Note: Descriptions are shown in the official language in which they were submitted.
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RETROVIRAL VECTOR PARTICLES PRODUCED IN A BACULOUIRUS EXPRESSION SYSTEM
~ The present invention relates to a composition.
In particular, the present invention relates to a novel system for producing
retroviral
particles.
More in particular, the present invention relates to a composition that is
capable of
expressing a retroviral particle that is capable of delivering a nucleotide
sequence of interest
io Uhereinafter abbreviated to "NOI") - or even a plurality of NOIs - to a
site of interest.
More in particular, the present invention relates to a composition useful in
gene therapy.
Gene therapy includes any one or more of: the addition, the replacement, the
deletion, the
~ s supplementation, the manipulation etc. of one or more nucleotide sequences
in, for example,
one or more targeted sites - such as targeted cells. If the targeted sites are
tareeted cells, then
the cells may be part of a tissue or an organ. General teachings on gene
therapy may be
found in Molecular Biology (Ed Robert Meyers, Pub VCH, such as pages 556-558).
~o Bv wav of further example. Gene therapy also provides a means by which any
one or more
of: a nucleotide sequence, such as a gene, can be applied to replace or
supplement a
defective gene; a pathogenic gene or gene product can be eliminated; a new
gene can be
added in order, for example, to create a more favourable phenotype; cells can
be
manipulated at the molecular level to treat cancer (Schmidt-Wolf and Schmidt-
Wolf, 1994,
~s Annals of Hematology 69:273-279) or other conditions - such as immune,
cardiovascular,
neurological, inflammatory or infectious disorders; antigens can be
manipulated andlor
introduced to elicit an immune response - such as genetic vaccination.
In recent years, retroviruses have been proposed for use in gene therapy.
Essentially,
~ 3o retroviruses are RNA viruses with a life cycle different to that of lytic
viruses. In this
regard, when a retrovirus infects a cell, its genome is converted to a DNA
form. In
otherwords, a retrovirus is an infectious entity that replicates through a DNA
intermediate.
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More details on retroviral infection etc. are presented later on.
There are many retroviruses and examples include: murine leukemia virus (MLV),
human
immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse
mammary
tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV),
Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR
MSV),
Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV),
Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus
(AEV).
o .-1 detailed list of retroviruses may be found in Coffin et al
("Retroviruses" 1997 Cold
Spring Harbour Laboratory Press Eds: JM Coffin. SM Hughes. HE Varmus pp 7~8-
763).
Details on the genomic structure of some retroviruses may be found in the art.
By way of
example, details on HIV may be found from the NCBI Genbank (i.e. Genome
Accession
is No. AF033819).
All retroviruses contain three major coding domains, gag, pol, env, which code
for
essential virion proteins. Nevertheless, retroviruses may be broadly divided
into two
categories: namely, "simple" and "complex". These categories are
distinguishable by the
0 organisation of their genomes. Simple retroviruses usually carry only this
elementary
information. In contrast, complex retroviruses also code for additional
regulatory proteins
derived from multiple spliced messages.
Retroviruses may even be further divided into seven groups. Five of these
groups represent
'S retroviruses with oncogenic potential. The remaining two groups are the
lentiviruses and
the spumaviruses. A review of these retroviruses is presented in
"Retroviruses" (1997
Cold Spring Harbour Laboratory Press Eds: JM Coffln, SM Hughes, HE Varmus pp 1-
25).
3o All oncogenic members except the human T-cell leukemia virus-bovine
leukemia virus
group (HTLV-BLV) are simple retroviruses. HTLV, BLV and the lentiviruses and
spumaviruses are complex. Some of the best studied oncogenic retroviruses are
Rous
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sarcoma virus (RSV), mouse mammary tumour virus (MMTV) and murine leukemia
virus
(MLV) and the human T-cell leukemia virus (HTLV).
The lentivirus group can be split even further into "primate" and "non-
primate" . Examples
of primate lentiviruses include the human immunodeflciency virus (HIV), the
causative
agent of human auto-immunodeficiency syndrome (AIDS), and the simian
immunodeficiency virus (SIV). The non-primate lentiviral group includes the
prototype
"stow virus" visna/maedi virus (VMV), as well as the related caprine arthritis-
encephalitis
virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently
described
~ o feline immunodeficiencey virus (FIV) and bovine immunodeficiencey virus
(BIV).
A distinction between the lentivirus family and other types of retroviruses is
that
lentiviruses have the capability to infect both dividing and non-dividing
cells (Lewis et al
1992 EMBO. J 11; 3053-3058, Lewis and Emerman 1994 J. Virol. 68: SIO-516). In
contrast, other retroviruses - such as MLV - are unable to infect non-dividing
cells such as
those that make up, for example, muscle, brain, lung and liver tissue.
During the process of infection, a retrovirus initially attaches to a specific
cell surface
receptor. On entry into the susceptible host cell, the retroviral RNA genome
is then copied
~o to DNA by the virally encoded reverse transcriptase which is carried inside
the parent
virus. This DNA is transported to the host cell nucleus where it subsequently
integrates
into the host genome. At this stage, it is typically referred to as the
provirus. The provirus
is stable in the host chromosome during cell division and is transcribed like
other cellular
proteins. The provirus encodes the proteins and packaging machinery required
to make
~5 more virus, which can leave the cell by a process sometimes called
"budding".
As already indicated, each retroviral genome comprises genes called gag, pol
and env
which code for virion proteins and enzymes. These genes are flanked at both
ends by
regions called long terminal repeats (LTRs). The LTRs are responsible for
proviral
3o integration, and transcription. They also serve as enhancer-promoter
sequences. In other
words, the LTRs can control the expression of the viral gene. Encapsidation of
the
retroviral RNAs occurs by ulnae of a psi sequence located at the 5' end of the
viral
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genome.
The LTRs themselves are indentical sequences that can be divided into three
elements, ,
which are called U3, R and U5. U3 is derived from the sequence unique to the
3' end of
s the RNA. R is derived from a sequence repeated at both ends of the RNA and
US is _
derived from the sequence unique to the 5'end of the RNA. The sizes of the
three elements
can vary considerably among different retroviruses.
For ease of understanding, a simple, generic diagram (not to scale) of a
retroviral genome
showing the elementary features of the LTRs, gag, pol and env is presented
below.
is
For the viral genome, the site of transcription initiation is at the boundary
between U3 and
R in the left hand side LTR (as shown above) and the site of poly (A) addition
(termination)
is at the boundary between R and U5 in the right hand side LTR (as shown
above). U3
'o contains most of the transcriptional control elements of the provirus,
which include the
promoter and multiple enhancer sequences responsive to cellular and in some
cases, viral
transcriptional activator proteins. Some retroviruses have any one or more of
the following
genes that code for proteins that are involved in the regulation of gene
expression: tar, rev,
tax and rex.
As shown in the diagram above, the basic molecular organisation of a
retroviral RNA
genome is (5') R - US - gag, pol, env - U3-R (3'). In a retroviral vector
genome gag, pol
and env are absent or not functional. The R regions at both ends of the RNA
are repeated
sequences. US and U3 represent sequences unique, respectively, to the 5' and
3' ends of
3o the RNA genome. These three sets of end sequences go to form the long
terminal repeats '
(LTRs) in the proviral DNA, which is the form of the genome which integrates
into the
genome of the infected cell. The LTRs in a wild type retrovirus consist of
(5')U3 - R - US
(3'), and thus U3 and US both contain sequences which are important for
proviral
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integration. Other essential sequences required in the genome for proper
functioning
include a primer binding site for first strand reverse transcription, a primer
binding site for
~ second strand reverse transcription and a packaging signal.
5 With regard to the structural genes gag, pol and env themselves and in
slightly more detail,
gag encodes the internal structural protein of the virus. Gag is
proteolytically processed
into the mature proteins MA (matrix), CA (capsid), NC (nucleocapsid). The gene
pol
encodes the reverse transcriptase (RT), which contains both DNA polymerise,
and
associated RNase H activities and integrase (IN), which mediates replication
of the genome.
r o The gene env encodes the surface (SU) glycoprotein and the transmembrane
(TM) protein
of the virion, which form a complex that interacts specifically with cellular
receptor
proteins. This interaction leads ultimately to fusion of the viral membrane
with the cell
membrane.
is The envelope giycoprotein complex of retroviruses includes two
polypeptides: an external,
glycosylated hydrophilic polypeptide (SU) and a membrane-spanning protein
(TM).
Together, these form an oligomeric "knob" or "knobbed spike" on the surface of
a virion.
Both polypeptides are encoded by the env gene and are synthesised in the form
of a
polyprotein precursor that is proteolytically cleaved during its transport to
the cell surface.
~o Although uncleaved Env proteins are able to bind to the receptor, the
cleavage event itself
is necessary to activate the fusion potential of the protein, which is
necessary for entry of
the virus into the host cell. Typically, both SU and TM proteins are
glycosylated at
multiple sites. However, in some viruses, exemplified by MLV, TM is not
glycosylated.
25 Although the SU and TM proteins are not always required for the assembly of
enveloped
virion panicles as such, they do play an essential role in the entry process.
In this regard,
the SU domain binds to a receptor molecule - often a specific receptor
molecule - on the
, target cell. It is believed that this binding event activates the membrane
fusion-inducing
potential of the TM protein after which the viral and cell membranes fuse. In
some viruses,
~ 3o notably MLV, a cleavage event - resulting in the removal of a short
portion of the
cytopiasmic tail of TM - is thought to play a key role in uncovering the full
fusion activity
of the protein (Brody et al 1994 J. Virol. 68: 4620-4627, Rein et al 1994 J.
Virol. 68:
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1773-1781). This cytoplasmic "tail", distal to the membrane-spanning segment
of TM
remains on the internal side of the viral membrane and it varies considerably
in length in
different retrovtruses.
s Thus, the specificity of the SU/receptor interaction can define the host
range and tissue .
tropism of a retrovirus. In some cases, this specificity may restrict the
transduction
potential of a recombinant retroviral vector. For this reason, many gene
therapy
experiments have used MLV. A particular MLV that has an envelope protein
called 4070A
is known as an amphotropic virus, and this can also infect human cells because
its envelope
i o protein "docks" with a phosphate transport protein that is conserved
between man and
mouse. This transporter is ubiquitous and so these viruses are capable of
infecting many
cell types. In some cases however, it may be beneficial, especially from a
safety point of
view, to specifically target restricted cells. To this end, several groups
have engineered a
mouse ecotropic retrovirus, which unlike its amphotropic relative normally
only infects
15 mouse cells, to specifically infect particular human cells. Replacement of
a fragment of an
envelope protein with an erythropoietin segement produced a recombinant
retrovirus which
then bound specifically to human cells that expressed the erythropoietin
receptor on their
surface, such as red blood cell precursors (Maulik and Patel 1997 "Molecular
Biotechnology: Therapeutic Applications and Strategies" 1997. Wiley-Liss Inc.
pp 45.}.
,o
In addition to gag, poi and env, the complex retroviruses also contain
"accessory" genes
which code for accessory or auxiliary proteins. Accessory or auxiliary
proteins are defined
as those proteins encoded by the accessory genes in addition to those encoded
by the usual
replicative or structural genes, gag, poi and env. These accessory proteins
are distinct
25 from those involved in the regulation of gene expression, like those
encoded by tat, rev, tax
and rex. Examples of accessory genes include one or more of vif, vpr, vpx, vpu
and nef.
These accessory genes can be found in, for example, HIV (see, for example
pages 802 and
803 of "Retroviruses" Ed. Coffin et al Pub. CSHL 1997). Non-essential
accessory proteins
may function in specialised cell types, providing functions that are at least
in pan
3o duplicative of a function provided by a cellular protein. Typically, the
accessory genes are
located between poi and env, just downstream from env including the U3 region
of the LTR
cr overlapping portions of the env and each other.
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The complex retroviruses have evolved regulatory mechanisms that employ
virally encoded
' transcriptional activators as well as cellular transcriptional factors.
These traps-acting viral
proteins serve as activators of RNA transcription directed by the LTRs. The
transcriptional
traps-activators of the lentiviruses are encoded by the viral tat genes. Tat
binds to a stable,
stem-loop, RNA secondary structure, referred to as TAR, one function of which
is to
apparently optimally position Tat to traps-activate transcription.
As mentioned earlier, retroviruses have been proposed as a delivery system
(ocher wise
t o expressed as a delivery vehicle or delivery vector) for inter alia the
transfer of a NOI, or a
plurality of NOIs, to one or more sites of interest. The transfer can occur in
vitro, ex vivo, in
vivo, or combinations thereof. When used in this fashion, the retroviruses are
rypicatly called
retroviral vectors or recombinant retroviral vectors. Retroviral vectors have
even been
exploited to study various aspects of the retrovirus life cycle, including
receptor usage,
reverse transcription and RNA packaging (reviewed by Miller, 1992 Curr Top
Microbiol
Immunol 158:1-24) .
In a typical recombinant retroviral vector for use in gene therapy, at least
pan of one or
more of the gag, pol and env protein coding regions may be removed from the
virus. This
3o makes the retroviral vector replication-defective. The removed portions may
even be
replaced by a NOI in order to generate a virus capable of integrating its
genome into a host
genome but wherein the modified viral genome is unable to propagate itself due
to a Iack of
structural proteins. When integrated in the host genome, expression of the NOI
occurs -
resulting in, for example, a therapeutic effect. Thus, the transfer of a NOI
into a site of
interest is typically achieved by: integrating the NOI into the recombinant
viral vector;
packaging the modified viral vector into a virion coat; and allowing
transduction of a site of
interest - such as a targeted cell or a targeted cell population.
It is possible to propagate and isolate quantities of retroviral vectors (e.g.
to prepare suitable
' 3o titres of the retroviral vector) for subsequent transduction of, for
example, a site of interest by
using a combination of a packaging or helper cell line and a recombinant
vector.
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In some instances, propagation and isolation may entail isolation of the
retroviral gag, pol
and env genes and their separate introduction into a host cell to produce a
"packaging cell
line" . The packaging cell line produces the proteins required for packaging
retroviral DNA
but it cannot bring about encapsidation due to the lack of a psi region.
However, when a
recombinant vector carrying a NOI and a psi region is introduced into the
packaging cell -
line, the helper proteins can package the psi-positive recombinant vector to
produce she
recombinant virus stock. This can be used to infect cells to introduce the NOI
into the
genome of the cells. The recombinant virus whose genome lacks all genes
required to
make viral proteins can infect only once and cannot propagate. Hence, the NOI
is
i o introduced into the host cell genome without the generation of potentially
harmful
retrovirus. A summary of the available packaging tines is presented in
"Retroviruses"
(1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE
Varmus pp
449). However, this technique can be problematic in the sense that the titre
levels are not
always at a satisfactory Level. Nevertheless, the design of retroviral
packaging cell lines has
t s evolved to address the problem of inter alia the spontaneous production of
helper virus that
was frequently encountered with early designs. As recombination is greatly
facilitated by
homology, reducing or eliminating homology between the genomes of the vector
and the
helper has reduced the problem of helper virus production.
~o More recently, packaging cells have been developed in which the gag, pol
and env viral
coding regions are carried on separate expression plasmids that are
independently
transfected into a packaging cell line so that three recombinant events are
required for wild
type viral production. This strategy is sometimes referred to as the three
plasmid
transfection method (Soneoka et al 1995 Nucl. Acids Res. 23: 628-b33).
Transient transfection can also be used to measure vector production when
vectors are
being developed. In this regard, transient transfection avoids the longer time
required to
generate stable vector-producing cell lines and is used if the vector or
retrovirai packaging
components are toxic to cells. Components typically used to generate
retroviral vectors
~o include a plasmid encoding the GagIPol proteins, a plasmid encoding the Env
protein and a
plasmid containing a NOI. Vector production involves transient transfection of
one or more
of these components into cells containing the other required components. If
the vector
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encodes toxic genes or genes that interfere with the replication of the host
cell, such as
inhibitors of the cell cycle or genes that induce apotosis, it may be
difficult to generate
' stable vector-producing cell lines, but transient transfection can be used
to produce the
vector before the cells die. Also, cell lines have been developed using
transient infection
s that produce vector titre levels that are comparable to the levels obtained
from stable
vector-producing cell lines (Pear er al 1993, PNAS 90:8392-8396).
In view of the toxicity of some HIV proteins - which can make it difficult to
generate stable
HIV-based packaging cells - HIV vectors are usually made by transient
transfection of
vector and helper virus. Some workers have even replaced the HIV Env protein
with that
of vesicular stomatis virus (VSV). Insertion of the Env protein of VSV
facilitates vector
concentration as HIVIVSV-G vectors with titres of 5 x 105 (10g after
concentration) were
generated by transient transfection (Naldini et al 1996 Science 272: 263-267).
Thus,
transient transfection of HIV vectors may provide a useful strategy for the
generation of
~ 5 high titre vectors (Yee et al 1994 PNAS. 9I : 9564-9568). A drawback,
however, with this
approach is that the VSV-G protein is quite toxic to cells.
Thus, and as indicated, retrovirat vectors are used extensively in biomedical
research and
for gene therapy. Current methods for the production of retroviral vectors
make use of the
~o fact that the two roles of the wild-type retrovirus genome. that is protein
encoding and as a
template for new genome copies, can be de-coupled (e.g. Soneoka et al 1995
Nuci. Acids
Res. 23, 628 and references therein). Protein that is required for the
assembly of new virus
panicles and for enzyme and regulatory functions can be produced by non-genome
sequences in, for example, a mammalian packaging cell line (e.g. Miller 1990
Hum. Gene
?5 Therapy 1, 5). A genome sequence lacking the protein encoding functions is
provided, so
that the resulting retroviral vector panicles are capable of infecting but not
of replicating in
a target cell. The genome sequence can also be designed for delivery and
integration of a
. therapeutic gene (V ile and Russet 1995 Brit. Med. Bull 51, 12). Standard
methods for
producing murine leukaemia virus (MLV)-based vectors, for example, include use
of cell
30 lines expressing the gag pot and env genes (the packaging components) of
MLV. These
will package a compatible retroviral vector genome introduced by transduction
or by
transfection with an appropriate plasmid. An alternative method involves
simultaneous
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transfection of gag pol, env, and vector genome plasmids into suitable cells.
Although the principles of these systems are well understood, in practice the
re-constructed ,
virus assembly system often fails to generate the quantity of vector particles
which will be
5 required in practice for use in gene therapy. Retroviral vector particles
are generally
harvested by removing supernatant from a culture of particle-producing cells.
The resulting
suspension may be concentrated with respect to the vector particles, using
physical
methods, but only to a limited degree as problems such as aggregation and
damage tend to
arise. Thus, it may only be possible to concentrate a suspension of vector
particles by up to
S o 100-fold.
The present invention seeks to provide an improved system for preparing viral
particles that
may be of subsequent use in medicine.
t 5 In particular. the present invention seeks to provide an improved system
for preparing a
high titre of viral particles that may be of subsequent use in medicine.
According to a first aspect of the present invention there is provided a
composition
comprising at least one baculoviral component and at least one retroviral
component,
~o wherein the retroviral component is capable of being packaged into a
retroviral particle.
According to a second aspect of the present invention there is provided a
composition
wherein the composition is a baculovirus expression system comprising at least
one
retroviral component, wherein the retroviral component is capable of being
packaged into a
35 retroviral particle.
According to a third aspect of the present invention there is provided a
retroviral particle
obtainable from expression of the composition according to the present
invention. ,
According to a fourth aspect of the present invention there is provided a
process for
preparing a retroviral particle comprising expressing the composition
according to the
present invention.
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.According to a fifth aspect of the present invention there is provided an
insect cell
- comprising the composition according to the present invention.
' S According to a sixth aspect of the present invention there is provided a
retroviral vector
particle production system comprising the composition according to the present
invention in
an insect cell.
According to a seventh aspect of the present invention there is provided a
retroviral vector
to particle produced by the retroviral vector particle production system
according to the
present mvenuon.
According to an eighth aspect of the present invention there is provided an
expression
vector comprising a polynucleotide sequence which encodes a retroviral vector
genome
15 having a S' and a 3' end, which retroviral vector genome is capable of
being expressed and
packaged into a retroviral vector particle in a baculovirus expression system.
Preferably the retroviral component corresponds to a retroviral genome.
?o Preferably the composition comprises an RNA transcription start site for
the retroviral
vector genome, and wherein the nucleotide sequence encoding the retroviral
component is
operably linked to a promoter comprising an upstream promoter component
located
upstream of the RNA transcription start site and a downstream promoter
component located
downstream of the RNA transcription start site.
Preferably the downstream promoter component is upstream of the polynucleotide
sequence
encoding the retroviral vector genome.
In one embodiment, preferably the promoter is a baculovirus promoter.
- 30
Preferably the promoter is the polyhedrin promoter, the p10 promoter andlor
the polh
promoter.
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In one embodiment, preferably the promoter is a non-baculovirus promoter.
Preferably the promoter is the T7 promoter or the sp6 Salmonella phage
promoter.
s
Preferably the composition comprises at least one RNA cleavage component.
Preferably manipultaion of at least one of the RNA cleavage comgonent(s) would
yield a
retroviral genome free of any baculoviral components.
~o
Preferably at least one of the RNA cleavage components) is located between the
promoter
and the sequence encoding the retroviral component.
Preferably at least one of the RNA cleavage components) is located immediately
adjacent
t s the sequence encoding the retroviral vector component for subsequent
cleavage at the 5' end
of the vector component.
Preferably at least one of the RNA cleavage components) is located downstream
of the
sequence encoding the retroviral component.
~o
Preferably the RNA cleavage components) has a cleavage site immediately
adjacent the
sequence encoding the retroviral vector component for subsequent cleavage at
the 3' end of
the vector component.
zs Preferably at least one of the RNA cleavage components) is a ribozyme
cleavage site for
subsequent cleavage thereof.
Preferably each RNA cleavage component is a ribozyme cleavage site for
subsequent
cleavage thereof.
The or each ribozyme cleavage site may be cleaved by a ribozyme which is
independently
derived from the composition. Preferably, however, any one or more of the
ribozyme
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cleavage sites is cleaved by a ribozyme derived from the second viral
component.
' Ribozymes are RNA molecules that can function to catalyse specific chemical
reactions
within cells without the obligatory participation of proteins. For example,
group I
ribozymes take the form of introns which can mediate their own excision from
self-splicing
precursor RNA. Other ribozymes are derived from self-cleaving RNA structures
which are
essential for the replication of viral RNA molecules. Like protein enzymes,
ribozymes can
fold into secondary and tertiary structures that provide specific binding
sites for substrates
as well as cofactors, such as metal ions. Examples of such structures include
hammerhead,
hairpin or stem-loop, pseudoknot and hepatitis delta antigenomic ribozymes
have been
described.
Each individual ribozyme has a motif which recognises and binds to a
recognition site in a
target RNA. This motif takes the form of one or more "binding arms" but
generally two
1 s binding arms. The binding arms in hammerhead ribozymes are the flanking
sequences
Helix I and Helix III which flank Helix II. These can be of variable length,
usually
between 6 to 10 nucleotides each, but can be shorter or longer. The length of
the flanking
sequences can affect the rate of cleavage. For example, it has been found that
reducing the
total number of nucleotides in the flanking sequences from 20 to 12 can
increase the
''o turnover rate of the ribozyme cleaving a HIV sequence, by 10-fold
lGoodchild, JVK, 1991
Arch Biochem Biophys 284: 386-391). A catalytic motif in the ribozyme Helix II
in
hammerhead ribozymes cleaves the target RNA at a site which is referred to as
the cleavage
site. Whether or not a ribozyme will cleave any given RNA is determined by the
presence
or absence of a recognition site for the ribozyme containing an appropriate
cleavage site.
Each type of ribozyme recognizes its own cleavage site. The hammerhead
ribozyme
cleavage site has the nucleotide base triplet GUX directly upstream where G is
guanine, U
is uracil and X is any nucleotide base. Hairpin ribozymes have a cleavage site
of
BCUGNYR, where B is any nucleotide base other than adenine, N is any
nucleotide, Y is
- 3o cytosine or thymine and R is guanine or adenine. Cleavage by hairpin
ribozymes takes
places between the G and the N in the cleavage site.
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More details on ribozymes may be found in "Molecular Biology and
Biotechnology" (Ed.
RA Meyers 1995 VCH Publishers Inc p831-8320 and in "Retroviruses" {Ed. JM
Coffin et
al 1997 Cold Spring Harbour Laboratory Press pp 683).
A broad aspect of this embodiment of the present invention relates to a
composition
comprising at least a first viral component obtainable from a first virus and
a second viral
component component obtainable from a second virus; wherein the first virus is
different
from the second virus; wherein the second viral component is flanked by at
least two
cleavage sites (which may be the same or different); wherein at least a pan of
the second
t o viral component is capable of being packaged into a viral particle; which
viral particle is
substantially free of any first viral component.
Here, preferably at least one of the cleavage sites is a ribozyme cleavage
site.
t s Here, preferably each of the cleavage sites is a ribozyme cleavage site.
In accordance with this aspect of the present invention, the ribozyme cleavage
site may be
cleaved by a ribozyme which is independently derived from the composition.
Preferably,
however, any one or more of the ribozyme cleavage sites is cleaved by a
ribozyme derived
from the second viral component.
Here, preferably the first virus is a baculovirus.
Here, preferably the second virus is a retrovirus.
?5
Preferably the downstream promoter component is located within the sequence
encoding the
retroviral component.
Preferably the downstream promoter component is located within the sequence
encoding the
30 retroviral vector.
Preferably the retroviral component comprises a retroviral R region at either
end of a
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sequence encoding a retroviral vector genome, wherein the downstream promoter
component is located in the 5' R region and has a counterpart sequence in the
3' R region.
In one embodiment, preferably the composition comprises in a downstream
direction: an
S upstream baculovirus promoter component, a downstream baculovirus promoter
component, a ribozyme sequence, a retroviral 5' R region, a retroviral US
region, a
retroviral vector region for insertion of one or more genes to be delivered by
the vector, a
retroviral U3 region, a retroviral 3' R region, and optionally a second
ribozyme sequence.
~ o In one embodiment, preferably the composition comprises in a downstream
direction: an
spstream baculovirus promoter component, a retroviral 5' R re;ion comprising a
downstream promoter component, a retroviral US region, a retroviral vector
reUion for
insertion of one or more genes to be delivered by the retroviral vector, a
retroviral U3
region, a retroviral 3' R region, and optionally a ribozyme sequence.
Preferably the composition comprises one or more nucleotide sequences encoding
one or
more packaging components for producing retroviral vector particles which
particles
comprise the retroviral component.
Preferably the composition further comprises at least one nucleotide sequence
of interest
{NOI).
Preferably the NOI is useful in medicine.
.5 Preferably the NOI is part of the retroviral component.
The term "composition" as used herein with reference to the present invention
may include
one entity (such as a composition of matter) or two or more entities. For
example, the
composition of the first or second aspect may be one entity - such as a
modified baculoviral
3o genome wherein at least a part of the baculoviral genome has been replaced
with the
retroviral component of the present invention. The term "modified" includes
actual
modification of a wild type genome or ab initio construction of an entity that
could have
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been modified from a wild type genome (such as by Iigating two or more
fragments so as to
form an entity corresponding to the modified genome). By way of further
example, the
packaging components of the present invention may be contained in an entity
different to ,
that just described. By way of further example, the composition of the present
invention
can be a retroviral particle producer and/or vector. ,
The composition may even be a kit comprising a first pan which includes the
baculoviral
component and a second pan which includes the retroviral component.
Optionally, the kit
may include one or more other parts, such as one or more suitable restriction
enzymes etc.
The term "retroviral vector ~enome" includes a retroviral nucleic acid which
is capable of
infection, but which is not capable, by itself, of replication. Thus it is
replication defective.
A retroviral vector genome carries or is capable of carrying polynucleotide
sequences of
non-retroviral origin, for delivery to target cells. A retroviral vector
genome may comprise
1 s further non-retroviral sequences, such as non-retroviral control sequences
in the U3 region
which influence expression of the genome once it is integrated as a provirus
into a target
cell. The retroviral vector genome need not contain elements from only a
single
retrovirus. WO 96!37623, for example, describes retroviral vectors having
hybrid LTRs
derived from two different retroviruses.
?0
The term "retroviral vector particle" refers to the packaged retroviral vector
genome, that
is preferably capable of binding to and entering target cells. The components
of the
particle, as already discussed for the vector genome, may be modified with
respect to the
wild type retrovirus. For example, the envelope proteins in the proteinaceous
coat of the
~5 particle may be genetically modified in order to alter their targeting
specificity or achieve
some other desired function.
If the retroviral component includes an env nucleotide sequence, then all or
pan of that
sequence can be optionally replaced with all or pan of another env nucleotide
sequence.
3o Replacement of the env gene with a heterologous env gene is an example of a
technique or -
strategy called pseudotyping. Pseudoryping is not a new phenomenon and
examples may be
found in WO-A-98105759, WO-A-98/05754, WO-A-97/17457, WO-A-96109400, WO-A-
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91/00047 and Mebatsion et al 1997 Cell 90, 841-847.
PCT/GB98/01626
' Pseudotyping can confer one or more advantages. For example, with the
lentiviral vectors,
the env gene product of the HIV based vectors would restrict these vectors to
infecting only
s cells that express a protein called CD4. But if the env gene in these
vectors has been
substituted with env sequences from other RNA viruses, then they may have a
broader
infectious spectrum (Verma and Somia 1997 Nature 389:239-242). By way of
example -
workers have pseudotyped an HIV based vector with the glycoprotein from VSV
(Verma
and Somia 1997 ibic~.
In accordance with the present invention we have surprisingly found that
functional
retroviral vector panicles can be produced in a baculovirus expression system
and those
particles can successfully deliver one or more NOI(s) to target cells.
is Another important advantage of the present invention is that higher titres
of vector panicles
may be produced than in the mammalian systems. Again, this is an unexpected
finding.
Moreover, there is the added advantage that baculovirus expression systems may
be free
from endogenous mammalian genetic material, so reducing the risk of mammalian
2o contaminants being present in the resulting retroviral vector preparation.
In this regard,
insect contaminants are very unlikely to be biologically active in a mammalian
system and
are therefore expected to present less of a problem than mammalian
contaminants.
As indicated, the composition of the present invention comprises a baculoviral
component.
In brief, baculoviruses are a diverse group of viruses found mainly in insects
with no
supposedly known arthropod hosts.(O' Reilly et al in Baculovirus Expression
Vectors, A
Laboratory Manual 1994, Oxford University Press). They can accommodate
relatively
large insertions of heterologous DNA - such as an NOI according to the present
invention
- 30 (O'Reilly et al. in Baculovirus Expression Vectors, A Laboratory Manual
1994, Oxford
University Press). They have the potential for very strong expression of
heterologous
genes DNA - such as an NOI according to the present invention - for example
expression
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levels of 25 to 50% of the total cellular protein have been reported.
In more detail, baculoviruses have large double-stranded, circular DNA genomes
within a
rod-shaped capsid. Within the capsid, the DNA is condensed into a
nucleoprotein stucture
known as the core. The capsid plus the core are collectively referred to as
the _
nucleocapsid.
The length of a baculoviral DNA is between 80 and 120 kilobasepairs (kbp). The
DNAs of
the baculoviruses commonly exploited as expression vectors, Autographa
California
to multiple nuclear polyhedrosis virus (AcMNPV) (Miller et al 1987 In Genetic
Engineering
vol 8, eds Setler, JK and Hollaender A Plenum Press, New York) and the
silkworm virus,
Bombyx mori nuclear polyhedrosis virus (BmNPV) (Maeda et al 1985 Nature 315:
592-594)
are both approximately 130kbp.
15 Nucleocapsids are made in the nucleus of the infected cells and are
subsequently enveloped
(that is, they acquire a membrane) by one of two processes. Nuclecapsids can
bud through
the plasma membrane of the infected cell or they can acquire an envelope
within the nucleus
where they are produced. Membrane-enveloped nucleocapsids are referred to as
virus
particles or virions. The plasma-membrane budded form of the virus is referred
to as the
-o budded virus (BV).
During normal infection, the viruses produce nuclear occlusion bodies which
comprise
enveloped nucleocapsid{s) embedded ~in a crystalline protein matrix, the major
component
of which is a virus-encoded protein called polyhedrin. Polyhedrin is the
product of a single
?s gene in the baculovirus genome and is produced, late in infection,
accounting for 50% or
more of all proteins being made by infected insect cells. Transcription of the
polyhedrin
(polh) gene is driven by an extremely active promoter, which is therefore
ideally suited as a
promoter for driving expression of foreign genes. Similiar levels of protein
production can
occur if the polyhedrin gene is replaced by a foreign gene. Construction of
expression
3o vectors has therefore consisted of inserting a foreign coding sequence just
downstream of
the polyhedrin promotor. However, this cannot be achieved directly because the
large size
(about 130kbp) fo the viral DNA precludes simple in vitro manipulation (Old
and Primose
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1989 "Principles of Genetic Manipulation" 4th Ed Blackwell Scientific
Publications pp290-
291 ).
The insect baculovirus expression system provides a eukaryotic environment
that is
generally conducive to the proper folding, disulfide bond formation,
oligomerisation and/or
other posttranslational modifications required for the biological activity of
some eukaryotic
proteins. Post-translational modifications that have been reported to occur in
baculovirus-
infected insect cells include signal cleavage, proteolytic cleavage, N-
glycosylation, O-
glycosylation, acylation, amidation, phosphorylation, prenylation, and
carboxyiation. The
to sites of such modifications are usually at identical positions on the
proteins produced in
insect and mammalian cells (O'Reilly er al ibis.
An advantageous feature of baculovirus expression vectors is the potential for
very strong
expression of a heterologous gene DNA - such as an NOI according to the
present
1 s invention. The highest expression levels reported using baculovirus
expression vectors is
25%-50%o of the total cellular protein. Such levels are equivalent to polh
gene (Polyhedrin)
expression and correspond to approximately 1 gram of protein product per 109
cells (eg a
litre culture).
All heterologous proteins, however. are not produced at the same level as
polyhedrin and
levels approaching 25 % of the total cellular protein have been achieved in a
few cases.
Most of these cases involved expression of structural genes of other virus
families, the
products of which are quite stable. Most heterologous proteins are produced at
levels
ranging from 10 mg to 100 mg per 109 cells. Nevertheless, in the cases where
different
as eukaryotic expression systems have been compared, the baculovirus system
has usually out-
performed other expression systems in overall protein production.
Thus, in one broad aspect, the present invention provides a production
facility (e.g. a vat,
or even an organism or cell thereof) for producing a quantity of an NOI or the
expression
3o product thereof; wherein the facility contains a medium comprising a
baculovirus
composition which comprises the NOI. Preferably, the baculoviral composition
is a
composition according to the first aspect of the present invention.
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The use of very late promoters (eg the polh and p10 promoters), which are
activated and
strongly transcribed during then unique occlusion phase of virus replication,
provides a
clear advantage for baculovirus-based expression systems. Because the
occlusion phase is
5 distinct from the late phase encompassing BV formation, expression of the
heterologous
gene DNA - such as an NOI according to the present invention - interferes
minimally with
BV production and there is very little selective pressure on the virus to
mutate toward
heterologous gene deletion or inactivation.
i o Very late expression may be particularly advantageous for the expression
of heterologous
genes DNA - such as an NOI according to the present invention - with a
deleterious effect
on essential cell functions such as cytotoxic gene products. The negative
aspect of
expressing genes during the very late phase of virus infection is that the
post-translational
modification capacity of cells appears to decline during the late phase.
The rod-shaped nucleocapsids of baculoviruses can extend to accomodate larger
viral DNA
genomes and it is likely that a baculovirus vector can accomodate an
additional 100 kbp of
DNA or more. The number of genes that might be expressed simultaneously using
baculovirus vector systems is also likely to be high. To make very large
insertions,
~o however, it may be necessary to construct a series of transfer plasmids
that allow the
building of the insert in successive increments. This limitation has more to
do with the
fragility of large DNAs in vitro rather than the vector system per se. Also,
if more than
three genes are to be expressed, additional transfer plasmids are required.
All characterised very-late genes of baculoviruses are unspliced but
efficiently expressed.
Thus, the baculovirus system is particularly useful for expressing unspliced
cDNA genes.
However, the baculovirus expression system does carry out at least some
splicing and its
usefulness in identifying and obtaining specific gene products from multigene
families has
been noted.
~o
Thus, in one broad aspect, the present invention provides the use of a
baculoviral
composition to express an NOI comprising at least one intron. Preferably, the
baculoviral
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composition is a composition according to the first aspect of the present
invention.
Bacuiovirus vectors are helper-virus independent and therefore relatively
simple to use.
Constructing a recombinant baculovirus is considerably faster and simpler than
constructing
s a cloned, high-expressing, recombinant eukaryotic cell line.
Although the basic technology for using AcMNPV- and BmNPV- based vectors is
similiar,
AcNPV- based systems have a number of advantages for most common applications.
The
cell lines sypporting AcMNPV replicaton are superior in growth characteristics
and
t o expression leves than cell lines supporting BmNPV replication. In
addition, the range of
transfer plasmids and parent viruses available for the AcMNPV-based system is
much
greater than that for the BmNPV-based system. Furthermore, the use of a
genetically
modified AcMNPV-based vector is advantageous because it replicates only in
insect cells
and is able to carry large (greater than l5kb inserts) (Boyce and Butcher1996
PNAS 93:
1s 2348-2352).
The cell lines that are most commonly used with AcNPV-based vectors are the S
podoptera
frugiperda (SF) cell lines because they are well-tested and have excellent
growth and
handling properties. These cells have been reported to produce some proteins
at levels
?o approachins 20% or more of the total cell protein, and it is unlikely that
any other
generally available cell line will provide more than threefold higher yields
of protein than
these. In particular, use of the Sf cell lines, such as the SF cell line IPBL-
SF-21 AE
(Vaughn et al., {1977) In Vitro, 13, 213-217) is preferred. The derivative
cell line Sf9 or
Sf8 may be preferred. However, other cell lines, such as Tricoplusia ni 368
(Kurstack and
?s Marmorosch, (1976) Invertebrate Tissue Culture Applications in Medicine,
Biology and
Agriculture. Academic Press, New York, USA} may be employed. These cell lines,
as
well as other insect cell lines suitable for use in the invention, such as
pBAC4x-lace
commercially available (e.g. from Stratagene, La Jolla, CA, USA and Novagen).
- 3o Although the host speficificity of the AcMNPV-based vectors is supposed
to be restricted to
arthropods, it has been demonstrated that recombinant AcMNPV virus can infect
a variety
of mammalian cell lines (Boyce and Butcher 1996 PNAS 93: 2348-2352). Unlike
retroviral
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and adenoviral vectors which do not allow for exclusive infection of the
liver, the
genetically modified AcMNPV baculovirus has been shown to demonstrate a strong
preference for hepatocytes (Sandig et al 1996 Human Gene Ther 7: 1937-1945;
Hofmann et
al 1995 PNAS 92: 10099-10103).
s
NOIs are generally introduced into baculovirus genomes by allelic replacement.
In allelic
replacement strategies, the foreign gene is inserted into a transfer plasmid
so that it is
downstream of the required viral promoter and flanked on both sides by viral
sequences that
will target the gene and promoter to a particular region in the viral genome.
The plasmid
i o and the parental viral DNA are cotransfected into host cells and enzymes
in the cells
recombine the DNAs. This primarily involves homologous recombination between
the
regions of the plasmid DNA flanking the foreign gene and their homologous
counterparts in
the viral genome.
t5 "Late" and "early" promoters are used in baculovirus expression systems.
The polh and
p10 promoters are examples of strong promoters which are expressed late in
infection. The
regulation of transciption from these promoters is presently not well
understood even
though a number of viral gene products that may be important for transcription
have been
identified (Hasnain et al 1997 Gene 190: 113-118). Promoters that drive gene
expression
2o earlier in the infection process are also being considered for nontoxic
proteins that are
slowly processed in baculovirus-infected cells. The baculovirus expression
system also has
an excellent track record for expressing genes as nonfusion proteins (that is>
for expressing
genes using their natural, translation initiation codon and N-terminal
sequence) but some
proteins are expressed to higher levels as fusions.
It is known that insect baculovirus expression systems have been used for the
production of
retroviral virus-like particles (VLPs) (for example, see Gheysen et al. 1989
Cell 59, 103;
Morikawa er al., 1991 Virol. 183, 288; Griffiths et al., 1993 J.Virol. 67,
3191;
Sommerfelt et al., 1993 Virol. 192, 298). Such insect baculovirus systems have
been used
3o extensively for the analysis of the Gag assembly reaction and a number of
the observations
made ate relevant to the production of gene therapy vectors. Co-expression of
the protease
and polymerase genes (using a gag pol construct) leads to maturation of the
virus particles
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to provide all the functional Gag and Pol antigens in the final virus particle
(Konvalinka er
al., 1995 Eur. J. Biochem. 228, 191; Wagner et al., 1992 Arch. Virol. 127,
117). Also
envelope antigens present on the cell surface of VLP-expressing cells are
incorporated into
VLPs (Yamshchikov et al., 1995 Virol. 214, S0; Gamier et al., 1995 J.Virol.
69, 4060).
However. it is to be noted that VLPs are proteinaceous particles without a
viral genome. In
this regard, VLPs are non-infectious and lack virus (or other DNA/RNA)
required for
replicaton. VLPs do not replicate in host cells. Sheep trials have shown that
VLPs are
more immunogenic than subunit vaccines (viral proteins) or viruses killed by
chemical
~ o inactivation. In addition, they are more effective at eliciting humoral.
cell-mediated and
mucosal immunities. 'LPs are also safe to produce and handle. The baculovirus
sector
and host cells used to make VLPs are not derived from a mammalian source.
Hence they
do not contain mammalian-derived pathogens (Roy, P 1996 Intervirology 39: 62-
7I). See
OBM 10 spec text page 2, lines 20-30 and page 3, lines 1-10).
Also, expression of the single gag gene has proven to be all that is required
for the
formation of retroviral VLPs and the amount of retroviral VLP produced appears
directly
proportional to the level of expression of the gag encoded precursor protein.
There are,
therefore, no secondary modifications that limit the budding process of the
retroviral VLPs
and yields of VLP outweigh the levels of baculovirus produced.
WO 95/22617 suggests using a baculovirus system for retroviral vector
production but.
importantly, it does not indicate any manner in which this might be achieved.
35 Thus, despite the observations relating to retroviral proteins, and despite
of the availability
of the data in the literature for several years, a baculovirus expression
system has never
- before now been used for the production of retroviral vectors. One reason
for this is that
while the protein components of retroviruses had been successfully produced in
insect
baculovirus systems, it was not expected that the requirements for a
functional retroviral
3o vector particle could be met. These requirements include the correct
packaging of the
vector genome, and an ability of the resulting vector particle to undergo
infection of and
proviral integration into the target cell. A particular consideration is the
need for a vector
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genome capable of undergoing accurate reverse transcription from RNA into
proviral DNA
in the target cell, and capable of producing proviral DNA which can
successfully integrate
into the target cell genome. This requires that the RNA contains the
appropriate priming
and other recognition sites required for reverse transcription (Reviewed in
Luciw and
Leung: The Retroviridae. Ed.J.A.Levy 1992 Plenum) and that the DNA product has
the
necessary terminal sequences for achieving integration (Reviewed in Luciw and
Leung.
Op.cit.; Cannon et al., 1996 J. Virol. 70, 8234). In addition the integrated
provirus must
contain the necessary control elements far gene expression upon subsequent
integration.
to The results of the present invention are highly surprising because
baculovirus expression
systems have certain unusual features which make the design of baculovirus
vectors for
producing retroviral vectors far from straightforward. The elements of a
baculovirus
promoter chat are, in part, required for its efficiency are downstream of the
RNA
transcription start sites (Posse and Howard, 1987 Nucl. Acids Res. 1~, 10233;
Rankin et
al, 1988 Gene 70, 39; Ooi et al., 1989 J.Mol.Biol. 210, 721). This means that
for the
efficient transcription that is required to provide the increase in vector
titre the retroviral
vector genome will necessarily have baculovirus sequences at its S' end. The
presence of
such non-retroviral sequences is in principle undesirable, given the need for
specific
retrovirus terminal sequences for reverse transcription and integration, and
can be expected
to interfere with normal retroviral vector genome function.
As indicated, the present invention overcomes the prior art problems by
providing a
baculovirus expression system useful for the production of retroviral vectors
andlor
retroviral particle (which may be capable of acting as a vector).
The retroviral particles produced by or from the composition of the present
invention
(which may otherwise be expressed as being "retroviral particle vectors") are
useful for the
delivery of one or more NOIs to cells in vivo and in vitro, in particular the
delivery of
therapeutically active NOI(s). One or more selected NOI(s) may be incorporated
in the
3o vector genome for expression in the target cell. The NOI(s) may have one or
more
expression control sequences of their own, or their expression may be
controlled by the
vector LTRs. For appropriate expression of the NOI(s), a promoter may be
included in or
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ZS
between the LTRs which is preferentially active under certain conditions or in
certain cell
types. The NOI may be a sense sequence or an antisense sequence. Furthermore,
if there
- is a plurality of NOIs then those NOIs may be sense sequences or antisense
sequences or
combinations thereof.
In a prefered aspect, the retroviral vector genome of the present invention
may generally
comprise LTRs at the 5' and 3' ends, one or more NOI(s) (including
therapeutically active
genes and/or marker genes), or suitable insertion sites for inserting one or
more NOI(s). In
some cases, there may be present a packaging signal to enable the genome to be
packaged
i o into a vector particle in a producer cell. There may even be suitable
primer binding sites
and interation sites to allow reverse transcription of the vector RNA to DN.A,
and
integration of the proviral DNA into the target cell genome. In a preferred
embodiment.
the retroviral vector particle has a reverse transcription system (compatible
reverse
transcription and primer binding sites) and an integration system (compatible
integrase and
~ 5 integration sites).
Ln accordance with the present invention, it is possible to manipulate the
viral genome or
the retroviral vector nucleotide sequence, so that viral genes are replaced or
supplemented
with one or more NOIs. The NOI(s) may be any one or more of selection gene(s),
marker
~o ~~ene(s) and therapeutic gene(s). Many different selectable markers have
been used
successfully in retroviral vectors. These are reviewed in "Retroviruses" (1997
Cold Spring
Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 444) and
include,
but are not limited to, the bacterial neomycin and hygromycin
phosphotransferase genes
which confer resistance to 6418 and hygromycin respectively; a mutant mouse
25 dihydrofolate reductase gene which confers resistance to methotrexate; the
bacterial gpt
gene which allows cells to grow in medium containing mycophenolic acid,
xanthine and
aminopterin; the bacterial hisD gene which allows cells to grow in medium
without
histidine but containing histidinol; the multidrug resistance gene (mdr) which
confers
resistance to a variety of drugs; and the bacterial genes which confer
resistance to
3o puromycin or phleomycin. All of these markers are dominant selectable and
allow
chemical selection of most cells expressing these genes.
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The composition of the present invention may be useful for inter alia medical
applications -
such as diagnostic or therapeutic applications.
s Thus, in accordance with the present invention, the NOI can be a therapeutic
gene - in the
sense that the gene itself may be capable of eliciting a therapeutic effect or
it may code for a
product that is capable of eliciting a therapeutic effect.
Non-limiting examples of therapeutic NOIs include genes encoding tumour
supressor
t o proteins, cytokines, anti-viral proteins, immunornodulatory molecules,
antibodies,
engineered immunoglobulin-like molecules. fusion proteins, hormones, membrane
proteins,
vasoactive proteins or peptides, growth factors, ribozymes, antisense RNA,
enzymes, pro-
drugs, such as pro-drug activating enzymes, cytotoxic agents, and enzyme
inhibitors.
~ s Examples of prodrugs include but are not limited to etoposide phosphate
(used with alkaline
phosphatase; 5-fluorocytosine (with cytosine deaminase); Doxorubin-N-p-
hydroxyphenoxyacetamide (with Penicillin-V-Amidase); Para-N-bis (2-
chloroethyl)aminobenzoyl glutamate (with Carboxypeptidase G2); Cephalosporin
nitrogen
mustard carbamates (with B-Iactamase); SR4233 (with p450 reductase);
Ganciclovir (with
2o HSV thymidine kinase); mustard pro-drugs with nitroreductase and
cyclophosphamide or
ifosfamide (with cytochrome p450).
Diseases which may be treated include, but are not limited to cancer, heart
disease, stroke,
neurodegenerative disease, arthritis, viral infection, bacterial infection,
parasitic infection,
25 diseases of the immune system, viral infection, tumours, blood clotting
disorders, and
genetic diseases - such as chronic granulomatosis, Lesch-Nyhan sysndrome,
Parkinson's
disease, empysema, phenyiketonuria, sickle cell anaemia, a-thalasemia, (3-
thalasemia,
Gaucher disease.
3o Target cells for gene therapy using retroviral vectors include but are not
limited to _
haematopoietic cells, (including monocytes, macrophages, lymphocytes,
granulocytes, or
progenitor cells of any of these); endothelial cells, tumour cells, stromal
cells, astrocytes,
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or glial cells, muscle cells, epithelial cells, neurons, fibrobiasts,
hepatocyte. astrocyte, and
lung cells.
Within the retroviral vector of the present invention, the one or more NOIs
can be under
the transcriptional control of the viral LTRs. Alternatively, a combination of
enhancer-
promoter elements can be present in order to achieve higher levels of
expression. The
promoter-enhancer elements are preferably strongly active or capable of being
strongly
induced in the target cells. An example of a strongly active promoter-enhancer
combination
is a human cytomegalovirus (HCMV) major intermediate early (MIE)
promoterlenhancer
i o combination. The promoter-enhancer combination may be tissue or temporally
restricted in
their activiy. Examples of a suitable tissue restricted promoter-enhancer
combinations are
those which are highly active in tumour cells such as a promoter-enhancer
combination
from a MUC1 gene or a CEA gene.
Hypoxia or ischaemia regulatable expression may also be particularly useful
under certain
circumstances. Hypoxia is a powerful regulator of gene expression in a wide
range of
different cell types and acts by the induction of the activity of hypoxia-
inducible
transcription factors such as hypoxia inducibIe factor-1 (HIF-1) (Wang and
Semenza 1993
PNAS. (USA) 90: 430) which bind to cognate DNA recognition sites, the hypoxia
~o responsive elements (HREs) on various gene promoters. A multimeric form of
HRE from
the mouse phosphoglycerate kinase-1 (PGK-1} gene has been used to control
expression of
both marker and therapeutic genes by human fibrosarcoma cells in response to
hypoxia in
vitro and within solid tumours in vivo (Firth er al 1994, PNAS 91(14): 6496-
b500; Dachs et
al 1997 Nature Med. 5: 515). Alternatively, the fact that glucose deprivation
is also
?5 present in ischaemic areas of tumours can be used to activate heterologous
gene DNA -
such as an NOI according to the present invention - expression especially in
tumours. A
truncated 632 base pair sequence of the grp 78 gene promoter, known to be
activated
specifically by glucose deprivation, has been shown to be capable of driving
high level
expression of a reporter gene in murine tumours in vivo (Gazit et al 1995
Cancer Res. 55:
30 1660.).
The retroviral vector genomes of the present invention for subsequent use in
gene therapy
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preferably contain the minimum retroviral material necessary to function
efficiently as
vectors. The purpose of this is to allow space for the incorporation of the
NOI(s), and for
safety reasons. Retroviral vector genomes are preferably replication defective
due to the
absence of functional genes encoding one or more of the structural (or
packaging)
components encoded by the gag pol and env genes. The absent components
required for
particle production are provided in traps in the producer cell. The absence of
virus
structural components in the genome also means that undesirable immune
responses
benerated against virus proteins expressed in the target cell are reduced or
avoided.
Furthermore, possible reconstruction of infectious viral panicles is
preferably avoided
to ~.vhere in vivo use is contemplated. Therefore, the viral structural
components are
preferably excluded from the genome as far as possible, in order to reduce the
chance of
any successful recombination.
The retroviral vector particles of the present invention are typically
generated in a suitable
producer cell.
Thus, the present invention also relates to a producer cell comprising the
composition of the
present invention.
2o A producer cell may be a packaging cell containing the virus structural
genes, normally
integrated into its genome. The packaging cell is then transfected with a
nucleic acid
encoding the vector genome, for the production of infective, replication
defective vector
particles. Alternatively the producer cell may be co-transfected with nucleic
acid sequences
encoding the vector genome and the structural components, andlor with the
nucleic acid
sequences present on one or more expression vectors such as plasmids,
adenovirus vectors,
herpes viral vectors, vaccinia viral vectors, or any method known to deliver
functional
DNA into target cells.
Suitable producer cells include insect cells and mammalian cells, but can be
other suitable
3o cells. Preferably, the producer cells are insect cells. ,
The present inver~ion also provides a pharmaceutical composition for treating
an individual
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by gene therapy, wherein the composition comprises a therapeutically effective
amount of
the composition according to the present invention or a viral particle
produced by or from
same. The pharmaceutical composition may be for human or animal usage.
Typically, a
physician will determine the actual dosage which will be most suitable for an
individual
s subject and it will vary with the age, weight and response of the particular
patient.
The composition may optionally comprise a pharmaceutically acceptable carrier,
diluent,
excipient or adjuvant. The choice of pharmaceutical carrier, excipient or
diluent can be
selected with regard to the intended route of administration and standard
pharmaceutical
to practice. The pharmaceutical compositions may comprise as - or in addition
to - the
carrier. excipient or diluent any suitable binder(s), lubricant(s), suspending
agentls),
coating agents j, solubilising agent(s), and other carrier agents that may aid
or increase the
viral entry into the target site (such as for example a lipid delivery
system).
i 5 Where appropriate, the pharmaceutical compositions can be administered by
any one or
more of: inhalation, in the form of a suppository or pessary, topically in the
form of a
lotion, solution, cream, ointment or dusting powder, by use of a skin patch,
orally in the
form of tablets containing excipients such as starch or lactose, or in
capsules or ovules
either alone or in admixture with excipients, or in the form of elixirs,
solutions or
.o suspensions containing flavouring or colouring agents, or they can be
injected parenterally,
for example intracavernosally, intravenously, intramuscularly or
subcutaneously. For
parenteral administration, the compositions may be best used in the form of a
sterile
aqueous solution which may contain other substances, for example enough salts
or
monosaccharides to make the solution isotonic with blood. For buccal or
sublingual
35 administration the compositions may be administered in the form of tablets
or lozenges
which can be formulated in a conventional manner.
The present invention provides in one aspect an expression vector comprising a
polynucleotide sequence which encodes a retroviral vector genome having a 5'
and a 3'
30 end, which retroviral vector genome is capable of being expressed and
packaged into a
retroviral vector particle in a baculovirus expression system. Hence, the 5'
and 3' ends
function in reverse transcription and integration.
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Three strategies are described herein for overcoming the problem of the
baculovirus
promoter having components upstream and downstream of the RNA transcription
start site.
These strategies are illustrated in Figures I to 3.
s
In a first strategy (see Figure 1 ), the downstream component of the promoter
is
incorporated into the R region at the upstream end of the sequence encoding
the retrovirai
vector genome (referred to as the 5' R region). This places the RNA
transcription start site
directly in front of the sequence encoding the retroviral vector genome.
Provided that a
1 o counterpart to the downstream component of the promoter is also
incorporated into the R
region at the downstream end of the sequence encoding the retroviral vector
aenome
(referred to as the 3' R region), so that the two R regions are identical or
sufficiently
similar for conversion of the vector RNA genome to the DNA provirus, the
vector genome
will function correctly.
In a second strategy (see Figure 2), a sequence capable of being cleaved in
the RNA
transcript is included in the vector between the bacuiovirus promoter and the
sequence
encoding the retroviral vector genome. The downstream component of the
baculovirus
promoter can thus be cleaved off once tile primary RNA transcript has been
produced.
3o Preferably. a cleavage site is present immediately adjacent the sequence
encoding the
retroviral vector genome, so that suitable vector genome ends are produced.
Ribozymes
are RNA enzymes which can perform such a function and can be engineered into
DNA
constructs. The structure and function of suitable ribozymes has been well-
studied (e.g.
Cech 1992 Curr. Op. Struct. Biol. 2, 605). Examples include hammerhead,
hairpin and
?5 hepatitis delta antigenomic ribozymes. Inclusion of a ribozyme sequence as
the RNA
cleavage sequence, with a cleavage site at the end of the sequence encoding
the retroviral
vector genome, can be used to give end-perfect RNA for all transcripts. Each
transcribed
molecule will contain the ribozyme sequence, and a single cleavage event in
ciS will be all
that is required for each ribozyme for correct template production.
In a third strategy (see Figure 3), a non-baculovirus promoter is employed.
Examples of
suitable promoters include the T7 phage promoter and the spb salmonella phage
promoter.
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For the T7 promoter, the terminal G residue of the 5' R region could be
positioned
precisely at the transcription start site of the T7 promoter, to give an
authentic 5' end
residue in the retroviral vector genome. A source of T7 polymerase would need
to be
provided, for example by a recombinant baculovirus expressing T7 polymerase
s (Polkinghorn and Roy 1995 Nucl. Acids Res. 23, 188).
Preferably, the expression vector encoding the retroviral vector genome is a
baculovirus
expression vector, that is, a vector based on a recombinant baculovirus
genome. Virus
vectors are in general easier to handle than non-viral vectors. For example,
the transfer of
o a virus vector into a cell is more reliably performed than transfection of a
cell with a DNA
plasmid. However, the expression vector may alternatively be a non-baculovirus
expression vector, or a non-viral expression vector, such as a DNA plasmid.
In an expression vector according to any of the three strategies described
above, there may
~ 5 be a RNA cleavage sequence located downstream of the sequence encoding the
retroviral
vector, to ensure correct termination of the RNA transcript and a correct 3'
end for the
vector genome. Preferably, the cleavage sequence has a cleavage site
immediately adjacent
the sequence encoding the retroviral vector genome, for cleaving at the 3' end
of the
genome. Ribozymes can provide suitable RNA cleavage sequences, as already
discussed
.o herein. Ribozymes may have a cleavage site either ~' or 3' to the ribozyme
sequence.
Thus, where there is a ribozyme for each end of the vector genome, these will
usually be
different. For example, the hammerhead ribozyme cleaves to the right of itself
and will
thus be suitable for the 5' end of the vector genome, while the hairpin
ribozyme cleaves to
the left and will be suitable for the 3' end of the vector genome.
2s
The invention provides in further aspects, a retroviral vector particle
production system
comprising an expression vector as described herein, in an insect cell, and
retroviral vector
particles produced by such systems.
3o For the production of retrovira! vector particles, packaging components
will need to be
provided. These will usually be gag pol and env, which may be provided on one
or more
suitable expression vectors. The expression vector or vectors will need to be
capable of
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expressing the packaging components in a baculovirus expression system. Thus,
the
packaging components will be under the expression control of one or more
baculovirus
promoters. The expression vector or vectors carrying the packaging components
are
preferably bacuiovirus expression vectors. Alternatively they may be non-
baculovirus or
non-viral expression vectors, such as plasmids. Some or all of the packaging
components
may be provided on the same expression vector as the retroviral vector genome.
Suitable baculovirus systems for use in the invention are easily available and
well known in
the art. Baculovirus expression vectors are described in detail in O'ReilIy et
al. 1994 (Op.
o cit). One suitable baculovirus for use in a recombinant baculovirus vector
is the well-
studied ~lcuographa californica nuclear polyhedrosis virus fAcMNPV). A
commercially
available recombinant baculovirus vector such as pBAC4 x- I (Novagen) may be
used.
pBAC4x-1 contains four insertion sites, and could be engineered to accommodate
nucleic
acid sequences encoding the retrovirai vector genome and the packaging
components.
is Similarly, suitable baculovirus promoters for use in the invention are well
known in the an.
Commonly-used baculovirus promoters are the polyhedrin and p10 promoters. The
DNA
sequence for a polyhedrin promoter is shown in Figure 18. The present
invention also
includes mutants, variants, homologues or fragments of that sequence.
o The terms "variant", "homologue" or "fragment" in relation to the nucleotide
sequence coding
for the preferred enzyme of the present invention include any substitution of,
variation of,
modification of, replacement of, deletion of or addition of one (or more)
nucleic acid from or
to the sequence providing the resultant nucleotide sequence codes far or is
capable of coding
for a promoter, preferably being at least as biologically active as the
sequence shown as in
35 Figure 18. In particular, the term "homologue" covers homology with respect
to structure
andlor function providing the resultant nucleotide sequence codes for or is
capable of coding
for a promoter. With respect to sequence homology, preferably there is at
least 75 % , more
preferably at least 85 % , more preferably at least 90% homology to the
sequence shown as
SEQ ID No. 1. More preferably there is at least 95 %a , more preferably at
least 98 % ,
3o homology to the sequence shown in Figure I8. ,
In particular, the term "homology'' as used herein may be equated with the
term "identity"
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Relative sequence homology (i.e. sequence identity) can be determined by
commercially
available computer programs that can calculate %a homology between two or more
sequences.
~ typical example of such a computer program is CLUSTAL.
s The terms "variant", "homologue" or "fragment" are synonymous with allelic
variations of
the sequences.
The term "variant" also encompasses sequences that are complementary to
sequences that are
capable of hybridising to the nucleotide sequence presented herein.
Preferably, the term
~ o w variant" encompasses sequences that are complementary to sequences that
are capable of
hybridising under stringent conditions (e.g. 6~°C and O.IxSSC {lx SSC =
0.15 M NaCI,
0.015 Na3 citrate pH 7.0}) to the nucleotide sequence presented herein.
The present invention also covers nucleotide sequences that can hybridise to
the nucleotide
15 sequence of the present invention (including complementary sequences of
those presented
herein). In a preferred aspect, the present invention covers nucleotide
sequences that can
hybridise to the nucleotide sequence of the present invention under stringent
conditions (e.g.
65°C and O.IxSSC) to the nucleotide sequence presented herein
(including complementary
sequences of those presented herein).
~o
In accordance with the present invention, other baculovirus promoters - as
well as mutants,
variants, homologues or fragments of those promoters - may also be used.
Insect cell lines for use with the expression vectors according to the
invention are also
?s readily available. One example is the sf8 insect cell line. The insect
cells used need to be
compatible with the particular expression vector chosen. The insect cells need
to support
replication of the expression vector encoding the retroviral vector genome,
and to support
expression of the retroviral vector genome. Certain glycosylation-deficient
cell lines may
be particularly suitable, because of differences in glycosylation between
mammalian and
3o insect systems. The glycosylation differences are not expected to present a
problem;
examples of clinical use such as clinical trials with HIV gpI20 produced in
insect cells,
indicate that any differences in secondary modifcations are not significant.
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PCT/GB98/01626
The types of retrovirus which can be used in the invention are not limited to
any specific
retrovirus. Oncoretroviruses such as the murine type-C virus MLV, or
lentiviruses such as
HIV, or other well-known retroviruses such as ASLV, SNV and RSV could be used.
The
~ invention is particularly useful for retroviral vectors based on
lentiviruses, as these have _
been very difficult to produce in high titres. Titres of lentiviral vectors
are commonly two
orders of magnitude lower than the murine vectors such as MLV (e.g. Naldini et
al. 1996
Science 272, 263). In addition, because the retroviral RNA can be so
efficiently produced
in baculovirus expression systems, it may be possible to omit from the
retroviral vector
~o <Tenome lentiviral elements such as HIV Rev andr RRE (Gheysen et al. 1989
Op. cit.). It is
advantageous to avoid unnecessary retroviral elements in retroviral vectors,
particularly
when using HIV, or other lentiviruses, because of the possible adverse effects
of these
elements.
~ 5 The present invention will now be described by way of example only, and
with reference to
the following Figures - in which:
Figure 1 is a schematic diagram;
Figure 2 is a schematic diagram;
o Figure 3 is a schematic diagram:
Figure 4 is a schematic diagram;
Figure 5 is a schematic diagram;
Figure 6 is a schematic diagram;
Figure 7 is a schematic diagram;
?s Figure 8 is a schematic diagram;
Figure 9 is a schematic diagram;
Figure 10 is a schematic diagram;
Figure 11 is a schematic diagram;
Figure 12 is a schematic diagram;
3o Figure 13 is a schematic diagram;
Figure 14 is a schematic diagram;
Figure 15 is a schematic diagram;
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Figure I6 is a schematic diagram;
Figure 17 is a schematic diagram;
' Figure 18 is a schematic diagram;
Figure 19 is a schematic diagram;
s Figure 20 is a schematic diagram;
Figure 21 is a schematic diagram;
Figure 22 is a schematic diagram;
Figure 23 is a schematic diagram;
Figure 24 is a schematic diagram;
t o Figure 25 is a schematic diagram;
Figure 26 is a schematic diagram:
Figure 27 is a schematic diagram;
Figure 28 is a schematic diagram; and
Figure 29 is a schematic diagram.
35
Figures l, 2, 3 and 18 have been referred to in the above text.
EXAMPLE 1
PCT/GB98/01626
2o EXPRESSION OF MLV gag-pol AND ENVELOPE GENES IN A BACULOVIRUS
EXPRESSION SYSTEM
The strategy is to first insert the envelope sequences into pBAC4x-1
(Novagen), followed
by insertion of the MLV gag-pol sequences, which will first be reconstructed
in pBluescript
2s (Stratagene).
a) Env expression (MLV ecotropic or VSV G)
For the MLV ecotropic envelope sequence, the EcoRI fragment which contains the
entire
3o env sequence from pHIT123 (Soneoka et al Op.Cit) is isolated. For the VSV-G
sequence,
an EcoRI fragment from pHCMV-G (Yee et al., 1994 PNAS 91, 9564) is also
isolated.
These fragments are then inserted into the EcoRI site of pBAC4x-1 (Novagen)
(Figure 4) to
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produce pBAC4-env-a and pBAC4-env-v respectively. Any viral envelope gene
could be
inserted into this vector in similar ways using technology known in the field
of recombinant
DNA. Envelope genes might include those from any retrovirus or any other virus
capable
of pseudotyping or otherwise being incorporated into particles produced from
retroviral
gag pol genes.
b) Reconstruction of MLV gag-pol in pBluescript (Figure ~)
~' and 3' fragments from pgagpolgpt (Markowitz et al., 1988 J. Virol. 62, I
120) are
produced by PCR (see primer list). The 5' fragment is amplified up to the XhoI
site and it
creates kpnI and NotI sites at the beginning of the coding sequence. to
generate a 940bp
fragment. The 3' fragment is amplified from the SphI site of the gag pol
sequence to the
end of the pol coding sequence, generating a 700bp fragment. EcoRI sites are
created at
both ends of this fragment and a NotI site only at the 3' end, so that when
the entire gag-
s 5 pol sequence is reconstructed, it can be excised by digestion with NotI
and inserted into
pBAC4x-1 (Novagen) at the NotI site.
The 5' fragment is inserted into pBluescript at the KpnI/XhoI site, and the 3'
fragment into
the EcoRI site.
~o
The XhoI-SphI (30bp) fragment is then excised from the above plasmid and
replaced with
an XhoI-SphI fragment (3580bp) from pgagpolgpt to reconstruct the entire gag-
pol sequence
in pBluescript to create pBluescript-gagpol.
?s c) Insertion of MLV gag-pol sequences into pBAC4-env to create pBAC4-
gagpolenv
(Figure 6)
The NotI fragment containing the gag-pol sequences from pBluescript-gagpol is
isolated and
inserted into the NotI site of pBAC4-env-a and -v to create pBAC4mgagpol-env-a
and
3o pBAC4mgagpol-env-v for the MLV envelope version and the VSV-G version
respectively. ,
These plasmids are used as baculovirus transfer vectors using standard methods
as
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described in O'Reilly et al. (Op.Cit.). The resulting virus preparations are
designated
bBAC4mgagpol-env-a and bBAC4mgagpol-env-v respectively. When these viruses are
used to infect insect cells such as sf8 cells large numbers of VLPs are
produced.
EXAMPLE 2
EXPRESSION OF AN MLV-BASED VECTOR GENOME USING THE T7
STRATEGY (Figures 3 and 'n
In this example the vector genome is expressed from a plasmid that will be
used in a
~ o transient expression system. The genome could be expressed from the same
recombinant
baculovirus as the aaa-pol and env genes.
The MLV ~enome derived from pLZSN (Adams et al. , 1991 J. Virol. 65, 4985) is
inserted
into pEc-Hd (Polkinghorn 1996 DPhil. Thesis, University of Oxford) at the
EagIISmaI
sites. The genome that is placed into this vector has the following structure:
The 5' and 3' ends of the genome are amplified by PCR so that the T7 promoter
sequence
is placed immediately upstream of the 5' R region. The 3' end is amplified up
to the end of
the 3' R sequence, generating a blunt-ended product so that it will be
precisely fused to the
~o hepatitis delta antigenomic ribozyme motif. A proof-reading polymerase is
used for PCR to
generate the blunt-ended product.
The 5' end of the genome is amplified up to the EagI site in the packaging
signal by PCR.
An additional EagI site is created at the very 5' end for insertion into the
EagI site of pEc-
35 Hd. The 3' end of the genome is amplified from the SmaI site in the 3' R
sequence, up to
the very end of the R sequence.
The 3' fragment is inserted first into pEc-Hd at the SmaI site (a SmaI site
will not be
regenerated at the very 3' end), then the 5' fragment is inserted into the
EagI site.
The SrnaI fragment from above is excised and replaced with the SmaI fragment
(5850bp)
from pLZSN to create pEc-Hd-LZSN. The same expression cassette could be
assembled
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into a baculovirus transfer vector with the gag-pol and env sequences by
standard
procedures.
Plasmid pEc-Hd-LZSN can be used to transfect sf21 cells that are coinfected
with
s bBAC4mgagpol-env-a or bBAC4mgagpol-env-v and Bac-T7 which expresses the T7 »
polymerase. In these cells alt the components of a retroviral vector are
expressed and so
the production of functional vectors can be tested. High titres of functional
vectors are
produced that transfer the lacZ gene to target cells.
to Primers for ivILV gag-pol, env and VSV-G constructions:
To amplify the MLV gag-pol sequences:
IS
Forward (35MER NOTFOR)
5'-CGG .C~TA . C . ATGGGCCAGACTGTTACC-3
Kpn NotI
.o Reverse (22MER YHOREV)
5'-GGGCG ~'~~ GGGAAAAGCGG-3'
XhoI
2s Forward (27MER SPHFOR)
S'-CCG GAATT'C s~CArGC CTCAGGTATTGG-3
EcoRI SphI
Reverse (33MER NOTREV)
30 5'-TTT ~~ ~ ~ ~ TTAGGGGGCCTCGCGG-3'
EcoRI NotI
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To amplify the genome (pLZSI~:
39
s Forward (44MER EAGFOR )
5'-GCG T ~ ~ TAATACGACTCACTATA GCGCCAGTCTTCCGATAG-3'
EagI T7 promoter
Reverse (18MER EAGRE'~
i o 5'-TTG CGGCCG GGTGTTCAG-3'
EagI
3' fragment
1 s Forward (20MER SMAFOR)
5'-TCG CCCGGG TACCCGTGTAT-3'
SmaI
Reverse (22MER SMARE~
2o S'-TGCAACTGCAAGAGGGTTTATT-3'
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EXAMPLE 3
PCT/GB98/01626
EXPRESSION OF HIV gag-pol and VSV-G IN A BACULOVIRUS EXPRESSION
SYSTEM (Figure 8).
5
The source of the HIV gag-pol sequences is plasmid pRV664. This is a pWI3 (Kim
et al.,
1989 J. Virol. 63, 3708) derived gagpol vif expression pIasmid. The RRE of
pWI3
(Genbank Accession number: U26942) is inserted by blunt-ending the Sty IISty I
fragment
(7720-8050) into Sma I cut pBluescript KS+ (Stratagene) thereby creating
pBSRRlJ. The
t o Nar IIEco RI fragment of pWI3 (637-5743) is inserted into pBSRRE cut with
Cla I and Eco
RI to create pBSGPRREl. The Xho IlNot I fragment (containing gagpot and RRE)
is
inserted into the expression plasmid pCI-Neo to create pGR-RRE1.
A 5.6kb XhoI-NotI fragment containing the HIV-I gag-pol sequences from pRV664
is
t 5 isolated and blunt ended with the Klenow fragment of DNA polymerase I.
This is then
inserted into the SmaI site of pBAC4-env-v to produce pBAC4hgagpol-env-v. This
is used
in the same way as the plasmids described in Example 1 were used.
EXAMPLE 4
zo EXPRESSION OF AN HIV-BASED VECTOR GENOME USING THE T7
STRATEGY (Figures 3 and 9 - 12)
The genome from pH4nZ has the structure as shown in Figure 9. It is produced
as follows.
HIVdge is made from HIVgpt (Page et al. , I990 J. Virol. 64, 5270) by blunt-
ending the Cla
25 I site (829) to create a frameshift mutation. HIVdge is then cut with Bgl
II and Pst I (473-
1414) and inserted into pTIN406 (Cannon et al. Op. City. pTIN406 has an LTR
structure
of CMV, R (HIV) and US (MLV). This creates a hybrid LTR containing CMV, and R,
US
from HIV called pBSS' . To provide the plasmid with Rev and RRE the Eco RIlXho
I
fragment (5743-8897) is cut from HIVdgel.2 which is a HIVdge derivative
containing a
~o deletion from Nde I to Bgl II (6403-7621) and is inserting into pBSS' to
create pBSS'R.
The 3' LTR is provided by inserting the Not IlXho I fragment of pBS3' into
pBSS'R
creating pH2. pBS3' is created by a three way Iigation of the Xho IlHind III
fragment of
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pWI3, the Hind IIiIKpn I fragment of pTIN408 (Cannon et al. Op. Cit.) and
pBluescript
KS+ cut with Xho IIKpn I. A CMV promoter fragment (Sal IIXho I) from pSPCMV is
inserted into the unique Xho I site of pH2 making pH2CMV. pSPCMV is created by
inserting the Pst ilHind III fragment from pLNCX (Genbank Accession number:
M28246)
into pSP72 (Piomega). The ~i-galactosidase gene is inserted from pTIN414
(Cannon et al.
Op.Cit.) into pSP72 (Xho IISph I) to make pSPlacZ. A Xho IISaI I digest of
pSPlacZ gives
the (3-galactosidase coding region which is inserted into pH2-CMV to give
pH3Z. pH4Z is
constructed to create tat-deficient vector. The first 50 by of the tat-coding
region is
removed by replacing EcoRI (5743)I-SpeI fragment in pH3 with EcoRI (588I)-SpeI
PCR
o product amplified using PCR primers
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DELTS (5'-CGTGAATTCGCCTAAAACTGCTTGTACCA-3') and
DELT3 (5'-GAACTAATGACCCCGTAATTG-3')
s
to create pH4.
PCT/GB98/01626
The Nsi IISpe I fragment from pH4 is inserted into pH3Z to generate pH4Z.
to Two PCR reactions are performed to amplify the 5' and 3' ends of the genome
for
baculovirus expression. Subsequently, the entire genome is reconstructed by
inserting the
missing intervening sequences. A PCR reaction is performed to amplify the 3'
sequences
of the genome. The ScaI site in the 3' U3 sequence is changed to a SmaI site
and amplified
to the very end of the R sequence. This change creates a 3 base pair mutation
but should
t s not affect integration. A proof-reading polymerase is used to create the
900bp blunt-ended
product. This product is then inserted into the SmaI site of pEc-Hd (Figure
10). This
destroys the SmaI site at the junction of the R sequence and pEc-Hd.
The S' sequences are first amplified from the beginning of the R sequence up
to the first
2o EcoRI sequence located in the packaging signal. This produces a 900bp
fragment. An EagI
site is placed at both ends of the product and a XhoI site at the very 5' end
of the fragment.
This product is inserted into the XhoI-EcoRI site of pBluescript KS
(Stratagene) (Figure
I1).
2s The sequences spanning the EcoRI site and the SpeI site (in the CMV
promoter) are isolated
from pH4nZ and inserted into the EcoRI-SpeI site of the above plasmid (Figure
11).
The EagI-SpeI fragment containing the genome sequences is then isolated from
pBluescript
KS and inserted into the EagI-Spel sites of pEc-Hd (Figure I2).
The entire genome is finally reconstituted by insertion of the SpeI-ScaI
fragment (3.9kb)
isolated from pH4nZ into the SpeI-Smal site of pEc-Hd (Figure I2) to produce
pEc-Hd-
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H4nZ. This plasmid can be used in a similar way to pEc-Hd-LZSN but with a
baculovirus
vector derived from pBAC4hgagpol-env-v to produce HIV-based retroviral vectors
at very
. high titre.
s EXAMPLE 5
EXPRESSION OF EIAV gag-pol AND VSV-G IN A BACULOVIRUS SYSTEM
(Figure 13)
The starting molecule is pSPEIAVI9 (AC:U01866) which is a proviral clone of
EIAV.
i o The envelope encoding region is disrupted by cutting pSPEIAV 19 with Hind
III
(5835/6571) within the envelope region and self ligating to produce
pSPEIAVI9dH. This
creates an envelope minus proviral clone. pSPEIAV l9dH is then cut with Mlu I
(216/8124)
and the resulting fragment inserted into pCI-Neo (Promega) cut with Mlu I
(216) to make
pONY3.
is
The Mlul fragment from pONY3 containing the EIAV gag-pol, tat and rev coding
sequences is isolated and blunt ended with the Klenow fragment of DNA
polymerase I.
This is then inserted into pBAC4-VSVenv at the Smal site (blunt-ended) to
produce
pBAC4egagpol-env-v. This pIasmid is used in a similar way to that described in
Example 1
2o to produce a recombinant baculovirus that will produce high levels of EIAV
VLPs.
EXAMPLE 6
EXPRESSION OF AN EIAV-BASED VECTOR GENOME USING THE T7
STRATEGY (Figures 3 and 14-1'n
2s
The genome of plasmid pONY2.1 nlslacZ has the structure as shown in Figure 14.
This is
constructed as follows. The 5' LTR of EIAV clone pSPEIAV19 is PCR amplified
using
pfu polymerase with the following primers:
. 30 5'GCATGGACCTGTGGGGTTTTTATGAGG
3'GCATGAGCTCTGTAGGATCTCGAACAGAC
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The amplified fragment is blunt ended by 5' overhang fill-in and is inserted
into pBluescript
II KS+ which has been cut with Bss HII and blunt ended by 3' overhang removal.
This
construct is called pONYI and the orientation is 5' to 3' in relation to ø-
galactosidase of
pBluescript II KS+. Sequencing of pONYI revealed no mutations. Plasmid
pSPEIAV l9dH is cut with Mlu I (21618124) and the fragment is inserted into
pONY 1 Mlu I
cur (216) to make pONY2. A Bss HII digest (6191792) of pBluescript II KS+ is
carried
out to obtain the multiple cloning site. This is blunt ended by 5' overhang
fill-in and
ligated to pONY2 cut with Bgl II and Nco I (190114949) and blunt ended by 5'
overhang
o fill-in. The orientation is 3' to ~' in relation to the EIAV sequence. This
is called
pONY2.1 Plasmid pSPCMV is created by inserting the PstiIHindIII fragment from
pLNCX (Genbank Accession number: M28246) into pSP72 (Promega). The ø-
galactosidase gene is inserted from pTIN414 {Cannon et al. Op.Cit.) into pSP72
cut with
Xho I and Sph I to make pSPlacZ. The 5' end to the ø-galactosidase gene is
replaced by
is the SV40 T antigen nuclear localisation signal from pAD.RSVBgaI (Bloggs et
al., 1992
J.CIin.Invest. 90, 626). pAD.RSVbgal is cut with Xho II Cla I and the fragment
inserted
into Xho II Cla I cut pSPlacZ to make pSPnlslacZ. The Pst I fragment
containing the CMV
promoter driving the lacZ gene from pSPnlslacZ is inserted into the Pst I site
of pONY2.1
in the 5' to 3' orientation of EIAV. This is designated pONY2.1nIslacZ .
?0
Two PCR reactions are then performed using pONY2.1n1s1acZ as template to
amplify the
5' and 3' ends of the genome for the baculovirus expression cassette.
Subsequently, the
entire genome is reconstructed by inserting the missing intervening sequences.
25 The 3' PCR product is amplified from the SmaI site in the env sequence to
the very end of
the R sequence using a proof-reading polymerase to produce blunt-ended
products. The
product is then inserted into the SmaI site of pEc-Hd. The very 3' end loses
the SmaI site.
(Figure 15).
o The SmaI fr:~ment containing the CMVnlslacZ sequences is isolated from
pONY2.1n1slacZ
and inserted into the SrnaI site (Figure I6).
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The 5' PCR product is amplified up to the EagI site in the MCS and an EagI
site is added
at the 5' end. This product is then inserted into the EagI site to
reconstitute the pONY
' genome in pEc-Hd (Figure I7) thereby creating pEc-Hd-ONY2.1n1s1acZ. This can
be used
in conjunction with pBAC4egagpol-env-v to produce high titres of EIAV-based
vectors in a
5 similar way to that described in Examples 2 and 4.
EXAMPLE 7
EXPRESSION OF AN MLV-BASED VECTOR GENOME USING THE R-REGION
REPLACEMENT STRATEGY (FIGURES 1, 18 and 19).
~o
The plasmid containing the LZSN retroviral vector genome expressed from the
poiyhedrin
promoter (O'Reilly et at. Op.Cit.) is called pBHLZSN (Figure 19). This plasmid
is
constructed as follows: The starting template for a series of PCR reactions is
pHITlll
(Soneoka et al., 1996 Op.Cit.). The 5'LTR containing the poIyhedrin promoter
is created
15 using PCR primers in reaction A to produce a PCR product shown as PCR:A
(Figure 19).
A second reaction (B) creates a fragment that overlaps with PCR:A. Reaction C
then
produces a combined fragment that is cleaved with SaII and HinaZII. The
resulting
fragment is then inserted into pBluescriptKS+ to produce plasmid pBHR (Figure
18). In
order to construct the 3' LTR with the appropriate component of the polyhedrin
promoter
3o within the newly constructed R region PCR reactions D and E (Figure 19) are
carried out.
The products of these reactions are then used to produce a combination PCR
product
(PCR:F) which is cut with XbaI and NotI and the resulting fragment is inserted
into pBHR
to produce pBHU3HR (Figure 19). The final stage is to then insert the internal
region of
LZSN from pHITIll by cutting pHITlll with SpeI and XbaI and inserting the
resulting
25 fragment into pBHU3HR cut with the same enzymes (Figure 19).
Primers used for PCR reactions for construction of pBHLZSN
A.5' Primer (polyhAS)-TACT GTC'~ ATA ACC ATC TCG CAA ATA AAT
30 (Underline = Sal I); 3' Primer (PolyhA3)-CAG TCT ATC GGA AGA CTG GCG CT ATT
TAT AGG TTT TTT TAT
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B. 5' Primer (polyhBS)- ATA AAA AAA CCT ATA AAT AGC GC CAG TCT TCC GAT
AGA CTG;3' Primer (polyhB3)- GCTA AAGC'IZ TCC GCC AGA TAC AGA GCT
(Underline = Hind III)
C.Use primer polyhAS and PoIyhB3 with PCR products A and B to obtain the 5'
LTR
hybrid (polyderin.R)
D. 5' Primer (polyhDS)- AGTT T 'TA A GAA CCA TCA GAT GTT TCC AGG
(Underline = Xba I); 3' Primer (PolyhD3)- TAC AAA ACT GTT ACG AAA ACA GTA
t o AAA TAC TT C CCG AGT GAG GGG TTG TGG
E. S' Primer (polyhES)- TTC GTA ACA GTT TTG TAA TAA AAA AAC CTA TAA
ATA GCG CCA GTC CTC CGA TTG; 3' Primer (polyhE3)- GATC GCGG
AAT GAA AGA CCC CCG CTG (Underline = Not I)
is
F. Use primer polyhDS and PolyhE3 with PCR products D and E to obtain the 3'
LTR
hybrid (U3.Polyherin.R)
EXAMPLE 8.
EXPRESSION OF AN EIAV-BASED VECTOR GENOME USING THE R-REGION
REPLACEMENT STRATEGY.
The plasmid containing the pONY2.1n1s1acZ retroviral vector genome expressed
from the
polyhedrin promoter with appropriate R=-region replacements is called
pBONY2.1n1s1acZ.
This plasmid is constructed as follows: The starting template for a series of
PCR reactions
is pONY2.1n1slacZ (see Example 6 and GB patent application number 9727135.7
and GB
patent application number 9711578.6). The 5' LTR containing the polyhedrin
promoter is
created using PCR primers in reaction AE to produce a PCR product shown as
PCR:AE. A
3o second reaction (BE) creates a fragment that overlaps with PCR:AE. Reaction
CE then
produces a combined fragment that is cleaved with Sal I and Hind III. The
resulting
fragment is then inserted into pBluescript KS+ to produce plasmid pBEHR. In
order to
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constuct the 3'LTR with the appropriate component of the polyhedrin promoter
within the
newly constructed R region PCR reactions DE and EE are carried out. The
products of
these reactions are then used to produce a combination PCR product (PCR:FE)
which is cut
with Xba I and Not I and the resulting fragment is inserted into pBEHR to
produce
s pBEHU3HR. The final stage is to then insert the internal region of
pONY2.1n1s1acZ by
cutting it with Nar I and Nsp V and inserting the resulting fragment into
pBEHU3HR cut
with the same enzymes (see the commentary below and Figure 20). Plasmid
pBONY2.1n1s1acZ can be used in conjunction with pBAC4egagpol-env-v to produce
high
titres of EIAV-based vectors. (Reference may also be made to Figure 2I).
io
Construction of pONY2.InIsLacZ
A.
15 5' Primer (polyhAES)
TACT GTCGAC ATA ACC ATC TCG CAA ATA AAT (Underline = Sal I)
3' Primer (PolyhAE3)
zo
AGA CCG CAG AAT CTG AGT GCCC T ATT TAT AGG TTT TTT TAT
This PCR product will give the polyhedrin promoter with pan of R
25 B.
5' Primer (polyhBES)
ATA AAA AAA CCT ATA AAT AGG GCA CTC AGA TTC TGC GGT CTG
3' Primer (polyhBE3)
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ACTG AAGCTT CAG GTC CCT GTT CG S~~ A ACT G (Bold = Hind III,
Underline = Nar I)
This PCR will give part of polyhedrin with R.
C.
Use primer poIyhAES and PolyhBE3 with PCR products AE and BE to obtain the 5'
LTR
hybrid (polyderin.R)
Insert into pBluescrip KS+ II by cutting with Sal I and Hind III called pBEHR.
3' LTR (U3.polyhedrin.R.US).
D. Within U3 to R but including Polyhedrin promoter
5' Primer (polyhDES)
CTG TCTAGA A GA TTCGAA_GCG AAG GAGGAAA C... (Underline = Xba I, bold
?o NspV)
3' Primer (PolyhDE3)
TAC AAA ACT GTT ACG AAA ACA GTA AAA TAC TT
?5 ATTGTCAGAATACAAGCACT
This PCR will give U3 with part of polyhedrin promoter.
E. R TO US with polyhedrin promoter
5' Primer (polytlEES)
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TTCGTA ACA GTT TTG TAA TAA AAA AAC CTA TAA ATA
GGCACTCAGATTCTGCGGTC
3' Primer (polyhEE3)
s
GATC C~_(~C~~ CTGTAGGATCTCGAACAGACAAAC (Underline = Not I)
This PCR will give pan of polyhedrin promoter with R.
~o F.
Use primer polyhDES and PolyhEE3 with PCR products DE and EE to obtain the 3'
LTR
hybrid (U3.Polyhedrin.R)
Insert into pBEHR by cutting wtih Xba I and Not I called pBEHRU3HR
G.
Cut pONY2.inlslacZ with Nar I and Nsp V to obtain a 7.8 kb fragment and insert
into
pBEHRU3HR via Nar I and Nsp V to create pBEHONYnlslacZ.
?o EXAMPLE 9.
EXPRESSION OF AN HIV-BASED VECTOR GENOME USING THE R-REGION
REPLACEMENT STRATEGY
The plasmid containing the pH4Z retroviral vector genome expressed from the
polyhedrin
promoter is called pBH4Z. This plasmid is constructed as follows: The starting
template for
a series of PCR reactions is pH4Z (see PCTIGB 97/02857 and GB patent
application
- number: 9711578.6). The 5' LTR containing the polyhedrin promoter is created
using PCR
primers in reaction AH to produce a PCR product shown as PCR:AH. A second
reaction
' 30 (BH) creates a fragment that overlaps with PCR;AH. Reaction C then
produces a combined
fragment that is cleaved with Sal I and Hind III. The resulting fragment is
then inserted into
pBluescript KS+ to produce plasmid pBHHR. In order to constuct the 3'LTR with
the
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appropriate component of the polyhedrin promoter within the newly constructed
R region
PCR reactions DH and EH are carried out. The products of these reactions are
then used to
produce a combination PCR product (PCR: FH) which is cut with Xba I and Not I
and the
resulting fragment is inserted into pBHHR to produce pBHHU3HR. The final stage
is to
5 then insert the internal region of pH4Z by cutting it with Nar I and Sph I
and inserting the
resulting fragment into pBHHU3HR cut with the same enzymes (see the commentary
below
and Figure 22). Plasmid pBH4Z can be used in conjunction with pBAC4hgagpol-env-
v to
produce HIV-based vectors at high titres. (Reference may also be made to
Figure 23.)
i o Construction of pBH4Z
A.
5' Primer (polyhAHS)
TACT GTCC'~ ATA ACC ATC TCG CAA ATA AAT (Underline = Sal I)
3' Primer (PolyhAH3)
~o AGA CCG CAG AAT CTG AGT GCCC T ATT TAT AGG TTT TTT TAT
This PCR product will give the polyhedrin promoter with pan of R
B.
5' Primer (polyhBHS)
ATA AAA AAA CCT ATA AAT AGG GCA CTC AGA TTC TGC GGT CTG
3' Primer (polyhBH3)
ACTG AAGCTT GGT CCC TGT TCG ~~ AC
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PCT/GB9810I626
(Bold = Hind III, Underline = Nar I)
' This PCR will give pan of polyhderin with R.
C.
Use primer polyhAHS and PolyhBH3 with PCR products AH and BH to obtain the 5'
LTR
hybrid (polyderin.R)
1 o Insert into pBluescrip KS + II by cutting with Sal I and Hind III called
pBHHR.
3' LTR (U3.polyhedrin.R.US).
D. Within U3 to R but including Polyhedrin promoter
S' Primer (polyhDHS)
GATC TCTAGA A AAGCATGCCTGCAGGTCGAGGTCGAT... (Underline = Xba I,
bold Sph I)
3' Primer (PolyhDH3)
TAC AAA ACT GTT ACG AAA ACA GTA AAA TAC TT AGT ACA GGC AAA AAG
CAG CTG C
This PCR will give U3 with pan of polyhedrin promoter.
- E. R TO US with polyhedrin promoter
3o S' Primer (polyhEHS)
TTCGTA ACA GTT TTG TAA TAA AAA AAC CTA TAA ATA
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GGGTCTCTCTGGTTAGAC
3' Primer (polyhEH3)
52
PCT/GB98/01626
s GATC TGC TAG AGA TTT TCC ACA CTG (Underline = Not I)
This PCR will give pan of polyhedrin promoter with R.
F.
~ o Use primer polyhDHS and PolyhEH3 with PCR products DH and EH to obtain the
3' LTR
hybrid (U3.Polyherin.R)
Insert into pBHHR by cutting wtih Xba I and Not I called pBHHRU3HR
t s G.
Cut pH4Z with Nar I and Sph I to obtain a 5.7 kb fragment and insert into
pBHHRU3HR
via Nar I and Nsp V to create pBH4Z.
EXAMPLE 10
EXPRESSION OF FUNCTIONAL MLV, EIAV AND HIV VECTOR GENOMES
FROM A POLYHEDRIN PROMOTER: THE DOUBLE RIBOZYME CLEAVAGE
METHOD.
This strategy is achieved by the inclusion of hammerhead ribozymes downstream
of the
polyhedrin promoter sequence designed such that they delete both themselves
and the 5'
polyhedrin sequence from the transcript to which they are joined. Outlined in
Figures 24,
25 and 26 are schematic descriptions of how this is achieved for MLV, EIAV and
HIV
vectors respectively.
The strategy is the same in alI three instances with only the ribozyme helix i
sequence
differing to match the respective complementary R sequences of MLV, EIAV and
HIV.
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Upon cleavage by the ribozyme at the indicated positions the resulting RNA
transcripts now
contain the correct R sequences at their five prime end.
The cloning strategy for the construction of each of these self cleaving
polyhedrin promoter
s based retroviral genomic expression vectors will now be outlined in turn.
Each of these can
be used in conjunction with theeeir cognate gagpol and env expression systems
to produce
high titre retroviral vector preparations. In addition their genome expression
cassettes can
be inserted into baculovirus vectors by standard procedures.
~ o (i) Construction of the MLV based vector:
Primers polyhAMS and polyhAM3 (see Figure 27 - and the commentary below) are
used to
PCR amplify the S'LTR of pEc-Hd-LZSN to incorporate the required changes
(polyhedrin
promoter/ribozyme sequence addition flush to the 5' R sequence) into a PCR
fragment
is which is then cloned into pEc-Hd-LZSN by EcoRl- Spel digestion to produce
the finished
vector- pBHz-Hd-aR.
Construction of pBHz-HS-aR
5' Primer (polyhAMS)
actg g~ATAACCATCTCGCAAATAAATAAGTA
2s TTTTACTGTTTTCGTAACAGTTTTGTAATAAA.AA
AACCTATAAATA GGACTGGCGC CTGATGAGCG
GCCGAAAGCCCGCGAAACCTGCGTCGACACGC
' 30
AGGTC GCGCCAGTCCTCCGATTGACTGAGTC
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(Underlined = EcoR I site)
3' Primer (poIyhAM3)
54
s GTTAGCTA ACTAGT ACAGACGCAG (Underline = Spe I)
PCT/GB98/01626
This PCR will give polyhderin, hammerhead ribozyme, MSV R region, US and
leader.
This fragment can be cloned into pEc-Hd- LZSN at the EcoR I and Spe I sites to
give
t o pBHz-H8-aRLZSN.
(ii) Construction of the EIAV based vector:
Primers polyhAEMS and polyhAEM3 (see Figure 28 - and the commentary below) are
used
t5 to PCR amplify the 5'LTR of pEc-Hd-ONY2.1n1slacZ to incorporate the
required changes
(polyhedrin promter/ribozyme sequence addition flush to the 5' R sequence)
into a PCR
fragment which is then cloned into pEc-Hd-ONY2.1n1s1acZ by Eag 1 digestion to
produce
the finished vector- pBEHz-H8-aR.
?o Construction of pBEHz-H8-aR
5' Primer (polyhAEMS)
actg~ ATAACCATCTCGCAAATAAATAAGTATTTT
ACTGTTTTCGTAACAGTTTTGTAATA,~~AAAAACCTAT
AAATA CTGAGTGCCC CTGATGAGCGGCCGA
3o AAGCCCGCGAAACCTGCGTCGACACGCAGGTC
GGGCACTCAGATTCTGCGGTCTG
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(Underlined = Eag I site)
3' Primer (polyhAEM3)
' S
CTAGTTCTAGAG~S~CCAC (Underline = Eag I)
PCT/GB98101626
This PCR will give polyhedrin, hammerhead ribozyme, EIAV R region, US and
leader.
to This fragment can be inserted into pEc-Hd-ONY2.1n1slacZ at the Eag I and
Eag I sites to
dive pBEHz-H8-aR.
(iii) Construction of the HIV based vector:
15 Primers polyhAHMS and polyhAHM3 (see Figure 29 - and the commentary below)
are
used to PCR amplify the 5'LTR of pEc-Hd-H4nZ to incorporate the required
changes
(polyhedrin promter/ribozyme sequence addition flush to the 5' R sequence)
into a PCR
fragment which is then cloned into pEc-Hd-H4nZ by Eagl-Narl digestion to
produce the
finished vector- pBHHz-H8-aR.
Construction of pBHHz-H8-aR
5' Primer (polyhAHMS)
2s ACTGTTTTCGTAACAGTTTTGTAATA,AAAAAACCTAT
Hammer head Rz
AAATA AGAGAGACCC CTGATGAGCGGCCGA
AAGCCCGCGAAACCTGCGTCGACACGCAGGTC
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R
GGGTCTCTCTGGTTAGACCAGATC ...
(Underlined = Eag I site)
s
3' Primer (polyhAHM3)
56
CCCTGTTCGGGCGCCACTGC (Underline = Nar I)
PCT/GB98/01626
to This PCR will give polyhderin, hammerhead ribozyme, HIV R region, US and
leader.
This fragment can be inserted into pEc-Hd- H4nZ at the Eag I and Nar I sites
to give
pBHHz-H8-aRLZSN.
is Summary
Thus, the present invention provides a novel system for producing retroviral
vector
particles. In a highly preferred embodiment, the novel system uses a
bacuiovirus
expression vector encoding a retroviral vector genome. In another preferred
embodiment,
?o the present invention provides a baculovirus expression vector encoding a
retroviral vector
genome, and to retroviral vector panicles produced by the novel system of the
invention.
All publications mentioned in the above specification are herein incorporated
by reference.
Various modifications and variations of the described methods and system of
the invention
is will be apparent to those skilled in the art without departing from the
scope and spirit of the
invention. Although the invention has been described in connection with
specific preferred
embodiments, it should be understood that the invention as claimed should not
be unduly
limited to such specific embodiments. Indeed, various modifications of the
described
modes for carrying out the invention which are obvious to those skilled in
molecular
3o biology or related fields are intended to be within the scope of the
following claims.
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