Note: Descriptions are shown in the official language in which they were submitted.
~ 2 i 7 2 a i ~
w og~/08635 1 PCT/GBg4/02092
EXPRESSION OF VIRAL DECOY PROTEINS UNDER THE CONTROL OF A LOCUS
CONTROL REGION AND USES THEREOF
The present invention relates to an agent for anti-viral
therapy which possesses a protective effect when
administered to healthy individuals. In particular, the
agent of the invention may be a mutant HIV gene product.
TntrO~Uction
Human immunodeficiency virus (HIV) has been identified as
the etiological agent in human ac~uired immunodeficiency
syndrome (AIDS) (Barre-Sinoussi et al., 1983; Gallo et al.,
1984). Conventional therapeutic strategies have
concentrated on antiviral drugs such as AZT and on the
development of preventive vaccines. However, the
intracellular immunisation approach (Baltimore, 1988) has
lead to the development of molecular strategies for the
inhibition of HIV replication (Malim et al ., 1989, Trono et
al., 1989, Sczakiel et al., 1991, Sullenger et al., 1990).
Molecular systems for in vivo cell specific therapy have
been described where~y a gene encoding a toxic product can
be controlled in its expression by regulatory regions of
genes active only in particular cells.
Initial studies on cell-specific ablation therapy have
utilised cytotoxic agents such as diphtheria toxin A or
ricin A chain genes under the control of lens (Breitman et
al ., 1987; Landel et al ., 1988) or pituitary (Behringer et
al., 1988) specific promoters. After microinjection into
mouse embryos and the production of transgenic animals,
these constructs resulted in the destruction of either lens
or pituitary cells. However, when associated with leaky
promoter elements, these toxin genes are unsuitable for
somatic therapy because of the constitutive cell lethality
and the extreme sensitivity of mammalian cells to diphtheria
and ricin toxins.
~ r~ 2 1 7 2 0 7 3
WO 95/0863~ 2 PCT/GB94/02092
A more versatile toxin-encoding gene for potential use in
human ablative therapy has been described (Borelli et al.,
1988). The Herpes Simplex Virus type 1 thymidine kinase
(tk) gene product is a conditional cell lethal and has been
shown to be toxic to mammalian cells only in the presence of
nucleoside analogues such as acyclovir (ACV) or gancyclovir
(GCV). These analogues kill actively cycling cells because
they possess high affinity for the tk gene product with
little or no affinity for endogenous mammalian tk. Model
systems have demonstrated in vlvo lymphocyte specific
lethality by anti-herpetic drug treatment of tk transgenic
mice (Borelli et al ., 1988, Heyman et al ., 1989).
Specificity of conditional toxicity is due to lymphoid
specific transcriptional control elements and quantitative
flexibility is inherent within the levels of tk transgene
expression and/or administered drug dose. Upon withdrawal
of the drug in these studies, mature lymphocytes are
restored to normal numbers. Thus, the in vivo ablative
system is regenerative, reversible and does not affect stem
cells.
Even advanced ablative systems, however, have their
disadvantages in antiviral therapy. In particular, the
rationale of the system is flawed in that it relies on the
destruction of virally infected cells. Although this
prevents viral replication, the pathogenic effect of the
virus, the destruction of T-lymphocytes in the case of HIV,
is actually promoted. Therefore, ablative systems are
unlikely to be applicable to cases of established infection
and must rely on reaching substantially all cells infected
by the virus.
Another potential system for use in anti-HIV therapy
involves the expression in cells susceptible to HIV
infection of a decoy gene.
Decoy genes encode proteins which act as antagonists to
natural proteins involved in the replication of the HIV
2 1 7 ~ 3 7 ~
wo95lo863s 3 PCT/GB94/02092
virus. For example, a decoy gene may encode a defective
mutant of a transactivator protein which is capable of
binding to the transactivator-responsive site on the host or
viral genome, yet is incapable of activating transcription.
s
Transdominant mutations have been reported in a number of
viral transactivators which abolish or attenuate the ability
of the wild-type protein to transactivate the target gene.
Examples include transdominant mutations of ElA (Glen et
10 al., 1987), tax (Wachsman et al., 1987) and VM65 (Friedman
et al ., 1988). Similar mutations in HIV genes have been
described for the Tat transactivator (Pearson et al., 1990)
and the Rev transactivator (Bevac et al, 1992; Malim et al.,
1992).
Expression of such mutant proteins in a HIV-infected cell
line leads to competition with the natural transactivator
and resultant loss of transactivating activity. See, for
example, International patent application W0 9014427
(Sandoz). A potential disadvantage of the use of decoy gene
approaches is that when a decoy is expressed in the absence
of the infecting virus a negative biological effect may be
exerted on the host. For example, a host immune response
may result from the production of the decoy gene product,
leading to destruction of the host cell by, for example,
cytotoxic T-lymphocytes (CTL). Furthermore, it must be
borne in mind that decoy proteins are derived from
biologically active gene products of pathogenic organisms
and may therefore exert a deleterious effect on the host.
For example, certain allelic variants of the HIV nef gene
product have been shown to downregulate CD4 expression on
thymocytes and to reduce the numbers of CD4 thymocytes in
transgenic mice (see our copending U.K. Patent Application
No. g305759.4 and Guy e~ al., 1990).
In order to avoid these disadvantages it has been suggested
that transcription and expression of decoy gene products
should be restricted to cells actually infected by HIV, for
~ i 1 g ~ ~ ~ 2 1 7 2 ~ 7 3
W095/08635 4 PcT/GB91/02092
example by using a transactivatable expression system for
decoy expression which is transactivated by an HIV gene
product (see WO 9011359).
However, because the decoy gene product is only expressed in
cells after HIV infection, a considerable excess of decoy
gene product is required in order to successfully arrest
viral replication, since the virus has an effective head
start. The production of large amounts of decoy in infected
cells is likely to give rise to the aforementioned negative
~iological effects against such cells, leading to death or
incapacitation of infected cells and the concomitant
disadvantages of ablative systems discussed above.
An alternative approach, which has not been proposed in the
prior art, would be to ensure constitutive expression of
decoy proteins which do not give rise to a negative
biological effect in cells susceptible to HIV infection.
Locus control regions (LCRs) are elements which confer
position-independent, copy number-dependent expression of
genes in gene transfer approaches. They have also been
shown to permit high levels of expression of cloned genes
and to possess tissue-specific properties. First discovered
in globin genes (Grosveld et al, 1987) these elements are
believed to direct the creation of independent regulatory
domains within the chromatin structure of cell genomes,
thereby ensuring the activity of a co-transferred gene.
A number of LCRs other than those for globin genes have been
described, for example in the CD2 gene in T-lymphocytes
(Greaves et al., 1989) and the lysozyme gene in macrophages
(Bonifer et al., 1990) and B-cells (see European patent
application 460042).
The targets of HIV infection are primarily CD4 T-
lymphocytes, but also include macrophages and, dendritic
cells which are related to macrophages and of importance in
wos5/08635 2 1 7 2 0 i 3 PCT/GBg4/02092
initiating an immune response (reviewed in McCune, 1991).
These cells share very few common features except for being
derived from common hematopoietic stem cells and
susceptibility of HIV infection. Hematopoietic stem cells
are not infected by HIV (Molina et al., 1990; David et al.,
1991) -
We have now determined that by inserting a transcriptionunit encoding a decoy gene under the control of the CD2 LCR
into stem cells, constitutive expression of the decoy may be
achieved in T-lymphocytes in transgenic mice and passed
through the germ line, without disadvantageous effects on
the host, by selection of decoy genes having low deleterious
properties.
According to a first aspect of the invention, therefore,
there is provided a recombinant nucleic acid vector for the
delivery of nucleic acid to a host organism comprising a
transcription unit encoding a transdominant negative mutant
of a viral gene product which has been selected
substantially to avoid a negative biological effect under
the control of a DNA sequence active in cells normally
infected by a virus which is effective to confer
constitutive tissue-specific, integration site independent,
copy number dependent expression of the transcription unit.
By "substantially avoid a negative biological effect" it is
intended to denote that the decoy used in the present
invention has been selected or specifically modified to give
rise to a negligible adverse effect on host cells, which may
safely be disregarded in therapeutic situations, or,
preferably, no adverse effect at all. The negative
biological effect may be a biological impairment of cell
function or the raising of a CTL response, as set out above,
or both.
By "biological impairment of cell function" it is intended
to denote biological effects on host cells, such as
~ ~ C~ 2 1 7 2 0 7 3
W095/0863~ 6 PCTtGB94/02092
downregulation of CD4, which may have a negative effect on
the patient. In some cases the host cells may be affected
by the decoy in a manner similar to the pathologic activity
of the virus. In other cases, the decoy proteins may have
effects not normally associated with viral infection but
which are undesirable when associated with a constitutively
expressed foreign protein.
For example,where the virus is HIV, the decoy is selected to
avoid, inter alia, an impairment of immune function in the
host.
Impairment of immune function has been demonstrated in vitro
for certain allelic variants of nef (Guy et al., 1990).
These results are reinforced in vivo in our copending U.K.
Patent Application No. 9305759.4, which shows that nef
expression may play a role in CD4 downregulation and in
decreasing the numbers of CD4+ T-cells in transgenic mice.
CD4 downregulation and loss of CD4+ T-cells is one of the
pathogenic features associated with HIV infection and AIDS.
CTL response to SIV proteins has been observed in SIV-
infected macaques (Venet et al ., 1992) and in human
retroviral infection (Kannagi et al., 1983; Mitsuya et al.,
1983; Autran et al., 1991; Nixon and McMichael, 1991). CTL
responses to Nef antigens and Rev antigens are strong. CTL
responses to Tat antigens are, however, rare and thought to
be very weak (T~Amh~edi-cherradi et al., 1992).
We have shown that the HIV tat gene product does not impair
cell function in transgenic mice. The tat gene product,
therefore, appears to exert little or no impairment of cell
function, while at the same time is of very low CTL-inducing
activity. Preferably, therefore, the invention comprises
the constitutive expression of a Tat decoy.
The invention further provides the use of other natural
decoy gene products which possess the desired
characteristics displayed by tat, namely the absence of
-` - 21 7~D~3
substantial ne~ative bio~ogical e~fects.
AlternatiYely, the lnvention pr~ides for the use of a decoy
~ene product which ha~ been specifically mutated to reduce
S ~he incidence and str~ngth of n~gative ~iological resp~nses
t~er~to. This may be achieved, ~or exa~pLe, by mutation or
deletion cf cer~a~n ~omains of ~ dec~y gen~ prcduct. For
example, the cell-impairing eff~cts o~ z dccoy gene product
may be reduced ar el~minated by the introduction of point
~utations in the g~ne (see Guy et a7., 1~90).
For example, we have shown that, in tr~nsgenic mice,
expression c~ the Tat gen~ product under thQ control o~ the
cnz LCR gives rise to a three-fold incre~se in the levels of
~NA encoding certain cytoXines, namely TGF-~, IL4 ~eceptor
and ~NF~
~n contrast, when a mutated Tat gene product is used
comprisin~ a point ~ut~tion which aboli~hQs its effector
function, cy~okine mRNA lev~s are not affe~ted.
~dvantageously, t~erefore, A mutate~ Tat gene produ~t is
used in the present in~ention.
CT~ response to a p~otein ~ay be modi~ied, either by the
introduction of muta~ions at certain resi~ues involved in
bindin~ to thQ presenting ~LA ~.olecules ~r interacting with
the T-cell rcceptors ~sae ChQppin et al~, 1992; 1991a, b;
Gotch et al., 1988~. Furthermore, it is po~sible to ~odiy
a protein to reduce its rate of degrad~tion by t~e cell and
3~ thereby lower the incidence ~ pre~enta~ion ~f antigens
derived f~o~ the protein (~c~ichael et al., 1977; Bachmai~
et al., 19~6; Towns~nd et al., 1988; Morrison et al., lg92~.
It has be~n shown that ~I~ natura~ly mutates to ~vo~d CTL
responses ~n vlvo and that the sites of amino acid Yariation
tend ~o be conserved, ~'~ laast to a certain extent, in
dif~erent patient~ (Phillips et al., 1~ referably,
therefore, the dec~y of the invention may be muta~ed ~n
A~ENC)EC SHEET
2 1 7 2 0 7 3
WO 9S/08635 8 PCT/GB94/02092
accordance with naturally-derived HIV isolates which display
a reduced CTL response.
However, it should be pointed out that CTL responses will
vary between individuals due to variation in the antigen-
presenting molecules present. Therefore, although some
general mutations may be carried out to reduce CTL response
caused by common HLA types, it remains possible that certain
individuals may show a CTL response even to decoys which are
believed to be of low CTL-inducing activity. In this case,
the invention provides means to mutate the decoy on an
individual basis in order to reduce or eliminate the CTL
response in that individual.
By "transdominant negative mutant" it is intended to refer
to a gene product which is rendered functionally
transdominant over its viral analogue and is effective to
block the activity of the viral analogue. Therefore, the
term is to be interpreted functionally, and comprises
mutants in the normal sense of the term, having an altered
amino acid sequence, as well as mutants which are
alternatively processed or spliced, and mutants which differ
from the wild-type protein in patterns of expression. For
example, the Nef gene product of HIV is known to be a
transdominant inhibitor when expressed in excess.
Therefore, an overproduced Nef protein is included in the
term "transdominant negative mutant".
Constitùtive expression confers a particular advantage of
the invention, that is that the presence of the decoy in
healthy cells effectively prevents the infecting virus from
becoming established. If the decoy gene is only activated
after infection by the virus, there is the possibility that
the virus may become established before the decoy is able to
exert any significant anti-viral effect
The DNA sequence controlling the transcription unit of the
vector of the invention is preferably a Locus Control Region
~i S i ~ ~ 2 1 7~.2 07~
W095/08635 PCT/GB94/02092
(LCR). A number of LCRs have been described in the art and
the selection of an appropriate LCR is within the
capabilities of a person skilled in the art. In the case of
the treatment of HIV infections, however, the use of the CD2
and the macrophage-specific lysozyme LCR is preferred. Both
T-cells, in which the CD2 LCR is active, and macrophages are
targets for HIV infection.
In addition to the LCR, the vector of the invention is
equipped with a promoter which is constitutively active in
the target tissue type. For example, if the cells to be
targeted are T-cells, the CD2 promoter may be used. The
promoter, however, may be active in cells other than the
target tissue. In such a case, high-level expression in
non-target cells is unlikely, because the LCR is inactive in
these cells. Even if a certain amount of non-specific
expression cells does occur, such expression will not be
harmful as the gene product is selected to avoid negative
biological effects. In any event, non-specific expression
can be minimised by the use of efficient vector targeting
techniques to deliver the vector of the invention.
The nucleic acid vector may be any vector capable of
delivering nucleic acid to a cell. For example, the vector
may be a plasmid, a virus or a linear DNA fragment. The
vector may be naked, complexed with proteins or packaged in
a delivery system such as a liposome, virosome, or receptor
mediated complex.
The vector of the invention is preferably for use in the
transfection of stem cells. Therefrom stems a further
advantage of the invention, that is that the decoy is
expressed in all cells of a particular lineage.
When the vector encodes an HIV decoy, the stem cells may be
hematopoietic stem cells.
Alternatively, T-cells may be targeted directly. The
targeting of T-cells is desirable, for example, in the case
2 1 7 2 0 7 3
W095/08635 10 pcTlGBs~lo2o92
where HIV infection is already established but the virus has
not yet spread to the peripheral T-cell population. In this
instance, such cells may be effectively protected from viral
infection.
A number of protocols for the transfection of stem cells and
T-cells are known in the art. Some involve the isolation of
stem cells from total cell populations, as described in, for
example, European patent applications 0 455 482 and 0 451
611.
An improved process for the transfection of stem cells and
T-cells is described in our copending U.K. Patent
Application No. 9317380.5, the disclosure of which is
incorporated herein by reference.
The host organism may be a mammal, insect, fish, plant or
any other organism which it is desired to protect from viral
disease. Preferably, the host organism is man.
Where the decoy is a Tat decoy, the decoy may be prepared
following any of the protocols known in the art. For
example, the method of Pearson et al. (1990) may be used to
generate deletion mutants of Tat which lack the
transactivating function but retain the ability to bind to
the tat region. Such deletion mutants may be tested for
decoy activity as described in Pearson et al. or according
to the methods set forth in our copending U.K. Patent
Application No. 9305759.4 as well as International Patent
Application WO90/14427.
The potential toxicity of any such mutants may be tested by
the methods described hereinbelow. Should such mutants
prove to give rise to~a CTL response in a patient, they may
be further mutated to reduce this response, in accordance
methods known in the art.
r 2 1 7 2r~ 13
W095/0863~ 11 PCT/GB94/02092
The decoy gene may be derived from any virus which gives
rise to infection in man or other organisms, including
plants. Especially preferred, however, are HIV decoys such
as Tat, Rev or Nef decoys.
The decoy may be derived from the same virus as it is
intended to combat with the vector of the invention.
However, it is envisaged that decoys derived from viruses
other than one it is intended to treat may be used. For
example, it has been noted that an HTLVl Rex decoy is active
in suppressing HIV1 Rev function (Bohnlein et al., 1991).
Furthermore, it is envisaged that entirely artificial decoy
genes encoding specialised decoy proteins may be designed.
For example, an artificial decoy gene may be designed which
encodes the tar-binding domain only of the HIV Tat
transactivator, or an analogue of the tar-binding domain
which effectively competes for tar binding with wild type
Tat.
The vector of the invention is preferably for use in the
transfection of a patient's cells in vivo or ex vivo, for
the treatment of a viral disease. According to a second
aspect of the .invention, therefore, there is provided a
vector according to the first aspect of the invention for
use in therapy.
By "therapy", it is intended to denote both the prevention
and the attenuation or elimination of viral infection. As
set out hereinbefore, it is preferred that the vectors of
the invention be used for the prophylaxis of viral
infections because it is believed that it may be important
to avoid establishment of the viral infection in the host.
However, especially in the early stages of a viral
infection, it is envisaged that the vectors of the invention
may have a conventional therapeutic application effective to
attenuate and eventually eliminate the viral infection.
Preferably, the vector of the invention is used for the
21 72073
W095/08635 ~ 12 pcTlGBs~lo2o92
treatment of stem cells or T-cells ex vivo. Accordingly, in
a third aspect of the present invention there is provided a
vector according to the first aspect of the invention for
use in the treatment of stem cells or T-cells ex vivo.
Stem cells or T-cells may be isolated according to
procedures described in the prior art, as set out
hereinbefore. Alternatively, as is preferred, stem cells or
T-cells may be targeted using an efficient targeted
transfection techni~ue, such as that described in our
copending U.K. Patent Application No. 9317380.5. Using such
a technique, it is possible to transfect stem cells or T-
cells in whole blood obtained from patients with extremely
high efficiency.
According to a further aspect of the invention, there is
provided a method for treating or preventing a viral
infection comprising the steps of:
a) removing a cell from the body of a patient;
b) transfecting the cell with a vector according to the
first aspect of the invention; and
c) returning the cell to the body of the patient.
Preferably, the cell is a stem cell. For example, the cell
is a haematopoietic stem cell. Alternatively, the cell may
be a T-cell.
Haematopoietic stem cells and T-cells are easily removed
from the body of a patient, for example from cord blood,
peripheral blood or bone marrow aspirate.
Transfection of stem cells may be accomplished by any of the
protocols cited hereinbefore.
The transfected stem cells, once returned to the body of the
a~ ` t f"
2 1 7 2 0
woss/08635 13 ~PC~/GB94/02092
patient, will divide in the usual manner and populate the
patient with cell lineages carrying the heterologous gene
comprised in the vector of the invention. The cell lineages
thus derived will possess the antiviral capabilities
S conferred by the heterologous gene.
According to a still further aspect of the invention, there
is provided the use of a vector according to the invention
in the manufacture of a composition for use in the treatment
or prophylaxis of a viral disease.
Preferably, the composition comprises the vector of the
invention in a suitable buffer for use in the transfection
of cells either in vivo or ex vivo. When used in vivo, the
buffer will consist essentially of pharmaceutically
acceptable excipients, diluents or carriers. For use ex
vivo, the nature of the buffer will be determined by the
transfection protocol being employed. For example, if the
method described in our copending U.K. Patent Application
No. 9317380.5 is to be used, the buffer as described therein
is used.
In a still further aspect of the invention, there is
provided a cell comprising a transcription unit encoded by
the vector of the invention. Preferably, the cell is a stem
cell and advantageously it is a haematopoietic stem cell.
Alternatively, the cell may be a T-cell.
The invention further provides a method for the treatment or
prevention of a viral infection comprising administering to
a patient a pharmaceutically effective amount of a
composition comprising the vector of the invention in
admixture with a pharmaceutically acceptable excipient,
diluent or carrier.
The invention will now be described for the purpose of
illustration only in the following examples, with reference
to the Figures, in which:
WO 95/08635 ~ ~ W ~ ~ ~ S 2 1 7 2 0 7 3 PCT/GB94/02092
Figure 1 is a diagrammatic representation of the structure
of the CD2-Tat transgene;
Figure 2 shows the identification of the Tat DNA in
transgenic mice carrying the CD2-Tat transgene;
Figures 3 A and B show the identification of Tat RNA in
transgenic mice carrying the CD2-Tat transgene;
Figure 4 shows a FACS analysis of thymus tissue from
transgenic and non-transgenic mice;
Figure 5 shows a FACS analysis of spleen and lymph node
tissues from transgenic and non-transgenic mice;
Figure 6 shows the impact of the presence of the Tat
transgene on cytokine gene expression in transgenic mice;
Figure 7 is a slot blot showing the generation of transgenic
mice carrying a mutated Tat transgene in which Tyr 47 has
been mutated to Ala; and
Figure 8 is a slot blot which demonstrates that mutant Tat
has no effect on the expression of the TNF-~ gene.
METHODS
CD2-Tat mice
A DNA fragment comprising the Tat coding sequence (see
Figure 1) was ligated into a unique EcoR1 site in the first
exon of the CD2 gene in the p2629 CD2 expression plasmid,
which was obtained from Dr. D. Kioussis, NIMR, Mill Hill,
Great Britain. A 4.5 kb fragment containing the CD2 LCR was
isolated from p2694 (also obtained from Dr. Kioussis) and
ligated into the unique Bam H1 - Not 1 sites of p 2629.
The 12 kb Sal 1 - Not 1 fragment comprising the CD2-Tat
construct was then excised and microinjected into single-
21 72073
",.~
cell ~ouse embryos as previously described ~Grosveld et al.,1387~. ~ositi~e ~ounder ~nimals were bred w~th CBA x C5
B~J10 ~ice and lines maintained zs he~erozygotes.
D~A an~ ~Ypre3sion Analysis
T~il D~A (lo~g~ from founder animal~ was ~nalysed by
Southern blot an21ysis after d~gesti~n with KindIII or
Asp718 DNA was run on a 1~ ~garose~Tris-~cet~t~, ~DTA gel,
~0 blotted onto nitr~ce~lulose and p~obed with a rand~mly
primèd Ba~KI-Sma~ ~at fragment. A 1.2 Kb Thy-1.2 ~ragment
was used as a lo~ing con~rol probe.
Appropriate amounts o~ pCD2T~t spiked in 10 ~g genomic ~A
were used as a copy number controls. ~uantitati~n was
per~or~ed on the Molecular Dyna~ics PhosphorImager.
~A w~s prepared using the lithiu~ ch~oride/urca method
(~raser et al., l~9Vj. For Northern blot analysis tSambrook
20 et al., lg~g) lO~g of RNA was run on a 1% formaldehyde gel,
~lo~ted onto nitrocellulose and p~obed with a 8~ bp Bam~I-
5~aI ne~ fra~ment ~rom pTG1147. For RNA slot b~ot6
(S~m~rook et aJ . ~ lg89) 5~g o~ 3~NA wa~: hlc>tted onto
nitrocellulose and pro~ed as above. R~ from the Nef
producing CRIP 1 producer cell line (Schwartz ~t al., l~g2)
was used as a positi~e control.
~ample 1
Expression o~ C~-T~ in i~ransgenic Mice
Exon 1 ~encoding ~a 1-72) of the HIV-1 TAT ~ene was inserted
downstream of the transcriptional start ~ite in the first
exon of the human CD2 qene (Figures 1 ~nd 2). A stop codon
w~s con~tructed in the sequence of hum~n c~2 ~xon 2 so as to
eli~inate the production of CD2 p~ot~in. T~ hu~an c~2 LC~
element was li~ated to thQ 3' end of t~e construct. A Sall-
~otl fragment was injected in~o fertili2ed mouse eggs. ~t
~ME~DED S~IE~T
- - - 2 1 ;7 ~
. 16
least three transgenic lines were created. L~ne C ~2 on
figure 3A~ contains 70 copies and line E (4 on figure 3A)
contains 40 copies.
S1 nucleass RNA protoection was perfor~2d on various tissues
from a transgenic and a non-transgen~c mouse u~in~ a ~AT
exon 1 probe. As shown in f ig~re 3B only thymus expressed
TAT highly. Spleen expressed Tat only to low levels. No
expression was obser~ed in the ~idney or li~er of the
tranSgQniC mouse or in ~ny of the tissues o~ the control
non-`transgenic.
In order to determine wheth~r the CD4 and CDs ~ aells
subsets are affected by the overexpression of HI~-TA~,
L5 an~ibody staining and FACS analysis was per~o~ed on
~hymocytes, spleen ~n~ lymph node cells from C02-TAT
trans~enic ~ice ~ Figures 4 and 5 3 . Single cell suspensions
were pr~pared from the tissues Gf lin~ C ~nd line E
transgenic ~ice ~samples 3 and 4 ~espectiYely~ an~ their
non-transge~ic li~ter~ates ~samples 1 and 2). PE labeled
CD8 and ~I~C la~1ed CD4 an~ibodies were incu~ted ~ith the
ce~is ~nd FACS analysis performed. As shown in the contour
plots, no chang~s ~n thQ per~-entage o~ dou~le negative,
dou~le positive or single positive subsets wer~ found in the
thymus of transgenic mice. Furthermore, no char,ge~ in the
percentages ~ CD4 or C~8 sinsle posLtive su~sets were ~ound
in spleen or lymph nodes of the ~ran~genic mice when
comp~re~ to nDn-transgenics. Thus, high le~el eXpreSSiOn o~
HIV-TAT do~c not af f ec~ subse~ distribution in vivc~ in the
o lymphoid organs .
TAT induced tra~scriptional upregulation of ~NT-~ le~a~ to
ov~rproductio~ of ~unctlonal ~ as ~ea~ure~ ~y
cyto~oxicity.
~orthern ~lot an~lysis of RNA from TAT transgenic lines C
and E demonstrated an increa~e o ~3 fold in TN~
transcription (Flgure ~A~. In order to tes~ whether thls
~A~ D'.!:~ S'~
r~ ~11!_ . ." 2 1 7 2 a 7 3
resulted in increased TN~-~ protein production, we perfor~ed
cytotoxicity assays with cell lysates of T cells from TAT
transgenic mice (Cll, C~2, E+1 and ~+2~ and non-tr~nsgenic
littermates ~C- and E-~. Active equi~alents of T~F-
~
S protein in each sample was quanti~ated ag~inst a known TNF-,~
protein standard (i~ units~. As shown in ~i~ure 6~, all of
four transgenic mice pr~duced siqnificant~y higher leYel~ of
T~F-~ ~2-4 fold) as compared to the non-trans~enic controls.
Cytok~ne ge~a ~xpre~ion i3 a~fecte~ ~y ~a pressure of ~V-
~A~.
Northern blot analysis ~as per~ormed cn MA from CD2-TA~
tr~nsgenic mice to test for quantitative dtfferences ~n
LS cytckine gene expression ~Figure 6A~. Total mRNA was
prepared f rom thymocytes of line C ~ransgenic m~ ce ~C+l ~nd
C+2~ and a non-transgsnic llttermate ~C-) and fro~
thy~ocytes o~ line E transgenic mice (E+1 and ~+2) and a
non ~ransgenic littermate ~F.-). 10 ~g of RN~ was loaded per
2~ lan~ on a forTnaldehyde agarose gel. Rr~A was tr2~nsferred
on~o ~ f ilter and hy~ridi~ed WLt~ a ~-actin probe as an ~A
~uantitation control and a TAT probe ~or verification of
~ansgene expre~ion. ~he filter wa~ r~hybrLdized several
tlmes with pr~bes ~o~ cytokine genes T~F-~, IL-4R, TNF-~ and
TNF-~. Autoradiogra~s of the Northern ~Lot ~e~onstrate an
incr~a~;e in expression of TGF~ -4R and TN~-~ gene
expres~ion in the TAT transgenic ~ice. However,
hybridiz~tion signal with the TNF-c~ probe sugs~ests no changa
Qr a decrease in TN~ ene expression in the T~ positiv~
3 0 mice .
Tl~e results Oc this Northern blot were quantit~ted on a
Phosphorlma~r ~nd fold increas~s or decreases w~re
calculated. A~ 3hown in Tabl~ 1, wh~n ~i$nal was normalized
aqainst the ~-actin quantitation control, TGF-~, IL-4R and
TGF-~ steady state ~R~A was increased in the transgenic
mice. TGF-~ levels inc~ease~ on 2verage 2.8 fold, IL-4R
levels increased 3.5 fold and T~ levels increased ~.3
AMfNDED SHE~T
2 1 720~3
~ .... . ..... . ...... . .... .
lB
fold. TNF-a levels decrease by about 25~. Thus, the
expr~ssion o~ TAT n thymocytes has an ~ffoct on cytokine
gene exp~ession.
~able 1
QU~ntitation o~ Cytoki~e R~A~ i~ CD2-T~ ~r~n~g~ic ~ic~
Transgenic 1 i~e ~ Line C Line ~
Mouse 1 2 1 2
TGF--~ 2.~0 3.26 ~.74 2.88
IL-4R 3.4~ ~.76 4.03 3.76
TNF_B ~.29 2.3~ 2.20 2.22
TNF-~Y 0.81 0.67 0.74 .t:~.85
~old RNA change as compared to non-transgenic littermates.
Exam~le 2
EYP~e~3iOn Of C~2-TA~ t~7a~a~ in ~r~nsge~ic ~$c-.
~5 A 12kb S~lI-NotI fragment containing the CD2 pro~oter and
~CR ele~en~ ~nd exon 1 ~enc~ding aa 1-72~ of a mutant form
cf the HIV-1 TAT gene in which amino acid 47 had been
convert~d ~rcm a tyrosine into an alanine ~47 Tyr --~ ~la),
was injected into fertilized mouse eggs. Ac shown in figur~
~C ~, thrQe tr~nsgQnic ~ounders were cr~ted ~Lines ~6, A7 and
A8). In comparison with transgene c~py number con~rols frc~
o to 50 (Lanff B~, thes~ f~unders contained between 8 and 2S
~opies of the trans~ene.
3~ In order to d~termine whether thymocyte T cell ~u~sets are
af~ected ~y the expression of the ~ut~nt form ~f the ~r~-TAT
(4~al8), antibody stainir.g and FACS analygis was p~rforme~
on ~hymo~ytes from C~2_TAT (~7ala~ transgenic ~ice. Single
!
~ ~tt~r~ S~_T
2 1 72~7~3~
. ~i . . .... . . . . . . . . . .... . .. ~. .. . .. . . ... .. ... ... . . . .
-
19
cell suspensions were prepared from ~ous~ A6 and a non-
transgenic animal, A4. ~E-labelled CD8 and FITC-l~belled CD~
antib~dles were incu~ated with tha c~lls and FACS ana~ysis
was performed. As shown in Ta~le 2~ no changes were ~ound
in the total number of thymocytes derived from either
t~ansgenic or non-transgen~c mice. Furthermore, no
di~erence was observed Ln ~he rcla~ive percentages of
dou~le negative (DN), double positive (DP~, or single
positive (SP) C~4 ~r CD8 cells in the thy~s from the
~0 transgenic compared with the non-t~ansgenic mo~se.
Tabl~ 2
~hy~c ~ cell Subae~ in C~2-~AT ~7~1a) Trz~g~nic Mice
Tot~l DN DP SP SP
T-cells ICD4-CD8-~(CD4'1C~8-~ (CD4t) 1C~8t)
xlO~
Non-TransgeniC 2.5 4.l 80.7 11.5 3.7
~an~eni~ 2.2 7.4 75.g 12.6 4.1
Cyto~ine gene ~xpr-~sion tTNF-~ is ~ct af~3cted by th~
! pr-~ca o~ ~he ~uta~t HI~-TAT t~a~a)
RNA slot blot analysis was per~ormed on RNA ~rom C~2-TAT
~C ~47~1a) transgenic mice to ~es~ for quantLtative differences
in cytokine gene expression (Fi~u~e ~)~ Total RN~ was
prepared from the thymocytes G~ C~2-~AT ~47ala~ transgenic
mic~ A.~ and A.7, two non transgenic controls A.4 and A.5,
and ~ro~n two ~ransgenic mice, on~ from line c~c.l) and one
from line EtE.l) harbourir.g the wild type HIY-TA~.
Duplicat~d lO~g RNA samples ~era denatured and loaded onto
the filter and hybridized with a ~-actLn probe as an RNA
~uanti~ation control, znd then with a probe specific for the
s AMENDED ~ T
21 72~73
-
c~tokine TNF-~. Autor~diographs of the ~NA blots
demonstrate that while an increase in expression of TNF-~ i8
seen in t~ans~nic ~;ice containing the wild type HI~-TA~, no
increase in expressis~ of TNF-~ is o~served in mice
containing the mutant ~IV-TA~ (47ala~.
The results of t~is R~-A slot bl~t were quantitated on a
phosph~rImager and the fold chang~s in cytokine gene
expression determined. As ~hown in Ta~le ~, when the ~ignals
are normalized against the internal quan~itation control ~-
actin, although TNF-~ steady state m~NA was eleYated in
lin~s C and E, there was no increas~ in expression of this
cytokin~ in the nutant ~iIV-TAT (47al~ transgenic mice.
Thus, th~ expression ~c the mutant HI~-~AT ~4?ala~ has no
effec~ on TNF-~ cytokine expression.
IA3L~ 3
ZQ ~u~h~itAti~n of cytokin~ ~NF-~) RNA in ~IV-TAT
transgen~c mice
Transsenic mouse A.6 A.7 C.l E.l
TN~-~ 0.~? 0.89 1.~ 2~4
Fold ~NA chan~e. as co~pared with non-trans~enic mice.
2 1 72~73 ~
.. . . . .. . .. .. . . ..
21
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