U.S. patent application number 10/252404 was filed with the patent office on 2003-10-02 for endometrial function.
This patent application is currently assigned to Cambridge University Technical Services Limited. Invention is credited to Charnock-Jones, David Stephen, Heap, Robert Brian, Sharkey, Andrew Mark, Smith, Stephen Kevin.
Application Number | 20030186907 10/252404 |
Document ID | / |
Family ID | 26306279 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186907 |
Kind Code |
A1 |
Charnock-Jones, David Stephen ;
et al. |
October 2, 2003 |
Endometrial function
Abstract
Disclosed is a method of altering one or more characteristics of
at least some of the cells of the reproductive tract of a mammalian
individual by the introduction into said cells of a nucleic acid,
together with a composition comprising nucleic acid, for use in the
method.
Inventors: |
Charnock-Jones, David Stephen;
(Cambridge, GB) ; Smith, Stephen Kevin;
(Cambridge, GB) ; Sharkey, Andrew Mark;
(Cambridge, GB) ; Heap, Robert Brian; (Cambridge,
GB) |
Correspondence
Address: |
PILLSBURY WINTHROP, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Assignee: |
Cambridge University Technical
Services Limited
|
Family ID: |
26306279 |
Appl. No.: |
10/252404 |
Filed: |
September 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10252404 |
Sep 24, 2002 |
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08860047 |
Sep 18, 1997 |
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6472374 |
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08860047 |
Sep 18, 1997 |
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PCT/GB95/03008 |
Dec 21, 1995 |
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Current U.S.
Class: |
514/44R ;
424/93.21; 435/366; 435/455 |
Current CPC
Class: |
A61K 38/1793 20130101;
A61P 31/20 20180101; C12N 15/1136 20130101; A61K 2039/53 20130101;
A61P 5/38 20180101; A61K 48/00 20130101; A61P 15/02 20180101; A61P
35/00 20180101; A61P 37/02 20180101; C12N 2320/32 20130101; A61P
35/02 20180101; A61P 43/00 20180101; C12N 2310/111 20130101; C12N
2330/50 20130101; A61P 31/18 20180101; C12N 2310/11 20130101; A61K
38/18 20130101; A61P 5/24 20180101; A61P 1/16 20180101; A61P 31/04
20180101; A61K 38/204 20130101; A61P 7/04 20180101; A61P 15/00
20180101; A61P 15/08 20180101; A61P 3/10 20180101; A61P 13/02
20180101 |
Class at
Publication: |
514/44 ;
424/93.21; 435/455; 435/366 |
International
Class: |
A61K 048/00; C12N
015/85; C12N 005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 1994 |
GB |
9426380.3 |
Oct 12, 1995 |
GB |
9520879.9 |
Claims
1. A method of altering one or more characteristics of at least
some of the cells of the reproductive tract of a mammalian
individual by the introduction into said cells of a nucleic
acid.
2. A method according to claim 1, wherein the nucleic acid is
introduced into endometrial cells.
3. A method according to claim 1 or 2, wherein the nucleic acid is
introduced into the glandular epithelium of the endometrium.
4. A method according to any of the preceding claims, wherein the
introduced nucleic acid is DNA.
5. A method according to any one of the preceding claims, wherein
the nucleic acid is introduced in the period followint ovulation,
up to and including the point at which there is a peak in the level
of progesterone in the blood.
6. A method according to any one of the preceding claims, wherein
the nucleic acid is introduced in a liposome, virus, or other
carrier particle.
7. A method according to any one of the preceding claims, wherein
the nucleic acid is introduced as a plasmid or other construct.
8. A method according to any one of the preceding claims, wherein
the introduced nucleic acid comprises a promoter operably linked to
a sequence to be transcribed in a eukaryotic cell.
9. A method according to claim 8, wherein the promoter is
inducible.
10. A method according to any one of the preceding claims, wherein
the introduced nucleic acid sequence comprises a portion operably
linked in the antisense orientation to a promoter, so as to inhibit
the expression of a polypeptide in the cells into which the
sequence is introduced.
11. A method according to any one of the preceding claims, wherein
the characteristic altered by introduction of the nucleic acid
sequence results in an alteration of the fertility of the
individual.
12. A method according to any one of the preceding claims, wherein
the introduced nucleic acid directs the expression of at least an
effective portion of a cytokine or a growth factor.
13. A method according to any one of the preceding claims, wherein
the introduced nucleic acid directs the expression of at least an
effective portion of one of the following: an interleukin;
leukaemia inhibitory factor (LIF); vascular endothelial growth
factor (VEGF); epidermal growth factor (EGF); heparin-binding
epidermal growth factor (HBEGF); insulin-binding growth factors I
and II (IGF-I and IGF-II); amphiregulin; colony stimulating factor
(CSF); tumour necrosis factor (TNF); hepatocyte growth factor
(HGF); and fibroblast growth factor (FGF).
14. A method according to any one of claims 1 to 12, wherein the
introduced nucleic acid directs the expression of at least an
effective portion of an antagonist of a cytokine or an antagonist
of a growth factor.
15. A method according to claim 15, wherein the introduced nucleic
acid directs the expression of interleukin-1 receptor antagonist or
a soluble receptor for one of the following: transforming growth
factor (TGF) .alpha.; fibroblast growth factor (FGF);
platelet-derived growth factor (PDGF); interleukin-6; vascular
endothelial growth factor (VEGF); or hepatocyte growth factor
("Met" receptor).
16. A method according to any one of clains 1 to 11, wherein the
introduced nucleic acid directs the expression in and/or the cell
of at least an effective portion of a receptor for a growth factor,
a cytokine or a steroid hormone.
17. A method according to claim 16, wherein the introduced nucleic
acid sequence directs the expression of at least an effective
portion of a receptor for one of the following: EGF, transforming
growth factor (TGF) .alpha., or VEGF.
18. A method according to any one of claims 1 to 11, wherein the
introduced nucleic acid directs the expression of at least an
effective portion of a polypeptide having a local immunological
effect.
19. A method according to claim 18, wherein the introduced nucleic
acid directs the expression of at least an effective portion of an
antigen from a pathogen.
20. A method according to claim 18 or 19, wherein the introduced
nucleic acid directs the expression of at least an immunogenic
portion of a polypeptide from one of the following: HIV, papilloma
viruses, Chlamydia or N. gonorrhoea.
21. A method according to claim 18, wherein the introduced nucleic
acid directs the expression of an immunoglobulin or an effective
portion thereof.
22. A composition comprising nucleic acid, for use in the method of
any one of the preceding claims.
23. Use of a composition comprising nucleic acid, in accordance
with any one of claims 1 to 21.
24. Use of a composition comprising nucleic acid in the preparation
of a substance for altering one or more characteristics of at least
some of the cells of the reproductive tract of a mammalian
individual.
25. A method of making a composition for use in the method of any
one of claims 1 to 21, the method comprising mixing a nucleic acid
with a physiologically acceptable carrier substance.
Description
FIELD OF THE INVENTION
[0001] This invention relates, inter alia, to a method of altering
one or more characteristics of mammalian endometrial tissue.
BACKGROUND OF THE INVENTION
[0002] Endometrial Physiology
[0003] Two major events are required for the embryo to become
established in the mammalian uterus: firstly, the preparation of
the endometrium so that it is receptive to the presence of a
blastocyst which can then implant and acquire nutritive support
through the formation of the placenta; and secondly, the
modification of myometrial activity which must become quiescent and
thereby allow the blastocyst to become resident within the uterine
cavity without the danger of expulsion. Both these events are
controlled by the action of the hormones of pregnancy, of which
oestrogens and progesterone are particularly important. These
steroid hormones act on the endometrium and myometrium through
their receptors which are located in the nucleus of target cells.
Once activated, the steroid-nuclear receptor complex interacts with
specific regions within the DNA to stimulate, repress or de-repress
genes that code for proteins and polypeptides such as enzymes or
growth factors.
[0004] The initiation of implantation is brought about by a cascade
of biochemical and biophysical changes. Adhesion molecules (e.g.
CAM 105) have been implicated in the early stages or attachment of
the blastocyst to the wall of the uterus. Afterwards, the
blastocyst and endometrium adopt various stratagems to improve
intimacy between fetal and maternal tissues. In ungulates,
trophoblast cells which form the outermost layer of the blastocyst
migrate into the uterine epithelium with which they subsequently
fuse. Cell migration is carried a step further in women because it
is not only isolated or specific cell types that migrate but large
areas of trophoblast which insinuate between the uterine epithelial
cells. In order to do this, some of the trophoblast cells fuse
together to form a syncytium The process is very rapid and the
embryo becomes established in the uterine tissues without much
apparent degeneration in the uterine epithelium. In some species
the process of implantation is delayed, either to await
environmental cues which ensure the young are born at favourable
times of the year, or by physiological factors such as lactation so
that the mother has finished weaning the previous litter before the
next pregnancy becomes fully established.
[0005] The preparation of the uterus for implantation is regulated
by the secretion of ovarian hormones. The transport of the
fertilised egg through the oviduct has to be precisely timed so
that it arrives in the uterus at the correct time of development
and when the uterus is in a fit condition to receive it. Under most
conditions the uterus is hostile to the embryo, more hostile in
fact than some other areas of the body. The epithelial lining of
the uterus is, under most conditions, resistant to attachment and
invasion by trophoblast and it is only under very precise hormonal
states that this resistance is relaxed.
[0006] In mice and rats unmated animals do not have a full oestrous
cycle because they do not form a normal secretory corpus leteum
which produces increasing amounts of progesterone. If mating occurs
at oestrus, a time when high levels of oestrogens are secreted by
the ovarian follicle from which the ovum is shed, a corpus luteum
will form in the place of the ruptured follicle, rising
concentrations of progesterone are then secreted, implantation
occurs and pregnancy progresses (length, 21 days). If an infertile
mating occurs, similar events occur except that the corpus luteum
only lasts about 11 days and pseudopregnancy is curtailed.
[0007] The cellular and biochemical changes that take place in the
endometrium have been most thoroughly studied in the mouse and the
rat, though information about these aspects in women has increased
substantially in recent years. The endometrium in all species is
made up of three main tissues--luminal epithelium, glandular
epithelium and stroma. Cell proliferation occurs at different time
in the three tissues. Luminal cells proliferate just before oestrus
(proestrus) under the influence of the rising levels of oestrogens
produced by the follicles in the ovary. By day 1 of pregnancy (day
of copulation plug in rodents) they have ceased dividion but then
undergo a second, though smaller, burst of activity on day 3.
Glandular cells show most activity on day 4 and then decline.
Stromal cells do not proliferate until day 4 but thereafter, under
the influence of progesterone, they reach high levels of
proliferation by day 5. In women, less is known of these changes
which presage the process of implantation but there appears to be
peak proliferation in epithelial cells during the follicular phase
of the cycle and in stromal cells during the luteal or secretory
phase, as in the mouse and rat.
[0008] The purpose of endometrial cellular proliferation is not
fully resolved. It is believed to prepare the endometrium for
implantation by increasing the number of cells that will serve a
nutritive and secretory function (glandular epithelium) and that
participate in the very early stages of placentation
(decidualization). As a prerequisite of successful implantation,
cell mitosis may progress towards cellular differentiation and
therefore plays a crucial role in the early events of the
establishment of pregnancy. Evidence in support of this role is the
endometrial production of growth factors (mitogens), cytokines and
nuclear oncogenes. Many of these compounds are produced in
increased concentrations in response to ovarian hormones acting
through their receptors.
[0009] Amongst growth factors, much attention is currently given to
epidermal growth factor (EGF), heparin binding epidermal growth
factor (HBEGF), amphiregulin and insulin-binding growth factors
(IGF-I and IGF-II). Evidence for the importance of the local
(paracrine) action of at least one of these growth factors,
amphiregulin, has been provided by recent experiments in mice.
Inhibition of the implantation-specific and progesterone-regulated
gene for amphiregulin was achieved by the anti-progestin, RU486,
and this resulted in the prevention of implantation (Das et al.
Molecular Endocrinology 9, 691-705, 1995).
[0010] Amongst the cytokines, leukaemia inhibitory factor (LIF) and
colony-stimulating factor (CSF), which are also produced by the
mouse uterus at the time of implantation, have been found from gene
knockout studies to be indispensable, demonstrating that their
removal is incompatible with implantation and normal placentation
(Stewart et al. Nature 359, 76-79, 1992; Pollard et al.
Developmental Biology 148, 273-283, 1991).
[0011] Amongst the nuclear oncogenes, levels of c-jun and c-fos
(which are early indicators of gene transcription) increase in the
uterus after oestrogen administration, and are inhibited by
progesterone.
[0012] Important differences exist between various species in the
extent of trophoblast invasion at the time of implantation. In
women, the early trophoblast is highly invasive whereas in pigs,
which have a non-invasive form of implantation, the endometrial
epithelium is never breached throughout the three month gestation
period. Failure of implantation in both these species is high,
reaching about 60 and 30%, respectively. The reasons for this high
rate of failure are complex and incompletely understood. In women,
about half the loss is attributable to genetic abnormalities but in
pigs, as in other ungulates where the loss is also high, genetic
defects only account for a few precent of the total.
[0013] After implantation failure in women a fall in progesterone
secretion causes bleeding, as at the end of the normal menstrual
cycle; this does not occur in most other animals. Disorders of
menstruation, as well as of implantation, are common. In addition
menstrual bleeding, either as a consequence of sequential hormonal
therapy, or in conjunction with continuous eombined hormone
replacement therapy or progestin-only long-acting contraceptives,
is a significant cause of ill-health in women. The underlying
reasons for this bleeding are the focus of many current studies
into biochemical (e.g. prostaglandins, enzymes, polypeptides and
proteins, vasoactive compounds such as platelet-activating factor
PAF, and vascular endothelial growth factor VEGF) and cellular
mechanisms (e.g. migrating cells homing to the uterus that produce
immunosuppressive compounds).
[0014] Current understanding of reporductive processes largely
centres on the control of steroid hormone production and the
actions of these hormones on their target tissues. However
paracrine and autocrine factors are increasingly seen to be key
mediators of reproductive function, albeit interacting with
steroids (Benton, 1991 Current Opinion in Cell Biology 3, 171-175;
Rozengurt, 1992 Current Opinion in Cell Biology 4, 161-165;
Tartakovsky et al., 1991 Developmental Biology 146, 345-352;
Robertson et al., 1992 Current opinion in Immunology 4, 585-590;
Smith, 1994 Human Reporduction 9, 936-946; and Tabibzadeh, 1994
Human Reporduction 9, 947-967). The clearest example of this is
seen in the ovariectomized mouse. In this model the uterus
undergoes marked growth in response to a single dose of estradiol.
This effect can be blocked by anti TGF.alpha. antibody (TGF is
"transforming growth factor") suggesting that the mitogenic effects
of estrogen in this tissue are mediated by TGF.alpha. (Nelson et
al., 1992 Endocrinology 131, 1657-1664).
[0015] Consequently medical intervention in Gynaecology is largely
based on steroidal/antisteroidal regulation of the uterus (Yen
& Jaffe, 1991 in "Reproductive Endocrinology", Eds. Yen, Jaffe
& Benton, Pub. W B Saunders, Philadelphia; Baird, 1993 British
Medical Bulletin 49, 73-87). Despite the undoubted success of this
approach, no conceptual advances in contraceptive technology have
arisen for 20 years, no means identified to improve implantation,
no advances made in promoting placental growth and development and
no new approaches found to treat benign gynaecological disease
(menstrual dysfunction and fibroids).
[0016] A number of publications have been made in relation to the
use of "gene transfer" in mammals to alter the genotype of at least
some cells in a certain tissue or tissues. In particular, it is
known to attempt "gene therapy" of humans by the introduction into
recipients of nucleic acid sequences, with the aim of overcoming a
genetic deficiency in the recipient by the expression of
polypeptides encoded by the introduced nucleic acid sequences. Gene
therapy trials have been conducted, for example, in which DNA
sequences (incorporated within viral vectors) were introduced into
the airways of cystic fibrosis patients, so as to alter the
phenotype of at least some of the epithelial cells lining the
patients' respiratory tract. Thus far, there have been no published
attempts to introduce DNA into the mammalian endometrium, despite
the availability of suitable techniques therefor.
SUMMARY OF THE INVENTION
[0017] In one aspect, the invention provides a method of altering
one or more characteristics of at least some of the cells of the
reproductive tract of a mammalian individual by the introduction
into said cells of a nucleic acid.
[0018] In a second aspect the invention provides a composition
comprising nucleic acid, for use in altering one or more
characteristics of at least some of the cells of the reproductive
tract of a mammalian individual.
[0019] In a third aspect the invention provides for use of a
composition comprising nucleic acid for altering one or more
characteristics of at least some of the cells of the reproductive
tract of a mammalian individual.
[0020] In a fourth aspect the invention provides for use of a
composition comprising nucleic acid in the preparation of a
substance for altering one or more characteristicsof at least some
of the cells of the reproductive tract of a mammalian
individual.
[0021] In a fifth spect the invention provides a method of making a
composition for use in altering one or more characteristics of at
least some of the cells of the reproductive tract of a mammalian
individual, comprising mixing a nucleic acid with physiologically
acceptable carrier substance.
[0022] The present invention can in no way be considered as an
obvious extension of genetic therapy techniques already known to be
at least partially successful when applied to the lungs of cystic
fibrosis patients. Inherited genetic disorders are not thought to
be responsible for any of the known diseases of the endometrium so
there would have been no incentive for those skilled in the art to
apply gene therapy techniques to the endometrium. Further, the
epithelium of the endometrium is of different type (cuboidan,
derived from coelomic epithelium) compared to lung epithelium
(which is straified) and therefore could not have been predicted to
behave in an analagous manner. Moreover, at least in primates,
there os cyclinal shedding of the endometerial epithelium which
would tend to cause the loss of any transfected cells.Finally, the
inventors have found that there was no transfer of the introduced
DNA into the organs of the mother, nor into the placenta of the
embryo, either of which might have occured and could have caused
practical difficulties.
[0023] Typically the nucleic acid is introduced into a mammalian
female (preferably a women) and, in particular, into the
endrometrial cells therof. Desireably the nucleic acid is
introduced into the glandular epithelium of the endometrium. The
nucleic acid may encode a polypeptide which is already naturally
synthesised by the cells into which the nucleic acid is introduced,
such that the level of expression of that polypeptide is increased
via a gene dosage effect. Alternatively the method can be used to
induce the cells to express a polypeptide not previously
synthesised in those cells. The polypeptide could, for example, be
an "artificial" recombinant polypeptide which does not exist in
nature, such as a chimeric polypeptide comprising, wholly or in
part, functional domains from two or more different proteins
[0024] The nucleic acid is preferably DNA, but one could seek to
introduce RNA (either sense or non-sense strands). An antisense
molecule could be used to inhibit or otherwise interfere with the
expression of a polypeptide in the cells into which the nucleic
acid is introduced. The nucleic acid sequence introduced may be
antisense RNA, or may be a DNA sequence directing the synthesis,
intracellularly, or antisense RNA. Another way of achieving such
inhibition is to introduce into the cells a sequence directing the
synthesis of a ribozyme, which will then specifically cleave the
mRNA needed to synthesise the polypeptide whose expression is
sought to be inhibited.
[0025] The present inventors have found that the time of
administration of the nucleic acid (relative to the stage of the
reproductive cycle) greatly affects the efficiency of uptake of the
nucleic acid. The inventors have found that, in general, in order
to obtain the optimum degree of uptake of the administered nucleic
acid it is necessary for the administration to be made in the
period following ovulation, up to and including the day on which
there is a peak of progesterone level in the blood. The
progesterone level normally peaks at around a similar time to the
point at which an embryo, if present in the uterus, could become
implanted
[0026] Thus, for example, the inventors have found that maximal
uptake of administered DNA by mouse endometrium occurs at day 2-3
in the cycle (with day 1 taken as the day on which a vaginal plug
is first detected). In humans, ovulation typically occurs at day 14
of the cycle, and implantation is generally reckoned to occur in
the mid-luteal phase (although the exact time is poorly defined in
humans).
[0027] The nucleic acid may be administered in a naked form, or may
be bound or associated with other substances (e.g. liposomes).
Conveniently the nucleic acid is introduced into the cells of the
recipient mammal by simple transfection (with or without
liposomes), which has been found by the present inventors to be
suprisingly effective, ithout the need for sequence to be
introduced within a viral vector. Nevertheless, viral vectors may
be diserable, especially those which may be targeted to certain
cell types (e.g. as disclosed in WO 93/20221).
[0028] The nucleic acid will conveniently be intoduced as part of a
construct (e.g. a plasmid, cosmid or the like), which construct
will advantageously comprise a promoter, operable in a mammal, to
cause transcription of at least part of the introduced nucleic
acid. The promoter may be constitutive or, more preferably,
inducible so as to allow greater control of expression of the
introduced sequence.
[0029] In one particular method performed in accordance with the
invention, introduced of a nucleic acid molecule into the
endometerial cells of an individual mammalian female allows for the
up- or down-regulation of the fertility of the individual. The
invention may particulary be used to provide a method of
contraception for companion animals (e.g. cats and dogs) to pevent
unwanted litters. In other embodiments the invention provides a
method of improving the fertility of livestock species, such as
pigs, cattle, sheep, and the like.
[0030] Perferably the nucleic acid is introduced into the
reproductive tract via the vagina, which avoids the need for
invasive sugical techniques. However, if neccessary, the nucleic
acid could be introduced by means of surgical techniques directly
into the reproductive tract (e.g. into the uterus). The invention
offers the possibility of altering one or more characteristics by
the introduction of one or more of a very large number of differnt
nucleic acid sequences.
[0031] In one embodiment, the sequence introduced into the
reproductive tract cells directs the expression (preferably at high
levels) of an effective portion of a cytokine or growth factor (an
effective portion is that part of the molecule which retains the
biological activity particularly associated with the whole e.g.
binding to a specific ligand etc.). Examples of such polypeptides
which might be expressed by the introduced sequence include, but
are not limited to, the following: interleukins, leukaemia
inhibitory factor (LIF), vascular endothelial growth factor (VEGF),
epidermal growth factor (EGF), heparin-binding epidermal growth
factor (HBEGF), insulin-binding growth factors I and II (IGF-I and
IGF-II), amphiregulin, colony stimulating factor (CSF), and tumour
necrosis factor (TNF).
[0032] In another embodiment the introduced sequence may direct the
expression of an effective portion of an antagonist of a cytokine
or growth factor, such as the IL-1 receptor antagonist.
Advantageously, the antagonist may be a soluble receptor for the
cytokine or growth factor. Suitable examples include soluble
receptors for the following: transforming growth factor (TGF)
.alpha., fibroblast growth factor (FGF), platelet-derived growth
factor (PDGF), interleukin-6 (IL-6), and VEGF.
[0033] In another embodiment the introduced sequence may direct the
expression of an effective portion of a polypeptide having an
immunological effect. In particular, the polypeptide may prossess
immunogenic activity, thereby srving to stimulate a local immune
response. Advantageously the immunogenic polypeptide will be an
antigen from a mucosal pathogen. By virtue of the common mucosal
immune system, stimulation of antibody production in the
reproductive tract may result in the production of corresponding
antibodies at distal mucosal sites, such as the gastro-intestinal
tract, the respirtory tract, lachrymal glands and the like.
Preferably however the antigen will be one from a pathogen which
invades and/or colonises the reproductive tract, typically a
pathogen which causes a sexually transmitted disease. Examples
include viruses such as HIV. papilloma viruses (e.g. HPV.of various
types). chlamydia and bateria (e.g. N. gonorrhea). Alternatively,
the polypeptide having an immunoloigical effect may be an
immunoglobulin or effective portion thereof (such as an Fab. Fv. or
scFv fragment. or a single chain antibody). The immunoglobulin or
effective pofrtion thereof may be directed against a pathogen (such
as those mentioned above). or may be directed against some other
antigen, such as a steroid or other hormone. Thus immunoglobulins
or fragments thereof could be expressed locally to provide against
disease or to regulate fertility.
[0034] In another embodiment the introduced sequence may direct the
expression of a polypeptide, or an effective portion thereof, which
has an effect on menstration.
[0035] In another embodiment the introduced nucleic acid may direct
the expression, on the surface of the reproductive tract cells, of
an effective portion of a receptor molecule. The receptor could be
a receptor for a cytokine, a steroid hormone, or a growth factor
(such as the EGF receptor,the TGF.alpha. receptor, or the VEGF
receptor). A number of receptors are known which are described as
"orphan" receptors, in that the ligand which binds to the receptor
is unknown. Such orphan receptors are of considerable interest to
the pharmaceutical industry, as they may proide targets for novel
therapeutic or prophylactic compounds. Accordingly, in another
aspect the invention provided a method of characterising the
biological properties of a polypeptide, comprising introducing the
sequence encoding the polypeptide to be characterised into the
cells of the reproductive tact of a mammal, and assesing the
effects of the xpressed polypeptide.Preferably the mammal is a
laboratory animal, such as a mouse or rat. Convenietly, the
polypeptide to be characterised will be an orphan receptor and
typically at least part of the characterisation thereof will
comprise identification of the ligand therefor. Generally the
method will involve the analysis of histological sections taken
from the laboratory mammal, and processing thereof by any one of
various standard techniques (e.g. histochemical staining, in situ
hybridisation, immunological staining etc.)
[0036] The present invention thus offers a novel alternative to
steroidal regulation to endometrial function (and thus reproductive
capacity or fertility) by direct gene transfer in vivo. To achieve
this, genetic constructs would be designed to specifically modulate
cytokine action. This can be achieved in a variety of ways. For
example the ce;;s that produce a secreted cytokine could be
prevented from synthesizing the factor by blocking transcription
and translation using promoter driven antisense constructs or
ribozymes. Alternatively the action of the secreted factor can be
blocked by receptor antagonists. Naturally occuring soluble
receptors may scavenge and neutralize bioactive ligand thereby
acting as competitive receptor antatagonists. Alternatively there
are natural receptor antagonists, for portion of a polypeptide
having an immunological effect. In particular, the polypeptide may
possess immunogenic activity, thereby serving to stimulate a local
immune response, thus the invention can be used to provide a novel
method of immunisation. Advantageously the immunogenic polypeptide
will be an antigen from a mucosal pathogen. By virtue of the common
mucosal immune system, stimulation of antibody production in the
reproductive tract may result in the production of corresponding
antibodies at distal mucosal sites, such as the gastro-intestinal
tract, the respiratory tract, lachrymal glands and the like.
Preferably however the antigen will be one from a pathogen which
invades and/or colonises the reproductive tract, typically, a
pathogen which causes a sexually transmitted disease. Examples
include viruses such as HIV, papilloma viruses (e.g. HPV, of
various types), chlamydia and bacteria (e.g. N. gonorrhoea).
Alternatively, the polypeptide having an immunological effect may
be an immunoglobulin or effective portion thereof (such as an Fab,
Fv, or scFv fragment, or a single chain antibody). The
immunoglobulin or effective portion thereof may be directed against
a pathogen (such as those mentioned above), or may be directed
against some other antigen, such as a steroid or other hormone.
Thus immunoglobulins or fragments thereof could be expressed
locally to provide protection against disease or to regulate
fertility. example IL1Ra (interleukin-1 receptor antagonist),
intraperitoneal administration of this protein blocks blastocyst
implantation in the mouse (Simon et al., 1994 cited elsewhere).
[0037] There is considerable evidence to show that soluble growth
factors secreted by the oviduct and uterine epithelium can control
pre-implantation development of the mammalian embryo, by acting
directly through receptors expressed on the embryo (Pampfer et al.,
1990 In Vitro Cellular and Developmental Biology 26, 944-948). In
turn, developing embryos produce growth factors which may act in an
autocrine fashion, or on the endometrium to influence its
receptivity. For example, in mice, LIF expression (from maternal
tissues) is dramatically upregulated in glandular epithelium on day
4, just prior to implantation. LIF is able to act on
pre-implantation blastocysts, which express the LIF receptor
(LIF-R). This maternal expression of LIF is vital for implantation
since in LIF knockout mice, embryos will not implant, although they
will do so on transfer to pseudopregnant dams (Stewart et al., 1992
cited elsewhere)
[0038] The inventors have now extended this work to humans, and
shown by RT-PCR that human embryos express the mRNA encoding the
LIF-R, but do not themselves express LIF. LIF acts by binding to a
low affinity receptor LIF-R. High affinity binding arises when the
LIF/LIF-R complex interacts with the signal transducing accessory
protein gp130. Human embryos also contain mRNA encoding this
protein (Sharkey et al., 1995 Biology of Reproduction 53, 955-962).
The inventors have also shown LIF secretion in human glandular
epithelium is regulated by steroids, being maximal in the luteal
phase (around the expected time of implantation--Charnock-Jones et
al., 1994 cited elsewhere). Furthermore, administration of LIF to
human pre-implantation embryos in vitro, has been reported to
improve development. All this evidence supports the idea that LIF
may be important in human implantation as it is in the mouse
Clearly cytokines may mediate important communication between the
embryo in the uterine lumen, and the endometrium (in both
directions). The present invention allows the use of gene transfer
to disrupt or enhance this communication, leading to novel methods
of contraception, or conversely improved implantation.
[0039] Most current studies of the paracrine and autocrine
regulation of reproductive function are limited to a descriptive
analysis by the lack of effective methods to modulate local
cytokine/receptor levels. The evidence presented in this
application indicates that transfection of the uterine epithelium
in viro is feasible. This allowa the endometrium to be manipulated
experimentally and offers new therapeutic strategies. The work
outlined below describes the use of a reporter gene to demonstrate
the practicability of in vivo uterine gene transfer. In pratice a
gene (or other DNA construct), able to alter uterine function,
would be used. Exmples of these include receptor antagonists (e.g.
IL-1Ra, soluble VEGF receptors etc.) natural or modified cytokines
and growth factors, protease inhibitors or steroid receptors and
variety of ribozyme and antisense constructs. This work shows that
genes can be transferred to the endometrium in vivo and this will
find utility in many endometrial (and placental) conditions for
eample improving implantation in both animals and man, disrupting
implantation (i.e. contraception), endometriosis and mennorrhagia,
hyperplasia and adenocarcinoma.
[0040] Using the protocols we have developed the results described
below were obtained. They demonstate that gene constructs can be
transferred to the endometerium both in vivo (in mice) and in vitro
and that these constructs are transcriptionally and
(translationally) active.
[0041] The invention will now be further described by way of
illustrative example and with reference to the accompanying
drawings, in which:
[0042] FIGS. 1A and 1B show photomicrographs of histological
sections of mouse endometerium transfected with (A) a plasmid
construct directing the expression of a .beta.-galactosidase
reporter gene,or (B) a similiar plasmid lacking the reporter gene.
Transfected cells can clearly br identified by the intense dark
(blue) staining within the cytoplasm, which is absent in section
B;
[0043] FIG. 2 is a photomicrograph of in vitro human endometerial
cells which have been transformed with a the same plasmid as in
FIG. 1A--the dark (blue) staining due to expression of the reporter
gene is mainly associated with the remaining glandular structure,
while the surrounding cells stain more weakly;
[0044] FIG. 3 is a bar chart showing the results of a CAT assay (in
counts per tube) for endometrial cells successfully transfected
with a gene encoding chloramphenicol acetyl-transferase (pcDNA3CAT)
compared to cells transfected with a control plasmid (pcDNA3);
and
[0045] FIG. 4 is a bar chart showing the results of a luciferase
assay (in relative light units) for endometrial cells successfully
transfected with a gene encoding luciferase (pcDNA3LUC) compared to
cells transfected with a control plasmid (pcDNA3).
EXAMPLES
Example 1
Mice
[0046] Nulliparous mature BALB/cJ mice were housed in a light (14 h
light:10 h dark; lights off at 20.00 h) and temperature (22.degree.
C.) controlled Small Animal House and fed a mouse and rat diet
(Labsure: Christopher Hill Group, Poole, Dorset, UK). They were
placed with vasectomized males of the same stock overnight and
examined the next morning for the presence of a vaginal plug.
Mating was presumed to have occurred at 02.00 h, time 0 and day 1
was counted as the day on which the plug was first detected. Mated
females were housed individually prior to experimentation.
[0047] Laparotomy was performed using aseptic procedures under
Metafane anaesthesia (methoxyfluorane, C-Vet Ltd., Bury St.
Edmunds). The uterine horns were exposed by either mid-ventral or
bilateral incisions. Injections were made either into the tip of
the horn at the tubo-uterine junction or at the base of the horn at
the utero-cervical junction. Repeated studies showed that the
latter technique provided the best method of administration but for
some purposes the former was preferable when it was necessary to
minimise disturbance of the reproductive tract.
[0048] Injections of liposome/DNA (pcDNA3 construct, +/-
.beta.-galactosidase reporter gene), naked DNA or control solutions
(25-100 .mu.l) were performed by insertion of the tip of a flat
Stratatip into the base of the horn. Solutions were previously
drawn up into the tip by means of a Travesty applicator which was
also used to control the slow injection of solution into the
horn.
[0049] After injection, the incision was closed using interrupted
mattress sutures and the mice allowed to recover in thier cages and
provided with food and water ad libitum.
Plasmid constructs
[0050] The plasmid described in this application (by way of
example) are based on the commerically available vector pcDNA3
(Catalogue No. V790-20, from Invitrogen, San Diego, Calif., USA).
Plasmid pcDNA3, without any reporter gene, was used as a negative
control. Experimental plasmids pcDNA3-.beta.gal, pcDNA3-CAT,and
pcDNA3-Luc contained the .beta.-galactosidase, chloramphenicol
acetyltransferase and luciferase reporter genes respectively. These
plasmids contained the following genetic elements: Ampicillin
reistance gene, ColE1 original of replication, CMVpromoter,
[reporter gene], bovine growth hormone polyA addition site,
florigin of replication, SV40 origin of replication, neomycin
resistance gene and an SV40 polyA addition site, in operable
relationship such that the reporter gene might be expressible in
eukaryotic cells upon introduction of the plasmid. Plasmid DNA was
purified from E.coli by alkaline lysis and further purified using a
Qiagen ion exchange column (accordong to the manufacturer's
instructions).
Liposome preparation
[0051] The liposome used was a 3:1 (w/w) lipid formulation of DOPSA
(2,3-dioleyloxy-N-[(sperminecarboxamido)ethyl]-N-N-dimethyl-1-propanamini-
um trifluoroacetate) and DOPE (dioleoylphosphatidyl ethanolamine)
(lipofectAMINE; Gibco BRLPaisley, Scotland). A number of DNA: lipid
ratios and different injection volumes were used as shown in table
1. The DNA/liposomes were mixed immediately prior to each
experiment. 10 .mu.lof DNA solution was added to 10 .mu.l of lipid
solution, mixed gently and left for 15 minutes at room temperature.
80 .mu.l of PBS was then added to give the final concentration of
DNA and lipid as shown in table 1. This was then injected in to the
uterus of the pseudopregnant mice. (See section above for details
of mice and surgery.)
Histochemical Localisation of .beta.-Galactosidase
[0052] Animals were killed by carbon dioxide inhalation and uterine
horns dissected out, free of fat and mesentery. Each horn was
divided into 3 sections with the top and bottom section being
snap-frozen in liquid nitrogen, and stored at -70.degree. C. prior
to quantification of .beta.-galactosidase content. The middle
section of each uterine horn was fixed in 1.25% gluteraldehyde in
PBS for 15 minutes, rinsed in PBS twice, and placed in X-gal
staining solution (1 mg/ml X-gal, 5 mM K.sub.3Fe(CN).sub.6, 5 mM
K.sub.4Fe(CN).sub.6, 2 mM MgCl.sub.2, 0.02% NP40, and 0.01% sodium
deoxycholate) for 24 hours at room temperature. Sections were then
rinsed in PBS/3% DMSO (2.times.5 minutes), 70% ethanol (3.times.5
minutes) and placed in 100% ethanol. Tissues were embedded in
glycol methacrylate resin and 7 .mu.m sections were cut and
counterstained with neutral red prior to microscopic
examination.
Results
[0053] Table 1 below shows the various conditions employed and the
resulting staining intensity of uterine sections after
administration of DNA/liposome complexes. The results shown in the
Table demonstrate the criticality of the timing of administration
of the plasmid DNA, with administration on day 2 giving the best
levels of expression, administration on day 3 giving reasonable
levels, but administration on day 4 resulted in very little
expression of the reporter gene, presumably because the endometrial
cells would not take up the construct at this time point, for
reasons that are not entirely clear.
1TABLE 1 Day of administration DNA Lipid Injection volume
Histochemical (autopsy day) (.mu.g/ml) (.mu.g/ml) (.mu.l) staining
intensity 2(5) 2 20 50 ++ 3(5) 2 20 50 ++ 4(6) 2 20 50 - 2(6) 2 20
50 +++ 2(5) 8 20 20 ++ 4(6) 8 20 50 - 4(6) 30 20 50 + 4(6) 2 60 50
- Staining intensity +++ strong, ++ moderate, + weak, - none
Controls
[0054] An uninjected 6-day pseudopregnant mouse gave no uterine
staining for .beta.-galactosidase activity.
[0055] A pseudopregnant mouse (administration day 2; autopsy day 6)
injected with 50 .mu.l of pcDNA3 minus .beta.-galactosidase (2
.mu.g/ml) and lipid (20 .mu.g/ml) gave no uterine staining for
.beta.-galactosidase activity.
[0056] Examination of histological sections after staining with
X-gal showed that the glandular epithelium was strongly stained and
luminal epithelium was also stained but less strongly. The optimal
staining was seen in animals transfected with 2 .mu.g/ml DNA and 20
.mu.g/ml lipid in 50 .mu.l administered on day 2 of
pseudipregnancy. FIG. 1a shows a section from such an animal and
FIG. 1b a section from a control animal which received (under
identical conditions) a plasmid which lacked the .beta.-gal
gene.
Example 2
Transfection of Primary Cultures of Human Endometrium
[0057] The inventors have also demonstrated that human endometrial
epithelial cells can be transfected in vitro at high
efficiency.
[0058] The same plasmid (pcDNA3, +/- .beta.-galactosidase reporter
gene) and lipids as already described were used. Endometrial cells
were prepared by the method of Smith and Kelly (Smith et al., 1987
Prostaglandins 34, 553-561). Once the culture were established the
following transfection protocol was used. DNA (2 .mu.g) and
liposome (8 .mu.g) were each diluted in 100 .mu.l of serum free
medium (Opti-MEM1 BRL), mixed and incubated at room temperature for
15 min. Following this a further 800 .mu.l of Opti-MEM1 was added.
The cells (in 24 well plates) were washed with PBS followed by
washing with Opti-MEM1. The DNA/liposome mixture (0.5 ml) was then
added to the cells and incubated at 37.degree. C. for 3 hr in a
CO.sub.2 incubator after which 0.5 ml of culture medium containing
20% foetal calf serum was added. Cells were fixed (0.1%
gluteraldehyde), rinsed and stained with X-gal 24 h after
transfection.
[0059] This work shows that genes can be transferred to the
endometrium in vivo and this will find utility in many endometrial
(and placental) conditions, for example improving implantation in
both animals and man, disrupting implantation (i.e. contraception),
endometriosis and menorrhagia, hyperplasia and adenocarcinoma.
[0060] Additional data were obtained relating to the transfection
in vitro of purified human uterine epithelial cells. This
complements the in vivo mouse work and shows that similar cells,
after minimal time in culture, can be efficiently transfected with
the same liposome and DNAs used in vivo.
Example 3
Transfection of Human Endometrial Epithelium in Vitro
[0061] Human primary epithelial cells from endometrium were
isolated and cultured according to the method of Zhang et al., (J
Cell Science, 1995; 108:323-331). The cells were plated in standard
6 well tissue culture plates to achieve a density of 50% confluence
the next day. The cells were cultured for 5 days, then replated
into 24 well plates, at a density of 60,000 cells per well. The
next day the cells were transfected with DNA/liposome complexes
(DNA/LC). These were prepared as follows: Transfection procedure
Apparatus LipofectAMINE (Gibco Catalogue No. 18324-012), Opti-MEM I
(Gibco Catalogue No. 51985-018) 24 well culture dish Cell culture
medium was as described by Zhang et al (cited above). This consists
of DMEM/HEPES, 10% FCS, Endothelial Cell Growth Supplement (Sigma
Catalogue No. E-2759), at 30 ,g/ml heparin (Sigma Catalogue No.
H-3149), at 90 ,ugml, gentamycin (Sigma Catalogue No. G-1272), at 5
pg/ml, and fungizone (Gibco Catalogue No. 15290-018) at 1 ug/ml.
Also used were Mg++, Ca++free PBS and a 2 ml Eppendorf tube. Two
different plasmid constructs were used containing different
reporter genes. pcDNA3CAT was obtained from Invitrogen Corporation,
and contains a reporter gene encoding the enzyme chloramphenicol
acetyl transferase. The second plasmid pcDNA3]uc comprised the same
vector, but the CAT gene was replaced with the gene encoding the
firefly luciferase enzyme. Large scale DNA preparations of the
vectors were made using the Qiagen midiprep system. As a negative
control, pcDNA3 containing no reporter gene was used. Preparation
of DNA/Liposome complexes 1) Solution A Dilute lag of DNA into
.sup.100li Opti-MEM I in an Eppendorf tube. Use DNA at 1.mu.g/ml
final concentration in the transfection medium.
[0062] 2) Solution B
[0063] Dilute 4 .mu.l of LipofectAMINE into 100 .mu.l Opti-MEM 1 in
an Eppendorf tube. Use lipofectAMINE at 8 .mu.g/ml final
concentration in the transfection medium.
[0064] 3) Combine two solutions A and B into a new tube and mix
gently.
[0065] 4) Incubate at room temperature for 15 minutes.
[0066] Cell Rinsing
[0067] 1) Prior to transfection, rinse the cell monolayer
ro.mu.roughly three times in FRESH PBS without serum.
[0068] 2) Re-rinse the cells monolayer twice with Opti-MEM 1.
[0069] Transfection
[0070] 1) Add 800 .mu.l (total 1.0 ml) of Opti-MEM to each tube
containing the DNA-lipid mixture. Final DNA concentration 1
.mu.g/ml, and lipofectAMINE concentration 8 .mu.g/ml.
[0071] 2) Remove the Opti-MEM 1 in the cell monolayer.
[0072] 3) Mix DNA/LC mixture gently and overlay the diluted complex
solution onto the washed cells, 0.5 ml/24 well.
[0073] 4) Incubate the cell monolayer for 3 hrs at 37.degree. C. in
a CO.sub.2 incubator.
[0074] Further Cell Culture
[0075] 1) 3 hrs later, remove transfection mixture, and add 2 mls
of the Zhang medium to each well, and culture further. Ouantitative
assay 24 to 48 hours after transfection, the cells were extracted,
and assayed for CAT or luciferase reporter activity, as
appropriate. 1. Rinse cells three times in PBS. 2. Extract cells
with 300 microlitres of Lysis buffer (Promega Catalogue No. E-
3871), scraping cells off thoro.mu.ghly into Eppendorf tube. 3.
Freeze extract rapidly at -70.degree. C, and store until assay. 4.
To assay, thaw extracts and centrif.mu.ge at 13,000g for 5 minutes.
5. Repeat freeze/thaw/spin cycle once more. 6. Assay luciferase
reporter gene activity using Luciferase assay kit from Tropix
(Catalogue No. BC100L). Use 40ul of each extract per tube. 7. CAT
reporter gene activity was assayed using the Quan-t-CAT kit from
Amersham (Catalogue No. TRK 1012). Results Primary endometrial
epithelial cells were transfected in 24 well plates as described
above. Transfections were performed on triplicate wells with pcDNA3
(as control), pCDNACAT, and pcDNA3LUC. After 48 hours the cells
were harvested and assayed for luciferase or CAT activity. The CAT
enzyme catalyses the transfer of acetyl groups from acetyl coenzyme
A to chioramphenicol. Use of tritiated acetyl coA results in
transfer of radiolabel to chloramphenicol. The CAT activity in a
sample is directly proportional to the amount of tritiated
chloramphenicol produced. Results are therefore expressed in cpm
per tube. A standard curve can be produced using lysis buffer
containing known amounts of purified CAT. The results are shown in
FIG. 3, which is a bar chart showing the mean +sem for triplicate
determinations for a typical experiment. The results in numerical
form were as shown below, with CAT activity in the cells treated
with pcDNA3CAT about 6 fold higher than the control samples,
demonstrating successful transfection of the endometrial cells.
2 pcDNA3 pcDNA3CAT 710 4480 626 4134 662 3234 mean 662 .+-. 271
3951 .+-. 372
[0076] In the assay for luciferase, the cell extract containing
luciferase is mixed with its substrate luciferin, resulting in the
emission of light. The light signal intensity is proportional to
the luciferase enzyme present in the extract, and can be measured
by a luminometer. Initial results are given in relative light
units.
[0077] Results are shown in FIG. 4, which is a bar chart
illustrating the mean .+-.sem for triplicate determinations for a
typical experiment. The results in numerical form were as shown
below. The signal from cells treated with pcDNA3LUC was over 30
fold higher than the background signal of the control samples,
again demonstrating successful transfection of the endometrial
cells.
Examples of Possible Applications of Endometrial Gene Transfer
[0078] At least seven different types of gene construct could be
transfected into the endometrium to achieve a variety of different
effects. Each of these different types will be described in
turn.
[0079] 1) Over-expression of Cytokines and Growth Factors
[0080] These are conceptually the simplest types of constructs in
that they will be designed to over-express either a cytokine or a
growth factor in the uterine epithelial cells. Examples of suitable
cDNAs for such over-expression include those encoding LIF, VEGF,
EGF, CSF, TNF, Amphiregulin, and a variety of interleukins and
colony stimulating factors. These have been shown to be expressed
naturally in the endometrium and are tho.mu.ght to be important in
regulating endometrial function. (Stewart et al, 1992, Nature, 359,
76-79; Chamock-Jones et al, 1994, Journal of Reproduction and
Fertility, 101, 421-426; Charnock-Jones et al, 1993, Biology of
Reproduction, 48, 1120-1128; Das et al, 1995, Molecular
Endocrinology, 9, 691-; Tabibzadeh (1994, Human Reproduction
Update, 9, 947-967) has published an extensive review of this
field). These agents each affect different aspects of reproductive
function including implantation, blood vessel development and
leukocyte biology. Therefore possible indications for the
administration of such constructs would be where one wished to
improve fertility, particularly in livestock species, or prevent
conception of humans and their companion animals, and also to treat
a variety of menstrual disorders in humans. An example of an
experiment designed to improve fertilirv of livestock It has been
shown that LIF is essential for the process of implantation
(Stewart et al, 1992 cited above). This factor is produced by the
endometrium at the time of implantation. It is therefore possible
that in species where the rates of embryonic loss are high,
increasing the levels of LIF expression from the endometrium at the
time of implantation could reduce these rates of loss. Therefore,
the transfection of a gene construct designed to direct the
synthesis of LIF from the endometrium at the time of implantation
could improve fertility rates in such species. Constructs
transfected into the endometrium would need to contain appropriate
regulatory sequences to ensure that the LIF protein was produced at
the appropriate time. It is likely that this could be achieved by
using the promoter from the LIF gene from the species in question.
Treatments designed to alleviate menstrual dysfunction in women can
also be envisaged using endometrium gene transfer. An example of
this would be to alter the blood vessel development within the
endometrium by transfection of gene encoding angiogenic growth
factors, for example VEGF. A local increase in VEGF production
might be expected to enhance capillary growth and therefore may
promote endometrial thickening. Equally, increased levels of VEGF
may facilitate the repair of capillaries after menstruation and
thus alter bleeding patterns of patients treated with this type of
construct.
[0081] 2) Over-expression of Receptors
[0082] Increasing the number and type of receptors expressed by the
uterine epithelium would be anticipated to have significant
biological consequences. The types of receptors one may wish to
over express include, but are not limited to, growth factor and
cytokine receptors for e.g. EGF, TGF.alpha., VEGF and a variety of
colony stimulating factors and interleukins. Steroid hormone
receptors are also suitable for expression in the epithelial cells.
Such transfection would be expected to be useful where one wishes
to improve fertility, prevent conception, treat menstrual disorder
and also to elucidate the function of orphan receptors (orphan
receptors are receptors where the ligand is currently
unidentified). Orphan receptors represent an area of great interest
to the pharmaceutical industry, since characterisation of the
ligand may well lead to generation of new dr.mu.gs.
[0083] It is becoming increasingly recognized that the development
of the endometrium is a complex process mediated by an interaction
of many cytokines and their receptors and that the stimulatory
effects of ovarian steroids are frequently mediated thro.mu.gh
these cytokines. In particular, it has been shown (Nelson et al.
1992, Endocrinology, 131, 1657-1644) that TGF.alpha. is a potential
mediator of oestrogen action in the mouse uterus. Therefore
transfection of constructs directing synthesis of this factor might
be expected to promote endometrial growth, and therefore might
enhance fertility in situations where the endometrium had not
developed adequately. Similarly, this factor might be anticipated
to promote epithelial surface repair after menstruation, and
therefore be useful in the treatment of menorrhagia.
[0084] There are several members of the steroid hormone receptor
superfamily for which the ligand is currently unknown. Transfection
of such cDNAs into the endometrium could be of great benefit in
elucidating the biological function of these receptors, and
therefore may find application in the search for new pharmaceutical
agents which act upon these receptors, (Evans 1988, Science 240,
889-895).
[0085] 3) Transfection of Constructs Designed to Block or Prevent
the Action of Cytokine Growth Factors and Other Hormones The
transfection of natural antagonists to cytokine and growth factors
opens the possibility for modulating endometrial function. An
example of such antagonists would be the interleukin 1 receptor
antagonist (Hannum et al, 1990 Nature 343, 336). Administration of
this protein has been shown to block pregnancy in mice (Simon et
al, 1994 Endocrinology 134, 521-528). Other natural antagonists of
cytokines and growth factors include the natural soluble receptors.
Soluble receptors have been described in a variety of growth
factor/cytokine systems. For example TNF (Engelmann et al, 1990, J.
Biol. Chem. 265, 14497-14504), FGF (Givol et al, 1992, FASEB
Journal, 6, 3362-3369), PDGF (Tiesman & Hart, 1993, J. Biol.
Chem. 268, 9621-9628) and IL-6 (Novick et al, 1989, J. Exp. Med.
170, 1409-1414). The common feature is that the extracellular
ligand binding domain of the receptor is released from the cell as
a freely soluble factor. This is achieved either by proteolysis or
by alternative splicing which generates a truncated protein
molecule lacking the transmembrane and intracellular domains.
Kendall and Thomas (1993, Proc. Natl. Acad. Sci. USA, 90,
10705-10709) described a soluble variant of the VEGF receptor fit.
This protein was able to block the action of VEGF in vitro. We have
isolated three further cDNAs encoding additional soluble variants
(see PCTI/GB95/01213). The use of these natural agents has several
advantages over other antagonists (for example anti-VEGF
antibodies). Since they occur naturally in the body one would
anticipate that they would not elicit an immune response and should
be well tolerated. Also. since they are derived from the membrane
bound receptor, the binding characteristics will be very similar
and thus will compete very effectively for the ligand. It is
possible that other soluble receptors exist naturally or that they
could be engineered in vitro. It is also likely that if the ligand
binding domain from a member of the steroid hormone receptor family
was expressed, it could act as a dominant negative receptor in that
it would compete for the ligand if expressed within the cell at
high eno.mu.gh levels. Altematively, a non-activating but DNA
binding "receptor" could be used to block gene trancription. This
application would be useful for antagonizing the action on natural
steroids including those which are the as yet unidentified ligands
for orphan receptors (Pemrick et al, 1994 Leukemia 8, 1797-1806).
Signalling deficient receptors from the seven transmembrane domain
receptor family could also be engineered and transfected. The
soluble interleukin-1 receptor antagonist (Eisenberg et al, 1990
Nature 343, 341) has been shown to antagonize the actions of IL-1
in vivo (Simon et al. 1994 Endocrinology 134, 521-528) Thus
transfection of the endometrium with a cDNA construct designed to
direct the synthesis of antagonist would be expected to block
pregnancy in mice.
[0086] Other factors likely to antagonise growth factor or cytokine
action include soluble variants of natural receptors, for example
the soluble variant of the VEGF receptor for flt has been described
by Kendall & Thomas (1993, cited above) and also by Boocock et
al (1995 J. Natl. Cancer Ins. 87, 506-516). Local production of
such factors would be expected to antagonise the actions of VEGF
and may lead to useful therapeutic use in situations where there is
hyperproliferation of endothelial cells, for example in a variety
of menstrual disorders where it is desirable to reduce the
capillary density in the endometrium. This would include malignant
disease.
[0087] 4) Use of Antisense Methods to Prevent Local Production of a
Specific Protein (or Enzyme)
[0088] An alternative approach to block the action of cytokines,
growth factors and hormones would be to use antisense or ribozyme
technology to block either the production of the ligands or the
production of the receptors in the appropriate cells (James, 1991
Antiviral Chemistry and Chemotherapy, 2 191-214; Albert &
Morris, 1994 Trends in Pharmacological Sciences 15, 250-254).
[0089] Antisense technology relies on the binding of a so-called
antisense oligonucleotide or polynucleotide to a cellular mRNA.
This binding prevents the translation of this mRNA, and therefore
reduces the amount of the appropriate protein produced by the cell.
Synthetic oligonucleotides or polyribonucleotides have both been
used successfully for this approach. Liposome mediated transfection
of oligonucleotides or liposome mediated transfection of DNA
constructs which direct the synthesis of longer antisense
polyribonucleotides would be expected specifically and selectively
to reduce protein reduction by the transfected cells. Ribozymes
also prevent protein production by selectively cleaving the RNA
that encodes the specific protein in question. These too can be
transfected in as polyribonucleotides or as DNA constructs which
direct the synthesis of such polynucleotides (for reviews see James
1991, and Albert & Morris 1994, both cited above). An example
of such a use of an antisense ribozyme to prevent fertility would
be as follows. It has already been shown that LIF is essential for
the process of implantation in the mammalian pregnancy (Stewart et
al, 1992, cited previously). Therefore the transfection of either
oligonucleotides or DNA constructs directing the synthesis of
antisense polyribonucleotides, or ribozymes directed against the
LIF MRNA, would be expected to prevent the synthesis of this
factor. The lack of this factor should then lead to a failure of
implantation and therefore conception would be blocked. A similar
approach could be used to block the production of angiogenic growth
factors, for example VEGF, which would prevent the proliferation of
endothelial cells required for tumour growth. Therefore this type
of therapy might be particularly advantageous where malignant
disease is being treated. 5) Local production of Immunoglobulins
and fragments thereof It is possible using modern recombinant DNA
technology to generate single chain antibodies which have nearly
identical binding characteristics to the entire monoclonal antibody
from which they were derived. Such single chain antibodies have
successfully been expressed in bacteria (He et at, 1995 Immunology
84, 662-668). It is possible in principle to engineer a construct
which will direct the expression of a single chain antibody and
express this in epithelial cells. If this was carried out in the
endometrium in vivo one would anticipate that single chain
antibodies directed against a steroid hormone would bind to the
steroid and prevent its action in the epithelial cells. An example
of such an antibody would be the single chain antibody derived from
the antiprogesterone monoclonal antibody DB3 (He et at, 1995, cited
above). If this antibody were secreted into the uterine lumen it
would also bind progesterone and may have actions elsewhere in the
uterine compartment. Antibody directed against growth factors and
cytokines which are known to be active in the endometrium could
similarly block their function if produced locallv in this manner.
An additional application for locally produced single chain
antibodies would be in preventing or treating sexually transmitted
diseases. In this situation antibodies directed against the agent
in question (for example papilloma virus, HIV, chlamydia) would be
secreted into the uterine lumen and prevent infection by the agent
in question. Antibodies directed against sperm or oocyte antigens
could be envisaged to play a role in contraception.
[0090] 6) Active Immunization for Achieving Mucosal Immunity
[0091] An additional method which could be used to achieve local
immunity would be to design constructs which would direct the
secretion of an antigen into the lumen. This would elicit a local
immune response and thus site specific mucosal immunity would be
achieved. It has been known for many years (Howe, 1967 Journal of
Reproduction and Fertility 13, 563-566) that the uterine lumen
contains many leukocytes. It is possible that antigen produced by
transfected endometrial cells taken up by these leukocytes and
subsequently presented to elicit a mucosal immune response.
Delivery of antigens to the intestinal lumen has resulted in such
immunity and in some instances has been shown to be very effective
(for example vaccination against poliomyelitis).
[0092] 7) Blocking Pathogen Attachment Sites
[0093] Attachment of pathogens to mucosal surfaces is frequently an
essential prerequisite to the establishment of infection. Blocking
attachment of the pathogens to these sites may thus present a
method of protecting humans or animals against disease,
particularly sexually transmitted diseases. This applies not only
to bacterial pathogens (such as certain pathogenic strains of E.
coli, and N. gonorrhoea), but also to viral pathogens. Many viruses
when infecting a cell attach by a cell surface "receptor". The
local production of soluble receptors may be expected to compete
with the cell surface molecules and thus prevent viral infection.
Equally the saturation of the cell surface receptors with viral
mimics (which act like the viral "ligand") might also block
infection, as might local production of specific immunoglobulins or
effective binding portions thereof.
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