-based helper and transducing genome constructs, Corsini and coworkers
[91] developed a vector, pSinNS1, in order to provide NS1 from a Sindbis
RNA replicon. Electroporation of this plasmid into indicator cells, which
produce b-galactosidase in response to LuIII NS1, demonstrated that
SinNS1 RNA expressed protein that could transactivate the P38 promoter
lying upstream of the b-galactosidase gene. Co-transfection of this replicon
together with a DNA helper plasmid expressing LuIII structural genes and
a LuIII-luciferase transducing genome resulted in low but detectable levels
of luciferase-transducing virus. Improved yields were observed when the
helper plasmid was replaced with a construct that expressed NS2 in addition
to the capsid proteins, suggesting that NS2 is required to maximize the production of LuIII transducing virus in human cells. Although this study demonstrated the principal that providing an RNA replicon-derived compo-
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Complete Coding Replacement
nent into the packaging system can produce transducing virus, the authors
did not report on whether the production of RCV was reduced or eliminated in this system.
The LuIII-luciferase-transducing system was also used to explore transcriptional regulation by tissue-specific or inducible promoters. Replacing
the viral constitutive P4 promoter with a liver-specific promoter (alpha-1
protease-inhibitor gene) produced a transducing virus that resulted in 10- to
20-fold preferential expression of the luciferase reporter gene in human hepatoma (HepG2) cells when compared to HeLa cells [88]. This report also
explored the influence of viral sequences on transcriptional control elements
within the context of the parvovirus genome. Basal levels of luciferase expression from a construct containing the yeast GAL4 binding elements adjacent to the LuIII left terminus were markedly lowered when a trimer of the
SV40 polyadenylation signal was inserted between the two sequences to prevent readthrough from upstream promoters. Transducing virus generated by
co-transfection of helper plasmid with this modified GAL4-responsive transducing genome showed a stimulation of luciferase activity up to 4,000-fold
in the presence of GAL4.
In an elegant approach to fine control of transcriptional regulation the
same group [88] designed a LuIII-luciferase transducing recombinant virus
using the tetracycline regulated transactivator system developed by Gossen
and Bujard [92]. Fusing the transactivator domain of the herpes simplex virus
protein, VP16, with the repressor of the tetracycline resistance operator, as
the DNA binding domain, creates a strong transcriptional activator (tTA)
whose binding to the operator can be reduced or abolished by low levels of
tetracycline. Thus, the transducing genome contains a chimeric tetracycline
operator fused to a minimal CMV promoter. Luciferase activity was examined in transduced HtTA-1 cells, which were derived from HeLa and constitutively express the tTA. The expression of luciferase in cells transduced with
the modified LuIII luciferase vector could be diminished, in a dose-dependent
fashion, by the addition of tetracycline to the culture medium.
The first MVM vectors described in the literature [89] made use of the
MVMp infectious clone, pMM984 [93], into which cDNAs encoding either
human interleukin 2 (IL-2) or murine interleukin 4 (IL-4) were inserted in
capsid gene sequences downstream of the P38 promoter. The genomic termini required in cis for intracellular amplification of viral RF DNA, the
non-structural genes encoding NS1 and NS2, and the viral polyadenylation
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Capsid Replacement
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signal sequences were all maintained. The theoretical maximum coding capacity of such vectors would be about 2,220 nucleotides, sufficient for a
polypeptide 740 amino acids in length. Packaging of these transducing genomes was accomplished by co-transfecting with either an NS1-defective
helper or with the infectious clone, to provide capsid proteins. The NS1 supplied by the transducing genome facilitates amplification of the recombinant
transducing genome, increasing the copy number of the transgene, and
transactivates the P38 promoter to drive expression of the interleukin transgene as well as the capsid proteins from the helper plasmid.
Russell and coworkers [89] demonstrated that recombinant transducing
parvoviruses could be generated in this way that transferred functional IL-2
or IL-4 cDNAs into MVM-susceptible target cells and induced transient,
high-level secretion of the appropriate biologically active interleukin. In
keeping with the inherent oncotropic properties of the parental virus, IL-2
secretion was greatly enhanced in transformed rat and human fibroblasts
compared with the non-transformed parental cell lines. The authors hypothesized that the cycotoxic effects of NS1 combined with the immune
modulation provided by either IL-2 or IL-4 would enhance tumor cell killing, however they were unable to generate the high-titer stocks free of RCV
necessary to pursue such studies in vivo.
In a very similar approach Dupont and colleagues [90] used a P38-driven chloramphenicol acetyltransferase (CAT) transducing system, built on
the pMM984 version of the MVMp infectious molecular clone backbone, to
assay expression of this reporter gene in a variety of transformed and nontransformed human cells derived from different tissue origins. Significant
CAT expression was found to be transformation-dependent in fibroblasts,
epithelial cells, T lymphocytes, and macrophages. However, CAT expression
varied depending on the tissue origin, with little or no expression observed
in Burkitt's lymphoma cells. The authors suggested that this was due to absence of the surface receptor for MVM on this cell type, and that this poses
a limitation on the usefulness of these vectors to a subset of tumor cells efficiently expressing receptor for MVM. While this limitation clearly should
hold, lack of expression should not be equated necessarily with lack of receptor, since one has to take into account the issue of reciprocal host range
restriction, as observed between MVMp and MVMi. For instance, the low
or undetectable levels of CAT activity in T lymphocytes observed when Jurkat cells were compared with non-transformed human T-blasts would likely
be due to this host range restriction rather than low receptor expression or
the transformation phenotype of the T lymphocyte. Had the same transduction experiments been carried out with vector packaged in the MVMi coat,
the results would likely have been significantly more favorable.
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Interestingly, the CAT expression was not correlated with vector DNA
replication. Immortalized HaCat keratinocytes efficiently replicated the recombinant genome but resulted in only moderate CAT expression, whereas
significant CAT expression was observed in both HeLa cells and transformed macrophages, despite a failure to replicate the recombinant viral
genome in either cell type. This has been observed elsewhere [94] and probably reflects the influence of varying host cell transcription and replication
factors available from one cell type to another. These observations do suggest, however, that simpler, non-replicative (NS1±) vectors could be effective
for transduction of genes into certain cell types thereby providing more
cloning space for cell- or tissue-specific regulatory sequences and/or larger
transgenes.
A recurring problem in these studies has been the observation that cotransfection of transducing constructs with helper constructs produced a low
titer of virus stock significantly contaminated with variable (50±80%)
amounts of RCV. This vector strategy conserves sequences flanking the
transgene that are homologous with those found in the helper/packaging
plasmid. In particular there are sequences in the 3'-end of the capsid genes
reported to enhance replication from MVM replication origins dramatically,
at least in the context of minigenome cassettes [95±97]. These internal replication sequences (IRS) create a significant region of sequence homology between vector and helper plasmids, and could contribute to the generation of
RCV by homologous recombination. The Dupont group has since undertaken an heroic effort to mutagenize every third nucleotide in the capsid gene
sequences within the vector to minimize the recombinogenic potential of
the plasmid pair. They have reported a significant diminution in the generation of RCV generated by co-transfection, however, apparent recombination
continues to produce low level contamination of vector virus stocks with
RCV [98].
Since packaging requires recovery of vector from plasmid form following co-transfection, it became clear that any manipulations of the infectious
clone which increased the efficiency of viral excision from the plasmid form
would increase recovery of packaged vector. The processing of multimeric
replication intermediates of MVM to give unit length precursors for progeny genomes is complex. In particular, the resolution of dimer junctions to
regenerate the left-hand end of the monomer, and subsequently the 3' end
of the packaged genome, is an asymmetric process, resulting from activation
of the nickase of NS1 at only one of the two potential DNA replication origins located either side of the dyad symmetry axis [99, 100]. The initial cloning of the MVM genome in an infectious plasmid form [93] failed to capture
all of the essential sequence for this active origin, this junction has been re-
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built into the infectious clone, so that it now contains this functional origin.
The resulting plasmid is over 100-fold more infectious than the original [42].
Kestler and coworkers [101] showed that H-1-based vectors constructed
from the infectious clone with this improved left-hand end were recovered
with similarly elevated efficiency. They went on to examine the influence of
transgene insertion and the deletion of structural gene sequences, as well as
the overall size of the recombinant genome, on production of recombinant
viruses capable of transducing and expressing foreign genes. By using a
standard co-transfection system, in which a helper plasmid supplies structural proteins in trans, they observed a 10-fold reduction in the overall encapsidation yield with as little as a 6% increase in genome size. Moreover, production of H-1 viral vectors was dramatically reduced when more than 800
bp of the structural gene sequence was removed. Maintaining genome
length by substitution of non-viral stuffer DNA for capsid gene sequences
resulted in a 50-fold reduction in vector titers when compared to genomes
in which stuffer DNA was not substituted for the residual capsid sequence.
Interestingly, progressive deletions of up to 1,600 bp of capsid gene sequence, as well as insertions of foreign DNA in replacement of those sequences, did not significantly affect the ability of these recombinant genomes to replicate [101]. Elimination of the IRS had a negative influence on
DNA amplification and an up to 100-fold reduction in vector virus titers.
However, even in vector genomes suffering an 800 bp deletion in capsid
gene sequences, measurable RCV was produced by recombination with
helper plasmid sequences.
In a similar study, Brandenburger and colleagues [102] have explored
the influence of sequence and size of vector genomes on the efficiency of
packaging in an MVM system. They found that vectors with genomes smaller than 91% of wildtype MVM and larger than 117% were produced very
inefficiently, or as in the case of the larger genome, not packaged at all.
Comparing vector genomes of the same size, all containing a 500 bp insert
of IL-2 coding sequence, but differing depending on viral capsid gene content or foreign DNA, they concluded that when phage k was used to replace
capsid gene sequences, significantly lower titers of vector virus was produced. These results imply that simple replacement of parvoviral DNA with
stuffer DNA from any source is not adequate, but that maintaining cis-acting elements within genomic DNA, such as NS1-binding sites as discussed
below, may play a critical role in conserving both replication and packaging
efficiencies intrinsic to the authentic viral DNA sequence.
These studies underscore the important considerations necessary for optimal vector design and demonstrate that size as well as sequence play important roles in efficient packaging. Until distinctly defined cis-acting packaging
Fig. 3. RCV Suppression Strategy. Diagram of the vector/packaging plasmids, designed to eliminate RCV formation, described in the text. Symbols are as described for figure 2. Sequences derived from MVM are shaded
and those from LuIII are shaded
. Two sets of SV40 elements within plasmid sequences, the DNA replication origin
.
(ori) and two upstream polyadenylation sites (pA), are shown with horizontal hatching
bg-pA denotes the polyadenylation signal sequence derived from the rabbit b-globin gene,
and GFP indicates the position of the transgene, encoding the humanized, enhanced green
fluorescent protein.
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signals are identified for parvoviruses, care must be taken to conserve parvovirus sequences in these genomes that will serve to promote DNA replication
and efficient packaging, while at the same time suppressing the homologous
recombination between vector and helper plasmids that produce RCV.
With these aims in mind, the two plasmid system outlined in figure 3
has been constructed in our laboratory [103]. Working on the assumption
that RCV arise due to homologous recombination between the vector and
helper plasmid, these sequences have been re-configured to minimize or
eliminate significant regions of homology between them. The transgene is
EGFP (enhanced green fluorescent protein), which was fused into the transducing plasmid at the N-terminus of VP2, conserving the Kozak translation
initiation sequence found in the parental virus. A translation termination codon (amb) was inserted in the VP1 N-terminus to eliminate the expression
of N-terminally-extended forms of the transgene and to further suppress the
formation of viable recombinants between this construct and the helper
plasmid. Importantly, MVM sequences in analogous regions of the two plasmids were reciprocally substituted with sequences from the related rodent
parvovirus LuIII. This was done to place the transgene, as far as possible, in
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a parvoviral genome context. There are up to 40 binding sites for NS1 distributed throughout the MVM genome [104], and it appears that NS1, which
is expressed during infection at a level more than 100-fold greater than that
required for DNA replication [41], may serve a histone-like function in the
atypical chromatin formed on intracellular viral DNA [105]. Thus, it may be
critical, for efficient replication and/or packaging, to maintain as many of
these sites as possible in the transducing genome. The NS1 molecules of
MVM and LuIII cross-transactivate their P38 promoters, and the distribution and specificity of NS1 binding sites are essentially superimposable.
However, throughout the majority of their sequences they have significant
enough disparity to be predicted to undergo minimal recombination. In order to suppress opportunities for RCV generation by recombination further,
each transgene construct is inserted at the VP2 N-terminus. The remaining
sequences are `shunted' down toward the right-hand end, and the excess
over unit genome length truncated at the VP2-3'UTR junction. This has the
effect of further increasing the number of illegitimate recombination events
required to regenerate wild-type MVM.
The recombination potential of the plasmid pair was further reduced by
deleting the MVM polyadenylation signal sequence in the helper plasmid
and replacing it with the polyadenylation signal sequence of the rabbit b
globin gene. Packaged vector was produced by co-transfecting the two plasmids into CMT4 cells, which inducibly express SV40 T-antigen from the
metallothionein promoter [106]. Optimal conditions for driving the replication of the helper plasmid through its SV40 DNA replication origin have
been established, balancing the ratio of packaged virion to total capsid production. Recombinant virus generated from this system efficiently transduces GFP into both murine and human T cells, and can be blocked by preincubation with anti-MVMi capsid-specific monoclonal antibody, demonstrating that GFP transduction was particle-mediated [103]. Virus titers of
approximately 107 transducing particles per milliliter of crude producer cell
extract have been attained. However, this extract contains a significant
amount of empty capsids that interfere with transduction efficiencies when
compared with isopycnic gradient-purified stocks.
Originally these MVM capsid replacement vectors were constructed to
include the IRS, which is located just inboard from the right-hand terminus
[95±97]. Even with the additional strategies adopted to suppress recombination, these vectors continued to yield significant RCV. A second generation
vector was constructed that removes the MVM IRS. Surprisingly, although
this vector appears to be at a slight disadvantage for packaging when compared with the original vector, but replicates faster in the absence of capsid
protein expression and assembly [103]. This suggests that the influence of
the IRS, studied in minigenomes deleted of most of the internal MVM sequences, may diminish as these sequences, or their LuIII equivalents, are restored. More significantly, extensive testing with sensitive assays, including
low dilution blind passage in permissive cells and Southern blots of inoculated monolayers, indicate that these IRS-deleted vectors do not generate
detectable RCV, allowing production of RCV-free vector stocks with titers
high enough to conduct experiments in animals.
Promoter Replacement
Experiments designed to delineate the minimal origin of DNA replication present at the left-hand end of the MVM genome indicated that its inboard boundary lies eight nucleotides downstream of the NS1 nick site and
just upstream of the promoter-proximal elements of P4 [100]. In order to
test whether this was indeed the border of the cis-acting elements required
for viral DNA replication, the region between this position and the P4
TATA box was modified by creating a chimera with analogous promoter
elements from the HIV-1 long terminal repeat [107]. Regions of HIV-1 extending from the most upstream of its three Spl binding sites through its
TATA box and the entire stem structure of the TAR element, were substituted for the equivalent regions of MVMi DNA. This insert replaces the
E2F, Ets-1 and Spl binding sites present in the MVM P4 promoter, its TATA
box and much of the 5' untranslated region of the MVM P4 transcripts. This
plasmid construct was recovered as live, replication-competent virus by
transfection into 324 K cells engineered to express HIV-1 tat constitutively,
demonstrating that the substituted sequence was not required in cis for viral
replication, at least in its exact MVM form. Unlike its MVMi parent, the resulting virus was restricted, in its NS1 gene expression, and subsequent
DNA replication and cell killing ability, to tat-expressing human cells of
both T cell and non-T cell origin. Such chimeras could be developed as vectors for delivering, into human T cells, therapeutic molecules whose own expression would be dependent upon the expression of tat from an incoming
or resident HIV provirus [107].
Autonomously replicating parvoviruses do not integrate, therefore are
not viable candidates as vectors for conventional gene replacement therapy.
Rather than regarding this as a limitation, we encourage consideration of
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Limitations and Prospects
these viruses as vectors for use under circumstances where targeted, high
level transient expression is required and cell death is either desired or inconsequential. Likely scenarios for their use, therefore, are in the fields of
cancer immunotherapy and vaccination. What has hampered progress toward their use in animal models are the cumbersome techniques required to
generate these vector viruses, free of RCV, at titers high enough for in vivo
studies.
Attempts to improve on the purification and concentration of autonomous parvovirus vectors have provided modest increases in vector yield
[108]. Development of a highly efficient packaging cell line would have a
profound influence on advancing these viruses as vehicles for gene transfer.
Although some progress has been made in the establishment of a packaging
line [109], this cell line produces rather low yields (< 103 infectious equivalents per ml). Such infections do not appear to produce RCV, even when
the vector stock is passaged once on the same cell line, although this attempt to amplify the vector did not result in an increase in transducing titer
[109]. The effects of RCV formation in packaging cell lines will likely be
even more disastrous than during transfection, since all evidence to date
suggests that RCV will have a competitive growth advantage over vector,
and in a very few rounds of replication will come to predominate in the resulting stock. Now that strategies designed to eliminate RCV formation are
meeting with some success, one should expect a substantial increase in efforts to establish packaging cell lines for these vectors.
Much of the future usefulness of vectors based on the autonomously replicating parvoviruses will result from further exploration of their biology.
For instance, a greater understanding of the basis of their tissue tropism and
predilection for growth in transformed cells, will likely point to modifications which will increase their effectiveness as therapeutic agents against
cancer. Likewise, understanding how these viruses persist in the host animal,
while driving a continual humoral immune response against their capsids
[4], could lead to their exploitation as vaccine vectors, where the antigen
driving the immune response is encoded by the transgene which replaces
the capsid gene in the vector.
Acknowledgments
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The authors acknowledge support from Public Health Service grants T32 CA09159
(to GAP) and CA 29303 (to PT) from the National Institutes of Health.
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Dr. Peter Tattersall, Department of Laboratory Medicine, Yale University School of Medicine,
333 Cedar Street, P. O. Box 20 80 35, New Haven, CT 06510-8035 (USA)
Tel. +1 203 785 45 86, Fax +1 203 688 73 40, E-Mail peter.tattersall@yale.edu
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