Note: Descriptions are shown in the official language in which they were submitted.
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8E~F-DELETING v~ O~S FOR GENE TU~PY
FIELD OF lr.v~ .lON
This invention relates to specially constructed vectors
which transduce transcriptional units into eukaryotic cells
such that preferably all sequences unrelated to the
transcriptional unit are eliminated upon integration. Only
one transcriptional unit comprising any natural or synthetic
promoter/enhancer sequence, protein coding sequence and
polyadenylation site is retained in the genome. By
eliminating themselves from the genome the vectors of the
invention circumvent the problems encountered with
conventional vectors, e.g. retroviruses and vectors thereof,
such as transcriptional interference with transduced genes
or genes adjacent to the integration site, activation of
cellular oncogenes, mobilization of endogenous retroviruses
and development of an immune response.
R~ ~a~ UND OF THE lNv~i.C.~lON
Retroviruses are RNA viruses that replicate through a DNA
intermediate. Flanking the ends of the viral RNA genome are
short sequence repeats (R) and unique sequences (U5 and U3)
that control DNA synthesis, integration, transcription, and
RNA processing. Between the control regions are coding
sequences for the major structural proteins of the virus
particle (gag and env) and for enzymes found in particles
(pol, protease, reverse transcriptase and integrase) (Figure
1) -
Shortly after infection, viral RNA is converted into DNA by
reverse transcriptase. The process is initiated by cellular
tRNA which by binding to a complementary region within the
viral genome serves as elongation primer. This region, also
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termed - primer binding site- (PBS) is located immediately
downstream of U5 and is essential for virus replication.
Prior to integration, terminal sequences of the viral genome
are duplicated such that the retroviral genome is flanked by
long terminal repeats (LTRs) each containing the U3, R and
U5 regions. Linear DNA molecules of this type integrate into
the genome (Figure 1).
LTR sequences are maintained in the integrated retrovirus,
also termed -provirus-, except that two nucleotides (nt) are
lost from each end. Cellular DNA sequences also are
unaltered except that upon integration, 4-6 nt are
duplicated such that the provirus is flanked at each end by
4-6 nt repeats. As a provirus, the retroviral genome is
replicated with cellular DNA and transcribed as a cellular
gene by RNA polymerase II. Provirus transcription is
controlled by promoter/enhancer sequences located in the U3
region of the 5'LTR. Polyadenylated transcripts initiate at
the junction between U3 and R (cap site) in the 5'LTR and
terminate in R of the 3'LTR that contains the signal for
polyadenylation. Full-length (genomic) RNA is transported
from the nucleus to the cytoplasm and either packaged into
virus particles that bud from the cell or are translated to
yield gag and pol proteins. A fraction of the RNA is
spliced to yield mRNA encoding env.
It is possible to adapt retroviruses to transduce genes into
-- ~lian genomes. Provided that certain control sequences
within the LTRs remain unaltered, the retroviral genome can
be deleted without impairing its ability to replicate in
cells that express proteins necessary for reverse
transcription, integration and particle formation. For this,
vector DNA is transfected into cell lines that contain
complete retroviral genomes or helper viruses. The helper
viruses are constructed such that they cannot assemble into
particles, due to a small deletion encompassing a sequence
(Y) between U5 and gag. Since the vector DNA does not
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contain the Y deletion, recombinant transcripts are packaged
and expelled from the cells as virus particles. In addition
to Y, gag sequences can also enhance the ability of the
vectors to be packaged.
To date, retroviral vectors are the most efficient means to
transduce foreign genes into mammalian cells. Accordingly,
retroviruses are used in over 80% of all approved gene
therapy trials. However, several factors undermine the
practical use of conventional retroviruses as gene therapy
vectors. First, transduced genes are often inactivated by
methylation or binding of transcriptional repressors to the
viral genome (1-3). Since these repressors were shown to
bind the primer binding site of several retroviruses, its
simple delètion would preclude virus replication. Second,
since retroviruses integrate mostly randomly throughout, the
genome, integrations sometimes result in mutations that
augment the expression of adjacent genes (4, 5). Activation
of adjacent proto-oncogenes followed by malignant
transformation of the infected cells has been described (6).
The activation m~h~n;cr involves transcriptional
enhancement either by upstream U3 promoters or nearby U3
enhancers. Third, viral vector sequences such as packaging
signals and leader sequences can potentially recombine with
endogenous retroviruses yielding new and unpredictable forms
of infectious virus (7-9). Finally, viral and non-viral
sequences of some vectors may trigger an immune response
which eliminates the transduced cells (10). A review on
gene therapy is given in (26).
The object underlying the present invention is to provide
novel vectors which do not show the disadvantages of prior
art vectors.
Said object is achieved by a vector system useful for gene
therapy comprising at least one coding sequence for a site-
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specific recombinase and at least one target sequence being
specifically recognized by said recombinase.
The vector system can be a DNA or RNA vector. Presently,
there are several vectors available which can be used for
gene therapy and which are suitable as a starting material
for the construction of a vector system according to the
invention.
The site-specific recombinase can be any recombinase which
specifically recognizes a target sequence. The prior art
provides several examples for site-specific recombinases and
the corresponding target sequence. The location of the
sequence coding for the recombinase and the target sequence
within the vector can be chosen by the skilled person
depending on which parts of the vector shall be deleted
after being incorporated into the mammalian genome.
Optionally, the site-specific recombinase can also be
encoded by a separate vector which is simultaneously
contained in the cell with the vector containing the target
sequence and the further sequences required for
incorporating said vector into the genome. In a further
alternative the recombinase can also be added as a protein
to the cell cont~;n;ng the vector with the target sequence.
In a preferred embodiment the combination of recombinase and
target sequence used is the recombinase Cre and the target
sequence loxP.
In a further preferred embodiment the recombinase is Flp and
the target sequence is frt.
The vector according to the present invention may comprise
any transcriptional unit, which transcriptional unit
comprises a gene coding for the desired function to be
introduced into the mammalian genome. In many cases said
gene will code for a protein which is not properly prepared
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by the cell which receives the vector according to the
invention.
~. .
In a further preferred embodiment the vector according to
the invention is a retroviral vector, most preferably a
retroviral DNA.
In case that a retroviral vector is used the target sequence
is preferably inserted into the U3 and/or U5 region, which
region may additionally contain the transcriptional unit.
In a further preferred embodiment the vector according to
the invention may also comprise a viral promoter and/or
enhancer.
As the result of the introduction of the vector according to
the invention into a mammalian cell a cell is obtained
which, after deletion of the non-desired vector parts from
the genome, contains at least one target sequence, which was
introduced by the vector. In most cases where the vector
also comprised the additional transcriptional unit said
additional transcriptional unit will be contained in the
mammalian cell genome. In the event that the
transcriptional unit contains a gene which normally is also
contained in the mammalian cell but for some reason non-
functional then the gene introduced into said cell via the
vector according to the invention will have a different
chromosomal environment compared to the gene as it naturally
occurs in the cell. When the vector sequences, e.g. the
proviral genome, is deleted from the chromosome in general a
few nucleotides of the proviral genome remain in the genome
of the cell. "Essentially no proviral genome" as used
herein, therefore, means that preferably less than 600 bp
nucleotides, more preferably less than 100 bp stay in the
genome.
By using the vector system according to the present
invention any DNA can be incorporated into the genome of a
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mammalian cell. The location of the target sequence within
the vector allows to predetermine which parts of the vector
shall be deleted from the genome after incorporation of the
vector.
The invention involves the development of novel vectors,
particularly retroviruses, to transduce transcriptional
units for therapeutic or non-therapeutic purposes into the
genome of eukaryotic cells. The vectors are equipped with a
site-specific recombination system including a recombinase
such as Cre or Flp and at least one specific target sequence
such as loxP or frt. The system is activated upon
integration such that all viral and non-viral sequences
unrelated to the transcriptional unit are eliminated.
Retroviruses of this type preferably circumvent all unwanted
side effects encountered with conventional retrovirus
vectors.
The preferred retroviruses of the invention contain a
transcriptional unit including a promoter, protein coding
sequence and polyadenylation sequence preferably in the U3
or U5 regions. Also preferably in the U3 or U5 regions, the
retroviruses of the invention contain at least one synthetic
or natural target sequence of a site specific recombination
system. In such a system, DNA fragments flanked by target
sequences that have the same orientation are eliminated by
the corresponding recombinase (Figure 2).
The retroviruses of the invention duplicate the target
sequences inserted into the U3 or U5 regions during
replication. This positions most of the viral genome
between identical target sequences and enables the
recombinase to delete the bulk of the provirus. Only one
copy of the transduced transcriptional unit that contains
the gene of interest remains in the genome. The expression
of the recombinase is either achieved in trans, by
introducing the protein or a recombinase-expressing plasmid
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into the transduced cells or, preferably in cis by
expressing the recombinase from the provirus itself (Figure
3,4). In this case, the recombinase coding sequences are
preferably placed between the target sequences outside of
the proviral control regions (LTRs). Their expression is
preferably controlled by a second synthetic or natural
promoter. A polyadenylation signal is preferably provided
by the R-region of the 3'LTR. In the event of an inverse
orientation of the recombinase cassette relative to the
provirus, transcripts may terminate in cryptic proviral
polyadenylation signals or in an additional synthetic or
natural polyadenylation sequence cloned into the virus in
inverse orientation.
The retroviruses of the invention are preferably enhancer
and/or promoterless. However, the retroviruses of the
invention may continue to contain their own promoter and
enhancer sequences.
The retroviruses of the invention are preferably used to
transduce therapy genes into mammalian cells, however, they
may also be used for basic research.
The retroviruses of the invention may also transduce
potentially hazardous sequences as long as these are deleted
in due time following integration. For example, a potential
application would be vectors based on human retroviruses
(HIV, HTLVI).
The invention includes mammalian cells containing at least
one site specific recombination target (e.g. loxP or frt)
and at least one protein coding sequence transduced by the
retroviruses of the invention. These cells are largely
devoid of retroviral sequences.
Finally, the invention includes procedures for transducing
cDNA sequences into the mammalian genome such as
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transfection of producer cells with the retroviruses of the
invention, infection of mammalian cells and expression of
site specific recombinases associated with deletion of the
recombinase-expressing cassettes.
BRIEF DP~~~TPTION OF THE FIGURES
Figure 1 is a schematic diagram or the genome of prior art
retroviruses.
Figure 2 is a schematic diagram of a site specific
recombination system.
~igure 3 and 4 is a schematic diagram of the retroviruses of
the invention where P = promoter; Ts = site specific target
sequence; PCS = protein coding se~uence, Rec = recombinase.
Figure S is a schematic diagram of the retrovirus vectors
U3pgklxtkneo and U3pgklxtkneoMCCre.
Figure 6 shows a Southern blot analysis of U3pgklxtkneo
expressing clones before and after transfecting MCCre. In
the first 6 lanes the DNA was digested with the enzymes NdeI
and XbaI that do not cut within the provirus. Lanes 7-12
contain DNAs digested with HindIII from Cre-expressing (+)
and non-expressing (-) clones.
Figure 7 shows a Southern blot analysis with DNAs derived
from U3pgklxtkneo (A) and U3pgklxtkneoMCCre (B) expressing
clones. Note the decrease in signal intensity of constant
HindIII fragments from Cre-expressing clones (B). Variable
bands correspond to the number of proviruses.
Figure 8 is a schematic diagram of the retrovirus vectors
pggSVCreU3lxpgkpuro and pggSVCreU3lxSVpuro.
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Figure 9 shows the recombination of U3lxpgkpuroSVCre
proviruses in NIH3T3 cells. (A) Predicted structure of
proviruses before and after site-specific recombination.
(B) Southern blot analysis of U31xpgkpuroSVCre proviruses.
Genomic DNAs were cleaved with EcoRI, fractionated on
agarose gels, blotted to nylon filters, and hybridized to a
32P-labeled pgk- probe. Lanes 1-14, U31xpgkruroSVCre
expressing clones 1-14. The ubiquitous 7 kb constant band
represents the endogeous pgk promoter. (C) PCR analysis of
U31xpgkpuroSVCre expressing clones. Genomic DNA was
amplified using Cre- (top) or b-actin- (bottom) specific
primers. Amplification products were resolved in 1% agarose
gels and visualized by Ethidium-bromide staining in lanes as
follows: M, molecular weight st~n~rds (1 kb BRL ladder); 1-
14, U31xpgkpuroSVCre expressing clones 1-14; 15, single copy
U3pgklxtkneo expressing clone (negative control); 16, single
copy U3pgklxtkneoMCCre expressing clone (positive control).
Figure 10 shows the recombination of U3lxSVpuroSVCre
proviruses in NIH3T3 cells. (A) Predicted structure of
proviruses before and after site-specific recombination.
(B) Southern blot analysis of U31xSVpuroSVCre proviruses.
Cell DNAs were cleaved with EcoRI (left) or Hind III
(right), processed as described in the legend to Figure 2,
and hybridized to a 32P-labeled SV40-probe. Lanes 1-12,
U31xSVpuroSVCre expressing clones 1-12. (C) PCR analysis of
U3lxSVpuroSVCre expressing clones. Genomic DNA was
amplified using Cre- (left) or b-actin- (right) specific
primers. Amplification products were resolved in 1% agarose
gels an visualized by Ethidium-bromide staining in lanes as
follows: M, molecular weight stAn~rds (BRL 1 kb ladder); 1-
12, U31xSVpuroSVCre expressing clones 1-12; 13-14, single
copy U3pgklxtkneo expressing clones (positive controls); 15-
16, single copy U3pgklxtkneoMCCre expressing clones
(negative controls).
The term "vector system" means at least one vector which can
introduce into the genome of a cell a coding sequence for a
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recombinase and a target sequence for said recombinase. The
recombinase encoding sequence and the target sequence can be
located on two different vectors but are preferably located
in one vector. Optionally, the recombinase can be
introduced as a protein into the cell, so that the vector
system needs not to comprise a coding sequence for the
recombinase.
The term "retrovirus" refers to any RNA virus that
replicates through a DNA intermediate. Such viruses can
include those that require the presence of other viruses,
such as helper viruses, to be passaged. Thus, retroviruses
are intended to include those containing substantial
deletions or mutations in their RNA.
The term "control region" refers to that region of a
retrovirus that is duplicated after infection and prior to
integration. Control regions include U3 and U5 regions.
Such regions also include LTR regions.
The term "transcriptional unit" refers to a sequence of
nucleic acids that includes a natural or synthetic promoter,
a protein coding sequence and a polyadenylation signal.
Promoters can include an enhancer.
The term "protein coding sequence" means a nucleotide
sequence encoding a polypeptide chain that has a therapeutic
value or interferes with the cellular metabolism in some
way. It also includes polypeptides which can be used to
distinguish cells expressing the polypeptide chain from
cells not expressing the polypeptide chain, commonly
referred to as "selectable markers".
The term "target sequence" or "site specific target
sequence" refers to synthetic or natural nucleotide
sequences that are recognized by a site-specific
recombinase. Examples of such sequences are loxP (11-13)
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derived from P1 phage or frt derived from S. cerevisiae (14-
16).
The term "recombinase" or "site specific recombinase" refers
to a synthetic, natural or recombinant enzyme that binds,
cleaves and recombines specific target sequences. Examples
for such enzymes are Cre-recombinase from P1 phage (13) or
Flp-recombinase from S. cerevisiae (14).
The present invention involves self-deleting vectors,
preferably retroviruses, that are preferably used to
transduce genes of therapeutic value into somatic cells.
Figure 5 shows two preferred embodiments of the invention.
In the upper vector -pggU3pgklxtkneo- a transcriptional unit
consisting of a murine phosphoglycerate kinase (pgk)
promoter (17), a loxP target sequence (ll) and a thymidine-
kinase/neomycinphosphotransferase (tkneo) fusion gene was
inserted into the 3'-U3 region of an enhancerless MoMuLV
retrovirus vector. In the lower vector an additional
transcriptional unit encoding MCCre (where Cre = Cre
recombinase, MC = HSV thymidine kinase promoter fused to a
Polyoma large T enhancer) was inserted between the LTRs into
the body of the virus. Virus replication and LTR-mediated
duplication places MCCre along with other viral and non-
viral se~uences between loxP sites enabling Cre recombinase
to delete most of the integrated provirus except for one
copy of a pgk-lx-tkneo containing LTR.
Figure 8 shows two additional preferred embodiments of the
invention. In both constructs the MC promoter was replaced
by the simian virus 40 (SV40) promoter and the tkneo fusion
gene by the puromycin resistance gene. In the upper vector -
pggSVCreU31xpgkpuro- the puromycin resistance gene is
controlled by a pgk promoter. In the lower vector -
pggSVCreU31xSVpuro the same U3 gene is controlled by an SV40
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promoter. In these examples the loxP site in the 3'hTR was
placed upstream of the transcriptional unit.
Although in the above preferred embodiments the
transcriptional unit and the site specific recombination
target are placed within U3, this is not obligatory. Both
transcriptional unit and target sequences can also reside in
the U5 region, or other parts of the vector, depending on
which parts of the vector shall be deleted.
Finally, Cre recombinase can also be provided in trans
either as a native protein or as a recombinase expressing
plasmid.
EXAMPLE 1
To investigate whether provirus sequences flanked by site
specific recombination targets can be deleted following
integration, we have inserted the transcriptional unit -pgk-
loxP-tkneo into the U3 region of an enhancerless Moloney
murine leukemia virus (Figure 5).
Plasmids
The sequences for Cre recombinase and loxP were derived from
pMCCre and pGEM30, respectively (11) The mouse
phosphoglycerate-kinase-promoter (pgk) was obtained from
ppgkCat (18) and the SV40/puromycin-acetyltransferase
cassette from pBABEpuro (19). The tkneo gene was obtained
by ligating an NheI/SpeI PCR amplification product of the
HSV thymidine kinase coding sequence (20) to an SpeI/NheI
amplification product of the neomycin-phosphotransferase
coding sequence (21). In frame fusion was achieved by
deleting the thymidine kinase stop codon and the Neo- AUG.
A pgklxtkneo cassette was cloned blunt ended into the unique
NheI site of pggU3en(-) to obtain pggU3pgklxtkneo. pggU3en(-
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) was derived from pggU3Neoen(-) (21) by deleting neo and
subcloning the viral sequences as an SstI fragment into the
backbone of pBABEpuro. pgklxtkneo was assembled in
pBluescriptIIKS (Stratagene) by inserting loxP as a
PstI/EcoRI fragment of pGEM30, pgk as an XbaI/BglII fragment
of pgkCAT and tkneo as a blunt ended fragment into the
corresponding sites of the pBluescriptIIKS polylinker. To
obtain pggU3pgklxtkneoMCCre, a PCR amplification product of
MCCre was cloned into the unique XhoI site of
pggU3pgklxtkneo.
Cells and viruses
NIH3T3 and BOSC23 (22) cells were grown in DMEM (Gibco)
medium supplemented with 10% fetal bovine serum (Gibco).
Helper virus free recombinant retroviruses were obtained by
transient transfection of BOSC23 cells as described by Pear
et al. (22). Infections were performed by incubating for
24 hours 105 NIH3T3 cells with filtered viral supernatants
in the presence of 4 ~g/ml polybrene (Aldrich). Provirus
expressing clones were isolated by selecting for 7 days in
medium containing 2 ~g/ml puromycin (Sigma) or 1 mg/ml G418
(Gibco).
Southern blot hybridization
DNA hybridizations were performed with 32P-labeled probes as
previously described (21). Southern blots were scanned with
a PhosphoImager (Molecular Dynamics) and analyzed with
ImageQuantNT software (Molecular Dynamics).
Results
Previous investigations have shown that the U3 region of
MoMuLV can tolerate relatively large amounts of extra-
sequences (up to 5 kb, (23)). These are duplicated along
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14
with viral sequences to generate long terminal repeats
(LTRs) that flank the integrated provirus (reviewed in
(24)). Therefore, we predicted that following insertion of
the target sequence for site-specific recombination -loxP-
into the 3'-U3 region of a retrovirus vector, LTR-mediated
duplication would place the viral genome between two loxP
sites, thus rendering it susceptible to excision by Cre-
recombinase.
To investigate this prediction, a transcriptional unit
consisting of a murine phosphoglycerate kinase (pgk)
promoter (17) a loxP target sequence (11), and a thymidine-
kinase/neomycinphosphotransferase (tkneo) fusion gene was
inserted into the 3'-U3 region of an enhancerless MoMuLV
retrovirus vector (pggU3en(-), (21)) to obtain -
pggU3pgklxtkneo- (Figure 5). This plasmid was transfected
into BOSC23 helper cells (22) to produce recombinant virus
that was used to infect NIH3T3 cells. LTR-mediated
duplication should generate in the infected cells proviruses
flanked by pgklxtkneo (Figure 6). To test this, twelve
neomycin-resistant clones obtained, after selecting in G418,
were analyzed by Southern blotting. LTR-mediated
duplication was confirmed in each case by using restriction
endonucleases that cleave the LTRs (data not shown). To
determine whether Cre-recombinase would delete the sequences
flanked by loxP and thus fuse the 5'pgk promoter to the
3'tkneo gene (Figure 6), two cell lines expressing
U3pgklxtkneo were co-transfected with the expression
plasmids MCCre (11) and pBABEpuro (19) encoding for Cre-
recombinase and puromycin-resistance, respectively. Genomic
DNAs extracted from cell pools obtained by selecting in
puromycin were digested with HindIII, an endonuclease that
cleaves the LTRs (Figure 6). When hybridized to a neo-
probe, non-recombined proviruses generate a constant band of
4.7 kb which accommodates the sequences flanked by loxP
(Figure 6, lanes 7-12). This band was significantly fainter
or disappeared completely in clones expressing Cre (Figure
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6, lanes 8, 9, 11, 12), indicating that most of the
proviruses have recombined. Thus, loxP sites inserted into
U3 enable Cre recombinase to excise most of the provirus
except for a single LTR. The part of proviral DNA left in
the chromosome after deletion of most of proviral DNA can be
easily examined by sequence analysis of the chromosomal
insertion site and sequence comparison with the vector used
for introducing the foreign gene.
EXAMPLE 2
Since we sought to develop a vector that carries all
required elements for self-deletion into mammalian cells, we
investigated whether loxP sites inserted into U3 undergo
recombination when Cre is expressed from the same provirus.
Coding sequences for Cre controlled by an MC promoter were
inserted between the LTRs into pggU3pgklxtkneo to obtain
pggU3pgklxtkneoMCCre. To obtain infectious virus,
pggU3pgklxtkneoMCCre was transfected into BOSC23 cells as
described in example 1. Neomycin resistant clones obtained
after infecting NIH3T3 cells and selecting in G418 were
analyzed by Southern blotting as described in example 1.
Proviruses derived from this vector were expected to excise
themselves after integration. Genomic DNA from several
independent U3pgklxtkneo and U3pgklxtkneoMCCre expressing
clones was digested with HindIII, a restriction endonuclease
that cleaves both proviruses downstream of tkneo. When
hybridized to neo, non-recombined proviruses maintain all
sequences flanked by loxP and as a result, generate a
constant band of 4.7 and 6 kb, respectively. Although this
band was still present in Cre expressing clones, it was 5-10
times fainter than in non-expressing clones (Figure 7, B-
lanes). Since both types of clones contained comparable
numbers of proviruses, the results indicate that a
significant number of U3pgklxtneoMCCre proviruses had
deleted the sequences flanked by loxP.
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16
EaU~MPLE 3
To improve the efficiency of self-deletion of proviruses
expressing Cre, two additional constructs were made using
alternative promoters and selectable marker genes.
Plasmids
pggSVCreU31x was constructed by sequentially inserting the
Cre region of MCCre, the SV40 promoter of pBABEpuro and the
loxP-fragment of pGEM30 which includes a downstream HindIII
site, as blunt ended fragments into the BamHI, XhoI and NheI
sites of pGgU3en(-), respectively.
pggSVCreU3lxpgkpuro and pggSVCreU3lxSVpuro were obtained by
ligating blunt ended pgk-puro- or SV40-puro expression
cassettes into the HindIII site of loxP.
Cells and viruses
as described for examples 1 an 2.
DNA Hybridizations and PCR
DNA hybridizations were performed with 32P-labeled probes as
previously described (21) Southern blots were scanned with a
PhosphoImager (Molecular Dynamics) and analyzed with
ImageQuantNT software (Molecular Dynamics). For PCR-assays,
150 ng of genomic DNAs were amplified for 40 cycles (94~C
30~', 60~C 1', 72~C 2' ) using the Cre-specific primers 5'-
TTAGCTAGCATGCCCAAG~A~-AAGAAG-3' and 5'-
GGAGCTAGCCTAATCGCCATCTTCCAG-3'. Control PCRs were performed
under the same condition except for using actin-primers as
previously described (25).
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Results
The vectors -pggSVCreU3lxpgkpuro- and -pggSVCreU3lxSVpuro-
(Figure 8) were transfected into BOSC23 cells to obtain
infectious virus. Recovered viruses were used to infect
NIH3T3 and puromycin resistant clones were isolated after 7
days of selection.
Genomic DNA of puromycin-resistant clones obtained with
either construct was analyzed by Southern blotting. To
identify recombination of U3 lxpgkpuroSVCre proviruses, the
DNA was digested with EcoR1, a restriction endonuclease that
cleaves both proviruses in front of each promoter (Figure
9A, lOA). When hybridized to pgk, non-recombined proviruses
maintain all sequences flanked by loxP, and as a result,
generate a constant band of 3.1 kb (Figure 9B, lanes 5, 9,
12-14). This band should disappear from Cre-expressing
clones (Figure 9A). Accordingly, the band was lost from 9
out of 14 U3 lxpgkpuroSVCre-expressing clones, indicating
that most proviruses have recombined (Figure 9B, lanes 1-4,
6-8, 10). Additional bands of varying sizes represent the
fragments extending from the EcoR1 sites of the 3'LTR to
sites in the flanking cellular DNA (Figure 9B). However all
clones, including those that appeared recombined on Southern
blots, generated Cre-specific amplification products when
analyzed by PCR (Figure 9C). Although recombined clones
generated 10 to 50 fold less amplification product (Figure
9C), the results suggest that the recombinase does not
uniformly achieve the threshold levels required for
recombination.
Significantly higher recombination efficiencies were
obtained with U3lxSVpuroSVCre. As shown in Figure 10B,
none of the clones generated a 3 kb restriction fragment
when hybridized to SV40, and only variable bands consistent
with proviral deletion were seen. To confirm recombination,
the DNA was digested with HindIII, which cleaves upstream of
CA 022281~9 1998-01-28
WO 97/07223 PCT/~1~6,'~761
18
puromycin (Figure lOA). As expected, only bands of varying
sizes were obtained when hybridized to SV40- or,
alternatively, to puromycin probes. Moreover, hybridization
patterns were unique, reflecting fragments ext~n~;ng from
the HindIII site of the 3'LTR, to sites in the 5' and 3'
flanking cellular DNA, respectively (Figure lOB and data not
shown). When analyzed by PCR, only 3 out of 12 clones
generated faint Cre-specific amplification products (Figure
lOC).
The vector system according to the invention can be used for
preparing a pharmaceutical composition which contains the
conventional carriers and/or diluents. Depending on
the disease to be treated the vector system contains in the
additional transcription unit the gene useful for curing the
disease.
CA 022281~9 1998-01-28
W O 97/07223 PCTAEP96/00761
19
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