Note: Descriptions are shown in the official language in which they were submitted.
2074188
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VECTOR WITH MULTIPLE TARGET RESPONSE
ELEMENTS AFFECTING GENE EXPRESSION
Field of the Invention
The present invention relates to a method of
effecting viral inhibition with DNA sequences encoding
multiple target response elements, and to constructs
suitable for use in same. In particular, the invention
relates to a method of inhibiting the Human
Immunodeficiency Virus (HIV).
Background Information
The tat protein of HIV transactivates viral gene
expression and is essential for virus production [Arya at
al, Science 229:69-73 (1985); Sodroski et al, Science
229:74-77 (1985); Dayton et al, Cell 44:941-947 (1986);
Fisher at al, Nature 320:367-371 (1986) ]. The tat
activation response element (termed TAR) has been
localized within the region of the first 44 nucleotides
downstream of the transcription initiation site [Chen,
C., and Okayama, H., Mol. Cell. Biol. 7:2745 (1987);
Rosen et al, Cell 41:813-823 (1985) ; Tong-Starksen et
al, Proc. Natl. Acad. Sci., USA 84:6845-6849 (1987) ;
Hauber et al, J. Virol. 62:673-679 (1988)]. This region,
present in all HIV-1 transcripts, forms an unusually
stable stem loop structure [Okamoto, T., and Wong-Staal,
F. Cell 47:29-35 (1986)], and several lines of evidence
suggest that the transcriptional effect of tat is
mediated through interaction with TAR region of viral RNA
[Sharp et al,
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Cell 59:229-230 (1989) ; Viscidi et al, Science 246:1606-
1608 (1989); Berkhout et al, Cell 59:273-282 (1989);
Garcia et al, EMBO J. 8:765-778 (1989); Feng, S. and
Holland, E. C. Nature 334:165-167 (1988); Southgate et
al, Nature 345:640-642 (1990)].
While tat binding to TAR RNA sequences has
been demonstrated [Rappaport, J. et al, Cold Spring
Xarbor, New York (1989b) ; Dingwall et al, Proc. Natl. Acad.
Sci USa 86:6925-6929 (1989)], the sequence requirements
for tat binding are not sufficient to explain the
sequence and structural requirements needed for
transactivation. Cellular factors also appear to play a
role in tat mediated transactivation which may confer
additional specificity [Marciniak et al, Proc. Natl. Acad.
Sci. 87:3624-3628 (1990)]. Tat appears to function
poorly in nonprimate cells and studies using
interspecific hybrids suggest that transactivation
potential is correlated with the presence of human
chromosome 12 [Hart et al, Science 246:488-491].
Several cellular TAR RNA as well as TAR DNA binding
proteins have been identified '[Gaynor, R. B. EHBO J.
8:765-778 (1989) ; Gatignol et al, Proc. Nsrl. acad. Sci. USA
86:7828-7832 (1989); Wu et al, EltBO J. 7:2117-2129
(1988); Jones et al, Science 232:755-758 (1986): Garcia
et al, F.HBO J. 8:?65-778 (1989); Marciniak et al, Proc.
Natl. Acad. Sci. 87:3624-3628 (1990) ] however the role of
these proteins in tat mediated transactivation,
however, is not yet clear.
In vitro, tat protein can be released and taken
up by cells [Frankel, A. D., and Pabo, C. O. Cell
55:1189-1193 (1988)], and has biological effects on the
WO 91/10453 PCT/US91/0017
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regulation of cellular proliferation in addition to its
role in HIV promoter activation. Recent studies
indicate that tat inhibits the antigen induced
lymphocyte proliferation [Viscidi et al, Science
246:1606-1608 (1989)] and has growth promoting activity
on cells derived from Kaposi Sarcoma lesions of AIDS
patients [Ensoli et al, Nature 340:84-86 (1990)]. In
contrast, tat does not cause significant reduction of
lymphocyte proliferation in response to mitogens.
Given the implication of HIV-I tat in the causation of
HIV associated diseases, interference with the tat
function might be therapeutically significant.
Transdominant mutations for HIV proteins have
been reported [Malim et al, Cell 58:205-214 (1989);
Torno et al, Cell 59:113-120 (1989); Marciniak et al,
Proc. Natl. Acad. Sci. 87:3624-3628 (1990) ] . These
proteins, produced constitutively from a strong
promoter, can antagonize the growth of HIV-I and
therefore, can be used to create cell lines "immunized"
to viral infection.
Since TAR RNA appears to interact wit tat
protein directly [Southgate et al, Nature 345: 640-642
(1990)] or through the combined activities of cellular
factors) [Marciniak et al, Proc. Natl. Acad. Sci. USA 87:
3624-3628 (1990)], Applicant hypothesized that TAR RNA,
produced in large amounts, might serve as a competitive
inhibitor of tat function. The results presented in
the Examples that follow indicate that overproduction
of TAR RNA downregulates tat mediated transactivation
in a dose dependent manner. The approach of
biologically controlled expression of high levels of
target RNA elements to sequester viral or cellular
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transactivators has general application in the
generation of a novel class of anti-viral reagents.
Inducible transcripts of poly-TAR, exemplified herein,
can be combined with coding sequences for trans-
dominant mutants to provide a synergestic effect for
intracellular immunization.
SUMMARY OF THE INVENTTON
It is an object of the present invention to
provide a means of down-regulating HIV-1 LTR directed
gene expression.
It is another object of the present invention
to provide a competitive inhibitor of tat function.
It, is a further object of the present
invention to provide a class of anti-viral reagents for
intracellular immunization.
Various other objects and advantages will
become apparent from the detailed description of the
invention and the drawings.
In one embodiment, the present invention
relates to a DNA construct comprising a vector and a
promoter operably linked to at least two target
response elements linked so that they are transcribed
in tandem. The construct may further comprises a DNA
segment encoding a ribozyme specific for a viral DNA or
a DNA segment encoding a transdominant negative mutant
of a viral protein.
In another embodiment, the present invention
relates to a method of treating viral infection. The
method involves obtaining cells from an viral infected
patient and transforming the cells with a construct of
the present invention. The cells are then introduced
back into the patient.
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In a further embodiment, the present invention
relates to a method of inhibiting viral replication
comprising introducing into a cell, infected with a
virus, the construct of the present invention. The
5 product of the virus regulates transcription of the
elements of the construct so that inhibition is
effected.
$RIEF DESCRIPTION OF THE DRAWINGS
Fig 1. shows the working hypothesis for
intracellular inhibition of HIV gene expression using
HIV-LTR-driven multiple TAR elements.
According to this model, tat expressed from
the viral'LTR activates the LTR driven transcription of
the multiple TAR elements. Multiple TAR RNA competes
for tat binding. Therefore, viral gene expression
together with tat expression decreases until an
equilibrium is reached, which is dependent on the
number of TAR elements in the construct, is reached.
Fig. 2A shows the construction of the multiple
TAR elements and the predicted secondary structure of
the transcript.
The figure shows the annealed oligonucleotide
containing the entire wild type TAR element with the
half palindromic sequence of DraI and SmaI. Arrows
represent the direction of transcription. The predicted
secondary structure of the multiple TAR RNA elements is
indicated as well.
B. shows the general structure of the plasmids
Plasmids used for this experiments contain
the CD7-LTR, which is derived from the wild type HIV-1-
LTR by deletion of the negative regulatory element
(NRE). All constructs contain the C-terminal part of
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the bacterial CAT gene (downstream from the NcoI site)
and SV-40 splice and polyadenylation signals.
Fig. 3 shows transcription of multiple TAR
elements downregulates transactivation.
A & B. COS cells were cotransfected with 4.4~Cg
different poly-TAR containing plasmid, lug RSV-
LUCIFERASE, 2.2~,g LTR-CAT and 0.48~g LTR-tat (Panel A)
or 0.48~g pSVL_ tat (Panel B) plasmids as indicated. +
and - represent the presence and absence of the
particular plasmids respectively in the cotransfection
assay. 48 hrs after transfection cell lysates were
analyzed for CAT expression and LUCIFERASE activity.
C. Total RNA was prepared (13) from COS cells
transfected with different plasmids indicated on the
figure. + and - represent the presence and absence of
the particular plasmids respectively in the
cotransfection assay. l0ug RNA was analyzed by Northern
blot hybridization. As Fig. 2 shows the constructs
used for cotransfections contain a short C-terminal
part of the CAT gene. A nick-translated CAT fragment
(Pharmacia) was used as 'ZP-labelled probe.
D. COS cells were cotransfected with 4.4~g of the
indicated different poly-TAR containing expression
plasmid, leg RSV-LUCIFERASE, 2.2~g HTLV-I-LTR-CAT,
0.48~tg FiTLV-I-LTR-TAX (13) , 0.48~g LTR-tat as
indicated. + and - represent the presence and absence
of each particular plasmids in the cotransfection
assay. 48 hrs after transfection, cell extracts were
assayed for CAT expression and LUCIFERASE activity.
Fig. 4 shows inhibition of transactivation is
dependent on the amount of TAR RNA transcripts.
WO 91 / 10453 PCT/US91 /00175
A. COS cells were cotransfected with 2.2~g LTR-CAT,
0.48ug LTR-tat and increasing amounts of LTR-SSTAR.
Decreasing amounts of LTR-OTAR (x indicates 0.22~g)
were used to keep the promoter concentration constant.
+ and - represent the presence and absence of each
particular plasmids in the cotransfection assay. 48 hrs
after transfection cell lysates were analyzed for CAT
expression.
8 & C. Total RNA was prepared from COS cells
transfected with different plasmids as indicated on the
figure. 10~,g RNA was analyzed by Northern blot
hybridization using a nick translated, 32P-labelled CAT
DNA fragment (Panel B). The CAT probe anneals to all
constructs illustrated in Fig. 1. A '2P-labelled tat
DNA probe was used to determine the amount of tat mRNA
expressed under different conditions (Panel C).
Fig. 5 shows the variation in the amount of
inhibition of transactivation depending upon the amount
of TAR RNA transcripts. The inhibition produced by
construct comprising OTAR to 50 TAR elements is
compared.
Figure 6 shows an example of a ribozyme-poly-
TAR construct for inhibition of viral replication.
Figure 7 shows an example of a nGAG-poly-TAR
construct for inhibition of FiIV replication.
DETAILED DESCRIPTION OF THE INVENTION
The specific aim of the studies leading to the
present invention was to establish an inducible vector
system which is activated by the action of tat protein
and which can concomitantly inhibit tat activity. The
approach of the present inventor was to inhibit tat
function by overproduction of the tat activation
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responsive (TAR) elements. Figure 1 depicts this
approach. Protected cells contain at least one copy of
the construct of the present invention and, after
infection, one copy of the integrated provirus. If the
HIV-LTR is activated, tat protein is made from the
proviral genome as an early gene product. Some of this
tat protein activates viral gene expression and some
activates the transcription of the multimerized TAR
elements from the construct. As the multiple TAR RNA
competes for tat binding, the viral gene expression
decreases. Since tat expression itself depends on the
presence of tat, its expression would slow down
together with all LTR directed gene expression until a
certain equilibrium, which is dependent on the number
of TAR elements in the multimer, is reached. This is
the first time that a construct, proposed for gene
therapy use, is under the control of a biological
regulation. The protective gene product will only be
expressed, if the cell becomes infected and tat is
made. Otherwise, the construct is silent in the
genome.
Inhibition of viral replication using multiple
TAR elements is effective against all HIV isolates
because it is a functional inhibition. The problem
most HIV vaccines encounter is the virus' high
mutational rate. The present construct is not limited
by retroviral mutations.
Accordingly, the present invention relates to
a DNA construct encoding at least one copy, and
preferably between 5 and 50 copies, and most preferably
more than 20 copies, of the TAR element. Constructs to
which the present invention relate comprise a DNA
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segment including multiple target response elements,
such as TAR elements, a promoter, such as LTR-HIV, and
a vector, such as pCD7. The multiple activation
response elements must be in tandem or if separated,
their transcription must not be interrupted by
separating sequences. The present inventor has found
that HIV inhibition increases with increasing TAR
elements until the TAR RNA contains 25 TARs at which
point further increases in the number of TAR elements
do not appear to increase inhibition. The determining
test was done in a transient expression assay and in
the case of a stable integration additional TARS would
provide further increases in inhibition.
In the construct of the present invention, the
promoter is operably linked to the multiple TAR DNA
segment so that the promoter controls the amount of TAR
RNA produced. While the following examples use the
HIV-LTR promoter, multiple TAR elements can be
transcribed from various other promoters. For example,
a promoter which can be activated by the tat protein
might be used. Other promoters (CMV, SV40 or tRNA
promoter) can be used, however, these promoters produce
a constitutive expression of the gene product. The
advantages using promoters such as HIV-LTR is that they
are inducible by the virus. This is more specific than
any other promoter. Further, tissue-specific promoters
could be useful in these constructs.
The vector used in the construct of the
present invention must ensure high efficiency gene
transfer to the in vivo target cell, for example,
retroviral vectors. Suitable vectors for use in the
present invention include vectors which contain a
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replication origin and a selection marker for
propagation in prokaryotes. Vectors may contain more
than one promoter. Further, vectors of the present
constructs can contain sequences which allow the site-
s specific integration of the construct into the
chromosome without disturbing the cell function. The
vectors can also contain "helper virus" sequences which
allow transmission of the construct into the target
cells and promote propagation of the vector through
further infection.
Using constructs of the present invention, the
inventor has shown that the degree of down-regulation
of HIV-1 gene expression is dependent on the number of
TAR elements in the constructs. This indicates that
the use of several excess target nucleotide sequences
can be used to down-regulate undesirable gene activity.
It is possible to combine TAR elements in
tandem with other elements which inhibit viral
expression or which act through inducing protective
proteins acting against destructive effects of the
viral proteins. For example, the constructs of figures
6 and 7, made by the method of the present invention,
can be used to down-regulate undesirable gene activity.
Examples of appropriate elements for incorporation into
the construct are transdominant regulatory proteins,
antisense sequences, coding sequences of antiviral
agents such as interferons or immunosystem stimulating
agents. The construct could contain different
activation or inhibition response elements as well.
The inhibitory activity of the constructs of
the present invention, as described above,can be
enhanced by including in the construct a transdominant
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negative mutant of a viral protein, for example a
mutant GAG, or the ribozyme-directed against a HIV
mRNA, such as, for example, GAGNAM. The constructs can
also include a rev-response element (RRE). The RRE
element of the construct functions to transport RNA
made from the construct out of the nucleus, into the
cytoplasm.
The combination of TARS with a ribozyme
against a viral mRNA in a single construct provides two
l0 different types of inhibition. While TARS inhibit HIV-
1 directed gene expression by sequestering tat, the
ribozyme inhibits protein translation by hybridizing to
the target RNA and cleaving it. Combining these two
inhibition mechanisms increases the possibility of
total inhibition. The ribozyme used in the following
examples, GAGNAM is directed against GAG mRNA, a
particularly good target since it is conserved in the
American HIV-isolates.
Constructs containing TARS and trans-dominant
mutants of HIV proteins, such as GAG, inhibit both HIV
gene expression and viral assembly. The combination of
TARS and mutant GAG provides pure functional inhibition
which the virus cannot overcome by mutations.
The constructs of the invention can be made by
appropriate means known in the art. The practitioner
can prepare multiple target response sequences using
purified response sequences which are then ligated in a
manner to allow tandem addition of the sequences to
provide multiple target response sequences. It should
be noted that, while constructs containing multiple
activation response sequences have been exemplified,
constructs can also contain multiple inhibitory
WO 91 / 10453 PCT/ US91 /0017;
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response sequences. The use of multiple inhibitory
sequences can be expected to allow the practitioner to
stimulate activity of a desired promoter. Such
constructs containing multiple inhibitory response
sequences in tandem can be used, for example, to
increase production of a desired product by stimulating
the promoter responsible for expression of the desired
protein.
The constructs of the present invention can be
used in gene therapy by known methods. The method
described by David Baltimore ( atur X5,:395-396
(1988)) known as "intracellular immunization" can be
used. For example, the constructs of the present
invention can be introduced into bone marrow cells,
including all hematopoietic stem cells. The blood
cells can be either of mixed population or of a
homogenous population such as lymphocytes. Using the
constructs exemplified, the cells of the HIV-infected
individual would be used. After introduction of the
gene, the cells would be injected back into the
patient. To make space for the growth of the implanted
cells, the marrow could be partially cleared by
irradiation or with a medication before the modified
cells are injected.
Blood cells from patients can also be
introduced with the vectors of the invention. The
cells with the construct would be re-introduced into
the patient. In the treatment of HIV infections, the
construct must be introduced into CD4+ cells. Since
the turn-over of these cells is relatively fast, re-
introduction of the protected cells is necessary so
long as the viral infection is present. The repeated
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introduction of such cells will be needed.
The multiple TAR constructs and or
TAR+ribozyme constructs of the present invention are
believed to produce only inhibitory RNA, not protein
products which could be important in gene therapy
strategies.
The use of the multiple TAR element construct
of the present invention is very advantageous. For
example, the construct provides for specific inhibition
l0 of HIV-1 directed gene expression. The expression of
the protective gene product is biologically controlled
which is distinctively advantageous since constitutive
expression of a TAR-containing transcript on normal
cell processes in vivo may be deleterious. The
usefulness of the construct is not limited by
variability between different HIV isolates. Further,
the use of the construct can be expanded by the
downstream insertion of sequences such as either
ribozymes or trans-dominant mutants of HIV proteins.
EXAMPLES
The following non-limiting examples are given
to further describe the present invention. While the
present invention is exemplified using the HIV system
and the TAR element, one skilled in the art will
appreciate that inhibition of other viruses can be
expected to be effected using the method of the present
invention.
Plasmid Construction
Plasmids containing different numbers of
unidirectional TAR elements under the control of HIV-
LTR were constructed. (See Figure 2).
"Multimerized" TAR sequences were cloned downstream of
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PCT/US91 /00175
the authentic TAR sequence of the 5~ HIV-1-LTR deletion
mutant CD7, lacking the negative regulatory element
(NRE) and having higher level of expression as compared
to the wild type HIV-I-LTR [Siekevitz et al, Science
238:1575-1578 (1987)]. Plasmid pCD7 (kindly provided
by Stepen Josephs) containing a part of the HIV-1 LTR
(-278 - +63) was digested with restrictions
endonucleases and ligated with the multiple, tandem TAR
elements. LTR-1TAR, LTR-4TAR and LTR-STAR contained
one, four and five copies of TAR elements,
respectively.
For the construction of plasmids LTR-STAR and
LTR-4TAR, two oligonucleotides containing the sequence
for the entire TAR element (+1 to +63) of HIV-I flanked
by half of the palindromic sequence for DraI and SmaI
restriction endonuclease recognition sites were
synthesized. The oligonucleotides were purified,
phosphorylated and annealed. The annealed DNA (TAR) was
ligated in the presence of DraI and SmaI allowing only
tandem (directional oriented) ligation of the TAR
elements. Plasmid CD7-CAT [Siekevitz et al, Science
238:1575-1578 (1987)] containing a part of the
HIV-I-LTR (-278 to +63) was digested with restriction
endonucleases HindIII and NcoI, ends were filled and
ligated with the multiple, tandem TAR elements. Of
several E. cola strain tested only one, Bj 5183: F-,
recBC, endoI, gal, met, str, thi, bio, hsd. (kindly
gift from F. Lacroute), was able to maintain these
plasmids without rearrangements.
LTR-1-TAR was constructed by digestion the
CD7-CAT with HindIII and NcoI, ends were filled and
relegated.
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Two classes of control plasmids were
generated: 5TAR having a deletion of the upstream
promoter sequences (TAR sequences are present but not
transcribed), and LTR-OTAR having no TAR sequences but
5 contains the upstream promoter sequences (Fig. 2).
LTR-0-TAR was made by digestion of CD7-CAT plasmid
[Siekevitz et al, Science 238:1575-1578 (1987)] with
PvuII and NcoI, blunt ended and relegated.
STAR plasmid was constructed by deleting the
10 5' part of the HIV-LTR from the plasmid LTR-STAR by
digesting with XbaI and PvuII, the ends were filled
and ligated. The LTR-tat was constructed by digesting
pSV~-tat [Rappaport et al, The New Biologist 1:101-110
(1989a)] with SalI and BamHI; the 350 Hp tat containing
15 fragment was isolated, and a blunt end ligation was
performed with vector CD?-CAT between HindIII and NcoI
sites.
All constructs were confirmed by restriction
mapping and sequencing.
The cloning strategy allows the formation of
direct, but not inverted repeats of the TAR element,
since inverted repeats are cleaved by the SmaI and DraI
enzymes during ligation. The correct orientation and
secondary structure of each element is presumably
important for the desired effect, since TAR functions
in transactivation only in a position dependant manner
[Peterlln et al, Proc. Natl. Acad. Sci. USA 83:9734-9738
(1986)].
Doxaregulatioa of transactivatioa is dependent on tb.
transcription of the TAR ~l~msats
To determine the effect of multiple TAR
elements on HIV-LTR directed gene expression, TAR
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PCT/US91 /00175
expression plasmids were cotransfected with LTR-CAT
[Siekevitz et al, Science 238:1575-1578 (1987)] and
LTR-tat or pSVy-tat [Rappaport et al, The New Biologist
1:101-110 (1989a)] in COS cells.
COS-1 cells were grown in Dulbecco~s Modified
Eagle Medium (DMEM) supplemented with 10% fetal calf
serum (GIBCO). 2 x 105 cells were plated in 3m1 media in
6 well tissue culture plates one day prior
transfection. 25 ~,g of total plasmid as used for 3
wells. The amount of the different plasmids are
indicated on the figures. Plasmid pBR322 was used as
carrier DNA. Transfections were carried out with
calcium phosphate procedure [Chen, C., and Okayama, H.
Hol. Cell. Biol. 7:27-45 (1987) J . 48h after transfection
cells were collected (SIGMA cell remover reagent) and
crude cellular extracts were made in PBS.
The plasmid RSV-Luciferase was included as an
internal control to detect the transfection efficiency.
The amount of CAT protein and relative levels of
luciferase activity were determined from extracts of
transfected cells. CAT protein was assayed with 5
PRIME 3 PRIME ELISA kit according to the manufacture
instruction: LUCIFERASE activity was measured according
to P.E. Stanley and S.G. Williams [Stanley, P. E. and
Williams, S.G. Anal. Biochem 29:381 (1969) J and
activities were expressed in arbitrary units (ARU).
As shown in Figure 3A, multiple TAR elements
transcribed from HIV-LTR inhibit HIV-LTR directed gene
expression in the presence of tat and the
downregulation observed is proportional to the number
of TAR elements in the construct. LTR-4TAR and
LTR-STAR inhibited transactivation an average of 70%
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and 80%, respectively. LTR-1TAR also has a measurable
effect resulting in up to 40% downregulation. This
reduction represents a cumulative effect of the
inhibition of both CAT and tat expression, since both
gene products are under the control of the HIV-I-LTR in
this experiment. Multiple TAR elements can suppress
transactivation when tat is expressed constitutively
from the SV40 late promoter (Fig. 38), albeit to a
reduced level. Results presented in Figures 2A and B
illustrate that CAT expression is reduced 82% when tat
is expressed from HIV-1 LTR compared to a 50% reduction
observed with the constitutively expressed tat.
Transcription of the multimerized TAR
sequence is required for efficient downregulation and
accumulation of steady state competitor RNA occurs only
in the presence of tat (See Fig. 3C). Sequences
upstream of the TAR element cannot account for the
observed effect. The LTR-OTAR plasmid containing no
TAR sequences or STAR plasmid having a deletion of the
upstream promoter sequences, produce no significant
effect on HIV-I LTR directed gene expression (See Fig.
3A, 3B, 3C) .
Cotransfection was performed with an another
human retroviral LTR to determine the specificity of
the effect of multimerized TAR RNA. HTLV-I-LTR-CAT
(kindly provided by M. Nerenberg) was used as a
reporter gene and HTLV-I-LTR-TAR plasmid was included
as transactivator of the HTLV-I LTR [Sodroski et al,
Science 225:381-385 (1984); Felber et al, Science 229:675-
679 (1985)]. LTR-tat was also supplied for the
transcription of the multimerized TAR elements.
Results, presented in Figure 3D, indicate that the
204188
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expression of multiple TAR RNA elements does not affect
HTLV-I promoter activity, suggesting that downregulation
of gene expression is specific to the HIV-LTR.
That the multiple TAR RNA elements can specifically
inhibit transactivation of the HIV gene expression is
also evidenced in figure 3 wherein the RSV promoter was
coupled with LUCIFERASE reporter gene. The construct was
used to verify that the specifically of the multiple TAR
RNA elements do not effect the use of heterologous
promoters. No significant difference of RSV promoter
activity could be detected with TAR RNA or DNA elements.
This indicates the relative promoter activities of the
HIV-LTR versus RSV promoter as the proportion of CAT and
LUCIFRASE expression. Inhibition of the transactivation
parallels the amount of TAR transcripts.
LTR-CAT and LTR-tat were cotransfected with
increasing amount LTR-STAR plasmid to determine the
effect of different amount of TAR transcripts on the
transactivation. RNA was isolated using CINNA/BIOTECX
RNAzol' reagent according to the company protocol. For
Northern analysis, RNA was electrophoresed through a 1%
formaldehyde/agarose gel. RNA was transferred onto
nitrocellulose paper and hybridizing with Nick translated
3zP labeled probe as previously described [Maniatis et al,
Cold Spring Harbor, New York: Cold Spring Harbor
Laboratory (1982)].
Figure 4A illustrates that increasing the amount of
LTR-STAR plasmid results in a proportional decrease in
CAT expression (up to 970). Inhibition cannot be due to
the competition for limiting
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transcription factors which associate with the
HIV-I-LTR since the amount of transfected LTR upstream
sequences was kept constant in this experiment.
Increasing amounts of transfected LTR-STAR plasmid
results in a similar increase of LTR-STAR transcripts
(See Fig. 4B). From these experiments, it is concluded
that downregulation of HIV-1 LTR directed gene
expression is dependent upon the relative amount of
expression plasmid DNA introduced into cells, in
l0 addition to the number of transcribed TAR elements
contained in the expression plasmid.
Northern blots show tat and CAT expression
appear to be reduced in parallel by multimerized TAR
RNA (Figure 4C), which is expected since they are both
driven by HIV-LTR.
The data of figure 4 supports the teaching
that the down-regulation of gene expression is
dependent on the number of transcribed target activator
nucleotide sequences. The evidence shows that HIV-1
transactivation by tai can be down-regulated using 7TAR
RNA elements in tandem up to 97%, and that the down-
regulation is a function of the amount of TAR RNA
transcription. The data would suggest that a construct
containing more than 7TAR elements would provide an
even more effective down-regulating effect.
Constructs Containing More Than 5 TAR 8lements
The multiple TAR construct was expanded and
plasmids containing up to 50 TARs were constructed and
tested (see Figure 5).
pSPTlB-polyTAR constructs containing between
15 and 45 TARs were constructed by cutting a pSPTl8
vector (Pharmacia) with XbaI. The ends were then
WO 91 / 10453
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blunt-ended with the Klenow enzyme and
dephosphorylated. The insert was prepared by cutting
pLTR-STAR with PvuII and ScaI and isolating the 5TAR
containing fragment (4558p).
5 The above vector and insert were then ligated
together and used to transform E. coli. Plasmid DNA was
prepared from single colonies and clones were selected
containing big inserts using methods well known in the
art. The orientation of the insert was checked with
10 restriction enzyme digestion using SspI and HindIII +
SspI.
Next LTR-STAR-CAT was cloned. The LTR-CAT
vector was cut with XbaI + HindIII and larger fragments
were isolated. The insert, LTR-STAR fragment, prepared
15 by the polymerase chain reaction, was prepared by
cutting the PCR fragment with XbaI + HindIII. The
vector and insert were ligated together and used to
transform E. coli. Single colonies were checked.
LTR-46TAR was prepared by cutting LTR-CAT with
20 HindIII + BamHI and isolating the larger fragment
containing the LTR-1TAR + pHR322. The pSPT45TAR,
prepared as described above, was cut with HindIII +
HamHI and the larger fragment containing the 45TAR was
isolated. The isolated fragments were ligated together
and E. coli transformed with the ligated product.
LTR-25TAR and LTR-50TAR were also prepared.
Vector LTR-STAR-CAT was cut with SalI + HamHI and the
fragments containing the LTR-STAR + pBR322 were
isolated. pSPT-20TAR or pSPT-45TAR were cut with SalI
+ BamHI and fragments containing the 20TAR or the 45
TAR were isolated. The vector and insert were then
WO 91 / 10453 PCT/US91 /00175
21
ligated together and E. coli transformed with ligated
product.
The results depicted in Figure 5 show that
inhibition of the HIV-1-LTR directed gene expression
increases with the number of TARS in the construct
until 25 TARs are used. Increasing the number of TARS
above 25 does not increase the inhibition. Thus it is
believe that the tat protein is saturated at this
point. It is also possible that the test is not
sensitive enough to detect further increases in
inhibition.
The LTR-50TAR can be transferred to a
retroviral vector, such as DC-vector [Hantzopaulos et
al, Proc. Natl. Acad. Sci. USA 86:3519 (1989) ] . The LTR-
50TAR is cut with XbaI, filled in with the Klenow ezyme
and inserted into the SnaBI site of the DC vector for
high efficiency gene transfer. Other vectors which
ensure high efficiency gene transfer would be
appropriate for use in the present invention.
Construction of Ribosyme-Poly-TAR
For the construction of pRRE-ribozyme, the
vector pRRE [ Daef ler et al , Proc . Natl. Acad . Sci . USA
87:4571-4575 (1990)] containing the RRE (rev-response
element under the control of a T7 promoter) was cut
with BamHI, dephosphorylated and purified. As the
insert, a 65Hp long ribozyme PCR fragment [Chang et al,
Clinical Biotechnology 2:23-31 (1990) ] flanked by BamHI
sites was cut by BamHI and purified. This ribozyme is
directed against HIV-1 GAG mRNA and was published in
Nature 247: 1222 (1990) by N. Sarver et al.
The vector and insert were ligated and an
aliquot of the ligation mix was transformed in E. coli.
WO 91/10453 2 ~ ~ 4 1 8 ~ PCT/US91/00175
22
Plasmids were prepared from individual transformants
and were tested by restriction enzyme digestion. 8
clones were found containing the insert. These clones
were tested in virro for biological activity and 3 of the
8 clones were found to have the ability to cut a
synthetic substrate (substrate gift of J. Rossi).
For construction of LRT-polyTAR-RRE-ribozyme
(see Fig. 6), the vector LTR-46TAR is cut with SalI,
the ends filled in with the Klenow enzyme and then cut
again with HindIII. The DNA is then purified. For the
preparation of the insert, pRRE-ribozyme is cut with
HindIII and SmaI. The 314Bp fragment is isolated by
gel electrophoresis. The vector and insert are then
ligated and used to transform E. coli. Individual
colonies will be checked.
The LTR-polyTAR-RRE-ribozyme is transferred to
a retroviral vector, such as DC-vector [Hantzopaulos et
al, Proc. Narl. Acad. Sci. USA 86:3519 (1989) ] , by cutting
with XbaI. The big XabI fragment is filled in by
Klenow enzyme and inserted in the SnaBI site of the DC-
vector. Putting the construct in a retroviral vector
is necessary for high efficiency gene transfer and the
DC vector is preferred. Nevertheless, any vector which
ensures high efficiency gene transfer would be
appropriate.
Conatructioa o! nal~G-Poly-TAR
pRRE-oGAG was constructed by cutting the pRRE
vector with BamHI, dephosphorylating the ends and
purifying the vector. The oGAG protein encoded in the
mutant viral DNA HT4(VI-DE-dhfr) [Torno et al, Cell 59:
113-120 (1989)], can dominantly interfere with the
replication of HIV-1. Plasmid DNA HT4(VI-oE-dhfr) was
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- 23 -
cut with BglII and a 1429 Bp fragment containing eGAG was
isolated from 1% agarose gel. The vector and insert were
ligated and an aliquot of the ligation mix was used to
transform E. coli. Plasmids were prepared from
individual transformants and were tested by restriction
enzyme digestion, EcoRI + SphI.
For construction of LRT-polyTAR-RRE-eGAG (see Fig.
7), the vector LTR-46TAR is cut with SalI, the ends
filled in with the Klenow enzyme. The DNA is then
purified. For the preparation of the insert, pRRE - eGAG
is cut with EcoRV and SmaI. The 1.6 kB fragment is
isolated by gel electrophoresis. The vector and insert
are then ligated and used to transform E. coli.
Individual colonies will be checked. The construct is
inserted in a DC-vector as described above.
A plasmid designated LTR-7TAR was deposited in E.
coli at the American Type Culture Collection in Bethesda,
Maryland on January 17, 1990 under the accession number
68203. Further, the LTR-50TAR plasmid was deposited in
E. coli at the American Type Culture Collection in
Bethesda, Maryland on October 12, 1990 under the
accession number 68446. The plasmids were deposited
under the terms of the Budapest Treaty.
While the foregoing invention has been described in
some detail for purposes of clarity and understanding, it
will be appreciated by one skilled in the art from a
reading of this disclosure that various changes in form
and detail can be made without departing from the true
scope of the invention.
